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 to test their effect on cell migration and invasion.The addition of these factors requires the strict maintenance of the liquid ECM at 4°C to prevent premature gelatinization.
To address this, the CoolBox XT™workstation is compatible with a variety of CoolRack® and CoolSink® modules to provides both greater sterility than a messy ice bucket under the hood and better temperature control of the ECM before adding it to the transwells.
Before embarking on a cell invasion or migration assay, the following points should be considered:
- 1) 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.
- 2) 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.
- 3) 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.
EXTRA-CELLULAR MATRIX (ECM) PREPARATION
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.
Current Method - Directly on Ice
Multi-well tissue culture plates on ice in a typical bucket results in uneven temperature of the extra-cellular matrix (ECM) due to uneven, insulating air pockets in the crushed ice. Additionally, risk of microbial contamination increases because of microbes present in ice and melt water.
CoolSink MethodThe_CoolSink thermoconductive plate holders offer better thermal control of tissue culture plates. Made of a thermo-conductive alloy, the module quickly adapts to the ice temperature (<4°C in 60-90 seconds) and keeps all tubes and plates uniform (+/- 0. 1°C), preventing the ECM from prematurely congealing.
Ice-free Coolbox XTMethod
Tissue culture plates are maintained at ~ 1°C for over 10 hours with an internal, reusable cooling core. The CoolSink module which sits inside the CoolBox XT offer better thermal control of tissue culture plates, without the additional ice-related problems. Using the CoolBox allows greater organization, thermal stability, and aseptic conditions.
NOTE: Cell Invasion and Migration assays can be performed in several plate formats. The CoolSink 48 thermo-conductive module is designed to be compatible with the standard sized 6-, 12-, 24- and 48-well tissue culture plates. The assay can also be performed in a high-throughput mode, using a 96-well tissue culture plate. There are two geometries available; the CoolSink 96F module is for flat-bottomed 96-well tissue culture plates, and the CoolSink 96U module fits the 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.
Additional independent application notes for a cell “wounding” assay, followed by automatic imaging by Essen Bioscience utilizing the CoolBox and CoolSink modules at both 4°C and 37°C, can be found below: