Díaz González, Giselle Yolanda2026-04-242026-04-242026https://hdl.handle.net/1828/23721Chronic wounds pose a serious and persistent public health challenge, resulting in devastating patient consequences and imposing a substantial economic strain on healthcare systems. Conventional treatments frequently fail because they target only single characteristics of the wound rather than addressing the complex, interconnected cycle where the microbiome and microenvironment play crucial roles in perpetuating inflammation. To develop effective biotherapies, advanced in vitro models that accurately replicate the complex skin microenvironment are urgently needed. Existing models, such as two-dimensional (2D) cultures and animal models, lack the necessary physiological complexity, are hindered by species-specific differences, and present ethical concerns. Three-dimensional (3D) bioprinting has emerged as a powerful alternative, enabling the creation of in vitro skin models that more closely mimic human in vivo conditions. The primary goal of this work was to investigate the clinical need, design, and verification of a 3D bioprinted stratified skin co-culture model suitable for studying the intricate interactions between the skin microbiome and cutaneous cells. Constructs were generated using extrusion-based bioprinting (EBB) with a high-viscosity, fibrin-based bioink containing co-cultured human keratinocytes (HEKa) and fibroblasts (HDFs), successfully creating a multi-layered structure replicating the epidermis and dermis. Crucially, epidermal stratification was induced through the Air-Liquid Interface (ALI) methodology to replicate the primary air-liquid barrier where bacteria reside and ensure comprehensive host-microbe dynamics. The functional maturity and interactions between the host-microbiome within the model were validated through biochemical signaling detection of cytokines through Human Cytokine/Chemokine Panel A 48-Plex Discovery Assay®, analyzing conditioned media collected pre- and post- inoculation with Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus), the results confirmed a colonization model capable of restoring the cutaneous barrier and maintaining a stable homeostatic environment through active molecular communication. Furthermore, achieving comprehensive analysis of this full-thickness, high-viscosity hydrogel construct required optimizing bioprocessing techniques. This process involved utilizing cryoslicing methodology, following specific protocols for cryopreservation infiltration using sucrose and optimal cutting temperature (O.C.T) solution, to preserve scaffold integrity and facilitate accurate microscopic evaluation. Optimized staining procedures, including Hematoxylin & Eosin (H&E) for scaffold stratification development, DAPI immunofluorescence staining for structural morphology, Safranin-O for extracellular matrix (ECM) components, and Gram staining for bacterial characterization, were then employed. This comprehensive framework provides a crucial standardization for creating and analyzing complex 3D skin models, offering a valuable, controllable, reproducible in vitro platform. Ultimately, this research aims to facilitate the efficient development of novel biotherapies that encompass microbiome homeostasis to effectively and efficiently address chronic wounds.enAvailable to the World Wide WebBioprintingMicrobiomeChronic woundsAir Liquid Interface3D Skin modelHistologyStratifiedCryoslicingHomeostasisWound healingHydrogelCryopreservationCell CultureStratum corneumStaphylococcus aureusStaphylococcus epidermidisKeratinocytesFibroblastsThesisDíaz GY, Da Silva VA, Kalantarnia F, Scheck K, Tschofen SA, Tuffs SW, Willerth SM. Using Three-Dimensional Bioprinting to Generate Realistic Models of Wound Healing. Advances in wound care, 10.1089/wound.2024.0138. 5 Mar. 2025, doi:10.1089/wound.2024.0138Díaz GY, Perry MA, Cárdenas LS, Da Silva VA, Scheck K, Tschofen SA, Tuffs SW, Willerth SM. Protocol for 3D Bioprinting a Co-culture Skin Model Using a Natural Fibrin-Based Bioink as an Infection Model. Bio-protocol vol. 15,14 e5380. 20 Jul. 2025, doi:10.21769/BioProtoc.5380