Monitoring cell culture 3D microenvironments: oxygen and pH nano-sensors within hydrogels

Ratiometric pH and O2 nanosensors were fabricated independently using a fluorophore that produces a signal proportional to the concentration of the analyte of interest, and a second fluorophore that produces a reference signal, insensitive to the analyte of interest. These fluorophores emit at different wavelengths and were either entrapped or covalently bound, within an inert optically transparent polymeric matrix. In this work uniform sized 3D micro-scaled alginate hydrogel constructs of approximately 100-300 $μ$m were fabricated. Through dual incorporation of HepG2 cells and nanosensors within these alginate constructs we aim to have a real-time, non-invasive method to measure microenvironmental pH and O2 content. Ratiometric fluorescent output from the microenvironment is used to monitor O2 concentrations and pH during cell culture. Measurements show that 3D micro-scaled constructs are suitable for cell growth and proliferation. Moreover O2 and pH values within the hydrogel cellularised microspheres are shown to have physiological values that enable the maintenance of the hepatic phenotype