U.S. patent application number 16/470180 was filed with the patent office on 2020-01-23 for methods of measuring exosomes using intrinsic fluorescence.
The applicant listed for this patent is Codiak BioSciences, Inc.. Invention is credited to Young Jun CHOI, Damian J. HOUDE, John D. KULMAN, Douglas E. WILLIAMS.
Application Number | 20200025685 16/470180 |
Document ID | / |
Family ID | 62559176 |
Filed Date | 2020-01-23 |
![](/patent/app/20200025685/US20200025685A1-20200123-D00001.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00002.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00003.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00004.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00005.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00006.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00007.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00008.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00009.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00010.png)
![](/patent/app/20200025685/US20200025685A1-20200123-D00011.png)
View All Diagrams
United States Patent
Application |
20200025685 |
Kind Code |
A1 |
HOUDE; Damian J. ; et
al. |
January 23, 2020 |
METHODS OF MEASURING EXOSOMES USING INTRINSIC FLUORESCENCE
Abstract
Described herein are novel rapid and reliable methods of
detection of extracellular vesicles and quantifying extracellular
vesicle concentrations and absolute number from various sources,
including raw cell harvest. The methods described herein comprise
detection of intrinsic fluorescence of extracellular vesicles in
biological samples. Extracellular vesicles analyzed by the methods
of this application have a stereotypical elution profile distinct
from known contaminants. The methods described herein are a
significant improvement over the state of the art and fulfills an
unmet need in the field of extracellular vesicle manufacturing and
quality control.
Inventors: |
HOUDE; Damian J.; (Plymouth,
MA) ; CHOI; Young Jun; (Cambridge, MA) ;
KULMAN; John D.; (Belmont, MA) ; WILLIAMS; Douglas
E.; (Boston, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Codiak BioSciences, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
62559176 |
Appl. No.: |
16/470180 |
Filed: |
December 14, 2017 |
PCT Filed: |
December 14, 2017 |
PCT NO: |
PCT/US17/66324 |
371 Date: |
June 14, 2019 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62434985 |
Dec 15, 2016 |
|
|
|
62542697 |
Aug 8, 2017 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 21/6486 20130101;
G01N 21/76 20130101 |
International
Class: |
G01N 21/64 20060101
G01N021/64; G01N 21/76 20060101 G01N021/76 |
Claims
1. A method of detecting extracellular vesicles, comprising:
obtaining a sample comprising extracellular vesicles; and
determining an intrinsic fluorescence emission signal from said
sample, wherein said intrinsic fluorescence emission signal is
indicative of the presence of said extracellular vesicles within
said sample.
2. The method of claim 1, wherein said intrinsic fluorescence
emission signal is generated using an excitation wavelength ranging
from 450 nm to 650 nm and an emission wavelength that is longer
than the excitation wavelength and ranging from 470 nm to 670
nm.
3. The method of claim 2, wherein said intrinsic fluorescence
emission signal is determined at an emission wavelength range of
500-600 nm.
4. The method of claim 3, wherein said intrinsic fluorescence
emission signal is determined at an emission wavelength range of
550-590 nm.
5. The method of claim 3, wherein said intrinsic fluorescence
emission signal is determined at an emission wavelength of 573
nm.
6. The method of claim 2, wherein said intrinsic fluorescence
emission signal is determined at an excitation wavelength range of
500-600 nm.
7. The method of claim 6, wherein said intrinsic fluorescence
emission signal is determined at an excitation wavelength range of
530-570 nm.
8. The method of claim 7, wherein said intrinsic fluorescence
emission signal is determined at an excitation wavelength of 556
nm.
9. The method of any one of the preceding claims wherein said
intrinsic fluorescence emission signal is determined at an
excitation wavelength of 550 nm and an emission of 570 nm.
10. The method any one of the preceding claims, wherein said sample
is separated into fractions prior to determining said intrinsic
fluorescence emission signal.
11. The method of claim 10, wherein said separation comprises a
column chromatography separation step.
12. The method of claim 10, wherein said separation comprises two
chromatography separation steps.
13. The method of claim 11 or 12, wherein one or both of said
chromatography steps is size-exclusion chromatography.
14. The method of claim 11 or 12, wherein one or both of said
chromatography steps is ion-exchange chromatography.
15. The method of claim 13, wherein said ion-exchange
chromatography is strong anion-exchange chromatography.
16. The method of claim 12, where said two chromatography steps are
anion-exchange chromatography and size-exclusion
chromatography.
17. The method of claim 16, wherein said anion-exchange
chromatography precedes said size-exclusion chromatography.
18. The method of any one of claims 10-15, wherein said intrinsic
fluorescence emission signal is determined using a flow cell.
19. The method of any one of the preceding claims, wherein said
sample is subjected to a filtration step prior to determining said
intrinsic fluorescence emission signal.
20. The method of any one of the preceding claims, wherein said
sample is subjected to a centrifugation separation step prior to
determining said intrinsic fluorescence emission signal.
21. The method of any one of the preceding claims, wherein said
sample is subjected to a sucrose density gradient step prior to
determining said intrinsic fluorescence emission signal.
22. The method of any one of the preceding claims, wherein said
sample is a subjected to a separation step comprising use of a
density gradient prior to determining said intrinsic fluorescence
emission signal.
23. The method of any one of the preceding claims, wherein said
sample is derived from a cell culture.
24. The method of claim 23, wherein said cell culture comprises
human embryonic kidney cells, mesenchymal stem cells or neuronal
cells.
25. The method of any one of the preceding claims, wherein said
sample is derived from a body fluid of an animal.
26. The method of any one of the preceding claims, comprising
determining an amount of said extracellular vesicles within said
sample comprising comparing said intrinsic fluorescence emission
signal to the intrinsic fluorescence emission signal of a
standard.
27. The method of any one of the preceding claims, comprising
quantifying the amount of said extracellular vesicles within said
sample, based upon the area under of the curve of a chromatogram
comprising said intrinsic fluorescence emission signal.
28. The method of any one of the preceding claims, comprising
determining an amount of said extracellular vesicles within said
sample comprising comparing said intrinsic fluorescence emission
signal to the luminescence signal of a standard.
29. The method of claim 28, wherein said standard is measured using
a luminescence proximity assay
30. The method of claim 29, wherein said standard is calculated
based on the relative abundance of one or more exosome-associated
proteins.
31. The method of claim 30, wherein said method is an AlphaScreen
assay.
32. The method of claims 30-31, wherein said one or more
exosome-associated proteins are selected from the group consisting
of CD9, CD81, CD63, and combinations thereof.
33. The method of any one of the preceding claims, wherein said
extracellular vesicle is an exosome.
34. The method of any one of claims 1-33, wherein said
extracellular vesicle is a nanovesicle.
35. The method of any one of the preceding claims, wherein said
extracellular vesicle comprises a therapeutic payload.
36. A method of detecting extracellular vesicles, comprising:
obtaining a sample comprising extracellular vesicles; and
determining a luminescence proximity signal from said sample,
wherein said luminescence proximity signal is indicative of the
presence of said extracellular vesicles within said sample.
37. The method of claim 36, wherein said luminescence proximity
signal is determined at an excitation wavelength of 680 nm.
38. The method of claim 37, wherein said luminescence proximity
signal is determined at an emission wavelength of 520-620 nm.
39. The method of claim 38, wherein said signal is determined by
excitation of ambient oxygen to induce chemiluminescence in a
fluorophore.
40. The method of claim 39, wherein said method is an
AlphaScreen.TM. assay.
41. The method of claim 40, wherein said method comprises a donor
substrate and an acceptor substrate conjugated to two binding
molecules.
42. The method of claim 41, wherein said binding molecules comprise
antibodies or fragments thereof.
43. The method of claim 42, wherein said antibodies recognize one
or more exosome-associated proteins.
44. The method of claim 43, wherein said one or more
exosome-associated proteins is selected from the group consisting
of CD9, CD63, CD81, and combinations thereof.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit under 35 U.S.C.
.sctn. 119(e) of U.S. Provisional Application No. 62/434,985 filed
on 15 Dec. 2016, and U.S. Provisional Application No. 62/542,697
filed on Aug. 8, 2017, both of which are incorporated by reference
herein in their entirety.
BACKGROUND OF THE INVENTION
Description of the Related Art
[0002] Exosomes and other small extracellular vesicles are
increasingly well-recognized biological particles. Although
extracellular vesicles are beginning to be used in commercial
processes and produced in industrial quantities, methods of
accurately and rapidly measuring extracellular vesicle
concentration and absolute number remain scarce. Current approaches
for the detection, isolation and purification of extracellular
vesicles derived from cell culture or other biological samples
requires laborious and time consuming methods. For example, current
ultra-centrifugation protocols are commercially unreproducible, as
they produce a heterogeneous mix of extracellular vesicles, other
cellular vesicles and macromolecular complexes and can lead to
vesicle aggregation. Therefore, novel methods for efficient,
low-cost and reliable purification and quantification of
extracellular vesicles are needed.
SUMMARY OF THE INVENTION
[0003] Disclosed herein are methods for the detection of
extracellular vesicles comprising detecting intrinsic fluorescence
of the extracellular vesicles without the use of additional dyes,
fluorophores, markers, or imaging compounds. In certain
embodiments, this application describes methods of detecting
extracellular vesicles comprising, obtaining a sample comprising
extracellular vesicles and determining an intrinsic fluorescence
emission signal from the sample, wherein the intrinsic fluorescence
emission signal is indicative of the presence of the extracellular
vesicles within the sample. In an aspect, the intrinsic
fluorescence emission signal is generated using an excitation
wavelength ranging from 450 nm to 650 nm and an emission wavelength
that is longer than the excitation wavelength and ranging from 470
nm to 670 nm. In an aspect, the intrinsic fluorescence emission
signal is determined at an emission wavelength range of 500-600 nm.
In an aspect, the intrinsic fluorescence emission signal is
determined at an emission wavelength range of 550-590 nm. In an
aspect, the intrinsic fluorescence emission signal is determined at
an emission wavelength of 573 nm. In an aspect, the intrinsic
fluorescence emission signal is determined at an excitation
wavelength range of 500-600 nm. In an aspect, the intrinsic
fluorescence emission signal is determined at an excitation
wavelength range of 530-570 nm. In an aspect, the intrinsic
fluorescence emission signal is determined at an excitation
wavelength of 556 nm. In an aspect, the intrinsic fluorescence
emission signal is determined at an excitation wavelength of 550 nm
and an emission of 570 nm.
[0004] In certain aspects, the sample is separated into fractions
prior to determining said intrinsic fluorescence emission signal.
In certain aspects, separation comprises a column chromatography
separation step. In certain aspects, separation comprises two
column chromatography separation steps. In an aspect, one or both
of the chromatography steps is size exclusion chromatography. In an
aspect, one or both of the chromatography is ion exchange
chromatography. In an aspect, said ion exchange chromatography is
strong anion exchange chromatography. In an aspect, said two
chromatography steps are anion-exchange chromatography and
size-exclusion chromatography. In an aspect, said anion-exchange
chromatography precedes said size-exclusion chromatography.
[0005] In certain aspects, the said intrinsic fluorescence emission
signal is determined using a flow cell. In certain aspects, the
sample is subjected to a filtration step prior to determining said
intrinsic fluorescence emission signal. In certain aspects, the
sample is subjected to a centrifugation separation step prior to
determining said intrinsic fluorescence emission signal. In certain
aspects, the sample is subjected to a sucrose density gradient step
prior to determining said intrinsic fluorescence emission signal.
In certain aspects, the sample is subjected to a separation step
comprising use of a density gradient medium prior to determining
said intrinsic fluorescence emission signal.
[0006] In certain aspects, the sample is derived from a cell
culture. In certain aspects, the cell culture comprises human
embryonic kidney cells, mesenchymal stem cells or neuronal cells.
In certain aspects, the sample is derived from a body fluid of an
animal.
[0007] In certain aspects, the method comprises determining an
amount of the extracellular vesicles within the sample comprising
comparing the intrinsic fluorescence emission signal to the
intrinsic fluorescence emission signal of a standard. In certain
aspects, the method comprises quantifying the amount of
extracellular vesicles within the sample, based upon the area under
of the curve of a chromatogram comprising the intrinsic
fluorescence emission signal. In certain aspects, the method
comprises determining an amount of the extracellular vesicles
within the sample comprising comparing the intrinsic fluorescence
emission signal to the luminescence signal of a standard. In
certain aspects, the luminescence signal is derived from a
luminescence proximity assay. In certain aspects, the luminescence
proximity assay is an AlphaScreen.TM. assay recognizing
exosome-associated proteins such as CD9, CD63, and/or CD81. In
certain aspects, the luminescence proximity assay is used to detect
exosomes independent of their intrinsic fluorescence.
[0008] In certain aspects, the extracellular vesicle is an exosome.
In certain aspects, the extracellular vesicle is a nanovesicle.
[0009] In certain aspects, the extracellular vesicle comprises a
therapeutic payload.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
[0010] These and other features, aspects, and advantages of the
present invention will become better understood with regard to the
following description, and accompanying drawings, where:
[0011] FIG. 1A illustrates a comparison of the absorbance profiles
from strong anion-exchange chromatography (AEX) of HEK293SF cell
harvest (red trace) and Optiprep.TM. density gradient purified
exosomes (black trace). Representative transmission electron
micrograph of the Optiprep.TM. purified exosome stock is shown.
[0012] FIG. 1B is a representative transmission electron micrograph
of the Optiprep.TM. purified exosome stock.
[0013] FIG. 2 is a representative comparison of the spectral
profiles from AEX of HEK293SF cell harvest. AEX was monitored with
both absorbance (254 nm, black trace) and fluorescence detectors.
Fluorescence was monitored with two different excitation (ex) and
emission (em) wavelengths; ex: 280 nm, em: 350 nm (grey trace) and
ex: 556 nm, em: 573 nm (red trace).
[0014] FIG. 3 is a comparison of the fluorescence profiles (ex: 556
nm, em: 573 nm) of density gradient purified exosomes from four
different cell lines: HEK293SF (red trace), HEK293T (black trace),
mesenchymal stem cells (blue trace), and AGE1.HN (grey trace).
[0015] FIG. 4A illustrates optimal fluorescent excitation
wavelengths for detection of Optiprep.TM. purified exosomes. The
emission wavelength was held constant at 573 nm and the excitation
wavelength was scanned from 280 to 560 nm.
[0016] FIG. 4B illustrates optimal fluorescent excitation
wavelengths for detection of Optiprep.TM. purified exosomes. The
excitation wavelength was held constant at 556 nm and the emission
wavelength was scanned from 570 to 700 nm.
[0017] FIG. 5A illustrates characterization of clodronate liposome
control samples. Nanoparticle tracking analysis (NTA) analysis of
clodronate liposomes after filtering through a 0.45 .mu.m filter is
shown.
[0018] FIG. 5B shows a representative transmission electron
micrograph of filtered clodronate liposomes.
[0019] FIG. 6 shows size-exclusion chromatography (SEC) analysis of
liposome and exosome samples monitored by UV at 254 nm.
Optiprep.TM. purified exosome samples (black trace) elute in the
column's void volume (V.sub.o) at approximately 7.5 min. The buffer
(teal trace) shows no interference and the liposome sample (red
trace) shows a broad elution profile.
[0020] FIG. 7 shows representative SEC profiles of buffer (teal
trace), exosome (black trace), and liposome (red trace) samples
with fluorescent detection at ex: 556 nm and em: 573 nm.
[0021] FIG. 8A shows the quantitation of exosomes by intrinsic
exosome fluorescence. Exosome stock sample was generated from
HEK293SF cells and purified by Optiprep.TM. density gradient.
Representative fluorescent traces of the serial dilutions of
exosome stock are shown.
[0022] FIG. 8B shows a linearity trace of the EV fluorescent peak
area versus dilution from stock.
[0023] FIG. 8C illustrates serial dilutions analyzed by NTA and
compared against the fluorescent peak area.
[0024] FIG. 9 shows AEX fluorescence traces of cell culture media
blank (grey trace) and a representative high (black trace) and low
(red trace) cell culture yield process from HEK293SF cells.
[0025] FIG. 10 shows the correlation between exosome quantity as
measured by fluorescence detection at ex: 556 nm and em: 573 nm and
AlphaScreen.TM. using antibodies against CD9 and CD81.
[0026] FIG. 11 shows a schematic for two-dimensional liquid
chromatography.
[0027] FIG. 12 is a representative transmission electron micrograph
of exosomes purified by anion-exchange chromatography or
anion-exchange chromatography followed by size-exclusion
chromatography.
[0028] FIG. 13A is a chromatogram of a dilution series of
Optiprep.TM.-purified exosomes analyzed by AEX at 280 UV/visual
detection at 200.times., 400.times., 800.times. or 1600.times.
dilution.
[0029] FIG. 13B is a chromatogram of a dilution series of
Optiprep.TM.-purified exosomes analyzed by SEC at ex: 556 nm and
em: 573 nm at 200.times., 400.times., 800.times. or 1600.times.
dilution.
[0030] FIG. 14A is a chromatogram of day 7-9 cell culture harvest
analyzed by AEX at 280 UV/visual detection.
[0031] FIG. 14B is a zoomed in chromatogram of day 7-9 cell culture
harvest analyzed by AEX at 280 UV/visual detection.
[0032] FIG. 14C is a chromatogram of day 7-9 cell culture harvest
analyzed by SEC at ex: 556 nm and em: 573 nm.
[0033] FIG. 15A is a chromatogram of Optiprep.TM.-purified exosomes
analyzed by SEC to detect fluorescent signals of proteins (ex:280
nm/em:350 nm), exosome intrinsic fluorescence (ex:556 nm/em:573
nm), and FITC fluorescence (ex:490 nm/em:525 nm).
[0034] FIG. 15B is a chromatogram of Optiprep.TM.-purified exosomes
pre-incubated with an anti-CD81_FITC antibody analyzed by SEC to
detect fluorescent signals of proteins (ex:280 nm/em:350 nm),
exosome intrinsic fluorescence (ex:556 nm/em:573 nm), and FITC
fluorescence (ex:490 nm/em:525 nm).
DETAILED DESCRIPTION
[0035] Advantages and Utility
[0036] Briefly, and as described in more detail below, is a rapid
and reliable method of quantifying extracellular vesicle
concentrations and absolute number from various sources, including
raw cell harvest without the use of additional dyes, fluorophores,
markers, or imaging compounds. Extracellular vesicle detection and
quantification typically relies on nanoparticle tracking assays and
other methods that are heavily dependent on the purity of
extracellular vesicle preparations, and often rely on the addition
of lipophilic dyes or other compounds and/or agents to aid in the
detection of vesicles. Extracellular vesicles analyzed by the
methods of the invention have a stereotypical elution profile as
measured by fluorescence distinct from known contaminants. The
methods described herein are a significant improvement over the
state of the art and fulfill an unmet need in the field of
extracellular vesicle manufacturing and quality control.
[0037] Definitions
[0038] Terms used in the claims and specification are defined as
set forth below unless otherwise specified.
[0039] As used herein, the terms "intrinsic fluorescence,"
"autofluorescence," and "auto-fluorescence" refer to the natural
emission of light by biological structures when they have absorbed
light, and are distinguished from light originating from
artificially added fluorescent markers, dyes, or fluorophores.
[0040] As used herein, the term "extracellular vesicle" refers to a
cell-derived vesicle comprising a membrane that encloses an
internal space. Extracellular vesicles comprise all membrane-bound
vesicles that have a smaller diameter than the cell from which they
are derived. Generally extracellular vesicles range in diameter
from 20 nm to 1000 nm, and may comprise various macromolecular
cargo either within the internal space, displayed on the external
surface of the extracellular vesicle, and/or spanning the membrane.
Said cargo may comprise nucleic acids, proteins, carbohydrates,
lipids, small molecules, and/or combinations thereof. By way of
example and without limitation, extracellular vesicles include
apoptotic bodies, fragments of cells, vesicles derived from cells
by direct or indirect manipulation (e.g., by serial extrusion or
treatment with alkaline solutions), vesiculated organelles, and
vesicles produced by living cells (e.g., by direct plasma membrane
budding or fusion of the late endosome with the plasma membrane).
Extracellular vesicles may be derived from a living or dead
organism, explanted tissues or organs, and/or cultured cells.
[0041] As used herein, the term "nanovesicle" refers to a
cell-derived small (between 20-250 nm in diameter, more preferably
30-150 nm in diameter) vesicle comprising a membrane that encloses
an internal space, and which is generated from said cell by direct
or indirect manipulation such that said nanovesicle would not be
produced by said producer cell without said manipulation.
Appropriate manipulations of said producer cell include but are not
limited to serial extrusion, treatment with alkaline solutions,
sonication, or combinations thereof. The production of nanovesicles
may, in some instances, result in the destruction of said producer
cell. Preferably, populations of nanovesicles are substantially
free of vesicles that are derived from producer cells by way of
direct budding from the plasma membrane or fusion of the late
endosome with the plasma membrane. The nanovesicle comprises lipid
or fatty acid and polypeptide, and optionally comprises a payload
(e.g., a therapeutic agent), a receiver (e.g., a targeting moiety),
a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar
(e.g., a simple sugar, polysaccharide, or glycan) or other
molecules. The nanovesicle, once it is derived from a producer cell
according to said manipulation, may be isolated from the producer
cell based on its size, density, biochemical parameters, or a
combination thereof.
[0042] As used herein, the term "exosome" refers to a cell-derived
small (between 20-300 nm in diameter, more preferably 40-200 nm in
diameter) vesicle comprising a membrane that encloses an internal
space, and which is generated from said cell by direct plasma
membrane budding or by fusion of the late endosome with the plasma
membrane. Generally, production of exosomes does not result in the
destruction of the producer cell. The exosome comprises lipid or
fatty acid and polypeptide, and optionally comprises a payload
(e.g., a therapeutic agent), a receiver (e.g., a targeting moiety),
a polynucleotide (e.g., a nucleic acid, RNA, or DNA), a sugar
(e.g., a simple sugar, polysaccharide, or glycan) or other
molecules. The exosome can be derived from a producer cell, and
isolated from the producer cell based on its size, density,
biochemical parameters, or a combination thereof.
[0043] As used herein, the terms "parent cell" or "producer cell"
include any cell from which an extracellular vesicle may be
isolated. The terms also encompasse a cell that shares a protein,
lipid, sugar, or nucleic acid component of the extracellular
vesicle. For example, a "parent cell" or "producer cell" may
include a cell which serves as a source for the extracellular
vesicle membrane.
[0044] As used herein, the terms "purify," "purified," and
"purifying" or "isolate," "isolated," or "isolating" or "enrich,"
"enriched" or "enriching" are used interchangeably and refer to the
state of a population (e.g., a plurality of known or unknown amount
and/or concentration) of desired extracellular vesicles, that have
undergone one or more processes of purification, e.g., a selection
or an enrichment of the desired extracellular vesicles composition,
or alternatively a removal or reduction of residual biological
products as described herein. In some embodiments, a purified
extracellular vesicles composition has no detectable undesired
activity or, alternatively, the level or amount of the undesired
activity is at or below an acceptable level or amount. In other
embodiments, a purified extracellular vesicle composition has an
amount and/or concentration of desired extracellular vesicles at or
above an acceptable amount and/or concentration. In other
embodiments, the purified extracellular vesicle composition is
enriched as compared to the starting material (e.g., biological
material collected from tissue, bodily fluid, or cell preparations)
from which the composition is obtained. This enrichment may be by
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%,
99%, 99.9%, 99.99%, 99.999%, 99.9999%, or greater than 99.9999% as
compared to the starting material.
[0045] Abbreviations used in this application include the
following: Size-exclusion chromatography (SEC), Anion Exchange
Chromatography (AEX), Two-dimensional liquid chromatography
(2D-LC), Nanoparticle tracking analysis (NTA), Resistive pulse
sensing (RPS), extracellular vesicles (EV or EVs), Phosphate
Buffered Saline (PBS) and Fluorescent Activated Cell Sorting
(FACS).
[0046] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
[0047] Methods of the Invention
Sources of Extracellular Vesicles
[0048] Described herein are methods for the detection and
quantification of extracellular vesicle from biological samples.
Biological samples can include, but are not limited to, raw cell
culture harvest, clarified cell culture medium, enriched
extracellular vesicle preparations, partially purified
extracellular vesicle preparations (e.g., by a single two-hour
ultracentrifugation step), or highly purified extracellular vesicle
preparations (e.g., extracellular vesicle preparations additionally
purified using a density gradient medium (e.g., sucrose density
gradient medium or medium comprising an iodixanol solution
(Sigma-Aldrich).
[0049] The parent cell can be cultured. Cultured parent cells can
be scaled up from bench-top scale to bioreactor scale. For example,
the parent cells are cultured until they reach saturation density,
e.g., 1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.7, or
greater than 1.times.10.sup.7 complexes per ml. Optionally, upon
reaching saturation density, the parent cells can be transferred to
a larger volume of fresh medium. The parent cells may be cultured
in a bioreactor, such as, e.g., a Wave-type bioreactor, a
stirred-tank bioreactor. Various configurations of bioreactors are
known in the art and a suitable configuration may be chosen as
desired. Configurations suitable for culturing and/or expanding
populations of parent cells can easily be determined by one of
skill in the art without undue experimentation. The bioreactor can
be oxygenated. The bioreactor may optionally contain one or more
impellers, a recycle stream, a media inlet stream, and control
components to regulate the influx of media and nutrients or to
regulate the outflux of media, nutrients, and waste products.
Enrichment of Extracellular Vesicle Preparations
[0050] With respect to purification or enrichment of extracellular
vesicles, it is contemplated that all known manners of purification
of extracellular vesicles are deemed suitable for use herein. For
example, physical properties of extracellular vesicles may be
employed to separate them from a medium or other source material,
including separation on the basis of electrical charge (e.g.,
electrophoretic separation, ion-exchange chromatography), size
(e.g., filtration, size-exclusion chromatography, molecular
sieving, etc.), density (e.g., regular or gradient centrifugation),
Svedberg constant (e.g., sedimentation with or without external
force, etc.). For ion-exchange chromatography, any suitable methods
known in the art may be used including, but not limited to,
anion-exchange chromatography, and strong-anion exchange
chromatography. For density gradient centrifugation, any
appropriate density gradient medium used in the art may be used,
including, but not limited to, sucrose density gradient medium and
mediums comprising, iodixanol solution, colloidal silica, inorganic
salts, polyhydric alcohols, polysaccharides, poly(vinyl alcohol),
iohexol and nonionic iodinated media. Purification of the
extracellular vesicles may be performed by manually loading columns
or other devices, or may be automated using devices such an
autosampler.
[0051] Alternatively, or additionally, isolation can be based on
one or more biological properties, and include methods that can
employ surface markers (e.g., precipitation, reversible binding to
solid phase, FACS separation, separation using magnetic surfaces,
specific ligand binding, immunoprecipitation or other
antibody-mediated separation techniques, non-specific ligand
binding such as annexin V, etc.). In yet further contemplated
methods, the extracellular vesicles can also be fused using
chemical and/or physical methods, including PEG-induced fusion
and/or ultrasonic fusion.
[0052] In certain embodiments, enrichment of extracellular vesicles
can be done in a general and non-selective manner (e.g., methods
comprising serial centrifugation), and can be performed by
aggregation where the extracellular vesicles are interlinked with
an interlinking composition (e.g., annexin V, fibrin, or an
antibody or fragment thereof against at least one of a tetraspanin,
ICAM-1, CD86, CD63). Alternatively, enrichment of extracellular
vesicles can be done in a more specific and selective manner (e.g.,
using tissue or cell specific surface markers). For example,
specific surface markers can be used in immunoprecipitation, FACS
sorting, bead-bound ligands for magnetic separation, etc.
[0053] In some embodiments, size exclusion chromatography can be
utilized to enrich the extracellular vesicles. Size exclusion
chromatography techniques are known in the art. Exemplary,
non-limiting techniques are provided herein. In some embodiments, a
void volume fraction is isolated and comprises extracellular
vesicles of interest. Further, in some embodiments, the
extracellular vesicles can be further isolated after
chromatographic separation by centrifugation techniques (of one or
more chromatography fractions), as is generally known in the art.
In some embodiments, for example, density gradient centrifugation
can be utilized to further enrich the extracellular vesicles. Still
further, in some embodiments, it can be desirable to further
separate the parent-cell derived extracellular vesicles from
extracellular vesicles of other origin. For example, the parent
cell extracellular vesicles can be separated from non-parent
cell-derived extracellular vesicles by immunosorbent capture using
an antigen antibody specific for the parent cell. For example,
anti-CD63 antibodies can be used.
[0054] In some embodiments, the isolation of extracellular vesicles
can involve combinations of methods that include, but are not
limited to, differential centrifugation, size-based membrane
filtration, concentration and/or rate zonal centrifugation, and
further characterized using methods that include, but are not
limited to, electron microscopy, flow cytometry and Western
blotting.
[0055] Extracellular vesicles can be extracted from the supernatant
of parent cells and demonstrate membrane and internal protein,
lipid, and nucleic acid compositions that enable their efficient
delivery to and interaction with recipient cells. Extracellular
vesicles can be derived from parent cells that may include, but are
not limited to, reticulocytes, erythrocytes, megakaryocytes,
platelets, neutrophils, tumor cells, connective tissue cells,
neural cells and stem cells. Suitable sources of extracellular
vesicles include but are not limited to, cells isolated from
subjects from patient-derived hematopoietic or erythroid progenitor
cells, immortalized cell lines, or cells derived from induced
pluripotent stem cells, optionally cultured and differentiated.
Cell culture protocols can vary according to compositions of
nutrients, growth factors, starting cell lines, culture period, and
morphological traits by which the resulting cells are
characterized. In some embodiments, the samples comprising
extracellular vesicles are derived from a plurality of donor cell
types (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 50, 100, 500, 1000, 5000,
or 10000 donor cell types) and are combined or pooled. Pooling may
occur by mixing cell populations prior to extracellular vesicles
extraction or by mixing isolated extracellular vesicles
compositions from subsets of donor cell types. Parent cells may be
irradiated or otherwise treated to affect the production rate
and/or composition pattern of secreted extracellular vesicles prior
to isolation.
[0056] In certain embodiments, the extracellular vesicles may be
derived from cell lines that are differentiated, proliferated and
cultured in-vitro. This enables controllable and reproducible
compositions of extracellular vesicles that are not subject to
constraints on isolation and purification of the requisite parent
cell type.
[0057] In certain embodiments, the samples comprising the
extracellular vesicles are obtained from raw cell harvest and the
intrinsic fluorescence is determined. In certain embodiments, the
raw cell harvest is clarified for larger cells and cellular debris
prior to determination of the intrinsic fluorescence. In certain
embodiments, the samples comprising the extracellular vesicles are
further purified using any of the above mentioned methods for
enrichment of the extracellular vesicles prior to determination of
the intrinsic fluorescence of the samples.
[0058] In certain embodiments, the methods comprise fractionating
the sample prior to determination of the intrinsic fluorescence. In
certain embodiments, the method comprises the steps of loading the
extracellular vesicle preparation on a size exclusion
chromatography (SEC) column (e.g., a sepharose resin SEC column).
In certain embodiments, the methods comprise the steps of loading
the extracellular vesicle preparation on an ion exchange
chromatography column. In certain embodiments, the methods comprise
the steps of loading the extracellular vesicle preparation on a
strong anion exchange chromatography column.
Detection of Intrinsic Florescence
[0059] In certain embodiments, the intrinsic fluorescence of the
eluted fractions from columns used for separation and/or
fractionation of the samples comprising the extracellular vesicles
are detected in a single step. In certain embodiments, the
intrinsic fluorescence of the eluted fractions from the columns
used for separation and/or fractionation is detected in multiple
steps. In certain embodiments, the detection of intrinsic
fluorescence of the eluted fractions is detected after the
fractions have been further processed or stored for a period of
time. In certain embodiments, the eluted fractions are analyzed for
intrinsic fluorescence on the same device as the device used for
separation and/or fractionation of the sample comprising the
extracellular vesicles. In certain embodiments, the fractions are
analyzed for intrinsic fluorescence on a separate device as the
device used for separation and/or fractionation of the sample
comprising the extracellular vesicles. In certain embodiments, the
sample fractions are collected using a flow-cell.
[0060] The relative amounts or concentrations of extracellular
vesicle are determined by measurement of intrinsic fluorescence
using standard techniques. Detection and/or measurement of
intrinsic fluorescence can be performed manually by fluorescent
microscopy or determined using automated systems for fluorescent
detection. In certain embodiments, intrinsic fluorescence of the
extracellular vesicle preparations or fractions of extracellular
vesicle preparation is determined using a microplate reader or any
other acceptable method known in the art for the detection and
measurement of fluorescence in a sample.
[0061] In certain embodiments, extracellular vesicle preparation
can be sorted by flow cytometry, e.g., bead-based flow cytometry as
described in Melo et al. (Nature, 2015 Jul. 9; 523[7559]:177-82)
based on intrinsic fluorescence at particular excitation and
emission spectra.
[0062] In certain embodiments, the intrinsic fluorescence profile
of extracellular vesicles is detected at a range between 450 and
550 nm absorbance wavelength and at a range between 470 to 570 nm
emission wavelength. In certain embodiments, the intrinsic
fluorescence profile of extracellular vesicles is detected at a
range between 450 and 460 nm, 460 and 470 nm, 470 and 480 nm, 480
and 490 nm, 490 and 500 nm, 500 and 510 nm, 510 and 520 nm, 520 and
530 nm and 540 and 550 nm absorbance wavelength. In certain
embodiments, the intrinsic fluorescence profile of extracellular
vesicles is detected at a range between 470 and 480 nm, 480 and 490
nm, 490 and 500 nm, 500 and 510 nm, 520 and 530 nm, 530 and 540 nm,
540 and 550 nm, 550 and 560 nm and 560 and 570 nm emission
wavelength. In an aspect, the intrinsic fluorescence emission
signal is generated using an excitation wavelength ranging from 450
nm to 650 nm and an emission wavelength that is longer than the
excitation wavelength and ranging from 470 nm to 670 nm. In an
aspect, the intrinsic fluorescence emission signal is determined at
an emission wavelength range of 500-600 nm. In an aspect, the
intrinsic fluorescence emission signal is determined at an emission
wavelength range of 550-590 nm. In an aspect, the intrinsic
fluorescence emission signal is determined at an emission
wavelength of 573 nm. In an aspect, the intrinsic fluorescence
emission signal is determined at an excitation wavelength range of
500-600 nm. In an aspect, the intrinsic fluorescence emission
signal is determined at an excitation wavelength range of 530-570
nm. In an aspect, the intrinsic fluorescence emission signal is
determined at an excitation wavelength of 556 nm. In an aspect, the
intrinsic fluorescence emission signal is determined at an
excitation wavelength of 550 nm and an emission of 570 nm.
[0063] In each embodiment, the excitation and emission wavelengths
are selected so that the excitation wavelength is shorter than the
emission wavelength. In certain aspects, the absorbance wavelength
varies according to the membrane composition and/or payload
composition of the extracellular vesicle. In certain aspects, the
emission wavelength varies according to the membrane composition
and/or payload composition of the extracellular vesicle. In certain
aspects, the emission wavelength and/or absorbance wavelength
varies according to the homogeneity of the extracellular vesicle
preparation. In certain aspects, the absorbance wavelength and/or
emission wavelength used to detect the extracellular vesicle varies
according to the type of producer cell from which the extracellular
vesicle is derived. In certain aspects, the absorbance wavelength
and/or emission wavelength used to detect the extracellular vesicle
varies according to the purity of the extracellular preparation
prior to detection of the extracellular vesicles.
Quantitation of Concentration of Extracellular Vesicle
Preparations
[0064] In certain aspects, the methods comprise displaying the
absorbance and/or emission spectra obtained from the sample on a
chromatogram. In certain aspects, the relative amounts or
concentrations of extracellular vesicles in the sample or a
fraction of the sample is obtained by calculating the area under
the resulting absorbance curve of the chromatogram and calculating
the same using a quantification standard, wherein the standard is
applied to a similar extracellular vesicle preparation. The
concentration of the extracellular vesicles in the quantification
standard can be measured by any of the known methods in the art
and, in certain embodiments, can be independently verified by more
than one technique, such as, but not limited to, electron
microscopy, flow cytometry analysis of extracellular vesicles
harboring exogenous fluorescent molecules, nanoparticle tracking
analysis, resistive pulse sensing, and determination of total
protein concentrations.
[0065] In certain aspects, the methods comprise determining the
relative amounts of extracellular vesicles in a sample by measuring
a luminescence signal. This signal can be used to directly measure
the amounts of extracellular vesicles, or can be used as a standard
based on the methods described herein used to measure the intrinsic
fluorescence signature of extracellular vesicle samples. In an
aspect, the luminescence signal is a luminescence proximity assay,
which relies on the generation of excited ambient oxygen species to
induce chemiluminescence in a nearby acceptor fluorophore. In
certain aspects, the luminescence assay comprises antibodies that
bind to exosomes-associate proteins such as CD9, CD63,CD81, and
combinations thereof. In certain aspects, the luminescence assay is
an AlphaScreen.TM. assay.
Further Assessments and Characterizations of Extracellular Vesicle
Preparations
[0066] The identity and concentration of the extracellular vesicles
in a preparation or fraction and/or the quantification standard
sample can be assessed and/or validated by in vitro assays. For
example, the identity and concentration of the extracellular
vesicles is determined by counting the number of complexes in a
population, e.g., by microscopy, by flow cytometry, or by
hemacytometry. Alternatively, or in addition, the identity and/or
concentration of the extracellular vesicles is assessed by analysis
of protein content of the complex, e.g., by flow cytometry, Western
blot, immunoprecipitation, fluorescence spectroscopy,
chemiluminescence, mass spectrometry, or absorbance spectroscopy.
In an embodiment, the protein content assayed is a non-surface
protein, e.g., an integral membrane protein, hemoglobin, adult
hemoglobin, fetal hemoglobin, embryonic hemoglobin, or a
cytoskeletal protein. In an embodiment, the protein content assayed
is a surface protein, e.g., a differentiation marker, a receptor, a
co-receptor, a transporter, a glycoprotein. In an embodiment, the
surface protein is selected from the list including, but not
limited to, glycophorin A, CKIT, transferrin receptor, Band3, Kell,
CD45, CD46, CD47, CD55, CD59, CR1, CD9, CD63 and CD81. In an
embodiment, the identity of extracellular vesicles is assessed by
analysis of the receiver content of the vesicle, e.g., by flow
cytometry, Western blot, immunoprecipitation, fluorescence
spectroscopy, chemiluminescence, mass spectrometry, or absorbance
spectroscopy. For example, the identity of the extracellular
vesicles can be assessed by the mRNA content of the complexes,
e.g., by RT-PCR, flow cytometry, or northern blot. The identity of
the extracellular vesicles can be assessed by nuclear material
content, e.g., by flow cytometry, microscopy, or southern blot,
using, e.g., a nuclear stain or a nucleic acid probe.
Alternatively, or in addition, the identity of the extracellular
vesicles is assessed by lipid content of the complexes, e.g., by
flow cytometry, liquid chromatography, or by mass spectrometry.
[0067] In some embodiments, the identity of the extracellular
vesicles is assessed by metabolic activity of the complexes, e.g.,
by mass spectrometry, chemiluminescence, fluorescence spectroscopy,
absorbance spectroscopy. Metabolic activity can be assessed by ATP
consumption rate and/or the metabolic activity is assessed
measuring 2,3-diphosphoglycerate (2,3-DPG) level in the parent
cells or extracellular vesicles. The metabolic activity can be
assessed as the rate of metabolism of one of the following,
including but not limited to, acetylsalicylic acid,
n-acetylcystein, 4-aminophenol, azathioprine, bunolol, captopril,
chlorpromazine, dapsone, daunorubicin, dehydroepiandrosterone,
didanosin, dopamine, epinephrine, esmolol, estradiol, estrone,
etoposide, haloperidol, heroin, insulin, isoproterenol, isosorbide
dinitrate, ly 217896, 6-mercaptopurine, misonidazole,
nitroglycerin, norepinephrine, para-aminobenzoic acid. In some
embodiments, the identity of the extracellular vesicles is assessed
by partitioning of a substrate by the complexes, e.g., by mass
spectrometry, chemiluminescence, fluorescence spectroscopy, or
absorbance spectroscopy. The substrate can be one of the following,
including but not limited to, acetazolamide, arbutine, bumetamide,
creatinine, darstine, desethyldorzolamide, digoxigenin
digitoxoside, digoxin-16'-glucuronide, epinephrine, gentamycin,
hippuric acid, metformin, norepinephrine, p-aminohippuric acid,
papaverine, penicillin g, phenol red, serotonin, sulfosalicylic
acid, tacrolimus, tetracycline, tucaresol, and vancomycin.
[0068] In some embodiments, the extracellular vesicles are assessed
for their basic physical properties, e.g., size, mass, volume,
diameter, buoyancy, density, and membrane properties, e.g.,
viscosity, deformability fluctuation, and fluidity. In an
embodiment, the diameter of the extracellular vesicles is measured
by microscopy or by automated instrumentation, e.g., a
hematological analysis instrument or by resistive pulse sensing. In
some embodiments, the extracellular vesicle has a longest dimension
between about 20-300 nm, such as between about 20-290 nm, 20-280
nm, 20-270 nm, 20-260 nm, 20-250 nm, 20-240 nm, 20-230 nm, 20-220
nm, 20-210 nm, 20-200 nm, 20-190 nm, 20-180 nm, 20-170 nm, 20-160
nm, 20-150 nm, 20-140 nm, 20-130 nm, 20-120 nm, 20-110 nm, 20-100
nm, 20-90 nm, 20-80 nm, 20-70 nm, 20-60 nm, 20-50 nm, 20-40 nm,
20-30 nm, 30-300 nm, 30-290 nm, 30-280 nm, 30-270 nm, 30-260 nm,
30-250 nm, 30-240 nm, 30-230 nm, 30-220 nm, 30-210 nm, 30-200 nm,
30-190 nm, 30-180 nm, 30-170 nm, 30-160 nm, 30-150 nm, 30-140 nm,
30-130 nm, 30-120 nm, 30-110 nm, 30-100 nm, 30-90 nm, 30-80 nm,
30-70 nm, 30-60 nm, 30-50 nm, 30-40 nm, 40-300 nm, 40-290 nm,
40-280 nm, 40-270 nm, 40-260 nm, 40-250 nm, 40-240 nm, 40-230 nm,
40-220 nm, 40-210 nm, 40-200 nm, 40-190 nm, 40-180 nm, 40-170 nm,
40-160 nm, 40-150 nm, 40-140 nm, 40-130 nm, 40-120 nm, 40-110 nm,
40-100 nm, 40-90 nm, 40-80 nm, 40-70 nm, 40-60 nm, 40-50 nm, 50-300
nm, 50-290 nm, 50-280 nm, 50-270 nm, 50-260 nm, 50-250 nm, 50-240
nm, 50-230 nm, 50-220 nm, 50-210 nm, 50-200 nm, 50-190 nm, 50-180
nm, 50-170 nm, 50-160 nm, 50-150 nm, 50-140 nm, 50-130 nm, 50-120
nm, 50-110 nm, 50-100 nm, 50-90 nm, 50-80 nm, 50-70 nm, 50-60 nm,
60-300 nm, 60-290 nm, 60-280 nm, 60-270 nm, 60-260 nm, 60-250 nm,
60-240 nm, 60-230 nm, 60-220 nm, 60-210 nm, 60-200 nm, 60-190 nm,
60-180 nm, 60-170 nm, 60-160 nm, 60-150 nm, 60-140 nm, 60-130 nm,
60-120 nm, 60-110 nm, 60-100 nm, 60-90 nm, 60-80 nm, 60-70 nm,
70-300 nm, 70-290 nm, 70-280 nm, 70-270 nm, 70-260 nm, 70-250 nm,
70-240 nm, 70-230 nm, 70-220 nm, 70-210 nm, 70-200 nm, 70-190 nm,
70-180 nm, 70-170 nm, 70-160 nm, 70-150 nm, 70-140 nm, 70-130 nm,
70-120 nm, 70-110 nm, 70-100 nm, 70-90 nm, 70-80 nm, 80-300 nm,
80-290 nm, 80-280 nm, 80-270 nm, 80-260 nm, 80-250 nm, 80-240 nm,
80-230 nm, 80-220 nm, 80-210 nm, 80-200 nm, 80-190 nm, 80-180 nm,
80-170 nm, 80-160 nm, 80-150 nm, 80-140 nm, 80-130 nm, 80-120 nm,
80-110 nm, 80-100 nm, 80-90 nm, 90-300 nm, 90-290 nm, 90-280 nm,
90-270 nm, 90-260 nm, 90-250 nm, 90-240 nm, 90-230 nm, 90-220 nm,
90-210 nm, 90-200 nm, 90-190 nm, 90-180 nm, 90-170 nm, 90-160 nm,
90-150 nm, 90-140 nm, 90-130 nm, 90-120 nm, 90-110 nm, 90-100 nm,
100-300 nm, 110-290 nm, 120-280 nm, 130-270 nm, 140-260 nm, 150-250
nm, 160-240 nm, 170-230 nm, 180-220 nm, or 190-210 nm.
[0069] In particularly preferred embodiments, the extracellular
vesicle described herein has a longest dimension between about
30-100 nm. In another preferred embodiment, the extracellular
vesicle has a longest dimension between about 20-300 nm. In another
preferred embodiment, the extracellular vesicle has a longest
dimension between about 40-200 nm. In another embodiment, a
population of the extracellular vesicles described herein comprise
a population wherein 90% of said extracellular vesicles have a
longest dimension 20-300 nm. In another embodiment, a population of
the extracellular vesicles described herein comprise a population
wherein 95% of said extracellular vesicles have a longest dimension
20-300 nm. In another embodiment, a population of the extracellular
vesicles described herein comprise a population wherein 99% of said
extracellular vesicles have a longest dimension 20-300 nm. In
another embodiment, a population of the extracellular vesicles
described herein comprise a population wherein 90% of said
extracellular vesicles have a longest dimension 40-200 nm. In
another embodiment, a population of the extracellular vesicles
described herein comprise a population wherein 95% of said
extracellular vesicles have a longest dimension 40-200 nm. In
another embodiment, a population of the extracellular vesicles
described herein comprise a population wherein 99% of said
extracellular vesicles have a longest dimension 40-200 nm. In other
preferred embodiments, the size of the extracellular vesicles or
population of extracellular vesicles described herein is measured
according to methods described, infra.
[0070] In an embodiment,the average buoyant mass of the
extracellular vesicles (pg/cell) is measured using a suspended
microchannel resonator or a double suspended microchannel resonator
(see e.g., Byun et al PNAS 2013 110(19):7580 and Bryan et al. Lab
Chip 2014 14(3):569). In an embodiment, the dry density of the
extracellular vesicles is measured by buoyant mass in an H2O-D20
exchange assay (see e.g., Feijo Delgado et al., PLOS One 2013
8(7):e67590). In some embodiments, the extracellular vesicles have
an average membrane deformability fluctuation of standard deviation
greater than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater
than 100 mrad as measured by spatial light interference microscopy
(SLIM) (see e.g., Bhaduri et al., Sci Reports 2014, 4:6211). In an
embodiment, the average membrane viscosity of a population of
extracellular vesicles is measured by detecting the average
fluorescence upon incubation with viscosity-dependent quantum yield
fluorophores (see e.g., Haidekker et al. Chem & Biol 2001
8(2):123). In an embodiment, the membrane fluidity of the
extracellular vesicles is measured by fluorescence polarization,
e.g., with BMG Labtech POLARstar Omega microplate reader.
EXAMPLES
[0071] Below are examples of specific embodiments for carrying out
the present invention. The examples are offered for illustrative
purposes only, and are not intended to limit the scope of the
present invention in any way. Efforts have been made to ensure
accuracy with respect to numbers used (e.g., amounts, temperatures,
etc.), but some experimental error and deviation should, of course,
be allowed for.
[0072] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of protein chemistry,
biochemistry, recombinant DNA techniques and pharmacology, within
the skill of the art. Such techniques are explained fully in the
literature. See, e.g., T. E. Creighton, Proteins: Structures and
Molecular Properties (W.H. Freeman and Company, 1993); A. L.
Lehninger, Biochemistry (Worth Publishers, Inc., current addition);
Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd
Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan
eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences,
18th Edition (Easton, Penn. Mack Publishing Company, 1990); Carey
and Sundberg Advanced Organic Chemistry 3.sup.rd Ed. (Plenum Press)
Vols A and B (1992).
Methods
[0073] Conditioned culture media is collected and centrifuged at
300-800.times. g for 5 minutes at room temperature to remove cells
and large debris. Media supernatant is then supplemented with 1000
U/L benzonase and incubated at 37.degree. C. for 1 hour in a water
bath. Supernatant is collected and centrifuged at 16,000.times. g
for 30 minutes at 4.degree. C. to remove residual cell debris and
other large contaminants. Supernatant is then ultracentrifuged at
133,900.times. g for 3 hours at 4.degree. C. to pellet the
exosomes. Supernatant is discarded and any residual media is
aspirated from the bottom of the tube. The pellet is resuspended in
200-1000 .mu.L PBS (--Ca--Mg).
[0074] To further enrich exosome populations, the pellet is
processed via density gradient purification (sucrose or
Optiprep.TM.). For sucrose gradient purification, the exosome
pellet is layered on top of a sucrose gradient as defined in Table
1 below:
TABLE-US-00001 TABLE 1 Working 65% Milli- Percentage (%) Stock Vol.
(mL) Q Vol. (mL) 50 3.85 1.15 40 3.08 1.92 25 1.92 3.08 10 0.46
2.54
[0075] The gradient is spun at 200,000.times. g for 16 hours at
4.degree. C. in a 12 mL Ultra-Clear (344059) tube placed in a SW 41
Ti rotor to separate the exosome fraction.
[0076] The exosome layer is gently removed from the top layer and
diluted in .about.32.5 mL PBS in a 38.5 mL Ultra-Clear (344058)
tube and ultracentrifuged again at 133,900.times. g for 3 hours at
4.degree. C. to pellet the purified exosomes. The resulting pellet
is resuspended in a minimal volume of PBS (.about.200 .mu.L) and
stored at 4.degree. C.
[0077] For Optiprep.TM. gradient, a 3-tier sterile gradient is
prepared with equal volumes of 10%, 30%, and 45% Optiprep in a 12
mL Ultra-Clear (344059) tube for a SW 41 Ti rotor. The pellet is
added to the Optiprep.TM. gradient and ultracentrifuged at
200,000.times. g for 16 hours at 4.degree. C. to separate the
exosome fraction. The exosome layer is then gently collected from
the top .about.3 mL of the tube.
[0078] The exosome fraction is diluted in .about.32 mL PBS in a
38.5 mL Ultra-Clear (344058) tube and ultracentrifuged at
133,900.times. g for 3 hours at 4.degree. C. to pellet the purified
exosomes. The pelleted exosomes are then resuspended in a minimal
volume of PBS (.about.200 .mu.L) and store at 4.degree. C.
[0079] For AlphaScreen.TM. assays, unconjugated acceptor beads,
streptavidin donor beads, universal buffer, and 1/2 area 96-well
white opaque plates were purchased from Perkin Elmer. Antibodies
used were tetraspanin specific anti-human CD9 (clone HI9a) and
biotinylated anti-human CD81 (clone 5A6) antibodies acquired from
Biolegend. The CD9 antibodies were conjugated to acceptor beads
following a protocol provided by Perkin Elmer. Sodium
cyanoborohydride and O-(Carboxymethyl)hydroxylamine
hemihydrochloride necessary for acceptor bead conjugation were
purchased from Sigma-Aldrich.
[0080] All samples, bead, and antibody solutions were diluted in
1.times. dilution buffer to the needed concentration. A standard
was created using Optiprep.TM. derived exosomes and serially
diluted with universal buffer to create concentrations from 1E11
P/mL to 1.6E9 P/mL. Liquid chromatography exosomes samples were
tested both neat and at 2.times. dilution.
[0081] In the half area plate, 10 uL of 1.5 nM biotinylated CD81
antibody, 10 uL of 50 ug/mL acceptor bead solution, and 5 uL of
samples/standards were added to each well. The plate was then
incubated in RT for 1 hour. Following incubation, 25 uL of a 80
ug/mL solution of streptavidin donor bead solution were added and
incubated in the dark for 1 hour. Finally, using an Alpha-capable
BMG CLARIOstar plate reader, the samples were read with an
excitation of 680 nm and emission band-range of 520-620 nm.
[0082] For affinity size-exclusion chromatography (SEC) assays,
exosome or exosome-antibody complex samples were analyzed on an
Acclaim 1000A 7.8.times.150 mm column run at 0.3 mL/min. Mobile
phase solution was 0.1M Na Phosphate, 0.2M NaCl, pH 7.2, and
samples were injected at 2.times.10.sup.11 particles/mL+/-170 ng
anti-CD81_FITC antibody in 100 .mu.L final volume.
Example 1: Strong Anion Exchange Chromatography Profiles of
Purified Exosomes and Cell Culture Harvest at Different Excitation
and Emmission Spectra
[0083] To understand fundamental properties of exosomes and develop
analytical tools for studying exosomes, Optiprep.TM. purified
exosome preparations and cell culture harvest were analyzed by
strong anion exchange chromatography (AEX). Exosomes from HEK293SF
cells grown in serum-free conditions were processed as described in
the methods above. For cell culture harvest, HEK293SF cells were
grown in serum-free medium, and 600 ml of supernatant were
collected. Cell culture harvest was then centrifuged at 1600 g for
10 minutes and filtered through a 0.8 .mu.m filter to remove
cellular debris before chromatographic analysis. AEX was monitored
at 210, 254, and 280 nm and was performed using a CIMac monolithic
QA-1 mL column from BIA Separations. The Optiprep.TM. purified
exosome stock concentration was measured to be 4.times.10.sup.12
particles/mL by nanoparticle tracking assay (NTA), and the sample
was diluted 100-fold prior to injection. As shown in FIG. 1A,
purified exosomes were detected in a strong peak at about 8.5
minutes using a detector set at 254 nm (a similar profile was
observed for all wavelengths monitored). Cell culture harvest
containing a heterogeneous population of particles including
exosomes displayed a broad peak at the same elution time point, but
also displayed a significantly more heterogeneous elution profile.
Optiprep.TM. purified exosomes were analyzed by transmission
electron microscopy (FIG. 1B) to verify their expected shape and
size.
[0084] To explore whether the exosomes contained within raw cell
culture harvest could be detected by AEX, HEK293SF culture
supernatant was processed and analyzed by AEX as described above
and monitored at various spectral settings (FIG. 2). The samples
were monitored by UV absorbance at 254 nm, and by two separate
fluorescence methods. The first fluorescence method used an
excitation wavelength (ex) of 280 nm and emission wavelength (em)
of 350 nm, which pertains to intrinsic protein fluorescence. The
second fluorescence method used an ex of 556 nm and an em of 573
nm. While all three spectral traces showed a peak corresponding to
exosomes at the expected 8.5 minute time point, only ex556/em573
showed a spectral trace with this peak as the dominant species.
This result suggests that exosomes within a heterogeneous cell
culture harvest milieu can be detected above background using a
simple and rapid AEX method.
[0085] To confirm that the peak detected by AEX at ex556/em573
shown in FIG. 2 corresponded to exosomes, Optiprep.TM. purified
exosome preparations resuspended in PBS from four different cell
types were analyzed using this method. As shown in FIG. 3, exosomes
from HEK293SF cells, HEK293T cells, mesenchymal stem cells, and the
human neuronal precursor cell line AGE1. HN were examined.
Importantly, all four of the exosome preparations displayed the
same spectral behavior with a discrete peak at 8.5 minutes when
analyzed by AEX at ex556/em573. Together, these data suggest that
there is an intrinsic fluorescence signature for exosomes, and that
this signature can be detected in purified exosome populations, or
in raw cell culture harvest.
Example 2: Determining the Optimal Fluorescence Spectrum for
Analyzing AEX-Purified Exosomes
[0086] To optimize the excitation and emission wavelengths for the
detection of exosomes, wavelength scanning on Optiprep.TM. purified
exosomes was carried out. To determine the optimal excitation
wavelength, Optiprep.TM. purified exosomes were analyzed by AEX,
while the emission wavelength was held constant at 573 nm, and the
excitation wavelength was scanned from 280 nm to 560 nm (FIG. 4A).
To determine the optimal emission wavelength, Optiprep.TM. purified
exosomes were analyzed by AEX, while the excitation wavelength was
held constant at 556 nm, and the emission wavelength was scanned
from 570 nm to 770 nm (FIG. 4B). These results indicate that the
optimal wavelengths for detecting intrinsic fluorescence of
exosomes is an excitation wavelength of 556 nm and emission
wavelength of 573 nm.
Example 3: Synthetic Liposomes and Cell-Derived Exosomes Do Not
Have the Same Fluorescence Spectra
[0087] To determine if the fluorescence profile determined in
Example 1 was specific to cell-derived exosomes, synthetic
liposomes were also analyzed by AEX at ex556/em573. Synthetic
clodronate liposome controls formulated in PBS were purchased. The
liposomes were composed of a mixture of phosphatidylcholine and
cholesterol and formulated to be between 0.15-3 .mu.m in size.
Before analysis, the liposomes were filtered through a 0.45 .mu.m
cellulose acetate filter to remove the larger sized liposomes. The
resulting filtered liposomes were analyzed by NTA (FIG. 5A) and
transmission electron microscopy (FIG. 5B) confirming that they are
similar in size and shape to cell-derived exosomes (see FIG.
1B).
[0088] To confirm that the synthetic liposomes had similar
size-dependent characteristics of cell-derived exosomes, both
populations were analyzed by size-exclusion chromatography (SEC)
and monitored by UV absorbance. As shown in FIG. 6, liposomes and
Optiprep.TM. purified exosomes were suspended in PBS, and SEC
separation was performed using a Tosoh Biosciences G4000SW.sub.XL
7.8 mm.times.30 cm column operated at 0.8 mL/min. Optiprep.TM.
purified exosome samples (black trace) eluted in the void volume at
approximately 7.5 minutes. The buffer (teal trace) showed no
interference, and the liposome sample (red trace) showed a broad
elution profile peaking near 8 minutes, which is consistent with
the large particle size distribution from NTA (FIG. 5A). The peaks
at approximately 17 minutes corresponded to one column volume.
These data demonstrate that purified liposomes and Optiprep.TM.
purified exosomes have similar elution profiles, presumably due to
their similar size and lipid composition.
[0089] To determine whether the purified liposomes described above
also have an intrinsic fluorescence profile, the samples from FIG.
6 were analyzed by AEX at ex556/em573. Surprisingly, only the
Optiprep.TM. purified exosomes (black trace) showed the expected
peak at about 8 minutes (FIG. 7; see also FIG. 3) while neither the
purified liposomes (red trace) nor a buffer sample (teal trace)
could be detected using this method. These results clearly
demonstrate that the fluorescence spectra determined by AEX at
ex556/em573 is specific to cell-derived exosomes, and not all
lipid-based vesicles.
Example 4: AEX at ex556/em573 is Useful for Determining Exosome
Concentration Number
[0090] To explore the performance characteristics of AEX at
ex556/em573, exosomes at a range of concentrations were analyzed
according to this method. Optiprep.TM. purified exosomes from
HEK293SF cells were purified as described above to a concentration
of 4.times.10.sup.12 particles/ml as determined by NTA. The stock
sample was serially diluted into HEK293SF culture harvest and
analyzed by AEX at ex556/em573 as described above. As shown in FIG.
8A, serially diluted exosome preparations could be differentially
detected. Relative fluorescence, as measured by the area under the
curve of the spectral plot, was linearly correlated with the
exosome dilution series (FIG. 8B). Additionally, particle number of
the dilution series was linearly correlated with the fluorescence
peak area (FIG. 8C). Together, these results indicate that AEX at
ex556/em573 can quantitatively measure exosome concentration and
raw particle count, even when diluted many orders of magnitude into
heterogenous cell culture supernatant.
Example 5: AEX at ex556/em573 Can be Used to Semi-Quantitatively
Measure Exosome Yield in Cell Culture Harvest
[0091] To determine if AEX at ex556/em573 can be used to directly
measure exosome levels in cell culture harvest, various samples
from HEK293SF culture medium were analyzed. As shown in FIG. 9,
cell culture blank media (grey trace) was not detectable, while
cell culture harvest from a low yield culture (red trace) was
detected at a lower peak fluorescence than cell culture harvest
from a high yield culture (black trace). These results demonstrate
that a rapid, single-step AEX method can semi-quantitatively
differentiate exosome yield directly from cell culture harvests
grown under different conditions.
Example 6: Exosomes Quantited by Intrinsic Fluorescence Correlate
with AlphaScreen.TM. Quantitation
[0092] To validate that the exosome quantitation methods using
intrinsic fluorescence are accurate, the yield estimates were
compared to exosome amounts as measured by AlphaScreen.TM.. As
shown in FIG. 10, estimated exosome yield determined by AEX
ex556/em573 correlates well with exosome yield determined by
exosome CD9/CD81 AlphaScreen.TM.. The agreement between these
orthogonal methods suggests that the absolute exosome yield
determined by ex556/em573 is accurate.
Example 7: Two-Dimensional Liquid Chromatography Improves Exosome
Detection and Purity
[0093] To further improve the ability to detect exosomes from cell
culture harvest, a two-dimensional liquid chromatography (2D-LC)
method was established, 2D-LC SEC ex556/em573 (FIG. 11). This
method applies an exosome sample to an AEX column where total
protein is analyzed by a UV/visual light detector. The peak
fraction that contains exosomes is then applied to an SEC column
and analyzed by ex556/em573. Exosomes that were analyzed by 2D-LC
SEC ex556/em573 had higher purity than exosomes analyzed by AEX
alone as judged by electron microscopy. (FIG. 12).
[0094] To determine whether 2D-LC could accurately quantitate
exosome titers, Optiprep.TM.-purified exosomes were spiked into PBS
at dilutions of 200.times. to 1600.times.. After AEX alone, the
exosomes were detected as a minor species at .about.3.5 minutes.
After 2D-LC SEC ex556/em573, the exosomes were detected as the only
signal by ex556/573 in a concentration-dependent manner (FIG.
13A-B).
Example 8: Two-Dimensional Liquid Chromatography Improves Exosome
Quantitation from Heterogeneous Samples
[0095] To determine whether exosomes could be accurately identified
and quantitated from a heterogeneous sample, cell culture media
samples from HEK293SF cells grown for 7-9 days were analyzed by
2D-LC SEC ex556/em573. As shown in FIG. 14A, samples analyzed by
AEX and UV/visual detector alone failed to readily identify
exosomes from the cell culture media samples. Higher magnification
of the chromatogram was able to uncover the exosome traces, but
they were a very minor population and could not be distinguished
from adjacent peaks (FIG. 14B). In contrast, the samples analyzed
by 2D-LC SEC ex556/em573were readily distinguished from background
signal and detected in a concentration-dependent manner, where
higher exosome yields were detected from older cultures, (i.e., Day
9 (D9)>Day 8 (D8)>Day 7 (D7)) (FIG. 14C). These results
demonstrate that 2D-LC relying on AEX and SEC can readily detect
exosomes from homogeneous and substantially heterogeneous
preparations.
Example 9: Affinity-SEC Demonstrates Co-Migration of Exosome
Markers and ex556/em573 Intrinsic Fluorescence Signature
[0096] To determine whether the exosome intrinsic fluorescence
signal (ex556/em573) detected in exosome populations could
physically associate with known exosome markers, purified exosome
populations were analyzed by affinity SEC. First, HEK293SF exosomes
were purified by Optiprep.TM. density-gradient ultracentrifugation
as described above. These exosomes naturally contain high levels of
CD81 protein on their surface, which can be readily detected by
Western blotting or bead-based exosome cytometry (data not shown).
Next, either (1) purified exosome populations or (2) purified
exosomes populations pre-incubated with a FITC-labeled anti-CD81
antibody were analyzed by affinity SEC. As shown in FIG. 15A,
unlabeled exosomes were detected at .about.3.4 minutes by protein
fluorescence (ex280/em350) or by exosome intrinsic fluorescence
(ex556/em573), while no signal was detected in the FITC channel
(ex490/em525). In contrast, when purified exosomes were
pre-incubated with an excess of anti-CD81_FITC antibody, a strong
ex490/em525 signal was detected at .about.3.4 min (FIG. 15B),
strongly suggesting that the CD81 signal is exosome-associated and
that the other fluorescence signals at .about.3.4 minutes are
exosome-specific. Free unbound antibody was detected in both the
ex490/em525 and the ex280/em350 channels at .about.5.4 minutes.
Together, these results indicate that exosomes containing a known
surface marker protein co-migrates with the ex556/em573 intrinsic
fluorescence signal, demonstrating that monitoring ex556/em573 can
be used for tracking exosome abundance and purity.
[0097] While the invention has been particularly shown and
described with reference to a preferred embodiment and various
alternate embodiments, it will be understood by persons skilled in
the relevant art that various changes in form and details can be
made therein without departing from the spirit and scope of the
invention.
[0098] All references, issued patents and patent applications cited
within the body of the instant specification are hereby
incorporated by reference in their entirety, for all purposes.
* * * * *