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

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.

Message in a Bottle: Upgrading Cardiac Repair into Rejuvenation

“…The first evidence of this has been recently performed for CPC-EVs. Indeed, human CPCs cultured in xeno-free conditions showed production of EVs with similar features as in research-grade conditions [228]. The GMP method guaranteed high exosome yield (>1013 particles) by isolating through a closed system of ultrafiltration, and consistent removal (≥97%) of contaminating proteins.…”

“…Moreover, one of the most relevant therapeutic advantage of EVs, compared to the parental cells, is their potential to "escape" to the immune system. The absence of costimulatory molecule MHC-II gives EVs the potential to escape recognition by CD4 lymphocytes [90,216,228], although this aspect needs more detailed investigation.…”

“…The GMP method guaranteed high exosome yield (>1013 particles) by isolating through a closed system of ultrafiltration, and consistent removal (≥97%) of contaminating proteins. Thus, such standardised production method for large-scale manufacturing of CPC-EVs may offer a good demonstration of the clinical translation of EV biology for human therapeutic applications for the treatment of acute myocardial infarction syndrome [228].…”

Prospects and challenges of extracellular vesicle-based drug delivery system: considering cell source

“…Recently, Pachler et al (2017) developed a GMP standard protocol for human MEVs. More recently, a GMP-grade method for the large-scale preparation of EVs from human cardiac progenitor cells was described (Andriolo et al, 2018). Additionally, Mendt et al (2018) reported a bioreactor-based, large-scale production protocol of clinical-grade exosomes employing GMP standards.…”

Engineering Extracellular Vesicles as Nanotherapeutics for Regenerative Medicine

“…Following isolation, current assessments of exosome identity involve an extensive profiling of characteristic surface proteins on both the exosomes and parental cells. Western blotting or flow cytometry can be used to assess the presence of exosome surface proteins (e.g., CD9, CD63, CD81) on the isolated particles, while parental cells are characterized for viability and cell surface marker profiles [33]. Microscopy techniques, such as electron microscopy (EM) and atomic force microscopy (AFM), may be useful for additional morphology and size distribution characterization [32].…”