Development and validation of hypoxic chambers combined with an oxygen diffusion model for controlled radiobiological experiments

Tumor hypoxia is a hallmark of cancer that drives radioresistance, metastasis and poor clinical outcomes. In vitro modelling of hypoxic conditions is therefore critical for understanding tumor biology and testing therapeutic strategies. Commercially available hypoxic chambers, while very efficient at maintaining atmospheric conditions of samples, are often not portable, unsuitable for irradiation experiments and have a significant cost.In this study, we developed and tested two in-house hypoxic chambers (a portable chamber and a glove chamber), combined with a 1-D oxygen diffusion model for performing and validating biological experiments under controlled hypoxic environment. The systems were validated under physiologically relevant oxygen conditions (0-4% O2) through continuous monitoring using PICO-O2 and LuminOX sensors integrated via Arduino/Bluetooth. A 1-D diffusion model based on Fick's law was developed, predicting the exposure time required to achieve a target oxygen concentration as a function of the medium height. For a full end-toend test of the system, we conducted immunofluorescence analysis to verify the hypoxic status of the cells and we irradiated triple negative breast cancer cells (MDA-MB-231) with photons and protons at doses ranging from 2.5-12.5 Gy under normoxic (21% O₂ concentration) and hypoxic (4% O₂ concentration) conditions, followed by clonogenic survival assay to test the cytotoxicity and radioresistance.Our results showed the ability of both chambers to properly maintain the target oxygen levels and the 1D-diffusion model reproduced hypoxia establishment times with <5% deviation from direct measurements in the medium (PICO-O2). Immunofluorescence analysis confirmed effective induction of cellular hypoxia, evidenced by increased hypoxia-associated fluorescence and elevated HIF-1α expression under hypoxic conditions compared with normoxia. Additional biological results confirmed hypoxia-induced morphological changes and increased radioresistance, with consistent survival outcomes across both chambers.Together, the two chamber designs and the diffusion model provide a low-cost system for studying hypoxia-induced radioresistance in vitro.