Organoids have extensive therapeutic potential and are increasingly opening up new avenues within regenerative medicine. However, their clinical application is greatly limited by the lack of effective GMP-compliant systems for organoid expansion in culture. Here, we envisage that the use of extracellular matrix (ECM) hydrogels derived from decellularized tissues (DT) can provide an environment capable of directing cell growth. These gels possess the biochemical signature of tissue-specific ECM and have the potential for clinical translation. Gels from decellularized porcine small intestine (SI) mucosa/submucosa enable formation and growth of endoderm-derived human organoids, such as gastric, hepatic, pancreatic, and SI. ECM gels can be used as a tool for direct human organoid derivation, for cell growth with a stable transcriptomic signature, and for in vivo organoid delivery. The development of these ECM-derived hydrogels opens up the potential for human organoids to be used clinically.

Despite the tremendous technical advancements in 3D bioprinting, the concept of fabricating 3D structures and functional tissues directly in live animals remains a visionary challenge. We show that 3D cell-laden hydrogels can be efficiently bioprinted across tissues and within tissues of living animals.We developed photo-sensitive polymers that allow in vitro and in vivo fabrication of hydrogels into pre-existing structures, by bio-orthogonal two-photon cycloaddition and crosslinking at wavelengths longer than 850 nm, without byproducts. By this technique, that we name intravital 3D bioprinting, after injection of these polymers in vivo it is possible to fabricate complex 3D structures inside tissues of living mice, including the dermis across epidermis, the skeletal muscle across epimysium or the brain across meninges. The use of commonly available multi-photon microscopes allows accurate (XYZ) positioning and orientation of bioprinted structures into specific anatomical sites. Finally, we show that intravital 3D bioprinting of donor muscle-derived stem cells allows de novo formation of myofibers in host animals. We envision that this strategy will offer an alternative in vivo approach to conventional bioprinting technology, holding great promises to substantially change the paradigm of 3D bioprinting for pre-clinical and clinical use.

Microengineering human "organs-on-chips" remains an open challenge. Here, we describe a robust microfluidics-based approach for the differentiation of human pluripotent stem cells directly on a chip. Extrinsic signal modulation, achieved through optimal frequency of medium delivery, can be used as a parameter for improved germ layer specification and cell differentiation. Human cardiomyocytes and hepatocytes derived on chips showed functional phenotypes and responses to temporally defined drug treatments.