Multilayer human keratinocyte cultures increasingly are used to model human epidermis. Until now, studies utilizing epidermal equivalents (HEE) have been limited due to their lack of a functional epidermal permeability barrier. In this report, we describe a protocol that yields HEE that closely match human postnatal epidermis and demonstrate that these HEE develop and repair a functional permeability barrier. These cultures display low transepidermal water loss (TEWL), and Ca2+ and pH gradients that match those seen in human epidermis. These cultures upregulate lipid synthetic enzymes and make normal-appearing lipid lamellar bilayers. The epidermal permeability barrier of these cultures can be perturbed, using the identical tools previously described for human skin, and recover in the same time course seen in in vivo barrier recovery. These cultures will be useful for assaying epidermal penetration, and also for testing components of epidermal permeability barrier homeostasis.
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