Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

The overall goal of this procedure is to isolate and propagate circulating tumor cells from the whole blood of a syngeneic mouse tumor model with hepatocellular carcinoma metastasis. This is accomplished by first centrifuging, the whole blood collected from the mice and isolating the buffy coat. Next, the red blood cells in the buffy coat are lysed and circulating tumor cells are collected in a pellet.

The cells are then washed, suspended in complete culture medium, and propagated in cell culture. The rapidly growing tumor cells remain viable through numerous passages and can be verified using standard molecular diagnostic techniques. The significance of isolating circling tumor cells using our technique is that it provides a reproducible model, which can be optimized for potential use in a clinical setting.

Demonstrating the procedure with me will be Michelle Nadu, an undergraduate student from our lab To begin harvest. Whole blood from a Ciea Kamau tumor model with hepatocellular carcinoma metastasis as described in a separate J of vertical centrifuge. The collected whole blood at 1, 167 times G for five minutes at room temperature.

Once separated into layers, remove the top plasma layer carefully and transfer it into a separate 1.5 milliliter rogen free tube for further experiments, such as detection of cell-free nucleic acids. Next, transfer the Buffy code layer into a separate 1.5 milliliter ROGEN free tube in preparation for red blood cell lysis. This is necessary to remove residual red blood cells in the Buffy coat layer prior to the isolation of circulating tumor cells.

Start by preparing a one liter solution of red blood cell lysis buffer with a final concentration of 155 millimolar ammonium chloride, and 10 millimolar triss. Adjust the pH of the solution to 7.5. Take it about two to four milliliter aliquot from the stock, one liter red blood cell lysis buffer mix.

Then add one milliliter of the red blood cell lysis buffer to the tube containing the collected buffy coat. Mix the contents of the tube by inverting it multiple times. Then incubate the tube for five minutes at room temperature on the bench.

Next centrifuge the mixed contents for five minutes. At 1, 167 times G after centrifugation, a whitish pellet will be obtained. Discard the reddish supernat slowly and carefully into 10%bleach without any disruption to the pellet.

After discarding the supernatant, wash the whitish pellet in one milliliter of PBS. Then pellet the cells again and resuspend them in complete culture medium. See the resuspended cells into a cell culture dish and incubate the cells at 37 degrees Celsius in a humidified incubator with air and 5%carbon dioxide.

Change the cell culture medium every two to three days. After five to seven days circulating tumor cells will have adhered to the cell culture dish and started to proliferate. In order to verify that the hepatocellular carcinoma circulating tumor cells were correctly isolated.

Perform PCR for a specific segment of the mouse beta globin gene, as described and validated by Stu. Then immunostain the cells with an antibody specific for the hepatocyte specific marker. Cyclic A MP responsive element binding protein three like three using standard techniques shown here are representative images of circulating tumor cells from two separate mice.

In culture from an orthotopic syngeneic mouse model of hepatocellular carcinoma. Metastasis tumor cells adhere to the dish and rapidly proliferate. They can be expanded beyond 25 passages and stand up to repeat freeze thaw cycles.

So after watching this video, you should have a good understanding of how to isolate and propagate circular tumor cells that are viable, as well as to characterize them both molecularly and functionally.