Development and characterization of a polymer–polymer aqueous two‐phase system <scp>3D</scp> cell culture platform for <scp>MDA</scp>‐<scp>MB</scp>‐231 cancer cell spheroid analysis

Abstract BACKGROUND Aqueous two‐phase systems (ATPS) are generated by mixing immiscible solutions of polymer–polymer combinations that above certain concentrations form two phases, enabling selective partition of biological particles, such as cells. Leveraging this, novel biomedical applications have emerged using ATPS for three‐dimensional (3D) cell culture due to their ability to replicate tumor in vivo conditions. Current approaches require sophisticated equipment and trained personnel for tumor cell in vitro production. To address this, a 3D cell culture method using ATPS and basic laboratory equipment for culturing MDA‐MB‐231 cancer cell spheroids (CCS) has been designed. RESULTS CCS configuration is achieved through the confinement of cells within droplets of 12.8% w/w dextran (DEX) 500 000 g mol −1 that are subsequently submerged in 5% w/w polyethylene glycol 35 000 g mol −1 . Confining MDA‐MB‐231 cells in DEX at different cell densities (5.0 × 10 3 to 1.6 × 10 5 cells) produced CCS of diameters ranging from 276.43 ± 17.08 to 734.28 ± 88.85 μm after 48 h of culture, indicating that CCS of increasing size are achieved. Furthermore, 8‐day examination of CCS viability showed reduced cell metabolic activity compared to their 2D culture ( P ‐value &lt; 0.05) and consistent viability from day 4 of culture ( P ‐value &gt;0.05), reflecting diminished diffusion of nutrients and enabling assessment of long‐term biological processes. Finally, CCS showed a deviation from a sigmoidal response to paclitaxel, displaying an increase of 31% in maximum cell viability inhibition ( E max ), in contrast to their 2D counterpart, signifying drug resistance. CONCLUSION These results indicate that the ATPS‐3D approach can produce in vitro models of consistent, predictable signals for endpoint assays and biologically complex responses to drugs. © 2025 Society of Chemical Industry (SCI).