AUTHOR=Andriolo Gabriella , Provasi Elena , Lo Cicero Viviana , Brambilla Andrea , Soncin Sabrina , Torre Tiziano , Milano Giuseppina , Biemmi Vanessa , Vassalli Giuseppe , Turchetto Lucia , Barile Lucio , Radrizzani Marina 

TITLE=Exosomes From Human Cardiac Progenitor Cells for Therapeutic Applications: Development of a GMP-Grade Manufacturing Method

JOURNAL=Frontiers in Physiology

VOLUME=Volume 9 - 2018

YEAR=2018

URL=https://www.frontiersin.org/journals/physiology/articles/10.3389/fphys.2018.01169

DOI=10.3389/fphys.2018.01169

ISSN=1664-042X

ABSTRACT=Exosomes, nanosized membrane vesicles secreted by cardiac progenitor cells (Exo-CPC), inhibit cardiomyocyte apoptosis under stress conditions, promote angiogenesis in vitro and prevent the early decline in cardiac function after myocardial infarction in vivo in pre-clinical rat models.
The recognition of exosome-mediated effects has moved attempts at developing cell-free approaches for cardiac repair. Such approaches offer major advantages including the fact that exosomes can be stored as ready-to-use agents and delivered to patients with acute coronary syndromes.

The aim of the present work was the development of a Good Manufacturing Practice (GMP)-grade method for the large-scale preparation of Exo-CPC as a medicinal product, for a future clinical translation.

A GMP-compliant manufacturing method was set-up, based on large scale cell culture in xeno-free conditions, collection of up to 8 L of exosome-containing conditioned medium and isolation of Exo-CPC through tangential flow filtration. 
Quality control tests were developed and carried out to evaluate safety, identity and potency of both cardiac progenitor cells as cell source and Exo-CPC as final product (GMP-Exo-CPC).

Cardiac progenitor cells, cultured in xeno-free conditions, showed a lower doubling-time than observed in research-grade condition, while producing exosomes with similar features. Cells showed the typical phenotype of mesenchymal progenitor cells (CD73/CD90/CD105 positive, CD14/CD20/CD34/CD45/HLA-DR negative), and expressed mesodermal (TBX5/TBX18) and cardiac-specific (GATA4/MESP1) transcription factors. 
Purified GMP-Exo-CPC showed the typical Nanoparticle Tracking Analysis profile and expressed main exosome markers (CD9/CD63/CD81/TSG101). 
The GMP manufacturing method guaranteed high exosome yield (>1013 particles) and consistent removal (≥97%) of contaminating proteins.
The resulting GMP-Exo-CPC were tested for safety, purity, identity and potency in vitro, showing functional anti-apoptotic and pro-angiogenic activity. The therapeutic efficacy was validated in vivo in rats, where GMP-Exo-CPC ameliorated heart function after myocardial infarction.
Our standardized production method and testing strategy for large scale manufacturing of GMP-Exo-CPC open new perspectives for reliable human therapeutic applications for acute myocardial infarction syndrome and can be easily applied to other cell sources for different therapeutic areas.