Josh P. Roberts has an M.A. in the history and philosophy of science, and he also went through the Ph.D. program in molecular, cellular, developmental biology, and genetics at the University of Minnesota, with dissertation research in ocular immunology.
Nearly all cells undergo some sort of migration—from bacteria pushing themselves along by flagella or cilia, to embryonic cells grouping together to form nascent tissues, to metastatic tumors breaking away from their organ of origin and invading a distal site. Not surprisingly, many researchers are interested in discovering the context and cues involved in these processes. To do so, they look to cell-migration assays.
Cellular-migration assays enable investigators to answer basic questions regarding whether a pharmacological, genetic or other perturbation changes a cell’s migratory (or invasive) potential, notes Jun Ma, product manager for cell structure assays at EMD Millipore.
These experiments are performed in cell culture, and the most popular approaches can be broken into two broad camps: gap-closure assays and chemotactic chamber assays. Other commercial and home-brew methods—sometimes newer, trickier, more expensive or specialized, and perhaps requiring specialized equipment or expertise—are also used. Here we break down your options.
Boyden chamber
The classic Boyden chamber assay has been a mainstay of cell-migration analyses for years and is still by far the most published method, notes Ken Rosser, director of marketing and sales for Cell Biolabs. A typical setup uses a tissue-culture plate with a hanging membrane insert. A gradient is created by placing a chemoattractant in the well below the insert; cells seeded on top of the insert can then migrate through the membrane in response to the gradient.
Home-brew assays can be visualized by fixing, staining and visually quantifying cells that have migrated through to the bottom of the membrane; cells that stayed put must be removed, generally with a cotton swab, prior to counting. Several vendors offer kits that release the migrated cells and prepare them for colorimetric or fluorescence quantification in a plate reader. Alternatively, fluorescently labeled cells can be detected by a bottom-reading fluorescence plate reader, without the need to remove or release any cells, by using BD Biosciences’ FluoroBlok inserts, which prevent the transmission of 490- to 700-nm light.
Boyden chamber assays require cells to be in suspension. It’s also important to “choose a pore size that’s slightly smaller than the diameter of the cell,” Rosser advises. “[Cells] will actively push through if they’re incentivized with that gradient.”
Only about 5% to 10% of even the most migratory cells will migrate through, and most of these will do so within the first three to six hours of this endpoint assay, Rosser says. For the most sensitivity, and to maximize the difference seen between experimental and control cells, he recommends seeding as many cells as possible and restricting the run time.
Variations on Boyden chamber assays include microfluidic chambers that allow cells to choose among chemoattractants. These assays can be used with small numbers of rare cells and diminutive amounts of precious compounds, and they are typically visualized by microscopy. But, they also tend to be comparatively expensive to set up and have much steeper learning curves than more traditional methods, says Didier Dreau, assistant professor of biology at the University of North Carolina in Charlotte.
Gap closure
Traditional quick-and-dirty “wound-healing” assays begin by making a scratch across a layer of confluent cells, usually with a pipette tip, leaving a gap to be filled in by migrating cells. Typically used to compare the migration potential of treated to untreated cells (via microscopy), these assays are inexpensive and technically nondemanding . But they also have a downside: It’s difficult to create uniform gaps, cells are damaged in the process, and residue from the damaged cells and substrate all conspire against achieving well-to-well consistency.
EMD Millipore’s Cell Comb Scratch assay offers a more reproducible alternative. The assay uses a comb to create “a lot of uniform, high-density, precise scratches, creating more surface area for the cells to recognize that there is a wound there, and therefore more cells will migrate,” Ma explains.
Systems have also been described in which wounds are created by laser or electrical current. The electric cell-substrate impedance sensing (ECIS) system from Ibidi, for instance, detects cell migration by changes in electrical impedance as well.
Alternatively, by laying down a monolayer around a removable barrier, the need for a “wound” can be eliminated. Platypus Technologies’ Oris™ cell-migration assay uses tapered silicon stoppers to create an exclusion zone in the center of a well, and Cell Biolabs’ CytoSelect™ assay uses a 0.9-mm polycarbonate barrier to bifurcate the well. After cells are seeded and adhered to the plate, the barriers are removed and the cells can begin to migrate into the remaining gap.
Both companies also offer a dissolvable barrier. The Oris Pro assays have “biocompatible gels that are solid when dry; add media and they dissolve,” explains Gopal Krishnan, manager and team leader for cell biology at Platypus. “By the time the gel dissolves, cells are already stuck to the bottom of the well.” In the case of Cell Biolab’s Radius kits, a hydrogel barrier is removed by adding a gel-removal system to the well after the cells have adhered.
Invasion
Invasion, in which cells are required to destroy or modulate an extracellular matrix (ECM) or cell-cell interaction, is closer to what’s going on in a cancer setting, says Dreau. Adding a layer of ECM protein can turn a transwell assay into an invasion assay, for example, “or you can make it more complicated by adding an endothelial layer (which we do some of),” he points out. Inserts coated with different ECM proteins are available from many vendors, including Corning and BD Biosciences. ECM proteins can also be added to gap-closure and microfluidics assays.
The ability of a cell to remodel the ECM can be tested in EMD Millipore's QCM™ Gelatin Invadopodia Assay, in which a GFP- or RFP-conjugated gelatin substrate loses its fluorescence as it digested. Similarly, Life Technologies offers quenched fluorescein-labeled collagen and gelatin substrates (under the DQ™ label) which fluoresce upon being digested.
Of course, the whole point of these assays is to model cell behavior in vivo. But many researchers say they lack physiological relevance. In the past five years of so, for instance, it has become clear that parameters such as the rigidity and microarchitecture of the substratum have profound influences on the way cells migrate, notes Alan “Rick” Horwitz, the Harrison Distinguished Professor of Cell Biology at the University of Virginia.
“What people are doing now is either putting cells into 3D or imaging in vivo,” Horwitz says. “Because some of the mechanisms that are prominent on a plastic dish or in a transwell are not seen as prominently as in vivo, that’s where the assay systems are going to have to go to become the most meaningful.”
Image: The Boyden chamber technique (Credit: EMD Millipore)