Journal of Laboratory Automation
The online version of this article can be found at:
2012 17: 59 Journal of Laboratory Automation
Reza Riahi, Yongliang Yang, Donna D. Zhang and Pak Kin Wong
Advances in Wound-Healing Assays for Probing Collective Cell Migration
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Society for Laboratory Automation and Screening
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Journal of Laboratory Automation
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Journal of Laboratory Automation
17(1) 59 –65
© 2012 Society for Laboratory
Automation and Screening
Collective cell migration is a highly regulated cellular activity
involved in numerous normal physiological processes, such
as embryogenesis, tissue repair, angiogenesis, and wound
healing.1,2 Recently, collective cell migration also has been
suggested to play essential roles in invasion and metastasis of
malignant tumors.3 During collective migration, cells often
form integrated monolayers and interact mechanically and
biochemically through cell–cell adhesions. Cell proliferation,
cell–microenvironment interaction, and cell signaling are
also involved in this complex process. To study the dynamic
collective cell migration process, various in vitro techniques,
such as wound healing assays, Boyden chambers, and sprouting
assays, have been developed.4,5 Among these techniques,
wound-healing assays are some of the most common
approaches in studying collective cell migration due to
their simplicity and ability to visualize the cells during cell
In a typical wound-healing assay, a cell-free region is intro-
duced in a cell monolayer by physically removing cells in
the monolayer. The introduction of cell-free region induces
various cellular responses, such as cell growth and cell migra-
tion.6 The dynamics of collective cell migration toward the
cell-free region can then be observed using time-lapse
microscopy, and the migration rate and interactions between
the cells can be analyzed. This is one of the earliest methods
to study cell migration in vitro and has been applied in
various cell types. Although widely used, there are several
limitations in the conventional wound-healing assay. First,
the scraping speed and the geometry of the wounded region
can vary among different experiments. These factors could
potentially make it difficult to compare between experiments.
Second, the scraping process involves mechanical injury to
the cells, and the cellular contents can be released into the
surroundings. The extent of cell injury is difficult to con-
trol and could complicate the cell migration process. Third,
modifying the surface using extracellular matrix (ECM) coat-
ings is challenging because the scratching process might
break the coating. Furthermore, the throughput, reproducibil-
ity, and multiplexability of the conventional wound-healing
assay can often be limited.
With recent advancements in micromachining, optics,
chemistry, and electronics in the past decades, researchers
Riahi et al.Journal of Laboratory Automation
1Department of Aerospace and Mechanical Engineering, The University
of Arizona, Tucson, AZ, USA
2Department of Pharmacology and Toxicology, The University of
Arizona, Tucson, AZ, USA
3Agricultural & Bioseystems Engineering, Biomedical Engineering and
Bio5 Institute, The University of Arizona, Tucson, AZ, USA
*These authors contributed equally to this work.
Received Jul 27, 2011.
Pak Kin Wong, PhD, Department of Aerospace and Mechanical
Engineering, University of Arizona, P.O. Box 210119,
Tucson, AZ 85721, USA
Advances in Wound-Healing Assays
for Probing Collective Cell Migration
Reza Riahi1,*, Yongliang Yang1,*, Donna D. Zhang2,
and Pak Kin Wong1,3
Collective cell migration plays essential roles in a wide spectrum of biological processes, such as embryogenesis, tissue
regeneration, and cancer metastasis. Numerous wound-healing assays based on mechanical, chemical, optical, and electrical
approaches have been developed to create model “wounds” in cell monolayers to study the collective cell migration processes.
These approaches can result in different microenvironments for cells to migrate and possess diverse assay characteristics
in terms of simplicity, throughput, reproducibility, and multiplexability. In this review, we provide an overview of advances in
wound-healing assays and discuss their advantages and limitations in studying collective cell migration.
wound healing, collective cell migration
Journal of Laboratory Automation 17(1)
have developed novel approaches to implement the wound-
healing assay in a more controllable manner. In this review,
we describe the advances in wound-healing techniques for
studying collective cell migration. These assays are catego-
rized according to their wounding principles. In particular,
mechanical approaches, including scraping and physical
blocking, are first described. Then, wounding techniques
based on nonmechanical methods such as chemical, elec-
trical, and optical approaches are reviewed. Finally, key
issues that should be considered when implementing a
wound-healing assay for a collective cell migration study
Mechanical Wounding Assay
The most common method for studying collective cell migra-
tions is mechanical wounding because of its simplicity and
cost-effectiveness. In a mechanical wounding assay, cells are
disrupted physically, and the collective cell migration process
is observed. Mechanical wounding assays can be categorized
into mechanical scratching and stamping assays.
In a typical mechanical scratching assay, cells are allowed
to grow until they reach confluence, and a mechanical wound
is created by physical scraping, as shown in Figure 1A.
Mechanical scraping can be performed using a pipette tip,
needle, bladder, razor, rubber policeman, cotton bud, or
Teflon spatula for creating a single scratch or a plastic hair
comb or wounder for creating multiple scratches simultane-
ously.7-13 To standardize the process and increase the through-
put, the scratching assay can be implemented using a robotic
system.14 To improve the reproducibility and minimize the
surface damage, a drill press assay has been developed.15,16
In this method, a stabilized, rotating, silicone-tipped drill
press is used to create uniform circular lesions in an intact
cell monolayer. In addition, the ECM (e.g., Matrigel) can be
incorporated with the assay to create a pseudo-matrix barrier
on the cell-free surface or wounding sites. Another reproduc-
ible scratching method for cell–substrate interaction studies is
the hydrophilic polydimethylsiloxane (PDMS) slab assay.17,18
This creates a wounded cell layer near the boundary and
minimizes cell debris and surface damage in the free region
of the substrate. In this method, a short hydrophilic PDMS
slab with a desired shape is embedded onto the substrate pre-
coated with the material of interest, allowing cells to grow
on top of both substrate and PDMS. After a confluent cell
monolayer is formed, the removal of PDMS wounds repro-
ducibly the cells near the boundary and allows cells to migrate
onto the debris-free surface.
Another approach for mechanical wounding is the stamp-
ing assay (Fig. 1B). In this method, a confluent cell monolayer
is punched by a stamp to form a reproducible and uniform
pattern. Advantages of stamping assays include creation of
wounds with arbitrary shapes and study of cell migration in
the presence of cell debris. For example, a stamping assay
has been reported to study phagocytosis of cell debris in the
wound site during wound healing.19 In this assay, a PDMS
mold pattern is placed on the cell monolayer to punch cells
and creates wounded cell areas. Because of the presence of
damaged cells and cell debris in the wound site, chemotactic
reagents can be released locally, which influences the cell
migration in directional studies. Another stamping assay for
studying cell regeneration is the stamp-sliding assay.20
Figure 1. Wound-healing assays based on (A) scratching, (B) stamping, (C) solid barrier, (D) liquid barrier, (E) droplet chemical assay,
(F) microfluidic chemical assay, (G) electrical assay, and (H) laser ablation.
Riahi et al.
In this method, a cell monolayer is stamped by a Neoprene
pattern under pressure control followed by rotary or lateral
movement of the stamp to create desired cell patterns, such
as circles and islands. The method is able to make uniform
and narrow cell bands, which expedites the activation of the
entire remaining cell population for collective migration.
Physical Barrier Assay
A physical barrier is another method to create cell-free regions
for collective cell migration. Instead of removing cells in the
monolayer, a physical barrier is applied before cell seeding to
block cell attachment, and the removal of the physical barrier
initiates the cell migration process. The repeatability and
standardization of the physical barrier assay are more
achievable compared with the scratch assay. The use of a
physical barrier also minimizes injury to the cells and keeps
the cell-free region intact for ECM deposition. This could
allow investigation of different molecular mechanisms and
signaling pathways underlying cell migration and elucidation
of the specific functions of ECM components and growth
factors. Using physical barriers to minimize cell damage is
likely to be important for studying collective migration pro-
cesses that do not involve cell injury, such as cancer metas-
tasis, inflammation, and angiogenesis. The barrier assays
can be divided into solid barriers and liquid barriers.
Solid barriers have been used for decades to study cell
migration, as shown in Figure 1C.21 A solid barrier is typi-
cally composed of biocompatible materials. One of the early
solid barriers for cell migration studies is the Teflon fence.22
In this assay, a Teflon fence is placed in a tissue culture dish,
and cells are plated at high density. After forming a conflu-
ent cell monolayer, migration and proliferation of cells are
studied after the release of the Teflon fence. Other solid stop-
pers include the Flexiperm disc23,24 and microstensil.25 These
solid barriers are made of silicone or PDMS with desired
shapes. These materials can adhere to smooth surfaces with-
out glue and are able to create multiple wounding sites. Their
biocompatibility, ease of use, and high adhesiveness render
them useful in numerous tissue culture applications. In addi-
tion, the barrier can have a closed or open shape.21 In the closed
shape, the stopper has no opening holes and acts like a solid
barrier.26 Cells are seated around the stopper and grown until
confluence, after which the stopper is removed. The open-
shape barriers, such as circular rings, consist of inner and
outer compartments in which cells can be seated either inside
or outside of the barrier.27-29 The open-shape assay can have
two different inside and outside coatings based on the exper-
Another physical barrier assay for collective cell migration
studies is called a “detachable substrate.”30,31 This method con-
sists of two complementary parts composed of PDMS and
polystyrene and fabricated by replica molding. In this setup, the
PDMS substrate has a convex edge that is complementary to
the concave edge of the polystyrene substrate, allowing two
parts to be attached and detached repeatedly. After seeding
cells and forming a cell monolayer on one substrate, the
intact complementary substrate is attached and allows cells
to migrate onto the untouched surface for cell migration
studies. This method can also be used for studying cell–cell
interactions. In this case, two different cell types are being
cultured in each substrate separately until they become con-
fluent. Then two parts are joined in discrete configurations
such that two cell types are adjacent to one another—for the
dynamic manipulation of cell–cell adhesion and investiga-
tion of heterotypic cell interactions.
Liquid stoppers are another barrier assay enrolling liquid
or gel barriers in outward cell migration and proliferation
studies in which less substrate and cellular damages are
required near the boundary (Fig. 1D). One of the earliest
liquid stoppers is agarose gel.32 Cells are introduced to aga-
rose gel after being centrifuged into pellets. To isolate cell
suspension, a droplet of the agarose gel–cell suspension is
delivered to the microwell plate surface and cooled down
until the gel solidifies. The medium is then gently added to
wells and is incubated for cell migration studies. To study
cell migration on the various surface coatings without cellu-
lar damage, a multichannel migration device, which takes
advantage of surface tension, can also serve as a liquid stop-
per in microchannels.33 In this technique, cells are first seated
in the main chamber to establish a confluent cell monolayer.
The fluid is prevented from entering the side microchannels
connected to the main chamber due to surface tension con-
trolled by appropriate channel dimensions and material prop-
erties. After forming a confluent cell layer, the fluid in the main
chamber enters the microchannels when they are backfilled
with a syringe, and this is done to investigate the impact of
different ECM coatings on collective cell migration inside
the microchannels. Another method to study sheet migra-
tion on a defined surface is the oil droplet migration assay.34
This is optimized for cell migration and proliferation studies
due to minimum disturbance on seated cells. In this method,
an aqueous drop of medium–cell suspension is pipetted into
a light mineral oil, and the drop falls on the matrix surface
due to the density difference. After a confluent monolayer is
formed, cells are released from the liquid stopper by aspi-
rating the oil out of the dish. In addition, a method called the
bullseye35 is served as a wound-healing assay to observe cell
behavior within the gelled ECM. In this method, cells sus-
pended in the collagen introduced to the central bore of a
disc shape Teflon support. Then, cell-free collagen is pipetted
over Teflon support, and the completed assay peels off the
support after almost a 1-h incubation for radial cell migration
in vitro. This technique provides a useful approach for study-
ing the polymeric ECM encountered by cells in gel-based
Journal of Laboratory Automation 17(1)
The cell monolayer can also be wounded using chemical
methods. For instance, sodium hydroxide has been applied
to lyse cells and create model wounds chemically.36 In this
approach, as shown in Figure 1E, a small droplet of sodium
hydroxide is pipetted onto the cell monolayer to selectively
remove cells in contact with the droplet. The size of the
wound is controlled by the volume of the chemical applied.
To control the location of cell removal, the chemical wound-
ing method can also be implemented using microfluidics.
The flow in a microchannel is generally laminar due to the
low Reynolds number of fluid flows in the microscale, which
does not mix across different streams. Based on the lami-
nar nature of a low Reynolds number flow, a microfluidic
device has been developed to selectively remove cells in a
microchannel.37 The concept of this assay is presented in
Figure 1F. The channel has three inlets, which are respon-
sible for cell seeding, wounding, and wound-healing pro-
cessing. During the experiment, trypsin solution is applied
in one or two inlets depending on the size and location
of the cell-free region. To improve the throughput of this
method, a fully integrated microfluidic system is used for
the wound-healing assay. This system is composed of a
24-well dish and computer controlled. The wounding and
observation are fully automated, and the fluids are precisely
controlled.38 The microchannel also allows cell stimulation
mechanically or chemically during the wounding process.
In particular, fluid flow can be applied to investigate the role
of shear stress in wound healing, and chemical gradients can
be created in the microchannel to study their chemotactic
Compared with mechanical wounding methods, electrical
wounding methods, based on the electrical cell–substrate
impedance sensing (ECSI) technique, can create wounds
with different geometries and measure the electric imped-
ance data of the cell monolayer, which indicates the cell move-
ment. The principle of this method is shown in Figure 1G.
The electrical wound-healing system typically is composed
of a working electrode for cell adhesion and a counter-
electrode. By applying a relatively large voltage between
the electrodes, the cells on the electrode can be electroper-
meablized permanently to generate a model wound in the
Figure 2. Characteristics of wound-healing assays.
Riahi et al.
monolayer.39 After wounding, the variation of impedance
on the cell–adhesion electrode is recorded to monitor the cell
migration. Because of the insulating property of the cell
plasma and membrane, the cell movement can be character-
ized by measuring the electric impedance of the cell mono-
layer. The wounding and measurement processes can be
strongly influenced by the electric signals applied between
the electrodes. For example, the polarity of the cell–adhesion
electrode determines the wounding characteristics if a direct-
current (DC) pulse is applied.40 If the cell adhesion electrode
is positive, the cell wounding is extended beyond the elec-
trode. If the polarity inverses, it will result in a higher uncer-
tainty in the impedance measurement. A high-frequency AC
signal can be applied to improve the electrical wounding
method. With AC electric potential, the cells permanently
electroporated can be confined on the electrode. Besides the
electroporation principle, the electrical wound methods can
also be combined with the physical blocker principle to gen-
erate a cell-free area for collective cell migration. In addition,
a self-assembled inhibitive monolayer can block the cell from
settling onto the electrode after cell seeding.1 When the cell
sheet is confluent and the cell edge forms around the elec-
trode, a DC signal is applied to remove the inhibitive mono-
layer through an electrothermal effect. After that, the cells are
able to migrate onto the cell-free electrode. Compared with
electropermeablization, this method can exclude the cell
debris in the cell-free region. A major advantage of the electri-
cal method is the ability to automate the process for high-
throughput study. An array of wounds can be monitored
simultaneously and automatically based on the impedance.
This can significantly increase the throughput compared with
manual optical characterization with a microscope.
Wounding of the cell monolayer can also be performed
using optical methods. A laser can mediate photothermal,
photochemical, and photomechanical effects on cells.41 The
laser-mediated effects can be used to wound the cell mono-
layer, as shown in Figure 1H. In this method, the cells in the
predefined wound region were eliminated by laser ablation
based on the photomechanical effect.42 The motion of the
laser pulse and the microscopy observation can be com-
puter controlled, and thus this automated method is suitable
for multiwell assays with high efficiency. Other advantages
of optical wounding methods include the ability to create
repeatable wounds, create wounds with arbitrary shapes,
and perform automated microscopic observation using the
Cell migration is an essential component of various pathologi-
cal and physiological processes, including wound healing,
cancer metastasis, embryonic development, and tissue regen-
eration. The requirements of collective cell migration studies
may vary according to the biological question of interest.
Thus, the selection of an appropriate cell migration assay
depends on the various features, including wound geometry,
surface properties, and other method limitations. These impor-
tant criteria are summarized in Figure 2. When cell injury is
involved, such as the wound-healing study, a scratch assay
is commonly used. To examine the influence of cell injury
at the boundary in the collective cell migration process, a
scratch wound-healing assay can be performed in conjunction
with noninjury assays, such as the physical barrier assay.21,40
The scratch assay is easy to perform and cost-effective, but
the result can vary between different laboratories. Other
wound-healing assays such as stamp, laser, and electrical
assays might be used when reproducibility and geometric
control are major concerns. The geometry of the wounded
region in these assays can be well defined and executed in
a reproducible manner. Furthermore, the optical and physi-
cal barrier assays with proper surface modification provide
useful approaches to investigate the roles of ECM proteins
in the collective migration process.33,42 When cells are
introduced to a free region without injury, such as those in
vitro applications dealing with tumor invasion and tissue
regeneration, a barrier assay may eliminate the effect of cell
injury.43 This method is also useful for those research topics
concerning the surface coating effects on collective cell
migration. When the geometry of wounds is of interest, the
solid barrier, optical wounding, and electrical blocker assays
are suitable choices to control the wounding geometry.
Nevertheless, a liquid barrier assay is a good choice where
confluent cells need to migrate onto an intact substratum of
endogenous ECM or purified matrix component, such as in
embryonic development and in angiogenesis during tumor
growth.34,35 The electrical blocker wounding method can also
be considered in this situation. Moreover, the microfluidic
method will benefit the cell migration study involving shear
stress and a chemical gradient, such as in angiogenesis and
osteogenesis. The fluid in the microchannel exerts a shear
stress on the cell monolayer, which mimics the in vivo
condition. To better represent the physiological envi-
ronment and study the role of the ECM, the bullseye
method can be used to avoid rigid substrate for cell migra-
tion in vitro.
In addition to the biological considerations, the assay char-
acteristics, such as the repeatability, throughput, and cost,
should also be considered when selecting a wound-healing
assay. Considering the throughput and repeatability, automated
wound-healing assays are often good choices. For instance, the
scratch assay can be performed automatically by incorporating
robotic techniques to provide assays with high throughput and
repeatability.14 With the high resolution of lasers and the micro-
fabrication of electrodes, the optical and electrical methods are
automated assays with precise control and repeatability. For
Journal of Laboratory Automation 17(1)
the electrical assay, cell movement can be measured through
the changes of the impedance of the cell-covered region.
Multiple wounding assays can be performed and measured in
parallel to increase the throughput. The cost of the assay should
be taken into consideration. The scratch, blocker, and chemical
assays are some of the cost-effective choices for research
applications. Although the optical and electrical methods are
generally more expensive, they can provide high resolution,
high throughput, and flexibility. A general summary of these
considerations is provided in Figure 2.
Numerous ongoing research directions have been performed
to develop novel wound-healing assays for studying collec-
tive cell migration. For instance, research has shown that
cells can behave differently in 2D and 3D conditions.
Research has shown that cells can behave differently in 2D
and 3D conditions. To mimic the physiological condition of
cells, researchers are developing 3D wound-healing assays.
A wound-healing assay in 3D will significantly benefit the
investigation of angiogenesis. In addition, the particle image
velocimetry and other cell motion tracking methods should
be further developed to analyze the characteristics of the col-
lective cell migration process in a systematic manner. On the
other hand, many mechanical and biochemical factors play
important roles in the dynamics of collective cell migration,
such as the stress among the cell in the monolayer, the intra-
cellular gene expression, and the mechanical interaction
between the cells and the substrate. Wound-healing assays
with novel sensing and actuation modules should be devel-
oped to investigate these aspects of collective cell migration.
In addition, the micro- and nano-techniques are versatile for
manipulating the cell microenvironment.44 The incorporation
of novel micro- and nano-techniques in a wound-healing
assay can potentially further enhance the resolution and con-
trollability of wound-healing assays. For example, lithographic
techniques, such as soft lithography and plasma lithography,
can be used to pattern cell monolayers in defined geometry,45
enabling researchers to study the geometric control of col-
lective cell migration.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect
to the research, authorship, and/or publication of this article.
The author(s) disclosed receipt of the following financial support
for the research and/or authorship of this article: R.R. is partially
supported by the University of Arizona TRIF Imaging fellowship.
This work is supported by the National Institutes of Health
Director’s New Innovator Award (1DP2OD007161-01), the National
Science Foundation (0855890), and the James S. McDonnell
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