Chapter title |
Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.
|
---|---|
Chapter number | 7 |
Book title |
Cell-Secreted Vesicles
|
Published in |
Methods in molecular biology, January 2023
|
DOI | 10.1007/978-1-0716-3203-1_7 |
Pubmed ID | |
Book ISBNs |
978-1-07-163202-4, 978-1-07-163203-1
|
Authors |
Andriolo, Gabriella, Provasi, Elena, Brambilla, Andrea, Panella, Stefano, Soncin, Sabrina, Cicero, Viviana Lo, Radrizzani, Marina, Turchetto, Lucia, Barile, Lucio |
Abstract |
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 (~1013 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. |
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