The overall goal of this invasion assay is to quantify the migratory and invasive capacities of rare and sensitive cells in a 3D setting without dislodging the cells from their micro-environment. This method can help answer key questions in the cancer research field about the invasion capability of sensitive and oray events, such as cancer cell fusion product. The main advantage of this technique is that the cells are analyzed in their original micro-environment using a 3D setting.
The implications of this technique extend toward therapy of cancer because it can be used to screen pharmacological agents capable of inhibiting cancer cell invasion and metastasis. Visual demonstration of this technique is critical. Aspiration in the temperature and pH of the collagen mixture affect its polymerization and rough handling causes the gel to detach.
Begin by treating a 35 millimeter culture dish with a 10 millimeter glass bottom well with one milliliter of one molar hydrochloric acid to lower the hydrophobicity of the glass. After 15 minutes, wash the plate two times with one milliliter of PBS per wash, followed by one wash with one milliliter of 70%ethanol. Next, rinse the plate with one milliliter of culture medium specific to the cells of interest to be used.
And seed the plate with the cells of interest. Then place the plate in a cell culture incubator. When the cells reach 100%confluency combine 114.5 microliters of freshly prepared rat-tail collagen, 16.6 microliters of 10 X DMEM medium, and 33.1 microliters of culture medium supplemented with chemo attractant of interest in a microcentrifuge tube on ice.
Neutralize the collagen mixture with 14 microliters of sodium bicarbonate and overlay the cells with 80 microliters of gel. Allow the collagen to polymerize in a humidified 37 degree Celsius and five percent carbon dioxide incubator for 30 minutes. Then layer two milliliters of reduced two percent serum cell culture medium over the gel and carefully return the cells to the incubator for the appropriate experimental time period without dislodging the collagen.
At the end of the incubation, gently remove the medium and carefully rinse the gel with one milliliter of PBS. Fix the cells with one milliliter of four percent paraformaldehyde for 15 minutes followed by two washes in one milliliter of PBS per wash. Then label the cells with DAPI solution for 20 minutes at room temperature and image the in vitro three dimensional collagen invasion by confocal microscopy.
Here, quantification of the invasion of the collagen gel by a breast cancer cell line and breast cancer cell fusion products in response to a chemo attractant over a 48-hour migration period, is shown. Notably, the migration of rare breast cancer cell fusion products into the collagen gel in response to the chemo attractant was also observed. The cells remain healthy throughout the collagen gel experiment, as evidenced by their maintenance of robust cell numbers and their morphology at 48 hours after gel polymerization.
This vertical collagen gel assay also allows the kinetics of cell migration at invasion, to be analyzed by imaging the cell migration in Z-series as well as the morphology of the migrating cells to be captured in situ. When attempting this procedure, it is important to remember to use a culture dish that is compatible with your microscope. Following this procedure, other variables like modifying the strength of collagen or using different chemo attractants can be introduced to answer additional questions about the effects of ECM engagement on cell invasion or the of different chemo attractant on cell invasion, respectively.
After its development, this technique paved the way for researchers in the field of biology and cancer to screen for pharmacological inhibitors of metastasis as well as to explore wound healing in all inflammatory responses in vivo. After watching this video, you should have a good understanding of how to assess the migratory and invasive capability of rare and sensitive cells, in a 3D setting, without distorting the cells on the micro involvement. Don't forget that walking with human cancer cells, hydrochloric acid, paraformaldehyde, and DAPI can be extremely hazardous and that precautions, such as using a number two bio septic cabinet and a fume hold should always be taken while performing this procedure.
