Preparation of Human Meningeal Cell (HMC) Conditioned Media and Collection of Cerebrospinal Fluid for the Culture of Circulating Tumor Cells

To begin, precoat a T175 flask with Poly-L-Lysine. Place the flask in an incubator at 37 degrees Celsius for one hour. Now use a sterile serological pipette to aspirate the lysine solution.

Pipette 30 milliliters complete meningeal culture medium containing human meningeal cells into the flask. Incubate the cells at 37 degrees Celsius under 5%Carbon dioxide supplementation. When cells reach approximately 75%to 80%confluency, collect and save the HMC cultured media in a 50 milliliter conical tube.

Then add complete meningeal culture medium to the tube in a one-to-one ratio. Finally, pipette the growth factors into the tube to create the HMC conditioned media. To process the cerebral spinal fluid, first place it in a 15 milliliter conical tube on ice to cool it.

Centrifuge the fluid in a pre-cooled centrifuge at 257 G for five minutes at four degrees Celsius. Pipette out the supernatant without disturbing the cell pellet and make aliquots of it in different tubes. Now add one milliliter of PBS to the cell pellet to resususpend it.

Centrifuge the cells at 1, 500 G for five minutes at four degrees Celsius. Aspirate the PBS supernatant while leaving about 50 microliters of it at the bottom of the tube. Perform a cell count with the cell suspension before culturing the cells.

Several growth factors were upregulated in the media cultured with human meningeal cells.