Protocol for 3D Bioprinting a Co-culture Skin Model Using a Natural Fibrin-Based Bioink as an Infection Model.

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 Staphylococcus aureus and Staphylococcus epidermidis 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.