Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.

Autor:	Andriolo G; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Provasi E; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Brambilla A; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Panella S; Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland., Soncin S; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Cicero VL; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Radrizzani M; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Turchetto L; Lugano Cell Factory, Istituto Cardiocentro Ticino, Ente Ospedaliero Cantonale, Lugano, Switzerland., Barile L; Cardiovascular Theranostics, Istituto Cardiocentro Ticino, Laboratories for Translational Research, Ente Ospedaliero Cantonale, Bellinzona, Switzerland. lucio.barile@eoc.ch.; Faculty of Biomedical Sciences, Università Svizzera italiana, Lugano, Switzerland. lucio.barile@eoc.ch.
Jazyk:	angličtina
Zdroj:	Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2023; Vol. 2668, pp. 69-98.
DOI:	10.1007/978-1-0716-3203-1_7
Abstrakt:	The development of an extracellular vesicles (EV)-based therapeutic product requires the implementation of reproducible and scalable, purification protocols for clinical-grade EV. Commonly used isolation methods including ultracentrifugation, density gradient centrifugation, size exclusion chromatography, and polymer-based precipitation, faced limitations such as yield efficiency, EV purity, and sample volume. We developed a GMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving, tangential flow filtration (TFF). We applied this purification method for the isolation of EV from conditioned medium (CM) of cardiac stromal cells, namely cardiac progenitor cells (CPC) which has been shown to possess potential therapeutical application in heart failure. Conditioned medium collection and EV isolation using TFF demonstrated consistent particle recovery (~10 13 particle/mL) enrichment of small/medium-EV subfraction (range size 120-140 nm). EV preparations achieved a 97% reduction of major protein-complex contaminant and showed unaltered biological activity. The protocol describes methods to assess EV identity and purity as well as procedures to perform downstream applications including functional potency assay and quality control tests. The large-scale manufacturing of GMP-grade EV represents a versatile protocol that can be easily applied to different cell sources for wide range of therapeutic areas. (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze:	MEDLINE
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