We describe a protocol to label macrophage-derived small extracellular vesicles with PKH dyes and observe their uptake in vitro and in the spinal cord after intrathecal delivery.
Small extracellular vesicles (sEVs) are 50-150 nm vesicles secreted by all cells and present in bodily fluids. sEVs transfer biomolecules such as RNA, proteins, and lipids from donor to acceptor cells, making them key signaling mediators between cells. In the central nervous system (CNS), sEVs can mediate intercellular signaling, including neuroimmune interactions. sEV functions can be studied by tracking the uptake of labeled sEVs in recipient cells both in vitro and in vivo. This paper describes the labeling of sEVs from the conditioned media of RAW 264.7 macrophage cells using a PKH membrane dye. It shows the uptake of different concentrations of labeled sEVs at multiple time points by Neuro-2a cells and primary astrocytes in vitro. Also shown is the uptake of sEVs delivered intrathecally in mouse spinal cord neurons, astrocytes, and microglia visualized by confocal microscopy. The representative results demonstrate time-dependent variation in the uptake of sEVs by different cells, which can help confirm successful sEVs delivery into the spinal cord.
Small extracellular vesicles (sEVs) are nanosized, membrane-derived vesicles with a size range of 50-150 nm. They originate from multi-vesicular bodies (MVBs) and are released from cells upon fusion of the MVBs with the plasma membrane. sEVs contain miRNAs, mRNAs, proteins, and bioactive lipids, and these molecules are transferred between cells in the form of cell-to-cell communication. sEVs can be internalized by recipient cells by a variety of endocytic pathways, and this capture of sEVs by recipient cells is mediated by the recognition of surface molecules on both EVs and the target cells1.
sEVs have gained interest due to their capacity to trigger molecular and phenotypic changes in acceptor cells, their utility as a therapeutic agent, and their potential as carriers for cargo molecules or pharmacological agents. Due to their small size, the imaging and tracking of sEVs can be challenging, especially for in vivo studies and clinical settings. Therefore, many methods have been developed to label and image sEVs to assist their biodistribution and tracking in vitro and in vivo2.
The most common technique to study sEV biodistribution and target cell interactions involves labeling them with fluorescent dye molecules3,4,5,6,7. EVs were initially labeled with cell membrane dyes that were commonly used to image cells. These fluorescent dyes generally stain the lipid bilayer or proteins of interest on sEVs. Several lipophilic dyes display a strong fluorescent signal when incorporated into the cytosol, including DiR (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindotricarbocyanine iodide), DiL (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine perchlorate), and DiD (1, 1′-dioctadecyl-3, 3, 3′, 3′-tetramethyl indocarbocyanine 4-chlorobenzenesulfonate salt)8,9,10,11.
Other lipophilic dyes, such as PKH67 and PKH26, have a highly fluorescent polar head group and a long aliphatic hydrocarbon tail that readily intercalates into any lipid structure and leads to long-term dye retention and stable fluorescence12. PKH dyes can also label EVs, which allows the study of EV properties in vivo13. Many other dyes have been used to observe exosomes using fluorescence microscopy and flow cytometry, including lipid-labeling dyes14 and cell-permeable dyes such as carboxyfluorescein diacetate succinimidyl ester (CFDA-SE)15,16 and calcein acetoxymethyl (AM) ester17.
Studies of sEV-mediated crosstalk between different cells in the CNS have provided important insights on the pathogenesis of neuroinflammatory and neurodegenerative diseases18. For example, sEVs from neurons can spread beta-amyloid peptides and phosphorylated tau proteins and aid in the pathogenesis of Alzheimer's disease19. Additionally, EVs derived from erythrocytes contain large amounts of alpha-synuclein and can cross the blood-brain barrier and contribute to Parkinson's pathology20. The ability of sEVs to cross physiological barriers21 and transfer their biomolecules to target cells makes them convenient tools to deliver therapeutic drugs to the CNS22.
Visualizing sEV uptake by myriad CNS cells in the spinal cord will enable both mechanistic studies and the evaluation of the therapeutic benefits of exogenously administered sEVs from various cellular sources. This paper describes the methodology to label sEVs derived from macrophages and image their uptake in vitro and in vivo in the lumbar spinal cord by neurons, microglia, and astrocytes to qualitatively confirm sEV delivery by visualization.
NOTE: All procedures were performed in compliance with the NIH Guide for the Care and Use of Laboratory Animals and approved by the Institutional Animal Care & Use Committee of Drexel University College of Medicine. Timed-pregnant CD-1 mice were used for astrocytic culture, and all dams were received 15 days after impregnation. Ten-twelve weeks old C57BL/6 mice were used for in vivo uptake experiments.
1. Isolation of sEVs from RAW 264.7 macrophage cells
2. Characterization of sEVs
3. Labeling of sEVs
4. Uptake of sEVs by Neuro-2a cells
5. Primary astrocytic cultures
6. Uptake of sEVs by astrocytes
7. Immunofluorescence
8. In vivo uptake of sEVs
9. Immunohistochemistry
After the isolation of sEVs from RAW 264.7 conditioned media via centrifugation, NTA was used to determine the concentration and size distribution of the purified sEVs. The average mean size of RAW 264.7-derived sEVs was 140 nm, and the peak particle size was 121.8 nm, confirming that most detectable particles in the light scattering measurement fell within the size range of exosomes or sEVs at 50-150 nm (Figure 1A). As suggested in the minimal information for studies of extracellular vesicles 2018 (MISEV2018)23, we analyzed a set of proteins that should be present or excluded from distinct EV populations. Western blotting of sEVs, cell lysate, and exo-depleted media demonstrated that sEV-derived protein samples contained the sEV marker proteins Alix, CD81, and GAPDH. The cell lysate fraction was enriched with the endoplasmic reticulum resident protein, calnexin, which was absent in the sEVs. Thus, calnexin served as a negative marker for cellular contamination (Figure 1B).
We next performed dose-response and time-course experiments for sEV uptake in vitro. Neuro-2a cells were incubated with a single 1 µg dose of PKH67-labeled sEVs for 1, 4, and 24 h, following which the uptake of different concentrations of sEVs (1, 5, and 10 µg) was examined at 1 h. The results of the NTA indicated that 1 µg of protein on average was equal to ~1 x 109 particles. In parallel, PBS, unlabeled sEVs, and dye-alone controls were also tested. We observed that uptake of sEVs occurred at 1 h (Figure 2A) and for the 1, 5, and 10 µg sEVs (Figure 2B). Fluorescence could be detected at 4 h for 5 and 10 µg of sEVs (Figure 2C) post incubation. Next, uptake of PKH26-labeled sEVs by primary astrocytes was examined (Figure 3). Maximal fluorescence from sEV uptake in primary cortical astrocytes occurred at 24 h. Unlabeled sEVs did not show fluorescence, demonstrating that sEV autofluorescence does not significantly contribute to false positives (Supplemental Figure S1A).
Next, labeled sEVs were intrathecally injected into mice to assess the delivery and uptake of sEVs by different cells in the spinal cord using immunohistochemistry and confocal microscopy. We stained for MAP2 as a neuronal marker, GFAP as an astrocytic marker, and IBA1 as a microglial marker. Neurons (Figure 4), astrocytes (Figure 5), and microglial cells (Figure 6) all took up PKH26-labeled sEVs, and maximal sEV fluorescence was observed at 6 h post-injection. While the sEVs did not always colocalize with the cellular markers, we did not observe any differential uptake by CNS cells. Intrathecal injection with 5 µg of unlabeled RAW 264.7 sEVs or dye control did not show significant fluorescence (Supplemental Figure S1B). Fluorescent signals were observed in the meninges, both 6 h and 18 h after the injection of sEVs (Supplemental Figure S1C).
Figure 1: Characterization of purified RAW 264.7 sEVs. (A) Size and concentration of sEVs were determined using NanoSight NS300. The particles were tracked and sized based on Brownian motion and diffusion coefficient. The size distribution of sEVs is shown in nm. The concentration of sEVs was expressed as particles/mL. (B) Western blot of proteins derived from purified sEVs, cell lysate, and exosome-depleted media using sEV markers ALIX, GAPDH, and CD81. The endoplasmic reticulum protein marker, calnexin, serves as a control to monitor cellular contamination in sEV preparations. (C) Transmission electron microscopy demonstrated the size and morphology of sEVs. Scale bar = 100 nm. Abbreviations: sEVs = small extracellular vesicles; ALIX = Alpha-1,3/1,6-Mannosyltransferase (ALG-2)-interacting protein X; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; CD81 = cluster of differentiation 81. Please click here to view a larger version of this figure.
Figure 2: Uptake of labeled RAW 264.7 sEVs by Neuro-2a cells. (A) PKH67-labeled sEVs (1 µg) were added to the cultured Neuro-2a cells for 1, 4, or 24 h. sEV uptake was observed at all time points with confocal microscopy. (B) PKH67-labeled sEVs (1, 5, or 10 µg) were added to Neuro-2a cells for 1 h. (C) PKH67-labeled sEVs (5 or 10 µg) were added to Neuro-2a cells for 4 h. sEV uptake was observed in all dosage groups with confocal microscopy. Negative control groups treated with PKH dye alone did not show sEV staining (Supplemental Figure S1). Neuro-2a cells were immunostained with MAP2A (probed with Alexa Fluor 594, shown in red), while cell nuclei were stained with DAPI (shown in blue) and sEVs with PKH67 (shown in green). Scale bar = 50 µm. Abbreviations: sEVs = small extracellular vesicles; MAP2A = microtubule-associated protein 2A; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 3: Uptake of PKH26-labeled RAW 264.7 sEVs by primary mouse cortical astrocytes. One µg of sEVs was labeled with PKH26 dye and added to the primary astrocyte culture medium. Uptake of sEVs was observed at 1 and 24 h post-addition using a confocal laser scanning microscope. Astrocytes were stained with GFAP (probed with Alexa Fluor 488, shown in red), while cell nuclei were counterstained with DAPI (shown in blue), and sEVs were previously stained with PKH26 (shown in green). Scale bar = 20 µm. PKH26 dye alone served as a negative control for sEV staining. Abbreviations: sEVs = small extracellular vesicles; GFAP = glial fibrillary acidic protein; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 4: Uptake of RAW 264.7 sEVsin neurons. PKH26-labeled sEVs were injected intrathecally in mice; 6 and 18 h later, the mice were perfused with 4% PFA, and the spinal cord was isolated and sectioned at 30 µm. Spinal cord sections were immunostained with a cell marker (probed with Alexa Fluor 488, shown in red) and DAPI nuclear counterstain (shown in blue), while sEVs were previously labeled with PKH26 (shown in green). Spinal cord sections were immunostained for MAP2A to visualize the neurons (red). Confocal microscopy shows sEVs in MAP2A-positive neurons at different time points. The negative control, PKH26 dye-alone group, did not show sEV staining. Scale bar = 20 µm. Abbreviations: sEVs = small extracellular vesicles; PFA = paraformaldehyde; MAP2A = microtubule-associated protein 2A; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 5: Uptake of RAW 264.7 sEVs in astrocytes. PKH26-labeled sEVs were injected intrathecally in mice; 6 and 18 h later, the mice were perfused with 4% PFA, and the spinal cord was isolated and sectioned at 30 µm. Spinal cord sections were immunostained with a cell marker (probed with Alexa Fluor 488, shown in red) and DAPI nuclear counterstain (shown in blue), while sEVs were previously labeled with PKH26 (shown in green). Spinal cord sections were immunostained for GFAP to visualize the astrocytes (red). Confocal microscopy shows sEVs in GFAP-positive astrocytes at different time points. The negative control, PKH26 dye-alone group, did not show sEV staining. Scale bar = 20 µm. Abbreviations: sEVs = small extracellular vesicles; PFA = paraformaldehyde; GFAP = glial fibrillary acidic protein; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 6: Uptake of RAW 264.7 sEVs in microglia. PKH26-labeled sEVs were injected intrathecally in mice; 6 and 18 h later, the mice were perfused with 4% PFA, and the spinal cord was isolated and sectioned at 30 µm. Spinal cord sections were immunostained with a cell marker (probed with Alexa Fluor 488, shown in red) and DAPI nuclear counterstain (shown in blue), while sEVs were previously labeled with PKH26 (shown in green). Spinal cord sections were immunostained for IBA1 to visualize the microglia (red). Confocal microscopy shows sEVs in IBA1-positive microglia at different time points. The negative control, PKH26 dye-alone group, did not show sEV staining. Scale bar = 20 µm. Abbreviations: sEVs = small extracellular vesicles; PFA = paraformaldehyde; IBA1 = ionized calcium-binding adaptor molecule 1; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Supplemental Figure S1: Uptake of labeled RAW 264.7 sEVs by primary mouse cortical astrocytes and in spinal cord. (A) Controls for the uptake of PKH26-labeled RAW 264.7 sEVs by primary mouse cortical astrocytes. One µg of unlabeled sEVs resuspended in PBS or an equal volume of PBS was added in parallel to the culture medium of astrocytes. No fluorescence was observed at 1 h for PBS and the unlabeled control using a confocal laser scanning microscope. Astrocytes were stained with GFAP (probed with Alexa Fluor 488, shown in red), while the nuclei were counterstained with DAPI (blue), and unlabeled sEVs were visualized under the same Alexa Fluor 546 channel as PKH26-labeled sEVs. Scale bar = 50 µm. (B) Controls for the uptake of PKH26-labeled RAW 264.7 sEVs by mouse spinal cord in vivo. Five µg of unlabeled sEVs or dye control were injected intrathecally in mice. Again, fluorescent signals were not observed for unlabeled sEVs or dye-alone control using a confocal laser scanning microscope. Astrocytes were stained with GFAP (probed with Alexa Fluor 488, shown in red), while nuclei were counterstained with DAPI (blue), and unlabeled sEVs were visualized under the same Alexa Fluor 546 channel as PKH26-labeled sEVs. Scale bar = 50 µm. (C)Representative images reveal the presence of RAW 264.7 sEVs in mouse spinal meninges 6 h and 18 h after intrathecal delivery. Five µg of sEVs were labeled with PKH26 dye (shown in green), and the nuclei were counterstained with DAPI (shown in blue). Asterisks indicate the anterior spinal artery. Scale bar = 50 µm. Abbreviations: sEVs = small extracellular vesicles; PBS = phosphate-buffered saline; GFAP = glial fibrillary acidic protein; DAPI = 4′,6-diamidino-2-phenylindole. Please click here to download this File.
In this protocol, we showed the labeling of sEVs with PKH dyes and the visualization of their uptake in the spinal cord. PKH lipophilic fluorescent dyes are widely used for labeling cells by flow cytometry and fluorescent microscopy3,5,6,12,24,25. Due to their relatively long half-life and low cytotoxicity, PKH dyes can be used for a wide range of in vivo and in vitro cell-tracking studies26,27. Although excellent membrane retention and biochemical stability are advantageous, the intercalation of fluorescent probes with lipoprotein contaminants purified with sEVs can compromise the interpretation of sEV internalization and functional studies. Thus, the purification and labeling of sEVs are critical steps in the protocol because the persistence of the dyes with contaminants can lead to misinterpretation of the in vivo distribution28. The inclusion of controls is critical to avoid false-positive fluorescence signals due to non-specific labeling of particles and the long half-life of these dyes.
Aggregation and micelle formation of lipophilic dyes may also yield false signals. We addressed the problem of free or unbound dye by including a dye-alone control and visualizing the EV uptake at earlier time points. An important limitation reported for PKH labeling is that numerous PKH26 nanoparticles are formed during PKH26 dye labeling of sEVs. Although not included in this protocol, it is reported that PKH26 nanoparticles can be removed by a sucrose gradient29. Another study evaluated the effect of PKH labeling on the size of sEVs by NTA and reported an increase in size following PKH labeling30. Nevertheless, PKH dyes serve as a pragmatic and valuable tracer to show where the sEVs have traversed. Another limitation of this study is that we did not quantify sEVs as this protocol focuses on the confirmation of cellular uptake after intrathecal delivery. Novel cyanine-based membrane probes have been developed recently for highly sensitive fluorescence imaging of sEVs without altering the size or generating artifacts, such as the formation of PKH nanoparticles31, and will undoubtedly improve future labeling studies.
Although macrophages play important roles in neuroinflammation, they also exert neuroprotective functions by delivering their cargo via exosomes32. Our studies show that labeled macrophage-derived sEVs are taken up by Neuro-2a cells, primary astrocytes, and in the lumbar spinal cord after intrathecal administration. The results indicate that a longer incubation time can lead to lower sEV signal intensity, which could be attributed to the degradation of sEVs or cell division by Neuro-2a cells in culture33,34. Although low-throughput, this protocol for visualizing labeled sEVs in the spinal cord can be used for initial validation studies that confirm sEV uptake before investigating the functional impact of sEVs delivered intrathecally. As we observed generally similar sEV uptake in several CNS cell types, the uptake process appears to be non-selective. If autofluorescence is an issue in imaging, unlabeled sEVs can be used as an additional control to negate sEV autofluorescence during imaging of tissues and cultures. Although the dose and the route of administration of sEVs can influence the pattern of biodistribution11, this protocol is not optimized for the quantitative analysis of sEV uptake. Several different approaches and various imaging strategies are being employed to investigate sEVs, and these are being continually refined and optimized for in vivo tracking of sEVs2.
This protocol is meant to be just one approach to confirm sEV uptake. As with all protocols, cross-validation using multimodal approaches can be beneficial. Specifically, the uptake of sEVs can be confirmed by investigating biomolecular cargo transfer to recipient cells and tissues. If the investigator knows the miRNA composition of delivered sEVs, an alternate approach to confirm sEV transfer would be to check for miRNA changes in the recipient cells or determine the changes in expression levels of target genes for the miRNAs transferred. PBS-treated samples can be used as a control for this approach. Overall, these results support the concept that macrophage-derived sEVs are taken up by CNS cells in vitro and in vivo. This protocol can be used to investigate the role of sEVs in spinal disorders, pain, and inflammation and to determine whether sEVs can be developed as cellular vehicles for the delivery of therapeutic small molecules, RNA, and proteins.
The authors have nothing to disclose.
This study was supported by grants from NIH NINDS R01NS102836 and the Pennsylvania Department of Health Commonwealth Universal Research Enhancement (CURE) awarded to Seena K. Ajit. We thank Dr. Bradley Nash for critical reading of the manuscript.
Amicon Ultra 0.5 mL centrifugal filters | MilliporeSigma | Z677094 | |
Anti-Alix Antibody | Abcam | ab186429 | 1:1000 |
Anti-Calnexin Antibody | Abcam | Ab10286 | 1:1000 |
Anti-CD81 Antibody | Santa Cruz Biotechnology | sc-166029 | 1:1000 |
Anti-GAPDH Monoclonal Antibody (14C10) | Cell Signaling Technology | 2118 | 1:1000 |
Anti-Glial Fibrillary Acidic Protein Antibody | Sigma-Aldrich | MAB360 | 1:500 for IF; 1:1000 for IHC |
Anti-Iba1 Antibody | Wako | 019-19741 | 1:2000 |
Anti-MAP2A Antibody | Sigma-Aldrich | MAB378 | 1:500 |
Bovine Serum Albumin (BSA) | VWR | 0332 | |
Cell Strainer, 40 μm | VWR | 15-1040-1 | |
Centrifuge Tubes | Thermo Scientific | 3118-0050 | 12,000 x g |
Coverslip, 12-mm, #1.5 | Electron Microscopy Sciences | 72230-01 | |
Coverslip, 18-mm, #1.5 | Electron Microscopy Sciences | 72222-01 | |
DAPI | Sigma-Aldrich | D9542-1MG | 1 µg/mL |
DC Protein Assay | Bio-Rad | 500-0116 | |
Deoxyribonuclease I (DNAse I) | MilliporeSigma | D4513-1VL | |
Donkey Anti-Rabbit IgG H&L (HRP) | Abcam | ab16284 | 1:10000 |
Donkey Anti-Rabbit IgG H&L, Alexa Fluor 488 | Invitrogen | A-21206 | 1:500 |
Double Frosted Microscope Slides, #1 | Thermo Scientific | 12-552-5 | |
DPBS without Calcium and Magnesium | Corning | 21-031-CV | |
Dulbecco's Modified Eagle Medium (DMEM) | Corning | 10-013-CV | |
Exosome-Depleted Fetal Bovine Serum | Gibco | A27208-01 | |
Fetal Bovine Serum (FBS) | Corning | 35-011-CV | |
FluorChem M imaging system | ProteinSimple | ||
FV3000 Confocal Microscope | Olympus | ||
Goat Anti-Mouse IgG H&L (HRP) | Abcam | ab6789 | 1:10000 |
Goat Anti-Mouse IgG H&L, Alexa Fluor 488 | Invitrogen | A-11001 | 1:500 |
Goat Anti-Mouse IgG1, Alexa Fluor 594 | Invitrogen | A-21125 | |
Hank's Balanced Salt Solution (HBSS) | VWR | 02-0121 | |
HEPES | Gibco | 15630080 | |
HRP Substrate | Thermo Scientific | 34094 | |
Intercept blocking buffer, TBS | LI-COR Biosciences | 927-60001 | |
Laemmli SDS Sample Buffer | Alfa Aesar | AAJ61337AC | |
Micro Cover Glass, #1 | VWR | 48404-454 | |
Microm HM550 | Thermo Scientific | ||
NanoSight NS300 system | Malvern Panalytical | ||
NanoSight NTA 3.2 software | Malvern Panalytical | ||
Neuro-2a Cell Line | ATCC | CCL-131 | |
Normal Goat Serum | Vector Laboratories | S-1000 | |
O.C.T Compound | Sakura Finetek | 4583 | |
Papain | Worthington Biochemical Corporation | NC9597281 | |
Paraformaldehyde | Electron Microscopy Sciences | 19210 | |
Penicillin-Streptomycin | Gibco | 15140122 | |
PKH26 | Sigma-Aldrich | MINI26-1KT | |
PKH67 | Sigma-Aldrich | MINI67-1KT | |
Protease Inhibitor Cocktail | Thermo Scientific | 1862209 | |
PVDF Transfer Membrane | MDI | SVFX8302XXXX101 | |
RAW 267.4 Cell Line | ATCC | TIB-71 | |
RIPA Buffer | Sigma-Aldrich | R0278 | |
Sodium Chloride | AMRESCO | 0241-2.5KG | |
Superfrost Plus Gold Slides | Thermo Scientific | 15-188-48 | adhesive slides |
T-75 Flasks | Corning | 431464U | |
Tecnai 12 Digital Transmission Electron Microscope | FEI Company | ||
TEM Grids | Electron Microscopy Sciences | FSF300-cu | |
Tris-Glycine Protein Gel, 12% | Invitrogen | XP00120BOX | |
Tris-Glycine SDS Running Buffer | Invitrogen | LC26755 | |
Tris-Glycine Transfer Buffer | Invitrogen | LC3675 | |
TrypLE Express | cell dissociation enzyme | ||
Triton X-100 | Acros Organics | 327371000 | |
Trypsin, 0.25% | Corning | 25-053-CL | |
Tween 20 | |||
Ultracentrifuge Tubes | Beckman | 344058 | 110,000 x g |