Abstract
The development and adoption of cell therapies has been largely limited by difficulties associated with their safety, handling, and storage. Extracellular vesicles (EV) have recently emerged as a likely mediator for the therapeutic effect of cells, offering several advantages over cell therapies. Due to their small size and inability to expand and metastasize, EV are generally considered safer than cell transplantation. Nevertheless, few studies have scrutinized the toxicity profile of EV, particularly after repeated high-dose administration. The present study aimed to evaluate a preparation of small EV obtained from umbilical cord blood mononuclear cells (UCB-MNC-sEV) for its cytotoxicity in different cell lines, as well as its differential accumulation, distribution, and toxicity following repeated intravenous (IV) administrations in a rodent model. In vitro, repeated sEV exposure in concentrations up to 1 × 1011 particles/mL had no deleterious impact on the viability or metabolic activity of peripheral blood mononuclear cells, THP-1 monocytes, THP-1-derived macrophages, normal dermal human fibroblasts, or human umbilical vein endothelial cells. DiR-labelled sEV, injected intravenously for four weeks in healthy rats, were detected in clearance organs, particularly the kidneys, spleen, and liver, similarly to control dye. Moreover, repeated administrations for six and twelve weeks of up to 1 × 1010 total particles of sEV dye were well-tolerated, with no changes in general haematological cell counts, or kidney and liver toxicity markers. More importantly, unlabelled sEV likewise did not induce significant alterations in cellular and biochemical blood parameters, nor any morphological changes in the heart, kidney, lung, spleen, or liver tissue. In sum, our data show that UCB-MNC-sEV have no significant toxicity in vitro or in vivo, even when administered repeatedly at high concentrations, therefore confirming their safety profile and potential suitability for future clinical use.
Highlights
Extracellular vesicles (EV), secreted by most cell types, are described as key mediators of intercellular communication through the transport of a wide array of bioactive molecules such as proteins, RNA, and DNA [1,2]
Three UCB-mononuclear cells (MNC)-small EV (sEV) concentrations (1 × 1010, 5 × 1010, and 1 × 1011 particles/mL) were tested on blood/immune system cells, endothelial cells (HUVECs), and fibroblasts (NHDF). These concentrations were chosen based on previous efficacy tests, which demonstrated that 1 × 1010 particles/mL are therapeutically active
The metabolic activity of total Peripheral blood mononuclear cells (PBMC), THP-1 monocytes and macrophages, Normal human dermal fibroblasts (NHDF), and human umbilical vein endothelial cells (HUVEC) was not reduced when compared with control, indicating that no measurable cell death occurred during this timeframe
Summary
Extracellular vesicles (EV), secreted by most cell types, are described as key mediators of intercellular communication through the transport of a wide array of bioactive molecules such as proteins, RNA (including microRNAs), and DNA [1,2]. Being such a heterogeneous group, EV are catalogued into different subsets according to their cellular origin and size, ranging from the micron to the sub-micron dimension [3,4]. EV isolated from mesenchymal stromal cells (MSC) and mononuclear cells (MNC) have been demonstrated to successfully replace cell-based therapies, improving the function of damaged organs in animal models of ischemic diseases such as stroke [11,12], cardiovascular diseases [10,13,14], and chronic wounds [15,16]
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