Stabilization of enzyme-immobilized hydrogels for extended hypoxic cell culture

Abstract

In this work, glucose oxidase (GOx)-immobilized hydrogels are developed and optimized as an easy and convenient means for creating solution hypoxia in a regular incubator. Specifically, acrylated GOx co-polymerizes with poly(ethylene glycol) diacrylate (PEGDA) to form PEGDA-GOx hydrogels. Results show that freeze-drying and reaction by-products, hydrogen peroxide, negatively affect oxygen-consuming activity of network-immobilized GOx. However, the negative effects of freeze-drying can be mitigated by addition of trehalose/raffinose in the hydrogel precursor solution, whereas the inhibition of GOx caused by hydrogen peroxide can be prevented via addition of glutathione (GSH) in the buffer/media. The ability to preserve enzyme activity following freeze-drying and during long-term incubation permits facile application of this material to induce long-term solution/media hypoxia in cell culture plasticware placed in a regular CO₂ incubator.

Keywords: Hypoxia; cancer; enzyme immobilization; glucose oxidase; hydrogel.

Figure 1.. Solution hypoxia induced by freshly prepared or freeze-dried PEGDA-GOx hydrogels.

(A) Schematic of preparing hypoxia-inducing hydrogel by crosslinking PEGDA with GOxPEGA. Gels were either freshly-prepared (B) or freeze-dried (C) before swelling/reconstituting in 1 mL PBS overnight, followed by incubation in buffer solution to induce hypoxia. Gel volume: 30 μl. [PEGDA_2kDa] = 15 wt%. Buffer: DPBS with 25 mM β-d-Glucose and 25 mM HEPES (***p<0.001. Mean ± SEM, n ≥ 3).

Figure 2.. Effect of cryoprotectants on solution hypoxia induced by freeze-dried PEGDA-GOx hydrogels.

(A) Chemical structure of trehalose. (B) Chemical structure of raffinose. (C) Oxygen consumption induced by freeze-dried PEGDA-GOx hydrogels in the absence or presence of trehalose or raffinose (3 mg/ml) during gelation. Hydrogel volume: 30 μl. [PEGDA_2kDa] = 15 wt%. LAP: 1mM. [GOxPEGA] = 0.8 mg/ml. Oxygen tensions were measured at room temperature in DPBS with 25 mM β-d-Glucose, 5 mM Glutathione and 25 mM HEPES (**p<0.01. Mean ± SEM, n ≥ 4).

Figure 3.. The effects of gel replacement and buffer volume and on oxygen consumption by PEGDA-GOx hydrogels.

(A) Timeline of the gel replacement study. (B) Effect of replacing PEGDA-GOx gel on oxygen consumption. (C) Timeline of the buffer volume study. (D) Effect of buffer volume on oxygen consumption by the same PEGDA-GOx gels. Hydrogel volume: 30 μl. [PEGDA_2kDa] = 15 wt%. LAP: 1mM. [GOxPEGA] = 0.8 mg/ml. Oxygen tensions were measured at room temperature in DPBS with 25 mM β-d-Glucose and 25 mM HEPES (*p<0.05. ***p<0.001. Mean ± SEM, n ≥ 3).

Figure 4.. Inhibitory effects of GOx reaction by-products on oxygen consumption.

Hydrogen peroxide (A) or gluconic acid (B) was added to the buffer containing PEGDA-GOx hydrogels. Hydrogel volume: 30 μl. [PEGDA_2kDa] = 15 wt%. LAP: 1mM. [GOxPEGA] = 0.8 mg/ml. Oxygen tensions were measured at room temperature in DPBS with 25 mM β-d-Glucose and 0 or 25 mM HEPES for gluconic acid and hydrogen peroxide experiments, respectively. (*p<0.05. ***p<0.001. Mean ± SEM, n ≥ 3).

Figure 5.

Effect of GSH on oxygen consumption. (A) Oxidation reaction mechanism of GSH by H2O2. (B) PEGDA-GOx hydrogel induced solution hypoxia was prolonged by the addition of GSH. (C) GSH consumption in the presence or absence of PEGDA-GOx hydrogel. (D) Solution hypoxia induced by PEGDA-GOx hydrogel and exogenously added GSH (5mM, added only at the beginning of experiment or periodically at 6 or 24 hours). Hydrogel (30 μl) were polymerized with 15 wt% PEGDA_2kDa, 0.2 mg/ml GOxPEGA, 3 mg/ml trehalose and 1mM LAP. (***p<0.001. Mean ± SEM, n ≥ 3).

Figure 6.

Effect of PEGDA-GOx gel induced hypoxia (A) and gas-controlled chamber induced hypoxia on viability of Mol14 cells over time. 25 mM glucose, 25 mM HEPES, and 5 mM GSH were added to PEGDA-GOx gel containing media. Additional GSH (5mM) was added at 6-hour and 24-hour. PEGDA-GOx gels: 30 μl; 15 wt% PEGDA_2kDa, 0.8 mg/ml GOxPEGA, 3 mg/ml trehalose. (Mean ± SEM, n ≥ 4. n.s.: Not significant. ***p<0.001).

Figure 7.

Effect of enzyme induced hypoxia on cell fate of COLO 357 cell- laden gels. (A) COLO-357 cell morphology under normoxia (control) or hypoxia. (B) mRNA expression. Housekeeping gene: Ribosomal 18s. (C) Cell size distribution. Hypoxia was induced by 30 μl hydrogels polymerized with 15 wt% PEGDA_2kDa, 0.2 mg/ml GOxPEGA, 3 mg/ml trehalose and 1mM LAP. GOxPEGA gel was placed in the same well as the cell-laden hydrogel (*p<0.05, **p<0.01. Mean ± SEM, n ≥ 3).