Development of a novel technology for long-term culture and live imaging of excised human tissue

An integrated understanding of the structure and reactivity of cells, extracellular matrices, and appendages is important for elucidating their functions and mechanisms in our bodies. Three-dimensional imaging using immuno-fluorescent staining with decolorization technology aids in comprehending the internal structure of human organs. However, live imaging of skin dynamics using animal models is highly invasive and unsuitable for humans. The aim of this study was to establish a non-invasive live imaging method for excised human tissue. In this study, to maintain excised human skin tissue in a state similar to a living body, we developed a novel microneedle-based culture technique. This method was evaluated for cytotoxicity detection, inflammatory cytokine release, and tissue morphology. Using microneedles, we cultured excised skin tissue and observed cellular organelles, reactive oxygen species (ROS), and fibrous structures via fluorescent probes and autofluorescence. The microneedle technique prevented cell death and inflammation, enabling long-term culturing. We live-imaged various skin cells, extracellular matrices, and appendage structures, visualizing epidermal cell membranes, mitochondria, and ROS. Collagen and elastin fibers were observed using autofluorescence and second harmonic generation. This approach enabled live imaging for 5 d, providing insights into skin metabolism, regeneration, and responses to stimuli and drugs, ultimately advancing dermatological research.