Encyclopedia of Experiments: Cancer Research
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Transcript
Certain organs produce soluble factors and become preferential sites for breast cancer metastasis. A conditioned medium prepared from these harvested organs cells may help identify and study such factors.
Begin by placing a harvested organ such as a liver in a tube containing cold PBS and flipping the tube up and down to remove the organ's residual blood. Place the organ in a Petri dish. And using a scalpel, dice the organ into small pieces.
Resuspend these pieces into a tube containing a basal medium supplemented with an antibiotic to support cell growth and prevent contamination. Now, pour the cell suspension into a culture plate and incubate at 37 degrees Celsius with a continuous carbon dioxide supply for the required duration.
During incubation, these cells release metabolites, growth factors, extracellular matrix proteins into the medium. Next, transfer the cell suspension to a centrifuge tube and add fresh medium to dilute it. Centrifuge the tube and filter the supernatant, which is the conditioned medium, into a fresh tube.
In the following protocol, we will prepare conditioned media from the mouse lungs, and brain to study the metastatic behavior of breast cancer cells.
To generate the lung conditioned medium, invert the tubes of lung tissue three times to remove any residual blood from the organs. Then replace the wash with fresh cold PBS and repeat until the saline is blood-free.
Next, transfer the lungs into one 60 square millimeter glass Petri dish per mouse and use two sterile scalpel blades to mince the tissues with repeated back and forth slicing.
When the tissue fragments are approximately 1 millimeter cubed in size, resuspend the pieces in the appropriate volume of DMEM/F-12 medium supplemented with antibiotics and incubate the tissues in one well of a 6-well plate per mouse at 37 degrees Celsius and 5% CO2.
After 24 hours, transfer the entire contents of each well into individual 50 milliliter conical tubes and dilute the conditioned media with three equivalent volumes of fresh DMEM/F-12 medium per tube.
Centrifuge the tubes to remove any large pieces of tissue debris. Then pool the conditioned media through a 0.22 micron syringe strainer into a single 50 milliliter conical tube.
