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Non-invasive cell culture monitoring technology is crucial to improve the manufacturing efficiency of cell products. We have found that extracellular vesicles (EVs) are secreted into the culture supernatants in the differentiation process from human induced pluripotent stem cells (iPSCs) to dopaminergic progenitor cells, and that the composition of EVs changes in accordance with the differentiation processes. In this study, we hypothesized that it is possible to evaluate the cultured cellular states by detecting compositional changes of EVs secreted from cultured cells with label-free Raman spectroscopy in a non-invasive manner. Therefore, Raman signal analysis derived from EV fractions isolated from culture supernatants throughout the differentiation process was conducted. iPSCs cultures were simultaneously implemented under a standard condition (control) and an artificial deviation condition inducing reductions in pluripotency by depleting FGF2 in culture medium (-FGF2), which is indispensable for maintaining the pluripotency. Subsequently, the differentiation step was conducted for each iPSCs culture under the same condition. As a result, it was found that under -FGF2, the expression level of the pluripotency marker NANOG decreased compared to that of the control and correlated with the identification results based on Raman signals with a correlation coefficient of 0.77. Lipid-derived Raman signals were extracted as identification factors, suggesting that changes in the lipid component of EV occur depending on the cellular states. From the above, we have found that the change in composition of EVs in the culture supernatant by detecting Raman signals would be a monitoring index of the cellular state of differentiation and pluripotency., (Copyright © 2024 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)