Transfilter Cell Invasion Assays
The ability to invade surrounding extracellular matrices and tissues is an important phenotype of malignant tumor cells. To determine the invasive properties of malignant cells, several invasion assays have been developed. For example, organ fragments (Nicolson et al., 1985), reconstituted tissue spheroids (Mareel et al., 1988), membranous tissues (Nabeshima et al., 1988; Yagel et al., 1989), cultured cell monolayers (Kramer and Nicolson, 1979; Waller et al., 1986), or extracellular matrices (Albini et al., 1987; Schor et al., 1980) have been used as tissue or matrix for cell invasion studies. Commercially available Matrigel, a mouse EHS tumor extract consisting of major basement membrane components polymerized into a gel, has been the most commonly used material to determine the invasiveness of various types of cells (Albini et al., 1987). The apparatus of choice for measuring cell invasion has been a modified Boyden chamber or Transwell. Briefly, the apparatus contains two chambers (upper and lower) that are separated by a microporous polycarbonate filter, the upper surface of which is coated with a thin layer of Matrigel. Tumor cells are placed into the upper chamber where they settle by gravity onto the Matrigel layer. In the lower chamber a chemotactic agent can be placed to stimulate directional cell migration of cells that invade the Matrigel layer. The invasive ability of these cells can be expressed as the number of cells invading through the Matrigel layer and filter with time. For the most part, the invading cells are found on the lower surface of the filter and are not released into the fluid of the lower chamber. Although there are some exceptions to this (Simon et al., 1992), the invasive abilities of tumor cells in the transfilter invasion assay are usually related to their in vivo invasion behavior (Albini et al., 1987; Repesh, 1989; Hendrix et al., 1987). This article describes the basic protocol of the Matrigel transfilter invasion assay.
II. MATERIALS AND INSTRUMENTATION
Culture medium DME/FI2 (Cat. No. 11330-032), fetal bovine serum (FBS), phosphate-buffered saline (PBS) (Cat. No. 14040-133), and trypsin (2.5%, Cat No. 15090-046) are from GIBCO-BRL. Matrigel (Cat. No. 40234) and fibronectin (Cat. No. 40008) are from Collaborative Biomedical Products. Bovine serum albumin (BSA) (Cat. No. 810661, fraction V) is from ICN. EDTA (Cat. No. 423-384) is from CMS. Hematoxylin (Cat. No. GHS-1-16), eosin (Cat. No. HTIIO-3- 16), and 10% neutralized formaldehyde solution (Cat. No. HT50-1-128) are from Sigma.
Transwells (Cat. No. 3421, 6.5-mm diameter, 5-µm pore size) and 24-well culture plates (Cat. No. 25820) are from Corning Costar Corporation. Additional equipment includes general tissue culture supply and equipment, forceps, cotton swabs, a reticle (1/10-mm measurement), and a cell-counting device (Coulter counter, hemocytometer, or equivalent equipment).
A. Matrigel Coating on the Microporous Transwell Filter
1:30 diluted Matrigel solution (sterile): To make 900 µl Matrigel solution for 12 Transwells (50 µl X the number of Transwells), thaw Matrigel at 4°C overnight and mix 30µl with 870µl of ice-cold PBS. Keep ice cold until the solution is applied to the Transwell. Aliquot the remaining Matrigel (e.g., 1 ml each), freeze, and store at -80°C.
1:100 diluted Matrigel solution (sterile): To make 2 ml, mix 20µl of Matrigel with 2ml of ice-cold PBS. Keep solution ice-cold.
B. Setting up the Invasion Assay
2% BSA stock solution: To make 100ml, slowly dissolve 2 g of BSA in ice-cold PBS for 20min and sterilize by filtration through a 0.22-µm filter.
Invasion buffer, 0.1% BSA in DME/F12: To make 40ml, mix 2ml of 2% BSA stock solution with 38 ml of DME/FI2 medium.
EDTA stock solution (200 mM): To make 100 ml, dissolve 7.44g EDTA with double-distilled H2O and adjust the pH to 7.4.
Trypsin-EDTA (0.25%): To make 100 ml, add 10ml of 2.5% trypsin solution and 1 ml of EDTA stock solution into 89ml of PBS.
10% FBS-DME: To make 20 ml, mix 2 ml of FBS with 18 ml of DME/FI2 medium.
Fibronectin solution (10µg/ml): To make 10ml, mix 100µl of fibronectin stock solution (1 mg/ml) with 10ml of invasion buffer. In the example here, fibronectin is used as a chemoattractant.
C. Staining Invading Cells with Hematoxylin-Eosin
D. Counting Invading Cells Using a Phase- Contrast Microscope (or Coulter Counter)
Steps 1. Place the Transwell into a 24-well culture place. Make sure that there is moisture on the inside of the culture plate. Count the cells in the 16 fields indicated in Fig. 2 at 200 × magnification. 2. Using a reticle with 1/10-mm guides (Bausch & Comb Inc.), place the cell suspension onto the stage of a phase-contrast microscope, and measure the diameter of the field (r; mm) with the magnification at 40×. The total number of invading cells (1) can be calculated with the formula
where c is the average number of invading cells per ×200 magnification field and R is the diameter of the filter (6.5 mm). For example, the value of r of a Diaphot (Nikon) is 4.45. Therefore, (5 × R/r)2 = 53.3. If the cells are too dense to count the entire field, a photo as shown in Fig. 2 can be used. Using a Diaphot (Nikon), the areas of a, b, and c in Fig. 2 are 47, 17, and 4.3% of the total area of the field, respectively.
E. Reusing Transwells
Although tumor cells in vivo usually invade connective or parenchymal tissue, a number of reports indicate that the invasive and metastatic phenotypes of cells correlate with their in vitro Matrigel invasion capacity (Albini et al., 1987; Repesh, 1989; Hen&ix et al., 1987). This may be due to the fact that invasion in vivo into connective or parenchymal tissue is limited by the capability of tumor cells to undergo basement membrane invasion, a key event in malignant progression. The invasive process involves at least three events (adhesion, degradation, and migration) (Liotta, 1986); for tumor cells to be invasive, they must be at least somewhat proficient in each step (Nicolson, 1989).
The incubation time for the invasion assay is usually 24 to 72h. We feel that shorter incubation times are better as long as a significant number of cells invade the filter. Using longer incubation times, other parameters, such as the growth of the cells on the Matrigel, loss of cell viability, or the detachment of cells from the underside of the filter, must be taken into account. The incubation time can be varied by changing the amount of Matrigel used in coating the filter, usually in the range of 5 to 50µg (or 10µg in our case).
The very thin Matrigel coating on the lower surface of the filter should be completely dried before adding the 1:30 Matrigel solution in the upper chamber. This will avoid leaks in the filter.
The overlay of 200 µl of double-distilled H2O on the Matrigel solution is necessary for an even coating of Matrigel on the filer.
The number of cells applied onto the filter of the Transwell should not exceed the number that will yield 100% confluency on the filter. Usually the cells are 50-80% confluent on the filter after settling.
The cell suspension volume in the upper chamber should be 200µl instead of 100µl to reduce the meniscus effect that can cause uneven settling of the cells onto the Matrigel layer. Thus the volume of the lower chamber should be increased to 800 btl (from the usual 600µl).
The treatment with 10% FBS medium after harvesting the cells by trypsinization is necessary for the quantitative recovery of the cells.
Several other methods can be used to count the invading cells. For example, cells can contain fluorogenic substrates (Garrido et al., 1995), radioisotopes (Garrido et al., 1995; Muir et al., 1993), or dyes (Imamura et al., 1994). These labels are suitable when dealing with a large number of samples; however, visual cell counting is still necessary for optimizing the methods or for checking reproducibility of the methods. When the number of invading cells is large enough (>1000), the cells can be counted using a Coulter counter after collecting the invading cells by trypsinization of the filter.
Transwells are normally reusable four or five times or until their polycarbonate filters become cracked.
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