Development of full-thickness human skin equivalents with blood and lymph-like capillary networks by cell coating technology.

We developed a human skin equivalent (HSE) containing blood and lymph-like capillary networks using a cell coating technique, which is a rapid fabrication technology of three-dimensional cellular constructs by cell surface coating using layer-by-layer assembled nanofilms of extracellular matrices. The thickness of dermis consisting of normal human dermal fibroblasts was easily controlled from approximately 5 to 100 µm by altering the seeded cell number. Keratinocytes as a major cell population showed homogeneous differentiation on the surface of the dermis by lifting to air-liquid interface. Histological analysis revealed four distinct layers such as basal layer, spinous layer, granular layer, and cornified cell layer in the epidermis. Interestingly, the measurement of transepithelial electrical resistance (TEER) indicated prolongation of the attainment time for maximum value by increasing the number of the dermal fibroblasts, and the HSEs with six layers of dermis revealed the longest period maintaining over 500 Ω cm(2) of TEER. The co-sandwich culture of human umbilical vein endothelial cells and normal human dermal lymphatic microvascular endothelial cells within eight-layered dermis showed in vitro co-network formation of individual blood and lymph-like capillaries inside the dermis. This is the report for homogeneous full-thickness HSEs with blood and lymph capillary networks, which will be useful for biomedical and pharmaceutical applications.