Differential taxonomic analyses were conducted at the level of species. Most significantly affected species (ANOVA, p-value < 0.05) included many undermined bacteria except Streptococcus and Staphylococcus in mouse skin at 1 Gy (Supplemental Table 4). However, most of them belonged to Clostridia, Bacilli, and Gammaproteobacteria classes in the skin in both mice and coHSE exposed to radiation although in different proportions (Fig. 5b). We observed a decreasing trend in Clostridia species in both coHSE and mouse skin at 4 Gy compared to 1 Gy, particularly in the ones belonging to the family of the Lachnospiraceae. Conversely, an increase in species belonging to the Actinobacteria and Bacteroidia classes were observed in in both coHSE and mouse skin at 4 Gy compared to 1 Gy.

This study evaluated a coHSE model as an innovative and long-lasting in vitro platform for studying the effects of radiation exposure on the skin. By incorporating natural mixed microbial colonization into a bioengineered human skin model, our research bridges significant gaps in existing radiobiological studies that rely primarily on animal models or 2D cultures. Time since radiation exposure is a critical criterion for patient triage in the scenario of a nuclear explosion. The Labskin model used in this study demonstrated robust culture stability for at least 25 days post-inoculation, including a 4-week post-irradiation period, supporting its utility in extended experimental timelines. While the combination of microbial colonization and subsequent irradiation posed potential challenges, this dual-stress paradigm had not been previously investigated to our knowledge. Despite these stresses, the model remained structurally and functionally competent throughout the experimental period. This was evidenced by consistent cell survival, preserved tissue integrity, and multi-omic signatures that closely align with known in vivo responses to radiation. These results support the suitability of the coHSE system as a durable and translational in vitro platform for investigating the effects of radiation on human skin.

The skin, as the first barrier exposed to radiation, represents a promising avenue for biodosimetry due to its accessibility and its role in systemic health. However, previous models, including rodent skin or 2D cultures, have inherent limitations, such as structural and microbiological differences from human skin. The colonized HSE model described in this study overcomes these barriers by providing a full-thickness skin equivalent capable of prolonged experimental designs while allowing microbial colonization. Our findings reinforce its potential to serve as a model of predilection for radiation research on human skin and the development of non-invasive radiomitigation measures.

Our results demonstrate that radiation exposure of up to 4 Gy, does not significantly impair the viability of coHSE or introduce opportunistic microbial overgrowth beyond manageable levels when simple procedural modifications are implemented. This validates the model's suitability for extended experiments. Our findings also revealed that radiation exposure may induce alterations in the epidermal layer (Figs. 1c and 2a), potentially mimicking radiation-induced desquamation observed in humans. To further evaluate tissue integrity, we assessed delamination scores, epidermis-to-dermis ratios, dermal cell density, and PCNA expression. These metrics are important indicators of morphological stability, which can directly influence swab sample yield consistency and the interpretability of downstream multi-omics analyses. In this context, the absence of statistically significant differences in delamination scores between 1, 2, and 4 Gy-exposed coHSEs indicates preserved tissue integrity across doses. Similarly, we observed no significant changes in epidermis/dermis ratios during the first week post-irradiation, nor in dermal cell density at weeks 1 and 4, suggesting minimal architectural disruption following photon exposure. This homogeneity ensures that such alterations do not introduce significant bias when assessing molecular and microbial profiles. This is a crucial observation, as it validates the reliability of the coHSE model for non-invasive sampling methods, such as swabbing, and subsequent molecular and microbial profiling, even in the presence of radiation damages such as skin desquamation.

Importantly, microbial inoculation has been shown to decrease cell proliferation and increase cell differentiation benefiting the skin barrier function. Consistently, PCNA expression significantly declined over time at all radiation doses, indicating a time-dependent reduction in proliferative activity. However, this occurred without any measurable loss in dermal cell density which was maintained within naturally ranges (10 cell.mm in 1 mm thick skin) further supporting the structural resilience of the coHSE model. Collectively, these analyses demonstrate that coHSEs retain sufficient morphological stability post-irradiation to support reproducible, swab-based molecular investigations over time.

Dyslipidemia and impaired lipid metabolism are frequently reported events after radiation exposure in various tissues including the skin. While substantial structural and physiological differences exist between mouse and human skin, such as thinner epidermis, higher hair follicle density, and differing immune and lipid profiles, we observed high overlapping lipidomic perturbations in both models following radiation exposure, with 60% of affected lipid subclasses being commonly found in both coHSE and mouse skin in response to radiation, which point out the existence of a well conserved lipidomic response to radiation and that robust biomarkers of radiation exposure might be found in the coHSE lipidome. Consistently, lipid metabolism was also prevalent in metabolomic analyses, with arachidonic acid (AA) metabolism and fatty acid metabolism being the most affected pathways in the skin in both models. Although murine skin is not directly homologous to human skin, the recurrence of these metabolic signatures accross species suggests the existence of conserved lipid remodeling in response to radiation and potential lipid bimarkers of radiation exposure in the skin. While acknowledging the limits inherent to interspecies extrapolation afore mentioned, we interpret these findings as cross-model molecular convergence that strengthens the plausibility of the mechanisms observed in the human-engineered tissue and its translational framework.

Mechanistically, radiation-induced enrichment of AA metabolism and associated glycerophosphocholine alterations observed in our study align with well-characterized inflammatory and cytoprotective lipid signaling pathways. Increased accumulation of lipids, especially unsaturated fatty acids, is reported as a pro-survival mechanism that can dampen radiation induced oxidative stress and DNA damge in glioblastoma. As such, enriched fatty acid metabolism pathways in the skin after radiation may reflect an underlying protective mechanism, such as the accumulation of unsaturated fatty acids. The skin's lipid bilayer, rich in phosphatidylcholine, is particularly susceptible to oxidative remodeling under ionizing radiation. Upon radiation exposure, cytosolic phospholipase A2 (cPLA2) is activated and hydrolyzes membrane phospholipids, especially phosphatidylcholine, which release AA and lysophosphatidylcholine (LPC). These lipid mediators act as potent signaling molecules, modulating inflammation, DNA damage response, cell viability, and tissue regeneration. Interestingly, AA release via cPLA2 is critical for maintaining viability of irradiated vascular endothelial cells through ERK1/2 and Akt signaling. Consistently, we found increased expression of the proliferation marker PCNA with dose exposure levels and maintained cell density over time in all groups. Oxidative degradation of AA leads to the formation of reactive α,β-unsaturated aldehydes such as the 4-hydroxy-2-nonenal (4-HNE), a major product of n-6 fatty acid peroxidation capable of promoting both cell death and cell survival. 4-HNE is a highly reactive and genotoxic electrophile that can induce the formation of DNA adducts while inhibit nucleotide excision repair (NER) pathway proteins, thereby reducing cellular capacity to resolve radiation-induced DNA lesions. However, due to its oxidant status, 4-HNE can also induce cellular defense mechanisms against oxidative stress, triggering its own detoxification thereby promoting cell survival. In addition to its roles in inflammation and cyto-protection, AA has been shown to actively promote skin regeneration through the GPR40/ERK signaling axis in keratinocytes. This suggests that radiation-induced release of AA may not only initiate inflammatory cascades but can also promote epidermal viability trough both cell survival and tissue repair mechanisms which are critical for maintaining skin barrier function after injury. As such, the AA and fatty acid oxidation metabolism pathways enrichment observed in our coHSE model may reflect a complex response to radiation-induced oxidative stress in the skin, including both inflammation processes and compensatory protective pathways that contribute to the increased cell survival and the preservation of tissue integrity following irradiation observed in our study. In both the coHSE and mouse models, the metabolomic and lipidomic profiles capturing these signatures may thus point to a coordinated response involving inflammation, tissue remodeling, and repair processes, all of which are relevant to both acute and delayed manifestations of radiation-induced skin toxicity. Collectively, these findings support the notion that AA pathway alterations may constitutes an early molecular indicator of radiation-induced skin remodeling with potential application for skin-based biodosimetry.

Overall, shared pathway enrichments, suggest conserved molecular responses, reinforcing the model's translational applicability while the unshared observed in coHSEs may represents the missing part of the physiological picture uncaptured in mice models due to inherent structural and cellular differences between human and mouse skin as well as laboratory environmental pressures. Additionally, microbial diversity analyses revealed dose-dependent shifts in microbial composition, with most of species belonging to specific microbial classes, such as Actinobacteria and Clostridia. Lachnospiraceae are commonly found on the skin and play a critical role in maintaining barrier integrity, primarly through the production of short-chain fatty acids (SCFAs) like butyrate and by directly competing with pathogenic organism. Additionaly, Cutibacterium (elevated in Labskin 1 Gy and mouse 4 Gy groups) and Corynebacterium (elevated in Labskin 4 Gy), two lipophilic genera that are part of the natural skin microflora, were among the most upregulated taxa in both models. These bacteria possess lipase activity that allows them to hydrolyze triglycerides in sebum, releasingfree fatty acids and SCFAs, including monounsaturated fatty acids. These lipid products help maintain a low skin pH, inhibiting the growthof pathogenic microorganisms. Beyond their antimicrobial effects, free fatty acids also enhance innate skin immunity by simulating the expression of human β-defensin 2 (hBD-2), one of the most abundant antimicrobial peptides (AMPs) in human skin. SCFAs, in particular, exert well-documented anti-inflammatory and immunomodulatory effects, supporting host resilience and reducing inflammation in epithelial tissues. Furthermore, Micrococcaceae, a family of Gram-positive bacteria also part of the natural bacterial population on human skin, are also amongst the most affected taxa in our study. Interestingly, members of the Micrococcaceae family are known for their radiation resistance, particularly some species found in skin like Micrococcus and Kocuria species. In our dataset, Kocuria species were downregulated 3- to 4-fold in coHSE, and an unidentified Micrococcus species was consistently downregulated in both models. Collectively, these findings underscore the sensitivity of skin microbial communities to radiation exposure and suggest that both beneficial commensals (e.g., Lachnospiraceae, Lactobacillaceae) and radiation-resistant taxa (e.g., Micrococcaceae) undergo reproducible alterations after radiation exposure. These microbial changes likely influence host immune tone, barrier function, and tissue repair capacity. The consistent detection of similar taxonomic alterations in both coHSE and murine models reinforces the translational potential of the coHSE system as a platform to study radiation-induced microbiome remodeling and its implications for personalized risk stratification in exposed populations.

Thus, the coHSE is a promising model for radiation research including various applications, from biodosimetry to radiotherapy optimization and space radiation research. Approximately 95% of cancer patients undergoing radiotherapy develop some form of radiation-induced dermatitis, including erythema, ulceration, edema, pruritus, and desquamation. These conditions increase pain and lead to complications including decreased self-esteem and image, stigma associated with radiation exposure, increased financial burden associated with treatment, impaired ability to complete daily activities, delay in treatment. Additionally, increased skin cancer risk has been reported in various exposed groups, including bomb survivors uranium minersas well as early 20th century radiologists with a risk up to 57% higher n radiotherapy treated breast cancers patients. Better understanding of the molecular response to radiation of the skin is essential to mitigate radiation-induced skin injuries including elevated cancer risks, delays in treatment, diminished aesthetic appeal, and reduced quality of life. The coHSE system, as an ethical and scalable alternative to animal models, could also potentially greatly benefit the field of space research, where research payload size and mass are determinant funding criteria.

Despite the promising results and translational relevance of the coHSE model, several limitations must be acknowledged. First, although X-rays provide a biologically relevant and technically accessible surrogate to gamma rays, they do not replicate the full spectrum of radiation qualities encountered in mixed-field nuclear incidents or space environments, which often include high-linear energy transfer (high-LET) radiation such as protons, alpha particles, or heavy ions. Additionally, the photon energy used in this study (up to 320 kVp) represents a lower energy spectrum than that encountered in real-world scenarios such as nuclear detonations or solar particle events, potentially affecting depth dose distribution and tissue interaction. Furthermore, the 1-4 Gy dose range used, although clinically and operationally relevant, does not encompass ultra-low-dose chronic exposures or high-dose acute exposures (> 6 Gy) relevant to certain radiological emergencies or therapeutic settings. Future studies should expand to include a broader spectrum of radiation energies and qualities to fully evaluate the versatility and robustness of the coHSE to model physiological responses under diverse radiation exposure scenarios.

Receiving a substantial amount of the blood flow, it is very likely that skin conditions might reflect internal health status, as it is already well documented for cardiovascular conditions. While the current system enables the coculture of the HSE insert with another cell type within the same well, future studies should also explore the combination of the coHSE model with other engineered systems to address the systemic limitation. Recently, a tissue-chip system has been developed which is fully ready-applicable, including human heart, liver, bone and a full thickness HSE linked by a circulating vascular flow allowing for the recapitulation of interdependent organ functions. Although the system did not include a microbial component which is critical for a true recapitulation of skin physiology, it relied on a HSE insert system highly similar to the one presented in this study, suggesting straightforward compatibility and enabling immediate implementation for future studies. The field of bioengineering is continuously evolving to closely mirror the natural biological system. Labskin HSEs containing melanocytes are now available. This represents an additional critical asset for the future of radiation research since melanocytes and melanin production are heavily studied to decipher their protective properties against radiation and develop biomimicking countermeasures.

Overall, this study shows that the coHSE model represents a significant step forward in radiation biology research, closely recapitulating the biological mechanisms underlying the radiation effects in the skin, enabling a better understanding of radiation biology and the development of better tailored medical tools accordingly, while advancing ethical research practices. While skin research is relatively nascent in radiation biology, more attention and efforts should continue to fully characterize the molecular dynamism of the human skin in response to radiation, as it represents the most convenient platform for rapid biodosimetry. Skin studies and skin biomarker discovery will provide critical insights for the development of real-time biosensing and health status monitoring. We advocate for a systematic implementation of coHSE models as a standard experimental method in radiation studies involving the skin.