by Caitlin Smith
Like herds of wild animals, cells migrate from one place to another. Although the reasons for cell migration are often unknown, we are gaining a better understanding of migration during normal development, cell growth and differentiation, inflammation, wound healing and dysfunctions such as cancer metastasis. The special case of cell invasion refers to the migration of cells through a three-dimensional extracellular matrix. At the cellular level, migration and invasion occur when a cell forms focal adhesions (integrin-mediated attachment sites at the leading edge of cellular movement). Using a focal adhesion as an anchor point to generate traction, the cell pulls itself through the matrix toward its destination. To study the complex signals that govern these processes, several varieties of cell migration assay have been developed; they measure the number (and sometimes the dynamics) of cells that move from a starting location to an ending location. The four main types of cell migration assays are the transmembrane/Boyden chamber assay, the scratch assay, the cell-exclusion zone assay (also called a 2D cell migration assay or a gap-closure assay) and assays based on microfluidic technology [1]. Choosing which assay to use requires some knowledge of these types and a clear idea of what kind of data you require (e.g., do you need a simple endpoint assay or real-time migration dynamics?). Asking the questions below with respect to your experiments may prove helpful.
When should I use a transmembrane/Boyden chamber assay?
One of the most common assays, the Boyden chamber assay is named after its creator from the 1960s, S.V. Boyden. This assay consists of two fluid-containing chambers separated by a microporous membrane (often coated with extracellular matrix proteins)—usually a polycarbonate cell culture insert nested inside a culture plate well, with the membrane at the bottom of the insert. You put cells in one chamber (or seeded on one side of the membrane) and chemotactic agent (or inhibitor) for testing in the other chamber. Cells respond to the chemotactic agent and migrate (or don’t) through the pores in the membrane. You then count the cells that migrated using a microscope. Advantages of this assay type are that you can use adherent or nonadherent cells, and you can test migration with a chemotactic gradient. Disadvantages are that the concentration gradient of the chemotactic agent is unknown and variable, and traditionally this assay has been an endpoint assay for the most part, because it is hard to see the cells during migration (although this is changing, as described below).
Manual counting cells after migration is tedious, so various means of labeling migrated cells are being developed. “We caution against choosing an assay where cells are pre-labeled,” says Ken Rosser, director of business development at Cell Biolabs. “While this is often marketed as a convenient way to eliminate manual cell counting, such pre-labeling may affect the migratory properties of the cells and lead to false conclusions about their true migratory nature. Cell Biolabs’ CytoSelect™ Cell Migration Assays similarly eliminate manual cell counting but are run without pre-labeling of cells, featuring instead the addition of a fluorescent dye only after the migration assay is complete.”
Another way to solve the cell counting problem is offered by BD Biosciences, which offers a unique membrane material to separate the two chambers. The BD FluoroBlok™ Cell Culture Inserts (which fit inside cell culture wells to create a Boyden chamber) contain a “light tight” membrane that blocks the transmission of light from 490 to 700 nm. Thus, you can detect fluorescence in the bottom chamber, but the light will not reach the top chamber. “In contrast to the BD FluoroBlok membrane, clear membrane inserts must be dismantled for sample processing and analysis, or the cells must be dissociated from the underside of the membrane to measure cell migration or invasion activity,” says Marshall Kosovsky, technical support manager at BD Biosciences. With the BD FluoroBlok system, fluorescently labeled cells migrate through the membrane and are detected using a bottom-reading fluorescence plate reader or fluorescence microscope. This method can be used to observe the migration dynamics of cells in real time for kinetic assays, which means that with today’s technology Boyden chambers are no longer limited to endpoint assays.
BD Biosciences offers variations on its FluoroBlok technology, too. “For example, endothelial cell migration can be examined using the BD BioCoat™ Angiogenesis System: Endothelial Cell Migration, which is comprised of BD FluoroBlok Inserts that are uniformly coated with human fibronectin,” says Kosovsky. “BD FluoroBlok Insert Systems are frequently used for tumor cell migration and invasion, endothelial cell migration and invasion, transendothelial cell migration of leukocytes or tumor cells and blood cell chemotaxis.”
When should I use a scratch assay?
Scratch (also known as wound-healing) assays used to be completely “home brew”—a researcher used a pin-type point to draw a line through a confluent monolayer of cells. Boundaries of the newly created cell-free area were measured under a microscope and observed as cells migrated in to fill the scratched region. More recently, companies such as Essen BioScience have offered an automated robotic pin tool that makes reproducible scratches. Essen’s CellPlayer™ Migration Assay is a 96-well plate system for scratch assays in greater volume. The advantages of scratch assays are that they are simple, and cell movements can be recorded in real time. The disadvantages of the scratch assay are that different labs have different methods for making scratches, and it is hard to control the precise scratch area even with a robotic pin tool, so your control vs. experimental scratches might not be equivalent.
Scratching also can damage the extracellular matrix underneath the cells. “Scratch assays can be useful in studying cytoskeletal structure and cell polarity but are highly variable as well, since the tools and technique for producing scratches have not been standardized,” says Jane Lo, product manager at EMD Millipore. “Wounds can thus be of different sizes and widths, which affects reproducibility and consistency of results. Cells at the edge of the scratch can be damaged, which leads to dysfunction in migration. Also, gradients of soluble factors and chemoattractant effects cannot be studied.” Damaged cells also may release into the media unidentified factors that affect the migration of other cells.
When should I use a gap-closure/cell-exclusion zone assay?
Cell-exclusion zone, or gap-closure, assays are similar to scratch assays in that they measure the migration of cells into a cell-free zone, except that in this case there is no “wound” involved. Cells are grown on a well bottom around something that prevents them from growing in one particular region—for example, a stopper placed in the middle of the well. The experiment begins when the stopper is removed, and you can study how the cells migrate in to fill the void in the monolayer. The advantage of the gap-closure assay is that the cell movements can be studied continuously in real time without the possible complications of wound-related factors, and you can add a matrix overlay to create a three-dimensional cell migration assay. Disadvantages include that you cannot do this with nonadherent cells or a chemotactic gradient (as you can with a Boyden chamber assay).
Platypus Technologies and Cell Biolabs offer gap-closure assays using multiwell plates. Platypus just came out with a high-throughput-compatible assay format that can be read by microscopes or high-content imaging instruments. “We recently launched our Oris™ Pro 384 Cell Migration Assays that are completely automatable, with both a tissue culture-treated surface, as well as a collagen I-treated surface in a 384-well format,” says Keren Hulkower, technical and applications manager at Platypus Technologies. “These represent improvements over our existing 96-well and stopper-based assay formats in that the Oris Pro 384 Cell Migration Assay is a denser format that’s suitable for screening in high-throughput labs and is automation-friendly. The biocompatible gel sits low in the well, and there are no stoppers present that interfere with dispensing cells or require a manual removal step.” Hulkower recommends cell migration assays that allow you to study the movement of your cells in real time. “With the advent of all these high-content analysis instruments and multiparametric readouts, you’re going to want to look at the phenotype of your cells.”
When should I use a microfluidic assay?
Cell migration assays based on microfluidic devices are gaining popularity. The devices vary depending on how they are constructed, but generally such a device will have two connected by an internal channel. You add the cells to the opening on one side and let them adhere to the bottom. Then you create a gradient by adding chemotactic agents to the opening on the other side and observe migration using a microscope. The advantage of microfluidic assays is that they conserve precious reagents and cells (expensive drugs or rare primary cells, for instance); they are also capable of generating linear concentration gradients (as opposed to Boyden chambers for example). The disadvantage is that to maintain healthy cells, the media within the device likely will need more frequent changing because of the small volumes.
EMD Millipore recently released a microfluidic migration assay that can be used alone or to complement its Boyden Chamber QCM™ migration and invasion assay kits. “We recently introduced the Millicell® µ-migration Assay Kit that offers real-time visualization of single-cell migration in a slide-based platform,” says Lo. “Capable of measuring parameters such as cell velocity, directionality and migration index, the assay supports the formation of a stable linear concentration gradient that lasts more than 48 hours, so researchers can distinguish between chemotaxis and random movement of both slow and fast migrating cells. And this kit can be used with fluorescently labeled cells.”
In June, EMD Millipore will release the QCM Invadopodia Assay Kit, “the first and only commercially available kit for visualizing and quantifying various modulator effects on ECM degradation by cancerous invadopodia, which are localized protrusions that lead to tumor cell invasion,” says Jun Ma, product manager at EMD Millipore. “Available with either green or red pre-conjugated fluorescent matrix, this new kit will provide optimized reagents and protocols to enable consistent and reproducible analysis of invadopodia degradation for this historically nonstandardized assay. The kits will also provide for co-localization of invadopodia degradation sites with the actin cytoskeleton to help clearly identify this often difficult-to-visualize event.”
Hulkower says that because most of Platypus’ customers are studying anti-cancer mechanisms, “we have to make our assays much more physiologically relevant. They’ve got to be more predictive of what will happen in an animal model, using these assays as part of a screening paradigm for drug discovery.” Three-dimensional cell migration assays might lend more physiological relevance. “Cells at different interfaces get different signals from the matrix,” says Hulkower. “Like a surface swimmer, where part of their body is in the water and part in the air, cells can have some surfaces exposed to the matrix and others exposed to liquid medium. Our invasion assays are more like swimming totally underwater, where your whole body, or in this case the cell, is completely surrounded by the matrix. There are lots of things to keep in mind when you’re trying to develop models.”
References:
[1] Hulkower, K.I.; Herber, R.L. (2011) Cell migration and invasion assays as tools for drug discovery. Pharmaceutics, 3:107-124.