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Biologia

培养的人间充质干细胞细胞外囊泡的分离、表征和治疗应用

Published: September 23, 2022
doi:
10.3791/64135
, , , , , , ,
1College of Life Science,Northwest University, 2State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi International Joint Research Center for Oral Diseases, Center for Tissue Engineering, School of Stomatology,Fourth Military Medical University, 3Xi’an Institute of Tissue Engineering and Regenerative Medicine, 4School of Basic Medicine,Fourth Military Medical University, 5Department of Orthodontics, School of Stomatology,Fourth Military Medical University

Summary

本协议描述了用于从培养的人MSC中分离和表征代表性EV（外泌体和微囊泡）的差异离心。本文还解释了这些电动汽车的进一步应用。

Abstract

细胞外囊泡（EVs）是大多数细胞类型释放的异质膜纳米颗粒，它们越来越被认为是机体稳态的生理调节因子和病理的重要指标;与此同时，它们在建立可及和可控制的疾病疗法方面的巨大潜力正在显现。间充质干细胞（MSCs）可以在培养物中释放大量EV，这已显示出有望启动有效的组织再生并促进具有良好可扩展性和可重复性的广泛治疗应用。对收集和应用 MSC-EV 的简单有效的协议的需求不断增长。在这里，提供了一个基于差速离心的详细方案，以从培养的人MSC，外泌体和微囊泡中分离和表征代表性的EV，以用于进一步的应用。该方法在标记、局部移植和全身注射等一系列下游方法中具有适应性。该程序的实施将解决在转化研究中对简单可靠的MSC-EV收集和应用的需求。

Introduction

干细胞是未分化的多能细胞，具有自我更新能力和翻译潜力1。间充质干细胞（MSCs）在实验室中易于分离、培养、扩增和纯化，这在多次传代后仍然是干细胞的特征。近年来，越来越多的证据支持了MSCs在治疗用途中以旁分泌模式起作用的观点2，3。特别是细胞外囊泡（EVs）的分泌在介导MSCs的生物学功能中起着至关重要的作用。作为从大多数细胞类型释放的异质膜纳米颗粒，EV由称为外泌体（Exos），微囊泡（MV）甚至更大的凋亡体的亚类别组成4，5。其中，Exos是研究最广泛的EV，尺寸为40-150nm，具有内体起源，在生理条件下活跃分泌。MVs是通过直接从直径为100-1，000nm的细胞质膜表面脱落而形成的，其特征在于磷脂酰丝氨酸的高表达和供体细胞表面标志物的表达6。EV含有RNA，蛋白质和其他生物活性分子，它们具有与亲本细胞相似的功能，并在细胞通讯，免疫反应和组织损伤修复中发挥重要作用7。MSC-EVs作为再生医学中强大的无细胞治疗工具已被广泛研究8。

MSC衍生电动汽车的分离和纯化是研究和应用领域的常见问题。目前，差分和密度梯度超速离心9、超滤工艺10、免疫磁分离11、分子排阻色谱12和微流控芯片13是电动汽车分离纯化中广泛使用的方法。由于每种方法的优点和缺点，收集的电动汽车的数量、纯度和活性不能同时满足14，15。在本研究中，详细显示了从培养的MSC中分离和表征EV的差异离心方案，该方案支持了有效的治疗用途16，17，18，19，20。该方法对荧光标记、局部移植和全身注射等一系列下游方法的适应性已被进一步举例说明。实施该程序将解决在转化研究中简单可靠地收集和应用MSC-EV的需求。

Protocol

所有动物程序均由第四军医大学动物护理和使用委员会批准，并按照美国国立卫生研究院实验动物护理和使用指南进行。使用8周龄的C57Bl / 6小鼠（对雌性或雄性都没有偏好）。用于本研究的冷冻保存的人脐带衍生MSCs（UCMSCs）是从商业来源获得的（见 材料表）。人体细胞的使用得到了第四军医大学伦理委员会的批准。 1. 人间充质干细胞（hMSCs）培养…

Representative Results

来自培养的人UCMSC的MV和Exos按照实验工作流程分离（图1）。NTA结果表明，来自人类MSC的Exos尺寸范围为40 nm至335 nm，峰尺寸约为100 nm，MV的尺寸范围为50 nm至445 nm，峰尺寸为150 nm（图2）。MSC衍生的Exos的形态学特征表现出典型的杯形（图3）。EV通过PKH26有效地标记，通过标记颗粒的总视图和荧光显微镜观察（图4?…

Discussion

电动汽车正在各种生物活动中发挥重要作用，包括抗原呈递、遗传物质转运、细胞微环境修饰等。此外，它们的广泛应用为诊断和治疗疾病带来了新的方法和机会21.电动汽车治疗应用的实现基于成功的分离和表征。然而，由于缺乏标准化的分离纯化方法和低提取效率，电动汽车的研究受到了阻碍。本文主要介绍了一种易于操作的EVs从培养的人间充质干细胞中提取和表征方案及其?…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了中国国家自然科学基金（32000974，81930025和82170988）和中国博士后科学基金（2019M663986和BX20190380）的资助。感谢国家基础医学实验教学示范中心（AMFU）和空军医科大学军事医学创新中心分析测试中心实验室的帮助。

Materials

10% povidone-iodine (Betadine) Weizhenyuan 10053956954292 Wound disinfection
Calibration solution Particle Metrix 110-0020 Calibrate the NTA instrument
Carprofen Sigma 53716-49-7 Analgesic medicine
Caudal vein imager  KEW Life Science KW-XXY Caudal vein imager
Centrifuge Eppendorf 5418R Centrifugation
Fatal bovine serum Corning 35-081-CV Culture of UCMSCs
Formvar/carbon-coated square mesh PBL Assay Science  24916-25 Transmission electron microscope
Heating pad Zhongke Life Science Z8G5JBMz Post-treatment care of animals
Heparin Solution StemCell 7980 Systemic injection
Isoflurane RWD Life Science R510-22 Animal anesthesia
Minimum Essential Medium Alpha basic (1x) Gibco C12571500BT Culture of UCMSCs
Nanoparticle tracking analyzer Particle Metrix ZetaView PMX120 Nanoparticle tracking analysis
PBS (1x) Meilunbio MA0015 Resuspend EVs
Penicillin/Streptomycin Procell Life Science PB180120 Culture of UCMSCs
Phosphotungstic acid Solarbio 12501-23-4 Transmission electron microscope
Pipette Eppendorf 3120000224
PKH26 Red Fluorescent Cell Linker Kit Sigma-Aldrich MINI26 Labeling EVs
Skin biopsy punch Acuderm 69038-10-50 Skin defects
Software ZetaView Particle Metrix Version 8.05.14 SP7 
Thermostatic equipment Grant v-0001-0005 Water bath
Transmission electron microscope HITACHI HT7800 Transmission electron microscope
UCMSCs Bai'ao  UKK220201 Commercially UCMSCs
Ultracentrifuge Beckman XPN-100 Centrifugation
Ultrapure filtered water purification system Milli-Q IQ 7000 Preparation of ultrapure water
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Tags

Keywords: Extracellular VesiclesMesenchymal Stem CellsIsolationCharacterizationDifferential CentrifugationCell CultureTherapeutic ApplicationPKH26 DyeParticle Tracking Analysis
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Xing, S., Zhang, K., Tang, S., Liu, L., Cao, Y., Zheng, C., Sui, B., Jin, Y. Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells. J. Vis. Exp. (187), e64135, doi:10.3791/64135 (2022).
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