Enzyme-Immobilized Hydrogels for Hypoxic In Vitro Cancer Cell Culture
Camron S Dawes,1 Heiko Konig,2 Chien-Chi Lin.1
1
Dept. of Biomedical Engineering, Indiana University-Purdue University Indianapolis, Indianapolis, IN 46202
2
Dept. of Medicine, Division of Hematology/Oncology, Indiana University School of Medicine, Indianapolis, IN 46202
Statement of Purpose: Hypoxia is an important
physiological condition implicated in many healthy and
diseased tissues in the body. For example, oxygen
concentration is around 20% in the lungs; ~13% in the
alveoli; ~5% in the circulation system and the bone
marrow; and below 5% in multicellular tissues including
solid tumor.[1, 2] Given its critical role in regulating normal
and pathological physiology, oxygen concentration should
be considered as a critical experimental condition when
performing in vitro cell studies.[1, 3-5] This project aims at
creating a hypoxic environment through enzymatic
reactions. Here, we describe the development of glucose
oxidase (GOX)-immobilized poly(ethylene glycol)diacrylate (PEGDA) hydrogels to create solution hypoxia
for up to 24 hours under ambient conditions. The effects
of enzyme-induced hypoxia were studied using Molm14
cells, an acute myeloid lymphoma (AML) cell line, as
well as Huh7 cells, a hepatocellular carcinoma cell line.
Methods: Macromer PEGDA (2 kDa) and photoinitiator
lithium arylphosphonate (LAP) were synthesized
according to established protocols.[6, 7] Primary amine
groups on GOX were modified with acrylate groups using
acrylate-PEG-succinimidyl valerate. PEG-acrylate
modified GOX (GOXPEGA) was co-polymerized with
PEGDA through UV-initiated photopolymerization to
yield GOX-immobilized hydrogels.[8] Cancer cell lines
suspension cells Molm14 or adherent cells Huh7 were
seeded in 24-well plates. To allow for adequate time for
attachment, Huh7 cells were cultured for 48 hours prior to
placing GOXPEGA gels in the wells. Molm14 cells were
seeded and GOXPEGA gels were placed immediately
thereafter. In select wells, cobalt chloride or catalase
(CAT) was added as control. Oxygen contents were
measured by an oxygen sensing optical probe (Microx4,
PreSens), which was positioned 1-2 mm above the gel and
4-5mm from the liquid-air interface. At selected culture
time following the placement of GOXPEGA gels, cells were
collected for RNA isolation, reverse transcription, and
quantitative real time PCR analysis.
Results: Efficacy of the enzyme system to induce
hypoxia in the presence of Molm14 or Huh7 cells was
evaluated by monitoring oxygen tension (Fig. 1A and 1B)
in the presence of cells. O2 content dropped to ~8% or
lower within one hour. Low O2 content (< 5%) was
maintained for both cell types up to 24 hours. At 48 hours
it can be seen that O2 levels were the same as the control
value, suggesting that enzyme activity was lost. This
could be attributed to the buildup of reaction by-products
(H2O2 or gluconic acid). Control experiments showed that
addition of substrate (i.e., glucose) did not reduce the O2
level (data not shown). The system was cytocompatible
for at least 24 hours (data not shown). The accumulation
of GOX reaction by-products was likely the cause for
decreased cell viability past 24 hours. Expression level of
hypoxia associated gene carbonic anhydrase 9 (CA9) for
Molm14 cells is shown in Fig. 1B. In the presence of
GOX-immobilized gel, CA9 expression was significantly
upregulated for both Molm14 (Fig. 1C) and Huh7 cells
(Fig. 1D). The addition of cobalt chloride (CoCl2), a
chemical compound commonly used to stabilize hypoxiainducible factor (HIF) and simulate cellular hypoxic
response,[9] failed to upregulate CA9 expression in the
first 24 hours. After the same period of time in culture,
the use of GOX-immobilized hydrogel led to a ~20-fold
increase in CA9 expression in Huh7 cells. After 48 hours,
the addition of CoCl2 caused ~15-fold upregulation in
CA9 expression, which was much lower than that induced
by the GOX-immobilized hydrogel (~80-fold higher, Fig.
1D).
Figure 1. Hypoxia induced by GOX-immobilized
hydrogel in the presence of Molm14 cells (A) or Huh7
cells (B). CA9 expression in Molm14 cells (C) or Huh7
cells (D) in the presence of different media components
(i.e., media only, GOX-gel + CAT, or media
supplemented with CoCl2). Ribosomal 18S was used as
the house-keeping gene. (**p < 0.01. ***p < 0.001. Mean
± SEM, n ≥ 3).
Conclusions: We have developed enzyme-immobilized
hydrogels for inducing hypoxia for in vitro cell culture.
The system was able to cause hypoxic cellular response
for 24-48 hours, a time frame appropriate for many
biological assays. Enzyme-immobilized hydrogels
provide a simple and more efficient option to induce
realistic hypoxia compared with the use of chemical
compounds. Future work will focus on developing oxygen
gradients using hypoxia inducing enzyme-immobilized
hydrogels.
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Vaupel P. J Natl Cancer Inst. 2001;93:266-76. [5] Liu L, Simon MC.
Cancer Biol Ther. 2004;3:492-7. [6] Hao Y, Lin CC. J Biomed Mater
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[9] An WG et al. Nature. 1998;392:405-8.