2.4. Tumor Spheroid Fabrication

MDA-MB-231 cell cultures were detached with TrypLE (Thermo Fisher Scientific, Waltham, MA) to generate a single-cell suspension and diluted to 40,000 cells/mL in ice-cold growth medium. A volume of 50 μL was then added to each well of a 96-well v-bottom plate (Corning, Corning, NY). To prevent cell attachment, the plate was previously coated with 50 μL/well 1% Poly-HEMA in 95% ethanol and air dried at 37°C overnight in a biological safety cabinet. The cells were pelleted into the v-bottom of each well by centrifugation at 1000×g for 10 minutes at 4°C using an Eppendorf 5810R centrifuge (Eppendorf, New York, NY). After centrifugation, 50 μL of ice cold growth medium supplemented with 0.35 mg/mL Matrigel (BD Biosciences, San Jose, CA) was gently layered over each well. The plate was then incubated under standard culture conditions for 24 hours, yielding spheroids with an average diameter of 150–200 μm.