Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

The overall goal of this procedure is to study breast cancer tumor agenesis in mice. First, the preparation of primary cells from mouse breast tumors is demonstrated. Next, how to extract the mouse lung for visualizing lung metastasis is presented.

Finally, the preparation of mouse embryonic fibroblasts from mouse embryos is shown ultimately, as these methods demonstrate mice transgenic for the mouse mammary tumor virus polyoma middle T antigen can be used to investigate breast cancer tumor agenesis. Though this method can provide insight into breast cancer, it can also be applied to the investigation of other solid tumors. Visual demonstration of this method is critical, as it is important to isolate only tumors lot other tissue during the tumor illation steps Begin by using scissors to open the skin from the urethral orifice to the neck of a mouse mammary tumor virus polyoma middle T antigen transgenic mouse.

Then pin down the skin and isolate the mammary breast tumor. Wash the tumor in situ with cold PBS and place the tumor in a sterile 10 centimeter tissue culture plate. Next, transfer the plate to a sterile laminar flow hood and use a sterile razor blade and scissors to chop the tumor into one cubic millimeter pieces.

Incubate the pieces with collagenase on a rocker at 37 degrees Celsius. After two hours, spin down the resulting tumor tissue suspension. Wash the pellet three more times in PBS Resus, suspending the pellet in two milliliters of tripsin EDTA after the last wash for five minutes at 37 degrees Celsius with mutating.

Then wash the cells three more times and resuspend them in normal growth medium. Finally, incubate the cells on a 10 centimeter tissue culture dish, changing the media every day for the next three days until the desired cell number is obtained. Before extracting the lung, remove the liver and diaphragm and dislodge the ribs.

Then open the upper part of the trachea, close to the jugular vein and use a catheter to inject the appropriate agent through the trachea to fill the lungs, remove the lungs, and then if using Z fix, embed the tissue in paraffin. If using India ink detain the tissue inec solution for five minutes, the normal lung tissue will appear black in color while the tumor nodules will appear. White classify any tumor nodules visualized at secondary sites with a greater than or equal to 0.5 millimeters in diameter as metastases to generate mouse embryonic fibroblasts.

First, open the skin of a pregnant mouse from the urethral orifice to the neck as just demonstrated taking care not to disturb the embryos. Next, remove the uterine horns and rinse them in sterile PBS. Then carefully separate the embryos from the placenta and one at a time.

Place each embryo in a 10 centimeter culture dish containing ice cold PBS. After removing the head, liver, and gut rinse the remaining portion of the embryo in one well of a 12 well tissue culture plate containing PBS cut the body into one cubic millimeter pieces. Transfer to another well containing five milliliters of tripsin EDTA and incubate the tissue at 37 degrees Celsius.

After five minutes, add one milliliter of FBS to inactivate the digestion. Then spin down the resulting cell suspension and resuspend the pellet. In one milliliter of warm DMEM, finally transfer the cells to a 10 centimeter culture dish containing 10 milliliters of DMEM supplemented with antibiotics and culture the cells until the desired cell number is obtained.

The tumor progression stage at which to sacrifice the animal depends on the particular study and I-A-C-U-C guidelines. For example, this 100 day old FVB mouse with the mouse mammary tumor virus polyoma middle T antigen was sacrificed at three months after isolating a tumor as just demonstrated mouse breast. Primary tumor cells were obtained.

Since many genes mediate breast cancer metastasis to the lung, the lungs also were extracted as just demonstrated to study the metastatic process. The lungs were then inflated with Z fix and stained with India ink To visualize the tumor nodules. Here, mouse embryonic fibroblasts, isolated from FVB strain mouse embryos are shown.

After watching this video, you should have a good of how to isolate primary mouse tumor cells and mouse embryonic fibroblasts, and extract the mouse lung. Don't forget that working with ISO fluorine can be extremely hazardous under that precautions, such as working with a chemical F hood should always be attacking while you're performing this procedure.