A successful method for creating human skin substitutes using xeno-free isolation, expansion, and cryopreservation of human endothelial cells, fibroblasts, pericytes, and keratinocytes. The skin substitutes were printed using a xeno-free bioink containing human collagen type I and fibronectin and a biocompatible polyglycolic acid mesh. When implanted in immunodeficient mice, the skin substitutes developed a mature stratified epidermis with rete ridge-like structures and human EC-lined perfused microvessels. This demonstrates the feasibility of a xeno-free approach to complex tissue engineering.

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