UNIT 3.22
Isolation and Characterization of
Exosomes from Cell Culture Supernatants
and Biological Fluids
Exosomes are small vesicles secreted by most cell types in culture. Exosomes form
intracellularly by inward budding of the limiting membrane of endocytic compartments,
leading to vesicle-containing endosomes, called multivesicular bodies (MVBs). MVBs
eventually fuse with the plasma membrane, thus releasing their internal vesicles (i.e.,
exosomes) into the extracellular medium. What, then, may the physiological function of
exosomes be? On one hand, exosome secretion could be a function per se, e.g., exosome
secretion by reticulocytes allows the elimination of proteins such as transferrin receptor or
integrins, which are useless in differentiated red blood cells (Pan et al., 1985; Vidal et al.,
1997). On the other hand, exosomes could be involved in intercellular communication,
allowing exchange of proteins and lipids between the exosome-producing cells and target
cells. Such a function has been exemplified in the immune system where exosomes allow
exchange of antigen or major histocompatibility complex (MHC)-peptide complexes
between antigen-bearing cells and antigen-presenting cells (e.g., dendritic cells; Wolfers
et al., 2001; Andre et al., 2002, 2004; Th
´
ery et al., 2002). Nevertheless, the physiological
functions of exosomes remain a matter of debate.
The purpose of this unit is to give simple and reliable methods for purifying and char-
acterizing exosomes. Cell culture supernatants (conditioned media; CM) contain several
types of shed membrane fragments and vesicles; therefore, before performing any func-
tional analysis, it is critical to ensure that the purified vesicles are exosomes and not other
contaminating material. The first part of this unit describes the most common protocols
used to purify exosomes from cell culture conditioned media or from physiological flu-
ids, and the second part describes different methods for characterizing and assessing the
purity of the isolated exosomes.
Exosomes have been successfully purified from cell culture conditioned medium or
bodily fluids. Support Protocols 1 and 2 provide all details and precautions to take in
collecting materials from which exosomes will be purified. Starting from this material, the
original and most commonly used protocol for exosome purification (Raposo et al., 1996)
is described in Basic Protocol 1. It involves several centrifugation and ultracentrifugation
steps. In some cases, the first centrifugation steps can be replaced by a single filtration
step: this option is described in an Alternate Protocol. A slightly modified version of
Basic Protocol 1, designed for purifying exosomes from viscous fluids (e.g., plasma)
is described in Basic Protocol 2. An extra purification step that provides extremely
pure exosomes can be added to these protocols, and is described in Support Protocol
3. A different purification procedure, involving trapping exosomes on beads bearing
an antibody specific for exosomal surface molecules, has more recently been described
(Clayton et al., 2001) and is provided in Basic Protocol 3. It is easy to use and useful for
rough characterization of exosomes, but it is not intended for purification of large amounts
of exosomes. An additional new method for purifying exosomes by ultrafiltration instead
of ultracentrifugation is not described in this unit. This method employs ultrafiltration
cartridges and pumps and is especially useful for purifying exosomes from large volumes
(>1 liter) of conditioned medium. It is suitable for clinical applications of purified
exosomes, but it is not the easiest option for laboratory applications. Interested readers
should see Lamparski et al. (2002) for details.
Contributed by Clotilde Th
´
ery, Aled Clayton, Sebastian Amigorena, and Grac¸a Raposo
Current Protocols in Cell Biology (2006) 3.22.1-3.22.29
Copyright
C
2006 by John Wiley & Sons, Inc.
Subcellular
Fractionation
and Isolation of
Organelles
3.22.1
Supplement 30
Isolation and
Characterization
of Exosomes
3.22.2
Supplement 30 Current Protocols in Cell Biology
Identification of membrane vesicles as exosomes requires morphological analysis. Given
their small size (50 to 90 nm), exosomes can be visualized only with an electron micro-
scope; thus, evaluation of the purity of exosome preparations as well as their character-
ization should be determined by electron microscopy (see Support Protocols 4, 5, and
6). Separation of exosomes by SDS-PAGE, followed by Coomassie blue staining of all
exosomal proteins also gives an idea of the quality of the exosome preparation: exosomes
should contain a large number of proteins, i.e., at least a few dozen, many of which are
distinct from the proteins detected in whole cell lysates. Further characterization in-
volves analysis of the physical properties of exosomes on a continuous sucrose gradient
(Support Protocol 7) and of protein quantity (Support Protocol 9) and composition by
immunoblotting (Support Protocol 8) and FACS analysis (Support Protocol 10).
BASIC
PROTOCOL 1
PURIFICATION OF EXOSOMES BY DIFFERENTIAL
ULTRACENTRIFUGATION
The general idea of exosome purification by ultracentrifugation is depicted in Figure
3.22.1. The first steps are designed to eliminate large dead cells and large cell debris
by successive centrifugations at increasing speeds (steps 1 to 5 below). At each of these
steps, the pellet is thrown away, and the supernatant is used for the following step (Fig.
3.22.1). The final supernatant is then ultracentrifuged at 100,000 × g to pellet the small
vesicles that correspond to exosomes. The pellet is washed in a large volume of PBS, to
eliminate contaminating proteins, and centrifuged one last time at the same high speed.
Figure 3.22.1 Flow chart for the exosome purification procedure based on differential ultracen-
trifugation. 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.
Subcellular
Fractionation
and Isolation of
Organelles
3.22.3
Current Protocols in Cell Biology Supplement 30
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 × g
rpm for
12,000 × g
rpm for
100,000 × g
rpm for
110,000 × g
SW 41 or 40
(swinging
bucket)
Polyallomer 12 72 7,500 8,200 24,000 25,000
SW 28 or 32
(swinging
bucket)
Polyallomer 30 180 7,500 8,200 24,000 25,000
70 Ti Polycarbonate
bottle
22 180 10,000 11,000 31,000 32,000
45 Ti Polycarbonate
bottle
68 400 9,000 10,000 30,000 31,000
TLA-100.3 Thick-walled 3 18 13,000 15,000 43,000 45,000
TLA-110 polycarbonate 5 40
Materials
Conditioned medium (Support Protocol 1, step 5), 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
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.
Remove cells, dead cells, and cellular debris
1. Transfer the cleared, conditioned medium to 50-ml centrifuge tubes.
2. Centrifuge 20 min at 2,000 × g,4
◦
C.
3. Pipet off the supernatant, and transfer it to polyallomer tubes or polycarbonate
bottles appropriate for the ultracentrifugation rotor to be used.
Isolation and
Characterization
of Exosomes
3.22.4
Supplement 30 Current Protocols in Cell Biology
Ensure that none of the pellet is collected and contaminates the supernatant. Use a
pipet, rather than pouring off the supernatant, and leave behind half a centimeter of
liquid above the pellet.
4. Mark one side of each ultracentrifuge tube with a waterproof marker, orient the
tube in the rotor with the mark facing up, and centrifuge 30 min at 10,000 × g,4
◦
C
(see Table 3.22.1).
The mark is a reference for the location of a pellet at the end of the centrifugation.
Collect exosome fraction
5. Transfer the supernatant to a fresh tube or bottle the same size as in step 3.
Ensure that none of the pellet is collected and contaminates the supernatant.
There will probably not be a visible pellet at this step. For swinging-bucket rotors, the
pellet is at the bottom of the tube. For fixed-angle rotors, the pellet is on the side of the
tube facing up (marked by the marker pen) near the bottom of the tube.
When removing the supernatant with the pipet, hold the tube at an angle, so that the
pellet is always covered with supernatant, and stop removing supernatant when half a
centimeter of liquid is still covering the pellet.
6. Centrifuge at least 70 min at 100,000 × g,4
◦
C.
For this high-speed centrifugation, all tubes should be at least three-quarters full. If one
of the tubes is not three-quarters full, add PBS.
Centrifugation time is calculated to allow a full hour at 100,000 × g, i.e., ∼10 min for
the centrifuge to reach 100,000 × g plus 1 hr at the final speed. A longer time (up to
3 hr) will not damage the exosomes.
7. Remove the supernatant completely.
For fixed-angle rotors, at this step, pour off the supernatant rather than use a pipet.
For swinging-bucket rotors, remove the supernatant with a pipet and leave 2 mm of
supernatant above the pellet.
Wash exosomes
8. Resuspend the pellet in each tube in 1 ml PBS, using a micropipettor. Pool the
resuspended pellets from all the tubes containing materials from the same cells in
a single centrifuge tube. Then add PBS to fill the tube completely.
There will probably not be a visible pellet at this step. For fixed-angle rotors, resuspend
by flushing up and down where the pellet should be (upper side of the tube, towards the
bottom). For swinging-bucket rotors, flush the bottom of the tube.
9. Centrifuge 1 hr at 100,000 × g,4
◦
C.
10. Remove the supernatant as completely as possible. For fixed-angle rotors, pour off
the supernatant, keep the tube upside down, and aspirate the remaining liquid on
the sides and the mouth of the tube with a micropipettor. Proceed to step 11a or
step 11b
11a. To resuspend the pellet (i.e., exosomes): Add a small volume (50 to 100 µl) of PBS
or TBS and resuspend.
If the final volume of exosomes is too large (>1/2000 of the initial volume of conditioned
medium) or if there was no visible pellet at step 10, use step 11b instead.
11b. To concentrate the exosome preparation: Centrifuge the supernatant (step 10) 1 hr
at 100,000 × g,4
◦
C in a tabletop ultracentrifuge, using a TLA-100.3 rotor and the
corresponding thick-walled polycarbonate tubes. Remove most of the PBS above
the visible pellet and resuspend exosomes in 20 to 50 µl of fresh PBS.
Subcellular
Fractionation
and Isolation of
Organelles
3.22.5
Current Protocols in Cell Biology Supplement 31
12. Store exosomes up to 1 year at –80
◦
C in 100-µl aliquots. Avoid repeated freezing
and thawing.
ALTERNATE
PROTOCOL
ELIMINATION OF LARGE CELL DEBRIS AND MEMBRANES BY
FILTRATION
For some cells (e.g., mouse dendritic cells) it is possible to replace steps 1 to 6 of
Basic Protocol 1 with a single filtration step using a 0.22-µm filter; this will eliminate
dead cells and large debris while keeping small membranes for further purification by
ultracentrifugation. To determine whether this Alternate Protocol can be used, perform
Basic Protocol 1 once using the same volume of conditioned medium obtained from the
same cells at the same time in parallel with this protocol, to compare the yields and the
quality of exosomes by electron microscopy and immunoblotting (Support Protocols 4,
5, 6, 7, 8, 9, and 10), to ensure that the exosome preparations are identical.
Materials (also see Basic Protocol 1)
0.22-
µm filter sterilization device (e.g., Steritop; Millipore)
100-ml to 1-liter glass bottle, sterile
1. Filter sterilize the conditioned medium by passing through a vacuum-connected
0.22-µm filter on top of a sterile bottle, using a vacuum.
2. Store the filtered supernatant up to 1 week at 4
◦
C before proceeding to exosome
purification (Basic Protocol 1, steps 8 to 12).
SUPPORT
PROTOCOL 1
COLLECTING CULTURE SUPERNATANTS FOR EXOSOME
PURIFICATION
The exosome-containing conditioned medium from cultured cells is used as the starting
material for exosome purification. Total cell lysate of the cells that produce the condi-
tioned medium is prepared at the same time and is used later in parallel with exosomes
for immunoblot analysis (Support Protocol 8).
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)
Collect and clear conditioned medium
1. Grow cells of interest under the usual conditions (see UNIT 1.1) until they reach 70% to
80% confluency for adherent cells, or 60% to 70% of their maximum concentration
for cells grown in suspension.
Use as many cells as necessary to produce at least 70-ml of conditioned medium (i.e.,
a minimum of seven 10-cm dishes and up to twenty 15-cm dishes); it is always better
to purify exosomes from large volumes of conditioned medium because the yield of the
purification procedure increases with the starting volume.
