Enzyme-Immobilized Hydrogels to Create Hypoxia for in vitro Cancer Cell Culture

Hypoxia is a critical condition governing many aspects of cellular fate processes. The most common practice in hypoxic cell culture is to maintain cells in an incubator with controlled gas inlet (i.e., hypoxic chamber). This thesis describes the design and characterization of enzyme-immobilized hydrogels to create solution hypoxia under ambient conditions for in vitro cancer cell culture. The first objective of this thesis was to modify glucose oxidase (GOX) for copolymerization with poly(ethylene glycol)-diacrylate (PEGDA) to form GOX-immobilized PEG-based hydrogels. The effect of soluble GOX or acrylated GOX on sustaining hypoxia was evaluated under ambient air condition (i.e., with constant oxygen diffusion from the air-liquid interface). The second objective of this thesis was to use the GOX-immobilized hydrogel system to create hypoxia for in vitro culture of cancer cells, including Molm14 (acute myeloid leukemia (AML) cell line) and Huh7 (hepatocarcinoma cell (HCC) line). Under ambient air conditions required for cell culture, the GOX-immobilized hydrogels were able to establish and sustain in vitro hypoxic conditions (\u3c5% O2) for 6 to 24 hours. Additionally, cell viability and the expression of hypoxia associated genes, including carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX), were evaluated in the presence of GOX-immobilized hydrogels. The third objective of this thesis was to establish hypoxic gradients using the enzyme immobilized hydrogels, which were placed in reservoirs of a commercially available channel slide. The combination of the devices geometry and the enzyme-immobilized hydrogel that served as an O2–sink permitted the generation of an O2-concentration gradient within the channel connecting the two reservoirs