An oxygen gradient chamber with simple geometry for generating oxygen gradients in hydrogel-based cell cultures...

Oxygen gradients strongly influence cellular behaviour in 3D tissues, but their controlled implementation in standard cell culture remains technically challenging. Here, we present a simple 3D-printed oxygen gradient chamber for hydrogel-based cell culture. The system uses a screw-mounted glass cover to locally restrict oxygen exchange between the gelatine methacryloyl (GelMA) hydrogel and its surrounding medium, thereby generating a diffusion- and consumption-driven radial oxygen gradient without pumps, gas channels, introduction of additional chemicals or complex microfluidics. Hypoxia reporter mesenchymal stem cells embedded within the GelMA hydrogel showed distinct hypoxia sensor signalling underneath the glass-covered region after 24 h. Direct oxygen measurements confirmed gradient formation, with oxygen levels stabilising around 2% O₂ in the middle of the construct and increasing to 5%-6% O₂ in its peripheral regions. As a proof of concept, the hypoxia-activated prodrug tirapazamine was applied in 2D cell culture, 3D hydrogel discs of two different stiffnesses and within the oxygen gradient chamber. The results showed oxygen-dependent toxicity in 2D MSC culture, with IC₅₀ values of 148.7 μM at atmospheric oxygen and 20.4 μM at 2.5% oxygen. In 3D hydrogel discs, the calculated IC₅₀ value at atmospheric oxygen was >400 µM and 47.3 μM at 2.5% oxygen. In the oxygen gradient chamber, tirapazamine-induced cell death was enhanced in the central hypoxic region. Thus, the presented chamber provides an accessible and well-plate-compatible platform of simple geometry for studying spatiotemporal effects of hypoxia and oxygen-dependent drug effects in 3D hydrogel cultures.