Cell migration, the movement of cells from one area to another, is a multi-step process that plays an important role in wound repair, cell differentiation, embryonic development and the metastasis of tumors. Cell invasion encompasses cell migration; however, it requires a cell to migrate through an extracellular matrix (ECM) to become established in a new location.
The ECM, also known as basement membrane extract, is comprised of many factors: proteoglycans, sulfates, non-proteoglycan polysaccharides, collagens, elastins, fibronectins, and laminas. Many commercially available transwells come pre-coated with ECM.
Frequently, however, original research requires novel additions into the extra-cellular matrix (ECM) to test their effect on cell migration and invasion. Commercially available ECM, such as Matrigel™ from Beckton-Dickenson, Culturex® from Trevigen, or other sources of ECM must to be maintained at 4°C to prevent premature solidifying or gelatinization.
ECM is fluid at 4°C, but rapidly congeals at higher temperature. Keeping the ECM at 4°C ensures that the layer of ECM in each well is identical and thus reduces well-to-well variability. Chill pipette tips, transwell, ECM and additives thoroughly, preferably overnight before beginning this assay.
- Choose which cell line to use: ideal growth conditions and densities will vary from cell type to cell type and may need to be re-established with each new line; American Tissue Culture Collection (ATCC) is an invaluable source for the proper care of various cell culture lines
- Determine the chemicals to be tested via addition to the ECM: ranging from matrix metalloproteinases, to drugs that inhibit ECM break down, to signaling proteins that potentiate cell invasion
- Reduce variables: the success of the assays requires attention to variables such as temperature, and sterile technique must be employed to maintain the sterility required for tissue culture
To address this, CoolBox workstations used together with a variety of CoolRack and CoolSink modules, provide both greater sterility than a messy ice bucket under the hood and better temperature control of the ECM before adding it to the transwells.
|Ice Bucket Method|
Cell Invasion and migration assays can be performed in several plate formats:
- The assay can be performed in a high-throughput mode, using a 96-well tissue culture plate
- There are two geometries available: the CoolSink 96F module, for flat-bottomed 96-well tissue culture plates; and the CoolSink 96U module, for 96-well round-bottom tissue culture plates
- Once the appropriate amount of ECM is added to the transwells, move to a 37°C incubator
- To ensure uniform heat transfer at 37°C, a CoolSink module can again be used
- At 37°C, the ECM will rapidly congeal depending on the preparation
- The prepared tissue culture cells are then added on top of the solidified ECM
- Following incubation at 37°C in a tissue culture incubator, cell invasion into the ECM can be evaluated