U.S. patent application number 13/765677 was filed with the patent office on 2013-10-17 for methods and compositions for exosome isolation.
This patent application is currently assigned to LIFE TECHNOLOGIES CORPORATION. The applicant listed for this patent is LIFE TECHNOLOGIES CORPORATION. Invention is credited to Richard Conrad, Mu Li, Alexander VLASSOV, Emily Zeringer.
Application Number | 20130273544 13/765677 |
Document ID | / |
Family ID | 47755029 |
Filed Date | 2013-10-17 |
United States Patent
Application |
20130273544 |
Kind Code |
A1 |
VLASSOV; Alexander ; et
al. |
October 17, 2013 |
METHODS AND COMPOSITIONS FOR EXOSOME ISOLATION
Abstract
Disclosed are methods, compositions and kits for the isolation
of exosomes from biological fluids and tissues. Volume-excluding
polymers are used to precipitate exosomes from biological samples
thereby allowing exosome isolation by low-speed (benchtop)
centrifugation or filtration. Further fractionation of exosomes
after precipitation is also described.
Inventors: |
VLASSOV; Alexander; (Austin,
TX) ; Li; Mu; (Austin, TX) ; Zeringer;
Emily; (Austin, TX) ; Conrad; Richard;
(Austin, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LIFE TECHNOLOGIES CORPORATION |
Carlsbad |
CA |
US |
|
|
Assignee: |
LIFE TECHNOLOGIES
CORPORATION
Carlsbad
CA
|
Family ID: |
47755029 |
Appl. No.: |
13/765677 |
Filed: |
February 12, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61625562 |
Apr 17, 2012 |
|
|
|
Current U.S.
Class: |
435/6.12 |
Current CPC
Class: |
G01N 33/92 20130101;
G01N 33/5005 20130101; G01N 33/5076 20130101; C12Q 1/6806 20130101;
G01N 1/34 20130101 |
Class at
Publication: |
435/6.12 |
International
Class: |
G01N 1/34 20060101
G01N001/34 |
Claims
1. A method for the isolation of exosomes from a biological fluid
sample comprising: a) adding a volume-excluding polymer to the
biological fluid sample, b) incubating the biological fluid sample
with the volume-excluding polymer; and c) isolating the
precipitated exosomes from the biological fluid sample.
2. The method of claim 1, wherein the molecular weight of the
volume-excluding polymer is from 1000 to 1,000,000 daltons.
3. (canceled)
4. (canceled)
5. The method of claim 1, wherein the concentration of
volume-excluding polymer upon mixing with the biological fluid is
from 1% to 90%.
6. The method of claim 5, wherein the concentration of
volume-excluding polymer upon mixing with the biological fluid is
from 3% to 15%.
7. (canceled)
8. The method of claim 1, further comprising the step of clarifying
the biological fluid sample before adding the volume-excluding
polymer.
9. The method of claim 1 further comprising the step of isolating
nucleic acid and protein from the exosomes.
10. The method of claim 1, wherein the biological fluid is from
prokaryotes, eukaryotes, bacteria, fungi, yeast, invertebrates,
vertebrates, reptiles, fish, insects, plants or animals.
11. The method of claim 1, wherein the biological fluid is selected
from the group consisting of blood serum, plasma, whole blood,
urine, saliva, breast milk, tears, sweat, joint fluid,
cerebrospinal fluid, semen, vaginal fluid, ascitic fluid, amniotic
fluid, and cell culture media.
12. (canceled)
13. The method of claim 1, further comprising fractionating the
isolated exosomes.
14. The method of claim 1, wherein the volume-excluding polymer is
polyethylene glycol, dextran, dextran sulfate, dextran acetate,
polyvinyl alcohol, polyvinyl acetate, or polyvinyl sulfate.
15. (canceled)
16. The method of claim 1, further comprising contacting the
biological fluid sample prior to the addition of the
volume-excluding polymer with a protease, under conditions suitable
for digestion of protein.
17. (canceled)
18. A method for the isolation of exosomes from a biological tissue
sample comprising: a) lysing the biological tissue sample, b)
clarifying the lysed sample c) adding a volume-excluding polymer to
the clarified sample, d) incubating the clarified sample with the
volume-excluding polymer, and e) isolating the precipitated
exosomes.
19. The method of claim 18, wherein the molecular weight of the
volume-excluding polymer is from 1000 to 1,000,000 daltons.
20. (canceled)
21. (canceled)
22. The method of claim 18, wherein the concentration of
volume-excluding polymer upon mixing with the biological sample is
from 1% to 90%.
23. The method of claim 22, wherein the concentration of
volume-excluding polymer upon mixing with the biological sample is
from 3% to 15%.
24. (canceled)
25. The method of claim 18, wherein the volume-excluding polymer is
polyethylene glycol, dextran, dextran sulfate, dextran acetate,
polyvinyl alcohol, polyvinyl acetate, or polyvinyl sulfate.
26. (canceled)
27. The method of claim 18 further comprising the step of isolating
nucleic acid and protein from the exosomes.
28. The method of claim 18, wherein the biological sample is from
prokaryotes, eukaryotes, bacteria, fungi, yeast, invertebrates,
vertebrates, reptiles, fish, insects, plants or animals.
29. The method of claim 18, wherein the biological sample is
selected from the group consisting of surgical samples, biopsy
samples, tissues, feces, plant tissue, insect tissue, fungi,
bacteria, parasites and cultured cells.
30. The method of claim 18, further comprising contacting the
biological tissue sample with a protease, under conditions suitable
for digestion of protein, prior to the addition of the
volume-excluding polymer.
31. (canceled)
Description
FIELD
[0001] The disclosure generally relates to the isolation of
exosomes from biological tissues and fluids.
BACKGROUND
[0002] Cells continuously secrete a large number of microvesicles,
nanovesicles, macromolecular complexes, and small molecules into
the extracellular space. Exosomes are small secreted vesicles
(typically about 30-150 nm) which may contain or have present in
their membrane nucleic acid, protein, or other biomolecules and may
serve as carriers of this cargo between diverse locations in the
body (Mittelbrunn & Sanchez-Madrid, Nature Reviews 13 (2012)
328-335; Thery et al. Nat. Rev. Immunol. 2 (2002) 569-579; Valadi
et al. Nat. Cell. Biol. 9 (2007) 654-659). Exosomes are secreted by
all types of cells in culture, and also found in abundance in body
fluids including blood, saliva, urine, and breast milk (Kosaka et
al., Silence 3 (2010) 1-7; Mitchell et al., PNAS 105 (2008)
10513-10518; Palanisamy et al., PLoS One 5 (2010) e8577).
[0003] Currently, the control of exosome formation, the makeup of
the "cargo", biological pathways and resulting functions are
incompletely understood. One of their most intriguing roles is
intercellular communication. Exosomes are thought to function as
messengers, delivering various effector or signaling macromolecules
between cells.
[0004] The accepted protocol for isolation of exosomes includes
ultracentrifugation (Thery et al., Cum Protoc. Cell. Biol., Chapter
3, Unit 3: 22 (2006)), often in combination with sucrose density
gradients or sucrose cushions to float the relatively low-density
exosomes. Isolation of exosomes by sequential differential
centrifugations is complicated by the possibility of overlapping
size distributions with other microvesicles or macromolecular
complexes. Furthermore, centrifugation may provide insufficient
means to separate vesicles based on their sizes. However,
sequential centrifugations, when combined with sucrose gradient
ultracentrifugation, can provide high enrichment of exosomes.
[0005] Isolation of exosomes based on size, using alternatives to
the ultracentrifugation routes, is another option. Successful
purification of exosomes using ultrafiltration procedures that are
less time consuming than ultracentrifugation, and do not require
use of special equipment have been reported (Cheruvanky et al., J.
Physiol. Renal Physiol. 292 (2007) 1657-1661.) Similarly, a
commercial kit is available (ExomiR, Bioo Scientific) which allows
removal of cells, platelets and cellular debris on one microfilter
and capturing of vesicles bigger than 30 nm on a second microfilter
using positive pressure to drive the fluid. For this process, the
exosomes are not recovered, their RNA content is directly extracted
off the material caught on the second microfilter, which can then
be used for PCR analysis. HPLC-based protocols could potentially
allow one to obtain highly pure exosomes, though these processes
require dedicated equipment and are difficult to scale up. A
significant problem is that both blood and cell culture media
contain large numbers of nanoparticles (some non-vesicular) in the
same size range as exosomes. For example, Wang et al. (Nucleic
Acids Res. 38 (2010) 7248-7259.) found that large number of miRNAs
are contained within extracellular protein complexes rather than
exosomes. As a consequence, the above methods are best described as
allowing one to obtain exosome-enriched samples, rather than pure
exosomes.
[0006] Volume-excluding polymers such as PEGs can sometimes be used
for precipitation of viruses and other small particles (Yamamoto et
al. Virology 40 (1970) 734-744; Adams, J. Gen. Virol. 20 (1973)
391-394; Lewis et al., Applied and Environmental Microbiol. 4
(1988) 1983-1988). We have unexpectedly found that despite exosomes
being noticeably less dense than viruses due to the lack of a
protein coat and variable, though probably lower (for their size),
nucleic acid content, volume-excluding polymers are capable of
differentially precipitating exosomes thereby allowing exosome
isolation by low-speed (benchtop) centrifugation or filtration.
SUMMARY
[0007] Discussed herein are methods and compositions using
volume-excluding polymers such as polyethylene glycols (PEG) to
precipitate exosomes from biological samples. The precipitated
exosomes can be isolated using low-speed centrifugation, filtration
or other method for isolating precipitated material. Some
embodiments are for a method for the isolation of exosomes from a
biological fluid sample comprising: a) adding a volume excluding
polymer to the biological fluid sample, b) incubating the
biological fluid sample with the volume-excluding polymer, and
isolating the aggregated/precipitated exosomes from the biological
fluid sample.
[0008] In other embodiments, non-limiting examples of volume
excluding polymers include polyethylene glycol, a dextran including
dextran sulfate and dextran acetate, or a hydrophilic polymer such
as polyvinyl alcohol, polyvinyl acetate or polyvinyl sulfate.
[0009] In particular embodiments, the volume-excluding polymer has
a molecular weight of between 1000 and 1,000,000 daltons, from 3000
to 20000 daltons, from 4000 to 20000 daltons, from 6000 to 20000
daltons, from 1000 to 10000 daltons, from 3000 to 10000 daltons,
from 3000 to 8000 daltons, from 4000 to 8000, or from 3000 to 6000
daltons.
[0010] In other embodiments, the volume-excluding polymer is added
to the biological fluid sample to a final concentration of from 1%
to 90%, 2% to 50%, 2% to 20%, 2% to 15%, 2% to 12%, 3% to 15%, 4%
to 15%, 6% to 15%, 8% to 15%, 4% to 12%, 6% to 12%, or 8% to 12%
(weight/volume).
[0011] In some embodiments the biological fluid sample may be
clarified prior to the addition of the volume-excluding polymer.
Clarification may involve, but is not limited to, centrifugation,
ultracentrifugation, filtration, and ultrafiltration.
[0012] In other embodiments, biological material may be isolated
from the exosomes after they are isolated. Biological materials
that may be isolated from exosomes include, but are not limited to,
nucleic acids such as RNA, DNA, proteins and peptides.
[0013] In further embodiments, non-limiting examples of biological
fluid samples include serum, plasma, whole blood, urine, saliva,
breast milk, tears, sweat, joint fluid, cerebrospinal fluid, semen,
vaginal fluid, ascitic fluid, amniotic fluid, and media taken from
cultured cells ("conditioned media"). In some embodiments, the
biological fluid sample may be obtained from a mammal such as a
mouse, rat, guinea pig, rabbit, dog, cat, bovine, horse, goat,
sheep, primate or human.
[0014] An alternate embodiment may be a method for the isolation of
exosomes from a biological tissue sample comprising: a) lysing the
biological tissue sample, b) clarifying the lysed sample, c) adding
a volume-excluding polymer to the clarified sample, d) incubating
the clarified sample with the volume-excluding polymer, and e)
isolating the precipitated exosomes.
[0015] In further embodiments, non-limiting examples of biological
tissue samples include surgical samples, biopsy samples, tissues,
feces, plant tissue, insect tissue, and cultured cells.
[0016] In some embodiments, exosomes isolated using a
volume-excluding polymer may be further fractionated based on their
size, density or proteins exposed on the surface of the
exosome.
[0017] Some embodiments provide for a kit for the isolation of
exosomes from biological fluids or tissues. Kits may comprise one
or more vessels containing one or more volume-excluding polymers,
one or more buffers or one or more solutions for performing density
gradient centrifugation of exosomes. The kit may further comprise a
filtration device for separating exosomes by their size. The kit
may also comprise one or more antibodies or other ligands which
bind to a protein or other ligand exposed on the surface of the
exosome and one or more solid supports which bind directly or
indirectly to the exosomes.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 Shows the results of an analysis comparing PEG size
and concentration with the efficiency of exosome recovery from
conditioned cell media.
[0019] FIG. 2 Shows a comparison of different salt and buffer
concentrations with the efficiency of exosome recovery from
conditioned cell media.
[0020] FIG. 3 Shows a comparison of PEG-mediated exosome isolation
from three cell types with isolation of exosomes using the
ultracentrifugation protocol.
[0021] FIG. 4 Shows analysis of an exosome preparation isolated
from HeLa cells. The X axis depicts the size distribution of the
detected nanoparticles (in nanometers). The Y axis depicts the
concentration of nanoparticles per mL of sample
(.times.10.sup.6).
[0022] FIG. 5 Shows the results of an analysis comparing PEG size
and concentration with the efficiency of exosome recovery from
blood serum.
[0023] FIG. 6 Shows the comparison of PEG6000 of different
concentrations for isolation of exosomes from blood serum.
[0024] FIG. 7 Shows a comparison of small scale versus large scale
exosome isolation from serum.
[0025] FIG. 8 Shows the analysis of exosomal RNA by qRT-PCR.
[0026] FIG. 9 Shows analysis of an exosome preparation isolated
from human blood serum. The X axis depicts size distribution of the
detected nanoparticles (in nanometers). The Y axis depicts
concentration of nanoparticles per mL of sample
(.times.10.sup.6).
[0027] FIG. 10 Shows Electron Microscopy analysis of exosomes
isolated from cell culture media with 10% PEG6000.
[0028] FIG. 11 Shows Western blot and qRT-PCR analysis for exosomes
derived from HeLa cell media with 10% PEG6000.
[0029] FIG. 12 Shows Western blot and qRT-PCR analysis for exosomes
derived from serum with 5% PEG6000.
[0030] FIG. 13 Shows efficiency of exosomes recovery from urine
with PEG6000 of different percentages.
[0031] FIG. 14 Shows efficiency of exosomes recovery from amniotic
fluid with PEG6000 of different percentages.
[0032] FIG. 15 Shows efficiency of exosomes recovery from
cerebrospinal fluid (CSF) with PEG6000 of different
percentages.
[0033] FIG. 16 Shows efficiency of exosomes recovery from plasma
with 5% PEG6000 and effects of Proteinase K on removal of Albumin
from the sample.
DETAILED DESCRIPTION
[0034] Disclosed herein are methods and compositions using
volume-excluding polymers such as polyethylene glycol (PEG) to
precipitate exosomes from biological samples. The precipitated
exosomes can be isolated using low-speed centrifugation, filtration
or other methods for separating precipitated material.
[0035] As used herein, exosomes are small secreted vesicles
(typically about 30-150 nm) which may contain, or have present in
their membrane, nucleic acid, protein, or other biomolecules and
may serve as carriers of this cargo between diverse locations in a
body or biological system. When concentrations of volume excluding
polymers are used they are presented as % weight/volume (w/v)
calculated using the formula: weight of solute (g)/volume of
solution (mL)*100.
[0036] The term biological fluid, as used herein, means any fluid
isolated or derived from an organism including prokaryotes,
eukaryotes, bacteria, fungi, yeast, invertebrates, vertebrates,
reptiles, fish, insects, plants and animals. Media taken from
cultured cells ("conditioned media", cell media, cell culture
media) may be a biological fluid.
[0037] The term biological tissue, as used herein, means a
collection of cells from prokaryotes, eukaryotes, bacteria, fungi,
yeast, invertebrates, vertebrates, reptiles, fish, insects, plants
and animals. Cultured cells may be a biological tissue.
[0038] Exosomes may be isolated from a variety of biological
sources including mammals such as mice, rats, guinea pigs, rabbits,
dogs, cats, bovine, horses goats, sheep, primates or humans.
Typically, exosomes are isolated from biological fluids such as
serum, plasma, whole blood, urine, saliva, breast milk, tears,
sweat, joint fluid, cerebrospinal fluid, semen, vaginal fluid,
ascetic fluid and amniotic fluid. Exosomes may also be isolated
from experimental samples such as media taken from cultured cells
("conditioned media", cell media, cell culture media).
[0039] Exosomes may also be isolated from tissue samples such as
surgical samples, biopsy samples, tissues, feces, plant tissue,
insect tissue, and cultured cells. When isolating exosomes from
tissue sources it may be necessary to homogenize the tissue in
order to obtain a single cell suspension followed by lysis of the
cells to release the exosomes. When isolating exosomes from tissue
samples it is important to select homoginazation and lysis
procedures that do not result in disruption of the exosomes.
[0040] Exosomes may be isolated from freshly collected samples or
from samples that have been stored frozen or refrigerated. Although
not necessary, higher purity exosomes may be obtained if fluid
samples are clarified before precipitation with a volume-excluding
polymer, to remove any debris from the sample. Methods of
clarification include centrifugation, ultracentrifugation,
filtration or ultrafiltration.
[0041] Precipitation of exosomes from the sample may be
accomplished using a water-soluable volume excluding polymer.
Examples of suitable polymers include polyethylene glycol (PEG),
dextrans and derivatives such as dextran sulfate, dextran acetate,
and hydrophilic polymers such as polyvinyl alcohol, polyvinyl
acetate and polyvinyl sulfate.
[0042] Suitable volume-excluding polymers typically have a
molecular weight between 1000 and 1,000,000 daltons. Polymers with
higher molecular weights may be too viscous. In general, when
higher concentrations of exosomes are present in a sample, lower
molecular weight polymers may be used.
[0043] Volume-excluding polymers may be used at a final
concentration of from 1% to 90% (w/v) upon mixing with the sample.
For example, when one volume of 40% (w/v) polymer solution is mixed
with equal volume of the biological fluid--the resulting polymer
concentration in the sample is 20%. Higher concentrations of the
volume-excluding polymers are useful when the concentration of the
exosomes in the sample is low.
[0044] A variety of buffers commonly used for biological samples
may be used for incubation of the exosome sample with the
volume-excluding polymer including phosphate, acetate, citrate and
TRIS buffers. The pH of the buffer may be any pH that is compatible
with the sample, but a typical range is from 6 to 8. The buffer may
have a pH from 4 to 10, 4 to 6, 4 to 8, 6 to 10, 6 to 8, or 8 to
10. The salt concentration can be any concentration that is
compatible with the sample, but typically ranges from 10 mM to 500
mM. The salt concentration may be 10 mM to 500 mM, 10 mM to 50 mM,
10 mM to 100 mM, 10 mM to 200 mM, 10 mM to 300 mM, 10 mM to 400 mM,
50 mM to 500 mM, 100 mM to 500 mM, 200 mM to 500 mM, 300 mM to 500
mM, or 400 mM to 500 mM.
[0045] Incubation of the sample with the volume-excluding polymer
may be performed at room temperature (about 20.degree. C.) or
higher temperature, but precipitation of the exosomes will
generally occur more quickly and completely if the incubation is
performed at a reduced temperature such as 4.degree. C. Incubation
may be performed at 1.degree. C. to 40.degree. C., 1.degree. C. to
4.degree. C., 4.degree. C. to 10.degree. C., 10.degree. C. to
20.degree. C., 20.degree. C. to 30.degree. C., or 30.degree. C. to
40.degree. C.
[0046] The time of incubation of the sample with the
volume-excluding polymer may be any, typically in the range 1
sec-24 hours, more typically in the range 5 min-14 hours. The
incubation time may be 1 sec-24 hours, 1 minute-24 hours, 5
minutes-24 hours, 10 minutes-24 hours, 20 minutes-24 hours, 30
minutes-24 hours, 1 hour-24 hours, 6 hours-24 hours, 12 hours-24
hours, 1 minute-12 hours, 1 minute-6 hours, or 1 minute-4 hours.
The incubation time is influenced by, among other factors, the
concentration of the volume-excluding polymer, the molecular weight
of the volume-excluding polymer, the temperature of incubation and
the concentration of exosomes and other components in the sample.
High concentrations of high molecular weight volume-excluding
polymers in general will precipitate exosomes more quickly. Samples
with low concentrations of exosomes may require longer incubation
times for efficient precipitation of exosomes.
[0047] After completion of the incubation of the sample with the
volume-excluding polymer the precipitated exosomes may be isolated
by centrifugation, ultracentrifugation, filtration or
ultrafiltration. Exosomes may be further fractionated using
conventional methods such as ultracentrifugation with or without
the use of a density gradient to obtain higher purity.
Sub-populations of exosomes may also be isolated by using other
properties of the exosome such as the presence of surface markers.
Surface markers which may be used for fraction of exosomes include
but are not limited to tumor markers and MHC class II markers. MHC
class II markers which have been associated with exosomes include
HLA DP, DQ and DR haplotypes. Other surface markers associated with
exosomes include CD9, CD81, CD63 and CD82 (Thery et al. Nat. Rev.
Immunol. 2 (2002) 569-579; Valadi et al. Nat. Cell. Biol. 9 (2007)
654-659).
[0048] As an example, exosomes having CD63 on their surface may be
isolated using antibody coated magnetic particles. Dynabeads.RTM.
are super-paramagnetic polystyrene beads which may be conjugated
with anti-human CD63 antibody, either directly to the bead surface
or via a secondary linker (e.g. anti-mouse IgG). The beads may be
between 1 and 4.5 .mu.m in diameter.
[0049] The antibody coated Dynabeads.RTM. may be added to an
exosome sample prepared using a volume-excluding polymer and
incubated at 2-8 .degree. C. or at room temperature from 5 minutes
to overnight. Dynabeads.RTM. with bound exosomes may then be
collected using a magnet. The isolated, bead bound exosomes may
then be resuspended in an appropriate buffer such as phosphate
buffered saline and used for downstream analysis (qRT-PCR,
sequencing, Westerns, flow cytometry etc.). Similar protocols may
be used for any other surface marker for which an antibody or other
specific ligand is available. Indirect binding methods such as
those using biotin-avidin may also be used.
[0050] Once an isolated exosome sample has been prepared, the
contents of the exosome may be extracted for study and
characterization. Biological material which may be extracted from
exosomes includes proteins, peptides, RNA and DNA, lipids. For
example the mirVana.TM. PARIS Kit (AM1556, Life Technologies) may
be used to recover native protein and RNA species, including small
RNAs such as miRNA, snRNA, and snoRNA, from exosomes.
[0051] Total RNA may be extracted using acid-phenol:chloroform
extraction. RNA may then be purified using a glass-fiber filter
under conditions that recover small-RNA containing total RNA, or
that separate small RNA species less than 200 nucleotides in length
from longer RNA species such as mRNA. Because the RNA is eluted in
a small volume, no alcohol precipitation step may be required for
isolation of the RNA.
[0052] Kits may comprise one or more vessels containing one or more
volume-excluding polymers, one or more buffers or one more
solutions for performing density gradient centrifugation of
exosomes. The kit may further comprise a filtration device for
separating exosomes by their size. The kit may also comprise one or
more antibodies or other ligands which bind to a protein or other
antigen exposed on the surface of the exosome and one or more solid
supports which bind directly or indirectly to the exosomes.
[0053] Preparation of polyethylene glycol (PEG) stock solutions
[0054] For preparation of 50 mL stock of 40% (w/v) PEG8000 in
water, 20 grams of PEG8000 powder (Sigma) may be weighed and
transferred into a 50 mL conical tube (Ambion). 20 mLs of
nuclease-free water (Ambion) may then be added, and the tube placed
into a Sarstedt M200 mixer (Germany) and dissolved for 1 hour or
until the solution becomes clear. The contents of the tube may then
be transferred into a graduated cylinder and water added to bring
the final volume to 50 mL. The contents of the graduated cylinder
may then be transferred back into the conical tube, and mixed for
another 10 min to ensure a homogenous 40% PEG solution. Solutions
containing 10-50% PEG3000-PEG20,000, with or without PBS or other
buffer or NaCl, may be prepared in a similar fashion. PEG solution
stocks may be stored at room temperature or 4.degree. C. long term.
Antibacterial agents such as sodium azide may be added to PEG stock
solutions to prevent bacterial growth.
Extraction of Exosomes from Blood Serum
[0055] Blood serum samples are removed from storage and placed on
ice. When serum is frozen it may be thawed slowly at room
temperature in lukewarm water until the sample is completely
liquid. Samples may be stored on ice until needed. The serum
samples may be centrifuged at 2,000.times.g for 30 minutes to
remove cell debris. Next, the supernatant containing the cell-free
serum may be transferred to a fresh container and held on ice until
precipitation.
[0056] For exosome precipitation, 100 .mu.L to 1 mL (or other
preferred volume) of cell-free serum may be transferred to a new
tube and combined with the desired volume of PEG precipitation
reagent.
[0057] For example, to achieve a 10% final concentration of
PEG8000, 33.3 .mu.L of 40% (w/v) PEG stock may be added to 100
.mu.l of serum. The serum/reagent mixture may then be mixed well
either by vortexing or pipetting up and down until there is a
homogenous solution. (Solution may have a cloudy appearance). The
samples may then be incubated at 4.degree. C. for 5 minutes to 2 h.
After incubation, the samples may be centrifuged at room
temperature or 4.degree. C. at 2,000.times.g to 10,000.times.g for
5-20 min. The supernatant is aspirated and discarded. Exosomes will
be contained in the pellet at the bottom of the tube. The pellet
may be resuspended in a convenient volume of phosphate bufferd
saline (PBS) buffer, e.g. 50 .mu.L for 100 .mu.L serum input. The
pellet in certain cases may be difficult to resuspend so a pipet
tip may be used to completely resuspend it in the solution.
Alternatively, the pellets may be incubated for 30 minutes at
37.degree. C. then vortexed. Once the pellet has been resuspended,
it may be stored at 4.degree. C. short term or at -20.degree. C.
long term. Solutions containing PEG3000 to PEG20,000, at 3-15%
final concentration (when mixed with serum sample), with or without
PBS buffer or NaCl, may be used in a similar fashion, with serum
input typically ranging from several microliters to several
milliliters.
Quantification and Sizing of Exosomes with Nanosight LM10
Instrument.
[0058] Exosomes purified from blood serum and cell media by PEG
precipitation may be quantified and sized using a Nanosight LM10
instrument (Nanosight, UK), following the manufacturer's protocol.
This instrument uses a laser light source to illuminate nano-scale
particles introduced to a viewing unit. Particles appear
individually as point-scatterers moving under Brownian motion. The
image analysis NTA software allows the user to automatically track
and size nanoparticles on an individual basis.
[0059] Typically, exosome samples may be diluted 10-1000.times.
with PBS buffer in order to achieve the nanoparticle concentration
range suitable for analysis by Nanosight. A 300-500 uL volume may
then be analyzed as recommended in the manufacturer's protocol.
EXAMPLES
Example 1
Evaluation of PEGs of Different Length and Percent for Capability
to Isolate Exosomes from Cell Media
[0060] Polyethylene glycol (PEG) of different length and percent
were evaluated for capability to isolate exosomes from cell culture
media (CM, cell media, conditioned media). HeLa cells were grown in
a CO.sub.2 incubator under normal conditions, in the recommended
cell media containing 10% FBS, until .about.80% confluency was
reached. Then, cells were washed with PBS, and media was replaced
with fresh media with 10% exosome-depleted FBS (exosomes were
removed from FBS by ultracentrifugation) and cells were allowed to
grow for another 12 h. The cell media was then harvested and
centrifuged at 2,000.times.g for 30 minutes to remove cell debris.
Next, the supernatant containing the cell-free cell media was
transferred to a fresh container and held at room temperature until
precipitation. For exosome precipitation, 1 mL of cell media was
combined with the PEG precipitation reagent.
[0061] In this experiment, polyethylene glycol (PEG) of four
different lengths was evaluated: PEG 3000, PEG 6000, PEG 8000, PEG
20000; all at three percentages: 4%, 8%, 12%. NaCl solution was
added to the final concentration of 300 mM.
[0062] The samples (PEG+cell media) were incubated at 4.degree. C.
for 14 h, then the samples were spun down at 10,000 g for 1 h. The
supernatant was discarded, and the exosome pellet was resuspended
in PBS and number of exosomes was quantified on a Nanosight LM10
instrument. The number of exosomes in the samples (30-150 nm size
range) was compared to the number of nanoparticles in the same size
range in the original total cell media sample to analyze efficiency
of recovery.
[0063] Results are shown in FIG. 1. As can be seen, all PEGs were
capable of isolating exosomes from the cell media samples. The
longer PEGs, 6000-20000 performed best, while PEG3000 was somewhat
less efficient. A higher percentage of PEG was beneficial. At 8-12%
PEG, recovery of exosomes was very efficient. At a 4% PEG
concentration (for all PEGs 3000-20000) exosome recovery from cell
media was less efficient. Note that at the optimal conditions (eg
PEG 8000 12%) recovery of the exosomes was quantitative, as seen
from comparison with the total cell media sample. In a similar
fashion, exosomes can be isolated from the media from any other
cell type, grown to any confluency, in a wide range of sample
volume inputs.
Example 2
Investigation of Salt Effects on the Efficiency of PEG-Mediated
Isolation of Exosomes from Cell Media
[0064] Effects of salt and buffer on the efficiency of PEG-mediated
isolation of exosomes from cell media were investigated. HeLa cells
were grown in a CO.sub.2 incubator under normal conditions, in the
recommended cell media containing 10% FBS, until reaching about 80%
confluency. Then, fresh media was added, without FBS, and cells
were allowed to grow for another 12 h. The cell media was then
harvested and centrifuged at 2,000.times.g for 30 minutes to remove
cell debris. Next, the supernatant containing the cell-free cell
media was transferred to a fresh container and held at room
temperature until precipitation. For exosome precipitation, 1 mL or
5 mL of cell media was combined with the PEG precipitation
reagent.
[0065] In this experiment, 8% PEG6000 was utilized, at several NaCl
or PBS concentrations. The samples (PEG+cell media) were incubated
at 4.degree. C. for 14 h, then the samples were spun down at
10,000.times.g for 1 h. The supernatant was discarded, and the
exosome pellet was resuspended in PBS and the number of exosomes
was quantified on a Nanosight LM10 instrument. The number of
exosomes in the samples (30-150 nm size range) was compared to the
number of nanoparticles in the same size range in the original
total cell media sample.
[0066] Results are shown in FIG. 2. As can be seen, exosome
isolation worked well at all conditions tested: PBS buffer only (no
extra salt); PBS+0.15 M NaCl; PBS+0.5 M NaCl; water (no buffer; all
buffering components and salts coming from cell media only); 0.15 M
NaCl; 0.5 M NaCl; small scale (1 ml cell media input) and larger
scale (5 mL cell media input).
[0067] Note that in this experiment cells were grown for the last
12 h prior to harvesting in media without FBS--to ensure high
purity of exosomes. This results in a lower number of exosomes
compared to cells grown in the presence of 10% exosome-depleted FBS
(or 10% whole FBS). Lower numbers of exosomes in the original
sample translates into a lower efficacy of isolation.
Example 3
Comparison of PEG-Mediated Isolation of Exosomes with
Ultracentrifugation Protocol on Cell Media for Three Cell Types
[0068] PEG-mediated isolation of exosomes was compared with
ultracentrifugation protocol on cell media for three different cell
types. HeLa, THP-1, Jurkat cells were grown in the CO.sub.2
incubator under the normal conditions, in the recommended cell
media containing 10% FBS, until about 80% confluency. Then, media
was replaced with fresh media with 10% exosome-depleted FBS, and
cells were allowed to grow for another 12 h. The cell media was
then harvested and centrifuged at 2,000.times.g for 30 minutes to
remove cell debris. Next, the supernatant containing the cell-free
cell media was transferred to a fresh container and held at room
temperature until precipitation. For exosome precipitation, 1 mL of
cell media was combined with the PEG precipitation reagent.
[0069] In this experiment, 8% PEG6000 was utilized. The samples
(PEG+cell media) were incubated at 4.degree. C. for 14 h, then the
samples were spun down at 10,000 g for 1 h. The supernatant was
discarded, and the exosome pellet was resuspended in PBS and the
number of exosomes was quantified on a Nanosight LM10 instrument.
The number of exosomes in the samples (30-150 nm size range) was
compared to the number of nanoparticles in the same size range in
the original total cell media sample.
[0070] Results are shown in FIG. 3. As can be seen, for all three
types of cells, with PEG6000 used at final concentration of 8%,
extremely efficient isolation of exosomes was achieved.
Ultracentrifugation, currently the "gold standard" procedure for
isolation of exosomes, results in significantly lower number of
exosomes isolated from the same volume of cell media.
Ultracentrifugation is also a very lengthy multi-step procedure
requiring special instrumentation and training. PEG enables very
fast and efficient isolation of exosomes from cell culture media,
the protocol is robust and superior to ultracentrifugation in terms
of the yield of exosomes obtained.
[0071] The representative analysis of the exosome sample derived
from the HeLa cell media is shown in FIG. 4. Exosomes isolated from
5 mL of HeLa cell media by 12% PEG6000 were resuspended in 200
.mu.L of PBS, diluted 10 fold and analyzed using a Nanosight LM10
instrument. The X axis depicts the size distribution of the
detected nanoparticles (in nanometers). The Y axis depicts the
concentration of nanoparticles per mL of sample (.times.10.sup.6).
As can be seen, the majority of microvesicles isolated by PEG
precipitation are in the size range typical for exosomes: 30-150
nm.
Example 4
Evaluation of PEGs of Different Length and Percent for Capability
to Isolate Exosomes from Blood Serum
[0072] Polyethylene glycol (PEG) of different length and percent
were evaluated for capability to isolate exosomes from the blood
serum.
[0073] The human blood serum samples were removed from storage and
placed on ice. When serum was frozen, it was thawed slowly at room
temperature in lukewarm water until the sample was completely
liquid. Samples were stored on ice until needed. The serum samples
were first centrifuged at 2,000.times.g for 30 minutes to remove
cell debris. Next, the supernatant containing the cell-free serum
was transferred to a fresh container and held on ice until
precipitation.
[0074] For exosome precipitation, 100 .mu.L of cell-free serum was
transferred to a new tube and combined with the PEG precipitation
reagent.
[0075] In this experiment, polyethylene glycol (PEG) of four
different lengths was evaluated: PEG 3000, PEG 6000, PEG 8000, PEG
20000; all at two percentages: 8% and 15% (final PEG percent, upon
mixing with the sample).
[0076] The samples (PEG+serum) were incubated at 4.degree. C. for 1
h, then the samples were spun down at 10,000 g for 20 min. The
supernatant was discarded, and the exosome pellet was resuspended
in PBS and the number of exosomes was quantified on a Nanosight
LM10 instrument. The number of exosomes in the samples (30-150 nm
size range) was compared to the number of nanoparticles in the same
size range in the original total serum sample to analyze the
efficiency of recovery.
[0077] Results are shown in FIG. 5. As can be seen, all PEGs
3000-20000 were capable of isolating exosomes from the serum
samples with about similar efficiency. Concentrations of PEG of 8%
and 15% produced very similar results. Note that at the optimal
conditions (eg PEG 6000 8%) recovery of the exosomes was
quantitative as seen from comparison with the total serum sample.
On the practical side, PEG 20000 is significantly more viscous and
hard to pipet compared to shorter PEG 3000-8000. Also, PEG stock
solutions 20-30% (weight/volume) are easier to work with compared
to stock solutions of higher concentration (40-60% w/v) that are
very viscous and hard to handle.
Example 5
Investigation of PEG6000 of Different Percent for Isolation of
Exosomes from Blood Serum
[0078] PEG6000 of different percent was investigated for efficiency
of isolation of exosomes from blood serum. The human blood serum
samples were removed from storage and placed on ice. When serum was
frozen it was thawed slowly at room temperature in lukewarm water
until sample was completely liquid. Samples were stored on ice
until needed. The serum samples were first centrifuged at
2,000.times.g for 30 minutes to remove cell debris. Next, the
supernatant containing the cell-free serum was transferred to a
fresh container and hold on ice until precipitation.
[0079] For exosome precipitation, 100 .mu.L of cell-free serum was
transferred to a new tube and combined with the PEG precipitation
reagent.
[0080] In this experiment, polyethylene glycol PEG 6000 was used,
at six percentages: 3%, 4%, 5%, 6%, 7%, 8% (final PEG percent, upon
mixing with the biological samples).
[0081] The samples (PEG+serum) were incubated at 4.degree. C. for 1
h, then the samples were spun down at 10,000 g for 20 min. The
supernatant was discarded, and the exosome pellet was resuspended
in PBS and number of exosomes was quantified on a Nanosight LM10
instrument. The number of exosomes in the samples (30-150 nm size
range) was compared to the number of nanoparticles in the same size
range in the original total serum sample to analyze efficiency of
recovery.
[0082] Results are shown in FIG. 6. As can be seen, PEG6000 at all
concentrations from 3%-8% was capable of isolating exosomes from
serum samples with high efficiency. PEG at 3% and 4% was somewhat
less efficient compared to PEG at 5-8%. Recovery of exosomes was
essentially quantitative starting from 4% PEG6000 as seen from
comparison with the total serum sample. In a similar fashion,
exosomes can be isolated from any other type of body or biological
fluid, in the wide range of sample volume inputs.
Example 6
Comparison of Small Scale vs Large Scale Exosome Isolation, and
Sample to Sample Variation
[0083] Comparison of small scale vs large scale exosome isolation
from blood serum was performed, and sample to sample variation was
studied. Human blood serum samples were removed from storage and
placed on ice. When serum was frozen, it was thawed slowly at room
temperature in lukewarm water until sample was completely liquid.
Samples were stored on ice until needed. Serum samples were first
centrifuged at 2,000.times.g for 30 minutes to remove cell debris.
Next, the supernatant containing the cell-free serum was
transferred to a fresh container and held on ice until
precipitation.
[0084] For exosome precipitation, 100 .mu.L or 1 mL of cell-free
blood serum was transferred to a new tube and combined with the PEG
precipitation reagent.
[0085] In this experiment, 5% PEG 6000 was used. The samples
(PEG+serum) were incubated at 4.degree. C. for 1 h, then the
samples were spun down at 10,000 g for 10 min. The supernatant was
discarded, and the exosome pellet resuspended in PBS and the number
of exosomes was quantified on a Nanosight LM10 instrument. The
number of exosomes in the samples (30-150 nm size range) was
compared to the number of nanoparticles in the same size range in
the original total serum sample to analyze efficiency of
recovery.
[0086] Results are shown in FIG. 7. As can be seen, 5% PEG6000
enabled efficient isolation of exosomes from the serum for both 100
.mu.L sample input (small scale) as well as 1 mL input (large
scale). In both cases, isolation efficiency is very high. This
experiment was carried out in duplicate, and the variation between
replicates was relatively small, indicating that the process of
PEG-mediated exosomes isolation is robust. In a smilar fashion,
isolatin of exosomes from other biological fluids can be performed,
for any sample volume inputs (typically ranging from microliters to
milliliters, but in certain cases up to liters--especially for cell
culture media).
Example 7
Isolation of the Exosomal RNA Cargo and Analysis by qRT-PCR
[0087] Following exosomes isolation from serum by PEG6000 (6%, 8%
or 10%), total exosomal RNA was extracted and qRT-PCR analysis was
performed. The human blood serum samples were removed from storage
and placed on ice. When serum was frozen it was thawed slowly at
room temperature in lukewarm water until sample was completely
liquid. Samples were stored on ice until needed. The serum samples
were first centrifuged at 2,000.times.g for 30 minutes to remove
cell debris. Next, the supernatant containing the cell-free serum
was transferred to a fresh container and hold on ice until
precipitation.
[0088] For exosome precipitation, 100 .mu.L of cell-free serum was
transferred to a new tube and combined with the PEG precipitation
reagent. In this experiment, polyethylene glycol PEG 6000 was used,
at three percentages: 6%, 8%, 10%. The samples (PEG+serum) were
incubated at 4.degree. C. for 1 h, then the samples were spun down
at 10,000 g for 20 min. The supernatant was discarded, and the
exosome pellet was resuspended in PBS. RNA was isolated using the
miRVana.TM. PARIS (Life Technologies) serum protocol and then
qRT-PCR analysis was performed for GAPDH mRNA and miR26a.
[0089] Results are shown in FIG. 8. PEG6000 at 6%-10% was capable
of isolating exosomes from the serum samples with high efficiency
as seen from comparison with the total serum sample. RNA isolation
using the miRVana.TM. PARIS kit was successful. Both GAPDH and
miR26a quantification by TaqMan.RTM. assays was robust and
efficient.
[0090] The representative analysis of the exosome sample derived
from human blood serum is shown in FIG. 9. Exosomes isolated from
100 uL of serum by 8% PEG6000 were resuspended in 25 .mu.L of PBS,
diluted 1000 fold and analyzed using a Nanosight LM10 instrument.
The X axis depicts size distribution of the detected nanoparticles
(in nanometers). The Y axis depicts concentration of nanoparticles
per mL of sample (.times.10.sup.6). As can be seen, majority of
microvesicles isolated by PEG precipitation are in the size range
typical for exosomes: 30-150 nm.
Example 8
Electron Microscopy Analysis of Exosomes Isolated from Three Cell
Lines with 10% PEG6000
[0091] Exosomes were isolated from Ramos, Sudh14, SW480 and cell
culture media. 5 mL cell media aliquots (after 30 min 2000 g
centrifugation to remove cell debris) were combined with 2.5 mL
(1/2 vol) of 30% (w/v) PEG6000 stock solution, resulting in 10%
final PEG concentration. The samples (PEG6000+cell media) were
mixed and incubated at 4.degree. C. for .about.14 h, then the
samples were spun down at 10,000 g for 1 h. The supernatant was
discarded, and the exosome pellet was used for downstream analysis
by Electron Microscopy (EM)--in the unlabeled form, and also
labeled with two types of antibodies conjugated to gold
nanoparticles.
[0092] For immunolabelling, exosome loading was precipitated
undiluted at room temperature for 15 min to grids. Next, blocking
with 0.5% BSA was performed for 10 min. Labeling with anti-CD81 and
anti-CD63 antibodies (the standard exosomal surface markers) was
carried out for 30 min. Following washing steps, Prot A Au 10 nm
were added and incubated for 15 min. After PBS and water wash
steps, embedding in 0.3% Uranyl acetate in methyl cellulose was
finally performed, followed by Electron Microscopy analysis.
[0093] The results of analysis of immunolabeled, negative stain
exosomes--recovered from the media of three cell lines with the
PEG6000 reagent, are shown in FIG. 10. Ramos (unlabeled and labeled
with anti-CD81 antibodies), Sudh14 (labeled with anti-CD63
antibodies), SW480 (labeled with anti-CD63 and anti-CD81
antibodies) exosomes are shown. Exosomes are in the 100 nm range,
and gold particles (attached to antibodies) .about.10 nm in
size.
[0094] The exosomes recovered with 10% PEG6000 have typical
appearance and size, and immunolabeling with anti-CD81 and
anti-CD63 antibodies (which are well known exosomal
markers)--clearly demonstrate that PEG6000 enabled isolation of
clean population of high quality exosomes.
Example 9
Comparison of RNA and Protein Content of Exosomes Isolated from
HeLa Cell Culture Media with 10% PEG6000 Versus Ultracentrifugation
Protocol
[0095] Exosomes were isolated from HeLa cell culture media: 1 mL
cell media aliquots (after 30 min 2000 g centrifugation to remove
cell debris) were combined with 0.5 mL of 30% PEG6000 stock
solution (w/v), resulting in 10% final PEG concentration. The
samples (PEG6000+cell media) were carefully mixed and incubated at
4.degree. C. for .about.14 h, then the samples were spun down at
10,000 g for 1 h. The supernatant was discarded, the exosome pellet
resuspended in PBS buffer and analyzed for presence of typical RNA
and protein markers. Exosomes were also recovered from the same
starting HeLa cell culture media samples, following the standard
ultracentrifugation protocol with sucrose gradient (C. Thery et
al., Current Protocols in Cell Biol (2006) 3.22.1-3.22.29)--for
comparison purposes.
[0096] First, presence of well known CD63 exosomal marker in the
samples obtained with PEG6000 and ultracentrifugation was analyzed
by Western Blots. Exosome samples were mixed with 2.times.
non-reducing Tris-glycine SDS sample buffer (Novex.RTM.), then
heated at 75.degree. C. for 5 min and loaded onto a 1.5 mm.times.15
well 4-20% Tris-Glycine gel (Novex.RTM.). Benchmark prestained
protein ladder (Invitrogen) was added to one well as a control to
monitor the molecular weight of the protein samples. The gel was
run under denaturing conditions at 150 V for 1.5 h then transferred
to a membrane using the iBlot instrument (Life Technologies). After
transfer, the membranes were processed on the BenchPro.RTM. 4100
(Life Technologies) with CD63 antibody diluted 100.times. to 20 ml
(Abcam). The WesternBreeze Chemiluminescence kit was utilized on
the next step, membranes were exposed to X-ray film for 1-10 min
and the film was analyzed.
[0097] The results are shown in FIG. 11 (left panel), in
triplicate. Exosomes isolated with ultracentrifugation protocol and
10% PEG6000 were both positive for CD63 marker, and the differences
were marginal if any. This indicates that polyethyleneglycol (PEG),
of optimized length and percentage, allows recovery of high quality
exosomes from the biological samples.
[0098] Next, RNA was isolated from the exosomes obtained with
PEG6000 and ultracentrifugation, using mirVana.TM. Paris kit (Life
Technologies), and the presence of certain RNA was analyzed by
Reverse transcription and Quantitative real-time PCR (qRT-PCR).
Reverse Transcription (RT) Master Mix was prepared for each sample
using the TaqMan.RTM. MicroRNA Reverse Transcription Kit reagents
and protocol (Applied Biosystems) with gene specific RT primers for
the RNA targets. 10 .mu.l of the RT Master Mix was added to
corresponding wells in a 96-well plate, and 5 .mu.l of each sample
was added to the master mix. Plates were covered with adhesive
(non-optical) cover and spun down to remove air bubbles then placed
into a 9700 thermocycler and incubated as follows: 4.degree. C. for
5 min; 16.degree. C. for 30 min; 42.degree. C. for 30 min; and
85.degree. C. for 5 min. Reactions were kept at 4.degree. C. until
use.
[0099] qPCR master mixes were prepared for each of five microRNAs
(let7e, miR26a, miR16, miR24 and miR451) and two mRNAs (GAPDH and
18S) by combining 5 .mu.l of AB Universal PCR Master Mix II, 3.5
.mu.l of nuclease-free water, and 0.5 .mu.l of the 20.times.
Taqman.RTM. Assay. After mixing, 8 .mu.l of each master mix was
placed into wells in a 384-well plate (enough for triplicate
reactions for each isolation replicate). 2 .mu.l of each RT
reaction was added in triplicate to the master mix of each target
and the plates were sealed with optical adhesive cover. Plate were
spun down to remove air bubbles then placed into a 7900HT
instrument and run using the following thermocycler protocol:
95.degree. C. for 10 min; (95.degree. C. for 15 s; 60.degree. C.
for 60 s) 40 cycles. Once the run was complete, automatic Ct
analysis was performed with SDSv2.3 software and average and
standard deviation was calculated for each set of isolations and
qPCR reactions for each target.
[0100] The results are shown in FIG. 11 (right panel). The levels
of five microRNAs (let7e, miR26a, miR16, miR24 and miR451) and two
mRNAs (GAPDH and 18S) (earlier reported to be present in exosomes
by Valadi et al. (Valadi et al., Nature Cell Biol 9 (2007)
654-659)), were quantified by qRT-PCR. Exosomes isolated with
ultracentrifugation protocol and 10% PEG6000 both contain all these
RNAs, in very similar levels (PEG recovers somewhat more material).
This indicates that polyethyleneglycol, of optimized length and
percentage, allows recovery of high quality exosomes from cell
media samples.
Example 10
Comparison of RNA and Protein Content of Exosomes Isolated from
Serum with 5% PEG6000 Versus Ultracentrifugation Protocol
[0101] Exosomes were isolated from human serum: 100 .mu.L serum
(after 30 min 2000 g centrifugation to remove cell debris) were
combined with 20 .mu.L of 30% (w/v) PEG6000 stock solution,
resulting in 5% final PEG concentration. The samples
(PEG6000+serum) were carefully mixed and incubated at 4.degree. C.
for .about.30 min, then the samples were spun down at 10,000 g for
10 min. The supernatant was discarded, the exosome pellet
resuspended in PBS buffer and analyzed for presence of typical RNA
and protein markers. Exosomes were also recovered from the same
starting serum samples, following the standard ultracentrifugation
protocol with sucrose gradient (C. Thery et al., Current Protocols
in Cell Biol (2006) 3.22.1-3.22.29)--for comparison purposes.
[0102] First, presence of well known CD63 exosomal marker in the
samples obtained with PEG6000 and ultracentrifugation was analyzed
by Western Blots. Protocol was the same as described in Example 9.
The results are shown in FIG. 12 (left panel), in triplicate.
Exosomes isolated with ultracentrifugation protocol and 5% PEG6000
were both positive for CD63 marker, and the differences were
marginal if any. This indicates that polyethyleneglycol, of
optimized length and percentage, allows recovery of high quality
exosomes from the biological samples.
[0103] Next, RNA was isolated from the exosomes obtained with
PEG6000 and ultracentrifugation, using mirVana.TM. Paris kit (Life
Technologies), and the presence of certain RNA was analyzed by
Reverse transcription and Quantitative real-time PCR (qRT-PCR).
Reverse Transcription (RT) Protocol was the same as described in
Example 9. The results are shown in FIG. 12 (right panel). The
levels of five microRNAs (let7e, miR26a, miR16, miR24 and miR451)
and two mRNAs (GAPDH and 18S) (earlier reported to be present in
exosomes by Valadi et al. (Valadi et al., Nature Cell Biol 9 (2007)
654-659)), were quantified by qRT-PCR. Exosomes isolated with
ultracentrifugation protocol and 5% PEG6000 both contain all these
RNAs, in very similar levels (PEG recovers somewhat more material).
This indicates that polyethyleneglycol, of optimized length and
percentage, allows recovery of high quality exosomes from blood
serum samples.
Example 11
Recovery of Exosomes from Urine with 10-20% PEG6000
[0104] Exosomes were isolated from human urine: 5 mL urine samples
from three healthy donors (after 30 min 2000 g centrifugation to
remove cell debris) were combined with PEG6000 stock solution,
resulting in 10%, 12%, 15% and 20% final PEG concentration. The
samples (PEG6000+urine) were carefully mixed and incubated at
4.degree. C. for .about.14 h, then the samples were spun down at
10,000 g for 1 h. The supernatant was discarded, and the exosome
pellet was resuspended in PBS and the number of exosomes was
quantified on a Nanosight LM10 instrument. The number of exosomes
in the samples (30-150 nm size range) was compared to the number of
nanoparticles in the same size range in the original whole urine
sample and cell-free sample (whole urine, subjected to 30 min 2000
g centrifugation to remove cell debris)--to analyze the efficiency
of recovery. The entire population of nanoparticles in the size
range 0-2000 nm was tracked as well.
[0105] The results are shown in FIG. 13. For all three donors (A,
B, C) PEG6000 at concentrations 10-20% allows very efficient
recovery of exosomes from urine. Higher PEG concentrations, in
particular 15-20%, results in more efficient extraction.
Example 12
Recovery of Exosomes from Amniotic Fluid with 2-14% PEG6000
[0106] Exosomes were isolated from human amniotic fluid: 1 mL
amniotic fluid samples (after 30 min 2000 g centrifugation to
remove cell debris) were combined with PEG6000 stock solution,
resulting in 2%, 4%, 6% , 8%, 10%, 12% and 14% final PEG
concentration. The samples (PEG6000+amniotic fluid) were carefully
mixed and incubated at 4.degree. C. for .about.14 h, then the
samples were spun down at 10,000 g for 1 h. The supernatant was
discarded, and the exosome pellet was resuspended in PBS and the
number of exosomes was quantified on a Nanosight LM10 instrument.
As always, following the manufacturer's protocol the concentrated
samples were diluted to ensure the concentration range of
nanoparticles is suitable for accurate analysis by Nanosight LM10
instrument. The number of exosomes in the samples (30-150 nm size
range) was compared to the number of nanoparticles in the same size
range in the original amniotic fluid sample (subjected to 30 min
2000 g centrifugation to remove cell debris)--to analyze the
efficiency of recovery. The entire population of nanoparticles in
the size range 0-2000 nm was tracked as well.
[0107] The results are shown in FIG. 14. PEG6000 at concentrations
2-14% allows very efficient recovery of exosomes from amniotic
fluid. In this particular setting, the maximal recovery was
accomplished with 4-6% PEG6000 (w/v).
Example 13
Recovery of Exosomes from Cerebrospinal Fluid (CSF) with 2-14%
PEG6000
[0108] Exosomes were isolated from human cerebrospinal fluid (CSF):
1 mL CSF samples (after 30 min 2000 g centrifugation to remove cell
debris) were combined with PEG6000 stock solution, resulting in 2%,
4%, 6%, 8%, 10%, 12% and 14% final PEG concentration (w/v). The
samples (PEG6000+CSF) were carefully mixed and incubated at
4.degree. C. for .about.14 h, then the samples were spun down at
10,000 g for 1h. The supernatant was discarded, and the exosome
pellet was resuspended in PBS and the number of exosomes was
quantified on a Nanosight LM10 instrument. The number of exosomes
in the samples (30-150 nm size range) was compared to the number of
nanoparticles in the same size range in the original CSF sample
(subjected to 30 min 2000 g centrifugation to remove cell
debris)--to analyze the efficiency of recovery. The entire
population of nanoparticles in the size range 0-2000 nm was tracked
as well.
[0109] The results are shown in FIG. 15. PEG6000 at concentrations
2-14% allows very efficient recovery of exosomes from CSF. In this
particular setting, the maximal recovery was accomplished with
8-14% PEG6000.
Example 14
Recovery of Exosomes from Plasma with 5% PEG6000 and Effects of
Proteinase K on Removal of Protein from the Sample
[0110] Exosomes were isolated from human plasma: 100 .mu.L plasma
samples from three healthy donors (after 30 min 2000 g
centrifugation to remove cell debris) were combined with 1/5th
volume of 30% (w/v) PEG6000 stock solution, resulting in 5% final
PEG concentration. The samples (PEG6000+plasma) were carefully
mixed and incubated at 4.degree. C. for 30 min, then the samples
were spun down at 10,000 g for 30 min. The supernatant was
discarded, and the exosome pellet was resuspended in PBS and the
number of exosomes was quantified on a Nano sight LM10
instrument.
[0111] Another set of 100 .mu.L plasma samples from three healthy
donors (after 30 min 2000 g centrifugation to remove cell debris)
was treated with Proteinase K (in order to remove protein from the
sample and thus enhance the purity of the exosomes), and then
combined with 1/5th volume of 30% PEG6000 stock solution, resulting
in 5% final PEG concentration--for precipitation of exosomes,
following above protocol.
[0112] The number of exosomes in the samples (30-150 nm size
range), obtained by Nanosight LM10 quantification, was compared to
the number of nanoparticles in the same size range in the original
plasma sample--to analyze the efficiency of recovery. Data for all
samples were normalized for sample volume and dilution factor.
[0113] The results are shown in FIG. 16 (Left panel). For all three
donors (donor 1, 2, 3) 5% PEG6000 allows very efficient, near
quantitative, recovery of exosomes from plasma. Proteinase K
treatment does not affect the number of exosomes recovered, but
removes extracellular proteins that might be present at low levels
in the preparations. All the proteins and protein complexes are
typically below 20 nm in size, and thus undetectable with Nanosight
instrument, which reliably measures only the larger nanoparticles,
in the range 30-1000 nm. The fact that Proteinase K treatment did
not result in reduction of the nanoparticle numbers provides
additional proof that what was traced with the Nanosight LM10
instrument is truly of exosome origin, and not the protein
complexes.
[0114] Next, the original plasma sample was treated with Proteinase
K and compared to the untreated plasma sample by western blot
analysis, using anti-Albumin antibodies--to monitor levels of
Albumin, the major protein found in blood. The results are shown in
FIG. 16 (Right panel). Lanes 1 and 2--untreated plasma samples;
lanes 3-6: plasma treated with Proteinase K.
[0115] Results clearly indicate that proteinase K treatment allows
elimination of the majority of protein from the plasma sample. With
subsequent PEG precipitation, it would be possible to recover
exosomes of higher purity, containing minimal--if any--protein
contamination. This additional step of Proteinase K treatment might
be utilized when ultra pure exosome preparations are required, and
in case body fluids rich in protein content are utilized- thus
making it challenging to recover clean exosome population.
[0116] Further exemplary embodiments are provided in the following
numbered clauses.
[0117] 1. A method for the isolation of exosomes from a biological
fluid sample comprising: [0118] a) adding a volume-excluding
polymer to the biological fluid sample, [0119] b) incubating the
biological fluid sample with the volume-excluding polymer; and
[0120] d) isolating the precipitated exosomes from the biological
fluid sample.
[0121] 2. The method of clause 1, wherein the molecular weight of
the volume-excluding polymer is from 1000 to 1,000,000 daltons.
[0122] 3. The method of clause 2, wherein the molecular weight of
the volume-excluding polymer is from 3000 to 10000 daltons.
[0123] 4. The method of clause 3, wherein the molecular weight of
the volume-excluding polymer is from 4000 to 8000 daltons.
[0124] 5. The method of clause 4, wherein the concentration of
volume-excluding polymer upon mixing with the biological fluid is
from 1% to 90%.
[0125] 6. The method of clause 5, wherein the concentration of
volume-excluding polymer upon mixing with the biological fluid is
from 3% to 15%.
[0126] 7. The method of clause 6, wherein the concentration of
volume-excluding polymer upon mixing with the biological fluid is
from 4% to 12%.
[0127] 8. The method of clause 1, further comprising the step of
clarifying the biological fluid sample before adding the
volume-excluding polymer.
[0128] 9. The method of clause 1 further comprising the step of
isolating nucleic acid and protein from the exosomes.
[0129] 10. The method of clause 1, wherein the biological fluid is
from prokaryotes, eukaryotes, bacteria, fungi, yeast,
invertebrates, vertebrates, reptiles, fish, insects, plants or
animals.
[0130] 11. The method of clause 1, wherein the biological fluid is
selected from the group consisting of blood serum, plasma, whole
blood, urine, saliva, breast milk, tears, sweat, joint fluid,
cerebrospinal fluid, semen, vaginal fluid, ascitic fluid, amniotic
fluid, and cell culture media.
[0131] 12. The method of clause 1, wherein the biological fluid
sample is from a human.
[0132] 13. The method of clause 1, further comprising fractionating
the isolated exosomes.
[0133] 14. The method of clause 1, wherein the volume-excluding
polymer is polyethylene glycol, dextran, dextran sulfate, dextran
acetate, polyvinyl alcohol, polyvinyl acetate, or polyvinyl
sulfate.
[0134] 15. The method of clause 14, wherein the volume-excluding
polymer is polyethylene glycol.
[0135] 16. The method of clause 1, further comprising contacting
the biological fluid sample prior to the addition of the
volume-excluding polymer with a protease, under conditions suitable
for digestion of protein.
[0136] 17. The method of clause 16, wherein the protease is
proteinase K.
[0137] 18. A method for the isolation of exosomes from a biological
tissue sample comprising: [0138] a) lysing the biological tissue
sample, [0139] b) clarifying the lysed sample [0140] c) adding a
volume-excluding polymer to the clarified sample, [0141] d)
incubating the clarified sample with the volume-excluding polymer,
and [0142] e) isolating the precipitated exosomes.
[0143] 19. The method of clause 18, wherein the molecular weight of
the volume-excluding polymer is from 1000 to 1,000,000 daltons.
[0144] 20. The method of clause 19, wherein the molecular weight of
the volume-excluding polymer is from 3000 to 10000 daltons.
[0145] 21. The method of clause 20, wherein the molecular weight of
the volume-excluding polymer is from 4000 to 8000 daltons.
[0146] 22. The method of clause 18, wherein the concentration of
volume-excluding polymer upon mixing with the biological sample is
from 1% to 90%.
[0147] 23. The method of clause 22, wherein the concentration of
volume-excluding polymer upon mixing with the biological sample is
from 3% to 15%.
[0148] 24. The method of clause 23, wherein the concentration of
volume-excluding polymer upon mixing with the biological sample is
from 4% to 12%.
[0149] 25. The method of clause 18, wherein the volume-excluding
polymer is polyethylene glycol, dextran, dextran sulfate, dextran
acetate, polyvinyl alcohol, polyvinyl acetate, or polyvinyl
sulfate.
[0150] 26. The method of clause 25, wherein the volume-excluding
polymer is polyethylene glycol.
[0151] 27. The method of clause 18 further comprising the step of
isolating nucleic acid and protein from the exosomes.
[0152] 28. The method of clause 18, wherein the biological sample
is from prokaryotes, eukaryotes, bacteria, fungi, yeast,
invertebrates, vertebrates, reptiles, fish, insects, plants or
animals.
[0153] 29. The method of clause 18, wherein the biological sample
is selected from the group consisting of surgical samples, biopsy
samples, tissues, feces, plant tissue, insect tissue, fungi,
bacteria, parasites and cultured cells.
[0154] 30. The method of clause 18, further comprising contacting
the biological tissue sample with a protease, under conditions
suitable for digestion of protein, prior to the addition of the
volume-excluding polymer.
[0155] 31. The method of clause 30, wherein the protease is
proteinase K.
[0156] All publications, patents and patent applications mentioned
in this Specification are indicative of the level of skill of those
of ordinary skill in the art and are herein incorporated by
reference to the same extent as if each individual publication,
patent, or patent applications was specifically and individually
indicated to be incorporated by reference.
[0157] The invention being thus described, it will be obvious that
the same may be varied in many ways. Such variations are not to be
regarded as a departure from the spirit and scope of the invention,
and all such modifications as would be obvious to one of ordinary
skill in the art are intended to be included within the scope of
the following claims.
* * * * *