3D culture and organoid technologies have been developing rapidly in the last decade, and already found widespread applications in biology and medicine. While cells, and stem cells in particular, have tremendous self-organization potential, most applications benefit from further engineering of the cellular microenvironment in order to guide the morphogenesis.
We will describe several recently developed technologies that exploit dynamic physical processes to support or guide morphogenesis in defined 3D cultures. In one instance, we formed hydrogels with reversible, dynamic bonds, in order to impart polyethylene glycol (PEG) hydrogels with stress relaxation and self-healing properties. The resulting hydrogels could support intestinal stem cell expansion as well as differentiation to budding organoids in a single defined hydrogel. In another instance, we exploited a dynamic aqueous-aqueous phase separation triggered by PEG cross-linking in order to gain control over the pore structure of the PEG gels. The method is simple, cost-effective, compatible with injection and cell encapsulation, yields clear hydrogels, and easily tuned to obtain pore sizes of any relevant size, from less than 1 to more than 100 micrometers. These hydrogels proved optimal to support the formation of functional 3D neural networks in a defined environment. Finally, we will show how morphogenesis can further be guided with dynamic patterning of morphogens / growth factors, using a recently developed two-photon patterning method, compatible with sensitive biomolecules, live cells, and both natural and synthetic extracellular matrices.
We believe such tailored microenvironments, exploiting dynamic processes to guide morphogenesis, will be key to the development of 3D engineered tissues with a higher degree of cellular organization.