Label-free characterization of extracellular vesicles from human Mesenchymal Stem Cells by Raman spectroscopy

Introduction: Extracellular vesicles (EVs) from human mesenchymal stromal cells (hMSCs) have been extensively proposed as a powerful and versatile therapeutic tool for regenerative medicine. They are a valid alternative to cell therapy because of remarkable therapeutic potential, suitability for drug delivery, reduced risk of ectopic tissues formation and immune rejection compared to cells. Nonetheless, the heterogeneity of EVs makes often the comparison of experimental results biased. We propose herein Raman spectroscopy as a label-free non-destructive method suitable for EV characterization prior to their use in complex disease models and as an alternative to mass spectrometry or flow cytometry, both suggested as minimal requirements by ISEV (J Extracell Vesicles. 2014;3:26913). Methods: After the standard mesenchymal characterization procedure, hMSCs (2nd- 4th passage) from bone marrow and adipose tissue were cultured until 90% confluency in medium containing 10% EV-deprived fetal bovine serum. hMSCs were then maintained 72h in serum free conditions. Conditioned medium was centrifuged to remove possible cell debris and apoptotic bodies before being ultracentrifuged twice for 1h at 100000 g (Bekman L7–65; Rotor 55.2 Ti). Freshly isolated EVs were analyzed by confocal Raman microspectroscopy with both 532 nm and 633 nm laser sources. Results: Raman spectra revealed the concomitant presence of phospholipids, cholesterol, ceramids, proteins, and nucleic acids in EVs from both cell sources. The general chemical composition of both samples shown by Raman microspectroscopy is in accordance with the reported ultrastructure of EVs, making it a suitable method for basic characterization of EV samples. Even more interestingly, beside the presence of common Raman peaks, the comparison of EVs from hMSCs of different tissues cultured under the same experimental conditions highlighted a specific Raman fingerprint dependent from the cell of origin. Summary/Conclusion: The herein reported preliminary results support the use of Raman spectroscopy as a valid tool for a fast EV characterization without vesicle damaging and chemical/physical fixation. Thanks to the avoidance of sample preparation and labeling, Raman spectroscopy can provide the overall EV biochemical fingerprint that facilitates EV applied research, overcoming difficulties in research data comparison. For these reasons, Raman-based methods have the potentialities to become new tools for the routine inspection of EV samples before in vitro or in vivo use.