Modification of the Boyden chamber to improve uniformity of cell invasion of matrigel-coated membranes.

The ability of cells to invade basement membranes is critical to a number of important biological processes, such as cell metastasis, embryo implantation and early development, and inflammation. Several in vitro invasion assays have been developed so that the invasiveness of cells can be measured. These assays are also used to monitor the altered cell response to stimuli or inhibitors, such as in the evaluation of potential inhibitors of metastasis and other chemotherapeutic drugs. One of the most rapid assays is performed in a modified blind-well Boyden chamber (1,4) containing a filter coated with Matrigel, which is a commercially available reconstituted basement extract from the Engelbreth-Holm-Swarm murine sarcoma (Becton Dickinson, Franklin Lakes, NJ, USA). The Matrigel acts as a barrier on the surface of a porous filter, separating cells in one compartment from a chemoattractant in another compartment. Boyden chambers can be purchased from a company such as Neuro Probe (Gaithersburg, MD, USA) or constructed in the workshop of a typical university or research institute. In brief, an appropriate filter such as one made of a polycarbonate membrane (Osmonics, Minnetonka, MN, USA) is coated with the Matrigel and then placed over the lower well containing a chemoattractant solution. A retainer is screwed into the chamber to secure the membrane and creates the upper compartment to which cells are added, minus the chemoattractant. An important aspect of performing invasion assays in these chambers is the difficulty associated with the quantitation of the results. This is addressed in numerous technical papers that have been published, each describing a new mode of visualizing or quantitating the cells (e.g., References 5 and 6). Many laboratories use a procedure that requires counting or analyzing the cells present on the lower side of the polycarbonate filter by subdividing the membrane into a predetermined grid (4). Therefore, it is essential that the invasion has occurred in a uniform fashion from field to field of the membrane so that counting a defined number of subsections is representative of the invasion that has occurred over the entire membrane. The more uniform the invasion is, the fewer fields that must be counted to yield accurate results. If the invasion occurs nonuniformly, then the selected fields will contain a wide variation in cell counts, leading to nonreproducible results between chambers and between experiments performed on different days. In extreme cases, nonuniform invasion can cause the formation of a ring around the outside edge of the membrane, once it has been stained. In less obvious cases, the nonuniform invasion is apparent from the extreme variation in cell counts between the fields present in different parts of the membrane. In both cases, the invasion patterns are not the result of general leakage between the two compartments. We have tested both commercially available and workshop-made versions of the Boyden chambers, and each is capable of showing the nonuniform invasion effects in a random fashion. Whereas one solution may be to perform additional invasion assays, we have modified the chambers to eliminate this problem. Although manufacturers claim that an O-ring is not required in the commercially available Boyden chambers, we found that the best solution was to include a silicone rubber washer similar to that used in certain leukocyte chemotaxis assays (2). The rubber washer has square edges and provides an efficient seal to prevent leaking. In addition, unlike a traditional O-ring, the use of the washer with square edges reduces the buckling of the flexible polycarbonate membrane while one is screwing down the upper chamber (2). Our procedure differs from the technique used by Bignold (2) in that the filters are also coated with a smooth layer of Matrigel. Thus, the dimensions and quality of the washer are quite important because they must complement the Matrigelcoated surface. While the diameter of the rubber washer will depend on the size of the Boyden chamber being tested, washers to our specifications will fit inside the Neuro Probe chambers and equivalent custom-made versions. The washer is made of silicone rubber, with an outer diameter of 13 mm, an inner diameter of 6 mm, and a thickness of 1.55 mm (Ludowici Rubber and Plastics, Brisbane, Australia). As noted above, the rubber washer has square edges to maintain an accurate seal between the two chambers. These rubber washers are cleaned with the same detergent that we use to clean the Boyden chambers (Tergazyme®; Alconox, White Plains, NY, USA). The experimental methodology used to assess the effectiveness of the washers was as described in detail by Price et al. (4). A 13 mm diameter polycarbonate filter with 12-μm pores was coated with a 1:30 dilution of Matrigel (17 μg/50 μL) and placed over the lower well of the Boyden chamber that had been filled with RPMI containing 10% FCS. The upper compartment was then created by screwing in the retainer either with or without the rubber washer. If the rubber washer was included, then it was placed above the Matrigel-coated membrane. MDA-MB-231 breast cancer cells were grown to a logarithmic growth phase, counted, and resuspended in serum-free RPMI medium, and 3 × 105 cells were added to the upper comBenchmarks