Development of a Multidose Irradiation Protocol for Clonogenic Assays in Cell Culture Plates

Abstract

PurposeIn vitro experimental radiobiology is a fundamental tool for understanding the cellular and molecular mechanisms involved in the response to ionizing radiation. Conventional experimental designs require separate irradiations to achieve different absorbed doses, thereby introducing experimental variability between irradiation sessions due to inter-session variability in both culture conditions and irradiation geometry. Here, we developed a multidose irradiation system for multiwell cell culture plates that enables the simultaneous delivery of three distinct dose levels within a single plate, thereby reducing resource consumption and operating time MethodsThe system was designed using a clinical linear accelerator delivering 6 MV X-rays and a 3D conformal irradiation approach based on CT imaging. Dose calculations for 200, 400 and 600 cGy were performed using Monaco Version 5.11. The irradiation geometry was optimized to achieve distinct and well-separated dose regions while preserving dose uniformity within each dose level. Treatment planning showed good agreement between estimated and prescribed dose levels, with mean dose deviation ranging from 0.35-1.63%. Physical verification using TLDs and radiochromic films demonstrated high dosimetric accuracy, showing deviations of 0.5-1.0% and 0.57-3.0%, respectively, expressed as the deviation of the measured mean dose from the nominal administered dose levels. Biological assessment included clonogenic assays in human tumor cell lines and a metabolic assay. Clonogenic assays showed a high concordance between the multidose and single-dose irradiation, with comparable linear-quadratic model fits (extra sum-of-squares F-test, p = 0.0923), confirming preservation of the intrinsic radiobiological response under simultaneous irradiation. ConclusionsThese results support the reliability of the proposed multidose irradiation system for in vitro radiobiology. This robust and reproducible system enables efficient characterization of radiobiological parameters.