Self-Pumping Spheroid Membrane Enabling Long-Term Spheroid Culture and Label-Free Immune Cytotoxicity Screening

Paper-based hanging-drop platforms have emerged as low-cost biocompatible systems for tumor spheroid culture, supporting medium exchange and drug screening without scaffolds. However, conventional designs fabricated using wax printing and hydrophobic coatings prevent immune cell transport and dead cell removal, limiting their use in immunotherapy evaluation. To address these challenges, a self-pumping spheroid membrane (SPSM) with a precisely tuned pore structure and vertical wettability gradient was developed, enabling size-selective cell transport and passive dead cell separation. Fabricated via parylene C deposition and O 2 plasma treatment, the SPSM supports long-term spheroid culture (>14 days) with high viability (>95%) and minimal handling. Its utility is validated in cytotoxicity assays using doxorubicin (DOX) and chimeric antigen receptor (CAR) T cells, where label-free efficacy assessment based on spheroid size correlates with fluorescence staining in commercial ultralow attachment (ULA) plates. This platform provides a robust and quantitative approach for evaluating drug and immune cell therapies, readily adaptable for broader immuno-oncology investigations.