Mixing vs. layering: How to make an in vivo like skin model

Three-dimensional human skin models are widely used to study the physiology, development, and pathological conditions of the skin as well as to test drugs and cosmetics. However, their production protocols are laborious and time consuming. Cell spheroids derived from keratinocytes and fibroblasts are considered promising tools for mimicking human skin in vitro because of their ease of production, sufficient relevance, and scalability of spheroid-based tissue models. In this study, we produced keratinocyte and fibroblast spheroids using different formation methods, mixing and layering, and thoroughly examined them. Four groups of spheroids were described: two groups obtained from either keratinocytes or fibroblasts (monospheroids) and two groups formed by a combination of both cell types in two different ways (heterospheroids). Furthermore, we investigated various biological properties of the spheroids, including diameter changes, their ability to spread when placed in adhesive conditions, biomechanical properties, localization, expression and content of marker proteins, and epidermal differentiation processes of keratinocytes within heterospheroids. We conclude that heterospheroids formed by layering keratinocytes on a fibroblast core can serve as the simplest in vitro skin models because of their skin-like architecture. Conversely, heterospheroids produced by mixing keratinocytes and fibroblasts are more suitable biological models for studying cell self-organization in living tissues.