Stabilization of enzyme-immobilized hydrogels for extended hypoxic cell culture

Abstract

Embodiments of the current invention include a hydrogel formed from crosslinked polyethylene glycol into which acrylated glucose oxidase has been immobilized through crosslinking to the gel. These hydrogels can be used to create hypoxia under ambient conditions for at least 72 hours and can be used to create hypoxic gradients. These embodiments permit the study of cells under a variety of hypoxic conditions.

Claims

1. A photopolymerized and freeze-dried composition, comprising: a hydrogel formed from a polymer building block selected from the group consisting of: polyethylene glycol; (polyethylene-glycol)-diacrylate; polyvinyl alcohol; polyglycerol; collagen; gelatin; chitosan; heparin; fibrinogen; hyaluronic acid; chondroitin sulfate; pullulan; xylan; dextran; alginate; silk fibroin; or derivatives of these polymers; an acrylated oxygen consuming enzyme selected from the group consisting of: glucose oxidase; bilirubin oxidase tyrosinase; laccase; lysyl oxidase; monoamine oxidase; xanthine oxidase; NADPH; or cytochrome P450 oxidase, wherein the acrylated oxygen consuming enzyme is crosslinked to the hydrogel through covalent bonds and retains at least some of its catalytic activity; and D-trehalose; wherein the acrylated oxygen consuming enzyme is immobilized in the hydrogel, and wherein the composition produces an oxygen gradient in a solution in contact with the composition.

2. The composition of claim 1 wherein the at least one enzyme includes acrylated glucose oxidase and where in a least a portion of the acrylated glucose oxidase is crosslinked to the hydrogel through covalent bonds.

3. The composition of claim 1, wherein the at least one polymer building block is polyethylene glycol.

4. The composition according to claim 1, wherein the hydrogel and the at least one enzyme immobilized in the hydrogel are lypophilized.

5. The composition according to claim 1, wherein the acrylated glucose oxidase is present in the range of about 1 mg/mL to about 50 mg/mL.

6. The composition according to claim 1, wherein the trehalose is present in the range of about 1 mg/mL to about 50 mg/mL.

7. A photopolymerized and freeze-dried composition in contact with a solution, comprising: a hydrogel formed from a polymer building block selected from the group consisting of: polyethylene glycol; (polyethylene-glycol)-diacrylate; polyvinyl alcohol; polyglycerol; collagen; gelatin; chitosan; heparin; fibrinogen; hyaluronic acid; chondroitin sulfate; pullulan; xylan; dextran; alginate; silk fibroin; or derivatives of these polymers; an acrylated oxygen consuming enzyme selected from the group consisting of: glucose oxidase; bilirubin oxidase tyrosinase; laccase; lysyl oxidase; monoamine oxidase; xanthine oxidase; NADPH; or cytochrome P450 oxidase, wherein the acrylated oxygen consuming enzyme is crosslinked to the hydrogel through covalent bonds and retains at least some of its catalytic activity, wherein the acrylated oxygen consuming enzyme is immobilized in the hydrogel; and D-trehalose; wherein the solution in contact with the composition comprises glutathione ranging from about 2 mM to about 10 mM.

8. The composition according to claim 1, further including glucose or other enzyme substrate is present in the range of 1 about mM to about 25 mM.

9. The composition according to claim 1, wherein the concentration of crosslinked polymer falls in the range of about 5% to about 30%.

10. The composition of claim 1, wherein the composition produces an oxygen concentration gradient from solution in contact with the composition outward to surrounding solution.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1A. Brief Description: Reaction scheme of GOX modification using Acryl-PEG-SVA. Protein structure for GOX was obtained from the RCSB Protein Data Bank (PDB-ID, 3QVP).

(2) FIG. 1B. Brief Description: O.sub.2 consumption profile using soluble GOX or GOX.sub.PEGA, 9.8 μg/mL CAT, and 25 mM β-D-Glucose.

(3) FIG. 1C. Brief Description: Reaction velocity of O.sub.2 consumption by GOX or GOX.sub.PEGA as a function of substrate β-D-glucose concentration. Values were generated from using 0.260 μM GOX or GOX.sub.PEGA with 0.30-25 mM of 3-D-glucose. All reactions were carried out in pH 7.4 PBS with constant stirring at 25° C. (Mean±SEM, n≥3).

(4) FIG. 2A. Brief description: Long term solution hypoxia induced by GOX or GOX.sub.PEGA in the absence of catalase (CAT).

(5) FIG. 2B. Brief description: Long term solution hypoxia induced by GOX or GOX.sub.PEGA in the presence of catalase (CAT). Between GOX and GOX.sub.PEGA groups, O.sub.2 content was very similar for all time points. The addition of CAT did not affect O.sub.2 content, which was below 5% in the first 24 hours for both GOX and GOX.sub.PEGA.

(6) FIG. 3A. Brief Description: Schematic of O.sub.2 measurement within and outside of a PEGDA hydrogel. The sensor probe was fully extended from the needle for measuring O.sub.2 tension exterior to the hydrogel (left). To measure O.sub.2 content at the interior of the hydrogel (right), the optic fiber was recessed within its needle housing to prevent damage of the gel matrix to the probe. After penetration the fiber was extended to the tip of the needle cannula so that it was exposed to the interior of the hydrogel.

(7) FIG. 3B. Brief Description: O.sub.2 consumption at the interior or exterior of GOX-immobilized hydrogels (120 μL of 8 wt % PEGDA gel with 4 mg/mL GOX.sub.PEGA). (***p<0.001. Mean±SEM, n≥3).

(8) FIG. 4. Brief Description: Effect of trehalose on solution hypoxia induced by freeze-dried GOX immobilized hydrogels. Trehalose was added at 3 mg/ml during gelation. Hydrogels (30 μl) were polymerized with 15 wt % PEGDA.sub.2kDa, 0.8 mg/ml GOX.sub.PEGA, and 1 mM LAP.

(9) FIG. 5A. Brief Description: Percent O.sub.2 measured as a function of time for a GOX.sub.PEGA hydrogel in an ibidi channel slide and a control with no hydrogel. The hydrogel is 20 total volume formed by 15% weight PEGDA with 0.4 mg/mL GOX.sub.PEGA. (***p<0.001. Mean±SEM, n≥3).

(10) FIG. 5B. Brief Description: Empirical mesh-modeling of Ficks 1D-diffusion equation for O.sub.2 concentration as a function of time and distance across a channel in a 50 mm channel slide. The boundaries were set to normoxia (19% O.sub.2) on one end of the channel and 3% at the other.

(11) FIG. 5C. Brief Description: Percent O.sub.2 measured as a function of time for a GOX.sub.PEGA hydrogels in an ibidi channel slide with a control lacking a hydrogel. The hydrogel is 20 μL total volume formed by 15% weight PEGDA with either 0.2 or 0.4 mg/mL GOX.sub.PEGA (one hydrogel per reservoir). (*p<0.05. Mean±SEM, n≥3).

(12) FIG. 5D. Brief Description: Empirical mesh-modeling of Ficks 1D-diffusion equation for O.sub.2 concentration as a function of time and distance across a channel in a 50 mm channel slide. The boundaries were set to 5% O.sub.2 on one end of the channel and 3% at the other.

(13) FIG. 6A. Brief Description: Oxidation reaction mechanism of glutathione (GSH) by H.sub.2O.sub.2.

(14) FIG. 6B. Brief Description: GSH consumption in the presence of GOX gel.

(15) FIG. 6C. Brief Description: Solution hypoxia prolonged by GSH. Hydrogels (30 μl) are polymerized with 15 wt % PEGDA.sub.2kDa, 0.2 mg/ml GOX.sub.PEGA, 3 mg/ml trehalose and 1 mM LAP. (***p<0.001. Mean±SEM, n≥3).

(16) FIG. 7A. Brief Description: Solution hypoxia induced by freshly prepared GOX-immobilized hydrogels. Hydrogels (30 μl) are polymerized with 15 wt % PEGDA.sub.2kDa and 1 mM LAP. All reactions are carried out at room temperature in DPBS with 25 mM β-D-Glucose and HEPES. (***p<0.001. Mean±SEM, n≥3).

(17) FIG. 7B. Brief Description: Solution hypoxia induced by freeze-dried GOX-immobilized hydrogels. Freeze-dried gels are reconstituted in 1 mL DPBS overnight. Hydrogels (30 μl) are polymerized with 15 wt % PEGDA.sub.2kDa and 1 mM LAP. All reactions are carried out at room temperature in DPBS with 25 mM β-D-Glucose and HEPES.

(18) FIG. 8A. Brief Description: Cytocompatibility of enzyme-free (i.e., PEGDA only) hydrogels. Molm14 cell viability is maintained above 95% over the course of 48 hours in the presence of an enzyme-free PEGDA hydrogel.

(19) FIG. 8B. Brief Description: The Molm14 cells proliferate over time, as indicated by steady increase in cell density.

(20) FIG. 8C. Brief Description: When a GOX.sub.PEGA gel is placed together with Molm14 cells (with media-supplemented CAT), cell viability in the initial 24 hours is comparable to that in the media-only control (around 90%).

(21) FIG. 8D. Brief Description: After 48 hours of in vitro culture, Molm14 cell viability declined sharply to ˜55%. In addition to the decreased cell viability after 48 hours, a similar trend can be seen with cell density over time. There is no significant difference in cell density between the control and experimental group at 6 hours (i.e., ˜3.6×10.sup.5 cells/mL). By 48 hours the Molm14 cell density in the media-only control group had increased to ˜5.5×10.sup.5 cells/mL, whereas the Molm14 cell density in the GOX-immobilized hydrogel group decreased significantly to ˜2.2×10.sup.5 cells/mL.

(22) FIG. 9A. Brief Description: Effect of enzyme induced hypoxia on cell fate of COLO 357 cell-laden gels. COLO-357 cell morphology is shown under normoxia (control) or hypoxia.

(23) FIG. 9B. Brief Description: mRNA expression Ribosomal 18s as the housekeeping gene.

(24) FIG. 9C. Brief Description: Cell size distribution. Hypoxia is induced by 30 μl hydrogels polymerized with 15 wt % PEGDA.sub.2kDa, 0.2 mg/ml GOX.sub.PEGA, 3 mg/ml trehalose and 1 mM LAP. GOX.sub.PEGA gel is placed in the same well as the cell-laden hydrogel (*p<0.05, **p<0.01. Mean±SEM, n≥3).

DESCRIPTION

(25) For the purposes of promoting an understanding of the principles of the novel technology, reference will now be made to the preferred embodiments thereof, and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the novel technology is thereby intended, such alterations, modifications, and further applications of the principles of the novel technology being contemplated as would normally occur to one skilled in the art to which the novel technology relates are within the scope of this disclosure and the claims.

(26) Herein is disclosed an immobilized-enzyme strategy for inducing hypoxia within and surrounding a PEG-based hydrogel for in vitro cancer cell culture. Immobilization of O.sub.2-consuming GOX within covalently crosslinked hydrogels provides an easy method to control solution O.sub.2 tension without the use of external devices. Furthermore, GOX-immobilized hydrogels can be readily added to or removed from cell culture without disturbing cells. The crosslinked PEG hydrogel network also provides opportunities for immobilizing multiple proteins/enzymes or other functional molecules for other biomedical applications.

(27) The flexibility and stability of the GOX-immobilized hypoxia-inducing hydrogel system is increased by the invention. GOX-immobilized hydrogels to which trehalose has been added are lyophilized for long-term storage. Trehalose is added into the gel formulation owing to its demonstrated ability to increase thermo-stability of proteins. Trehalose has been used as a soluble excipient to stabilize proteins under heat or lyophilization treatments. In the current invention, unmodified trehalose is used to preserve the activity of reconstituted freeze-dried GOX-immobilized hydrogels.

(28) In the current invention, glutathione (GSH) is used instead of CAT in conjunction with GOX to sustain hypoxia. For each mole of CAT undergoing the reaction, one-half mole of oxygen is produced, which offsets the oxygen consumption ability of GOX. GSH however, reduced hydrogen peroxide to water, without molecular oxygen as a by-product, thereby solving this problem.

(29) Fabrication of enzyme-immobilized hydrogels capable of inducing hypoxia is accomplished by functionalizing the primary amine groups on GOX with Acryl-PEG-SVA. TNBSA assay results showed an average of 93±1.7% (Mean±SEM, n=5) of the primary amines on enzyme surface were functionalized with Acryl-PEG. The acrylate moieties on the surface of Acryl-PEG-GOX (i.e., GOX.sub.PEGA) permit its homopolymerization with PEGDA to afford enzyme-immobilized hydrogels. As shown in FIG. 1B., while un-modified GOX caused rapid O.sub.2 reduction (from ˜20% to ˜3.2% in 5 min) in solution, the ability of GOX.sub.PEGA to consume O.sub.2 was slightly hindered after Acryl-PEG-SVA modification (from ˜20% to ˜5.9% within 5 minutes). To quantify the impact of polymer modification on its enzyme activity, reaction velocities of GOX and GOX.sub.PEGA were measured and compared in FIG. 1C. Michaelis-Menten enzyme kinetic parameters were listed in Table 1. Maximum reaction velocity, V.sub.max, was reduced for GOX.sub.PEGA to 0.664 mM min.sup.−1, or approximately 75% of that for GOX (0.880 mM min.sup.−1). Additionally, K.sub.m, an estimate of the dissociation constant for enzyme and substrate, was also decreased for GOX.sub.PEGA at 1.173 mM versus GOX at 4.380 mM.

(30) TABLE-US-00001 TABLE 1 Michaelis-Menten constants of GOX and GOX.sub.PEGA V.sub.max (mM min.sup.−1) K.sub.m (mM) GOX 0.880 ± 0.045 4.380 ± 0.900 GOX.sub.PEGA 0.664 ± 0.033 1.173 ± 0.275

(31) To evaluate the ability of the enzyme system to maintain hypoxia, O.sub.2 content measurements were carried out for 72 hours. FIG. 2. shows long term solution hypoxia induced by GOX or GOX.sub.PEGA in the absence (FIG. 2A.) and presence (FIG. 2B.) of CAT. Between GOX and GOX.sub.PEGA groups, O.sub.2 content was very similar for all time points. Within the first 24 h, O.sub.2 was maintained below 5% but gradually increased to ˜13% by 72 hours (FIG. 2A.). The addition of CAT did not affect O.sub.2 content, which was below 5% in the first 24 hours for both GOX and GOX.sub.PEGA. The O.sub.2 content in both conditions rose to ˜16% and ˜18% at 50 and 72 hours, respectively.

(32) GOX.sub.PEGA was covalently immobilized within PEGDA hydrogels to provide a simple method for inducing solution hypoxia. The ability of the immobilized enzyme to reduce O.sub.2 in the surrounding solution and within the gel was measured with a needle type optical probe as shown in FIG. 3A. With the needle type O.sub.2 probe, it was possible to measure O.sub.2 content outside (left panel) or inside (right panel) the GOX.sub.PEGA immobilized hydrogels (FIG. 3B.). Control experiments using hydrogels without enzyme immobilization (i.e., (−) GOX.sub.PEGA) showed that O.sub.2 content remained close to normoxia (17-20% O.sub.2). Furthermore, there was no significant difference between O.sub.2 content within or outside of the enzyme-free hydrogels. With the use of GOX.sub.PEGA immobilized PEG hydrogels, however, there was a rapid drop in the ‘exterior’ (i.e., outside of the GOX.sub.PEGA immobilized hydrogel) O.sub.2 tension within one hour, a level similar to that with soluble enzyme (FIG. 2A.). O.sub.2 tension was roughly at ˜8% O.sub.2 for 48 hours in solution with the GOX.sub.PEGA hydrogels. Conversely the O.sub.2 tension within the GOX.sub.PEGA hydrogel quickly reached and maintained near anoxia (˜0% O.sub.2) for 48 hours. The O.sub.2 tension at the gel exterior had increased to ˜15% by 120 hours, while that in the gel interior was still below 2%.

(33) In one embodiment of the current invention, glucose oxidase (GOX) is acrylated and copolymerized with poly(ethylene glycol)-diacrylate (PEGDA) in the presence of trehalose to form GOX-immobilized PEG-based hydrogels.

(34) In another embodiment, hypoxia gradients are created by placing the enzyme-immobilized gels into a channel slide.

(35) In still another embodiment, glutathione is added to the buffer solution so as to extend hypoxia within the hydrogel for at least 72 hours.

(36) In yet another embodiment, the enzyme-immobilized hydrogels are lyophylized for longer term storage.

(37) In another embodiment, the enzyme-immobilized hydrogels are used for the cancer cell studies.

EXPERIMENTAL

(38) Materials and Methods

(39) Linear PEG (Mn=2 kDa) was purchased from Sigma-Aldrich. Glucose oxidase (0243-500KU) and catalase (LS001847) were purchased from Amresco and Worthington Biochemical, respectively. Acrylate-PEG-succinimidyl valerate (Acryl-PEG-SVA, MW 3400 Da) was obtained from Laysan Bio Inc. Zeba Spin Desalting Columns (7 K MWCO), 2,4,6-trinitrobenzene sulfonic acid (TNBSA), and β-D-glucose were purchased from Thermo Scientific. Penicillin-streptomycin, antibiotic-antimycotics, fetal bovine serum (FBS), Roswell Park Memorial Institute media (RPMI), and Dulbecco's modified Eagle's medium (DMEM) were acquired from Life Technologies. HEPES and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Lonza. Membrane culture plate inserts (PIXP012-50) were purchased from EMD Millipore. Trypan blue and AlamarBlue® reagents were purchased from Mediatech and Fisher Scientific, respectively.

(40) Linear PEG (Mn: 2 kDa) was purchased from Sigma-Aldrich. Glucose oxidase (0243-500KU) and acrylate-PEG-succinimidyl valerate (Acryl-PEG-SVA) were obtained from Laysan Bio and Amresco, respectively. β-D-glucose and glutathione were purchased from Thermo Scientific. D-trehalose was acquired from Acros Organic. Penicillin-streptomycin, antibiotic-antimycotics, fetal bovine serum (FBS), and Dulbecco's modified Eagle's medium (DMEM) were acquired from Life Technologies. HEPES and Dulbecco's phosphate-buffered saline (DPBS) were purchased from Lonza. Membrane culture plate inserts (PIXP-012-50) were purchased from EMD Millipore. Ellman and AlamarBlue® reagents were purchased from Fisher Scientific.

Example 1

(41) Macromer Synthesis and Characterization

(42) PEG-diacrylate (PEGDA) is synthesized according to an established protocol and characterized with .sup.1H NMR (Bruker 500). The degree of PEGDA functionalization is around 89% (FIG. S1). Photoinitiator lithium aryl phosphonate (LAP) is synthesized as described elsewhere. To facilitate enzyme immobilization within hydrogels, glucose oxidase is acrylated using Acryl-PEG-SVA using the method according to Choi et al. Briefly, the enzyme is first dissolved at 20 mg/mL in PBS supplemented with 2 mM EDTA (pH 8.5) and 50 mM sodium carbonate. Acryl-PEG-SVA is added in 200× molar excess to enzyme concentration and the reaction proceeds at room temperature for 2 hours with stirring. During the reaction, primary amines on the surface of the enzyme react with SVA groups to afford PEG-acrylate (PEGA)-modified GOX (GOX.sub.PEGA). Unreacted macromers are removed using size exclusion chromatography columns (Zeba Spin Desalting column). Un-modified GOX at the same concentration is also passed through the columns and used as a control to account for any loss/entrapment of enzyme within the columns. Following synthesis, both GOX and GOX.sub.PEGA are assayed using TNBSA assay to determine the degree of PEGA functionalization. For each assay, enzyme samples are diluted to 30-35 μg/mL. A series of lysine hydrochloride solutions (0-10 μg/mL, 200 μL/well) are used as standards. 100 μL of 0.01% TNBSA reagent is added into wells of a 96-well plate, which is sealed and incubated at 37° C. for 2 hours, followed by cooling for 5 minutes. Absorbance at 335 nm is measured using a microplate reader (SynergyHT BioTek). The degree of PEGA functionalization on GOX is determined as the concentration of remaining amine groups on GOX.sub.PEGA over that of the un-modified GOX.

Example 2

(43) Characterization of Enzymatic Activity of GOX.sub.PEGA

(44) To examine the enzyme activity, O.sub.2 consumption in the presence of the enzyme and glucose is quantified. The changes in O.sub.2 content over time in the presence of GOX or GOX.sub.PEGA (V.sub.o=Δ [O.sub.2]/ΔTime) is defined as the reaction velocity. The enzyme is dissolved PBS (pH 7.4) at 0.13 μM in a 2 mL microtube with constant stirring at 25° C. The O.sub.2 consumption reactions are carried out under ambient air with constant O.sub.2 diffusion from the air to mimic actual cell culture conditions. Stock β-D-glucose solution is injected at the start of every measurement to give starting concentrations of 0.30-25 mM [S].sub.I. Dissolved O.sub.2 concentration is monitored for 3 minutes using an O.sub.2 probe and meter (Microx4, PreSens; see Example 6). O.sub.2 contents are plotted as a function of time and the initial linear portion of the curve was used for V.sub.o calculation (change in substrate concentration over time). Non-linear regression analysis and curve fitting is applied to paired V.sub.o and [S].sub.I using the equation V.sub.o=V.sub.max[S]/(K.sub.m+[S]). In the equation, V.sub.max is the theoretical maximum enzyme reaction velocity and K.sub.m is the Michaelis-Menten constant, the equilibrium dissociation constant (i.e., affinity) for the enzyme and the substrate.

Example 3

(45) Synthesis and Characterization of Enzyme-Immobilized Hydrogels

(46) All macromer solutions are sterilized by passing through 0.22 μm syringe filters. PEGDA hydrogels (15 wt %) are polymerized aseptically through radical mediated photopolymerization in the absence or presence of GOX.sub.PEGA monomer (6 mg/mL), and LAP (1 mM) as the photoinitiator. 60 μL gels are injected between two glass slides separated by Teflon spacers (2 mm) and gelation is initiated with a UV lamp (365 nm, 5 mW/cm.sup.2, 2 minutes exposure). Following photopolymerization, hydrogels (˜3.1 mm dia.×2 mm thickness) are incubated in DPBS for 24 hours at 37° C.

Example 4

(47) Synthesis of Trehalose-Stabilized Enzyme-Immobilized Hydrogels

(48) Trehalose is added at 3 mg/ml during gelation. Hydrogels (30 μl) are polymerized with 15 wt % PEGDA.sub.2kDa, 0.8 mg/ml GOX.sub.PEGA, and 1 mM LAP. All reactions are carried out at room temperature in DPBS with 25 mM β-D-Glucose and HEPES.

Example 5

(49) Effect of Solution GSH Content on Sustained Hypoxia

(50) To mitigate the adverse effect of the accumulated H.sub.2O.sub.2, glutathione (GSH) is added to the buffer. GSH is a strong reducing agent for reactive oxygen species, such as H.sub.2O.sub.2. Specifically, GSH reduces H.sub.2O.sub.2 into water without producing additional oxygen as does catalase. Indeed, when GSH is added to the solution at 2.5 mM or 5 mM, the oxygen contents in the buffers remain at around 2-3% for 24 hours (FIG. 6C). By 48-hour measurements, the oxygen contents in the solutions increases to ˜6% and ˜3.5% for solution added with 2.5 mM and 5.0 mM of GSH, respectively. By 72-hour measurement, the solution oxygen level remains at around 5% for buffer added with 5 mM GSH (FIG. 6C).

Example 6

(51) Measurement of O.sub.2 Concentration

(52) O.sub.2 concentration in solution is measured with a dipping-type O.sub.2 sensor (Microx4, PreSens). For solution based measurements, the probe is extended to ˜2 mm above the bottom of the 24 well plate or 1 mm above the gel (˜2 mm from the liquid-air interface). To measure the H.sub.2O.sub.2 produced during the reactions, 10 μL aliquots of the solutions are collected and quantified with a Quantichrom Peroxide Assay Kit following the manufacturer's protocol (BioAssay Systems).

Example 7

(53) Establishing Hypoxia Gradients Using Enzyme Immobilized Hydrogels

(54) Specialized channel slides are available from ibidi (GmbH, Munich, Germany, catalogue number 80111, μ-Slide-1). The slides contain two reservoirs connected by a 50 mm channel. O.sub.2 diffusion within the channel is simulated numerically with a finite difference approximation of a one-dimensional diffusion equation. For boundary conditions, O.sub.2 concentration is held constant at each end of the channel (i.e., in the reservoirs) to represent either O.sub.2-sinks or O.sub.2-sources. For initial conditioning within the channel, O.sub.2 concentration is assumed to be normoxic (about 19% O.sub.2) at the left reservoir and 3% at the right at 0 hours of the simulation. At 4 hours of simulated O.sub.2 diffusion in the channel, normoxic concentration of O.sub.2 are predicted from the 0-35 mm mark, while from the 35-50 mm mark, O.sub.2 concentration drops from 18.1% to 3%. At 12 hours, from the 25-50 mm mark, O.sub.2 concentration decreases monotonically from 18.1% to 3%. By 36 hours, O.sub.2 concentration drops from 17.3% to 3% at the 10-50 mm marks. Finally, at 48 hours, a channel-wide gradient from 19% to 3% O.sub.2 is predicted. In contrast, a second simulation using 5% and 3% O.sub.2 concentration in the two reservoirs of the channel slide gives a peak O.sub.2 concentration at the center of the channel (from the 15-35 mm marks) with monotonically decreasing values to either the left or the right.

(55) Hydrogels with either 0.2 or 0.4 mg/mL GOX.sub.PEGA (20 μL volume) are used within the reservoirs of the channel slide to generate O.sub.2 consumption over time. FIG. 5 shows measured O.sub.2 values as well as results of the numerical simulations of O.sub.2 gradients within the channel.

Example 8

(56) Cell Culture and Viability Assays Using Molm 14 Cells

(57) A suspension cell type, human acute myeloid leukemia (AML) cells Molm14, are commercially available through the Leibniz Institute, German Collection of Microorganisms and Cell Cultures. Cells are maintained in RPMI media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin 25 mM HEPES, and 25 mM β-D-Glucose. 400,000 cells/mL of Molm14 cells are seeded per well in non-treated 24 well plates. GOX.sub.PEGA gels (15 wt % PEGDA, 6 mg/mL GOX.sub.PEGA, 60 μL per gel) are added to half of the wells (one gel per well) containing 0.54 mg/mL catalase. Remaining wells are placed with gels without immobilized enzyme. In vitro O.sub.2 concentration is measured 1 mm above the hydrogel with a dipping-type O.sub.2 sensor (PreSens). Molm14 cell viability and density are characterized by tryphan blue staining and counting with a hemocytometer. The survival and progression of these cells, just like many other cancer cell types, are significantly affected by O.sub.2 tension. GOX.sub.PEGA immobilized hydrogels were prepared and added to anchorage-independent Molm14 cells cultured directly. Solution hypoxia is rapidly induced and maintained below 5% O.sub.2 from 6 to 24 h. By 48 h, however, O.sub.2 concentration rises to near normoxia (17-20% O.sub.2).

(58) RNA isolation is carried out using NucleoSpin RNA II kit (Clontech). Briefly, 600 μL of lysis buffer is added to each well containing cells. Cell lysates are snap frozen and stored in 80° C. until assay. After thawing the lysates, 600 μL of 70% RNase free ethanol is added, pipetted vigorously, and then run through NucleoSpin RNA columns. After desalting/purification steps, RNA is eluted with DNase/RNase-free H.sub.2O and quantified by spectroscopy (NanoDrop 2000, Thermo Scientific). Isolated RNA is stored at −80° C. Complementary DNA is generated from the isolated total RNA by using PrimeScript RT reagent kit (Clontech, TaKaRa). Gene expression is analyzed by real time quantitative PCR using SYBR Premix Ex Taq II Kit (Clontech, TaKaRa). The kit components, cDNA, and primers are mixed in a PCR plate and analyzed on a 7500 Fast Real-Time PCR machine (Applied Biosystems). Thermocycling parameters were one cycle at 95° C. for 30 s, followed by 95° C. for 3 s, 60° C. for 30 s, and repeated for 45 cycles. Gene expression results are analyzed using 2.sup.−ΔΔCT methodology. For each experimental condition, cycle count is first standardized to ribosomal 18S housekeeping gene (ΔCT level) and then normalized with respect to the media control group for that specific time point (ΔΔCT level; media control values are set as one-fold).

(59) The expression of hypoxia associated gene carbonic anhydrase 9 (CA9) in Molm14 cells is evaluated at 6 and 24 hours of culture in the presence of a GOX-immobilized hydrogel. Enzyme-induced hypoxia increased the expression of CA9 significantly compared with control groups (˜3-fold and ˜10-fold higher at 6 and 24 hours of culture, respectively).

Example 9

(60) Cell Culture and Viability Assays Using Huh7 Cells

(61) Adherent cell type human hepatocarcinoma cells (Huh7) are grown in high glucose DMEM supplemented with 10% FBS, 1% antibiotic antimycotics, and 25 mM HEPES. Cells are seeded on treated 24 well plates with 1 mL per well of cell suspension (60,000 cells/mL) and allowed to grow/spread for 48 h prior to the onset of the experiments, at which time (labeled as 0 h) culture media is refreshed in all wells. At the onset of the experiment, membrane inserts containing GOX.sub.PEGA gels are placed in the wells and the medium is supplemented with 0.54 mg/mL CAT. Half of the wells only have media refreshed and are used as control groups for the experiment (no enzyme added). AlamarBlue® reagent (10× dilution in media) is used for assaying metabolic activity of Huh7 cells. After a 90 min incubation, 200 μL from each well is transferred to a clear 96-well microplate and read for fluorescence (excitation/emission: 560/590 nm). GOX.sub.PEGA-immobilized hydrogels are placed in a standard transwell device and co-cultured with the cells adhered to the surface of a multi-well plate. The purpose of using a transwell device is to prevent direct contact of the gel with the cells, which could mechanically disrupt cell attachment. The O.sub.2 profile development was similar to that for Molm14 cells. Low O.sub.2 concentration was reached quickly and maintained up to 24 h. By 48 h, the O.sub.2 content had returned to almost normoxia. RNA isolation and analysis is carried out as described in Example 8. The expression of carbonic anhydrase 9 (CA9) and lysyl oxidase (LOX) is examined after the cells are exposed to the enzyme-immobilized hydrogel (Note: no detectable LOX expression was found in Molm14 cells). In selected groups, CoCl.sub.2 was added as another control for chemically stimulated hypoxic response. CoCl.sub.2 failed to upregulate CA9 expression in the first 24 hours. After the same period of time in culture, the use of GOX.sub.PEGA gels+CAT led to a ˜20-fold increase in CA9 expression in Huh7 cells. After 48 hours, the addition of CoCl.sub.2 caused ˜15-fold upregulation in CA9 mRNA expression, which was much lower than that induced by the enzyme-immobilized hydrogel group (˜80-fold higher). In Huh7 cells, LOX mRNA expression was upregulated only in cells co-cultured with a GOX.sub.PEGA gel (˜2.5 fold, FIG. S4). The addition of CoCl.sub.2 did not increase the expression of LOX in Huh7 cells.

Example 10

(62) COLO-357, a pancreatic cancer cell line, was maintained in high glucose DMEM supplemented with 10% FBS, 1% antibiotic antimycotics, and 25 mM HEPES. Cells are encapsulated in gelatin-norbornene (GelNB)-thiolated hyaluronic acid (THA) hybrid hydrogels via thiol-norbornene photopolymerization as described previously. Cell-laden hydrogel is cultured in the presence of GOX-immobilized hydrogel for 2 weeks with periodical exchange of GOX-immobilized gel to maintain solution hypoxia.

(63) While the novel technology has been illustrated and described in detail in the figures and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiments have been shown and described and that all changes and modifications that come within the spirit of the novel technology are desired to be protected. As well, while the novel technology was illustrated using specific examples, theoretical arguments, accounts, and illustrations, these illustrations and the accompanying discussion should by no means be interpreted as limiting the technology. All patents, patent applications, and references to texts, scientific treatises, publications, and the like referenced in this application are incorporated herein by reference in their entirety.

APPENDIX

References

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