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Isolation and Characterization of Exosomes from Cell Culture Supernatants and Biological Fluids

Clotilde Théry1,  Sebastian Amigorena1,  Graça Raposo1,  Aled Clayton2

1Institut Curie, Paris, France, France
2Cardiff University, Whitchurch, Cardiff, United Kingdom


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
UNIT 3.22
DOI: 
10.1002/0471143030.cb0322s30
Online Posting Date: 
April, 2006
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Abstract

Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in various experimental systems suggest their involvement in multiple biological processes. Because both cell-culture supernatants and biological fluids contain different types of lipid membranes, it is critical to perform high-quality exosome purification. This unit describes different approaches for exosome purification from various sources, and discusses methods to evaluate the purity and homogeneity of the purified exosome preparations.

Keywords: Exosomes; multivesicular bodies; purification; characterization; exosome markers; immunoblot; sucrose gradient; immunoisolation; electron microscopy; immunogold labeling

     
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Purification of Exosomes by Differential Ultracentrifugation
  • Alternate Protocol: Elimination of Large Cell Debris and Membranes by Filtration
  • Support Protocol 1: Collecting Culture Supernatants for Exosome Purification
  • Support Protocol 2: Preparing Exosome-Production Medium
  • Basic Protocol 2: Purifying Exosomes from Viscous Fluids
  • Support Protocol 3: Preparation of Exosomes on a 30% Sucrose Cushion
  • Basic Protocol 3: Purification of Exosomes by Immunoisolation
  • Support Protocol 4: Electron Microscope Analysis of Whole-Mounted Exosomes
  • Support Protocol 5: Immunogold Labeling of Whole-Mount Exosomes
  • Support Protocol 6: Preparation of Formvar-Carbon Coated Grids
  • Support Protocol 7: Determination of the Density of an Exosome Preparation on a Continuous Sucrose Gradient
  • Support Protocol 8: Immunoblot Analysis of Exosomes
  • Support Protocol 9: Measuring the Protein Content of Exosomes Using the Bradford Assay
  • Support Protocol 10: Analysis of Exosomes by FACS of Labeled Exosomes Bound to Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
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Materials

Basic Protocol 1: Purification of Exosomes by Differential Ultracentrifugation

 Materials
  • Conditioned medium (Support Protocol 1, step ), cleared
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Tris-buffered saline (TBS; appendix 2A), optional
  • Refrigerated centrifuge
  • 50-ml polypropylene centrifuge tubes
  • Ultracentrifuge and fixed-angle or swinging-bucket rotor (see Table 3.22.1)
  • Polyallomer tubes or polycarbonate bottles, appropriate for the ultracentrifuge rotor (see Table 3.22.1)
  • Micropipettor (e.g., Pipetman)
  • Tabletop ultracentrifuge (e.g., Beckman TL-100)
  • –80°C freezer
     
    Table 3.22.1 Ultracentrifuge and Rotor Information for Exosome Purification
    Rotor (Beckman)Tubes (Beckman)Max vol/tube (ml)Max vol/rotor (ml)rpm for 10,000 × grpm for 12,000 × grpm for 100,000 × grpm for 110,000 × g
    SW 41 or 40 (swinging bucket)Polyallomer12727,5008,20024,00025,000
    SW 28 or 32 (swinging bucket)Polyallomer301807,5008,20024,00025,000
    70 TiPolycarbonate bottle2218010,00011,00031,00032,000
    45 TiPolycarbonate bottle684009,00010,00030,00031,000
    TLA-100.3Thick-walled31813,00015,00043,00045,000
    TLA-110polycarbonate540

NOTE: All centrifugations should be performed at 4°C.

NOTE: It is only necessary to use sterile equipment if the final use of exosomes is going to require sterility (e.g., functional in vivo or in vitro assay). If only biochemical analyses will be performed (e.g., immunoblot, proteomics, FACS analysis), very clean, but not necessarily sterile, tubes are required.

NOTE: If sterility is required, sterile centrifuge and ultracentrifuge tubes must be used, and all steps up to the time when CM-containing tubes or tube holders are closed must be performed in a tissue culture hood. To sterilize ultracentrifuge tubes, wash the clean tubes and their lids briefly in 70% ethanol, rinse twice in sterile PBS, pour PBS off, and drain the remaining drops of PBS with a pipet before use. The rotor lid (45Ti rotor) or the lid for each tube holder (SW41, SW28 rotors) must also be cleaned with 70% ethanol before closing the rotors.



Alternate Protocol: Elimination of Large Cell Debris and Membranes by Filtration

 Materials (also see Basic Protocol 1)
  • 0.22-µm filter sterilization device (e.g., Steritop; Millipore)
  • 100-ml to 1-liter glass bottle, sterile

Support Protocol 1: Collecting Culture Supernatants for Exosome Purification

 Materials
  • Cells in culture (unit 1.1)
  • Exosome-production medium (Support Protocol 2)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Cell lysis buffer (see recipe)
  • Refrigerated centrifuge
  • 50-ml polypropylene centrifuge tubes
  • 500-ml or 1-liter glass bottle, sterile
  • 1.5-ml microcentrifuge tubes
  • Additional materials for growing and counting cells in culture (unit 1.1) and clearing conditioned medium by filtration (Alternate Protocol; optional)

Support Protocol 2: Preparing Exosome-Production Medium

 Materials
  • Culture medium complete with required nutrients (e.g., antibiotics, l-glutamine, HEPES, 2-mercaptoethanol, FBS
  • Ultracentrifuge and fixed-angle or swinging-bucket rotor (see Table 3.22.1)
  • Polyallomer tubes or polycarbonate bottles, appropriate for the ultracentrifuge rotor (see Table 3.22.1)
  • 0.22-µm filter-sterilization device (e.g., Steritop; Millipore)
  • 100-ml to 1-liter glass bottle, sterile

Basic Protocol 2: Purifying Exosomes from Viscous Fluids

 Materials
  • Fluid (e.g., plasma: separate from blood cells by Ficoll centrifugation; lymph, serum, urine, bronchiolar lavage, or tumor ascites)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Refrigerated centrifuge
  • 50-ml polypropylene centrifuge tubes
  • 0.22-µm filter device (e.g., Steritop, Millipore)
  • Ultracentrifuge and fixed-angle or swinging-bucket rotor (see Table 3.22.1)
  • Polyallomer tubes or polycarbonate bottles, appropriate for the ultracentrifuge rotor (see Table 3.22.1)

NOTE: All centrifugations should be performed at 4°C.

Support Protocol 3: Preparation of Exosomes on a 30% Sucrose Cushion

 Materials
  • Partially purified exosome pellet (Basic Protocol 1, step or Basic Protocol 2, step )
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Tris/sucrose/D2O solution (see recipe)
  • Ultracentrifuge with SW 28 and 45 Ti rotors
  • Polyallomer tubes appropriate for the SW 28 rotor (Table 3.22.1)
  • Thick-walled polycarbonate tubes appropriate for the 45 Ti rotor (Table 3.22.1)
  • 5-ml syringe
  • 18-G needle

NOTE: All centrifugations should be performed at 4°C.

Basic Protocol 3: Purification of Exosomes by Immunoisolation

 Materials
  • Conditioned medium from cultured cells (Support Protocol 1)
  • Phosphate-buffered saline (PBS; appendix 2A) containing 3 mg/ml bovine serum albumin (BSA), filter sterilized and stored up to 1 month at 4°C
  • 50-ml centrifuge tubes, sterile
  • Refrigerated centrifuge
  • 4.5 µM paramagnetic Dynabeads M-450 (Dynal), ready-coated with antibodies (e.g., anti-human MHC II)
  • Magnet (Dynal)
  • Test tube rolling machine holding 50-ml tubes

Support Protocol 4: Electron Microscope Analysis of Whole-Mounted Exosomes

 Materials
  • 100,000 × g exosome pellet or frozen concentrated exosome preparations (Basic Protocol 1 or Support Protocol 3)
  • 2% or 4% (w/v) paraformaldehyde (PFA; see recipe)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 1% glutaraldehyde (see recipe)
  • Uranyl-oxalate, pH 7 (see recipe)
  • Methyl cellulose-UA, pH 4: 9 parts 2% methyl cellulose (see recipe) and 1 part 4% uranyl acetate (see recipe) mixed just before use
  • Formvar-carbon coated EM grids (Support Protocol 6; also see Video 11 for unit 4.7 at http://www.currentprotocols.com)
  • Parafilm
  • Forceps (Dumont no. 5), clean
  • Glass dish
  • Stainless steel loops (homemade; see Video 1 for unit 4.7 at http://www.currentprotocols.com), slightly larger than grids
  • Whatman no. 1 filter paper
  • Grid storage boxes (e.g., PELCO; http://tedpella.com)
  • Transmission electron microscope (TEM)

Support Protocol 5: Immunogold Labeling of Whole-Mount Exosomes

 Materials
  • 100,000 × g exosome pellet (Basic Protocol 1)
  • 2% or 4% (w/v) paraformaldehyde (PFA; see recipe)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • PBS/50 mM glycine or PBS/50 mM NH4Cl
  • Blocking buffer: PBS/5% (w/v) BSA, PBS/10% (v/v) fetal calf serum (FCS), or PBS/1% (w/v) cold-water fish skin gelatin (CFG; Sigma Aldrich)
  • Primary antibody
  • Antibody diluent: PBS/1% (w/v) BSA, PBS/5% (v/v) FCS, or PBS/1% (w/v) cold-water fish skin gelatin
  • Washing buffer: PBS/0.1% (w/v) BSA, PBS/0.1% (v/v) FCS, or PBS/0.1% (w/v) CFG
  • Secondary (bridging) antibody (optional; e.g., Dakopatt)
  • PBS/0.5% (w/v) BSA
  • Protein A–gold conjugates (Cell Microscopy Center, Utrecht, The Netherlands)
  • 1% glutaraldehyde (see recipe)
  • Formvar-carbon coated EM grids (Support Protocol 6 or Pelco International, http://www.pelcoint.com)
  • Parafilm
  • Clean forceps (Dumont no. 5)
  • Additional reagents and equipment for contrasting, embedding, and electron microscopy (Support Protocol 4)

Support Protocol 6: Preparation of Formvar-Carbon Coated Grids

 Materials
  • EM grids, 200 mesh/copper-palladium, hexagonal specimen (e.g., PELCO; http://tedpella.com)
  • Warming plate or 37°C drying oven
  • 100-ml volumetric flask: rinse with chloroform before use
  • Funnel-shaped glass column with a stopcock (e.g., PELCO; http://tedpella.com; Electron Microscopy Sciences): rinse with chloroform before use
  • Glass microscope slides
  • Lens tissue
  • Deep glass dish: rinse with chloroform before use
  • Clean forceps
  • Razor blades
  • Adhesive label (e.g., address label) or Parafilm
  • Petri dishes with tops
  • Filter paper
  • Carbon vacuum evaporator (Bal-tec, http://www.bal-tec.com)

Support Protocol 7: Determination of the Density of an Exosome Preparation on a Continuous Sucrose Gradient

 Materials
  • Exosome preparation (Basic Protocol 1, step or Basic Protocol 2, step )
  • HEPES/sucrose stock solution (see recipe)
  • HEPES stock solution (see recipe)
  • 4× SDS sample buffer (appendix 2A)
  • 30-ml gradient maker
  • Ultracentrifuge with SW 41 rotor
  • 3-ml ultraclear tubes
  • Tabletop ultracentrifuge with TLA-100.3 rotor
  • 3-ml thick-walled polycarbonate ultracentrifuge tubes
  • Refractometer
  • Additional reagents and equipment for SDS-PAGE (unit 6.1), protein staining (unit 6.6), and immunoblotting (unit 6.2)

Support Protocol 8: Immunoblot Analysis of Exosomes

 Materials
  • Exosomes (pellet from Basic Protocol 1, step ; Basic Protocol 2, step ; or Basic Protocol 3, step )
  • Whole cell lysates: prepared from same cell source as exosomes (Support Protocol 1, step )
  • Phosphate-buffered saline (PBS; appendix 2A)
  • 4× SDS sample buffer, reducing or nonreducing (i.e., with or without DTT or 2-mercaptoethanol; appendix 2A)
  • Additional reagents and equipment for quantifying protein (Support Protocol 9), SDS-PAGE (unit 6.1); and western blots (immunoblotting; unit 6.2)

Support Protocol 9: Measuring the Protein Content of Exosomes Using the Bradford Assay

 Materials
  • BSA standards (see recipe)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • Cell lysates (Support Protocol 1, step )
  • Exosome preparations (Basic Protocol 1, step or Basic Protocol 2, step )
  • Bradford concentrate solution (Bio-Rad)
  • 0.5-ml microcentrifuge tubes
  • Flat-bottom 96-well plates
  • Microplate reader with 590 nm filter

Support Protocol 10: Analysis of Exosomes by FACS of Labeled Exosomes Bound to Beads

 Materials
  • Purified exosomes (Basic Protocol 1, step ; or Basic Protocol 2, step ; or Basic Protocol 3)
  • 3.9-µm latex beads, surfactant-free aldehyde/sulfate, 4% solids (Interfacial Dynamics 12-4000; http://www.idclatex.com)
  • Phosphate-buffered saline (PBS; appendix 2A)
  • PBS/1 M glycine
  • PBS/0.5% (w/v) BSA
  • Fluorochrome-conjugated primary or secondary antibodies
  • Test tube rotator wheel for 1.5-ml microcentrifuge tubes
  • Microcentrifuge
  • Flow cytometer (e.g., FACScan; BD)
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Figures

  • Figure 3.22.1
    Flow chart for the exosome purification procedure based on differential ultracentrifugation. The speed and length of each centrifugation are indicated to the right of the arrows. After each of the first three centrifugations, pellets (cells, dead cells, cell debris) are discarded, and the supernatant is kept for the next step. In contrast, after the two 100,000 × g centrifugations, pellets (exosomes + contaminant proteins, exosomes) are kept, and supernatants are discarded.

    View Image
  • Figure 3.22.2
    Typical characteristics of exosomes. (A) Electron-microscopic observation of whole-mounted exosomes purified from mouse dendritic cells. Arrows indicate exosomes, arrowheads point to smaller nonexosomal vesicles. Insert: Immunogold labeling of MHC class II molecules (10-nm gold particles). Scale bar = 100 nm. (B) Coomassie blue–stained SDS polyacrylamide gel after separation of 30 µg of total cell lysates (Cell) or exosomes (Exo) from mouse dendritic cells. Molecular weight markers were loaded in the first lane (kDa).

    View Image

Literature Cited

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