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O2-controllable hydrogels for studying cellular responses to hypoxic gradients in three dimensions in vitro and in vivo

Nature Protocols volume 12, pages 1620–1638 (2017)Cite this article

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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.

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Figure 1: Schematic representation of the Gel-HI hydrogels for in vitro and in vivo applications.
Figure 2: Gtn-HI hydrogel preparation for in vitro 3D tissue/biopsy culture.
Figure 3: Real-time oxygen monitoring system.
Figure 4: Tubulogenesis in Gel-HI.
Figure 5: Tumor encapsulation and tracking.
Figure 6: Tumor encapsulation and tracking.
Figure 7: Subcutaneous and intramuscular in vivo oxygen sensing.
Figure 8: Application of Gtn-HI hydrogels for in vivo vascular tissue recruitment.

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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

Author notes

  1. Kyung Min Park

    Present address: Present address: Division of Bioengineering, Incheon National University, Incheon, Republic of Korea.,

  2. Daniel M Lewis and Michael R Blatchley: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Chemical and Biomolecular Engineering, Johns Hopkins Physical Sciences–Oncology Center and Institute for NanoBioTechnology,

    Daniel M Lewis, Michael R Blatchley, Kyung Min Park & Sharon Gerecht

  2. Johns Hopkins University, Baltimore, Maryland, USA

    Daniel M Lewis, Michael R Blatchley, Kyung Min Park & Sharon Gerecht

  3. Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland, USA

    Michael R Blatchley

  4. Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, Maryland, USA

    Sharon Gerecht

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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

Correspondence to Sharon Gerecht.

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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)

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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

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