60 mm ultra low attachment culture dish

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.

The use of teeth and the study protocols were approved by the Institutional Ethics Committee Board of the Guanghua School of Stomatology, Sun Yat-sen University (KQEC-2019-06). Informed consent was obtained from the donor patient family. We obtained exfoliated third molars from healthy donors 18-24 years in age who provided informed consent. DPSCs were isolated as previously described. Briefly, the pulp in the chamber and canals was gently removed using various instruments and cut into small fragments. The dental pulp tissue was added to a solution of collagenase type I (4 mg·mL⁻¹; Sigma-Aldrich, MO, USA) and dispase (4 mg·mL⁻¹; Sigma-Aldrich). The solution was placed at 37 °C for 30 min, with the tube inverted at 10-min intervals. The single-cell suspension was cultured in low-glucose Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY, USA) containing 20% fetal bovine serum (Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich) in a 60 mm culture flask (Corning, Cambridge, MA, USA) or 60 mm ultra-low-attachment culture dish (Corning) at 37 °C in a 5% CO₂ humidified atmosphere.