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). Here, we describe the design and characterization of
enzyme-immobilized hydrogels to create
solution hypoxia under ambient conditions for in vitro
cancer cell culture. Specifically,
glucose oxidase (GOX) was acrylated and co-polymerized with
poly(ethylene glycol)-diacrylate (PEGDA) through photopolymerization to form GOX-immobilized PEG-based
hydrogels. We first evaluated the effect of soluble GOX on inducing
solution hypoxia (O2<5%) and found that both unmodified and acrylated GOX could sustain
hypoxia for at least 24h even under ambient air condition with constant
oxygen diffusion from the air-liquid interface. However, soluble GOX gradually lost its ability to sustain
hypoxia after 24h due to the loss of
enzyme activity over time. On the other hand, GOX-immobilized
hydrogels were able to create
hypoxia within the
hydrogel for at least 120h, potentially due to enhanced
protein stabilization by
enzyme 'PEGylation' and immobilization. As a proof-of-concept, this GOX-immobilized
hydrogel system was used to create
hypoxia for in vitro culture of Molm14 (
acute myeloid leukemia (AML) cell line) and Huh7 (
hepatocellular carcinoma (HCC) cell line). Cells cultured in the presence of GOX-immobilized
hydrogels remained viable for at least 24h. The expression of
hypoxia associated genes, including
carbonic anhydrase 9 (CA9) and
lysyl oxidase (LOX), were significantly upregulated in cells cultured with GOX-immobilized
hydrogels. These results have demonstrated the potential of using
enzyme-immobilized hydrogels to create hypoxic environment for in vitro
cancer cell culture.