Keratinocyte Culture Techniques in Medical and Scientific Applications

Human skin is a complex organ essentially organized in a thin non-vascular epidermis, a thick collagenous dermis, and subcutaneous fat tissue called hypodermis. The epidermis is subdivided into five layers termed: stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum germinativum. Furthermore, skin appendages, like hair follicles and glands, are derived from the epidermis but project deep into the dermis (Priya et al, 2008). They fulfil specialized functions for body homeostasis and temperature control. Cellular differentiation and growth follow precise temporal and spatial patterns. In the epidermis constant replacement of cells lost by desquamation in the outer layers by cells of the basal layers takes place (Atiyeh & Costagliola, 2007). The basal layers of the epidermis retain two cell populations with different proliferation capacity: stem cells with high potential for self-renewal have low level proliferation rates but retain their ability to generate daughter cells throughout life; other cells display high frequencies of cell divisions but finally are destined for terminal differentiation. These cells are called transit-amplifying (TA) cells. Epidermal stem cells have been found in the bulge region of hair follicles and in the epidermal bottom of the deep rete ridges (Lorenz et al, 2009). After wounding, keratinocyte stem cells contribute to wound closure by giving rise to daughter cells which migrate to the site of defect (Ito et al, 2005). Human keratinocyte stem cells and TA cells can be differentiated by their panel of cell surface marker gene expression; while stem cell markers CD29 and CD49f are highly expressed in both cell types, the transferring receptor CD71 is expressed in higher amounts in the rapidly proliferating TA cells (Lorenz et al, 2009). Moreover, keratinocyte stem cells have the ability to grow clonogenically with the number of colony-forming units, obtained per mm2 skin biopsies, varying between 94 and 3190. The growth potential of clonogenic keratinocytes can be differentiated by the morphological appearance of the colonies: colonies with significant growth potential (holoclones and meroclones) contain mostly small cells and have a regular round appearance, paraclones, however, contain large, terminally differentiated cells and appear rather irregular (Ronfard et al, 2000). Due to the restricted growth potential of terminally differentiated cells colony-forming units increase when kept under ideal culture conditions. Conditions supporting keratinocyte differentiation, however, decrease growth potential of the culture.