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Wound healing assays: Cell outgrowth assays

Cell outgrowth assays, also referred to as nest assays or radial migration assays, can be used to investigate collective cell migration in fields such as oncology and drug discovery 1–5. The outgrowth assay is essentially the opposite of the cell exclusion zone assay and wound healing assay in that cells expand outward from a nest as opposed to inward to close a void6,7. A further difference between the outgrowth assay and the wound healing assay is that it reflects the normal cell microenvironment better8. Both cell outgrowth assays and cell exclusion zone assays resemble the normal cell microenvironment; therefore, these two assays share many of the same advantages. The two main benefits of the outgrowth assay are that the cell monolayer is not wounded and the cell-free surface can be defined before migration9,10.

There are a few disadvantages to the outgrowth assay that should be taken into consideration when designing an experiment. Firstly, the initial amount of cells in the nest cell number may have an effect on the rate of migration. Therefore it is important to repeat assays with different initial cell densities so that these effects can be observed and quantified11. In addition, the geometry of the initial cell area affects cell migration and, therefore, the outcome of the assay9. For most experiments, however, the geometries used in outgrowth assays are either square or circular7. Well-designed experiments can mitigate these disadvantages. The following sections will elaborate on the various outgrowth assays available to the researcher. An overview is summarized in figure 1.

Figure 1 | Examples of cell outgrowth assays: A) explant B) solid barrier C) liquid barrier D) air barrier E) spheroid F) microcarrier bead.

1. Explant outgrowth assays

One assay that lies between in vivo studies and in vitro experiments is the explant outgrowth assay. The method involves the collection of tissue samples from an organism, culturing tissue specimens, and monitoring the outward migration of cells from these explants12. This assay has been used to monitor the outgrowth of keratinocytes from skin explants, study glomerular diseases using outgrowth from kidney explants, investigate tenocyte migration from tendon explants, and examine lymphangiogenesis using lymphatic duct explants12–15. The advantage of using explants in cell outgrowth assays is that the unique features present within the diseased tissue are preserved12.

2. Solid barrier

The most common outgrowth assay features a solid barrier to initially confine cultured cells before migration. This migration assay is most commonly known as the fence assay16. Cells are limited to an area of the microplate with the use of a solid barrier. The barrier is usually cylindrical, with the cells added inside the cylinder and allowed to adhere to the surface of the microplate. The cylinder can be made from glass or metal-silicone8,16–19. Alternatively, stencils made from poly-dimethylsiloxane (PDMS) can be used to create uniform cell islands on the surface20,21. Before the experiment, the barrier is removed, and any unattached cells are rinsed off. After that, the remaining attached cells migrate onto the surrounding cell-free substrate8,16–19.

The advantages of solid barriers include that they are reusable and that they can be adapted for high throughput automated imaging systems3,20. An example of a high throughput system is the injection-molded gaskets developed by Oliver et al. (2020), which enable the performance of 24 radial migration assays simultaneously. However, drawbacks of this system are the non-uniformity of clamping pressure of the gaskets, the formation of bubbles, and thorough cleaning that is required after each experiment3. To date, only commercial cylindrical barriers are available such as the metal-silicone barriers from Aix Scientifics and Pyrex® Cloning Cylinders available from Fisher Scientific and Sigma8,11,18.

3. Liquid barrier

The use of aqueous two-phase systems (ATPS) can be applied to both outgrowth and cell exclusion zone assays. 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 cells22. In addition to ATPS, the use of other immiscible liquids has been applied to cell exclusion zone assays though this has not been widely used23.

To set up an outgrowth assay using ATPS droplets of DEX containing mammalian cells can be printed onto a substrate and covered with a solution of PEG. The cells included in the DEX phase are excluded from entering the PEG phase due to PEG/DEX interfacial tension. A cell-free surface is maintained outside the DEX droplets that can then be used for the assay22,24.

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 throughput22,24,25. The geometry of the cell-free surface can also be controlled in more sophisticated setups26. 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 added22.

4. Air barrier

The liquid-gas interphase can also be used to generate cell-free surfaces for cell outgrowth assays. In this method, droplets of cells are added to a dry surface and are allowed to adhere (between 30-60 minutes). Following adherence, the surface is then entirely covered in cell medium, enabling the cells to migrate out from original droplets27.

This technique is simple to set up and only requires standard cell culture materials. Limitations of this system include the requirement that cells must adhere rapidly, the cell-free surface is initially dry, and due to the necessity of a short adherence time to maintain cell viability in the droplets, the cell patterning can vary27.

5. Spheroids outgrowth

Another regularly used assay is the flat surface spheroid migration assay or simply the spheroid migration assay28–31. This assay is a combination of three-dimensional (3D) cell spheroids and the two-dimensional outgrowth assay. In this assay, spheroids, produced from cells in suspension culture, are attached to the microplate surface and measured for outgrowth2,4,5,32. The surface of the plate can be coated to alter attachment affinity. Laminin, which is a strong adhesion protein for epithelial cells, can be used to increase attachment, whereas, agarose can be used to prevent attachment to the surface2,33.

A significant advantage of using spheroids is that, because of their 3D structure, they more closely represent the tissue physiology being studied, such as small cancer clusters7. Another advantage of starting with spheroids is that they can be produced with a consistent diameter. This uniformity makes the assay accurate and easily miniaturized and automated for high content imaging2. A limiting factor to this assay is that not all cells can form spheroids, and experience with spheroid formation is required7. Commercial plates such as the 15-well µ-Slide Angiogenesis plates by ibidi have been used to prepare gels for spheroid attachment4.

7. Micro-carrier beads

The use of micro-carrier beads is a wholly different approach to tackle reproducibility. Pre-seeded micro-carrier beads provide a highly reproducible basis from which cells can migrate outwards. This assay can be used to study cell migration via analysis of spreading from the point of contact between the bead and culturing surface, or invasion when the bead is embedded in a layer of gel such as fibrin gels34. Beads can be made from hydrated collagen-coated dextran beads35. Commercial beads are available such as Dextran-coated Cytodex 3 microcarrier beads from Amersham Biosciences36. This method yields highly reproducible results; however, there are a few drawbacks. The disadvantages include the cost of the beads, and some beads will be insufficiently coated in cells and must be removed7.

7. Conclusion

A multitude of outgrowth assays is available to the researcher to investigate collective cell migration. These can range in sophistication from spheroid outgrowth assays and ATPS systems to air and solid barriers to create cell nests for expansion. All these assays, except explants, spare the cell monolayer from wounding. The scientist can also specify the substrate onto which cells migrate. The outgrowth can be used in parallel with other cell migration assays. Information relating to collective cell migration techniques, specifically cell removal and exclusion, and single-cell migration assays form part of this series of articles covering methods used in cell migration.




Commercial products



Preserves features of diseased tissue

Requires ethics approval, wounding



Solid barrier

Reusable, automatable

Incomplete contact with the surface

Metal-silicone barriers (Aix Scientifics) Pyrex® Cloning Cylinders (Fisher Scientific and Sigma)


Liquid barrier

Basic setup, automatable

DEX viscosity increase variability



Air barrier

Basic setup

Fast adhering cells required



Spheroids outgrowth

Consistent spheroid size, closer to in vivo physiology, automatable

Limited to a cell that forms spheroids, experience required

µ-Slide Angiogenesis plates (ibidi)


Microcarrier beads





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