Evaluating Anticancer Drug Efficacy in 2D and 3D Cell Models

Transfected cells were cultured for 48 h and were then maintained alone or in combination with paclitaxel (10 nM) or doxorubicin (10 nM). On day 7, cells were fixed with 4% PFA for 5 min followed by washing twice in PBS and stained with crystal violet (0.1% in 10% EtOH) for 10 min at room temperature. The images were taken and compared with appropriate controls. Subsequently, plates were air-dried at room temperature, and CFUs were quantified by dissolving crystal violet in 5% SDS and measuring absorbance at 590 nm. The experiments were repeated twice, and data are represented as mean ± SD from four technical replicas.
The 3D organoid cultures were conducted as we previously described [21 (link)]. Briefly, 250,000 transfected cells were mixed with overnight thawed Matrigel (Corning cat. no. 356231; Growth Factor Reduced (GFR) Basement Membrane Matrix). Subsequently, multiple drops of cell suspension were plated in pre-warmed (37 °C) 60 mm Ultra-Low Attachment Culture Dish (Corning; 3261). Dishes were then placed upside-down in a 37 °C, 5% CO₂ cell culture incubator to allow the droplets to solidify for 20 min, before adding 4–5 mL of expansion medium alone or supplemented with paclitaxel (10 nM) or doxorubicin (10 nM). Seven days later, organoid formation was observed under the microscope.