Three-Dimensional Epidermal Model from Human Hair Follicle-Derived Keratinocytes

Three-dimensional (3D) epidermal models reconstructed from human skin-derived keratinocytes have been utilized as an alternative to animal testing and models, not only in toxicology, but also in skin biology. Although there are currently several reconstructed human epidermis (RHE) models commercially available, the donors of the keratinocytes are not identified in these models. A tailor-made system is needed to investigate the individual differences in RHE derived from each donor.It is possible to make an individual RHE using each donor's keratinocytes, which are usually obtained by invasive procedures such as skin excision or biopsy. To overcome this drawback, we established an RHE model using keratinocytes derived from plucked hair follicles as a less invasive procedure under conditions without feeder cells, serum, or matrix proteins. In this chapter, we provide a method of isolation and two-dimensional (2D) culture of keratinocytes derived from adult human plucked hair follicles including the outer root sheath (ORS). We also provide a detailed protocol for establishing an RHE model by culturing the keratinocytes under a 3D culture condition. We believe that our less invasive technique will provide a useful tool for investigating individual RHE in both normal and disease settings.