3D bioprinting of flap-tissue with heterogenous cells and flexible perfusable vessels

Despite advancements in bio-manufacturing, the fabrication of large-scale vascularized tissue with heterogeneous cells remains a daunting challenge. To address this challenge, the reverse engineered structured tissue (REST) three-dimensional (3D) bioprinting method was proposed. This strategy enables the seeding and subsequent assembly of multiple tissue-specific constituent cells onto a robust, flexible, 3D biomimetic vascular scaffold with long-term perfusion capability. The material choices for the vessel network and seeding cells could be decoupled, and separate co-culture of multiple seeding cells in a customized bioreactor could be realized. The cellular layers could be reassembled into an engineered tissue by manually folding the robust vessels. Centimeter-scale vascularized skin-flap-like tissues that comprise epidermal, dermal, and adipose layers were engineered with an in vivo-like communicating vascular network using this strategy. The engineered tissue exhibits three-layered cellular heterogeneity (containing HaCaT cells, fibroblasts, and adipose-derived stem cells) and perfusable tubes (containing HUVECs and VSMCs). The complex tissue could be remolded through in vitro perfusion of vessel network and specific culture/differentiation medium supplied to tissue-specific cellular layers. After in vivo transplantation for three months, the tissue construct formed a viable complex tissue with a rich network of blood vessels. Our results demonstrate that "REST" bioprinting technology can be used to fabricate multicellular tissues, ranging from millimeter to centimeter scales with perfusable vessels, opening new avenues for functional artificial organ bioprinting.