The isolation, culture-expansion, cryopreservation, characterization, and properties of umbilical cord-derived mesenchymal stromal cells and their extracellular vesicles

Abstract

Mesenchymal stromal cells (MSCs) are of therapeutic interest due to their immunomodulatory and regenerative properties. As a therapeutic, MSCs have limitations such as variability among tissue and species source, low survival, and risk of thrombosis or embolism following intravenous administration. Preclinical data supports MSCs as a therapeutic but has not translated to consistent, successful clinical trial results possibly due in part to aforementioned limitations. Extracellular vesicles (EVs) have been shown to be involved with physiological cell signaling and communication and may play a role in MSC’s therapeutic effect. In addition, EVs purified from cell culture conditioned media have been shown to retain some properties of the parent cell type, such as cargo and protein surface marker expression, thus making EVs a potential therapeutic target. Although a cell-free product, EVs come with their own limitations such as the inability to produce sufficient and consistent EVs. Similar to MSCs from other species, EVs purified from these cell types are not well understood. Here, an optimized protocol for the isolation, expansion, cryopreservation, and characterization of canine umbilical cord-derived MSCs (UC-MSCs) is presented. This protocol addresses shortcomings in the canine MSC field by employing the coating of tissue culture surfaces to increase cellular adherence and the use of basic fibroblast growth factor in cell culture medium to allow canine MSCs to be maintained in culture longer than published methods before senescing. In addition, the effect of storage temperature of human UC-MSC conditioned media (CM) on subsequent purified EVs is presented demonstrating that comparable numbers of EVs could be isolated from CM following storage at room temperature, 4ºC, -20ºC, and -80ºC compared to fresh CM. Storage of CM at -80ºC resulted in a more homogeneous population of particles, with similar surface potential and hydrodynamic size. Although the presence of EVs were confirmed in all CM storage conditions by transmission electron microscopy, only EVs from CM stored at -80ºC exhibited similar morphology and size to EVs purified from fresh CM. EVs from CM stored at -80ºC displayed stronger overall protein expression of tetraspanins CD9, CD63, and CD81, as well as heat shock protein 70, indicating that storage of CM at -80ºC is comparable to fresh CM for downstream EV purification. Lastly, it is demonstrated here that EVs purified from canine and human UC-MSC CM retain surface tissue factor expression from parental cells and display tissue factor-mediated procoagulant activity in the form of FXa generation. Thus, EV administration is a safety concern and poses a risk of thromboembolism. This is concerning since MSCs, and possibly MSC-EVs, are being investigated as a therapeutic, specifically with respiratory complications associated with COVID-19. We suggest that the procoagulant activity of EVs may serve as a safety screening tool in clinical use.