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Wound healing assays: Cell exclusion assays

Cell exclusion zone assays differ fundamentally from wounding assays; instead of removing part of the culture, they provide an artificial barrier to the culturing area. This assay is currently the only method that can investigate the effects of extracellular matrix proteins on cell motility 1. The cell exclusion zone assay also provides researchers with several benefits over traditional wounding assays.

The main advantage is that the assay does not destroy the cell monolayer. Damage to the monolayer during wounding assays has several important implications. Firstly, cells that are damaged release intracellular contents into the medium and generate reactive oxygen species2–4. Cells at the border of the mechanical injury also often transiently retract, and this not observed when exclusion zone assays are used2. Mechanical wounding can also trigger other processes such as anoikis, cell membrane repair, phagocytosis, and cytokine production. These additional processes can increase experimental noise that can affect data analysis3,5.

The second advantage of using cell exclusion zone assays focuses on the cell-free surface that is made when the barrier is removed. The exact geometry of the surface can be easily defined and reproduced based on the design of the barrier. Multiple barriers also allow for parallel testing in the same cell monolayer2,5. When the barrier is removed, the cell-free surface that is created has delineated borders in comparison to those produced in wounding assays, and the defined boundaries improve overall assay reproducibility2. The chemistry of the cell-free surface can also be configured in cell exclusion assays1,2,6. In contrast, the surface chemistry generated in wounding assays is dependent on the extracellular matrix that is deposited by the cell monolayer2,5.

Cell exclusion zone assays are also cost-effective when compared to more repeatable and commercial scratch assays that create wounds, and fewer handling are required5. Several ways exist in creating cell-free zones, as will be discussed below and are summarized in table 1 and are shown in figure 1.

Figure 1 | subtypes of cell removal assays: A) solid barriers B) degradable gels C) aqueous 2 phase systems D) magnetically attachable barriers

1. Solid barriers

Zones of cell exclusion can be created with the use of solid barriers such as stoppers and stencils. These barriers are placed on the microplate surface before plating the cells. Cells are then seeded into the microplate and reach confluency in the presence of the barrier. Once a monolayer has formed, the barrier can then be removed to produce a cell-free surface into which migration can occur2,3,5–7. Solid barriers can be designed to meet the requirements of the researcher.

Barriers require two essential characteristics, namely that they have a high degree of adhesiveness to the surface of the dish and that they can be easily removed after attachment1. To meet these requirements, the material used in the design of the barrier can vary from polydimethylsiloxane (PDMS), silicon, silicone rubber, Teflon, and agarose1–5,7–9. These materials are then generally formed into circular or rectangular barriers capable of being inserted in microplates depending on the experiment10. The use of solid barriers is cost-effective as the barriers can be made to a predefined size negating the need to collect premigration images. In the case of silicon stoppers, these can be sterilized and reused3,7. Assays that make use of solid barriers are compatible with high-content imaging (HCI) techniques, and commercial kits are available such as the Oris™ Cell Migration Assay manufactured by Platypus Technologies and Culture-Inserts by ibidi5,11.

The disadvantage of using mechanical barriers is that there are underlying physiological differences in cell migration based on mechanical wounding or the sudden availability of space when a barrier is removed3,4. These physiological differences must be taken into consideration when designing an experiment. The type of material used for the solid barrier may under high confluency cause the cells to attach to the barrier itself12. When the barrier is removed sheets of cells are then pulled from the monolayer affecting reproducibility. The manual removal of the stopper also limits this assay for high throughput screening (HTS) operations1,5. Barrier removal can circumvent altogether using microfluidics and pneumatic control of barriers13.

2. Degradable gels

The manual removal of solid stoppers can also be made redundant with the use of degradable gel stoppers. As with cell exclusion zone assays that make use of solid barriers, the gel barrier is placed onto the surface of the microplate before cell seeding. The gel barrier either degrades when cell media is added, or a gel removal solution is used to remove the barrier once a cell monolayer has formed. Complete degradation of the gel stopper can take 30-60 minutes. Cells then migrate into the newly exposed cell-free surface14,15.

These water-soluble gels can be made using poly(N‐acryloylmorpholine), amongst other polymers16. Drawbacks with this system can be that cells need to adhere rapidly, cells can settle on the degrading barrier, or cells can detach when the gel is being removed. Cell exclusion zone assays that use degradable gel barriers are well suited for HTS operations 14,17. There are commercial kits available for this assay, including the Radius™ Cell Migration Assay by Cell Biolabs, Inc, Oris™ Pro Cell Migration Assays by Platypus Technologies, and Cell migration BioGel assay by Enzo Life Sciences 14,18–20.

3. Aqueous two-phase systems

The use of aqueous two-phase systems (ATPS) can be applied to the cell exclusion zone assay. In this technique, an ATPS can be produced when solutions of two incompatible polymers are mixed at threshold concentrations. The most well understood ATPS is the polyethylene glycol (PEG)/dextran (DEX) system. PEG and DEX phase separation occurs at low polymer concentrations and under non-denaturing conditions making it viable for mammalian cells21. In addition to ATPS, the use of other immiscible liquids has been applied to cell exclusion zone assays though this has not been widely used22.

To set up a cell exclusion zone assay using ATPS droplets of DEX can be printed onto a substrate and covered with a solution of PEG containing mammalian cells. The cells included in the PEG phase are excluded from entering the DEX phase due to PEG/DEX interfacial tension. A cell-free surface is maintained in the DEX droplet that can then be used for the assay21,23.

The advantages of ATPS are that these systems are inexpensive to establish and do not require sophisticated equipment. The assay is rapid, compatible with a variety of cell types, and can be automated for high throughput21,23,24. The geometry of the cell-free surface can also be controlled in more sophisticated setups25. The drawbacks of this approach are that the viscosity of the DEX phase can result in increased variability of pipetted volumes, and the DEX droplets can be disrupted when the PEG solution is added21.

4. Magnetically attachable barriers

Cell migration is very much dependent on the underlying matrix onto which cells move. Given the importance of the matrix, in vitro assays are needed to evaluate the effects of surface composition, organization, and presentation on cell migration. Magnetically attachable barriers can be used to preserve the underlying substrate of the cell-free surface. To date, these barriers have been produced using PEG and magnetized with iron ferrous microparticles26,27.

Magnetically attachable barriers allow researchers to evaluate unique surfaces such as micropatterned proteins, nano-textured surfaces, and pliable hydrogels26,28. The barriers also do not affect cell viability 27. A drawback to this method is that these barriers are time-consuming to produce 10.

5. Conclusion

Cell exclusion zone assays use physical barriers to prevent cell migration. The underlying surface is then exposed by removing the barriers, creating a void that the surrounding cells then close. This technique makes use of a variety of barriers to exclude cells. These include solid and degradable barriers to ATPS systems and magnetically attachable stencils. The cell exclusion zone assay provides the scientist with a technique of investigating cell migration without wounding the cells and for experimenting with the cell-free surface chemistry. Other cell migration assays include the wound healing and outgrowth assays used to study collective migration, as well as single-cell migration assays. Details of these assays form part of this series of articles on cell migration.

Table 1 | Methods in cell-exclusion zone creation.




Commercial products


Solid barriers

Low expertise required

Limited throughput

Oris™ Cell Migration Assay (Platypus Technologies)

Culture-Inserts (ibidi)


Degradable gels

High throughput

No barrier removal step

Cells must adhere when gel degrades

Proprietary technology limits the availability of surface coatings

Radius™ Cell Migration (Cell Biolabs, Inc)

Oris™ Pro Cell Migration Assays (Platypus Technologies)

Cell migration BioGel assay (Enzo Life Sciences)


Aqueous two-phase systems

Broad cell range can be tested

High throughput

Delicate surfaces can be used

Phase viscosity can affect variability



Magnetically attachable barriers

Unique cell-free surfaces can be investigated

Time-consuming barrier production



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