Isolation of intact extracellular vesicles from cryopreserved samples.
Isolation of intact extracellular vesicles from cryopreserved samples.
复制标题
DOI：
10.1371/journal.pone.0251290
复制
发表时间：
2021
期刊：
影响因子：
3.7
通讯作者：
Stott SL
中科院分区：
文献类型：
--
作者：
Tessier SN;Bookstaver LD;Angpraseuth C;Stannard CJ;Marques B;Ho UK;Muzikansky A;Aldikacti B;Reátegui E;Rabe DC;Toner M;Stott SL
细胞外囊泡（EV）已成为生物标志物发现和诊断的有前途的候选者。在脂质双层的保护下，不同生物流体中EV的分子内容物受到保护，免受周围环境中可能快速降解目标靶标的RNA酶和蛋白酶的影响。尽管如此，将含EV的样品冷冻保存至-80 ° C可能会使脂质双层暴露于物理和生物应激源，这可能导致冷冻损伤并导致EV产量、功能或分子货物的变化。在目前的工作中，我们系统地评估了与成对的新鲜对照相比，在-80 ° C下冷冻保存相对较短的储存时间（长达12天）对血浆和培养基来源的EV颗粒计数和/或RNA产量/质量的影响。平均而言，我们发现储存样品的血浆衍生EV浓度降低至新鲜样品的23%。此外，EV颗粒计数的这种显著降低与RNA产率的相应显著降低相匹配，由此血浆来源的储存样品仅含有来自新鲜样品的总RNA的47-52%，这取决于所使用的提取方法。类似地，培养基来源的EV显示RNA产率的统计学显著降低，其中储存的样品是来自新鲜样品的总RNA的58%。相比之下，我们没有获得明确的证据，RNA质量下降，通过分析RNA的痕迹。这些结果表明，储存长达12天的样品确实可以产生高质量的RNA;然而，我们注意到，当直接比较新鲜与冷冻保存的样品时，没有冷冻保护剂，总RNA有显着损失。最后，我们证明了添加常用的冷冻保护剂DMSO，以及更好地控制冷却/升温速率，可以拯救EV免受破坏性冰形成并提高RNA产量。
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.
登录
查看更多内容
影响因子：
3
作者：
Massie, Isobel;Selden, Clare;Morris, G. John
通讯作者：
Morris, G. John
影响因子：
3.7
作者：
Enderle D;Spiel A;Coticchia CM;Berghoff E;Mueller R;Schlumpberger M;Sprenger-Haussels M;Shaffer JM;Lader E;Skog J;Noerholm M
通讯作者：
Noerholm M
影响因子：
3.4
作者：
KARLSSON, JOM;CRAVALHO, EG;TONER, M
通讯作者：
TONER, M
影响因子：
9.3
作者：
El-Hefnawy, T;Raja, S;Godfrey, TE
通讯作者：
Godfrey, TE
影响因子：
5.8
作者：
Sokolova, Viktoriya;Ludwig, Anna-Kristin;Glebel, Bernd
通讯作者：
Glebel, Bernd