Generating Organotypic 3D Skin Cultures as an in-vitro Model of the Human Epidermis

Skin is the largest organ in the human body and serves numerous functions including protection, the absorption of substances, and thermoregulation. Skin has a complex stratified structure consisting of three main layers: 1) the tightly packed cells of the epidermis form a barrier against intruders and water loss, and includes an outer layer called the stratum corneum, consisting of overlapping, nonviable corneocytes that protect against threats such as UV radiation and pathogens; 2) the dermis, which supports the epidermis and contains nerve endings, sweat glands, sebaceous glands, hair follicles, and blood vessels. The basal layer of the dermis is where keratinocytes constantly undergo mitosis to replenish cells; and 3) the subcutaneous layer, or hypodermis, which contains mostly adipocytes and acts to protect underlying tissues, as an energy reserve, and to provide some thermoregulation via insulation.

Historically, animal models have been used to test the toxicity and efficacy of cosmetics¹ and transdermal drug absorption.² Due to ethical concerns, however, these animal models have gradually been replaced by “cruelty-free” in vitro organotypic skin models that use primary human cells and cell culture inserts to recapitulate the stratified epidermal architecture.³ In 2013, the European union was the first to ban animal testing of cosmetics entirely (Cruelty Free International). Here, we describe a step-by-step culture protocol that can be used to generate human epidermal skin models using primary human keratinocytes (PHK), dermal fibroblasts (primary human fibroblasts, PHF), collagen-coated MilliCell^® culture inserts and the proprietary 3dGRO™ Skin Differentiation Medium which supports the robust differentiation of keratinocytes during the air:liquid interface culture steps.