*3.1. Experimental Design*

A functional approach was designed to model the impact of ionizing radiation (IR) on the regenerative capacity of human epidermal keratinocyte precursor cells (Figure 1). The cellular material used in this study is defined as holoclone keratinocytes (Figure 1A), which correspond to the progeny of single keratinocyte stem cells [11]. These cells exhibit extensive long-term growth potential in bidimensional (2D) culture, as well as genomic stability and efficient epidermal regeneration, as assessed by in vitro epidermis reconstruction and in vivo xenografting [11,12], thus showing functional characteristics of a highly immature population of cultured precursors. Three-dimensional (3D) skin substitutes were bioengineered (Figure 1B) according to a preclinical process [20]. Skin reconstructs were either exposed or not exposed to ionizing radiation (IR) (Figure 1C) at an early stage of epidermis development, corresponding to a non-stratified keratinocyte basal monolayer (see Figure 1B). Samples were submitted to a single exposure to IR at a low dose (50 mGy) or at a higher dose (2 Gy). The next day, irradiated and non-irradiated tissue samples were xenografted onto recipient nude mice (Figure 1D) [21]. This experimental process enabled the study of human epidermis regeneration in an in vivo context up to complete differentiation. Fully differentiated human epidermis substitutes were then characterized (Figure 1E). The model repeatedly gave rise to normally organized and differentiated epidermises.
