Linoleic acid concentration at the air-liquid interface is key for optimal incorporation into skin model lipidome.

Linoleic acid is an essential fatty acid required for skin barrier formation. However, linoleic acid-deficient media remains widely used in cell culture and reconstructed skin production, potentially contributing to suboptimal barrier function in skin models. In this study, we investigated the effects of varying linoleic acid concentrations in culture media on lipid composition, bioactive lipid mediator production, and gene expression of lipid-metabolizing enzymes in self-assembled skin substitutes. High-concentration linoleic acid significantly improved barrier function, as evidenced by reduced testosterone permeability, and enhanced incorporation into phospholipids, triacylglycerols, and diacylglycerols. Unexpectedly, ω-hydroxy-sphingosine ceramide proportions were reduced, reflective of downregulation of key enzymes in ω-O-acylceramide synthesis. These findings reveal a complex interplay between linoleic acid availability, lipid remodeling, and enzymatic regulation, offering new insights into optimizing reconstructed skin models for improved barrier function and lipid homeostasis.