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Here, we developed a three-dimensional culture system that enables human microvascular units (MVUs) to expand and self-organize into vascularized microtissues containing well-formed microvascular structures and stem-cell-derived functional stroma. Moreover, the vascularized microtissue is customizable in both shape and size. The functionality of vascularized microtissues was evaluated in various tissue defect models including spinal cord injury, skin defects, and femoral bone defects. In vivo evidence confirmed that transplantation of customized vascularized microtissues efficiently enhanced nerve function and motor recovery in the spinal cord injury model, accelerated wound closure, facilitated skin appendage regeneration in the skin defect model, and boosted bone regeneration in the femoral defect model. 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