Monte Carlo simulation‐guided design for size‐tuned tumor spheroid formation in <scp>3D</scp> printed microwells

This study focuses on the design and fabrication of micromold templates for forming multicellular tumor spheroids in a controlled and high-throughput manner, using polydimethylsiloxane-based micromolds created via soft lithography with 3D-printed molds. The spheroid formation was tested with GFP-expressing HEK-293 cells, achieving high circularity (>0.8) and cell viability (>90%) after 7 days. Spheroids reached sizes of approximately 454-459 μm depending on microwell diameter, with cell seeding concentration at 1500 cells/microwell. Factors such as microwell spacing and surfactant treatment were crucial for uniform spheroid formation, and centrifugation was essential for cell collection. Monte Carlo simulations validated the seeding conditions, enhancing understanding of tumor growth mechanisms.