Generation of Microtumors: An In Vitro Culture Technique to Culture 3D Microtumors to Analyze Response to Drug Treatment

To generate the microtumors, spin down the dissociated patient-derived glioblastoma xenoline cells and resuspend the pellet at 50,000 cells per 2 microliters of FBS. Next, dilute the cells in ice-cold, high-density human biogel at a 1 to 4 cell to biogel ratio. Then, use an electronic multichannel pipette to dispense 10 microliters of cell mixture per pin onto a 96-pin steel plate with hydrophobic coating.

It's important to prevent bubbles when mixing the cells, medium, and biogel, while still working quickly and efficiently to generate the microtumors before the human biogel begins to solidify on the pins.

When all of the tumors have been plated, transfer the 3D tumors into a tissue culture incubator for 20 minutes to gelate the tumor beads. At the end of the incubation, transfer the microtumors to a 10-centimeter culture dish containing complete neurobasal medium and return the tumors to the incubator.

After 1 to 2 days, use a wide-mouth dispensing pipette to transfer the microtumors into the wells of a 96-well culture plate containing 50 microliters of neurobasal medium per well and add 50 microliters of the appropriate dosing solution to each well. Maintain the treated microtumors in the tissue culture incubator for 1 to 14 days, feeding the tumors twice weekly with fresh medium and drug solution.