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Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms

Nature Protocols volume 11, pages 727–746 (2016)Cite this article

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Abstract

Progress in advancing a system-level understanding of the complexity of human tissue development and regeneration is hampered by a lack of biological model systems that recapitulate key aspects of these processes in a physiological context. Hence, growing demand by cell biologists for organ-specific extracellular mimics has led to the development of a plethora of 3D cell culture assays based on natural and synthetic matrices. We developed a physiological microenvironment of semisynthetic origin, called gelatin methacryloyl (GelMA)-based hydrogels, which combine the biocompatibility of natural matrices with the reproducibility, stability and modularity of synthetic biomaterials. We describe here a step-by-step protocol for the preparation of the GelMA polymer, which takes 1–2 weeks to complete, and which can be used to prepare hydrogel-based 3D cell culture models for cancer and stem cell research, as well as for tissue engineering applications. We also describe quality control and validation procedures, including how to assess the degree of GelMA functionalization and mechanical properties, to ensure reproducibility in experimental and animal studies.

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Figure 1: Overview of the GelMA-based hydrogel preparation protocol.
Figure 2: GelMA-based hydrogel preparation and 3D cell culture.
Figure 3: Application of GelMA-based hydrogels as cell delivery vehicles for an intraperitoneal animal model.
Figure 4: Application of GelMA-based hydrogels in a breast cancer bone colonization animal model.
Figure 5: Formation of a humanized vascular network in GelMA-based hydrogels.
Figure 6: Microfluidic channels coated with GelMA-based hydrogels for use in a cardiomyocyte culture model.
Figure 7: Effect of Teflon casting mold depth on mechanical and swelling properties of GelMA-based hydrogels.

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References

  1. Wolf, K. et al. Collagen-based cell migration models in vitro and in vivo. Semin. Cell Dev. Biol. 20, 931–941 (2009).

    Article  CAS  Google Scholar 

  2. Schwarzbauer, J. Basement membranes: putting up the barriers. Cur. Biol. 9, R242–R244 (1999).

    Article  CAS  Google Scholar 

  3. Hynes, R.O. The extracellular matrix: not just pretty fibrils. Science 326, 1216–1219 (2009).

    Article  CAS  Google Scholar 

  4. Frantz, C., Stewart, K.M. & Weaver, V.M. The extracellular matrix at a glance. J. Cell Sci. 123, 4195–4200 (2010).

    Article  CAS  Google Scholar 

  5. Lu, P., Takai, K., Weaver, V.M. & Werb, Z. Extracellular matrix degradation and remodeling in development and disease. Cold Spring Harb. Perspect. Biol. 3, a005058 (2011).

    Article  Google Scholar 

  6. Kutys, M.L. & Yamada, K.M. An extracellular-matrix-specific GEF-GAP interaction regulates Rho GTPase crosstalk for 3D collagen migration. Nat. Cell Biol. 16, 909–917 (2014).

    Article  CAS  Google Scholar 

  7. Bissell, M.J. & Radisky, D. Putting tumours in context. Nat. Rev. Cancer 1, 46–54 (2001).

    Article  CAS  Google Scholar 

  8. Wirtz, D., Konstantopoulos, K. & Searson, P.C. The physics of cancer: the role of physical interactions and mechanical forces in metastasis. Nat. Rev. Cancer 11, 512–522 (2011).

    Article  CAS  Google Scholar 

  9. Pickup, M.W., Mouw, J.K. & Weaver, V.M. The extracellular matrix modulates the hallmarks of cancer. EMBO Rep. 15, 1243–1253 (2014).

    Article  CAS  Google Scholar 

  10. Levett, P.A. et al. A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. Acta Biomater. 10, 214–223 (2014).

    Article  CAS  Google Scholar 

  11. Engler, A.J., Sen, S., Sweeney, H.L. & Discher, D.E. Matrix elasticity directs stem cell lineage specification. Cell 126, 677–689 (2006).

    Article  CAS  Google Scholar 

  12. Huebsch, N. et al. Harnessing traction-mediated manipulation of the cell/matrix interface to control stem-cell fate. Nat. Mater. 9, 518–526 (2010).

    Article  CAS  Google Scholar 

  13. Loessner, D. et al. Bioengineered 3D platform to explore cell-ECM interactions and drug resistance of epithelial ovarian cancer cells. Biomaterials 31, 8494–8506 (2010).

    Article  CAS  Google Scholar 

  14. Nichol, J.W. et al. Cell-laden microengineered gelatin methacrylate hydrogels. Biomaterials 31, 5536–5544 (2010).

    Article  CAS  Google Scholar 

  15. Chaudhuri, O. et al. Extracellular matrix stiffness and composition jointly regulate the induction of malignant phenotypes in mammary epithelium. Nat. Mater. 13, 970–978 (2014).

    Article  CAS  Google Scholar 

  16. Hutmacher, D.W. Biomaterials offer cancer research the third dimension. Nat. Mater. 9, 90–93 (2010).

    Article  CAS  Google Scholar 

  17. Hutmacher, D.W. et al. Can tissue engineering concepts advance tumor biology research? Trends Biotechnol. 28, 125–133 (2010).

    Article  CAS  Google Scholar 

  18. Loessner, D., Holzapfel, B.M. & Clements, J.A. Engineered microenvironments provide new insights into ovarian and prostate cancer progression and drug responses. Adv. Drug Deliv. Rev. 79–80, 193–213 (2014).

    Article  Google Scholar 

  19. Malda, J. et al. 25th anniversary article: engineering hydrogels for biofabrication. Adv. Mater. 25, 5011–5028 (2013).

    Article  CAS  Google Scholar 

  20. Annabi, N. et al. 25th anniversary article: rational design and applications of hydrogels in regenerative medicine. Adv. Mater. 26, 85–123 (2014).

    Article  CAS  Google Scholar 

  21. Bichsel, C.A. et al. Diagnostic microchip to assay 3D colony-growth potential of captured circulating tumor cells. Lab Chip 12, 2313–2316 (2012).

    Article  CAS  Google Scholar 

  22. Bray, L.J. et al. Multi-parametric hydrogels support 3D in vitro bioengineered microenvironment models of tumour angiogenesis. Biomaterials 53, 609–620 (2015).

    Article  CAS  Google Scholar 

  23. Loessner, D. et al. A bioengineered 3D ovarian cancer model for the assessment of peptidase-mediated enhancement of spheroid growth and intraperitoneal spread. Biomaterials 34, 7389–7400 (2013).

    Article  CAS  Google Scholar 

  24. Szot, C.S., Buchanan, C.F., Freeman, J.W. & Rylander, M.N. 3D in vitro bioengineered tumors based on collagen I hydrogels. Biomaterials 32, 7905–7912 (2011).

    Article  CAS  Google Scholar 

  25. Kleinman, H.K. & Martin, G.R. Matrigel: basement membrane matrix with biological activity. Semin. Cancer Biol. 15, 378–386 (2005).

    Article  CAS  Google Scholar 

  26. Hutmacher, D.W. & Cukierman, E. Engineering of tumor microenvironments. Adv. Drug Deliv. Rev. 79–80, 1–2 (2014).

    Article  Google Scholar 

  27. Ehrbar, M. et al. Biomolecular hydrogels formed and degraded via site-specific enzymatic reactions. Biomacromolecules 8, 3000–3007 (2007).

    Article  CAS  Google Scholar 

  28. Serebriiskii, I., Castello-Cros, R., Lamb, A., Golemis, E.A. & Cukierman, E. Fibroblast-derived 3D matrix differentially regulates the growth and drug-responsiveness of human cancer cells. Matrix Biol. 27, 573–585 (2008).

    Article  CAS  Google Scholar 

  29. Kenny, H.A., Krausz, T., Yamada, S.D. & Lengyel, E. Use of a novel 3D culture model to elucidate the role of mesothelial cells, fibroblasts and extra-cellular matrices on adhesion and invasion of ovarian cancer cells to the omentum. Int. J. Cancer 121, 1463–1472 (2007).

    Article  CAS  Google Scholar 

  30. Kenny, H.A. et al. Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy. Nat. Commun. 6, 6220 (2015).

    Article  CAS  Google Scholar 

  31. Chwalek, K., Tang-Schomer, M.D., Omenetto, F.G. & Kaplan, D.L. In vitro bioengineered model of cortical brain tissue. Nat. Protoc. 10, 1362–1373 (2015).

    Article  CAS  Google Scholar 

  32. Jenkins, J., Dmitriev, R.I., Morten, K., McDermott, K.W. & Papkovsky, D.B. Oxygen-sensing scaffolds for 3-dimensional cell and tissue culture. Acta Biomater. 16, 126–135 (2015).

    Article  CAS  Google Scholar 

  33. Sieh, S. et al. Paracrine interactions between LNCaP prostate cancer cells and bioengineered bone in 3D in vitro culture reflect molecular changes during bone metastasis. Bone 63, 121–131 (2014).

    Article  CAS  Google Scholar 

  34. Gomez-Guillen, M.C., Gimenez, B., Lopez-Caballero, M.E. & Montero, M.P. Functional and bioactive properties of collagen and gelatin from alternative sources: a review. Food Hydrocolloid. 25, 1813–1827 (2011).

    Article  CAS  Google Scholar 

  35. Van Den Bulcke, A.I. et al. Structural and rheological properties of methacrylamide modified gelatin hydrogels. Biomacromolecules 1, 31–38 (2000).

    Article  CAS  Google Scholar 

  36. Sutter, M., Siepmann, J., Hennink, W.E. & Jiskoot, W. Recombinant gelatin hydrogels for the sustained release of proteins. J. Control. Release 119, 301–312 (2007).

    Article  CAS  Google Scholar 

  37. Kaemmerer, E. et al. Gelatine methacrylamide-based hydrogels: an alternative three-dimensional cancer cell culture system. Acta Bomater. 10, 2551–2562 (2014).

    Article  CAS  Google Scholar 

  38. Yue, K. et al. Synthesis, properties, and biomedical applications of gelatin methacryloyl (GelMA) hydrogels. Biomaterials 73, 254–271 (2015).

    Article  CAS  Google Scholar 

  39. Schuurman, W. et al. Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. Macromol. Biosci. 13, 551–561 (2013).

    Article  CAS  Google Scholar 

  40. Chen, Y.C. et al. Functional human vascular network generated in photocrosslinkable gelatin methacrylate hydrogels. Adv. Funct. Mater. 22, 2027–2039 (2012).

    Article  CAS  Google Scholar 

  41. Bertassoni, L.E. et al. Hydrogel bioprinted microchannel networks for vascularization of tissue engineering constructs. Lab Chip. 14, 2202–2211 (2014).

    Article  CAS  Google Scholar 

  42. Paul, A. et al. Injectable graphene oxide/hydrogel-based angiogenic gene delivery system for vasculogenesis and cardiac repair. ACS Nano 8, 8050–8062 (2014).

    Article  CAS  Google Scholar 

  43. Thibaudeau, L. et al. Mimicking breast cancer-induced bone metastasis in vivo: current transplantation models and advanced humanized strategies. Cancer Metastasis Rev. 33, 721–735 (2014).

    Article  CAS  Google Scholar 

  44. Annabi, N. et al. Hydrogel-coated microfluidic channels for cardiomyocyte culture. Lab Chip 13, 3569–3577 (2013).

    Article  CAS  Google Scholar 

  45. Gong, J.P. Friction and lubrication of hydrogels: its richness and complexity. Soft Matter 2, 544–552 (2006).

    Article  CAS  Google Scholar 

  46. O'Brien, A.K., Cramer, N.B. & Bowman, C.N. Oxygen inhibition in thiol–acrylate photopolymerizations. J. Polym. Sci. A Polym. Chem. 44, 2007–2014 (2006).

    Article  CAS  Google Scholar 

  47. Bartnikowski, M., Bartnikowski, N.J., Woodruff, M.A., Schrobback, K. & Klein, T.J. Protective effects of reactive functional groups on chondrocytes in photocrosslinkable hydrogel systems. Acta Biomater. 27, 66–76 (2015).

    Article  CAS  Google Scholar 

  48. Levett, P.A., Hutmacher, D.W., Malda, J. & Klein, T.J. Hyaluronic acid enhances the mechanical properties of tissue-engineered cartilage constructs. PLoS ONE 9, e113216 (2014).

    Article  Google Scholar 

  49. Hollander, A.P. et al. Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J. Clin. Invest. 93, 1722–1732 (1994).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The work presented by the authors was supported by the Australian Research Council (Future Fellowship awarded to T.J.K. and D.W.H.; Discovery Project grants awarded to D.L., T.J.K. and D.W.H.), the National Health and Medical Research Council of Australia (D.W.H.), Cancer Council Queensland (D.L.), the European Union (Marie Curie Fellowship PIOF-GA-2010-272286 to F.P.W.M.), the US National Science Foundation (EFRI-1240443 to A.K.), IMMODGEL (602694 to A.K.) and the US National Institutes of Health (EB012597, AR057837, DE021468, HL099073, AI105024 and AR063745 to A.K.). We also acknowledge the National Breast Cancer Foundation (IN-15-047 to D.W.H.) Foundation (IN-15-047 to D.W.H.) and an Australia-Harvard Fellowship (to A.K. and D.W.H.) from Harvard Club of Australia Foundation. Australia-Harvard Fellowship (to A.K. and D.W.H.) Harvard Club of Australia Foundation.

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Author notes

  1. Daniela Loessner and Christoph Meinert: These authors contributed equally to this work.

Authors and Affiliations

  1. Queensland University of Technology (QUT), Brisbane, Queensland, Australia

    Daniela Loessner, Christoph Meinert, Elke Kaemmerer, Laure C Martine, Peter A Levett, Travis J Klein, Ferry P W Melchels & Dietmar W Hutmacher

  2. Division of Biomedical Engineering, Department of Medicine, Biomaterials Innovation Research Center, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts, USA

    Kan Yue & Ali Khademhosseini

  3. Division of Health Sciences and Technology, Harvard-Massachusetts Institute of Technology, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA

    Kan Yue & Ali Khademhosseini

  4. Department of Orthopaedics, University Medical Center Utrecht, Utrecht, the Netherlands

    Ferry P W Melchels

  5. Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, UK

    Ferry P W Melchels

  6. Wyss Institute for Biologically Inspired Engineering, Harvard University, Boston, Massachusetts, USA

    Ali Khademhosseini

  7. Department of Bioindustrial Technologies, College of Animal Bioscience and Technology, Konkuk University, Hwayang-dong, Gwangjin-gu, Seoul, Republic of Korea

    Ali Khademhosseini

  8. Department of Physics, King Abdulaziz University, Jeddah, Saudi Arabia

    Ali Khademhosseini

  9. Australian Prostate Cancer Research Centre-Queensland, Translational Research Institute, Queensland University of Technology, Brisbane, Queensland, Australia

    Dietmar W Hutmacher

  10. George W. Woodruff School of Mechanical Engineering, Georgia Institute of Technology, Atlanta, Georgia, USA

    Dietmar W Hutmacher

  11. Institute for Advanced Study, Technische Universität München, Munich, Germany

    Dietmar W Hutmacher

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Contributions

D.L., C.M., E.K., A.K. and D.W.H. conceived and designed the experiments. D.L., C.M. and E.K. performed the experiments and analyzed the data. D.L., C.M. and D.W.H. wrote the manuscript; E.K. and K.Y. partially wrote the manuscript. L.C.M. performed the breast cancer bone colonization animal study. C.M., P.A.L. and T.J.K. established the cartilage tissue engineering. F.P.W.M. established the GelMA polymer production and physico-chemical characterization. A.K. and D.W.H. supervised this project. All authors read and critiqued the manuscript extensively.

Corresponding authors

Correspondence to Daniela Loessner, Ali Khademhosseini or Dietmar W Hutmacher.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Hydrogel casting mould.

Technical drawing and dimensions for the custom-made Teflon casting mould. This mould produces hydrogel strips of 50 mm x 4 mm x 2 mm (length x width x height), which can be cut into smaller units using a cutting guide. All dimensions are in mm.

Supplementary Figure 2 Hydrogel cutting guide.

Technical drawing and dimensions for the custom-made Teflon cutting guide. This guide can be used to cut hydrogel strips obtained after polymerization in the Teflon casting mould into smaller units of 4 mm x 4 mm x 2 mm (length x width x height). All dimensions are in mm.

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Loessner, D., Meinert, C., Kaemmerer, E. et al. Functionalization, preparation and use of cell-laden gelatin methacryloyl–based hydrogels as modular tissue culture platforms. Nat Protoc 11, 727–746 (2016). https://doi.org/10.1038/nprot.2016.037

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