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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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.
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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