Wax-printing-free fabrication of paper-supported 3D cancer cell culture

Three-dimensional (3D) in vitro tumor models are critical for studying transport-limited drug efficacy in solid tumors; however, many existing platforms are technically complex and remain difficult to access. Stacked paper-based tumor models ("cells-in-gel-in-paper", CiGiP) address this challenge by enabling formation of diffusion-limited microenvironments while allowing direct access to cells from distinct tissue depths. Nevertheless, current CiGiP implementations rely on wax or hydrophobic barrier patterning of paper, which has become increasingly inaccessible. Here, we present a wax-printing-free approach for fabricating stacked paper-supported 3D tumor tissues using a simple 3D-printed press-fit enclosure that holds circular paper layers snugly together, thereby enforcing one-dimensional transport without lateral leakage. Using MDA-MB-231 breast cancer cells embedded in Matrigel, we demonstrate the formation of nutrient-limited microenvironments across the tissue depth, as evidenced by layer-dependent cell viability. The platform enables direct quantification of spatial and temporal drug responses, demonstrated using doxorubicin and paclitaxel, both individually and in combination. Layer-dependent cytotoxicity was measured, and combination treatment analysis revealed antagonistic interactions consistent with prior reports. By eliminating the need for hydrophobic patterning, this approach substantially lowers the technical barriers to constructing stacked paper tumor models and is expected to facilitate broader adoption of paper-supported 3D tissues for drug screening and mechanistic studies.