Large-Scale exosome isolation from Mesenchymal Stem Cells cultured in Spinner flask

ANTONELLA LOMBARDI; PAULA BUCCI; SANTIAGO GABRIEL MIRIUKA; MARÍA CELESTE BIANI; JORGE MONTANARI; CARLOS DANIEL LUZZANI; CYNTIA ABAN; GUSTAVO SEVLEVER

Mar del Plata

Congreso; S A I C . S A F E . S A B . SAP 2 0 1 9; 2019

Due to their potential for differentiation and their immunomodulatory capacity Mesenchymal Stem Cells (MSC) have been widely used in preclinical and clinical studies for various diseases and tissue regeneration. Multiple evidences show that their regenerative effects are mainly due to their paracrine function mediated by their secretome, which is composed of both, soluble factors and factors released within extracellular vesicles (EV). Exosomes are a type of EV that are thought to mediate cellular communication. Our group have previously shown that within the exosomal content there are lots of proteins involved in regeneration and immunomodulation processes, moreover, we observed positive effects on cell migration when using medium with MSC exosomes in wound healing assays. From the translational point of view, these microvesicles are very interesting since they could be produced in big amounts and at a lower cost than MSC, stored for later use and would allow developing cell-free therapies of easier application. The aim of this work was to isolate MSC exosomes on a large scale and characterize them for later use in functional tests. First, we needed to scale up MSC culture. For this, we set up the conditions to grow them in Spinner flasks, obtaining a big number of cells in a short time. These cells were checked and characterized by evaluating the expression of typical membrane integrins and extracellular matrix proteins by Flow cytometry and Real-Time PCR. Once we set up the culture conditions, we proceeded to isolate exosomes from it. After 24 hours of medium conditioning, we performed a series of centrifugations and separation of vesicles by SizeExclusion Chromatography. The eluted fractions were incubated with anti-CD9 and anti-CD81 following incubation with anti-CD63 magnetic beads. The flow cytometry results confirmed that we could effectively isolate exosomes, and TRPS?s quantification showed a large number of isolated vesicles. In the future we plan to assess a quality check of the isolated exosomes, as well as to analyze their effect in functional assays.