Cryopreservation of mesenchymal stromal cell-derived extracellular vesicles using trehalose maintains their ability to expand hematopoietic stem cells in vitro

Abstract

The multitude of clinical trials using mesenchymal stromal cells (MSCs) has underscored their significance as a promising cell source for regenerative therapies. Most studies have however shown that MSCs get entrapped into the microvasculature of lungs, liver and spleen. In addition to intercellular communication, MSCs exert their effects in a paracrine manner by secretion of extracellular vesicles (EVs). The therapeutic effects of MSC-derived EVs have been examined in several diseases such as hepatic failure, liver injury, hematopoiesis etc. Therefore, optimization of cryopreservation strategies for the long-term storage of functional EVs could help in the development of off-the-shelf biologics. The aim of this study was to develop an optimal cryopreservation strategy for the efficient storage of both types of EVs – Microvesicles (MVs) and exosomes, independently, and to further examine the effect of the cryopreserved EVs on the ex vivo expansion of HSCs. MVs and exosomes were separately cryopreserved at different temperatures using PBS or PBS supplemented with trehalose (pTRE), and these cryopreserved EVs were then assessed for their functionality after revival. We found that addition of trehalose during cryopreservation helped in maintaining the morphology and functionality of the EVs, as assessed by their HSC-supportive potential, ability to expand phenotypically defined HSCs and ability to maintain the chemotactic migration potential of the HSCs co-cultured with them. This strategy could prove to be beneficial for facilitating the use of EVs as cell-free ready-to-use biologics for the ex vivo expansion of HSCs and in regenerative medicine.