ACEA Biosciences
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Cell invasion and migration (CIM) are key facets of many critical biological processes. White blood cells (leukocytes) must squeeze between the cells of blood vessels to reach and attack infected tissues, for instance, while embryogenesis is marked by disciplined and well-timed migrations as cells rearrange themselves into tissues and complex structures. Wound healing also is accompanied by migrating cells, as is cancer metastasis.
In transwell-based cell invasion and migration assays, cells seeded in the top chamber of the apparatus migrate across a barrier in response to chemoattractants or other cues. The assay is scored by counting cells on the bottom side of the membrane.
These processes are of intense scientific interest. Among other applications, researchers and therapeutics companies hope to identify chemical compounds that can influence these processes—accelerating wound healing, for instance, or inhibiting cancer spread. Such studies require reliable assays for monitoring migration, and several are available, including the “scratch assay” and the “transwell assay.” ACEA Biosciences Inc. offers the “xCELLigence CIM assay” that combines the benefits of continuous, label free, impedance-based technology with the classic Boyden chamber. Here, we review the pros and cons of these different approaches.
The scratch assay
The simplest approach to monitoring cell migration is the so-called “scratch” assay. A pipette tip or other sharp object is used to gouge a scratch or “wound” in a confluent cell monolayer. Then a microscope is used to observe cells filling in or “repairing” the wound.
A related assay is the “stopper-based assay,” which creates a cell-free gap in a confluent cell monolayer not with a pipette tip but by growing cells in the presence of a stopper, which blocks cell growth in a given space. Once the cells reach confluence, the stopper is removed. Again, the question is, how quickly do the cells fill the gap.
The advantage of these assays is economical—no special hardware is required other than a microscope. However, since microscopic images need to be captured at regular time intervals (e.g., 2 hr, 4 hr, 8 hr, 12 hr, 24 hr, etc.) and the “in-between stages” are not always captured due to the differences in cell types and treatments, these assays are extremely time consuming and not reproducible. More significantly, as two-dimensional one-pot systems, they are incompatible with chemotactic studies—there simply is no way to establish a chemical gradient across the dish.
The transwell assay
Transwell assays come in many forms and throughputs, but all are based on the same basic idea: Cells are seeded in the top of porous insert in serum-free media, while serum or chemoattractant containing media is placed in the well blow. Cells that move through the porous membrane toward the chemoattractant gradients can be quantified by cell counting or using a plate reader. Cell invasion can also be accessed by simply adding a layer of ECM (extracellular matrix) on the top of the porous insert.
Transwell assay—such as the Boyden chamber assay—are compatible with both adherent and non-adherent cells, and most importantly permit chemotaxis studies. But because the cells usually must be stained in order to be quantified—which involves multiple steps including disassembling the apparatus, fixing and staining cells—they are endpoint assays, providing no kinetic data. This can be detrimental when studying primary cells, which vary widely in their response, meaning it can be difficult to know exactly when to stop the experiment.
Some transwell strategies use fluorescently labeled cells instead, which can be tracked over time. But those cells must be labeled prior to the assay, which complicates the experiment and could potentially influence cell behavior.
The xCELLigence CIM assay
The xCELLigence® Real-Time Cell Analysis (RTCA) DP Instrument in combination with CIM-Plate® 16 devices (ACEA Biosciences) allows label-free, automated quantification of cell migration and invasion in real time under physiological conditions. Each well in the CIM-Plate 16 is a modified Boyden chamber. The impedance microelectric sensor on the porous membrane automatically detects cells as they migrate through the porous membrane and attach to the impedance microelectrode in the lower chamber.
The xCELLigence RTCA DP system collects those migratory data over extended periods with no back-end sample processing—there is no need to disassemble the unit and stain and count the cells, and the system software creates the kinetic migration curves automatically. The DP system can hold up to three CIM-Plate 16 devices, resulting in a 48-well throughput.
Like other transwell assays, the xCELLigence CIM assay can be used to vet potential therapeutics and study the basic biology of migratory processes. But in contrast to traditional transwell assays, this CIM assay can track migration in real-time (at second or minute intervals) automatically without exogenous labels. This approach has been shown to be extremely beneficial when working with primary cells. In an epidermal wound healing study (PDF) led by Manton et al., the authors suggested that the CIM assay remove the variability and provide fast, high quality results for less time and consumable cost.
As a cautionary note, the migratory cells must attach to the microelectrode to be detected; therefore the CIM assay is not compatible with non-adherent cells. However, this might be overcome by coating the membrane with ECM proteins or using specific chemokine to enhance cell attachments. Iqbal et al. have successfully measured macrophage migration using this real-time CIM assay [1].
Additionally, the continuous real-time data also identifies optimal time points for performing parallel imaging studies and other functional analyses of cell migration and invasion.
The xCELLigence RTCA DP instrument is available for sale globally. Get a quote.
Reference
[1] Iqbal, AJ, et al., "A real time chemotaxis assay unveils unique migratory profiles amongst different primary murine macrophages," PLoS ONE, 8(3):e58744, 2013. [PubMed ID: 23516549]
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