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of cosmetics1 and transdermal drug absorption.2 Due to ethical concerns, however, these animal models have gradually been replaced by “cruelty-free” in vitro organotypic skin models that use primary human cells and cell culture inserts to recapitulate the stratified epidermal architecture.3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® cell culture inserts and the proprietary 3dGRO™ Skin Differentiation Medium which supports the robust differentiation of keratinocytes during the air:liquid interface culture steps.
Figure 1.Organotypic human skin culture protocol overview. Prepare a collagen/fibroblast (PHF) bed in Millicell® inserts. Seed and culture keratinocytes (PHK) on the bed for three days. Lift the insert to air:liquid interface to induce skin differentiation. Change skin culture medium every other day and harvest the culture on day 10.
Important note: The quality of the primary human keratinocytes (PHKs) and fibroblasts (PHFs) is critical to generating high quality 3D skin cultures. Use only primary cells that are at passage 1 or 2, and do not allow cells to become over-confluent.
A. H&E Staining of In-Vitro Organotypic Skin Culture
B. H&E Staining of Ex-Vivo Neonatal Skin Tissue
C. Filaggrin Staining of In-Vitro Organotypic Skin Culture
D. Filaggrin Staining of Ex-Vivo Neonatal Skin Tissue
Figure 2. Organotypic skin models have stratified epithelial morphology comparable to neonatal skin tissue. H&E staining of in vitro skin culture and ex vivo skin sections containing both dermal and epidermal stratified layers (A, B). Filaggrin staining identifies cornified keratinocyte layers (brown) (C, D).
A. Ki-67 Stain
B. H&E Stain
C. Filaggrin Stain
Figure 3. Organotypic skin models grown on collagen-coated Millicell® inserts with 0.4 µm hydrophilic PTFE membrane (PICM01250) contain actively proliferating keratinocytes. In vitro skin models stained with anti-Ki-67 antibody (275R-1) (brown) (A), H&E (B), and anti-filaggrin antibody (HPA030189) (brown) (C) identify cells differentiating and proliferating within the basal layer of the dermis.
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