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Extracellular vesicles (EVs), through their complex cargo, can reflect the state of their cell of origin and change the functions and phenotypes of other cells. These features indicate strong biomarker and therapeutic potential and have generated broad interest, as evidenced by the steady year-on-year increase in the numbers of scientific publications about EVs. Important advances have been made in EV metrology and in understanding and applying EV biology. However, hurdles remain to realising the potential of EVs in domains ranging from basic biology to clinical applications due to challenges in EV nomenclature, separation from non-vesicular extracellular particles, characterisation and functional studies. To address the challenges and opportunities in this rapidly evolving field, the International Society for Extracellular Vesicles (ISEV) updates its Minimal Information for Studies of Extracellular Vesicles, which was first published in 2014 and then in 2018 as MISEV2014 and MISEV2018, respectively. The goal of the current document, MISEV2023, is to provide researchers with an updated snapshot of available approaches and their advantages and limitations for production, separation and characterisation of EVs from multiple sources, including cell culture, body fluids and solid tissues. In addition to presenting the latest state of the art in basic principles of EV research, this document also covers advanced techniques and approaches that are currently expanding the boundaries of the field. MISEV2023 also includes new sections on EV release and uptake and a brief discussion of in vivo approaches to study EVs. Compiling feedback from ISEV expert task forces and more than 1000 researchers, this document conveys the current state of EV research to facilitate robust scientific discoveries and move the field forward even more rapidly.

Surface-enhanced Raman spectroscopy (SERS) is a powerful tool that provides valuable insight into the molecular contents of chemical and biological samples. However, interpreting Raman spectra from complex or dynamic datasets remains challenging, particularly for highly heterogeneous biological samples like extracellular vesicles (EVs). To overcome this, we developed a tunable and interpretable deep autoencoder for the analysis of several challenging Raman spectroscopy applications, including synthetic datasets, chemical mixtures, a chemical milling reaction, and mixtures of EVs. We compared the results with classical methods (PCA and UMAP) to demonstrate the superior performance of the proposed technique. Our method can handle small datasets, provide a high degree of generalization such that it can fill unknown gaps within spectral datasets, and even quantify relative ratios of cell line-derived EVs to fetal bovine serum-derived EVs within mixtures. This simple yet robust approach will greatly improve the analysis capabilities for many other Raman spectroscopy applications., Competing Interests: There are no conflicts to declare., (© 2024 Optica Publishing Group.)

Trophoblasts are placenta-specific epithelial cells that play an essential role in conducting nutrient, gas, and waste exchange between the fetus and the mother. Primary culture of human trophoblasts from donated term placentae is an important tool to study placental functions. Currently, there is a lack of general consensus of the optimal culture conditions for maintaining term trophoblast cells in vitro. A key problem with culturing trophoblasts from term placentae is overgrowth of the trophoblasts by rapidly proliferating cellular contaminants. Recently we reported a system to culture trophoblasts from term placentae which differentiate into syncytiotrophoblast-like multinucleated cells that can be maintained in culture for at least 30 days with minimal contamination. This chapter details our optimized approach for long-term, contaminant-free in vitro culture of primary trophoblasts from term placentae., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The efficient production of extracellular vesicles (EVs) from adherent cells in vitro can be challenging when using conventional culture flasks. Issues such as low cell density leading to low EV yield, and the inability to completely remove bovine serum EVs without starvation contribute to this challenge. By comparison, the two-chamber CELLine adherent bioreactor can produce significantly more EVs with improved time, space, and resource efficiency. Furthermore, it is highly accessible and can continually produce EVs using long term cultures without the need for passaging. Lastly, the 10 kDa semipermeable, cellulose acetate membrane separating the cell and media chambers allows for the continual use of bovine serum in the media chamber while preventing bovine EVs from contaminating the conditioned media., (© 2021. Springer Science+Business Media, LLC.)