GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Strubergasse 21, 5020 Salzburg, Austria; Research Program "Nanovesicular Therapies", Paracelsus Medical University (PMU), Strubergasse 21, 5020 Salzburg, Austria.
GMP Unit, Spinal Cord Injury and Tissue Regeneration Center Salzburg (SCI-TReCS), Paracelsus Medical University (PMU), Strubergasse 21, 5020 Salzburg, Austria; Department for Transfusion Medicine, SALK, Müllner Hauptstrasse 48, 5020 Salzburg, Austria.
Methods. 2020 May 1;177:67-73. doi: 10.1016/j.ymeth.2020.02.006. Epub 2020 Feb 17.
A major concern for the extracellular vesicle (EV) field is the current lack of accurate methods for EV quantification. Total protein measurement fails to reliably quantify EVs from serum-containing conditioned media and classical nanoparticle tracking analysis (NTA) allows quantification and size determination of particles, but fails to discriminate between membrane-bounded EVs, lipids and protein aggregates. However, EVs can be fluorescently labelled with non-specific membrane markers or with antibodies specifically recognizing EV surface marker proteins. Fluorescence-based NTA (F-NTA) is thus emerging as a method for counting and phenotyping of EVs. We have validated a differential NTA/F-NTA method using specific antibodies against surface markers in analogy to flow cytometric analyses.
EVs from umbilical cord mesenchymal stromal cells (UC-MSCs) were isolated by a combined tangential flow filtration and ultracentrifugation protocol. EV preparations from 2 × 10 cells were stained with AlexaFluor 488-conjugated specific antibodies or corresponding isotype controls. Amount and size of particles in normal scattering light mode (N mode) versus fluorescence mode (F mode, laser wavelength 488 nm) was measured using ZetaView Nanoparticle Tracking Analyzer (Particle Metrix). Cryo electron microscopy (EM) was used to verify the presence of membrane bilayer surrounded nanoparticles.
All UC-MSC-EV preparations were found positive for typical EV marker proteins and negative for MHC class I. Novel and improved devices that include more sensitive cameras for detection in the fluorescent mode further increase the detection limit.
Differential NTA/F-NTA facilitates determination of the percentage of EV marker protein-positive nanoparticles within a mixed particulate solution. The set of markers can be extended to other MSC-EV positive and negative surface proteins in order to establish F-NTA-based profiling as a supporting method for the quantification of EVs.
细胞外囊泡（EV）领域的一个主要关注点是目前缺乏准确的 EV 定量方法。总蛋白测量不能可靠地定量含有血清的条件培养基中的 EV，而经典的纳米颗粒跟踪分析（NTA）允许对颗粒进行定量和大小测定，但不能区分膜结合的 EV、脂质和蛋白质聚集体。然而，EV 可以用非特异性膜标记物或特异性识别 EV 表面标记蛋白的抗体进行荧光标记。因此，荧光 NTA（F-NTA）作为一种计数和表型分析 EV 的方法正在出现。我们已经使用针对表面标记物的特异性抗体验证了一种差分 NTA/F-NTA 方法，类似于流式细胞分析。
使用切向流过滤和超速离心联合方案从脐带间充质基质细胞（UC-MSCs）中分离 EV。用 AlexaFluor 488 缀合的特异性抗体或相应的同种型对照对 2×10 个细胞的 EV 制剂进行染色。使用 ZetaView 纳米颗粒跟踪分析仪（Particle Metrix）在正常散射光模式（N 模式）和荧光模式（F 模式，激光波长 488nm）下测量颗粒的数量和大小。使用冷冻电子显微镜（EM）验证存在被膜双层包围的纳米颗粒。
所有 UC-MSC-EV 制剂均被鉴定为典型的 EV 标记蛋白阳性，而 MHC 类 I 阴性。包括用于荧光模式下更敏感检测的新型和改进设备进一步提高了检测极限。
差分 NTA/F-NTA 有助于确定混合颗粒溶液中 EV 标记蛋白阳性纳米颗粒的百分比。可以将标记物集扩展到其他 MSC-EV 阳性和阴性表面蛋白，以建立基于 F-NTA 的分析作为 EV 定量的辅助方法。
