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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

Abstract

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

Introduction

Stem cells are undifferentiated pluripotent cells with self-renewal capability and translational potential1. Mesenchymal stem cells (MSCs) are easily isolated, cultured, expanded, and purified in the laboratory, which remains characteristic of stem cells after multiple passages. In recent years, increasing evidence has supported the view that MSCs act in a paracrine mode in therapeutic use2,3. Especially the secretion of extracellular vesicles (EVs) plays a crucial role in mediating the biological functions of MSCs. As heterogeneous membranous nanoparticles released from most cell types....

Protocol

All animal procedures were approved by the Animal Care and Use Committee of the Fourth Military Medical University and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Eight-week-old C57Bl/6 mice (no preference for either females or males) were used. Cryopreserved human umbilical cord-derived MSCs (UCMSCs), used for the present study, were obtained from a commercial source (see Table of Materials). The use of human cells was approved by the .......

Representative Results

MVs and Exos from cultured human UCMSCs are isolated following the experimental workflow (Figure 1). The NTA results demonstrate that the size of Exos from human MSCs ranges from 40 nm to 335 nm with a peak size of about 100 nm, and the size of MVs ranges from 50 nm to 445 nm with a peak size of 150 nm (Figure 2). Morphological characterization of MSC-derived Exos exhibit a typical cup shape (Figure 3). EVs are efficiently labeled b.......

Discussion

EVs are emerging to play an important role in diverse biological activities, including antigen presentation, genetic material transport, cell microenvironment modification, and others. Furthermore, their wide application brings new approaches and opportunities for diagnosing and treating diseases21. Implementation of therapeutic applications of EVs is based on successful isolation and characterization. However, due to the lack of standardized isolation and purification methods and the low extracti.......

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (32000974, 81930025, and 82170988) and the China Postdoctoral Science Foundation (2019M663986 and BX20190380). We are grateful for the assistance of the National Experimental Teaching Demonstration Center for Basic Medicine (AMFU) and the Analytical and Testing Central Laboratory of Military Medical Innovation Center of Air Force Medical University.

....

Materials

NameCompanyCatalog NumberComments
10% povidone-iodine (Betadine)Weizhenyuan10053956954292Wound disinfection
Calibration solutionParticle Metrix110-0020Calibrate the NTA instrument
CarprofenSigma53716-49-7Analgesic medicine
Caudal vein imager KEW Life ScienceKW-XXYCaudal vein imager
CentrifugeEppendorf5418RCentrifugation
Fatal bovine serumCorning35-081-CVCulture of UCMSCs
Formvar/carbon-coated square meshPBL Assay Science 24916-25Transmission electron microscope
Heating padZhongke Life ScienceZ8G5JBMzPost-treatment care of animals
Heparin SolutionStemCell7980Systemic injection
IsofluraneRWD Life ScienceR510-22Animal anesthesia
Minimum Essential Medium Alpha basic (1x)GibcoC12571500BTCulture of UCMSCs
Nanoparticle tracking analyzerParticle MetrixZetaView PMX120Nanoparticle tracking analysis
PBS (1x)MeilunbioMA0015Resuspend EVs
Penicillin/StreptomycinProcell Life SciencePB180120Culture of UCMSCs
Phosphotungstic acidSolarbio12501-23-4Transmission electron microscope
PipetteEppendorf3120000224
PKH26 Red Fluorescent Cell Linker KitSigma-AldrichMINI26Labeling EVs
Skin biopsy punchAcuderm69038-10-50Skin defects
Software ZetaViewParticle MetrixVersion 8.05.14 SP7 
Thermostatic equipmentGrantv-0001-0005Water bath
Transmission electron microscopeHITACHIHT7800Transmission electron microscope
UCMSCsBai'ao UKK220201Commercially UCMSCs
UltracentrifugeBeckmanXPN-100Centrifugation
Ultrapure filtered water purification systemMilli-QIQ 7000Preparation of ultrapure water

References

  1. Liu, S., et al. The application of MSCs-derived extracellular vesicles in bone disorders: Novel cell-free therapeutic strategy. Frontiers in Cell and Developmental Biology. 8, 619 (2020).
  2. Arthur, A., Zannettino, A., Gronthos, S.

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