Introduction The human skin is host to trillions of microorganisms including bacteria, viruses, and fungi. This natural flora which lives on our skin is called the skin microbiome. Its impact and influence on skin homeostasis have been largely described recently. Hence, new skincare products are now evolving to better preserve or enhance this natural ecosystem and suitable experimental models are then required for research on the skin microbiome. Currently, Human Reconstructed Epidermis (RHE) models are widely accepted as a valuable tool in dermatological research. On its MicroBIOS Platform, StratiCELL combines skin microbiota key components and 3D RHE, to study the efficacy of dermo-cosmetic ingredients on microbial homeostasis and disorders linked with dysbiosis. Methods StratiCELL is studying commensal and opportunistic strains of the skin including Staphylococcus epidermidis, Staphylococcus aureus, Cutibacterium acnes, Corynebacterium spp., Malassezia furfur and Trichophyton rubrum. The RHE are colonized with these microbiological strains and a two-tiered approach is developed to objectivate the influence of dermo-cosmetic actives. The first is the monitoring of the microorganism’s adhesion and growth on the stratum corneum through CFU counting upon harvest with a swab. The second is the study of the epidermal response to bacteria, yeasts and fungi with a transcriptomic tool based on the TaqMan Low Density Array (TLDA) technology. This TLDA evaluates the expression of 93 key genes selected from full-transcriptome analysis and involved in the skin response to micro-organisms. We also focus on specific biomarkers expression (RT-qPCR) and mediators release in the culture media (ELISA). Results For each bacterial or fungal species selected to infect RHE, a systematic methodology was applied. According to their characteristics and their metabolism, the protocols of culture, infection, harvest and counting of CFU were optimized. Their ability to form biofilm was also evaluated. The tissue response to infection was studied after a period of growth of the microorganism on the top of the RHE. For each species, we established a transcriptomic signature following infection. These expression profiles allowed us to identify multiple response genes shared for all micro-organisms and some specific to each of them. These genes are most often related to the antimicrobial response, inflammation and skin barrier (e.g. CXCL8, DEFB4, IL1, HBEGF, S100A7, CLDN17, etc.). The relevance and reliability of these infection biomarkers was then validated by qPCR. For some of them, a quantification by ELISA was performed to confirm that the changes in mRNA expression also affect protein abundance (e.g. IL-8, TNF-α, HßD2). Nevertheless, it should be noted that the development of each model was subject to adaptation of culture protocols, CFU enumeration, infection conditions of RHE, kinetics, recovery using swabs, etc. For example, the infection of RHE by Cutibacterium acnes requires an optimized medium enriched in cutaneous lipids in order to mimic the growth conditions of the bacteria in vivo in the sebaceous gland or in the presence of sebum. The phylotype of the strain, in this case IA1, is also essential to observe an acne-like inflammatory reaction. Kinetic considerations are also very important to observe a reliable skin response to infection. For example, when infecting RHE with the yeast Malassezia furfur, barrier alteration, as measured by Lucifer Yellow fluorescent probe penetration, or induction of pro-inflammatory or antimicrobial genes expressions are only observed after three days of contact. For dermatophyte infections aimed at modelling skin mycoses, the invasive nature of Trichophyton rubrum is a factor limiting the use of the model beyond four days post-infection. After this time, the keratinophilic fungus penetrates the viable layers of the tissue, which is no longer representative of an in vivo infection where it remains confined to the stratum corneum. Discussion and conclusion In conclusion, the infection of in vitro reconstituted epidermis by microorganisms constitutes an essential tool to approach the conditions of in vivo infection, allowing among other things the inoculation of strains in a topical way. The technical challenge is the adaptation of the different models to the specificities of the microorganisms of interest. The data related to the different infection models will be presented and the specificities related to each species will be documented and discussed.