Background

The human skin is the largest organ of the human body and functions as a physical barrier shielding the body from a number of harmful external agents such as air pollution or solar radiation (Gebhard et al., 2014). Solar radiation, e.g., induces increased production of reactive oxygen species (ROS) and thereby DNA damage and mutations in skin, which results in skin aging and skin cancer (Gebhard et al., 2014). Mitochondrial dysfunction resulting from DNA damage is thought to play an important role in these and other essential cellular processes. The accumulation of reactive oxygen species (ROS)-damaged mitochondrial DNA (mtDNA) and proteins can induce mitochondrial dysfunction within the electron transport chain (ETC) and in turn lead to an enhanced ROS production and increased mitochondrial dysfunction (Furda et al., 2012; Yoshida et al., 2012). According to the ‘mitochondrial theory of aging’, this vicious cycle is a major cause for cellular aging, tissue dysfunction and degeneration (Harman, 1972). Understanding the impact of harmful external influences on mitochondrial function is therefore essential for a better view on aging in general. Unfortunately, the measurement of mitochondrial respiration in cell cultures cannot completely reflect the real situation in skin. We therefore established an efficient method to measure the mitochondrial respiration ex vivo directly in human epidermis biopsies with the Seahorse XF24 Flux Analyzer and Seahorse XF24 Islet Capture microplates. The XF24 Analyzer is a multi-well plate system which measures oxygen consumption rates (OCR) and extracellular acidification rates (ECAR) by changes in the fluorescence of solid state fluorophores. In this system, up to four different ports can be used to inject compounds to obtain a mitochondrial respiration profile. The procedure described in this protocol includes the preparation of the biopsies, the preparation of the epidermis for insertion into the XF24 Analyzer and the measurement of mitochondrial parameters by the addition of specific mitochondrial inhibitors. This protocol was established for the testing of toxic substances and their influence on the mitochondrial respiration parameters in human epithelial tissue. In addition, age-related effects of various substances can be tested based on the age of the donor. The analysis of mitochondrial respiration in epidermis derived from skin biopsies represents an important addition to existing screenings.