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

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.