Human organotypic skin explant culture (hOSEC): an alternative approach to Staphylococcus aureus colonization assays

hOSEC provides a relevant ex vivo platform to study Staphylococcus aureus infection, preserving the structural and immune components of human skin while reducing animal use. This pilot study aimed to standardize hOSEC for S. aureus colonization and infection, supporting translational research and novel therapeutic approaches, compared to other models in offering a cost-effective platform.

hOSEC was established from surgical samples (N = 06) using dermatological punch biopsies. Tissue viability was assessed by TTC assay. Staphylococcus aureus ATCC^® 25923™ (susceptible), ATCC^® BAA-1026™ and NCTC 12,493™ (resistant) strains were isolated and cultured. Infection assays were performed by epidermal colonization and intradermal inoculation, followed by bacterial curve analysis (CFU counts). Antimicrobial treatments were applied, and efficacy was evaluated through quantitative bacteriological analysis.

The hOSEC model maintained structural integrity and mitochondrial activity throughout the study. Explants exposed retained high viability across antibiotic treatments (97.0%) and in untreated controls (98.0%). Intradermal inoculation (skin infection model) with 10⁶ CFU/mL led to bacterial proliferation (> 1 × 10⁸ CFU/mL) in untreated explants, while antibiotic-treated tissues showed strong suppression (average 2 × 10² CFU/mL). During epidermal colonization with wound and resistant strain, saline-treated explants maintained high bacterial counts: >10⁸ CFU/mL at 0-6 h, 4.5 × 10⁴ CFU/mL at 12 h (p = 0.002), and persisted longer but were significantly reduced by antibiotic treatment (from 1 × 10⁸ CFU/mL at 0-6 h to 2 CFU/mL at 24 h). Notably, viable bacteria were still detectable in explants for up to 7 days (2.5 × 10² CFU/mL). Histopathological analysis revealed tissue damage characterized by acantholysis, necrosis, and apoptosis.

The hOSEC model preserved tissue viability under bacterial inoculation and antibiotic exposure, supporting its use for ex vivo antimicrobial testing. Although intradermal inoculation (skin infection model) maintained high bacterial viability and effectively modeled deep tissue infection, injury-associated colonization (skin wound model) provided better spatial control of the infectious site by restricting bacterial growth to the injury area. Additionally, it was possible to identify histopathological alterations induced by bacterial infection, as histopathological analysis revealed acantholysis, necrosis, and apoptosis. These results demonstrate the model's utility for evaluating antibiotic penetration, local efficacy, and bacterial dynamics in controlled human skin-mimetic conditions.