Bioengineered tissues or organs produced using matrix proteins or components derived from xenogeneic sources pose risks of allergic responses, immune rejection, or even autoimmunity. Here, we report successful xeno-free isolation, expansion, and cryopreservation of human endothelial cells (EC), fibroblasts (FBs), pericytes (PCs), and keratinocytes (KCs). We further demonstrate the bioprinting of a human skin substitute with a dermal layer containing xeno-free cultured human EC, FBs, and PCs in a xeno-free bioink containing human collagen type I and fibronectin layered in a biocompatible polyglycolic acid mesh and subsequently seeded with xeno-free human KCs to form an epidermal layer. Following implantation of such bilayered skin grafts on the dorsum of immunodeficient mice, KCs form a mature stratified epidermis with rete ridge-like structures. The ECs and PCs form human EC-lined perfused microvessels within 2 weeks after implantation, preventing graft necrosis, and eliciting further perfusion of the graft by angiogenic host microvessels. As proof-of-concept, we generated 12 individual grafts using a single donor of all four cell types. In summary, we describe the fabrication of a bioprinted vascularized bilayered skin substitute under completely xeno-free culture conditions demonstrating feasibility of a xeno-free approach to complex tissue engineering.

中文翻译：

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植入式无异种血管化人体皮肤移植物的 3D 生物打印


使用异种来源的基质蛋白或成分生产的生物工程组织或器官会带来过敏反应、免疫排斥甚至自身免疫的风险。在此，我们报告了人内皮细胞 (EC)、成纤维细胞 (FB)、周细胞 (PC) 和角质形成细胞 (KC) 的成功无异源分离、扩增和冷冻保存。我们进一步证明了人类皮肤替代品的生物打印，其真皮层含有不含异种物质的培养的人类 EC、FB 和 PC，在含有人类 I 型胶原蛋白和纤连蛋白的无异种生物墨水中分层在生物相容性聚乙醇酸网中，随后接种无异源人类 KC 形成表皮层。将这种双层皮肤移植物植入免疫缺陷小鼠的背部后，KC 形成具有网状脊状结构的成熟分层表皮。 EC 和 PC 在植入后 2 周内形成人 EC 衬里的灌注微血管，防止移植物坏死，并引发血管生成宿主微血管对移植物的进一步灌注。作为概念验证，我们使用所有四种细胞类型的单一供体生成了 12 个单独的移植物。总之，我们描述了在完全无异种的培养条件下生物打印的血管化双层皮肤替代品的制造，证明了无异种的复杂组织工程方法的可行性。