The overall goal of this procedure is to build 3D co-culture models termed mammary architecture and microenvironment engineering, or MAM for use in live cell imaging of cell cell interactions in real time. This is accomplished by first coating cover slips with a fibroblast D quenched, or DQ collagen one. Collagen one matrix.
Next D quenched collagen.Four. Reconstituted basement membrane matrix is added on top of the solidified DQ collagen one, collagen one matrix with embedded fibroblasts. In the third step, an epithelial or tumor cell suspension is layered on top of the cover slips, allowing the cells to attach to the DQ collagen, four reconstituted basement membrane matrix, and then media is added.
Finally, confocal microscopy is used to perform four D imaging, which analyzes an image in three spatial dimensions, plus time of the live mam co cultures. Ultimately four D imaging allows visualization and analysis of the dynamic interaction among breast tumor cells and other cells of the tumor microenvironment that may impact progression to an invasive phenotype. A major advantage of this technique over existing methods like intra vital imaging, is that the interactions being observed in our model are between cells of one species that is human tumor cells and human tumor associated cells, rather than, for example, between human tumor cells and mouse stromal cells.
Generally, individuals new to this method will struggle because assembling the main core cultures need first to be visualized and then requires moderate skills and practice for success. Begin by placing one alcohol sterilized rectangular plastic cover. Slip into a 35 millimeter culture dish or use an IBI tree, 35 millimeter mu dish.
Then mix the desired number of fibroblasts with DQ collagen. Next, using a 100 microliter micro pipetter, carefully pipette and spread 70 microliters of fibroblast DQ collagen, one collagen one matrix over the entire surface of the cover slip and place the culture dish in the humidified 37 degrees Celsius incubator without carbon dioxide for 30 minutes. After the matrices have solidified, allow to equilibrate for 10 minutes in a 37 degree Celsius incubator with 5%carbon dioxide.
Then place the dish under a hood until it comes to room temperature. Add 60 microliters of DQ collagen, four reconstituted basement membrane matrix on top of the solidified DQ collagen, one, collagen one matrix with embedded fibroblasts. Then using a pipette tip carefully spread DQ collagen four reconstituted basement membrane matrix evenly avoiding scratching the fibroblasts DQ collagen one, collagen one matrix.
Then transfer the dish and cover slip to the 37 degrees Celsius incubator with 5%carbon dioxide for 10 minutes, while the DQ collagen four reconstituted basement membrane matrix is solidifying trypsin eyes and count the cells for the co culture. Now place 50 microliters of the cell suspension onto the coated cover slip and place the dish into a 37 degree Celsius incubator with 5%carbon dioxide for 40 to 60 minutes to allow the cells to attach to the DQ collagen. Four, reconstitute a basement membrane matrix after the cells have attached to the matrix, add two milliliters of culture medium containing 2%reconstituted basement membrane to the 35 millimeter dish.
Then incubate for the desired period of time before imaging after washing with PBS, incubate the co cultures with five micromolar cell tracker orange in MEGM medium in a 37 degrees Celsius 5%carbon dioxide incubator for 45 minutes after the incubation, wash the co cultures with PBS again and incubate them with pre warmed MEGM medium for 30 minutes in a 37 degree Celsius 5%carbon dioxide incubator image. The co cultures live in low magnification. For example, at 10 x with a water dipping lens on a confocal microscope, capture optical sections at 10 micron intervals throughout the entire depth of the structures.
Use the optical sections to reconstruct the images in 3D using velocity software or a similar program. Make movies of the 3D reconstructions so that the structures, cell cell interactions and interactions with the surrounding matrix can be visualized. In this representative schematic diagram of a maim co-culture, an IBI treat mu dish was first coated with collagen, one containing DQ collagen, one in fibroblasts as seen here in magenta.
Then a second layer of reconstituted basement membrane containing DQ collagen four was added. Next tumor cells in red were plated on top and 2%reconstituted basement membrane. In culture.
Media represented by the cream colored dots was overlaid with every change of media. The fluorescent cleavage products of DQ collagen one and four are represented in green. Over the 23 days of this representative MCF 10 DCIS, and to WS 12 TI co culture, there was proliferation of the cells imaged here and in the following figures, red as well as degradation of DQ collagen four and one observed here, and in the following figures, green and collagen matrices as indicated by the reduction in volume of the co cultures.
The same live co cultures were observed over the 23 days with images captured by confocal microscopy at days three 16 and 23 of co-culture. The interactions in the Mame co cultures are dynamic and can change over time. For example, as seen in this 3D model of the cell mam, co-culture on day three, the cells are just beginning to proliferate in the matrixes at this point in time, still quite stable as evidenced by the lack of green fluorescence.
However, by day 16, as the cells have continued to proliferate, they have also begun to degrade the matrix structure as observed here by the DQ collagen, one and four cleavage products in green, and the active breaking down of the matrix by the cells where the matrix and the cells co fluoresce as observed in yellow. By day 23, nearly half of the matrix has been degraded by the rapidly proliferating cells compared to day three when the co-culture volume was 110, 000 cubic micrometers. By day 16 to 23, the volume has been reduced to only 46, 250 cubic micrometers in this day.
16 co-culture, the proteolysis of DQ collagen four at the surface of the DCIS cells in red, and the diffuse proteolysis of DQ collagen, one in areas near the fibroblasts in white can be observed. The differential staining used in this co-culture also allows observation of the distinct migration and proliferation patterns of the two cell types After its development. This technique paved the way for researchers in the field of breast cancer to explore the effects of tumor macroenvironment on the progression of cancer, losing live cell imaging of dynamic interactions between cancer cells and surrounding stroma cells.
