Fully defined matrices for the culture and expansion of intestinal stem cells and organoids

摘要

Intestinal stem cells cultured in three-dimensional (3D) matrices are capable of self-organizing into ever-expanding epithelial structures, termed intestinal organoids, which faithfully mimic the histology and architecture of their native counterparts. Intestinal organoids reenact multiple aspects of intestinal development, function and disease, thus emerging as powerful tools in stem cell research, disease modeling and regenerative medicine. The applicability of organoids in both basic and Iranslational research, however, is curtailed by their dependence on animal-derived matrices, which are poorly defined, variable, uncondurive to controlled modifications, and potentially immunogenic. We created fully defined synthetic 3D matrices that allow for the sustained expansion of intestinal stem cells (ISCs), and their subsequent differentiation and morphogenesis into intestinal organoids. Aside from enabling the large-scale production of clinical-grade intestinal tissue and allowing for disease modeling in a chemically defined environment, these hydrogel-based matrices permit controlled perturbation of their biochemical and biophysical parameters, thus opening previously inaccessible directions in organoid-based research. We took advantage of the modularity of the systems to delineate the roles of key microenvironmental factors, including mechanical parameters and extracellular matrix components, during distinct stages of intestinal organoid formation, unveiling previously unforeseen effects. We found that matrix stiffness, proteolytic degradation and adhesion profoundly influence ISC fate, epithelial organization and morphogenesis. Notably, separate stages of the organoid formation process displayed differential mechanical and adhesion requirements. The design principles we uncovered in creating fully defined matrices for intestinal organoid culture can be readily extended and adapted to identify synthetic microenvironments optimal for the growth of other types of epithelial stem cells and organoids, thus expanding their applicability in basic and clinical research.