Impact of Storage Conditions on EV Integrity/Surface Markers and Cargos

Abstract

Extracellular vesicles (EVs) are small biological particles released into biofluids by every cell. Based on their size, they are classified into small EVs (<100 nm or <200 nm) and medium or large EVs (>200 nm). In recent years, EVs have garnered interest for their potential medical applications, including disease diagnosis, cell-based biotherapies, targeted drug delivery systems, and others. Currently, the long-term and short-term storage temperatures for biofluids and EVs are −80 °C and 4 °C, respectively. The storage capacity of EVs can depend on their number, size, function, temperature, duration, and freeze–thaw cycles. While these parameters are increasingly studied, the effects of preservation and storage conditions of EVs on their integrity remain to be understood. Knowledge gaps in these areas may ultimately impede the widespread applicability of EVs. Therefore, this review summarizes the current knowledge on the effect of storage conditions on EVs and their stability and critically explores prospective ways for improving long-term storage conditions to ensure EV stability.

Natural Killer Cell-Derived Extracellular Vesicles as a Promising Immunotherapeutic Strategy for Cancer: A Systematic Review

“…To achieve this, researchers need to device a storage condition that can preserve EV stability over a long period of time. As an acellular product, NK-EVs can be kept chill (4 °C) for immediate use or frozen (−20 °C or −80 °C) up to 12 months with acceptable loss of proteins and RNAs [ 106 , 107 , 108 , 109 , 110 ]. More importantly, the NK-EVs retain their anti-tumor effect.…”

Stability and Function of Extracellular Vesicles Derived from Immortalized Human Corneal Stromal Stem Cells: A Proof of Concept Study

“…The main protective effect of trehalose on EVs is during the lyophilization process or freeze-thaw cycling as explained above. EVs directly stored at RT have been shown to have lower DNA concentration, miRNA level, and CD63 expression after short-term storage (42). We therefore did not include EV stored at RT with trehalose.…”

Extracellular vesicles and their cells of origin: Open issues in autoimmune diseases

“…The quality of endogenous EV preparations ( 105 ), from body fluids, tissues, or intracellular spaces ( 40 ), is affected by the circadian clock ( 106 ). In addition, isolation method, purity, storage protocol ( 107 ), time and temperature ( 108 ), presence or absence of anti-coagulants, isotonic buffers, other reagents such as cryoprotectants ( 109 ), pH, and ionic strength ( 110 – 112 ) are the other influencing factors. The concentration and content of EVs are influenced by demographic factors, including sex, age and race to some extent ( 113 ) and more than that, the EV cargo could be deliberately engineered through microenvironment modifications ( 114 ).…”