Review of Some Techniques Available to Assess Cellular Motility

Subject: Science
Type: Classification Essay
Pages: 8
Word count: 2102
Topics: Stem Cell, Biology, Health, Medicine, Microbiology


Cell motility is defined as the ability of a cell to move spontaneously and actively from one location to another and perform mechanical work, characterised by the consumption of metabolic energy in the process (Wang et al 2014, p. 89). The term motility entails a number of different types of motion such as swimming, twitching, crawling, swarming and gliding. Cell invasion, on the other hand, is related to cell migration whereby it defines the ability of cells to become motile and intrude neighbouring tissues or move through the extracellular matrix in a tissue (Wang et al 2014, p. 93).Cell motility and invasion are associated with critical roles in an extensive range of biological functions such as various developmental stages, immune function and healing of wounds. However, it is also believed that deregulated motile behavior plays a significant contributory part in pathological processes such as atherosclerosis, metastasis and tumor angiogenesis.Therefore, it is also imperative to note that regulated motility is critical, the implication of which is that certain techniques are required to assess cellular motility. This paper will review some of the techniques that are currently available to assess cellular motility and invasion. It will cover transwell assay, wound healing assay and cell exclusion assay and discuss their principles and differences as well as the advantages and disadvantages of each.

Cell migration is essential for the healing of wounds as the cells of the inflammatory system populate wounds and initiate re-epithelialisation1 but the assessment of such migration is equally important (Schaffer et al 2015, p. 908). The currently available and most commonly used assay techniques are transmigration assays that use the modified Boyden Chambers (essentially with transwell inserts); wound-healing assays; and those that use mechanical barriers such as the cell exclusion zone assay to allow accurate timing of the initiation of cell migration (Dong et al 2017, p. 69). However, each has its own advantaged and drawbacks.

Transwell Assay

Also known as Boyden chamber assay, the transwell assay is used in the analysis of migratory response of endothelial cells to angiogenic inhibitors or inducers (Rutten et al 2015, p. 192). It is based on the principle of two medium containing chambers that are divided by a membrane. The membrane is essentially porous to allow the transmigration of cells and its size (of the membrane) is determined by the size of the cell to be assessed (figure 1A). Therefore, it is imperative that a pore diameter is selected such that it facilitates active transmigration. In the assay, endothelial cells are positioned on a cell culture insert’s the upper layer with porous membrane and a solution that contains the test agent is placed below the cell porous membrane. Cells that will have migrated through the porous membrane after a three to eighteen hour incubation period are then stained and counted. Typically, the membrane is coated with an extracellular matrix component such as collagen that facilitates not only migration but also adherence (Qiu et al 2014, p. 5375).

Available membranes in transwell assay typically range in diameter from three to 12µm. The cells are generally seeded in medium in the upper layer from where they can move vertically through the pores into the lower layer in which there is a higher content of serum in the medium. A notable determinant is the horizontal migration phase of the cells until a pore is actually reached. Two possibilities are available to distinguish and enumerate the migrated cells. The first one involves fixing the cells that go through the membrane then staining and enumerating them. Secondly, the migrated cells can fluorescently be stained and, using cell dissociation agents, be removed from the membrane through dissociation and enumerated by the use of a fluorescent reader.

The key advantage of the transwell assay is in the form of its detection sensitivity (Ma et al 2014, p. 83) since migration through the porous membrane can be originated by a significantly low degree of angiogenic inducers. However, it is considerably difficult to conduct prolonged studies because the test-agent concentration typically equalises rapidly between the compartments above and below the membrane (Qiu et al 2014, p. 5380). Another notable drawback of the transwell assay is the complexity involved in setting up the transwells although cell culture inserts that are commercially available have demonstrated the capability to alleviate the burden.

Wound-healing Assay

It is also known as scratch assay is arguably the most popular technique to study cell migration in vitro.Unlike the transwell assay, wound-healing assay is cheaper and less demanding technically (Jonkman et al 2014, p. 440). However, they both can be used in the assessment of cell motility on two-dimensional (2D) surfaces. In this technique, a confluent plate of attached cells is scrapped off cells usually with the tip of a plastic pipette to wound them, as illustrated in figure 1B. As the cells move from the “unwounded” into the “wounded” zones, it is then possible to microscopically monitor cell migration. The movement is then computed by quantifying how the uncovered area decreases at various time points until it is completely closed. The most common and significant information drawn from the wound-healing assay is the rate at which the gap closes, which essentially is a representation of the rate of the collective movement of the cells (Rodriguez-Menocal et al 2015, p. 24). When the plates are coated with agents such as basal membrane extract, fibronectin, collagen I or collagen IV before seeding of the cells presents an opportunity to assess the migration on various substrates (Chereddy et al 2014, p. 138). In typical tests, the rate of gap closure is analysed and measured under a variety of conditions. The conditions include modulating the composition of the extracellular matrix, altering environmental variables, for example substrate stiffness and treating cells with RNA interference.It is noted that it is not possible to differentiatebetween cell propagation and changes in cell survival through a long-term wound-healing assay (approximately greater than 24 hours) in cell mitosis (Rodriguez-Menocal et al 2014, p. 26). Cells can migrate as epithelial cells (collectively as sheets), as single cells or mesenchymal cells (loosely connected groups). 

A key advantage of the wound-healing assay is that is both easy to seat up and read out the analysis. However, while the wound-healing assay is relatively uncomplicated, it lacks standardisation in its use, the implication of which is that it is difficult not only to compare results but also reproduce experiments among researchers. Further, the scratch is usually of uneven thickness. In explanation, (Jonkman et al 2014, p. 444) notes that the speed of migration of the cell increases just before closure of the wound; thus, differences in the width of the gap before the migration of the cells have critical implications. Equally importantly, some cells remain attached to the scratch’s border following the wounding. They are then reattached to the plate after which they move into the wounded region, the inevitable result of which is adulterated results (Chereddy et al 2014, p. 146). 

Cell Exclusion Zone Assay

The cell exclusion zone assay is similar to the transwell assay and wound-healing assays in that they are all 2D cell migration methods that characteristically use a barrier to stop the engraftment of cultured cells (Farías et al 2015, p. 1222). The barrier is positioned after the culture dish is coated with extracellular matrix protein and seeding of the cells. The barrier is then removed after the cells are confluent, whereby it is possible to observe the resultant cell motility. The currently available exclusion zone assay kits consist of either a 384-well or 96-well plate and since the assay dimensions are small, cell motility area is the only investigable variable. Additionally, since the commercially available plates are already coated with specific extracellular matrix protein, it follows that there is a very limited choice of extracellular matrix proteins that can be assessed.

The wound-healing assay was the only widely known method for many years to assess cell motility. However, the method is known to destroy any extracellular matrix coating, which makes it ineffective in investigating the extracellular matrix and cell motility (Stampolidis et al 2015, p. 40). The cell exclusion zone assay presents a possible means to avoid the cell remnant issue described in the wound-healing assay above. This is achieved by the creation of a cell exclusion region during cell seeding with, for example, microstencils or the application of an electrical fence (Descatoire et al 2014, p. 999). For instance, small silicone stoppers have been designed to fit into the wells of a 96-well plate, in which the stoppers are positioned before the cells are seeded to create an exclusion zone using the stopper tip. The cell density is then adjusted such that the cells are completely confluent. The stoppers are then removed after cell adhesion to create a 2mm diameter circular region that is free of cells as illustrated in figure 1C. The cells will then migrate into this region.

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Comparison of the Techniques

Unlike the wound-healing assay, the cell exclusion zone assay is sufficiently standardised and easy to set up. Further, no special equipment is required for analysis, which makes it a preferred approach among researchers. However, it is only suitable for adherent cells and calls for particular care to ensure that the stoppers are attached tightly or risk cells infiltrating the exclusion zone below the silicone device and adulterating results. The transwell assay, unlike wound-healing and cell exclusion zone assays, is suitable for both suspension and adherent cells (Rutten et al 2015, p. 193). On the other hand, the wound-healing and cell exclusion zone assays are only suitable for adherent cells. All three techniques discussed in this paper do not require special equipment (although transwell assay is relatively complex) and are technically not demanding. For instance, any plate can be used in the wound-healing assay, although a key disadvantage it features unlike the other two is that cells and the extracellular matrix are damaged. Further, there is variation in the wound area in the wound-healing assay while the zone exclusion assay features a cell-free area that is clearly defined. A feature that distinguishes the transwell assay from the cell exclusion and wound-healing assay is the direction of motility: while the cells move horizontally then vertically in the transwell assay, they only move horizontally in the other two.

The transwell assay experiment is arguably more informative in the context of cell motility. According to (Jonkman et al 2014, p. 451), this argument is supported by knowledge that the fact that cells fill the wound simply by propagation cannot be excluded. In contrast, cells in the transwell assay must first digest the artificial extracellular matrix before they migrate in similar manner to the in vivo context. On the other hand, both the wound-healing and cell exclusion zone assays entail the creation of a cell-free region on a confluent cell layer (Farías et al 2015, p. 1231. Cells at the edge will then start moving inwards to cover the gap, although the wound-healing assay features remnant cells, an issue addressed in the cell exclusion zone assay.

The table below outlines a side by side comparison and contrasting of cell zone exclusion, wound-healing and transwell assays.


Cell Exclusion AssayWound-healing AssayTranswell Assay
MeasurementMigration areaMigration areaFluorescence, cell count
SubstrateGlass, plastic or coatedGlass, plastic or coatedPET membrane, PC or coated
Direction of MovementHorizontalHorizontalHorizontal then vertical
Type of AnalysisKineticKineticEndpoint
Recapitulated “in vivo” Migration ModeEMT, collective migration of epithelial sheetsEMT, collective migration of epithelial sheetsChemotaxis, single cell migration
ChemotaxisNot suitableNot suitableSuitable
Stable Chemokine GradientNot suitableNot suitableNot suitable
Live ImagingSuitableSuitableNot suitable
ImmunofluorescenceSuitableSuitableNot suitable
ImmunohistochemistrySuitableSuitableNot suitable
High Throughput Screening SuitableSuitableSuitable

Table 1: Comparison of cell exclusion, wound-healing and transwell assays.

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Cell motility has been described as playing critical roles in normal cellular processes as well as human diseases. Notably, cell migration is essential for the healing of wounds as the cells of the inflammatory system populate wounds and initiate re-epithelialisation1. All the three techniques discussed in this paper are two dimensional but feature marked differences in other areas such as measurement, substrate, direction of movement and type of analysis. While the transwell assay assesses cell motility through porous membranes, wound-healing and cell exclusion zone assays detect 2D movement of cells attached on solid surfaces. With the exception of the transwell analysis that is relatively complex, wound-healing and cell exclusion zone assays are straightforward. 

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  11. Stampolidis, P., Ullrich, A. and Iacobelli, S., 2015. LGALS3BP, lectingalactoside-binding soluble 3 binding protein, promotes oncogenic cellular events impeded by antibody intervention. Oncogene, 34(1), pp.39-52.
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