Characterizing oxygen transport in hydrogel on a 3D microfluidic chip

OOC models are microfluidic systems, designed to mimic the physiological processes of specific human organs. On a cellular level, sufficient O2 supply is crucial for the viability of cells in cell cultures. In culture medium, oxygen gradients form as soon as local changes in oxygen levels occur. Especially, in 3D cultures, oxygen sensing and control of concentrations, is inevitable for successful On-Chip cell studies. Hydrogels are commonly used in OOC systems, to create an environment more natural to the cells, and mimic a certain tissue type. For these reasons, there is need for methods to study oxygen transport mechanisms in hydrogels. The objective of this thesis was to experimentally characterize the oxygen diffusion in fibrin hydrogel. Moreover, results from the experiments were compared to simulated data. A microfluidic chip with a ratiometric imaging system was developed to measure the partial oxygen pressure in fibrin hydrogel. The device consisted of a bottom glass plate coated with an \gls{o2} sensing film, a vinyl cut tape layer, and laser cut top glass. The O2 sensing of the sensing film is based on fluorescent quenching in the presence of oxygen molecules. The chip layout consisted of a hydrogel area with a liquid reservoir on either side. Chip calibration was done before every measurement, and followed by the injection of fibrin hydrogel to the designated area. Then, water was added to the liquid reservoirs for constant hydration. Diffusion gradients within the hydrogel were established by repeatedly changing water to Na2SO4 scavenger liquid and back. Photon emission from the sensing film was detected by a color camera, followed by analysis of the red and green signal intensities. Through the ratiometric imaging method, the partial oxygen pressures in a grid-like array in the imaged area were calculated. Then, 2D maps of the pO2 levels were created and studied. Additionally, FEM simulations were conducted on a simple 2D side view model of the microfluidic device. pO2 values in the center region of the hydrogel from the experiment were compared to the simulated data. In this thesis, different experiments were conducted to characterize the O2 diffusion in fibrin hydrogel. Continuous diffusion from one or two sides was detected. Symmetrical and even diffusion profiles were observed in the center of the hydrogel. Stabilization of pO2 levels within the hydrogel was achieved within hours after the one-sided liquid exchange. The time until saturation was shortened in the two-side experiment. Thus, bubbles within the hydrogel, slowed diffusion processes down, while multiple liquid exchange repetitions in the same measurement area, doubled the diffusion speed. Furthermore, medium color dependency of the ratiometric imaging method was discovered. The presented microfluidic device provides a setup to measure 2D oxygen gradients in hydrogel at high resolution and enables characterization of O2 diffusion