US20130273544A1 - Methods and compositions for exosome isolation - Google Patents

Methods and compositions for exosome isolation Download PDF

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US20130273544A1
US20130273544A1 US13/765,677 US201313765677A US2013273544A1 US 20130273544 A1 US20130273544 A1 US 20130273544A1 US 201313765677 A US201313765677 A US 201313765677A US 2013273544 A1 US2013273544 A1 US 2013273544A1
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exosomes
volume
sample
biological
excluding polymer
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Alexander VLASSOV
Mu Li
Emily Zeringer
Richard Conrad
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Life Technologies Corp
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Life Technologies Corp
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Publication of US20130273544A1 publication Critical patent/US20130273544A1/en
Priority to US14/191,109 priority patent/US8901284B2/en
Priority to US14/523,764 priority patent/US9347087B2/en
Priority to US15/142,143 priority patent/US9671321B2/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/34—Purifying; Cleaning
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5076—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving cell organelles, e.g. Golgi complex, endoplasmic reticulum
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/92—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving lipids, e.g. cholesterol, lipoproteins, or their receptors

Definitions

  • the disclosure generally relates to the isolation of exosomes from biological tissues and fluids.
  • 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).
  • Exosomes are thought to function as messengers, delivering various effector or signaling macromolecules between cells.
  • 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.
  • 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).
  • volume-excluding polymers are capable of differentially precipitating exosomes thereby allowing exosome isolation by low-speed (benchtop) centrifugation or filtration.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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”).
  • 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.
  • 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.
  • non-limiting examples of biological tissue samples include surgical samples, biopsy samples, tissues, feces, plant tissue, insect tissue, and cultured cells.
  • 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.
  • 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.
  • FIG. 1 Shows the results of an analysis comparing PEG size and concentration with the efficiency of exosome recovery from conditioned cell media.
  • FIG. 2 Shows a comparison of different salt and buffer concentrations with the efficiency of exosome recovery from conditioned cell media.
  • FIG. 3 Shows a comparison of PEG-mediated exosome isolation from three cell types with isolation of exosomes using the ultracentrifugation protocol.
  • 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 ( ⁇ 10 6 ).
  • FIG. 5 Shows the results of an analysis comparing PEG size and concentration with the efficiency of exosome recovery from blood serum.
  • FIG. 6 Shows the comparison of PEG6000 of different concentrations for isolation of exosomes from blood serum.
  • FIG. 7 Shows a comparison of small scale versus large scale exosome isolation from serum.
  • FIG. 8 Shows the analysis of exosomal RNA by qRT-PCR.
  • 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 ( ⁇ 10 6 ).
  • FIG. 10 Shows Electron Microscopy analysis of exosomes isolated from cell culture media with 10% PEG6000.
  • FIG. 11 Shows Western blot and qRT-PCR analysis for exosomes derived from HeLa cell media with 10% PEG6000.
  • FIG. 12 Shows Western blot and qRT-PCR analysis for exosomes derived from serum with 5% PEG6000.
  • FIG. 13 Shows efficiency of exosomes recovery from urine with PEG6000 of different percentages.
  • FIG. 14 Shows efficiency of exosomes recovery from amniotic fluid with PEG6000 of different percentages.
  • FIG. 15 Shows efficiency of exosomes recovery from cerebrospinal fluid (CSF) with PEG6000 of different percentages.
  • FIG. 16 Shows efficiency of exosomes recovery from plasma with 5% PEG6000 and effects of Proteinase K on removal of Albumin from the sample.
  • volume-excluding polymers such as polyethylene glycol (PEG) to precipitate exosomes from biological samples.
  • PEG polyethylene glycol
  • 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.
  • 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.
  • biological fluid 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.
  • biological tissue 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.
  • 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).
  • 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.
  • 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.
  • Precipitation of exosomes from the sample may be accomplished using a water-soluable volume excluding polymer.
  • 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.
  • 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.
  • 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.
  • 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.
  • Incubation of the sample with the volume-excluding polymer may be performed at room temperature (about 20° 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° C. Incubation may be performed at 1° C. to 40° C., 1° C. to 4° C., 4° C. to 10° C., 10° C. to 20° C., 20° C. to 30° C., or 30° C. to 40° C.
  • 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.
  • 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.
  • CD9 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).
  • exosomes having CD63 on their surface may be isolated using antibody coated magnetic particles.
  • Dynabeads® 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 ⁇ m in diameter.
  • the antibody coated Dynabeads® may be added to an exosome sample prepared using a volume-excluding polymer and incubated at 2-8 ° C. or at room temperature from 5 minutes to overnight. Dynabeads® 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.
  • Biological material which may be extracted from exosomes includes proteins, peptides, RNA and DNA, lipids.
  • the mirVanaTM 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.
  • 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.
  • 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.
  • PEG3000-PEG20,000 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° C. long term.
  • Antibacterial agents such as sodium azide may be added to PEG stock solutions to prevent bacterial growth.
  • 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 ⁇ 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.
  • 100 ⁇ 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.
  • PEG8000 33.3 ⁇ L of 40% (w/v) PEG stock may be added to 100 ⁇ 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° C. for 5 minutes to 2 h. After incubation, the samples may be centrifuged at room temperature or 4° C. at 2,000 ⁇ g to 10,000 ⁇ 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 ⁇ L for 100 ⁇ L serum input.
  • PBS phosphate bufferd saline
  • the pellet in certain cases may be difficult to resuspend so a pipet tip may be used to completely resuspend it in the solution.
  • the pellets may be incubated for 30 minutes at 37° C. then vortexed.
  • the pellet Once the pellet has been resuspended, it may be stored at 4° C. short term or at ⁇ 20° 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.
  • 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.
  • exosome samples may be diluted 10-1000 ⁇ 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.
  • PEG Polyethylene glycol
  • CM cell culture media
  • FBS cell media
  • conditioned media Cell culture media
  • HeLa cells were grown in a CO 2 incubator under normal conditions, in the recommended cell media containing 10% FBS, until ⁇ 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 ⁇ 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.
  • PEG polyethylene glycol
  • the samples were incubated at 4° 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.
  • results are shown in FIG. 1 .
  • all PEGs were capable of isolating exosomes from the cell media samples.
  • a higher percentage of PEG was beneficial.
  • recovery of exosomes was very efficient.
  • 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.
  • exosomes can be isolated from the media from any other cell type, grown to any confluency, in a wide range of sample volume inputs.
  • HeLa cells were grown in a CO 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 ⁇ 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.
  • results are shown in FIG. 2 .
  • 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).
  • FIG. 4 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 ⁇ 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 ( ⁇ 10 6 ).
  • the majority of microvesicles isolated by PEG precipitation are in the size range typical for exosomes: 30-150 nm.
  • Polyethylene glycol (PEG) of different length and percent were evaluated for capability to isolate exosomes from the 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 the sample was completely liquid. Samples were stored on ice until needed. The serum samples were first centrifuged at 2,000 ⁇ 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.
  • PEG polyethylene glycol
  • the samples (PEG+serum) were incubated at 4° 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.
  • Results are shown in FIG. 5 .
  • 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.
  • PEG 20000 is significantly more viscous and hard to pipet compared to shorter PEG 3000-8000.
  • 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.
  • 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 ⁇ 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.
  • polyethylene glycol PEG 6000 was used, at six percentages: 3%, 4%, 5%, 6%, 7%, 8% (final PEG percent, upon mixing with the biological samples).
  • the samples (PEG+serum) were incubated at 4° 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.
  • results are shown in FIG. 6 .
  • 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.
  • exosomes can be isolated from any other type of body or biological fluid, in the wide range of sample volume inputs.
  • results are shown in FIG. 7 .
  • 5% PEG6000 enabled efficient isolation of exosomes from the serum for both 100 ⁇ 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.
  • 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).
  • 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 ⁇ 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.
  • 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 miRVanaTM PARIS kit was successful. Both GAPDH and miR26a quantification by TaqMan® assays was robust and efficient.
  • FIG. 9 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 ⁇ 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 ( ⁇ 10 6 ).
  • majority of microvesicles isolated by PEG precipitation are in the size range typical for exosomes: 30-150 nm.
  • 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° 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 used for downstream analysis by Electron Microscopy (EM)—in the unlabeled form, and also labeled with two types of antibodies conjugated to gold nanoparticles.
  • EM Electron Microscopy
  • 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.
  • 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.
  • 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° C. for ⁇ 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.
  • the membranes were processed on the BenchPro® 4100 (Life Technologies) with CD63 antibody diluted 100 ⁇ 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.
  • Reverse Transcription (RT) Master Mix was prepared for each sample using the TaqMan® MicroRNA Reverse Transcription Kit reagents and protocol (Applied Biosystems) with gene specific RT primers for the RNA targets. 10 ⁇ l of the RT Master Mix was added to corresponding wells in a 96-well plate, and 5 ⁇ l of each sample was added to the master mix.
  • 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 ⁇ l of AB Universal PCR Master Mix II, 3.5 ⁇ l of nuclease-free water, and 0.5 ⁇ l of the 20 ⁇ Taqman® Assay. After mixing, 8 ⁇ l of each master mix was placed into wells in a 384-well plate (enough for triplicate reactions for each isolation replicate). 2 ⁇ 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.
  • Exosomes were isolated from human serum: 100 ⁇ L serum (after 30 min 2000 g centrifugation to remove cell debris) were combined with 20 ⁇ 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° C. for ⁇ 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.
  • 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° 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 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.
  • 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° 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 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.
  • 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° C. for ⁇ 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.
  • CSF cerebrospinal fluid
  • 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.
  • Exosomes were isolated from human plasma: 100 ⁇ 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° 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.
  • 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.
  • a method for the isolation of exosomes from a biological fluid sample comprising:
  • the biological fluid is from prokaryotes, eukaryotes, bacteria, fungi, yeast, invertebrates, vertebrates, reptiles, fish, insects, plants or animals.
  • 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.
  • volume-excluding polymer is polyethylene glycol, dextran, dextran sulfate, dextran acetate, polyvinyl alcohol, polyvinyl acetate, or polyvinyl sulfate.
  • a method for the isolation of exosomes from a biological tissue sample comprising:
  • volume-excluding polymer is polyethylene glycol, dextran, dextran sulfate, dextran acetate, polyvinyl alcohol, polyvinyl acetate, or polyvinyl sulfate.

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US9347087B2 (en) 2016-05-24
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