Effect of human skin storage on stratum corneum drug penetration assessed by confocal Raman spectroscopy.

Fresh human skin is the preferred model for ex vivo dermal penetration studies. However, limited availability often necessitates frozen storage. We investigated whether storage for two months at -20 °C or -80 °C affects skin barrier function, selected Raman-derived skin properties, and drug penetration. For each of six donors, paired full-thickness abdominal skin samples were analyzed fresh or after frozen storage. Barrier integrity was assessed by transepidermal water loss, while quantitative confocal Raman spectroscopy was used to determine water concentration profiles and the amount of caffeine, diclofenac sodium, and salicylic acid within the upper 15 µm after application of 2% (w/w) hydrogels for 1 h. Transepidermal water loss varied across storage conditions, although neither frozen-storage condition differed significantly from fresh skin. Raman-derived water content was significantly higher after storage at -80 °C than in fresh or -20 °C-stored skin, whereas no consistent storage-related differences were detected in the fingerprint spectra or combined ceramide and fatty acid signal. Caffeine penetration was 18.57, 19.82, and 21.51 µg/cm2 in fresh, -20 °C-stored, and -80 °C-stored skin, respectively, with significantly greater penetration in -80 °C-stored skin than in fresh skin. No significant differences were detected for diclofenac sodium or salicylic acid. Under these conditions, storage at -20 °C did not significantly alter the Raman-derived stratum corneum parameters or drug penetration outcomes, whereas storage at -80 °C altered the superficial Raman-derived water signal and caffeine penetration. Storage conditions should therefore be standardized for each compound and experimental endpoint.