Storage stability and delivery potential of cytochalasin B induced membrane vesicles

Cell-free therapies based on extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) are considered as a promising tool for stimulating regeneration and immunomodulation. The need to develop a practical approach for large-scale production of vesicles with homogenous content led to the implementation of cytochalasin B-induced to induce microvesicles (CIMVs) biogenesis. CIMVs mimic natural EVs in size and composition of the surrounding cytoplasmic membrane. Previously we observed that MSC derived CIMVs demonstrate the same therapeutic angiogenic and immunomodulatory activity as the parental MSCs, making them a potentially scalabale cell-free therapeutic option. However, little is known about their storage stability and delivery potential. Therefore, in this study, we determined the effects of different storage conditions (+37°C in serum, +4°C,-20°, +25°C in saline, as well as freeze-drying prior to storage at-20°C) on the integrity and effective delivery of CIMVs derived from human MSCs. We determined that different storage conditions alter the protein concentration within the solution used to store CIMVs over time, this concided with a decrease in the amount of CIMVs due to gradual degradation. We established that freezing and storage CIMVs in saline at-20°C reduces degredation and prolongs their shelf life. Additionally, we found that freeze-thawing preserved the CIMVs morphology better than freeze drying and subsequent rehydration which resulted in aggregation of CIMVs. Collectively our data demonstrates for the first time, that the most optimal method of CIMVs storage is freezing at-20°C, to preserve the CIMVs in the maximum quantity and quality with retention of effective delivery. These findings will benefit the formation of standardized protocols for the use of CIMVs for both basic research and clinical application