Oxygen Concentration Microgradients for Cell Culture

There is a growing need for technology that can control microscale oxygen gradients onto a tissue or culture sample in vitro. This dissertation introduces the oxygen microgradient chip (OMA), which employs electrolysis to generate oxygen microgradients within cell culture without forming bubbles. Dissolved oxygen generated at noble microelectrodes patterned on a chip surface diffuses through a gas-permeable silicone membrane and is dosed into cell culture. The amount of generated oxygen is directly proportional to a current flowing across the electrodes and thus can be controlled with high precision. By real-time modulation of the current, spatial profile of the oxygen concentration microgradient can be modified during an experiment. Distinct microgradients generated by different electrodes can be superimposed to pattern arbitrary and multi-dimensional oxygen concentration profiles with microscale resolution. Design of the OMA is based on simulation results of diffusion and physical considerations. Microfabrication, assembly process, and improvements of the early OMA are presented. Use of an oxygen-sensitive fluorophore film verifies that the measured microgradients in OMA match simulation results. Generation of reactive oxygen species during the electrolysis is also considered and quantified. This technology enables the biological study of gradient-related effects. Three different, well-known biological models are introduced to demonstrate how the microtechnology enables new types of experiments. Lastly, we explore the effect of oxygen gradients on muscle progenitor cells having half-deficiency of superoxide dismutase-1 (SOD1) enzyme. Another type of oxygen gradient chip that utilizes atmospheric oxygen is developed and used in this experiment. Myotube formation of the SOD1 heterozygote myoblasts decreased significantly compared to that of wild-type myoblasts, indicating that the oxygen stress on SOD1 heterozygote myoblasts induces defective cellular function on differentiation.