This article discusses the generation of microtumors, which are three-dimensional in vitro tumor cell aggregates that closely mimic the in vivo tumor microenvironment. These microtumors serve as promising platforms for evaluating drug efficacy.
Microtumors - three-dimensional in vitro tumor cell aggregates - closely mimic the in vivo tumor microenvironment and serve as promising platforms for evaluating drug efficacy. To generate microtumors, begin with a uniform suspension of patient-derived tumor cells.
Next, dilute the cell suspension with the desired volume of a chilled human-derived extracellular matrix or ECM. Pipette small volumes of this cell-matrix mixture onto a customized metal plate containing pins with a hydrophobic coating. The hydrophobic coating on the pins allows the droplets to maintain their spherical shape.
Incubate to facilitate the gelation of the ECM, embedding the cells within. Now, transfer the cell-embedded gel beads to a large volume culture plate containing a suitable media. Incubate for the desired duration. In culture, the encapsulated cells begin to aggregate within the ECM matrix, which provides an environment for unstimulated cell growth.
Additionally, the media components diffuse through the matrix and promote cell proliferation, facilitating them to form three-dimensional microtumors within the beads. Subsequently, transfer the microtumor-containing beads into a multi-well plate containing media. Treat one set of the microtumors with the drug of interest and incubate.
Supplement the microtumors with media and drug solution biweekly. Determine the cell morphology in microtumors via live-cell imaging. The untreated microtumors display continuous growth, while the drug-sensitive microtumors show suppressed growth.
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.
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