Oxygen concentration measurement in 3D cell culture using multifocal optical projection microscopy

Control over molecular oxygen concentrations in cell cultures is vital for maintaining normal physiological functions and modelling pathological conditions. However, current approaches for measuring oxygen are often invasive or limited in their capability to assess oxygen distribution in large-volume 3D cell cultures beyond a few hundred microns in depth. In this work, we have developed an adaptable method utilizing multifocal optical projection microscopy and commercially available fluorescent microsensor beads. Fluorescent projection images of the beads were acquired while simultaneously measuring oxygen concentration with an optical fibre-based sensor. A Stern-Volmer calibration curve was then generated by depleting oxygen with sodium sulfite, allowing fluorescence intensity to be converted into oxygen concentration. The method was demonstrated to quantify oxygen concentrations at depths beyond typical 3D cell culture dimensions, up to 21 mm. Fibroblasts were cultured within agarose hydrogels at varying cell densities (200 000 to 700 000 cells per ml). The results revealed a significant decrease in oxygen concentration with increasing cell density and depth of the specimen, thus also highlighting the need for O₂ measurements in 3D cell cultures. Here we demonstrated that our method is well suited for minimally invasive quantification of oxygen levels and gradients, especially in large-volume hydrogel-based 3D cell cultures.