Fabrication of three-dimensional (3D) structures and functional tissues directly in live animals would enable minimally invasive surgical techniques for organ repair or reconstruction. Here, we show that 3D cell-laden photosensitive polymer hydrogels can be bioprinted across and within tissues of live mice, using bio-orthogonal two-photon cycloaddition and crosslinking of the polymers at wavelengths longer than 850 nm. Such intravital 3D bioprinting—which does not create by-products and takes advantage of commonly available multiphoton microscopes for the accurate positioning and orientation of the bioprinted structures into specific anatomical sites—enables the fabrication of complex structures inside tissues of live mice, including the dermis, skeletal muscle and brain. We also show that intravital 3D bioprinting of donor-muscle-derived stem cells under the epimysium of hindlimb muscle in mice leads to the de novo formation of myofibres in the mice. Intravital 3D bioprinting could serve as an in vivo alternative to conventional bioprinting.

直接在活体动物中制造三维 (3D) 结构和功能组织将使用于器官修复或重建的微创手术技术成为可能。在这里，我们展示了载有 3D 细胞的光敏聚合物水凝胶可以在活体小鼠组织内和组织内进行生物打印，使用生物正交双光子环加成和聚合物在波长超过 850 nm 的交联。这种活体 3D 生物打印——它不会产生副产品，并利用常用的多光子显微镜将生物打印结构准确定位和定向到特定的解剖部位——能够在活体小鼠的组织内制造复杂的结构，包括真皮、骨骼肌和大脑。我们还表明，小鼠后肢肌外膜下供体肌源干细胞的活体 3D 生物打印导致小鼠肌纤维从头形成。活体 3D 生物打印可以作为传统生物打印的体内替代品。