Data Sheet 2_An oxygen gradient chamber with simple geometry for generating oxygen gradients in hydrogel-based cell cultures within a well-plate-compatible format.pdf

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.