Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Summary

The proposed protocol includes guidelines on how to avoid contamination with endotoxin during the isolation of extracellular vesicles from cell culture supernatants, and how to properly evaluate them.

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

Introduction

Extracellular vesicles (EVs), according to the MISEV 2018 nomenclature, are a collective term describing various subtypes of cell-secreted membranous vesicles that play crucial roles in numerous physiological and pathological processes^1,2. Moreover, EVs show promise as novel biomarkers for various diseases, as well as therapeutic agents and drug delivery vehicles. However, realizing their full potential is difficult as there are still many technical obstacles related to their acquisition³. One such challenge is the isolation of endotoxin-free EVs, which has been neglected in many cases. One of the most common endotoxins is lipopolysaccharide (LPS), which is a major component of gram-negative bacterial cell walls and can cause an acute inflammatory response, owing to the release of a large number of inflammatory cytokines by various cells^4,5. LPS induces a response by binding to LPS binding protein, followed by interaction with the CD14/TLR4/MD2 complex on myeloid cells. This interaction leads to the activation of MyD88- and TRIF-dependent signaling pathways, which in turn triggers the nuclear factor kappa B (NFkB). Translocation of NFkB to the nucleus initiates the production of cytokines⁶. Monocytes and macrophages are highly sensitive to LPS, and their exposure to LPS results in a release of inflammatory cytokines and chemokines (e.g., IL-6, IL-12, CXCL8, and TNF-α)^7,8. The CD14 structure enables the binding of different LPS species with similar affinity and serves as a co-receptor for other toll-like receptors (TLRs) (TLR1, 2, 3, 4, 6, 7, and 9)⁶. The number of studies being conducted on the effects of EVs on monocytes/macrophages is still increasing^9,10,11. Especially from the perspective of studying the functions of monocytes, their subpopulations, and other immune cells, the presence of endotoxin and even their masked presence in EVs is of great importance¹². The overlooked contamination of EVs with endotoxins may lead to misleading conclusions and hide their true biological activity. In other words, working with monocytic cells requires confidence in the absence of endotoxin contamination¹³. Potential sources of endotoxins can be water, commercially obtained media and sera, media components and additives, laboratory glassware, and plasticware^5,14,15.

Therefore, this study aimed to develop a protocol for the isolation of low endotoxin-containing EVs. The protocol provides simple hints on how to avoid endotoxin contamination during EVs isolation instead of removing endotoxins from EVs. Previously, many protocols have been presented on how to remove endotoxins from, for example, engineered nanoparticles used in nanomedicine; however, none of them are useful for biological structures such as EVs. The effective depyrogenation of nanoparticles can be carried out by ethanol or acetic acid rinsing, heating at 175 °C for 3 h, γ irradiation, or triton X-100 treatment; however, these procedures lead to the destruction of EVs^16,17.

The presented protocol is a pioneering study focused on avoiding endotoxin impurities in EVs, unlike previous studies on the effect of EVs on monocytes⁹. Applying proposed principles to laboratory practice may help to obtain reliable research results, which can be crucial when considering the potential use of EVs as therapeutic agents in the clinic¹².

Protocol

1. Preparation of ultracentrifuge tubes

1. Use sterile, single-use tubes. If this is not possible, reuse the ultracentrifuge tubes after washing them with a detergent using a sterile Pasteur pipette or other single-use applicators. Remember that ultracentrifuge tubes should be dedicated to one type of centrifuged material (cell culture supernatant/serum/plasma) and species (human/mouse/etc.).
2. After a detergent wash, rinse the ultracentrifuge tubes with deionized, LPS-free water 3x.
   NOTE: Do not use low-quality water.
3. Dry the ultracentrifuge tubes, and then fill them up with 70% ethanol. Leave the tubes with 70% ethanol for overnight disinfection. Remove the ethanol and dry the tubes again.
4. Place the tubes and caps in sterilization packaging and close them tightly. Use the plasma or gas (ethylene oxide) sterilization method^18,19. Store the ultracentrifuge tubes enclosed in the sterilization packaging in a dry place, and use them before the expiration date.
   ​NOTE: Perform monthly monitoring of the LPS level in phosphate buffered saline (PBS) and the water used for EVs isolation. Monitoring should include the tubes as well (e.g., by testing the water [wash control] which has been stored in the ultracentrifuge tubes overnight at room temperature [RT]).

2. Preparation of EV-depleted low-endotoxin fetal bovine serum (EE-FBS)

1. Ensure to use ultra-low endotoxin FBS (commercially available; see Table of Materials; <0.1 EU/mL).
2. Place a bottle of ultra-low endotoxin FBS in a water bath and incubate at 56 °C for 30 min. This step is required to inactivate the complement system.
3. Pipette the inactivated FBS to ultracentrifuge tubes and centrifuge it at 100,000 x g for 4 h at 4 °C. Collect the supernatant into sterile 50 mL tubes, ensuring not to exceed 45 mL per tube to avoid cap contamination and wetting the ring of the tube.
4. Store the EE-FBS serum prepared in this manner at -20 °C. Check the concentration of LPS in EE-FBS (step 6). The LPS concentration should be at the same level as before ultracentrifugation.

3. Cell culture

1. For this study, culture SW480 and SW620 cell lines in Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/L glucose, L- glutamine, sodium pyruvate, and gentamicin (50 µL/mL) supplemented with 10% EE-FBS accordingly in 75 cm² culture flasks using aseptic technique. Seed nearly 4.5 x 10⁶ cells and 6.5 x 10⁶ cells per flask for SW480 and SW620, respectively.
2. Collect the supernatants twice a week (~10 mL per flask) when the cells reach full confluency (~13.5 x 10⁶ and 20 x 10⁶ cells per flask for SW480 and SW620, respectively), and split. Ensure that the viability of cells is not less than 99% and that the cells are cultured at 37 °C in a 5% CO₂ atmosphere.
3. Collect supernatants from the cell culture into appropriately labeled 15 mL tubes. Centrifuge the collected supernatants at 500 x g for 5 min at RT to remove cell debris.
4. Collect the supernatants carefully, avoid aspirating debris, place in labeled tubes, and centrifuge again at 3,200 x g for 12 min at 4 °C.
5. Collect supernatants from the second centrifugation step into sterile, labeled 50 mL tubes. Avoid wetting the edges of tubes. Ensure that the volume of supernatant does not exceed 45 mL and that the tubes are kept vertically.
6. Wrap the tube's cap with a sterile transparent film and store supernatants in the vertical position at -80 °C.
   ​NOTE: Perform monthly control of Mycoplasma spp. and LPS contamination in fresh culture supernatants (step 6). If the level of endotoxin in the supernatants exceeds 0.05 EU/mL, verify at which stage contamination might have occurred and restart the process from the beginning. If contamination of the cell culture by Mycoplasma spp. is detected, isolation of the EVs from such supernatants should be discontinued.

4. Isolation of EVs from cell culture supernatants

1. Prepare 0.22 µm syringe filters and syringes of proper volume. Prepare two tubes with culture supernatants (90 mL).
2. Fill the syringe with the supernatant and attach the filter to the needle adapter. Place the filled syringe with the filter over a 50 mL tube. Push on the plunger flange and collect ~90 mL of filtrate.
3. Pipette the filtrate (7 mL) to the ultracentrifuge tubes (ensuring that the same volume is pipetted to each tube for proper balance) and centrifuge at 100,000 x g for 2 h at 4 °C.
   NOTE: The efficiency of EVs pelleting depends on many factors (e.g., rotor type, its k-factor, migration path length, the viscosity of the medium, etc.), which need to be optimized for better recovery of EVs²⁰.
4. Discard the supernatant using a sterile Pasteur pipette. Collect the remaining pellets using long, filtered pipette tips and pool them into two ultracentrifuge tubes. Fill them up to 7 mL with filtered endotoxin-free PBS to rinse the EVs.
5. Centrifuge the PBS-resuspended pellets at 100,000 x g for 2 h at 4 °C. Completely remove the supernatant using a sterile Pasteur pipette, and add 200 µL of filtered, endotoxin-free PBS to both tubes to resuspend the EVs. Gently pipette the EVs pellet to collect all of the EVs.
6. Transfer the EVs suspension into a sterile 1.5 mL test tube (if possible, use a low protein binding tube). Retain 10 µL of the EV suspension for preparing a dilution for nanoparticle tracking analysis (NTA) and for other purposes (e.g., protein concentration measurement).
7. Dilute the EVs with filtered PBS (1:1,000) for measurements by NTA, according to the manufacturer's instructions. Secure the EV tubes by wrapping the cap with a sterile transparent film. Store the EVs at -80 °C.

5. Specific markers detection by western blotting

1. Determine the protein level in the samples (e.g., by Bradford assay), and prepare the samples (20 µg) with loading buffer. Prepare the loading buffer by mixing 5 µL of sample buffer (4x) and 2 µL of sample reducing agent (10x) to a total volume of 20 µL. Incubate the samples at 70 °C for 10 min.
2. Prepare polyacrylamide gels with SDS (10%-14%) and load the 20 µg of EVs to each well.
3. Run the electrophoresis with running buffer (30 g of Tris, 144 g of glycine, 10% SDS per 1 L) for 45 min at 150 V.
4. Perform semi-dry transfer of proteins from the gel onto the polyvinylidene difluoride (PVDF) membrane in a transfer machine with Towbin buffer (1.51 g of Tris, 7.2 g of glycine, 10% methanol per 0.5 L) at 25 V for 1 h.
5. Place the membrane in TBST buffer (1 mL of Tween, 100 mL of Tris-buffered saline (TBS 10x) per 1 L) for blocking with 1% bovine serum albumin (BSA) in TBST buffer, and incubate for 1 h on the rocker at RT.
6. Add diluted (1:1,000) antibodies, anti-CD9¹ (clone#D8O1A) or anti-Alix¹ (clone#3A9), in 1% BSA and incubate with the membrane overnight at 4 °C on the rocker. Remove antibodies and wash membrane 3x with 10 mL of 1x TBST for 10 minutes.
7. Add goat anti-rabbit or goat anti-mouse (dilution: 1:2,000) secondary antibody conjugated with horseradish peroxidase, depending on the primary antibody on the membrane, and incubate for 1 h on the rocker at RT. Remove antibodies and wash membrane 3x with 10 mL of 1x TBST for 10 minutes.
8. Mix the substrate and luminol at a 1:1 ratio to obtain a 1 mL solution. Pour the solution on the membrane. Place the membrane immediately in the measuring chamber of the imaging system, choose the chemiluminescence module, and visualize protein bands on the screen.

6. Measurement of endotoxin level by Limulus Amebocyte Lysate test (LAL)

1. Perform chromogenic LAL measurement of the endotoxin level, according to the manufacturer's recommendation. Briefly, use the method based on the interaction of the endotoxin with the limulus amebocyte lysate (LAL). The detection limit of this method is 0.005 EU/mL.
   NOTE: LAL assay, despite its limitations, currently remains the standard method for detecting and quantifying endotoxin contamination in different kinds of solutions and other products used in science or medicine⁸. The limiting factor of this kit is the stability of the reconstituted amebocyte lysate solution, which is stable for only 1 week at -20 °C if frozen immediately after reconstitution.
2. Use a tenfold dilution of EVs and other samples and a 50-fold dilution of serum. For further experiments, use only low-endotoxin reagents such as EE-FBS, DMEM, PBS, RPMI (<0.005 EU/mL), and LPS-free culture supernatants.

7. Detection of prokaryotic 16S rRNA gene in EV samples

1. Isolate DNA from the EV samples. Try to keep the reagents and the site of isolation aseptic. Measure the DNA concentration and quality (e.g., using a spectrophotometer).
2. Perform polymerase chain reaction (PCR), as described previously²¹. Prepare 1.5% agarose gel with ethidium bromide or SYBR green to visualize the PCR products in ultraviolet light (UV).
3. Run electrophoresis of the sample and weight marker with TRIS-acetate-EDTA buffer at 75 V for 45 min. Visualize DNA bands using the imaging system.

8. Determination of effective LPS concentration for stimulation in human monocyte model

1. Prepare 2 x 10⁶ monocytes/mL of monocyte suspension in RPMI 1640 supplemented with L-glutamine, glucose, and 2% EE-FBS. Add 50 µL of the suspension per well of a 96-well tissue culture plate²².
2. Add a proper volume of LPS or culture medium to obtain the following final concentrations: 0 pg/mL (control), 10 pg/mL, 50 pg/mL, 100 pg/mL, 1 ng/mL, and 100 ng/mL, in 100 µL total volume of cell suspension. Make triplicates of each LPS concentration.
3. Culture the cells for 18 h at 37 °C, 5% CO₂. Collect the supernatant.
4. Spin down the supernatant at 2,000 x g for 5 min. Transfer the supernatants into new tubes. Measure TNF^8,23 and IL-10²³ concentration in collected supernatants with the cytometric beads array (CBA) human cytokine kit, according to the manufacturer's procedure, with a flow cytometer.

Results

A prerequisite or obligatory step for this protocol is the exclusion of possible endotoxin contamination from reagents. All the reagents being used, such as FBS, DMEM, RPMI, PBS, and even ultracentrifuge tubes, must be endotoxin-free (<0.005 EU/mL). Maintaining the regime of no endotoxin contamination is not easy as, for example, the regular/standard serum for cell culture can be its rich source (0.364 EU/mL; see Table 1).

Although this protocol was developed to isolate EV...

Discussion

In the last few years, methods for proper EVs isolation have become increasingly important, enabling their further reliable analyses, for example, in the context of obtaining reliable omics and functional data²⁴. Based on previous research experience, it seems that not only the type of isolation method, but also other conditions during this procedure may be important. The use of EV-depleted FBS is widely recognized as a necessity^25,26; how...

Materials

Name	Company	Catalog Number	Comments
Alix (3A9) Mouse mAb	Cell Signaling Technology	2171
1250ul Filter Universal Pipette Tips, Clear, Polypropylene, Non-Pyrogenic	GoogLab Scientific	GBFT1250-R-NS
BD FACSCanto II Flow Cytometr	BD Biosciences
CBA Human Th1/Th2 Cytokine Kit II	BD Biosciences	551809
CD9 (D8O1A) Rabbit mAb	Cell Signaling Technology	13174
ChemiDoc Imaging System	Bio-Rad Laboratories, Inc.	17001401
DMEM (Dulbecco’s Modified Eagle’s Medium)	Corning	10-013-CV
ELX800NB, Universal Microplate Reader	BIO-TEK INSTRUMENTS, INC
Fetal Bovine Serum	Gibco	16000044
Fetal Bovine Serum South America Ultra Low Endotoxin	Biowest	S1860-500
Gentamicin, 50 mg/mL	PAN – Biotech	P06-13100
Goat anti-Mouse IgG- HRP	Santa Cruz Biotechnology	sc-2004
Goat anti-Rabbit IgG- HRP	Santa Cruz Biotechnology	sc-2005
Immun-Blot PVDF Membrane	Bio-Rad Laboratories, Inc.	1620177
LPS from Salmonella abortus equi S-form (TLRGRADE)	Enzo Life Sciences, Inc.	ALX-581-009-L002
Mini Trans-Blot Electrophoretic Transfer Cell	Bio-Rad Laboratories, Inc.	1703930
Nanoparticle Tracking Analysis	Malvern Instruments Ltd
NuPAGE LDS Sample Buffer (4X)	Invitrogen	NP0007
NuPAGE Sample Reducing Agent (10x)	Invitrogen	NP0004
Parafilm	Sigma Aldrich	P7793	transparent film
Perfect 100-1000 bp DNA Ladder	EURx	E3141-01
PierceTM Chromogenic Endotoxin Quant Kit	Thermo Scientific	A39552
PP Oak Ridge Tube with sealing caps	Thermo Scientific	3929, 03613
RPMI 1640	RPMI-1640 (Gibco)	11875093
SimpliAmp Thermal Cycler	Applied Biosystem	A24811
Sorvall wX+ ULTRA SERIES Centrifuge with T-1270 rotor	Thermo Scientific	75000100
Sub-Cell GT Horizontal Electrophoresis System	Bio-Rad Laboratories, Inc.	1704401
SuperSignal West Pico PLUS Chemiluminescent Substrate	Thermo Scientific	34577
SW480 cell line	American Type Culture Collection(ATCC)
SW480 cell line	American Type Culture Collection (ATCC)
Syringe filter 0.22 um	TPP	99722
Trans-Blot SD Semi-Dry Transfer Cell	Bio-Rad Laboratories, Inc.	1703940	Transfer machine
Transfer pipette, 3.5 mL	SARSTEDT	86.1171.001