Peripheral blood mononuclear cell (PBMC) separation is routinely performed by centrifugation with a density gradient medium, and is essential for immunological research and clinical applications. PBMC separation highly relies on laborious, time‐consuming blood layering and PBMC collection by careful pipetting. Current methods to address this problem use a physical barrier formed between the separation medium and a blood sample to avoid the need for delicate pipetting, but still pose a risk of red blood cell (RBC) contamination. Here, a blood divider (B‐DV) is proposed as a centrifuge tube insert that allows blood cells to pass through a separation medium in one direction, while blocking fluid passage in the opposite direction and thus preventing RBC contamination during PBMC harvest. The B‐DV is a 3D‐printed mesh in which a fluid can divide into two, above and below the mesh face as soon as inverting the centrifuge tube. Such complete fluid division enables minimizing the volume of the separation medium to the extent of RBC volume to be packed, thus reducing the sedimentation distance for RBCs, and facilitating rapid PBMC isolation by simple tube inverting. This technology enables rapid, easy PBMC separation without user‐to‐user variability on PBMC separation efficiencies.

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