A Simple Benchtop Filtration Method to Isolate Small Extracellular Vesicles from Human Mesenchymal Stem Cells

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 immunomodulation^2,3,4,5

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.......

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

Materials

Name	Company	Catalog Number	Comments
40% acrylamide	Nacalai Tesque	06121-95	Western blot
95% ethanol	Nacalai Tesque	14710-25	Disinfectants
Absolute Methanol	Chemiz	45081	To activate PVDF membrane (Western blot)
Accutase	STEMCELL Technologies	7920	Cell dissociation enzyme
ammonium persulfate	Chemiz	14475	catalyse the gel polymerisation (Gel electrophoresis
anti-CD 81 (B-11)	Santa Cruz Biotechnology	sc-166029	Antibody for sEVs marker
anti-TSG 101 (C-2)	Santa Cruz Biotechnology	sc-7964	Antibody for sEVs marker
Bovine serum albumin	Nacalai Tesque	00653-31	PVDF membrane blocking
bromophenol blue	Nacalai Tesque	05808-61	electrophoretic color marker
Centricon Plus-70 (100 kDa NMWL)	Millipore	UFC710008	sEVs isolation
ChemiLumi One L	Nacalai Tesque	7880	chemiluminescence detection reagent
CryoStor Freezing Media	Sigma-Aldrich	C3124-100ML	Cell cryopreserve
Dulbecco’s modified Eagle’s medium	Nacalai Tesque	08458-45	Cell culture media
ExcelBand Enhanced 3-color High Range Protein Marker	SMOBIO	PM2610	Protein molecular weight markers
Extra thick blotting paper	ATTO		buffer reservior (Western blot)
Glycerol	Merck	G5516	Chemicals for western blot
Glycine	1st Base	BIO-2085-500g	Chemicals for buffer (Western Blot)
horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgGκBP-HRP)	Santa Cruz Biotechnology	sc-516102	Secondary 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-13118	Antibody for sEVs marker
Nanosight NS300 equipped with a CMOS camera, a 20 × objective lens, a blue laser module (488 nm), and NTA software v3.2	Malvern Panalytical, UK
paraformaldehyde	Nacalai Tesque	02890-45	Sample Fixation during TEM
penicillin–streptomycin	Nacalai Tesque	26253-84	Antibiotic for media
phenylmethylsulfonyl fluoride	Nacalai Tesque	27327-81	Inhibit proteases in the sEVs samples after adding lysis buffer
phosphate-buffered saline	Gibco	10010023	Washing, sample dilution
polyvinylidene fluoride (PVDF) membrane with 0.45 mm pore size	ATTO		To hold protein during protein transfer (Western blot)
protease inhibitor cocktail	Nacalai Tesque	25955-11	Inhibit proteases in the sEVs samples after adding lysis buffer
Protein Assay Bicinchoninate Kit	Nacalai Tesque	06385-00	Protein measurement
sample buffer solution with 2-ME	Nacalai Tesque	30566-22	Reducing agent for western blot
sodium chloride	Nacalai Tesque	15266-64	Chemicals for western blot
sodium dodecyl sulfate	Nacalai Tesque	31606-62	ionic surfactant during gel electrophoresis
Tecnai G2 F20 S-TWIN transmission electron microscope	FEI, USA
tetramethylethylenediamine	Nacalai Tesque	33401-72	chemicals to prepare gel
tris-base	1st Base	BIO-1400-500g	Chemicals for buffer (Western Blot)
Tween 20	GeneTex	GTX30962	Chemicals for western blot
UVP (Ultra Vision Product) CCD imager			CCD imager for western blot signal detection