This protocol describes the enrichment of astrocyte-derived extracellular vesicles (ADEVs) from human plasma. It is based on the separation of EVs by polymer precipitation, followed by ACSA-1 based immunocapture of ADEVs. Analysis of ADEVs may offer clues to study changes in inflammatory pathways of living patients, non-invasively by liquid biopsy.
Extracellular vesicles (EVs) are biological nanoparticles secreted by all cells for cellular communication and waste elimination. They participate in a vast range of functions by acting on and transferring their cargos to other cells in physiological and pathological conditions. Given their presence in biofluids, EVs represent an excellent resource for studying disease processes and can be considered a liquid biopsy for biomarker discovery. An attractive aspect of EV analysis is that they can be selected based on markers of their cell of origin, thus reflecting the environment of a specific tissue in their cargo. However, one of the major handicaps related to EV isolation methods is the lack of methodological consensuses and standardized protocols. Astrocytes are glial cells with essential roles in the brain. In neurodegenerative diseases, astrocyte reactivity may lead to altered EV cargo and aberrant cellular communication, facilitating/enhancing disease progression. Thus, analysis of astrocyte EVs may lead to the discovery of biomarkers and potential disease targets. This protocol describes a 2-step method of enrichment of astrocyte-derived EVs (ADEVs) from human plasma. First, EVs are enriched from defibrinated plasma via polymer-based precipitation. This is followed by enrichment of ADEVs through ACSA-1-based immunocapture with magnetic micro-beads, where resuspended EVs are loaded onto a column placed in a magnetic field. Magnetically labeled ACSA-1+ EVs are retained within the column, while other EVs flow through. Once the column is removed from the magnet, ADEVs are eluted and are ready for storage and analysis. To validate the enrichment of astrocyte markers, glial fibrillary acidic protein (GFAP), or other specific astrocytic markers of intracellular origin, can be measured in the eluate and compared with the flow-through. This protocol proposes an easy, time-efficient method to enrich ADEVs from plasma that can be used as a platform to examine astrocyte-relevant markers.
Extracellular vesicles (EVs) are a heterogeneous group of membranous nanoparticles secreted by all types of cells, carrying proteins, lipids, and nucleic acids1. Microvesicles (100-1000 nm), exosomes (30-100 nm), and apoptotic bodies (1000-5000 nm) constitute the main EV types, as distinguished by their site of origin2,3. EVs regulate important physiological processes, such as antigen presentation and immune responses4, receptor recycling, metabolite elimination5, and cellular communication6. The regulation of these processes may occur by direct binding between proteins enriched in the EV cell membrane and targets in recipient cells, and/or through the internalization and release of their cargo in the cytoplasm of the recipient cell7. While EVs perform essential cellular functions, they have gained increasing interest from a pathological perspective in the fields of cancer and neurology. Indeed, several studies have shown EVs can help promote tumor cell migration8,9 or seed toxic protein aggregates in neurodegenerative diseases, such as Alzheimer's disease10,11.
EVs can be selected and enriched from biofluids based on cell surface markers related to their cell of origin, thus reflecting the environment of a specific tissue in their cargo12,13,14,15,16,17,18,19,20. In addition, given their presence in blood, cerebrospinal fluid (CSF), saliva, urine, and breast milk, EVs represent an excellent, non-invasive tool for diagnosis, and can be considered a liquid biopsy for biomarker discovery. This is of special interest in neurology, given the difficulties of studying brain analytes in accessible fluids other than CSF.
Astrocytes have gained rising interest, as they are at the intersection of neuro-vascular communication21. Under physiological conditions, they are responsible for the preservation of the blood-brain barrier, the recycling of neurotransmitters, the supply of nutrients and growth factors to neurons and other glial cells22,23,24 as well as neuro-immune defense, given their metabolic plasticity from pro-inflammatory to anti-inflammatory states and vice versa25,26,27. An important mechanism by which astrocytes accomplish their regulatory functions is by communication through EVs28,29. Reactive astrocytosis is a key hallmark of several neurodegenerative diseases, such as Alzheimer's disease,30 multiple system atrophy (MSA), progressive supranuclear palsy (PSP)31, and amyotrophic lateral sclerosis (ALS)32. Astrocyte reactivity may lead to altered EV cargo, release of inflammatory mediators, and aberrant cellular communication, thus facilitating the spread of pathology and leading to neurodegeneration10,11. Therefore, studying astrocyte derived EVs (ADEVs) and changes in their cargo is an attractive resource to examine neurodegenerative processes in a non-invasive manner.
Currently, several methodologies exist for the isolation of EVs, each with its corresponding advantages and disadvantages33. It is essential to consider which method is more suitable for a specific use, depending on the final application of interest. In the neurology field, and more specifically, in astrocyte studies, polymer-based precipitation followed by immunocapture has been the predominantly used method12,18,19,20,34. However, even when applying the same approach, there remains heterogeneity between studies in the different steps applied for EV isolation. Therefore, there is a need for a clear, step-by-step standardized methodology to facilitate astrocyte EV studies and study reproducibility. Polymer-based precipitation facilitates biomarker screening given that it is a fast, simple procedure that does not require complex equipment, leading to a high yield of EVs without affecting their biological activity35.
The present protocol describes a detailed, simple, two-step method for the enrichment of ADEVs from human plasma. It is based on a polymer-based precipitation of the total EV fraction, followed by an immunocapture of astrocyte EVs. Given the important functions of astrocytes, analysis of ADEVs may shed light for the discovery of biomarkers and brain inflammatory pathways that can be studied in a non-invasive manner.
The research described in this protocol has been conducted with human plasma samples from healthy adult donors of both sexes (age range 65.9-81.3 years, 45.5% females), from the Sant Pau Initiative on Neurodegeneration (SPIN) cohort, Barcelona, Spain36. Participants gave informed consent. The study has been conducted following the international ethical guidelines for medical research contained in the declaration of Helsinki and the Spanish law. The Sant Pau Research Ethics Committee (CEIC) reviewed and approved the protocol for the collection and storage of human plasma samples from the SPIN cohort (#16/2013).
1. Enrichment of astrocyte EVs from human plasma
NOTE: This protocol involves the use of human plasma samples. All details about reagents and laboratory material used in this protocol are included in the Table of Materials. No special equipment is required for this procedure, however, please review the safety considerations of each reagent, as specified individually by each manufacturer.
Figure 1: Schematic representation of the two-step procedure for the enrichment of astrocyte derived EVs. In the first step, EVs are enriched from defibrinated human plasma by polymer-based precipitation and centrifugation steps. After total EV resuspension, astrocyte EVs are then selected by immunocapture with biotinylated anti-GLAST (ACSA-1) antibodies and anti-biotin magnetic microbeads. Abbreviations: ACSA-1 = astrocyte cell surface antigen-1; ADEVs = astrocyte-derived extracellular vesicles; DPBS = Dulbecco's phosphate-buffered saline; EVs = extracellular vesicles; GLAST = glutamate-aspartate transporter; No ADEVs = non-astrocytic extracellular vesicles; No EVs = No extracellular vesicles (EV-depleted plasma); PIC = protease inhibitor cocktail; RT = room temperature. Figure created with BioRender. Please click here to view a larger version of this figure.
2. Protocol validation
3. Data analysis
The isolation of ADEVs from plasma collected from healthy donors was successfully accomplished. A polymer-based precipitation method was employed to obtain the total EV fraction, followed by an immunocapture with magnetic microbeads to obtain ADEVs.
Western blot analysis of the total EV fraction prior to the immuno-capture step indicated the lack of calnexin (cellular contamination marker) and the presence of Alix and the transmembrane protein CD9 in the EV preparations (Figure 2A).
Following immunocapture, the presence and enrichment of vesicular and astrocytic markers were validated in the ADEV fraction. GFAP concentrations were significantly higher in ADEVs compared with the no ADEV fraction (flow-through), showing a six-fold enrichment (p = 0.008). This confirms the predominantly astrocytic origin of the EVs in the ADEV fraction (Figure 2B). The CV of GFAP quantification across ADEV preparations from 10 identical samples was 25%.
In order to confirm the presence of vesicular markers in ADEV preparations, two markers were examined: Alix, a classical cytosolic vesicular marker42, and the transmembrane protein CD8144. Alix levels were higher in the ADEV fraction compared with free plasma (p = 0.001), as well as with EV-depleted plasma, referred to as no EVs (p = 0.0007; Figure 2C). ADEVs also showed the enrichment of CD81 compared with free plasma (p = 0.03; Figure 2D).
To further characterize the ADEV population, the size and count profile was analyzed by NTA and the morphology by cryo-EM. The NTA analysis showed a homogeneous profile of EVs with an average size of 93.7 ± 2.7 nm, consistent with the size of small EVs (microvesicles and exosomes). Moreover, the characterization by cryo-EM further confirmed the presence and expected morphology of EVs (Figure 2E).
Given that lipoproteins may co-precipitate with EVs, the levels of ApoB were analyzed across different fractions. Results show that after the immunocapture step, ApoB levels are minimal in ADEV preparations, being higher in plasma (p = 0.005; Figure 2F).
Figure 2: Characterization of the total EV and ADEV fractions. (A) Representative Western blot showing lack of calnexin and presence of Alix and CD9 in total EV preparations. (B) ADEV fraction showed increased levels of GFAP compared with No ADEVs (ADEV, n = 7; No ADEV, n = 7; p = 0.008). (C) Higher levels of Alix were detected in ADEVs compared with plasma (p = 0.001) and No EVs (p = 0.0007); plasma: n = 6; No EVs: n = 6; ADEVS: n = 16. (D) Higher levels of CD81 were detected in the ADEV fraction compared with plasma (p = 0.003); plasma: n = 5; No EVs: n = 1 (CD81 was undetectable in eight out of nine No EV samples); ADEVS: n = 17. (E) NTA analysis revealed the presence of particles with a median size of 93.7 ± 2.7 nm and whose vesicular characteristic was confirmed by cryo-EM. (F) ApoB was significantly lower in the ADEV fraction compared with plasma (p = 0.005); plasma: n = 4; EVs: n = 4; ADEVS: n = 4. Abbreviations: ADEVs = astrocyte-derived extracellular vesicles; Alix = programmed cell death 6 interacting protein (PDCD6IP); ApoB = Apolipoprotein B; EVs = total extracellular vesicle preparations after polymer-based precipitation; GFAP = glial fibrillary acidic protein; No ADEVs = non-astrocytic extracellular vesicles; No EVs = No extracellular vesicles (EV-depleted plasma). For (B), a two-tailed Mann Whitney test (**p < 0.01) was used and for (C,D,F), Kluskal-Wallis and Dunn's multiple comparisons tests (*p < 0.05, **p < 0.01 and ***p < 0.0001) were used. The graphs illustrate the mean and the error bars show the standard error. Please click here to view a larger version of this figure.
Finally, the applicability of this protocol was examined by using ADEVs to quantify inflammatory markers. All 25 inflammatory cytokines in the panel were detectable in ADEVs obtained from healthy donors (Figure 3).
Figure 3. Quantification of inflammatory cytokines in the ADEV fraction. A panel of 25 inflammatory cytokines in ADEVs from healthy donors revealed that all of them were detectable and differentially expressed (HC, n = 3). The graph illustrates the mean and the error bars show the standard error. Please click here to view a larger version of this figure.
EVs have gained strong interest in biomedical research due to their diagnostic and therapeutic potential. Currently, one of the major handicaps related to EV isolation methods is the lack of methodological consensus and standardized protocols. This study provides a detailed protocol for the enrichment of astrocyte EVs from human plasma via polymer-based precipitation and GLAST immunocapture.
Different methodologies exist for the isolation of EVs from body fluids, each with their own advantages and limitations. A polymer-based approach was chosen because of its simplicity, ease of use, non-time-consuming, no need of a specific instrument, high EV yield, and because it is widely used in EV biomarker studies in neurology12,18,19,20, allowing for comparison of results. However, alternative methods such as size exclusion chromatography could also be tested. A common criticism of polymer-based methods is the co-precipitation of lipoproteins together with the EVs. However, the additional immunocapture step with ACSA-1 antibodies and subsequent washes can overcome this limitation by selectively binding to ADEVs, minimizing the contamination with lipoproteins, as seen with the analysis of ApoB. When using this protocol for quantitative studies, the intra-assay CV should be considered, particularly with small sample sizes. Another limitation of this procedure is that minimal polymer traces could interfere with certain downstream analyses, such as mass spectrometry. Therefore, the combination of this protocol with subsequent analytical methods should be tested for each specific use.
The protocol described in this study has been validated for the enrichment of astrocytic and vesicular markers in the ADEV fraction. The astrocyte EV immunocapture was conducted using GLAST as a target and validated the astrocytic origin of the EVs as shown by the sixfold enrichment of an additional astrocytic marker, GFAP. Though this procedure has been tested and optimized for ADEV enrichment from human plasma of healthy older adults, certain steps in the protocol can be modified and adapted according to the downstream application to be used. For instance, the use of ultrapure water for pellet resuspension is preferred for subsequent immunoprecipitation. In addition, the incubation step of both antibody and magnetic beads (time and concentration) can be modified to concentrate or dilute ADEVs as desired.
Critical steps in the protocol include the mechanical resuspension of the EV pellet before the immunocapture step. Pelleted EVs must be very carefully treated, avoiding foaming during resuspension. Attention should also be paid to the use of 3x concentrated protease inhibitors in the buffers, to inhibit active proteases present in the plasma. Moreover, it is crucial not to push with the plunger onto the column in steps prior to the elution of ADEVs retained in the column.
While there are studies using this approach (polymer-based precipitation + immunoprecipitation) to isolate ADEVs from individuals with Alzheimer's disease and traumatic brain injury12,20,34, ADEVs remain under-investigated with respect to EVs from other cell origins (e.g., neurons) and merit more research.
Certain neurodegenerative diseases, like Alzheimer's disease and Down syndrome, have been associated with endosomal abnormalities and increased EV secretion45,46,47. One way to account for higher EV secretion is to quantitate the levels of an EV marker, such as Alix and CD81, as done in this study, or other tetraspanins, such as CD63 or CD9, to normalize the levels of the analyte of interest. Besides validating the enrichment of astrocytic and EV markers, the utility of the protocol to measure inflammatory cytokines in ADEVs was tested. Using a multiplex platform, detectable and quantitative concentrations of 25 cytokines and chemokines were obtained in the ADEV fraction.
Therefore, this procedure allows the collection of EVs that can be used as a biomarker discovery platform, as well as a non-invasive tool for the study of astrocyte-related molecular mechanisms and pathways. Indeed, the study of ADEVs has huge potential in the fields of neurological diseases, since the ADEV cargo can be used as a resource to explore the astrocyte phenotype and changes along different disease stages.
While this manuscript extensively describes the methodology, it is essential to consider the advantages and limitations of this method and the appropriate applicability. Indeed, there are important standardization efforts in the EV field, such as the extensive research recommendations and validation steps outlined by the MISEV guidelines from the International Society for Extracellular Vesicles48, and the EV-track platform for the reporting of methodological information about EV studies49.
The authors have nothing to disclose.
The authors would like to acknowledge the help of Soraya Torres, Shaimaa El Bounasri El Bennadi, and Oriol Sanchez Lopez for sample handling and preparation. We would also like to acknowledge the collaboration of José Amable Bernabé, from the ICTS "NANBIOSIS", unit 6 (Unit of CIBER in Bioingineering, Biomaterials & Nanomedicine) of the Barcelona Materials Science Institute, Marti de Cabo Jaume from the Electron Microscopy Unit at Universitat Autonoma de Barcelona, Dr. Marta Soler Castany and Lia Ros Blanco from the Flow Cytometry Platform at Sant Pau Biomedical Research Institute (IIB-Sant Pau), as well as Dr. Joan Carles Escolà-Gil from the Pathophysiology of lipid-related diseases group at IIB-Sant Pau for help with the NTA, cryo-EM, Luminex, and ApoB determinations, respectively.
The authors acknowledge financial support from the Jérôme Lejeune Foundation (Project #1941 and #1913 to MFI and MCI), Instituto de Salud Carlos III (PI20/01473 to JF, PI20/01330 to AL, PI18/00435 to DA, and INT19/00016 to DA), the National Institute of Health (1R01AG056850-01A1, R21AG056974, and R01AG061566 to JF), the Alzheimer's Association and Global Brain Health Institute (GBHI_ALZ-18-543740 to MCI), the The Association for Frontotemporal Degeneration (Clinical Research Postdoctoral Fellowship, AFTD 2019–2021) to ODI, and the Societat Catalana de Neurologia (Premi Beca Fundació SCN 2020 to MCI). This work was also supported by the CIBERNED program (Program 1, Alzheimer Disease to AL and SIGNAL study. SS is a recipient of a Postdoctoral grant “Juan de la Cierva-Incorporación” (IJC2019-038962-I) by the Agencia Estatal de Investigación, Ministerio de Ciencia e Innovación (Gobierno de España).
Anti-Alix primary antibody for Western blotting | EMD Millipore | ABC40 | |
µMACS Separator | Miltenyi Biotec | 130-042-602 | The µMACS Separator is used in combination with µ Columns and MACS MicroBeads. |
Anti-calnexin primary antibody for Western blotting | Genetex | GTX109669 | |
Anti-CD9 primary antibody for Western blotting | Cell Signaling | 13174 | |
Blocker BSA (10%) 200 mL | Thermo Fisher | 37525 | |
Bransonic 1510E-MT Ultrasonic bath | Branson | ||
COBAS 6000 autoanalyzer | Roche Diagnostics | Analyzer for immunoturbidimetric determination of ApoB; commercial autoanalyzer | |
cOmplete Protease Inhibitor Cocktail (EDTA-free) | Roche | 11873580001 | |
Digital Micrograph 1.8 | micrograph software | ||
Dulbecco's PBS Mg++, Ca++ free 500 mL | Thermo Fisher | 14190144 | |
EveryBlot Blocking Buffer | BioRad | 12010020 | |
Exoquick (exosome precipitation solution 5 mL) + Thrombin | System Bioscience | EXOQ5TM-1 | ExoQuick 20 mL can also be purchased (EXOQ20A-1) |
Gatan 895 USC 4000 | camera | ||
GeneGnome XRQ chemiluminiscence imaging system | Syngene | ||
Human CD81 antigen (CD81) ELISA kit | Cusabio | CSB-EL004960HU | |
Human Programmed cell death 6-interacting protein (PDCD6IP) ELISA kit | Cusabio | CSB-EL017673HU | |
Immun-Blot PVDF Membrane | BioRad | 1620177 | |
JEOL 2011 transmission electron microscope | JEOL LTD | Equipped with a CCD Gatan 895 USC 4000 camera (Gatan 626, Gatan, Pleasanton, USA) | |
Lavender EDTA BD Vacutainer K2E tubes | Becton dickinson | 367525 | |
Leica EM GP | Leica Microsystem | commercial plunge freezer | |
Low binding microtubes 1,5 mL | Deltalab | 4092.3NS | |
MACS µ Columns with plungers | Miltenyi Biotec | 130-110-905 | µ Columns with plungers are especially designed for isolation of exosomes from body fluids |
MACS Multistand | Miltenyi Biotec | 130-042-303 | |
MAGPIX plate reader | Luminex Corporation | 80-073 | Luminex's xMAP multiplexing unit (Luminex xPonent v 4.3 software) |
MicroBead Kit100 μL Anti-GLAST (ACSA-1)-Biotin, human, mouse, rat – small size; 100 μL Anti-Biotin MicroBeads | Miltenyi Biotec | 130-095- 825 | |
MILLIPLEX MAP Kit Human cytokine/Chemokine/Growth Factor Panel A magnetic bead panel | EMD Millipore | HCYTA-60K-25 | |
M-PER Mammalian Protein Extraction Reagent 25 mL | Thermo Fisher | 78503 | For certain applications like Western blot, more aggressive lysis buffers can be used (e.g. RIPA) |
MultiSkan SkyHigh Microplate Spectrophotometer | Thermofisher | A51119500C | |
NanoSight NS300 | Malvern Panalytical | NTA; 3.4 version | |
Pierce Halt Protease and Phosphatase Inhibitor Cocktail | Thermo Fisher | 78441 | |
Polypropylene syringe (G29) | PeroxFarma | 1mL syringe; 0.33x12mm-G29x1/2" | |
Secondary anti-rabbit antibody | Thermo Fisher | 10794347 | |
Simoa GFAP Discovery Kit | Quanterix | 102336 | |
Simoa, SR-X instrument | Quanterix | SR-X Ultra-Sensitive Biomarker Detection System; commercial biomarker detection technology | |
Specific Protein Test Apolipoprotein B – APOB (100 det) COBAS C/CI | Roche Diagnostics | 3032574122 | |
SuperSignal West Femto | Thermo Fisher | 34095 | Ultra-sensitive enhanced chemiluminescent (ECL) HRP substrate |
Trans-Blot Turbo Transfer System | BioRad | 1704150 |
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