Abstract:

Building upon the construction of a traditional 3D skin model, commensal skin bacteria from normal human skin were cultured and encapsulated via ionic cross-linking between chitosan and sodium tripolyphosphate, forming uniform microcapsules containing commensal bacteria （CS-bacteria-TPP microcapsules） that were subsequently applied to the surface of the 3D skin model. This established an ecological balance system in which the skin model coexists with microorganisms, thereby better mimicking the microenvironment of human skin. A dermal layer was constructed by isolating and culturing fibroblasts, while an epidermal layer was established using cultured keratinocytes, which were subsequently seeded onto the dermal layer to form a normal skin model. Target bacterial species, such as Staphylococcus epidermidis, Staphylococcus aureus, lactic acid bacteria, or Micrococcus, spp., were cultured separately. Using the ionic cross-linking principle, positively charged amino groups of chitosan and negatively charged tripolyphosphate ions interacted, forming stable, bacteria-loaded microcapsules. Spreading these bacteria-coated microcapsules onto the surface of the 3D skin model yielded a 3D simulated human skin model incorporating a microcolony system. The physiological and morphological characteristics, as well as bacterial viability and growth within the microcapsules, were evaluated using scanning electron microscopy （SEM）, hematoxylin and eosin （H&E） staining, transepithelial electrical resistance （TEER） measurement, light microscopy, and agar plate counting, respectively. Characterization results confirm that the 3D skin model exhibits a high and stable transepithelial electrical resistance, indicating the successful establishment of an intact skin barrier alongside microcapsules featuring uniform morphology and robust mechanical strength. Applying these microcapsules to the model surface successfully recreates a microecological environment resembling normal human skin. Overall, these findings demonstrate that through the successful colonization of commensal skin bacteria on a 3D skin construct, a highly representative in vitro model simulating the human skin microenvironment was established.

Key words: 3D skin model, skin commensal bacteria, microcapsules, microbial barrier