Designer matrices for intestinal stem cell and organoid culture

Abstract

Epithelial organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived organoids. Our approach overcomes multiple limitations of current organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and organoids.

Complementary, Semiautomated Methods for Creating Multidimensional PEG-Based Biomaterials

“…These biomaterials have been developed to either allow for cell culture on their surfaces (Two-dimensional (2D) hydrogels), or to allow for encapsulation within the material (Three-dimensional (3D) hydrogels). 3D hydrogel models have been used to independently tune biophysical and biochemical cues to determine their impact on intestinal stem cell organoid growth, 1 cancer cell invasion, 9 and drug response. 10 Moreover, the dynamics of microenvironments have been captured in some 3D models by using photoinitiated polymerization to change the hydrogel modulus and ligand density 11 and by creating patterns within a hydrogel to control the spatial arrangement of biochemical cues within the matrix to direct cell phenotype.…”

Mechanotransduction and Growth Factor Signalling to Engineer Cellular Microenvironments

“…A very recent study by Gjorevski et al describes the use of fully-defined, degradable PEG-based gels, enriched with fibronectin or laminin-111, for intestinal stem cell (ISC) and organoid culture. [1] Enzymatically degradable hydrogels did not support ISC expansion and non-degradable matrices were needed. However, for organoid development matrix softening through ester-based hydrolysis of hybrid PEG gels was required.…”

Take a deep breath and digest the material: organoids and biomaterials of the respiratory and digestive systems

“…More recently, synthetic hydrogels have been developed for intestinal organoid cultures in order to define the both the physical and adhesive properties of the scaffold and without the need for Matrigel [18] (Fig. 1).…”

“…[5,[14][15][16][17][18][19][20][21][22] Intestinal organoids have been used to model epithelial barrier function, interactions with microbes, infectious diseases and cancer. [23][24][25][26][27][28][29][30][31][32] Intestinal organoids derived from human samples were first grown from isolated small intestinal or colonic crypts, which are localized at the base of the intestinal and colonic epithelium, respectively.…”