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In This Article

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

Summary

This protocol demonstrates how to isolate human umbilical cord-derived mesenchymal stem cells' small extracellular vesicles (hUC-MSC-sEVs) with a simple lab-scale benchtop setting. The size distribution, protein concentration, sEVs markers, and morphology of isolated hUC-MSC-sEVs are characterized by nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively.

Abstract

The ultracentrifugation-based process is considered the common method for small extracellular vesicles (sEVs) isolation. However, the yield from this isolation method is relatively lower, and these methods are inefficient in separating sEV subtypes. This study demonstrates a simple benchtop filtration method to isolate human umbilical cord-derived MSC small extracellular vesicles (hUC-MSC-sEVs), successfully separated by ultrafiltration from the conditioned medium of hUC-MSCs. The size distribution, protein concentration, exosomal markers (CD9, CD81, TSG101), and morphology of the isolated hUC-MSC-sEVs were characterized with nanoparticle tracking analysis, BCA protein assay, western blot, and transmission electron microscope, respectively. The isolated hUC-MSC-sEVs' size was 30-200 nm, with a particle concentration of 7.75 × 1010 particles/mL and a protein concentration of 80 µg/mL. Positive bands for exosomal markers CD9, CD81, and TSG101 were observed. This study showed that hUC-MSC-sEVs were successfully isolated from hUC-MSCs conditioned medium, and characterization showed that the isolated product fulfilled the criteria mentioned by Minimal Information for Studies of Extracellular Vesicles 2018 (MISEV 2018).

Introduction

According to MISEV 2018, sEVs are non-replicating lipid bilayer particles with no functional nucleus present, with a size of 30-200 nm1. MSC-derived sEVs contain important signaling molecules that play important roles in tissue regeneration, such as microRNA, cytokines, or proteins. They have increasingly become a research "hotspot" in regenerative medicine and cell-free therapy. Many studies have shown that MSC-derived sEVs are as effective as MSCs in treating different conditions, such as immunomodulation2,3,4,5

Protocol

NOTE: See the Table of Materials for details about all materials, equipment, and software used in this protocol.

1. Human umbilical cord mesenchymal stem cells and culture

  1. Culture the hUC-MSCs at a seeding density of 5 × 103/cm2 in DMEM, supplemented with 8% Human Platelet Lysate and 1% Pen-Strep. Incubate the cells at 37 °C in 5% CO2. Replace the cell culture medium every 3 days to ensure proper cell grow.......

Representative Results

Figure 2 shows that hUC-MSC-sEVs have a particle size mode at 53 nm, while other significant peaks of particle size were 96 and 115 nm. The concentration of hUC-MSC-sEVs measured by NTA was 7.75 × 1010 particles/mL. The protein concentration of hUC-MSC-sEVs measured with the BCA assay was approximately 80 µg/mL.

In western blotting analysis, hUC-MSC-sEVs demonstrated positive bands for exosomal markers CD9, CD81, and TSG101, but were negative .......

Discussion

EVs are one of the important subsets of the secretome in MSCs that play a crucial role during normal and pathological processes. However, sEVs, with a size range between 30 to 200 nm, have risen as a potential tool for cell-free therapy in the past decade. Various techniques were developed to isolate sEVs from MSCs. However, differential ultracentrifugation, ultrafiltration, polymer-based precipitation, immunoaffinity capture, and microfluidics-based precipitation possess different advantages and disadvantages

Acknowledgements

The publication of this video was supported by My CytoHealth Sdn. Bhd.

....

Materials

NameCompanyCatalog NumberComments
40% acrylamideNacalai Tesque06121-95Western blot
95% ethanolNacalai Tesque14710-25Disinfectants
Absolute MethanolChemiz45081To activate PVDF membrane (Western blot)
AccutaseSTEMCELL Technologies7920Cell dissociation enzyme
ammonium persulfateChemiz14475catalyse the gel polymerisation (Gel electrophoresis
anti-CD 81 (B-11)Santa Cruz Biotechnologysc-166029Antibody for sEVs marker
anti-TSG 101 (C-2)Santa Cruz Biotechnologysc-7964Antibody for sEVs marker
Bovine serum albuminNacalai Tesque00653-31PVDF membrane blocking
bromophenol blueNacalai Tesque05808-61electrophoretic color marker
Centricon Plus-70 (100 kDa NMWL)MilliporeUFC710008sEVs isolation
ChemiLumi One LNacalai Tesque7880chemiluminescence detection reagent
CryoStor Freezing MediaSigma-AldrichC3124-100MLCell cryopreserve
Dulbecco’s modified Eagle’s mediumNacalai Tesque08458-45Cell culture media
ExcelBand Enhanced 3-color High Range Protein MarkerSMOBIOPM2610Protein molecular weight markers
Extra thick blotting paperATTObuffer reservior (Western blot)
GlycerolMerckG5516Chemicals for western blot
Glycine1st BaseBIO-2085-500gChemicals for buffer (Western Blot)
horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgGκBP-HRP)Santa Cruz Biotechnologysc-516102Secondary antibody (Western Blot)
Human Wharton’s Jelly derived Mesenchymal Stem Cells (MSCs)Centre for Tissue Engineering and Regenerative Medicine, Faculty of Medicine, The National University Malaysia
mouse antibodies anti-CD 9 (C-4)Santa Cruz Biotechnology sc-13118Antibody for sEVs marker
Nanosight NS300 equipped with a CMOS camera, a 20 × objective lens, a blue laser module (488 nm), and NTA software v3.2Malvern Panalytical, UK
paraformaldehydeNacalai Tesque02890-45Sample Fixation during TEM
penicillin–streptomycinNacalai Tesque26253-84Antibiotic for media
phenylmethylsulfonyl fluorideNacalai Tesque27327-81Inhibit proteases in the sEVs samples after adding lysis buffer
phosphate-buffered salineGibco10010023Washing, sample dilution
polyvinylidene fluoride (PVDF) membrane with
0.45 mm pore size
ATTOTo hold protein during protein transfer (Western blot)
protease inhibitor cocktailNacalai Tesque25955-11Inhibit proteases in the sEVs samples after adding lysis buffer
Protein Assay Bicinchoninate KitNacalai Tesque06385-00Protein measurement
sample buffer solution with 2-MENacalai Tesque30566-22Reducing agent for western blot
sodium chlorideNacalai Tesque15266-64Chemicals for western blot
sodium dodecyl sulfateNacalai Tesque31606-62ionic surfactant during gel electrophoresis
Tecnai G2 F20 S-TWIN transmission electron microscopeFEI, USA
tetramethylethylenediamineNacalai Tesque33401-72chemicals to prepare gel
tris-base1st BaseBIO-1400-500gChemicals for buffer (Western Blot)
 Tween 20GeneTexGTX30962Chemicals for western blot
UVP (Ultra Vision Product) CCD imagerCCD imager for western blot signal detection

References

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Small Extracellular VesiclesMesenchymal Stem CellsBenchtop FiltrationCentrifugal FiltrationNanoparticle Tracking AnalysisCell CultureCell Debris RemovalPhosphate buffered SalineConditioned MediumSyringe Filter

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