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Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture

Nature Protocols volume 12, pages 2263–2274 (2017)Cite this article

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Abstract

Growing cells within an extracellular matrix-like 3D gel is required for, or can improve, the growth of many cell types ex vivo. Here, we describe a protocol for the generation of well-defined matrices for the culture of intestinal stem cells (ISCs) and intestinal organoids. These matrices comprise a poly(ethylene glycol) (PEG) hydrogel backbone functionalized with minimal adhesion cues including RGD (Arg-Gly-Asp), which is sufficient for ISC expansion, and laminin-111, which is required for organoid formation. As such, the hydrogels present a defined and reproducible, but also tunable, environment, allowing researches to manipulate physical and chemical parameters, and examine their influence on ISC and organoid growth. Hydrogels are formed by an enzymatic cross-linking reaction of multiarm PEG precursors bearing glutamine- and lysine-containing peptides. PEG precursors containing either stable or hydrolytically degradable moieties are used to produce mechanically softening hydrogels, which are used for the expansion of ISCs or the formation of organoids, respectively. We also provide protocols for immunofluorescence analysis of cellular structures grown within these matrices, as well as for their dissociation and retrieval of cells for downstream use. Hydrogel precursors can be produced and their mechanical properties characterized to ascertain stiffness within 5–7 d. Hydrogel formation for ISC expansion or organoid formation takes 1–2 h. The materials described here can be readily adapted for the culture of other types of normal or transformed organoid structures.

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Figure 1: Overview of ISC and intestinal organoid culture in PEG-based hydrogels.
Figure 2: Preparation of PEG gels for rheometric characterization.
Figure 3: Typical results of rheometric characterization of PEG hydrogels.
Figure 4: ISC colonies and organoids formed in PEG-based hydrogels.

References

  1. Lancaster, M.A. & Knoblich, J.A. Organogenesis in a dish: modeling development and disease using organoid technologies. Science 345, 1247125 (2014).

    Article  Google Scholar 

  2. Hughes, C.S., Postovit, L.M. & Lajoie, G.A. Matrigel: a complex protein mixture required for optimal growth of cell culture. Proteomics 10, 1886–1890 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  4. Vukicevic, S. et al. Identification of multiple active growth factors in basement membrane Matrigel suggests caution in interpretation of cellular activity related to extracellular matrix components. Exp. Cell Res. 202, 1–8 (1992).

    Article  CAS  Google Scholar 

  5. Nelson, C.M. & Bissell, M.J. Of extracellular matrix, scaffolds, and signaling: tissue architecture regulates development, homeostasis, and cancer. Annu. Rev. Cell Dev. Biol. 22, 287–309 (2006).

    Article  CAS  Google Scholar 

  6. Gjorevski, N. et al. Designer matrices for intestinal stem cell and organoid culture. Nature 539, 560–564 (2016).

    Article  CAS  Google Scholar 

  7. Lutolf, M.P. & Hubbell, J.A. Synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering. Nat. Biotechnol. 23, 47–55 (2005).

    Article  CAS  Google Scholar 

  8. Saha, K., Pollock, J.F., Schaffer, D.V. & Healy, K.E. Designing synthetic materials to control stem cell phenotype. Curr. Opin. Chem. Biol. 11, 381–387 (2007).

    Article  CAS  Google Scholar 

  9. Tibbitt, M.W. & Anseth, K.S. Dynamic microenvironments: the fourth dimension. Sci. Transl. Med. 4, 160ps124 (2012).

    Article  Google Scholar 

  10. Caliari, S.R. & Burdick, J.A. A practical guide to hydrogels for cell culture. Nat. Methods 13, 405–414 (2016).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Lorand, L. & Graham, R.M. Transglutaminases: crosslinking enzymes with pleiotropic functions. Nat. Rev. Mol. Cell Biol. 4, 140–156 (2003).

    Article  CAS  Google Scholar 

  13. Sternlicht, M.D. & Werb, Z. How matrix metalloproteinases regulate cell behavior. Annu. Rev. Cell Dev. Biol. 17, 463–516 (2001).

    Article  CAS  Google Scholar 

  14. Lutolf, M.P. et al. Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: engineering cell-invasion characteristics. Proc. Natl. Acad. Sci. USA 100, 5413–5418 (2003).

    Article  CAS  Google Scholar 

  15. Sato, T. et al. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett′s epithelium. Gastroenterology 141, 1762–1772 (2011).

    Article  CAS  Google Scholar 

  16. Hennink, W.E. & van Nostrum, C.F. Novel crosslinking methods to design hydrogels. Adv. Drug Deliv. Rev. 54, 13–36 (2002).

    Article  CAS  Google Scholar 

  17. Lin, C.C. Recent advances in crosslinking chemistry of biomimetic poly(ethylene glycol) hydrogels. RSC Adv. 5, 39844–398583 (2015).

    Article  CAS  Google Scholar 

  18. Rosales, A.M. & Anseth, K.S. The design of reversible hydrogels to capture extracellular matrix dynamics. Nat. Rev. Mater. 1, 15012 (2016).

    Article  CAS  Google Scholar 

  19. DeForest, C.A. & Anseth, K.S. Advances in bioactive hydrogels to probe and direct cell fate,. Annu. Rev. Chem. Biomol. Eng. 3, 421–444 (2012).

    Article  CAS  Google Scholar 

  20. Wang, H. & Heilshorn, S.C. Adaptable hydrogel networks with reversible linkages for tissue engineering. Adv. Mater. 27, 3717–3736 (2015).

    Article  CAS  Google Scholar 

  21. Kloxin, A.M., Kasko, A.M., Salinas, C.N. & Anseth, K.S. Photodegradable hydrogels for dynamic tuning of physical and chemical properties. Science 324, 59–63 (2009).

    Article  CAS  Google Scholar 

  22. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    Article  CAS  Google Scholar 

  23. Yin, X. et al. Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny. Nat. Methods 11, 106–112 (2014).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank D. Hacker and the EPFL Protein Expression Core Facility (PECF) for the production of R-spondin, Noggin and EGF; D. Pioletti for rheometer use; and N. Brandenberg and A. Negro for technical assistance.

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Authors and Affiliations

  1. Laboratory of Stem Cell Bioengineering, Institute of Bioengineering, School of Life Sciences and School of Engineering, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland

    Nikolce Gjorevski & Matthias P Lutolf

  2. Institute of Chemical Sciences and Engineering, School of Basic Science, EPFL, Lausanne, Switzerland

    Matthias P Lutolf

Authors
  1. Nikolce Gjorevski
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  2. Matthias P Lutolf
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Contributions

N.G. conducted the experiments. M.P.L. and N.G. wrote the manuscript.

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Correspondence to Matthias P Lutolf.

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Competing interests

The École Polytechnique Fédérale de Lausanne has filed for patent protection on the technology described herein, and M.P.L. and N.G. are named as inventors on those patents; M.P.L. is a shareholder in QGel SA, which is active in the commercialization of similar synthetic matrices for cancer applications.

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Gjorevski, N., Lutolf, M. Synthesis and characterization of well-defined hydrogel matrices and their application to intestinal stem cell and organoid culture. Nat Protoc 12, 2263–2274 (2017). https://doi.org/10.1038/nprot.2017.095

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