Organ Conditioned Medium: Generating Medium from Harvested Organ Cells for Metastatic Cancer Studies

1. Generation of Lung- and Brain- Conditioned Media

1. In a sterile tissue culture hood, invert PBS tubes containing lung or brain three times to remove residual blood from organs and aspirate PBS containing blood. Repeat with fresh cold PBS until solution appears clear with no evidence of blood.
2. Place lungs and brains in separate 60 mm2 glass petri dishes. Using two sterile scalpel blades, mince lungs or brains by repeatedly slicing back and forth until tissue fragments are approximately ~ 1 mm3 in size.
3. Determine the amount of media needed to resuspend tissue fragments from the calculated weight difference.
   NOTE: Lung and brain tissue are weight normalized by resuspension in a 4:1 media:tissue ratio (vol/wt).
4. Resuspend tissue fragments in appropriate volume (determined previously in step 1.3) of Dulbecco's Modified Eagle's Medium (DMEM):F12 media supplemented with 1x concentrated mitogen supplement and penicillin (50 µg/ml)/streptomycin (50 µg/ml).
5. Add resuspended lung or brain tissue fragments to one well of a 6-well plate.
6. Incubate tissue fragments in media for 24 hr at 37 °C and 5% CO2. Following incubation, collect conditioned media for each tissue and further dilute by adding three equivalent volumes of fresh media in a 50 ml conical tube.
7. Centrifuge at 1,000 x g in diluted conditioned media for each organ at 4 °C for 15 min to remove large tissue debris. Collect media supernatant and filter through a 0.22 µm syringe filter.
8. Pool conditioned media from each organ (i.e., lung with lung and brain with brain) from multiple mice to account for mouse-to-mouse variability. Aliquot and store conditioned media at -80 °C until use.