Methodologies for Scalable Production of High-Quality Purified Small Extracellular Vesicles from Conditioned Medium.

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.

Combined delivery of bone marrow-derived mononuclear cells in chronic ischemic heart disease: rationale and study design.

BACKGROUND: Treatment with bone marrow-derived mononuclear cells (BM-MNC) may improve left ventricular (LV) function in patients with chronic ischemic heart disease (IHD). Delivery method of the cell product may be crucial for efficacy. HYPOTHESIS: We aimed to demonstrate that the combination of intramyocardial and intracoronary injection of BM-MNC is safe and improves LV function in patients with chronic IHD. METHODS: After a safety/feasibility phase of 10 patients, 54 patients will be randomly assigned in a 1:1:1 pattern to 1 control and 2 BM-MNC treatment groups. The control group will be treated with state-of-the-art medical management. The treatment groups will receive either exclusively intramyocardial injection or a combination of intramyocardial and intracoronary injection of autologous BM-MNC. Left ventricular function as well as scar size, transmural extension, and regional wall-motion score will be assessed by cardiac magnetic resonance imaging studies at baseline and after 6 months. The primary endpoint is the change in global LV ejection fraction by cardiac magnetic resonance from 6 months to baseline. RESULTS: The results, it is hoped, will have important clinical impact and provide essential information to improve the design of future regenerative-medicine protocols in cardiology. CONCLUSIONS: As cell delivery may play an important role in chronic IHD, we aim to demonstrate feasibility and efficacy of a combined cell-delivery approach in patients with decreased LV function.