Uptake of Fluorescent Labeled Small Extracellular Vesicles In Vitro and in Spinal Cord

Summary

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.

Abstract

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.

Introduction

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 molecules^3,4,5,6,7. 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.

Protocol

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

1. Culture RAW 264.7 cells in 75 cm² flasks in DMEM exosome-depleted medium containing 10% exosome-depleted fetal bovine serum (FBS) and 1% penicillin-streptomycin (pen-strep) for 24-48 h.
2. Collect 300 mL of conditioned medium and centrifuge at 300 × g for 10 min at 4 °C.
3. Collect the supernatant and centrifuge at 2,000 × g for 20 min at 4 °C.
4. Transfer the supernatant to centrifuge tubes, centrifuge for 35 min at 12,000 × g at 4 °C.
5. Collect the supernatant and filter through a 0.22 µm syringe filter.
6. Transfer to ultracentrifuge tubes and centrifuge for 80 min at 110,000 × g at 4 °C.
7. Store the supernatant (exosome-depleted medium), resuspend the pellet in 2 mL of 1x phosphate-buffered saline (PBS), and centrifuge for 1 h at 110,000 × g at 4 °C.
8. Resuspend the pellet in 100 µL of PBS for further characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) or in radioimmunoprecipitation assay (RIPA) buffer for western blotting.

2. Characterization of sEVs

1. Nanoparticle tracking analysis (NTA)
   NOTE: The size distribution and particle number/concentration of the purified sEVs from RAW 264.7 cells were measured by NTA.
   1. Dilute the sEVs in filtered PBS to obtain 20-60 vesicles per field of view for optimal tracking.
   2. Introduce the diluted sample into a flow cell using a syringe pump with a constant flow rate.
   3. Take 3-5 videos of 30 s each. Set the shutter speed and gain, and manually focus the camera settings for the maximum number of vesicles to be visible and capable of being tracked and analyzed.
   4. Advance the samples between each recording to perform replicate measurements. Optimize the NTA post-acquisition settings and keep the settings constant between the samples.
   5. Analyze each video using the NTA software to obtain the average size and concentration of the vesicles.
   6. Carry out all NTA measurements with identical system settings for consistency.
2. Western blot
   1. Quantify the total protein amounts in sEVs, cell lysates, and exosome-depleted media using a protein assay kit following the manufacturer's instructions.
   2. For cell lysate preparation, culture the RAW 264.7 cells in 75 cm² flasks until 80-90% confluent. Detach the cells with 0.25% trypsin for 10-15 min, neutralize the trypsin with culture media, and pellet the cells by spinning at 400 × g for 5 min. Resuspend the cells in fresh growth medium.
   3. Count the cells using a hemocytometer and transfer 1 × 10⁶ cells to another tube. Wash the cells with PBS twice using the same centrifugation conditions as above and add 50 µL of lysis buffer (RIPA buffer with protease inhibitor cocktail added) to the cell pellet from the final spin.
   4. Vortex the cells and keep them on ice for 20 min. Subject the mixture to centrifugation at 10,000 × g for 30 min at 4 °C, collect the supernatant (i.e., the lysate) in fresh microcentrifuge tubes, and keep at −80 °C until use.
   5. Concentrate 2 mL of exosome-depleted media to 100 µL using 3 kDa-cutoff centrifugal filters before quantifying the amount of protein. Mix the sEVs with lysis buffer in a 1:1 ratio, vortex for 30 s, and incubate on ice for 15 min to quantify the amount of protein.
   6. Mix equal amounts of protein (2 µg) of the sEVs, RAW 264.7 cell lysate, and exosome-depleted media with reducing sample buffer.
   7. Denature the samples at 95 °C for 5 min, keep them on ice for 5 min, and spin for 2 min at 10,000 × g. Load the samples on a 12% Tris-glycine protein gel and run the gel at 125 V for 45 min.
   8. Transfer the protein onto a polyvinylidene difluoride (PVDF) membrane at 25 V for 2 h.
   9. Following the transfer, block the PVDF membranes with blocking buffer (see the Table of Materials) for 1 h at room temperature.
   10. Incubate the blot with primary antibodies on a shaker overnight at 4 °C.
       NOTE: Primary antibodies used were anti-CD81 (1:1,000), anti-alpha-1,3/1,6-mannosyltransferase (ALG-2)-interacting protein X (Alix) (1:1,000), anti-Calnexin (1:1,000), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:1,000).
   11. Wash the blots 3 x 15 min with 1x Tris-buffered saline, 0.1% Tween 20 (TBST), and incubate at room temperature with goat anti-mouse IgG-horseradish peroxidase (HRP)- or donkey anti-rabbit IgG-HRP-conjugated secondary antibodies (1:10,000) for 1 h on the shaker.
   12. Wash the blots 3 x 15 min with 1x TBST, and detect the proteins using an HRP substrate.
   13. Analyze the blots by enhanced chemiluminescence using a western blot imager.
3. Transmission electron microscopy (TEM)
   1. Fix sEVs by resuspending them in 2% paraformaldehyde (PFA) in 0.1 M phosphate buffer (PB); vortex for 2 x 15 s.
   2. Place a drop of 10 µL of sEV suspension on clean parafilm. Float the carbon-coated formvar grid on the drop with their coated side facing the suspension. Let the membranes absorb for 20 min in a dry environment.
   3. Place the grids (membrane side down) on a drop of PB to wash for 3 x 2 min.
   4. Transfer the grids to 50 µL of 1% glutaraldehyde for 5 min.
   5. Wash the grids with 100 µL of distilled water for 8 x 2 min.
   6. Contrast the sample by placing the grids on a drop of 1% uranyl acetate for 2 min.
   7. Embed the sample with 50 µL of 0.2% uranyl acetate with 2% methylcellulose solution for 10 min on a parafilm-covered ice dish.
   8. Use stainless steel loops to hold the grids and remove excess fluid with filter paper.
   9. Air-dry the grid for 10 min while still on the loop.
   10. Observe under a transmission electron microscope at 80 kV.

3. Labeling of sEVs

1. Dilute 20 µg of sEVs in 1 mL of diluent buffer or the same volume of PBS in 1 mL of diluent buffer for dye control.
2. Dilute 3 µL of PKH67 or PKH26 dye in 1 mL of diluent buffer and mix by pipetting.
3. Add diluted PKH dye to the diluted sEVs and mix by pipetting. Incubate for 5 min in the dark at room temperature. For a dye control, mix the diluted dye with diluted PBS from step 3.1.
4. Add 2 mL of 1% bovine serum albumin (BSA) in PBS to the tube with the dye and the sEV mix and to the dye control tube to absorb excess dye.
5. Centrifuge for 1 h at 110,000 × g at 4 °C. Discard the supernatant, resuspend the pellet in 2 mL of PBS, and centrifuge for 1 h at 110,000 × g at 4 °C. Repeat the wash with PBS and resuspend the labeled sEVs or the dye control in an equal volume of PBS.
6. Quantify the amount of total protein by the Bradford method.

4. Uptake of sEVs by Neuro-2a cells

1. Place 18-mm coverslips in a 12-well plate and plate 10 × 10⁴ Neuro-2a cells in each well in a total of 1 mL of complete DMEM medium containing 10% FBS and 1% pen-strep.
2. Change the medium to DMEM exosome-depleted medium when the cell confluency is 80-90%. Add 1, 5, or 10 µg of labeled sEVs in each well for 1, 4, and 24 h for dose- and time-dependent uptake, or add an equal volume of dye control.

5. Primary astrocytic cultures

1. Anesthetize 4 postnatal pups 4 days after birth by inducing hypothermia.
2. Collect the brains in a 60-mm Petri dish containing ice-cold Hank's Balanced Salt Solution (HBSS) supplemented with 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).
3. Dissect both cortical lobes and remove the meninges. Mince the tissues with a sterilized blade.
4. Transfer the tissues to a 15 mL conical tube containing papain/deoxyribonuclease I dissociation buffer and incubate for 20 min at 37 °C. Swirl every 5 min.
   NOTE: For 4 mouse cortices, 9 mL of 7.5 U/mL papain in HBSS is activated at 37 °C for at least 30 min, filtered through a 0.22 µm syringe filter, and mixed with deoxyribonuclease I to a final concentration of 0.1 mg/mL.
5. Aspirate the supernatant and add 5 mL of complete DMEM to inactivate the enzyme activity. Carefully triturate to dissociate the tissues with a 5 mL glass serological pipette and a flame-polished Pasteur pipette.
6. Pass the cell suspension through a 40 µm cell strainer and centrifuge the cells at 250 × g for 5 min at 4 °C. Aspirate the medium and seed the cells in 10 mL of complete DMEM in a 75 cm² flask. Replace the supernatant medium with 15 mL of fresh DMEM medium 4 h after plating.
7. After 14 days in vitro, transfer the flask to an orbital shaker to detach the microglia and oligodendrocytes at 320 rpm for 6 h.
8. Trypsinize the remaining astrocytes using 5 mL of the cell dissociation enzyme (Table of Materials) for 10 min at 37 °C. Add 5 mL of complete DMEM to inactivate the enzymatic action and pellet the cells at 250 × g for 5 min at 4 °C.
9. Resuspend the cells in complete DMEM. Seed 5 × 10⁴ cells on 12 mm #1.5 coverslips in a 24-well plate.

6. Uptake of sEVs by astrocytes

1. When astrocytes reach 80-90% confluency, change the medium to DMEM exosome-depleted medium.
2. Add 1 µg of unlabeled, labeled sEVs, an equal volume of dye control, or PBS to the cells. Use the cells for staining 1 h and 24 h after sEV treatment.

7. Immunofluorescence

1. Rinse the cells with PBS 3x and fix them with 4% PFA in PB for 10 min at room temperature.
2. Wash the fixed cells 3 x 5 min with PB and permeabilize them using 0.1% Triton X-100 in PB for 10-15 min and wash with PB 3 x 5 min.
3. Block the cells with 5% normal goat serum (NGS) in PB for 1 h at room temperature.
4. Incubate the cells with primary antibodies: microtubule-associated protein 2 (MAP2A, 1:500) for Neuro-2a cells or glial fibrillary acidic protein (GFAP, 1:500) for primary astrocytes in fresh 5% NGS/PB overnight at 4 °C with gentle shaking.
5. Wash 3 x 10 min with PB and add fluorophore-conjugated secondary antibodies (Goat Anti-Mouse IgG1, Alexa Fluor 594; or Goat Anti-Mouse IgG H&L, Alexa Fluor 488) in 5% NGS and incubate for 2 h at room temperature on a rocker.
6. Wash 3 x 10 min with PB and incubate with 1 µg/mL of nuclear stain 4',6-diamidino-2-phenylindole (DAPI) for 10 min at room temperature. Wash the cells again 3x with PB.
7. Mount the coverslips on #1 slides using an antifade mounting medium. Let them dry overnight in the dark, and store the prepared glass slides at 4 °C until imaging on a confocal microscope.

8. In vivo uptake of sEVs

1. Perform intrathecal injection of 5 µg of unlabeled or labeled sEVs resuspended in 10 µL of PBS, or equal volume (10 µL) of dye control (as prepared in section 3) into C57BL/6 mice.
2. After 6 and 18 h post-injection of sEVs, deeply anesthetize mice by intraperitoneal injection of 100 mg/kg body weight of ketamine and 10 mg/kg body weight of xylazine.
3. Perform intracardial perfusion of mice with 0.9% saline to flush out blood, followed by freshly made ice-cold 4% PFA/PB.
4. Dissect the spinal cord and fix in 4% PFA/PB at 4 °C for 24 h. Cryoprotect the tissues in 30% sucrose in PB at 4 °C for 24 h or until the tissues sink. Store the tissues at 4 °C until immunohistochemistry.

9. Immunohistochemistry

1. Embed L4-L5 spinal cord in O.C.T compound. Freeze on dry ice until completely solidified.
2. Section the tissues at 30 µm (cross-sectionally for the spinal cord) using a cryostat, and collect the sections in a 24-well plate containing PB. Wash the sections 3 x 5 min with 0.3% Triton in PB.
3. Block non-specific binding sites with 5% NGS in 0.3% Triton/PB for 2 h at room temperature.
4. Dilute primary antibodies: Anti-MAP2A (1:500), GFAP (1:1,000), Iba1 for microglia (1:2,000) with 5% NGS in 0.3% Triton/PB, and incubate the sections overnight at 4 °C on a shaker.
5. Wash the sections 3 x 5 min with 0.3% Triton/PB, and add secondary antibodies (Donkey Anti-Rabbit IgG Alexa Fluor 488, 1:500, or Goat Anti-Mouse IgG Alexa Fluor 488, 1:500) in 5% NGS/PB for 2 h at room temperature.
6. Wash 3 x 5 min with PB, and incubate the sections in 1 µg/mL of DAPI for 10 min at room temperature. Wash the sections 3 x 5 min with PB.
7. Mount the sections on a clean adhesive slide (Table of Materials) with a fine paintbrush under a light microscope.
8. Wet the coverslip with mounting medium. Cure overnight in the dark at room temperature.
9. Image under a confocal microscope with the respective lasers.

Results

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 vesicl...

Discussion

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 microscopy^{3,5,6,12,24,25}. Due to their relatively long half-life and low cytotoxicity, PKH dyes can be us...

Materials

Name	Company	Catalog Number	Comments
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