细胞类型和培养条件对人骨髓间充质基质细胞衍生的细胞外囊泡促凝血活性的影响。
The effects of cell type and culture condition on the procoagulant activity of human mesenchymal stromal cell-derived extracellular vesicles.
机构信息
From the Blood and Coagulation Research Task Area, U.S. Army Institute of Surgical Research, (T.C.C., R.M.K., G.C.P., A.P.C., J.A.B.), Fort Sam Houston, San Antonio, Texas; Department of Biomedical Engineering, University of Texas at San Antonio, (T.C.C., C.R.R.) San Antonio, Texas.
出版信息
J Trauma Acute Care Surg. 2019 Jul;87(1S Suppl 1):S74-S82. doi: 10.1097/TA.0000000000002225.
BACKGROUND
Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) have great potential as a cell-free therapy in wound healing applications. Because EV populations are not equivalent, rigorous characterization is needed before clinical use. Although there has been much focus on their RNA composition and regenerative capabilities, relatively less is known regarding the effects of MSC cell type (adipose tissue [Ad-MSCs] or bone marrow [BM-MSCs]) and culture condition (monolayer or spheroid) on MSC-EV performance, including characteristics related to their ability to promote coagulation, which could determine EV safety if administered intravenously.
METHODS
The successful isolation of EVs derived from Ad-MSCs or BM-MSCs cultured in either monolayer or spheroid cultures was confirmed by NanoSight (particle size distribution) and Western blot (surface marker expression). Extracellular vesicle surface expression of procoagulant molecules (tissue factor and phosphatidylserine) was evaluated by flow cytometry. Extracellular vesicle thrombogenicity was tested using calibrated thrombogram, and clotting parameters were assessed using thromboelastography and a flow-based adhesion model simulating blood flow over a collagen-expressing surface.
RESULTS
The MSC cell type and culture condition did not impact EV size distribution. Extracellular vesicles from all groups expressed phosphatidylserine and tissue factor on their surfaces were functionally thrombogenic and tended to increase clotting rates compared to the negative control of serum-free media without EVs. On average, EVs did not form significantly larger or stronger clots than the negative control, regardless of cell source or culture condition. Additionally, EVs interfered with platelet adhesion in an in vitro flow-based assay.
CONCLUSION
Adipose-derived EVs were more thrombogenic and expressed higher amounts of phosphatidylserine. Our findings suggest that, like intact MSCs, source variability among EVs is an important factor when considering EVs for potential therapeutic purposes.
LEVEL OF EVIDENCE
Therapeutic care management, level II.
背景
间充质基质细胞（MSC）衍生的细胞外囊泡（EV）作为一种无细胞治疗方法，在伤口愈合应用中具有巨大的潜力。由于 EV 群体并不等效，因此在临床应用之前需要进行严格的表征。尽管人们已经对其 RNA 组成和再生能力进行了大量的关注，但对于 MSC 细胞类型（脂肪组织 [Ad-MSCs] 或骨髓 [BM-MSCs]）和培养条件（单层或球体）对 MSC-EV 性能的影响，包括与促进凝血能力相关的特征，相对了解较少，如果静脉内给药，这可能会决定 EV 的安全性。
方法
通过纳米粒子跟踪分析（粒径分布）和 Western blot（表面标志物表达）证实了成功分离源自单层或球体培养的 Ad-MSCs 或 BM-MSCs 的 EV。通过流式细胞术评估细胞外囊泡表面促凝分子（组织因子和磷脂酰丝氨酸）的表达。使用校准的血栓图测试细胞外囊泡的血栓生成性，使用血栓弹性图和模拟血流在表达胶原蛋白表面上的流动型粘附模型评估凝血参数。
结果
MSC 细胞类型和培养条件不会影响 EV 的粒径分布。所有组的 EV 均在其表面表达磷脂酰丝氨酸和组织因子，具有功能上的血栓生成性，并与不含 EV 的无血清培养基的阴性对照相比，倾向于增加凝血速率。平均而言，无论细胞来源或培养条件如何，EV 都不会形成比阴性对照明显更大或更强的血栓。此外，EV 在体外流动型测定中干扰血小板粘附。
结论
脂肪来源的 EV 更具血栓生成性，并表达更高量的磷脂酰丝氨酸。我们的研究结果表明，与完整的 MSC 一样，EV 之间的来源变异性是在考虑 EV 用于潜在治疗目的时的一个重要因素。
证据水平
治疗护理管理，二级。
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