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Current vascular replacement grafts used in congenital heart defect corrective surgery have poor longevity and growth potential. Recipient patients often require multiple reoperations. Tissue engineering has the promise to produce a graft with the potential to grow, remodel, and repair. In this study, we aimed at developing an amnion-based scaffold suitable for cardiovascular tissue engineering applications and in vivousage. The developed human amnion-based scaffold was made by an enzymatic decellularization process followed by freeze-drying as a single or multilayered structure. These structures were compared to native amnion for seeded cell viability and biomechanical properties then tested for in vivobiocompatibility. Our results demonstrated that while native amnion tissue supported little cell growth, the decellularized-amnion allowed cell engraftment and survival. In addition, preservation of the scaffold by freeze-drying as a single layer allowed cell engraftment and growth. Multilayering the freeze-dried amnion scaffolds resulted in a similar cell growth potential of the single layered construct but superior mechanical strength. The multilayered construct showed in vitrobiocompatibility with endothelial cells, smooth muscle cells, cardiac myocytes, and thymus and cord blood-derived mesenchymal stem cells (MSCs). When implanted in a piglet model of left pulmonary artery grafting, the multilayered construct showed its in vivosuitability and biocompatibility for vascular repair as demonstrated by the development of newly formed endothelium in the intima, a smooth muscle cell-rich medial layer and an adventitia containing new vasa vasorum. In conclusion, our developed amnion-derived scaffold represents an off-the-shelf biocompatible structure that can be seeded with the patient's own MSCs to produce an autologous vascular graft.Impact StatementThis study aimed at developing an amnion-based scaffold suitable for vascular tissue engineering applications and in vivousage. We successfully produced a multilayered scaffold with improved biomechanical properties and biocompatibility for in vivovascular implantation. Our approach not only offers an allogeneic “off-the-shelf” solution for clinical use but also it provides the possibility of personalized medicine using a patient's own amnion and stem cells for the production of tissue engineered grafts for reconstructive heart surgery.