In Vitro Culture and Expansion of Cerebro Spinal Fluid (CSF)-Circulating Tumor Cells (CTCs) for the Study of Leptomeningeal Disease

To begin, spin the thawed cultures of cryopreserved CSF-CTCs at 257 G for five minutes at four degrees Celsius. Aspirate the supernatant with the pipette. Add one milliliter of PBS to wash the cell pellet.

Aspirate the supernatant again. Next, resuspend the cells in 450 microliters of HMC-conditioned media in three separate wells of a 96 well plate. Pipette 150 microliters of the cell suspension into three different wells of a 96 well plate.

Top up the HMC-conditioned media with fresh media every three days. When the cell culture reaches 90%confluency, transfer the trypsinized cells of a well into an empty well in a 24 well plate. During the continued culture of the tumor cells, select the clones of the cells that transform and expand exponentially for further experimentation.