Purification of Extracellular Vesicles Secreted by Skin Fibroblasts and Keratinocytes

Extracellular vesicles (EVs) secreted by all cell types play crucial roles in intercellular communication through the transfer of bioactive molecules, including proteins, lipids, and nucleic acids, within tissues and throughout the organism. In cutaneous biology, EVs have emerged as key mediators in maintaining skin homeostasis and regulating pathological processes. However, obtaining sufficient quantities of EVs from primary cell cultures remains technically challenging due to inherent limitations in cell number, proliferative capacity, and EV production efficiency compared to immortalized cell lines. In this chapter, we present detailed, step-by-step protocols specifically optimized for isolating small EVs from primary human dermal fibroblasts and epidermal keratinocytes. These methodologies consistently yield sufficient EV quantities for comprehensive functional studies.