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 preclinical 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 (CPC) as cell source and Exo-CPC as final product (GMP-Exo-CPC). CPC, 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.
Highlights
Exosomes are extracellular vesicles (EV) of endosomal origin, with a diameter of 30–150 nm, involved in intercellular communication processes
The tissue was processed under sterile conditions (Class A laminar hood): after transfer to a sterile support (Petri dish, Corning, United States) two washings were performed with Dulbecco’s phosphate buffered saline without calcium and magnesium (DPBS, Gibco/Thermo Fisher Scientific, United States), the cardiac muscle tissue was isolated from the connective tissue and minced in small fragments
Cardiac progenitor cells were successfully isolated from heart tissue fragments in all the tested conditions, as shown in Figure 1A for a representative donor
Summary
Exosomes are EV of endosomal origin, with a diameter of 30–150 nm, involved in intercellular communication processes. Recent evidence suggests that human CPC release EV that phenotypically recapitulate Exo (Barile et al, 2012) Such vesicles inhibit cardiomyocyte apoptosis and promote angiogenesis in vitro, playing a fundamental role in the paracrine effect of CPC. In a preclinical model of AMI in rats, CPC-derived Exo (Exo-CPC) reduce tissue damage, inhibit cardiomyocyte apoptosis, and promote angiogenesis, improving post-infarction cardiac function (Barile et al, 2014) Key mediators of these effects have been identified in specific microRNAs such as miR-132 and miR-210 (Barile et al, 2014), as well as specific proteins such as the active form of pregnancy-associated plasma protein-A (Barile et al, 2018)
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