While organoid culture has the potential to revolutionize our understanding of human biology, current protocols rely on the use of a decellularized membrane from murine Engelbreth-Holm-Swarm sarcoma, a complex, heterogeneous material with large batch-to-batch variations that hinders reproducibility and materials control. In response, several groups have begun designing synthetic hydrogel systems to enable the reproducible culture of organoids. Recently, the matrix stress relaxation rate (i.e. the ability of a hydrogel to remodel its network connectivity in response to an applied stress) has been demonstrated to have profound effects on encapsulated cells. To date, the role of matrix stress relaxation on organoid cultures has not been explored. Here we present the design of a family of double-network hydrogels composed of protein-engineered block-copolymers and chemically-modified hyaluronan. The network undergoes two stages of crosslinking: the first stage uses reversibly dynamic covalent chemistry bonds, while the second stage reinforces the hydrogel through thermal-induced polymer aggregation. This double-network of physical interactions results in a gel with a broad dynamic range of tunable mechanical properties, where the gel stiffness is set by the number of crosslinks and the gel stress relaxation rate is independently set by the kinetics of the crosslinks. These novel, double-network hydrogels are being used to study the role of mechanotransduction in the culture of patient-derived, human primary intestinal organoids. In this system, we find that the organoid cultures display strong phenotypic responses to matrix stress relaxation, which are dependent on cell-matrix interactions with both CD44 and integrin cell-surface receptors.

Adaptable protein-engineered hydrogels for organoid culture

2019 Fall National Meeting

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