Construction of Three-Dimensional Dermo–Epidermal Skin Equivalents Using Cell Coating Technology and Their Utilization as Alternative Skin for Permeation Studies and Skin Irritation Tests

Abstract

In vitro generated human skin equivalents are generating interest as promising tools in basic study, as alternatives to animal testing, and for clinical applications in regenerative medicine. For prediction of skin irritation and corrosion, three-dimensional human skin equivalents consisting of differentiated human keratinocytes (KCs) have been developed and some models have been internationally accepted. However, more delicate assessments using full-thickness skin models, such as skin sensitization tests, cannot be performed due to the lack of a dermis containing fibroblasts or appendages. In a previous study, we developed dermo-epidermal human skin equivalents (DESEs) using a cell coating technique, which employs cell surface coating by layer-by-layer assembled extracellular matrix (ECM) films. The DESEs with dermis consisting of normal human dermal fibroblasts (NHDFs) and epidermis consisting of human KCs were easily fabricated by using this technology. In this study, the constructed DESEs were evaluated as an alternative skin for skin permeation and irritation tests. A good relationship of permeability coefficient of chemicals was observed between the DESEs and human skin data. We investigated whether the DESEs, a new in vitro skin model, are capable of identifying skin irritant and nonirritant substances among 20 reference chemicals. It was confirmed that the DESEs are applicable to skin irritation testing as defined in the European Centre for the Validation of Alternative Methods (ECVAM) Performance Standard (OECD Test Guideline 439). We further studied the construction of DESEs with density-controlled blood capillary networks using human umbilical vein endothelial cells (HUVECs). The results suggest that DESEs allowing incorporation of skin appendages are more promising alternatives to animal testing and can be applied to the design of physiologically relevant in vitro skin models.

A novel strategy to engineer pre-vascularized 3-dimensional skin substitutes to achieve efficient, functional engraftment

“…Moreover, we reported the fabrication of pre-vascularized, functional 3D tissue constructs by coculturing FN-G-coated fibroblasts, hepatocytes, or iPSC-derived cardiomyocytes with vessel-forming cells, such as tissue-derived microvascular endothelial cells 13–16 . Recently, we developed a dermo-epidermal skin equivalent in vitro with characteristics, such as permeability to chemicals, similar to those of human skin 17,18 . Therefore, our engineering technique, which creates a vascular network within the multicellular stratified tissues, may have many advantages over conventional approaches for the fabrication of functional, transplantable tissues for tissue replacement.…”

2D vs. 3D Cell Culture Models for In Vitro Topical (Dermatological) Medication Testing

“…Currently available HSEs are primarily epidermal substitutes composed of keratinocytes in 3D cell culture models such as Epiderm™, SkinEthic RHE™, and EpiSkin™, which are used for pharmaceutical and cosmetic testing of skin irritation from topical products [45,47,48]. As these skin substitutes are derived of only the epidermal layer and primarily keratinocytes, it limits their use for testing of products related to particular types of skin conditions that involve the immune system, including testing of products for wound healing [47]. Full thickness models consisting of both the epidermal and dermal layers are thus beneficial [8].…”

“…More recent human skin equivalent (HSE) cultures can even be used for up to 20 weeks [41]. Skin substitutes that are derived from only the epidermal layer and primarily keratinocytes are limited in their use for testing of products related to particular types of skin conditions that involve the immune system, including testing of products for wound healing [47]. Full thickness models consisting of both the epidermal and dermal layers are thus beneficial [8].…”

Skin toxicology and 3Rs—Current challenges for public health protection

“…More cell types can be included such as melanocytes and dendritic cells . Moreover, dermo‐epidermal human skin equivalents with vascularisation using four different cell types in a sandwich fabrication process show promising results . Lessons may also be learned from tissue construction by 3D‐printing which can be applied to construction of FT‐skin models on scaffolds .…”