间充质干细胞小细胞外囊泡的分离纯化方法比较

Comparison of the separation and purification methods of small extracellular vesicles in mesenchymal stem cells

目的 探讨在间充质干细胞上清液中分离纯化小细胞外囊泡的优化方法。方法 对比差速离心[differential(ultra)centrifugation,dUC]、聚乙二醇(polyethylene glycol,PEG)沉淀和QIAGEN试剂盒法3种标准细胞外囊泡分离程序,并基于标准程序分别进行超滤浓缩、0.22μm滤膜过滤或0.45μm滤膜过滤改良方案提取小细胞外囊泡,通过比较各分离方法的操作时长和简易程度,并分别用透射电镜、纳米颗粒跟踪分析技术(nanoparticle tracking analysis,NTA)和Western blot评估小细胞外囊泡的形态结构、粒径分布和标志蛋白表达情况,分析各分离方法提取的小细胞外囊泡质量和效率。采用CCK-8实验和Transwell迁移实验评估改良方法提取的小细胞外囊泡对乳腺癌细胞增殖和迁移能力的影响。结果 dUC法、PEG沉淀法和QIAGEN试剂盒法3种分离方法均可分离出小细胞外囊泡,其中QIAGEN试剂盒法操作时长最短,平均为48 min,添加超滤浓缩步骤后延长至93 min(P<0.0001)。PEG沉淀法的操作时间最长,平均为487 min,添加超滤浓缩步骤后延长至547 min(P<0.0001)。dUC法平均操作时间为217 min,添加超滤浓缩步骤后延长至274 min(P<0.0001)。各样本在透射电镜下均观察到典型的“茶托”样结构,粒径分布范围除QIAGEN试剂盒法在200 nm以上,其余均在30~200 nm之间,其中dUC+0.45μm滤膜过滤组一个视野中观察到的小细胞外囊泡典型结构最多且最完整。Western blot检测结果显示各方法提取的样本中均有阳性标志蛋白CD9、CD63、TSG101表达,不表达阴性标志物calnexin,但0.22μm滤膜过滤后,各方法的小细胞外囊泡标志蛋白条带均变浅。NTA结果显示,dUC+0.45μm滤膜过滤的小细胞外囊泡占比最高,达94.86%。不同方法提取的样本的粒径分布图显示,dUC法标准流程和dUC+0.45μm滤膜过滤组的NTA结果显示为单峰,曲线流畅。取dUC+0.45μm滤膜过滤的样本进行乳腺癌细胞表型实验,结果显示,细胞增殖和迁移能力均增强(均P<0.05)。结论 dUC法是一种有效的间充质干细胞小细胞外囊泡分离方法,在进行超速离心前对细胞上清液进行0.45μm滤膜过滤,可以提高小细胞外囊泡的质量。

Objective To explore the optimal method for separating and purifying small extracellular vesicles from the supernatant of mesenchymal stem cells.Methods Three standard extracellular vesicle separation procedures,including differential(ultra)centrifugation(dUC),polyethylene glycol(PEG)precipitation and QIAGEN kit method,were compared;small extracellular vesicles were extracted through ultrafiltration concentration and filtration with a 0.22μm or 0.45μm filter membrane,respectively,based on the standard procedure.The quality of the extracted extracellular vesicles and the efficiency of the separation methods were analyzed by comparing the operation duration and simplicity of the separation methods,and evaluating the morphological structure,particle size distribution and marker protein expression of small extracellular vesicles through transmission electron microscopy,nanoparticle tracking analysis technology(NTA)and Western blot,respectively.The CCK-8 assay and Transwell migration assay were used to evaluate the effects of small extracellular vesicles extracted by the modified methods on the proliferation and migration of breast cancer cells.Results Small extracellular vesicles could be isolated by all these three methods,and the QIAGEN kit method has the shortest operation duration,48 min on average,and up to 93 min after ultrafiltration concentration(P<0.0001).The PEG precipitation method had the longest operation duration,487 min on average,and up to 547 minutes with additional ultrafiltration concentration step(P<0.0001).The average operation duration of the dUC method was 217 minutes;after the addition of the ultrafiltration conentration step,it was up to 274 minutes(P<0.0001).The typical"saucer"structure was observed in all samples under transmission electron microscopy,and the particle sizes of each sample ranged between 30 and 200 nm except for that in the QIAGEN kit method,which was above 200 nm.Among them,the dUC+0.45μm filter membrane group had the largest number and the most complete small exbracellular vesicles structures in one field of view.Western blot results showed that positive marker proteins CD9,CD63 and TSG101 were expressed in the samples extracted by different methods,but the negative marker calnexin was not expressed.However,after 0.22μm filter membrane filtration,the bands of small extracellular vesicle marker proteins became shallow.The NTA results showed that the proportion of small extracellular vesicles filtered by dUC+0.45μm filter membrane was the highest,reaching 94.86%.The particle size distribution maps of samples extracted by different methods showed that the NTA results of dUC standard and the dUC+0.45μm filter membrane groups showed a unimodal and smooth curve.Samples filtered by dUC+0.45μm filter membrane were selected for breast cancer cell phenotype experiment,and the results showed that cell proliferation and migration were enhanced(all P<0.05).Conclusions dUC is an effective method for separating small extracellular vesicles from mesenchymal stem cells.The quality of small extracellular vesicles can be improved by filtering the cell supernatant with 0.45μm filter membrane before ultracentrifugation.