Thrombin is used to rapidly polymerize fibrinogen ( 17), whereas TG is a slow-acting Ca 2+-dependent enzymatic cross-linker that imparts the mechanical and thermal stability ( 18) needed for long-term perfusion. The thermally reversible gelation of the gelatin–fibrinogen network enables its use in both printing and casting, where gel and fluid states are required, respectively ( SI Appendix, Fig. The cell-laden inks must facilitate printing of self-supporting filamentary features under ambient conditions as well as subsequent infilling of the printed tissue architectures by casting without dissolving or distorting the patterned construct ( Fig. Specifically, these materials form a gelatin–fibrin matrix cross-linked by a dual-enzymatic, thrombin and transglutaminase (TG), strategy ( Fig. To satisfy the concomitant requirements of processability, heterogeneous integration, biocompatibility, and long-term stability, we first developed printable cell-laden inks and castable ECM based on a gelatin and fibrinogen blend ( 16). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation.Ĭentral to the fabrication of thick vascularized tissues is the design of biological, fugitive, and elastomeric inks for multimaterial 3D bioprinting. However, in both cases, the inability to directly perfuse these vascularized tissues limited their thickness (1–2 mm) and culture times (6 wk). ( 14) developed an alternate approach, in which multiple cell-laden, fugitive (vasculature), and extracellular matrix (ECM) inks are coprinted under ambient conditions. ( 15) reported an elegant method for creating vascularized tissues, in which a sacrificial carbohydrate glass is printed at elevated temperature (>100 ☌), protectively coated, and then removed, before introducing a homogeneous cell-laden matrix. Three-dimensional bioprinting is an emerging approach for creating complex tissue architectures ( 10, 11), including those with embedded vasculature ( 12– 15), that may address the unmet needs of tissue manufacturing. The ability to manufacture human tissues that replicate the essential spatial ( 1), mechanochemical ( 2, 3), and temporal aspects of biological tissues ( 4) would enable myriad applications, including 3D cell culture ( 5), drug screening ( 6, 7), disease modeling ( 8), and tissue repair and regeneration ( 9, 10). This longitudinal study of emergent biological phenomena in complex microenvironments represents a foundational step in human tissue generation. These thick vascularized tissues are actively perfused with growth factors to differentiate hMSCs toward an osteogenic lineage in situ. Specifically, we integrate parenchyma, stroma, and endothelium into a single thick tissue by coprinting multiple inks composed of human mesenchymal stem cells (hMSCs) and human neonatal dermal fibroblasts (hNDFs) within a customized extracellular matrix alongside embedded vasculature, which is subsequently lined with human umbilical vein endothelial cells (HUVECs). To improve their physiological relevance, we report a method for bioprinting 3D cell-laden, vascularized tissues that exceed 1 cm in thickness and can be perfused on chip for long time periods (>6 wk). To date, bioprinting methods have yielded thin tissues that only survive for short durations. The advancement of tissue and, ultimately, organ engineering requires the ability to pattern human tissues composed of cells, extracellular matrix, and vasculature with controlled microenvironments that can be sustained over prolonged time periods.

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