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.
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.
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 processes1,2. 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 cells4,5. 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 increasing9,10,11. 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 plasticware5,14,15.
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 EVs16,17.
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.
1. Preparation of ultracentrifuge tubes
2. Preparation of EV-depleted low-endotoxin fetal bovine serum (EE-FBS)
3. Cell culture
4. Isolation of EVs from cell culture supernatants
5. Specific markers detection by western blotting
6. Measurement of endotoxin level by Limulus Amebocyte Lysate test (LAL)
7. Detection of prokaryotic 16S rRNA gene in EV samples
8. Determination of effective LPS concentration for stimulation in human monocyte model
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 EVs with low endotoxin content, it is important to characterize the isolated EVs. In this study, the EVs were isolated from two colorectal cancer cell lines, SW480 and SW620. The expression of EVs markers, CD9 and Alix, was confirmed by western blot (Figure 1A). There was no difference in the mean size of EVs isolated from SW480 and SW620 cells (134 nm and 128.2 nm, respectively; Figure 1B). Additionally, there were no differences in the concentration of isolated EVs between these cell lines (Figure 1C). The exemplary distributions of EV size, as measured by NTA, are presented in Figure 1D. The time of ultracentrifugation was optimized according to the results of Cvjetkovic et al.20. Briefly, the mathematical formula for the conversion of centrifugal run parameters between two fixed angle rotors (70Ti and T-1270) was used. The effectiveness of small EVs depletion by spinning with a T-1270 rotor for 120 min at 100,000 x g was slightly less than that achieved by using a 70Ti rotor at 118,000 x g for 155 min; however, it was still in the range of effective RNA and protein pelleting. The calculation was confirmed by experimental data (Table S1), where the extended time of centrifugation (4 h vs. 2 h) had no meaningful impact on the number of EVs pelleted or still present in supernatants.
The level of LPS that caused a monocyte response was assessed. For this purpose, human monocytes were cultured with successive concentrations of LPS (0 pg/mL, 10 pg/mL, 50 pg/mL, 100 pg/mL, 1 ng/mL, and 100 ng/mL). After overnight culture, the level of IL-10 and TNF was determined in the culture supernatants. In this study, the lowest dose of LPS enabling the secretion of IL-10 and TNF in monocytes was 50 pg/mL, which corresponded to 0.5 EU/mL (Figure 2).
Finally, the last step was related to testing the level of LPS in the EVs isolated as above. The LPS contamination of the EV samples was around 0.5 EU/mL (50 pg/mL; Figure 3) for both cell lines (45.80 ± 20.39 pg/mL and 48.75 ± 7.412 pg/mL for SW480 and SW620, respectively). This means that 1 µL of EVs (around 109 EVs) contains less than 0.05 pg of endotoxin, and when added to 100 µL of monocyte suspension (ratio of 104 EVs per one monocyte) is diluted 100 times (the final concentration of LPS is around 0.5 pg/mL).
Additionally, the purity of EVs was tested by PCR for 16S rRNA gene21, which confirms the lack of bacterial contamination in EVs isolated from SW480 and SW620 cell lines (Supplementary Figure 1).
Figure 1: Characterization of isolated EVs. (A) Western blot analysis of the protein markers of EVs isolated from SW480 and SW620 cell lines. (B) NTA was used to measure the size of EVs isolated from SW480 and SW620 cell lines (nm). (C) NTA was used to measure the concentration of EVs isolated from SW480 and SW620 cell lines (EVs/mL). (D) Exemplary distributions of EVs size, as measured by NTA (blue numbers indicate particle size at a given point on the curve). Data in B and C are presented as mean ± SD (n = 10); t-test for statistical analysis was used. Please click here to view a larger version of this figure.
Figure 2: IL-10 and TNF secretion by monocytes stimulated with different doses of LPS. Secretion of cytokines by monocytes was determined after stimulation with LPS doses as indicated: 10 pg/mL, 50 pg/mL, 100 pg/mL, 1 ng/mL, and 100 ng/mL, in comparison to control monocytes (MO). Data are presented as mean ± SD (n = 4); Mann-Whitney U test was used. TNF and IL-10 concentrations were measured in culture supernatants by the cytometric beads array (CBA) method. Please click here to view a larger version of this figure.
Figure 3: Measurement of LPS level in isolated EV samples by chromogenic LAL test. LPS content in EV samples obtained from SW480 and SW620 cell lines (pg/mL). Data are presented as mean ± SD (n = 6). Please click here to view a larger version of this figure.
S.No. | sample | endotoxin level [EU/mL] | endotoxin level [pg/mL] | ||
1 | DMEM | <0.005 | <0.5 | ||
2 | RPMI1640 | <0.005 | <0.5 | ||
3 | FBS ultra low endotoxin | <0.005 | <0.5 | ||
4 | FBS ultra low endotoxin after 4 h centrifugation (EE-FBS) | <0.005 | <0.5 | ||
5 | regular FBS | 0.368 | 36.8 | ||
6 | filtered PBS | <0.005 | <0.5 | ||
7 | culture supernatant form SW480 cells after 2 h centrifigation | <0.005 | <0.5 | ||
8 | culture supernatant from SW620 cells after 2 h centrifugation | <0.005 | <0.5 | ||
9 | wash control (water stored in ultracentrifuge tubes) | <0.005 | <0.5 |
Table 1: LPS level in various reagents and samples determined by chromogenic LAL test. Measurements are presented as EU/mL and pg/mL. Samples included DMEM, RPMI, EE-FBS, FBS, filtered PBS, and wash control (water from ultracentrifugation tubes).
Supplementary Table 1: Representative examples of EVs concentration (EVs/mL) and size (nm) measurements in pellets and supernatants after 2 h or 4 h centrifugation of culture supernatants from SW480 and SW620 cell lines and PBS. Measurements were performed by NTA. Please click here to download this File.
Supplementary Figure 1: PCR results of pan-prokaryote 16S rRNA gene amplification in the following samples, from the left: molecular weight ladder (MW, 100-1000 bp), NC-negative control, EVs derived from SW480 and SW620, and PC (positive control-bacterial DNA) Please click here to download this File.
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 necessity25,26; however, the monitoring of endotoxin contamination in EVs is often neglected.
Nevertheless, all methods used for the isolation of EVs should have developed standards for rigorous aseptic handling and quality control at every stage of the procedure. This is particularly significant due to EVs' further downstream application. The proposed protocol is the result of previous experience with endotoxin-sensitive cells, such as monocytes and macrophages, which are endotoxin sensitive. CD14, a marker of monocytes, is capable of binding LPS at picomolar concentrations. Obtained results indicate that monocytes, after stimulation with 50 pg/mL (0.5 EU/mL) of LPS secrete TNF and IL-10. Yang et al., however, reported that even 0.1 EU/mL (10 pg/mL) of endotoxin might induce a potent reaction (upregulation of the inflammatory IL1B gene)27. Moreover, Chaiwut et al. demonstrated that the intracellular production of TNF and IL-6 in monocytes might be induced by even lower concentrations of LPS, 2.5 pg/mL or 5 pg/mL, respectively23. Alvarez et al. confirmed that the monocyte activation rate (calculated value) rises after a small concentration of LPS, such as 5 pg/mL28. Therefore, limiting endotoxin contamination in EVs at every possible stage of the isolation protocol is crucial for further experiments conducted with LPS-sensitive cells. Currently, ultracentrifugation is the most widely used method for EV isolation. However, to the best of our knowledge, there are no studies on the LPS contamination of EVs isolated by this method. Hence, the above protocol focuses on obtaining low-endotoxin EVs without external EVs contamination from the culture supernatants.
As mentioned above, endotoxin contamination may have various sources. First, it should be stressed that endotoxin is a tough oppponent, and not all methods of sterilization of glass or plasticware are effective in its removal or degradation. For example, the standard autoclaving procedure cannot eliminate LPS due to endotoxin's high heat stability27. Also, washing, even extensively, cannot remove endotoxin completely. By applying more depyrogenic methods to laboratory practice, such as plasma or ETO (ethylene oxide) sterilization18,19, endotoxin contamination can be limited. Next, permanent monitoring and prevention of possible contamination are crucial. The presented results indicate the importance of using ultra-low endotoxin reagents, such as serum, for culture supplementation. In addition, routine control of endotoxin contamination of all reagents (such as PBS, DMEM, and RPMI) and even plasticware (e.g., tubes) is highly recommended. It is also necessary to pre-check the culture supernatants before EVs isolation to prevent endotoxin accumulation. As presented above, an interesting alternative to the standard measurement of endotoxin levels by LAL assay is PCR amplification of the bacterial 16s rRNA gene. Determination of permissive (which does not induce monocyte stimulation) endotoxin levels of LPS in EVs is crucial. Considering culture conditions (concentration of monocytes and contamination with LPS of the applied EVs, the concentration of EVs; Figure 3), the final concentration of endotoxin that affects monocytes stimulated with EVs is not higher than 0.5 pg/mL (EVs are usually diluted 100-1,000 times). This dose is five times lower than that postulated by Chaiwut et al. as the least effective dose (2.5 pg/mL) able to induce the production of TNF by monocytes23. In the Chaiwut et al. study, cytokine production was determined using flow cytometry and presented as an MFI (mean fluorescent intensity) shift without quantification. Furthermore, monocyte stimulation with very low doses of LPS was performed in medium supplemented with 10% FBS, which may be an additional source of LPS23. This may suggest that the effective dose of LPS used for monocyte stimulation was higher. Thus, considering the presented results and literature data, it seems that the concentration of endotoxin up to 50 pg/mL (0.5 EU/mL) in EVs is permissive, and is not the cause of monocytes' activation. The next step in optimizing this protocol is further reduction of endotoxin contamination, even to the level undetected by LAL or cell-based assays. Recently, the importance of using biological models to detect masked endotoxin contamination, called low endotoxin recovery (LER), is strongly recommended8.
Thus, this protocol is innovative in this respect because it collects all aspects necessary for the isolation of EVs with the lowest possible endotoxin content, which has not yet been addressed in other studies.
As with each method, this protocol has some limitations. First, the proposed protocol reduces endotoxin contamination but does not eliminate it. Second, it is unknown whether the purity achieved is sufficient for other immune cells. Therefore, each protocol should be adapted for further application. Establishing such a protocol will allow obtaining results that will correspond more to the biologically active components of EVs than to their accidental contamination. Finally, the procedure is more expensive than usual because of the need to purchase several endotoxin testing kits and low endotoxin serum. As a promising alternative, Bussolati presented a new, sophisticated method for EVs isolation by tangential flow filtration. This method allows the acquisition of EVs with low endotoxicity (0.1-0.7 EU/mL; 10-70 pg/mL)29. Until now, this novel method has not been commonly used for EVs isolation, and requires specialized equipment in the form of a tangential flow filtration (TFF) system. Recently, Gałuszka et al. proposed a different way to eliminate endotoxin from air pollutants (particulate matter with the size of EVs but without membrane structure) by using polymyxin B30. However, the usefulness of this protocol for cell-derived vesicles needs to be verified through biological models, especially when thinking about in vivo experiments. Polymixin B and sodium deoxycholate (also applicable to endotoxin removal) have been previously described as neuro- and nephrotoxic31. Other possibilities are affinity columns with poly(ε-lysine), which selectively bind endotoxins. This method is quick and efficient, however, dedicated to protein samples/solutions; therefore, applying it to such complicated structures as EVs needs validation32. Moreover, these columns are intended for samples with high initial endotoxin levels, and their effectiveness in removing relatively small amounts of LPS is unknown and requires verification. The final anticipated concentration of LPS after purification with LPS-depleting columns may still be too high for immune cell tests (for example, <5 EU/mL).
In summary, this protocol proposed how to avoid endotoxin contamination by applying several principles to laboratory practice, such as rigorous aseptic technique during all steps of EVs isolation, using ultra-low endotoxin reagents, monitoring endotoxin impurities at all procedure steps, and changing the sterilization method. Moreover, the presented protocol provides hints on how to control endotoxin levels at every step of the isolation procedure. LPS present in EVs affects the function of immune cells, and therefore may cause false results in assays conducted with these cells.
The authors have nothing to disclose.
This work was supported by the National Science Centre, Poland, grant number 2019/33/B/NZ5/00647. We would like to thank Professor Tomasz Gosiewski and Agnieszka Krawczyk from the Department of Molecular Medical Microbiology, Jagiellonian University Medical College for their invaluable help in the detection of bacterial DNA in EVs.
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 |