3D printed microtissue cassettes enabling high throughput proton radiobiological assays

Uncertainties on proton relative biological effectiveness (RBE) across the spread out of Bragg peak (SOBP) (typically assumed to be 1.1) may lead to suboptimal treatment plan and unwarranted toxicity to organs-at-risk. Herein, we report a reliable analytical method to determine the proton RBE along the SOBP and distal fall-off region. The 3D microtissue cassette enables the high throughput assessment of biological assays including clonogenic assay and γ-H2AX assay following a single proton irradiation. Clonogenic assay shows the RBE of 1.6 (10% cellular survival) which is consistent with the deter-mined RBE of 1.58 using the γ-H2AX assay. Besides, we also show that the high spatial resolution of the cassette can distinguish the minute but significant foci changes (number, area) in response to small proton radiation dose fraction. The results validate the reliability of our setup in addressing critical proton radiobiological questions.