Abstract
Oxygen (O2) acts as a potent upstream regulator of cell function. In both physiological and pathophysiological microenvironments, the O2 concentration is not uniformly distributed but instead follows a gradient that depends on distance from oxygen-carrying blood vessels. Such gradients have a particularly important role in development, tissue regeneration, and tumor growth. In this protocol, we describe how to use our previously reported gelatin-based O2-controllable hydrogels that can provide hypoxic microenvironments in vitro. The hydrogel polymeric network is formed via a laccase-mediated cross-linking reaction. In this reaction, laccase catalyzes diferulic acid (diFA) formation to form hydrogels with an O2-consuming reaction. Cells, such as cancer or endothelial cells, as well as tumor/tissue grafts, can be encapsulated in the hydrogels during hydrogel formation and then analyzed for cellular responses to 3D hypoxic gradients and to elucidate the underlying mechanisms governing these responses. Importantly, oxygen gradients can be precisely controlled in standard cell/tissue culture conditions and in vivo. This platform has been applied to study vascular morphogenesis in response to hypoxia and to understand how oxygen gradients mediate cancer cell behavior. Herein, we describe the means to validate the assay from polymer synthesis and characterization—which take 1–2 weeks and include verification of ferulic acid (FA) conjugation, rheological measurements, and O2 monitoring—to the study of cellular responses and use in rodent models. Time courses for biological experiments using this hydrogel are variable, and thus they may range from hours to weeks, depending on the application and user end goal.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Brizel, D.M. et al. Tumor oxygenation predicts for the likelihood of distant metastases in human soft tissue sarcoma. Cancer Res. 56, 941–943 (1996).
Mori, R. et al. The relationship between proangiogenic gene expression levels in prostate cancer and their prognostic value for clinical outcomes. Prostate 70, 1692–1700 (2010).
Koukourakis, M.I. et al. Hypoxia-inducible factor (HIF1A and HIF2A), angiogenesis, and chemoradiotherapy outcome of squamous cell head-and-neck cancer. Int. J. Radiat. Oncol. Biol. Phys. 53, 1192–1202 (2002).
Bertout, J.A., Patel, S.A. & Simon, M.C. The impact of O2 availability on human cancer. Nat. Rev. Cancer 8, 967–975 (2008).
Lewis, D.M. et al. Intratumoral oxygen gradients mediate sarcoma cell invasion. Proc. Natl. Acad. Sci. 113, 9292–9297 (2016).
Chaturvedi, P. et al. Hypoxia-inducible factor-dependent breast cancer-mesenchymal stem cell bidirectional signaling promotes metastasis. J. Clin. Invest. 123, 189–205 (2013).
Eisinger-Mathason, T.K. et al. Hypoxia-dependent modification of collagen networks promotes sarcoma metastasis. Cancer Discov. 3, 1190–1205 (2013).
Carmeliet, P. et al. Role of HIF-1α in hypoxia-mediated apoptosis, cell proliferation and tumour angiogenesis. Nature 394, 485–490 (1998).
Carmeliet, P. & Jain, R.K. Angiogenesis in cancer and other diseases. Nature 407, 249–257 (2000).
Kvanta, A., Algvere, P., Berglin, L. & Seregard, S. Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor. Invest. Ophthalmol. Vis. Sci. 37, 1929–1934 (1996).
Lin, C.-H. et al. Silibinin inhibits VEGF secretion and age-related macular degeneration in a hypoxia-dependent manner through the PI-3 kinase/Akt/mTOR pathway. Br. J. Pharmacol. 168, 920–931 (2013).
Stefánsson, E., Geirsdóttir, Á. & Sigurdsson, H. Metabolic physiology in age related macular degeneration. Prog. Ret. Eye Res. 30, 72–80 (2011).
Lewis, D.M., Abaci, H.E., Xu, Y. & Gerecht, S. Endothelial progenitor cell recruitment in a microfluidic vascular model. Biofabrication 7, 045010 (2015).
Arnould, T., Michiels, C. & Remacle, J. Increased PMN adherence on endothelial cells after hypoxia: involvement of PAF, CD18/CD11b, and ICAM-1. Am. J. Physiol. 264, C1102–1110 (1993).
Luster, A.D., Alon, R. & von Andrian, U.H. Immune cell migration in inflammation: present and future therapeutic targets. Nat. Immunol. 6, 1182–1190 (2005).
Craig, R., Schofield, J. & Jackson, S. Collagen biosynthesis in normal human skin, normal and hypertrophic scar and keloid. Eur. J. Clin. Invest. 5, 69–74 (1975).
Botusan, I.R. et al. Stabilization of HIF-1α is critical to improve wound healing in diabetic mice. Proc. Natl. Acad. Sci. 105, 19426–19431 (2008).
Ramirez-Bergeron, D.L. & Simon, M.C. Hypoxia-inducible factor and the development of stem cells of the cardiovascular system. Stem Cells 19, 279–286 (2001).
Licht, A.H., Muller-Holtkamp, F., Flamme, I. & Breier, G. Inhibition of hypoxia-inducible factor activity in endothelial cells disrupts embryonic cardiovascular development. Blood 107, 584–590 (2006).
Krock, B.L., Skuli, N. & Simon, M.C. Hypoxia-induced angiogenesis good and evil. Genes Cancer 2, 1117–1133 (2011).
Kusuma, S., Peijnenburg, E., Patel, P. & Gerecht, S. Low oxygen tension enhances endothelial fate of human pluripotent stem cells. Arterioscler., Thromb., Vasc. Biol. 34, 913–920 (2014).
Lee, Y.M. et al. Determination of hypoxic region by hypoxia marker in developing mouse embryos in vivo: a possible signal for vessel development. Dev. Dyn. 220, 175–186 (2001).
Liu, Y., Cox, S.R., Morita, T. & Kourembanas, S. Hypoxia regulates vascular endothelial growth factor gene expression in endothelial cells identification of a 5′ enhancer. Circ. Res. 77, 638–643 (1995).
Covello, K.L. & Simon, M.C. HIFs, hypoxia, and vascular development. Curr. Top. Dev. Biol. 62, 37–54 (2004).
Pugh, C.W. & Ratcliffe, P.J. Regulation of angiogenesis by hypoxia: role of the HIF system. Nat. Med. 9, 677–684 (2003).
Marti, H.J. et al. Hypoxia-induced vascular endothelial growth factor expression precedes neovascularization after cerebral ischemia. Am. J. Pathol. 156, 965–976 (2000).
Oppegard, S.C., Nam, K.H., Carr, J.R., Skaalure, S.C. & Eddington, D.T. Modulating temporal and spatial oxygenation over adherent cellular cultures. PLoS One 4, 8 (2009).
Rexius-Hall, M.L., Mauleon, G., Malik, A.B., Rehman, J. & Eddington, D.T. Microfluidic platform generates oxygen landscapes for localized hypoxic activation. Lab Chip 14, 4688–4695 (2014).
Adler, M., Polinkovsky, M., Gutierrez, E. & Groisman, A. Generation of oxygen gradients with arbitrary shapes in a microfluidic device. Lab Chip 10, 388–391 (2010).
Chen, Y.A. et al. Generation of oxygen gradients in microfluidic devices for cell culture using spatially confined chemical reactions. Lab Chip 11, 3626–3633 (2011).
Funamoto, K. et al. A novel microfluidic platform for high-resolution imaging of a three-dimensional cell culture under a controlled hypoxic environment. Lab Chip 12, 4855–4863 (2012).
Oppegard, S.C. & Eddington, D.T. A microfabricated platform for establishing oxygen gradients in 3-D constructs. Biomed. Microdevices 15, 407–414 (2013).
DelNero, P. et al. 3D culture broadly regulates tumor cell hypoxia response and angiogenesis via pro-inflammatory pathways. Biomaterials 55, 110–118 (2015).
Rodenhizer, D. et al. A three-dimensional engineered tumour for spatial snapshot analysis of cell metabolism and phenotype in hypoxic gradients. Nat. Mater. 15, 227–234 (2016).
Wu, D. & Yotnda, P. Induction and testing of hypoxia in cell culture. J. Vis. Exp. (54), 2899 (2011).
Lin, X. et al. Oxygen-induced cell migration and on-line monitoring biomarkers modulation of cervical cancers on a microfluidic system. Sci. Rep. 5, 9643 (2015).
Allen, C.B., Schneider, B.K. & White, C.W. Limitations to oxygen diffusion and equilibration in in vitro cell exposure systems in hyperoxia and hypoxia. Am. J. Physiol. Lung Cell. Mol. Physiol. 281, L1021–L1027 (2001).
Park, K.M. & Gerecht, S. Hypoxia-inducible hydrogels. Nat. Commun. 5, 4075 (2014).
Hanjaya-Putra, D. et al. Controlled activation of morphogenesis to generate a functional human microvasculature in a synthetic matrix. Blood 118, 804–815 (2011).
Davis, G.E. & Senger, D.R. Endothelial extracellular matrix - biosynthesis, remodeling, and functions during vascular morphogenesis and neovessel stabilization. Circ. Res. 97, 1093–1107 (2005).
Davis, G.E., Bayless, K.J. & Mavila, A. Molecular basis of endothelial cell morphogenesis in three-dimensional extracellular matrices. Anat. Rec. 268, 252–275 (2002).
Chan, X.Y. et al. Three-dimensional vascular network assembly from diabetic patient-derived induced pluripotent stem cells. Arterioscler., Thromb., Vasc. Biol. 35, 2677–2685 (2015).
Blatchley, M., Park, K.M. & Gerecht, S. Designer hydrogels for precision control of oxygen tension and mechanical properties. J. Mater. Chem. B 3, 7939–7949 (2015).
Higuchi, A., Ling, Q.D., Hsu, S.T. & Umezawa, A. Biomimetic cell culture proteins as extracellular matrices for stem cell differentiation. Chem. Rev. 112, 4507–4540 (2012).
Paguirigan, A.L. & Beebe, D.J. Protocol for the fabrication of enzymatically crosslinked gelatin microchannels for microfluidic cell culture. Nat. Protoc. 2, 1782–1788 (2007).
Yung, C.W. et al. Transglutaminase crosslinked gelatin as a tissue engineering scaffold. J. Biomed. Mater. Res. Part A 83A, 1039–1046 (2007).
Loessner, D. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl-based hydrogels as modular tissue culture platforms. Nat. Protoc. 11, 727–746 (2016).
Minussi, R.C., Pastore, G.M. & Duran, N. Potential applications of laccase in the food industry. Trends Food Sci. Technol. 13, 205–216 (2002).
Mozaffarian, D. et al. Heart disease and stroke statistics-2016 update: a report from the American Heart Association. Circulation 133, e38 (2016).
Gaengel, K., Genove, G., Armulik, A. & Betsholtz, C. Endothelial-mural cell signaling in vascular development and angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29, 630–638 (2009).
Carmeliet, P. & Jain, R.K. Molecular mechanisms and clinical applications of angiogenesis. Nature 473, 298–307 (2011).
Semenza, G.L. Vascular responses to hypoxia and ischemia. Arterioscler. Thromb. Vasc. Biol. 30, 648–652 (2010).
Bayless, K.J., Kwak, H.I. & Su, S.C. Investigating endothelial invasion and sprouting behavior in three-dimensional collagen matrices. Nat. Protoc. 4, 1888–1898 (2009).
Yoon, S.S. et al. Efficacy of sunitinib and radiotherapy in genetically engineered mouse model of soft-tissue sarcoma. Int. J. Radiat. Oncol. Biol. Phys. 74, 1207–1216 (2009).
Kirsch, D.G. et al. A spatially and temporally restricted mouse model of soft tissue sarcoma. Nat. Med. 13, 992–997 (2007).
Wu, P.-H., Giri, A. & Wirtz, D. Statistical analysis of cell migration in 3D using the anisotropic persistent random walk model. Nat. Protoc. 10, 517–527 (2015).
Koh, M.Y. & Powis, G. Passing the baton: the HIF switch. Trends Biochem. Sci. 37, 364–372 (2012).
Acknowledgements
This work was supported by a fellowship from the National Cancer Institute (NCI; T-32 2T32CA153952-06), a Nanotechnology Cancer Research training grant (to D.M.L.), the American Heart Association (15EIA22530000), a President's Frontier Award from Johns Hopkins University, and Project 3 of the NCI Physical Sciences-Oncology Center (U54CA210173 to S.G.).
Author information
Authors and Affiliations
Contributions
D.M.L., M.R.B., and K.M.P. developed, tested, applied, and validated the Gel-HI hydrogel system; D.M.L., M.R.B., and K.M.P. performed the experiments; D.M.L., M.R.B., K.M.P., and S.G. designed the experiments, analyzed the results, and wrote the paper.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Characterization of Gtn–FA conjugate
1H NMR spectra of Gtn–FA (red) and Gtn (blue) (300 MHz, D2O, 25 °C): a, δ6.48 (d, 1H); b, δ7.45 (d, 1H); c, δ7.16 (d, 1H); d, δ6.99 (d, 1H); e, δ6.89 (d, 1H). Reproduced with permission from Park and Gerecht (ref. 38), Nature Publishing Group.
Supplementary information
Supplementary Figure 1.
Characterization of the Gtn–FA conjugate. 1H NMR spectra of Gtn–FA (red) and Gtn (blue) (300 MHz, D2O, 25 °C): a, δ6.48 (d, 1H); b, δ7.45 (d, 1H); c, δ7.16 (d, 1H); d, δ6.99 (d, 1H); e, δ6.89 (d, 1H). Reproduced with permission from Park and Gerecht (ref. 38), Nature Publishing Group. (PDF 350 kb)
Rights and permissions
About this article
Cite this article
Lewis, D., Blatchley, M., Park, K. et al. O2-controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo. Nat Protoc 12, 1620–1638 (2017). https://doi.org/10.1038/nprot.2017.059
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2017.059
This article is cited by
-
Middle-out methods for spatiotemporal tissue engineering of organoids
Nature Reviews Bioengineering (2023)
-
The extracellular matrix mechanics in the vasculature
Nature Cardiovascular Research (2023)
-
Current hydrogel advances in physicochemical and biological response-driven biomedical application diversity
Signal Transduction and Targeted Therapy (2021)
-
Optical projection tomography as a quantitative tool for analysis of cell morphology and density in 3D hydrogels
Scientific Reports (2021)
-
Mimicking tumor hypoxia and tumor-immune interactions employing three-dimensional in vitro models
Journal of Experimental & Clinical Cancer Research (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.