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Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47-52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield., Competing Interests: Drs. Stott, Tessier, and Toner are inventors on several provisional patents on the topic of blood stabilization and cryopreservation of EVs, cells, tissues, and organs (W02016022433A1, W02018005802A1, W02020163774A1). These do not alter our adherence to PLOS ONE policies on sharing data and materials. There are no restrictions on sharing of data and/or materials as a result of these competing interests. Researcher’s interests are managed by the MGH and Partners HealthCare in accordance with their conflict-of-interest policies.

Cryopreservation is of significance in areas including tissue engineering, regenerative medicine, and organ transplantation. We investigated endothelial cell attachment and membrane integrity in a microvasculature model at high subzero temperatures in the presence of extracellular ice. The results show that in the presence of heterogeneous extracellular ice formation induced by ice nucleating bacteria, endothelial cells showed improved attachment at temperature minimums of -6 °C. However, as temperatures decreased below -6 °C, endothelial cells required additional cryoprotectants. The glucose analog, 3- O -methyl-D-glucose (3-OMG), rescued cell attachment optimally at 100 mM (cells/lane was 34, as compared to 36 for controls), while 2% and 5% polyethylene glycol (PEG) were equally effective at -10 °C (88% and 86.4% intact membranes). Finally, endothelialized microchannels were stored for 72 h at -10 °C in a preservation solution consisting of the University of Wisconsin (UW) solution, Snomax, 3-OMG, PEG, glycerol, and trehalose, whereby cell attachment was not significantly different from unfrozen controls, although membrane integrity was compromised. These findings enrich our knowledge about the direct impact of extracellular ice on endothelial cells. Specifically, we show that, by controlling the ice nucleation temperature and uniformity, we can preserve cell attachment and membrane integrity. Further, we demonstrate the strength of leveraging endothelialized microchannels to fuel discoveries in cryopreservation of thick tissues and solid organs., Competing Interests: Notes The authors declare the following competing financial interest(s): S.N.T., M.T., S.L.S., K.U., and L.W. are inventors on several provisional patents on the topic of cryopreservation of cells, tissues, and organs, including high subzero preservation. Further, K.U. has a financial interest in Organ Solutions, a company focused on developing organ preservation technology. The interests of all researchers are managed by the MGH and Partners HealthCare in accordance with their conflict of interest policies.