Selective Cell‐Repellent 3D Scaffold Platform for Proton Radiobiology

In vitro models play a vital role in elucidating cellular behavior and disease mechanisms within reproducible, ethical, and physiologically relevant microenvironments. While 2D models are easier to use and cost‐efficient, they often alter physiological cell morphology and function. In contrast, 3D models more accurately recapitulate in vivo‐like architecture, cell–cell interactions, and tissue‐specific behavior. However, scaffold‐based 3D culture systems are commonly fabricated on planar substrates, which can lead to cell attachment and proliferation on the surrounding 2D surface, depending on factors such as substrate chemistry, surface functionalization, scaffold geometry, and culture conditions. Therefore, minimizing influence from 2D‐grown cells on devices including 3D‐engineered cell microenvironments is crucial in assays such as gene expression analysis, to avoid undesired 2D‐cell contributions in biological readouts. In this work, we report the combination of ad hoc biochemical cell‐repellent Lipidure coatings with two‐photon polymerized 3D‐engineered glioblastoma (GBM) microenvironments, able to substantially exclude the formation of a 2D GBM cell monolayer in the context of proton radiobiology studies. The proposed platform enables more reliable biological characterization of GBM cells in 3D, precise detection of DNA damage markers following proton radiotherapy, and sufficient RNA yield for gene expression profiling.