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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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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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.
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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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DOI: https://doi.org/10.1038/nprot.2016.037
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