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, enhancing osteogenesis⁶, diabetes mellitus^7,8, or vascular regeneration^9,10. As early phase trials progress, three main key issues in relation to the clinical translation of MSCs-EVs have been highlighted: the yield of the EVs, the purity of the EVs (free from cell debris and other biological contaminants such as protein and cytokines), and the integrity of the phospholipid bilayer membrane of the EVs after isolation.

Various methods have been developed to isolate sEVs, exploiting the density, shape, size, and surface protein of the sEVs¹¹. The two most common methods in sEVs isolations are ultracentrifugation-based and ultrafiltration-based techniques.

Ultracentrifugation-based methods are considered gold standard methods in sEVs isolation. Two types of ultracentrifugation techniques that are usually employed are differential ultracentrifugation and density gradient ultracentrifugation. However, ultracentrifugation methods often result in low yield and require expensive equipment for high-speed ultracentrifuge (100,000-200,000 × g)¹¹. Furthermore, ultracentrifugation techniques alone are inefficient in separating EV subtypes (sEVs and large EVs), resulting in an impure sediment layer¹¹. Lastly, density gradient ultracentrifugation could be also time-consuming and require additional precaution steps such as sucrose buffer addition to inhibit the gradient damage during acceleration and deceleration steps¹². Hence, ultracentrifugation usually leads to a relatively low yield and is not capable of discriminating between different populations of EVs¹³, which limits its application for large-scale EV preparation¹¹.

The second method of EV isolation is via ultrafiltration, which is based on size filtration. Ultrafiltration is relatively time- and cost-effective compared to ultracentrifugation, as it does not involve expensive equipment or long processing times¹⁴. Hence, ultrafiltration appears to be a more effective isolation technique than both aforementioned ultracentrifugation methods. The isolated products can be more specific based on pore sizes and higher yield¹⁵. However, the additional force incurred during the filtration process may result in the deformation or eruption of the EVs¹⁶.

The current paper proposed a cost- and time-effective benchtop protocol for isolating MSC-derived sEVs for downstream analysis and therapeutic purposes. The method described in this paper combined a simple filtration method with bench top centrifugation to isolate high-yield and good-quality EVs from hUC-MSCs for downstream analysis, including particle size analysis, biomarker assay, and electron microscopic imaging.

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 growth.
2. Expand the cells to passage 5 in T175 flasks for sEV isolation.
   ​NOTE: Many flasks are needed to harvest a high yield of sEVs (the yield increases with the number of cells).

2. Small extracellular vesicle isolation from hUC - MSCs

1. Replace the culture medium with fresh phenol-red free DMEM supplemented with 1% Pen-Strep [Conditioned medium (CM)] when the culture reaches 70%-80% confluency at passage 5.
   CAUTION: Use basal media only; avoid FBS and human platelet lysate supplement to avoid contamination by external sEVs.
2. After 24 h, centrifuge the CM at 200 × g for 5 min at 4 °C to remove cell debris. Collect and filter the supernatant through a 0.22 µm filter to remove particles larger than 220 nm.
3. Fill the centrifugal filter unit with 30 mL of phosphate-buffered saline (PBS) filtered through a 0.2 µm filter. Centrifuge the centrifugal filter unit at 3,500 × g for 5 min at 4 °C.
4. Fill the filtered CM into the centrifugal filter unit (each unit's maximum volume is 70 mL). Centrifuge the CM at 3,500 × g at 4 °C.
   NOTE: The duration of centrifugation should be monitored from time to time until the solution has reached the surface of the filter.
5. Discard the solution in the filtrate solution cup. Reverse centrifuge the sample filter cup with concentrate collection cup at 1,000 x g at 4 °C for 2 min.
6. Transfer the concentrated CM back to the centrifugal filter unit. Add 30 mL of filtered PBS into the centrifugal filter unit and centrifuge the unit at 3,500 × g at 4 °C.
7. Discard the solution in the filtrate solution cup. Install a concentrate collection cup on the filter unit and reverse-centrifuge at 1,000 × g for 2 min at 4 °C to obtain the purified sEVs.
8. Filter the sEVs through a 0.22 µm syringe filter.
9. Transfer the samples to new tubes and store them at -80 °C for further analysis.

3. Characterization of hUC-MSC-sEVs

1. Nanoparticle tracking analysis
   1. Dilute the isolated hUC-MSC-sEVs in filtered PBS to 20-100 particles/frame.
   2. Introduce 1 mL of the diluted sample into the NTA chamber using 1 mL disposable syringes.
   3. Set the measurement settings accordingly: adjust the camera level to level 14, determine the detection threshold to include as many particles as possible with the restriction that 10-100 red crosses are counted, and the blue cross count is limited to five.
   4. For each measurement, record five 1 min videos and analyze them using the NanoSight Software with a detection threshold of five.
   5. Take measurements in triplicate for each sample.
2. Western blotting analysis
   1. Lyse the hUC-MSC-sEVs with ice-cold radioimmunoprecipitation assay (RIPA) lysis buffer, incubate for 30 min at 4 °C, and centrifuge at 200 × g for 5 min at 4 °C. Collect the supernatant.
   2. Quantify the protein using a Bicinchoninic Acid (BCA) Protein Assay Kit. Assemble the gel electrophoresis set accordingly and perform gel electrophoresis at 90 V until the protein reaches the stacking gel's end. Change the voltage to 200 V to separate the proteins until the end of the resolving gel.
   3. Perform semidry transfer to transfer the proteins from the gel to a polyvinylidene difluoride (PVDF) membrane at 15 V for 1 h.
   4. Block the PVDF membrane with 3% bovine serum albumin (BSA)/Tris-buffered saline-0.1% v/v Tween 20 (TBS-T) for 1 h on a shaker at room temperature. Incubate the PVDF membrane with primary antibodies as follows: mouse anti-CD 9 monoclonal antibody (1:500), mouse anti-CD 81 monoclonal antibody (1:500), mouse anti-GRP 94 monoclonal mouse anti-TSG 101 monoclonal antibody (1:500) at 4 °C overnight with constant shaking.
   5. The next day, wash the PVDF membrane 5 x 5 min with TBS-T and further incubate with a secondary antibody: horseradish peroxidase-conjugated mouse IgG kappa binding protein (m-IgGκ BP-HRP) (1:5,000) for 1 h with agitation at room temperature.
   6. Again, wash the PVDF membrane with TBS-T five times, and visualize the membrane in a charge-coupled (CCD) imager using a chemiluminescence detection reagent. Analyze the expression level of the proteins using ImageJ software. First, open the image file in ImageJ (File | Open…). Enhance the quality of the image by adjusting the brightness and contrast (Image | Adjust | Brightness/Contrast). Adjust the image until the blots are clearly visible, click on the Apply button, and then save the images in TIFF format (File | Save As | TIFF…).
3. Transmission electron microscope
   1. Dilute one part of the hUC-MSC-sEVs sample with four parts of filtered PBS to a total volume of 10 µL and incubate on a carbon-coated copper grid for 15 min.
   2. Remove excess sample using a laboratory wipe and allow it to air-dry for 3 min.
   3. Incubate 10 µL of 1% phosphotungstic acid (PTA) solution to stain the sample for 3 min.
   4. Remove excess 1% PTA solution using a laboratory wipe and allow it to air-dry for 3 min.
   5. Use the sample for transmission electron microscope imaging.
      NOTE: Refer to Figure 1 for the summarized schematic experiment steps.

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 for GRP94 ...

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

List of materials used in this article

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