To date, extracellular vesicles (EVs) have been extensively investigated as potential substitutes for cell therapy. Research has suggested their ability to overcome serious risks associated with the application of these cells. Although, the translation of EVs into clinical practice is hampered by the lack of a cheap reasonable way to obtain a clinically relevant number of EVs, an available method for the large-scale production of EVs ensures vesicles’ integrity, preserves their biological activity, and ensures they are well reproducible, providing homogeneity of the product from batch to batch. In this review, advances in the development of methods to increase EVs production are discussed. The existing approaches can be divided into the following: (1) those based on increasing the production of natural EVs by creating and using high capacity “cell factories”, (2) those based on the induction of EVs secretion under various cell stressors, and (3) those based on cell fragmentation with the creation of biomimetic vesicles. The aim of this review is to stimulate the introduction of EVs into clinical practice and to draw attention to the development of new methods of EVs production on a large scale.

Extracellular vesicles (EVs) are promising therapeutic instruments and vectors for therapeutics delivery. In order to increase the yield of EVs, a method of inducing EVs release using cytochalasin B is being actively developed. In this work, we compared the yield of naturally occurring extracellular vesicles and cytochalasin B-induced membrane vesicles (CIMVs) from mesenchymal stem cells (MSCs). In order to maintain accuracy in the comparative analysis, the same culture was used for the isolation of EVs and CIMVs: conditioned medium was used for EVs isolation and cells were harvested for CIMVs production. The pellets obtained after centrifugation 2300× g, 10,000× g and 100,000× g were analyzed using scanning electron microscopy analysis (SEM), flow cytometry, the bicinchoninic acid assay, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA). We found that the use of cytochalasin B treatment and vortexing resulted in the production of a more homogeneous population of membrane vesicles with a median diameter greater than that of EVs. We found that EVs-like particles remained in the FBS, despite overnight ultracentrifugation, which introduced a significant inaccuracy in the calculation of the EVs yield. Therefore, we cultivated cells in a serum-free medium for the subsequent isolation of EVs. We observed that the number of CIMVs significantly exceeded the number of EVs after each step of centrifugation (2300× g, 10,000× g and 100,000× g) by up to 5, 9, and 20 times, respectively.

In order to improve the regeneration of full-layer skin defects, hydrogels were developed based on the combination of chitosan (Cs), Daba silk fibroin (DSF), and graphene oxide (GO): CS, DSF/Cs and DSF/Cs/GO. The biocompatibility of hydrogels with human dermis fibroblasts in vitro was evaluated using the MTS assay. To assess the regenerative potential of hydrogels, a model of a full-layer skin defect was reconstructed on the back of rats and closed the wound surface with CS, DSF/Cs and DSF/Cs/GO hydrogels. The morphological and morphometric characteristics of regenerate tissues were obtained by staining with hematoxylin-eosin, Heidengain azocarmine, and immunohistochemistry on days 7 and 14 of the experiment. It has been shown that the use of DSF/Cs and DSF/Cs/GO promotes enhanced healing and epithelization of a full-layer skin wound. The addition of GO to the hydrogel increased the synthetic activity of fibroblasts and improved the characteristics of the produced collagen fibers.

Extracellular vesicles (EVs) are membrane particles of cellular origin that play a major role in intercellular communication. EVs have a number of characteristics that make them promising agents for targeted delivery of drugs and biologically active factors. However, their use in clinical practice is limited due to the lack of an industrially adapted production method that would provide a high yield of vesicles while maintaining their biological activity. The aim of this article is to analyze the morphology, size and cytoplasmic content of ultrasound (US)-generated membrane vesicles (MVs) from producer cells. MVs were generated by US treatment of producer cells. The concentration and size of the resulting vesicles were analyzed with NTA (nanoparticle tracking analysis) and SEM (scanning electron microscopy). We also assessed whether US-MVs contain the cytoplasmic content of the parental cell or whether there is a loss of cytosolic content upon rupture of the cell membrane. To do this, we preliminarily stained parental cells – human mesenchymal stem cells (MSCs), with Calcein Violet 450 AM dye. We found that the average size and uniformity of the obtained vesicles changed depending on the mode of US treatment. The longer the US treatment lasts, the greater the yield and the more uniform vesicles we get. According to the SEM results, the average size of MVs is 123±85 nm. In addition, we found a fraction of small rounded structures, the average size of which is 16±6 nm. We believe that the visualized structures represent ribosomes. Also, we found that after US exposure to prestained cells, the level of fluorescence in the supernatant increased (42.77±5.66 RFU), while MVs contained an insignificant amount of dye (1.83±0.16 RFU). Thus, we found for the first time that as a result of US exposure to MSCs, the cell membrane was ruptured and the cytosolic content of the cells leaked out. The method of US treatment of producer cells makes it possible to obtain MVs. However, the MVs fraction is “contaminated” with cell organelles, which will require additional purification of the obtained MVs and an assessment of their immunogenicity and toxicity in vivo. This method of production does not allow the cytoplasmic contents of the parent cell to be enclosed in vesicles, which reduces the potential biological activity of МVs-MSCs. Therefore, one of the possible ways of US-generated vesicles application is to use them as a vector for drug delivery.