Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

Area of Science:

• Dermatology and in vitro modeling
• Tissue engineering and regenerative medicine
• Cell biology and skin physiology

Background:

• Developing accurate in vitro models of human epidermis is crucial for skin research.
• Existing models may not fully recapitulate the complex structure and function of native skin.
• Neonatal primary keratinocytes offer a viable cell source for reconstructing human epidermis.

Purpose of the Study:

• To present a detailed protocol for cultivating a 3D reconstructed human epidermis model.
• To characterize the morphological and functional properties of the reconstructed epidermis.
• To establish the utility of this model for assessing skin responses to stimuli.

Main Methods:

• Culturing neonatal primary keratinocytes on polycarbonate inserts at an air-liquid interface.
• Stimulating differentiation using growth factors, ascorbic acid, and high calcium medium for 14 days.
• Assessing stratification, barrier function (TEER), and protein expression (immunofluorescence) of differentiation markers.

Main Results:

• Histological analysis confirmed a fully stratified epidermis resembling native human skin.
• Immunofluorescence revealed appropriate expression of keratin 10, involucrin, loricrin, and filaggrin.
• The model demonstrated barrier integrity via transepithelial electrical resistance measurements and responded to inflammatory stimuli with cytokine release.

Conclusions:

• A straightforward and reproducible protocol for generating 3D reconstructed human epidermis was established.
• The model accurately mimics human epidermis morphology and barrier function.
• This reconstructed human epidermis serves as a valuable in vitro tool for environmental and skin-related studies.