A pump-free microfluidic device for integrated multi-functional testing of tumor spheroids

Three-dimensional (3D) multicellular ex vivo cultures have become central tools for cancer research and drug testing under physiologically relevant conditions. Organ-on-a-chip technologies based on microfluidics provide platforms for culturing and analyzing 3D tissues under flow. However, maintaining long-term continuous perfusion typically requires pumps and complex tubing networks, increasing operational complexity, cost, and limiting scalability for routine use. Pumpless approaches have been explored but often suffer from short flow duration, inconsistent unidirectional perfusion, and frequent reservoir replenishment. Here, we present a vertical pump-free fluidic platform designed for spheroid formation, culture, and biological testing. The system integrates molds for spheroid assembly with a modular 3D-printed culture chamber that allows direct sample access. A stackable cartridge-like design enables parallel assays under identical conditions, while the incorporation of commercially available syringes as structural elements improves standardization and reduces the footprint. Continuous perfusion over several days is achieved using a hydrogel-based flow resistor that generates passive pressure gradients. Using three human ovarian cancer cell lines (Ovcar-3, A2780, and Ovcar-8), we demonstrate the formation of uniform spheroids that maintain viability and metabolic activity for up to one week within the 3D-printed cartridges. Drug response was evaluated using paclitaxel, with measurable effects on spheroid growth and invasion. Flow simulations and experimental measurements confirm stable perfusion for approximately 3 days, followed by a gradual decline until cessation at day 7. Overall, this pump-free platform provides a scalable, modular solution for controlled 3D culture and multi-functional assays without external pumping systems.