Díaz Giselle Y, Perry Madeleine A, Cárdenas Laura S, Da Silva Victor A, Scheck Kali, Tschofen Silken A, Tuffs Stephen W, Willerth Stephanie M
Department of Mechanical Engineering, University of Victoria, Victoria, BC, Canada.
School of Engineering and Sciences, Tecnológico de Monterrey, Zapopan, Jalisco, Mexico.
Bio Protoc. 2025 Jul 20;15(14):e5380. doi: 10.21769/BioProtoc.5380.
The skin microbiome, a diverse community of microorganisms, plays a crucial role in maintaining skin health and homeostasis. Traditional studies have relied on two-dimensional (2D) models, which fail to recreate the complex three-dimensional (3D) architecture and cellular interactions of in vivo human skin, and animal models, which have species-specific physiology and accompanying ethical concerns. Consequently, both types of models fall short in accurately replicating skin physiology and understanding its complex microbial interactions. Three-dimensional bioprinting, an advanced tissue engineering technology, addresses these limitations by creating custom-designed tissue scaffolds using biomaterial-based bioinks containing living cells. This approach provides a more physiologically relevant 3D structure and microenvironment, allowing the incorporation of microbial communities to better reflect in vivo conditions. Here, we present a protocol for 3D bioprinting an in vitro skin infection model by co-culturing human keratinocytes and dermal fibroblasts in a high-viscosity, fibrin-based bioink to mimic the dermis and epidermis. The bioprinted skin tissue was co-infected with and to mimic bacterial skin disease. Bacterial survival was assessed through colony-forming unit enumeration. By incorporating bacteria, this protocol offers the potential to serve as a more representative in vivo 3D bioprinted skin infection model, providing a platform to study host-microbe interactions, immune responses, and the development of antimicrobial therapeutics. Key features • This protocol provides a detailed description of the cell culture process for both keratinocyte and fibroblast cells. • This protocol outlines step-by-step preparation of the high-viscosity fibrin bioink and chemical crosslinker. • The protocol uses an extrusion-based bioprinter, with an easy-to-follow methodology that clarifies the printing details, including the incorporation of skin cells into the bioink. • This protocol details how the bacteria are inoculated into the construct to achieve the co-infection 3D skin model.
皮肤微生物群是一个多样化的微生物群落，在维持皮肤健康和内环境稳定方面发挥着关键作用。传统研究依赖于二维（2D）模型，该模型无法重现体内人类皮肤复杂的三维（3D）结构和细胞相互作用，以及动物模型，动物模型具有物种特异性生理学和相关伦理问题。因此，这两种模型在准确复制皮肤生理学和理解其复杂的微生物相互作用方面都存在不足。三维生物打印是一种先进的组织工程技术，通过使用含有活细胞的基于生物材料的生物墨水创建定制设计的组织支架来解决这些局限性。这种方法提供了一个更具生理相关性的3D结构和微环境，允许纳入微生物群落以更好地反映体内条件。在这里，我们提出了一种通过在高粘度、基于纤维蛋白的生物墨水中共培养人角质形成细胞和真皮成纤维细胞来三维生物打印体外皮肤感染模型的方案，以模拟真皮和表皮。将生物打印的皮肤组织与 和 共感染以模拟细菌性皮肤疾病。通过菌落形成单位计数评估细菌存活情况。通过纳入细菌，该方案有可能作为更具代表性的体内三维生物打印皮肤感染模型，提供一个研究宿主-微生物相互作用、免疫反应和抗菌治疗方法开发的平台。关键特性 • 本方案详细描述了角质形成细胞和成纤维细胞的细胞培养过程。 • 本方案概述了高粘度纤维蛋白生物墨水和化学交联剂的逐步制备过程。 • 该方案使用基于挤压的生物打印机，采用易于遵循的方法，阐明了打印细节，包括将皮肤细胞纳入生物墨水中。 • 本方案详细说明了如何将细菌接种到构建体中以实现共感染三维皮肤模型。
