WO2013188832A1 - Procédés d'isolement et de retrait sélectif de microvésicule - Google Patents

Procédés d'isolement et de retrait sélectif de microvésicule Download PDF

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WO2013188832A1
WO2013188832A1 PCT/US2013/045997 US2013045997W WO2013188832A1 WO 2013188832 A1 WO2013188832 A1 WO 2013188832A1 US 2013045997 W US2013045997 W US 2013045997W WO 2013188832 A1 WO2013188832 A1 WO 2013188832A1
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serum
microvesicles
peg
precipitation solution
exosome
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PCT/US2013/045997
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Travis John ANTES
Kevin KWEI
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System Biosciences, Llc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/16—Blood plasma; Blood serum

Definitions

  • the invention relates to the field of cell biology, and in particular, to the study of circulating microvesicle structures that are produced by cells.
  • the invention relates to compositions and methods for the isolation of microvesicles produced by cells, finding use in biomedical research and potential therapeutic applications.
  • microvesicles also known as microparticles refers to a heterogeneous in vivo collection of membrane bound (i.e., encapsulated) biological structures. These structures are formed from lipid bilayer, which is the same lipid bilayer that comprises eukaryotic cell membranes. Microvesicles can reside within the cell, or in the extracellular environment. Microvesicle structures (intracellular and/or extracellular) are produced by nearly all mammalian cell types, as well as during in vitro cell culture.
  • microvesicles The molecular composition of microvesicles is diverse, containing and/or transporting a variety of nucleic acids, proteins and lipids.
  • Microvesicle molecular composition is generally reflective of the plasma membrane and antigenic content of the cell types, tissues and organs from which they originate. Mathivanan and Simpson, "Exosomes: extracellular organelles important in intercellular communication,” J. Proteomics 73(10):1907-1920 (2010).
  • protein composition of the microvesicles varies, most of these structures are enriched for various soluble protein markers, including HSP70, Hsc70, CD63, CD9, CD81 and others. Circulating microvesicles have also been reported to contain nucleic acids, including messenger NAs, and relatively high levels of small RNAs and microRNAs.
  • Circulating microvesicles are associated with numerous cell functions, including intercellular (cell-to-cell) communication, removal of metabolic byproducts and toxins (including misfolded proteins, cytotoxic agents and metabolic waste), angiogenesis, tissue regeneration, endocytic recycling of the plasma membrane, selective removal of plasma membrane proteins and regulation of immune functions such as antigen presentation.
  • Some microvesicles have been shown to transport messenger RNA (mRNA) and microRNA (miRNA), which is highly suggestive of microvesicles functioning as messengers that allow one cell type to regulate the activity of a distant cell type by acting as a shuttle that can merge with the distant cell and release its contents into that target recipient cell.
  • mRNA messenger RNA
  • miRNA microRNA
  • This microvesicle shuttle can utilize the body fluids to travel to distant sites and control the activity of distant target cells.
  • Circulating microvesicles or synonymously, extracellular microvesicles
  • eMVs describe an eclectic group of microvesicles that are released by cells, and therefore, exist in extracellular spaces and/or reside in body fluids.
  • the mammalian body fluids that are known or suspected to contain cMVs include, but are not limited to, blood, urine, ascites fluid and
  • cerebrospinal fluid secreted microvesicles are also found in cell culture media that has been exposed to cultured mammalian cells.
  • microvesicles are theorized to include (i) exocytosis of intracellular multivesicular bodies, (ii) outward budding, fission and shedding of plasma membrane, and (iii) byproducts of apoptosis.
  • the diverse collection of circulating microvesicle structures can range in size from about 20 nanometers (nm) to upwards of about 1,000 nm (i.e., 1.0 micrometer, micron, or ⁇ ) in diameter.
  • the first recognized subgroup of cMVs are those produced by direct plasma membrane budding, fission and shedding. Some sources describe these shed microvesicles as generally large, namely with lower sizes limits of at least 100 nm or 200 nm, and with an upper size limit of about 1,000 nm in diameter. Some have proposed that these structures be termed
  • ectosomes or “shedding microvesicles (SMVs).” Still other groups state that ectosome particles may be as small as 40 or 50 nm in diameter.
  • a second recognized subgroup of cMVs are exosomes, that is, the preformed microvesicles that are released from the cell following the exocytic fusion of intracellular multivesicular bodies with the plasma membrane.
  • exosome structures are generally smaller than ectosmoes, and have an upper size limit estimated to be about 100, 150 or 200 nm, and a lower size limit of about 40 nm or 50 nm.
  • various sources differ in their size-based definitions for exosomes, and this size distinction remains unresolved.
  • a third group of structures is the apoptotic blebs released by dying cells. These membrane structures have a less well defined size range, and may be anywhere from about 50 nm to about 5,000 nm in diameter.
  • microvesicles have been alternatively referred to as microparticles, nanoparticles, exosomes, ectosomes, epididimosomes, argosomes, exosome-like vesicles, promininosomes, prostasomes, dexosomes, texosomes, archeosomes, oncosomes, exosome-like vesicles, apoptotic blebs, and shedding microvesicles.
  • uses of these terms is conflicting or overlapping.
  • extracellular microvesicles are not clearly understood, although they are theorized to act as nano-shuttles for the transport and delivery of information from one location and/or cell type to distant locations and/or other cell types. Exosomes are theorized to be involved in a wide variety of physiological processes, including cardiac disease, adaptive immune responses to pathogens, and in tumor biology. It is suggested that microvesicles may play roles in tumor immune suppression, metastasis, and tumor-stroma interactions. Microvesicles are thought to play a role in immune system cellular communication, for example, involving dendritic cells and B cells.
  • microvesicles can potentially serve as tools in molecular medicine as measures of physiological state, disease diagnostics, and possibly therapeutic targeting.
  • NTA nanoparticle tracking analysis
  • FNTA fluorescent nanoparticle tracking analysis
  • centrifugation is the process whereby a centrifugal force is applied to a mixture, whereby more-dense components of the mixture migrate away from the axis of the centrifuge relative to other less-dense components in the mixture.
  • the force that is applied to the mixture is a function of the speed of the centrifuge rotor, and the radius of the spin.
  • a density-based separation or “gradient centrifugation” technique is used to isolate a particular species from a mixture that contains components that are both more dense and less dense than the desired component (e.g., OptiPrepTM).
  • the force that is applied is the product of the radius and the angular velocity of the spin, where the force is traditionally expressed as an acceleration relative to "g," the standard acceleration due to gravity at the Earth's surface.
  • the centrifugal force that is applied is termed the “relative centrifugal force” (RCF), and is expressed in multiples of "g” (or "x g").
  • centrifugation procedures that have been used to isolate circulating microvesicles can incorporate as many as five centrifugation steps, with at least two of these spins requiring centrifugal forces in excess of 100,000 x g for several hours.
  • ultracentrifugation is centrifugation conditions that produce forces in excess of 100,000 x g.
  • Size exclusion chromatography can also be used to isolate microvesicles, for example, by using a SephadexTM G200 column matrix. This approach is also time consuming and the yields are inconsistent.
  • Selective immunoaffinity capture can also be used to isolate circulating microvesicles, for example, by using antibodies directed against the epithelial cell adhesion molecule, a type-l transmembrane cell-surface protein (also known as EpCAM, CD326, KSA, TROP1).
  • EpCAM epithelial cell adhesion molecule
  • CD326, KSA, TROP1 a type-l transmembrane cell-surface protein
  • TROP1 type-l transmembrane cell-surface protein
  • the anti-EpCAM antibodies can be coupled to magnetic microbeads, such as
  • These minimal media contain various concentrations of defined components, including, for example, but not limited to, the 20 amino acids, purine and pyrimidine nucleotides and/or nucleotide precursors, phospholipids and phospholipid precursors, vitamins (as parts of coenzymes), lipoic acid, a carbon source such as glucose, and inorganic ions.
  • Some formulations add additional components, such as growth factors and hormones, and/or vary the concentrations of the various components.
  • the serum that is used to supplement the minimal defined medium can be from a variety of sources, for example, bovine, equine (horse), human, mouse, rat and goat. Bovine serum is most commonly used in laboratory settings. Serum supplements that are derived from age-staged animals can also be used, and may be desirable for their various growth properties.
  • bovine serum can be age-staged as fetal bovine serum (FBS), calf serum (CS), newborn calf serum, or adult bovine serum.
  • FBS fetal bovine serum
  • CS calf serum
  • Heat inactivated FBS is frequently used in many applications. Heat inactivated FBS is frequently used in combination with DMEM to form a complete growth media for many types of mammalian cells.
  • FBS contains an abundance of endogenous bovine microvesicles, including exosomes. These endogenous microvesicles can exert effects on cultured cells when the FBS is used as a supplement to make a complete culture medium.
  • Sakwe et a/. "Fetuin-A (alpha- 2HS-Glycoprotein) Is a Major Serum Adhesive Protein That Mediates Growth Signaling in Breast Tumor Cells," J Biol Chem. 285(53):41827-41835 (2010).
  • These endogenous microvesicles may impact the growth or differentiation of cells maintained in culture, and may skew or interfere with experimental results and experimental interpretation.
  • the endogenous microvesicles found in blood serum can copurify with microvesicles that are produced by the cultured cell lines of interest. Bhatnagar et al., "Exosomes released from macrophages infected with intracellular pathogens stimulate a proinflammatory response in vitro and in vivo," Blood Vol. 110, no. 9:3234-3244 (2007).
  • microvesicles specifically circulating microvesicles, for example, exosomes and microsomes.
  • these methods will utilize common laboratory reagents and apparatus, and will not require high speed centrifugation, such as ultracentrifugation.
  • high speed centrifugation such as ultracentrifugation.
  • What is needed in the art are methods for the isolation of circulating microvesicles, where the methods utilize low speed centrifugation that uses centrifugal forces significantly less than 100,000 x g.
  • the present invention provides methods for the rapid and inexpensive isolation of microvesicles, specifically circulating microvesicles such as exosomes. These methods utilize common laboratory reagents and apparatus, and do not require high speed centrifugation, such as ultracentrifugation. These methods of the invention also do not require the use of gradient sedimentation for the isolation of circulating microvesicles.
  • microvesicles can be used to isolate microvesicles from any source, including biofluids, such as but not limited to, blood serum, blood plasma, ascites fluid, urine, or cerebrospinal fluid (CSF). These methods can also be used to isolate microvesicles from cell culture media that has been used to culture mammalian cells in vitro.
  • biofluids such as but not limited to, blood serum, blood plasma, ascites fluid, urine, or cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the microvesicles isolated by the methods of the invention e.g., the exosomes, have the characteristics of true microvesicles, as assayed by protein markers, small RNAs and microscopic examination of size and structure of the microvesicles.
  • the invention provides methods for isolating secreted microvesicles from a liquid sample, where those methods comprise (i) combining the liquid sample with a precipitation solution that minimally contains polyethylene glycol (PEG) having an average molecular weight of about 8,000 Daltons, (ii) incubating the mixture; typically at 4°C, and typically overnight, (iii) centrifuging the mixture to form a pellet and a supernatant, most advantageously, in a low speed centrifugation, (iv) removing the supernatant after the spin, and (v) recovering the pellet by resuspending in a resuspension solution.
  • PEG polyethylene glycol
  • the secreted microvesicles that are isolated are exosomes.
  • isolation of exosomes is confirmed by determining whether or not the isolated material is enriched for protein or nucleic acid makers that are known to preferentially segregate with exosomes. Confirmation can also be obtained by physical analysis where microvesicles having an average diameter between about 40 nm and about 150 nm is consistent with exosome isolation.
  • the liquid sample is a conditioned cell culture media, i.e., a culture media that has been used to culture cells.
  • the liquid sample can also be any type of biofluid, for example but not limited to whole blood, blood serum, blood plasma, urine, saliva, sputum, breast milk, ascites fluid, synovial fluid, amniotic fluid, semen, cerebrospinal fluid, follicular fluid and tears.
  • the invention also provides method for producing serum that is depleted or partially depleted of endogenous microvesicles, or the microvesicles are below the limits of detection.
  • These methods comprise (i) combining the serum with a precipitation solution that minimally contains polyethylene glycol (PEG) having an average molecular weight of about either 8,000 or 10,000 Daltons, (ii) incubating the mixture, typically at 4°C, and typically overnight, (iii) centrifuging the admixture to form a pellet and a supernatant, most advantageously, in a low speed centrifugation, (iv) recovering the supernatant after the spin, and (v) transferring the supernatant to a suitable container, where the supernatant is the exosome-depleted serum. This supernatant then finds use as a supplement for complete cell growth media.
  • PEG polyethylene glycol
  • concentration of PEG that is between about 300 mg/mL and 500 mg/mL.
  • the serum and the precipitation solution are most typically combined in a volume ratio of about 5:1 (five parts serum, one part precipitation solution), although other volume ratios can be used as well, for example, in the range from 1:1 to 10:1.
  • the microvesicle-depleted serum comprises microvesicles in a concentration of not more than about 10 4 microvesicles per milliliter (mL).
  • the serum that is used to prepare the exosome-depleted serum is not limited.
  • the starting serum that is used to produce the exosome-depleted serum can be any serum including but not limited to bovine serum, horse serum, human serum, rat serum, mouse serum, rabbit serum, sheep serum, goat serum, lamb serum, chicken serum and porcine serum.
  • the serum can be fetal bovine serum.
  • FIG. 1 provides a histogram summarizing the results of an experiment that analyzes the ability of different PEG polymers and PEG polymer combinations to isolate total protein from conditioned cell culture media.
  • FIG. 5A provides a graphic showing the size distribution sorting analysis of microvesicles isolated from serum-free media used to culture the human HT1080 lung sarcoma cell line, as assessed using NanoSight instrumentation for nanoparticle tracking analysis (NTA).
  • FIG. 5B provides an optical microsope representative field screen capture of the particles characterized in FIG. 5A using the NanoSight LM10 instrumentation.
  • FIG. 6A provides a graphic showing the size distribution sorting analysis of microvesicles isolated from serum-free media used to culture the human embryonic kidney (HEK) cell line, as assessed using NanoSight instrumentation for nanoparticle tracking analysis (NTA).
  • FIG. 6B provides an optical microsope representative field screen capture of the particles characterized in FIG. 6A using the NanoSight LM10 instrumentation.
  • FIG. 7A provides a graphic showing the size distribution sorting analysis of microvesicles isolated from urine, as assessed using NanoSight instrumentation for nanoparticle tracking analysis (NTA).
  • FIG. 7B provides an optical microscope representative field screen capture of the particles characterized in FIG. 7A using the NanoSight LM10 instrumentation.
  • FIG. 9 provides a photographic image of a 15% TBE-urea gel visualized using SYB green staining for RNA. The supernatant and pellet samples are taken following exosome isolation from a human urine sample.
  • FIG. 11 provides a histogram showing ELISA results measuring the presence of bovine CD63 protein in untreated serum (FBS) and exosome-depleted serum (Exo-FBS).
  • FIG. 12 provides a histogram summarizing comparative cell growth data for the cell lines, where each of those lines was alternatively cultured in DM EM supplemented with either untreated fetal bovine serum (FBS) or exosome-depleted serum (Exo-FBS).
  • FBS fetal bovine serum
  • Exo-FBS exosome-depleted serum
  • FIG. 13A provides phase-contrast light microscope images of HT1080 and HEK293 cell cultures that were alternatively cultured in DMEM supplemented with either untreated fetal bovine serum (FBS) or exosome-depleted serum (Exo-FBS).
  • FIG. 13B provides phase-contrast light microscope images of HT1080 and PC-3 cell cultures that were alternatively cultured in DMEM supplemented with either untreated fetal bovine serum (FBS) or exosome-depleted serum (Exo-FBS).
  • FIG. 15 provides a histogram showing side-by-side testing data to assess and compare the efficacy of various methods for microvesicle isolation from urine samples.
  • FIG. 16 provides a graphic summary of qPCR testing for the presence and relative abundances of 12 NA sequences in standard (untreated) FBS, and in exosome-depleted FBS (Exo- FBS).
  • the present invention provides compositions and methods for producing preparations of isolated secreted microvesicles from a liquid sample.
  • the invention also provides methods for producing blood serum that has been at least partially depleted of microvesicles.
  • cells or “cell culture” or “cell lines” or “cell culture media” refers to eukaryotic cells, and more specifically (but not exclusively), to higher eukaryotic cells such as mammalian cells, as in human cells or mouse cells.
  • the description of the present invention does not pertain to prokaryotic cells such as eubacteria cells or Archaea cells.
  • microvesicle refers generally to any plasma membrane bound particle, that may reside within the cell, or in the extracellular environment. These structures are not limited in any way with regard to in vivo localization (e.g., intracellular or extracellular), in a body fluid, in a cell culture media, generated by in vitro cultured cells, mechanism of origin or size characteristics. In some embodiments, a microvesicle can range in size with a lower size limit of at least about 20 nanometers (nm) in diameter, or alternatively, 30 nm, or 40 nm, or 50 nm in diameter.
  • a microvesicle has an upper size limit of not more than about 1,000 nm (i.e., 1.0 micrometer, micron, or ⁇ ), or alternatively, not more than about 1,500 nm, about 2,000 nm or about 2,500 nm.
  • the term "secreted microvesicle” is used synonymously with
  • circulating microvesicle cMV
  • extracellular microvesicle e V
  • circulating microvesicle e V
  • extracellular microvesicle e V
  • a cMV of the invention can be produced by (i) exocytosis from multivesicular bodies to produce exosomes, (ii) budding, fission and shedding of microvesicles directly from a cytoplasmic membrane, and (iii) membranous blebs caused by programmed cell death leading to the formation of apoptotic bodies.
  • the term "cMV" is not limited to microvesicles of any particular size or size range.
  • exosome refers to a subset of circulating microvesicles that are preformed microvesicles that are released from the cell following the exocytic fusion of intracellular multivesicular bodies with the plasma membrane, i.e., exosomes have an endocytic origin. As used herein, it is not intended that an exosome of the invention be limited by any particular size or size range.
  • apoptotic body refers to a subset of circulating microvesicles that are produced as a result of apoptotic cell destruction. As used herein, it is not intended that an apoptotic body of the invention be limited by any particular size or size range.
  • the term "isolating,” or “to isolate,” refers to any artificial (i.e., not naturally occurring) process for treating a starting material, where the process results in a more useful form of a molecule or structure of interest that is in the starting material.
  • the "more useful form” of the molecule or structure of interest can be characterized in a variety of ways, no one of which is limiting.
  • the invention provides methods for isolating secreted microvesicles from conditioned cell culture media. Further, for example, the process for isolating can result in:
  • the starting material e.g., concentrating
  • any artificial process for separating a molecule or structure of interest from at least one other component with which it is normally associated e.g., purifying
  • isolated generally refers to the state of the molecule or structure of interest after the starting material has been subjected to a method for isolating the molecule of interest. That is to say, isolating a molecule of interest from a starting material will produce an isolated molecule.
  • the methods of the invention are used to produce preparations of isolated microvesicles. These preparations of microvesicles have been isolated from their natural source, for example, from urine, or from conditioned cell culture media.
  • purifying or “to purify” a molecule or structure of interest refers to a process for removing at least one impurity or contaminant from a starting material.
  • purifying a molecule of interest from a starting material refers to a process for removing at least one impurity from the starting material to produce a relatively more pure form of the molecule of interest.
  • substantially purified refers to molecules or structures of interest that are removed from their natural environment or from a starting material (i.e., they are isolated) and where they are largely free from other components with which they are naturally associated or substantially free of other components that may render future use or study sub- optimal, difficult or impossible.
  • purified refers to molecules or structures of interest that are removed from either (1) their natural environment, or from (2) a starting material (i.e., they are isolated), and where (a) at least one impurity from the starting material has been removed, or (b) at least one component with which the molecule is naturally associated has been removed.
  • a “purified” or “partially purified” molecule may still contain additional components that may render future use or study of the molecule sub-optimal, difficult or impossible.
  • enriching refers to a process whereby a molecule of interest that is in a mixture has an increased ratio of the amount of that molecule to the amount of other undesired components in that mixture after the enriching process as compared to before the enriching process.
  • the term "depleted” refers to a mixture containing an undesirable component, where that undesirable component has been (i) completely removed from the mixture, (ii) sufficiently removed from the mixture to be undetectable, or (iii) partially removed from the mixture such that its concentration in the mixture is significantly reduced.
  • a blood serum that has been depleted of endogenous microvesicles may contain no microvesicles, or may contain no detectible microvesicles, or may contain a reduced level of microvesicles compared to the untreated serum.
  • cell culture media refers to any growth media that can support in vitro cell growth of a designated cell line. Such media can be supplemented or non- supplemented, for example, with 10% by volume, heat-inactivated fetal calf serum.
  • minimal defined cell culture media or “minimal media” refers to any culture media where each component is defined by name and the
  • Minimal defined cell culture media generally does not contain a serum supplement.
  • Dulbecco's Modified Eagle's medium (DMEM) is a defined minimal cell culture media.
  • Minimal defined cell culture media generally can be used to culture cells in vitro, but not for extended periods of time.
  • the expression "complete cell culture media” refers to a culture media that comprises a defined minimal cell culture media, and in addition, also comprises a complex supplement that enhances the growth properties of the culture media.
  • a blood serum supplement is commonly added to a minimal media to produce a complete cell culture media.
  • Fetal calf serum (FBS or FCS) is a common supplement (10% by volume) that is added to a minimal media to produce a complete culture media.
  • Complete culture media are used to culture cells in vitro for indefinite (long) periods of time.
  • the expression "conditioned cell culture media” refers to any cell culture media (including complete media or minimal media) that has been exposed to live cells in culture. Conditioned cell culture media comprises not only the defined components of the minimal media and the serum supplement, but also contains additional components that the living cultured cells have produced. In many cases, conditioned cell culture media is a serum-free media.
  • the present invention provides methods for the isolation of secreted microvesicles from liquid samples.
  • the methods for isolating comprise the following steps.
  • the precipitation solution that is used is an aqueous solution comprising at least one polyethylene glycol (PEG) species that has a molecular weight of between 400 Daltons to 8,000 Daltons.
  • PEG polyethylene glycol
  • the PEG that is used is PEG-8,000.
  • the precipitation solution comprises PEG-10,000.
  • the nature of the solution that is used to make the PEG precipitation solution is not particularly limited, and can be, for example, water, or any physiological saline, such as phosphate buffered saline.
  • a precipitation solution is prepared by dissolving the PEG species (e.g., PEG-8,000) in an aqueous phase.
  • This precipitation solution can contain, for example, between 300 milligrams per milliliter (mg/mL) to about 500 mg/mL of the PEG polymer (i.e., 30%-50% PEG by weight).
  • a concentration of about 500 mg/mL of PEG in the precipitation solution was preferred in some embodiments.
  • the PEG is dissolved in any suitable aqueous solution, including simply water. It is not intended that the aqueous solution used to prepare the PEG precipitation solution be limited in any way.
  • the PEG precipitation solution is typically prepared in a standard phosphate buffered saline (PBS) solution, although the PBS is not strictly required.
  • PBS phosphate buffered saline
  • PBS is isotonic and physiologically compatible, and maintains pH and osmolality near physiological levels. It is widely used when handling cell cultures and complex biomolecules.
  • a variety of formulations of PBS are known in the literature, any of which can find use with the invention.
  • the PBS uses the following formulation, which is one common formulation for PBS.
  • the precipitation solution is added to the liquid sample (e.g., conditioned cell culture media), generally in a volume ratio of about one part of PEG precipitation solution to about 5 parts of liquid sample (1:5). It is not intended that the invention be limited to this ratio or quantity of PEG used to precipitate the exosomes. Depending on the liquid sample, other volume ratios may be useful, for example, any ratio (precipitation solution to liquid sample) between about 1:1 to about 1:10.
  • the tube or vessel with these two components is mixed or agitated to fully disperse the components. In some embodiments, gentle mixing, such as by swirling or inverting, is preferred.
  • the resulting mixture is then incubated.
  • the incubation can be with any degree of cooling, for example at 5°C, although such cooling is not required.
  • the incubation times can vary, and are not in any way limiting. For example, incubation can be anywhere between 30 minutes to overnight (e.g., 16 hours).
  • the incubation can be with or without mixing, and the mixing during the incubation period can be constant or intermittent.
  • the mixture is subjected to a centrifugation.
  • the centrifugation typically forms a pellet and a supernatant, although pelleted material may not be visible to the eye. In contrast to the prior art, this centrifugation does not require
  • ultracentrifugation e.g., does not require centrifugal forces in excess of 100,000 x g.
  • This centrifugation can be done at slower speeds, for example, to generate RCF values of not more than 30,000 x g, or not more than 20,000 x g, or not more than 12,000 x g, or not more than 10,000 x g, or not more than 5,000 x g, or not more than 2,000 x g, or not more than 1,500 x g.
  • RCF values not more than 30,000 x g, or not more than 20,000 x g, or not more than 12,000 x g, or not more than 10,000 x g, or not more than 5,000 x g, or not more than 2,000 x g, or not more than 1,500 x g.
  • a centrifugation producing 1,500 x g is preferred.
  • the length of time for centrifugation is not limiting. In some embodiments, the centrifugation is for 30 minutes.
  • the pellet is resuspended in any desired resuspension solution and collected for further analysis.
  • the resuspension solution can use either water, phosphate buffered saline (PBS), or any other suitable aqueous, such as any isotonic solution.
  • PBS phosphate buffered saline
  • the volume used for the resuspension is most typically the smallest possible practical volume, and is typically many times smaller than the volume of the original liquid sample comprising the secreted microvesicles. In some embodiments, the volume of the resuspension solution is smaller by at least one order of magnitude than the volume of the original liquid sample.
  • centrifuged material there may not be a visible pellet, there still may well be centrifuged material at the bottom of the centrifuge. In this case, carefully remove the supernatant, being careful not to disturb the bottom area of the centrifuged vessel. After removing the supernatant, add a reasonably small volume of resuspension liquid to the tube, and agitate to collect any centrifuged material, e.g., microvesicles.
  • preparations of isolated microvesicles where the preparations can comprise substantially purified or partially purified microvesicles, preparations enriched in microvesicles, and/or preparations comprising microvesicles that have been
  • the methods for isolating microvesicles can include additional step or steps prior to admixing the liquid sample with the precipitation solution.
  • the liquid sample can be subjected to an optional centrifugation or filtration step for the purpose of removing unwanted whole cells, cellular debris or other precipitates from the sample prior to mixing with the PEG precipitation solution.
  • the methods of the invention can be adapted to isolate intracellular membrane- bound structures (e.g., organelles or endosomes) from a preparation of cells following disruption of the cellular membrane. This can be accomplished by methods for cell membrane disruption that preserve the integrity of smaller intracellular membrane bound organelles, for example, by mechanical disruption such as shearing.
  • intracellular membrane- bound structures e.g., organelles or endosomes
  • the methods of the invention are optimized for the isolation of microvesicles from conditioned cell culture media, where the cell culture media can contain a serum supplement, or be serum-free.
  • the methods for microvesicles isolation can be optimized for various biofluids. For example, isolation of microvesicles from urine or conditioned cell culture media is optimized by the use of a precipitation solution comprising PEG-8,000. Also for example, the isolation of microvesicles from blood serum or other body fluids is optimized by the use of a precipitation solution comprising PEG-10,000.
  • the present invention provides methods for isolating circulating microvesicles from liquid samples. It is not intended that the nature of the liquid samples be in any way limited, and can be any liquid sample that contains microvesicles.
  • very small volumes of liquid sample can be used, for example, as little as 1.0 mL, or 2.0 mL. or 3.0 mL. or 5.0 mL of starting sample can be used.
  • the liquid sample can be conditioned cell culture media that has been used to culture a cell line in vitro that has produced microvesicles, and therefore, those microvesicles are now contained in the conditioned media.
  • the conditioned cell culture media can be a complete media (containing a serum supplement), or a serum-free culture media.
  • the conditioned cell culture media is a complete media comprising a serum supplement
  • the serum supplement that is used can be a serum that has been depleted of any endogenous circulating microvesicles prior to addition of the supplement to the defined minimal growth media.
  • the present invention also provides methods for producing such exosome-depleted serum.
  • the liquid sample is a biofluid (synonymous with body fluid).
  • body fluid that is used in the analysis is not particularly limited. Microvesicles can be isolated from any body fluid using the methods of the invention, even though a particular body fluid is not itemized herein, as it is intended that the present methods find use with any and all body fluids.
  • body fluids that can be analyzed by the methods of the invention include, but are not limited to, amniotic fluid, blood serum, blood plasma, breast milk, cerebrospinal fluid, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, semen, synovial fluid, tears, urine and ascites fluid.
  • the present invention provides methods for the isolation of microvesicles from liquid samples, where the methods utilize a precipitation solution, combined with the liquid sample, to initiate the microvesicle (or exosome) precipitation and isolation.
  • the precipitation solution that is used is an aqueous solution comprising at least one polyethylene glycol (PEG) species that has an average molecular weight between and including 400 Daltons and 8,000 Daltons.
  • PEG polyethylene glycol
  • the PEG that is used is PEG-8,000.
  • the precipitation solution comprises PEG-10,000.
  • the nature of the aqueous phase that is used to make the PEG precipitation solution is not particularly limited, and can be, for example, only water, or a physiological saline, such as phosphate buffered saline.
  • a physiological saline such as phosphate buffered saline.
  • PBS PBS
  • the resulting solution is termed ExoQuick-TCTM manufactured by System Biosciences, Inc. (SBI; Mountain View, California).
  • Combinations of PEG polymer species can also be used in the methods of the invention.
  • the PEG polymer that is used is not limited to any particular purity, and the choice of purity is dependent on the users' requirements. Regardless of the purity that is used, it remains a feature of the invention that the average molecular weight is defined, for example, as a PEG polymer with an average molecular weight (i.e., Daltons) of 8,000 Daltons, or alternatively, 10,000 Daltons. PEG polymers are available from a variety of commercial manufacturers, any of which find use with the invention.
  • the concentration of the PEG polymer in the precipitation solution is not particularly limited. As a guide, 300-500 mg/mL of the PEG species can be used as a guide, but users can define their own optimal conditions that may lie outside this range. Concentrations that lie outside this range are also within the scope of the invention. In some embodiments, PEG polymer is in a concentration of about 500 mg/mL in the precipitation solution. These weight concentrations can also be expressed as molar concentration equivalents, e.g., about 0.06 to 1.25 mol/L.
  • the invention provides methods for removing microvesicle structures from a blood plasma or a blood serum, thereby creating a microvesicle (or exosome)-depleted plasma or serum.
  • This method entails adding a precipitation solution to the plasma or serum, incubating the admixture, subjecting that mixture to a low speed centrifugation, and then isolating and preserving the supernatant.
  • This supernatant is now depleted of microvesicle structures, where the supernatant has no detectable microvesicle structures, or where the supernatant comprises significantly reduced levels of microvesicle structures.
  • the precipitation solution comprises one or a combination of PEG polymers.
  • the precipitation solution can comprise a PEG polymer of 8,000 Daltons, or about 10,000 Daltons, for example. Precipitation conditions similar to the conditions to producing isolated microvesicles are utilized.
  • the invention be limited to the generation of any one type of microvesicle-depleted plasma or serum.
  • plasma and serum are used for research purposes, and all of these plasma and sera have advantageous sues when they have been depleted of microvesicle structures.
  • the plasma or serum that can be produced as a microvesicle-depleted product includes, but is not limited to, bovine serum, horse serum, human serum, rat serum, mouse serum, rabbit serum, sheep serum, goat serum, lamb serum, chicken serum and porcine serum.
  • Such sera and plasma can also be age staged, for example, fetal bovine serum, calf bovine serum, newborn calf bovine serum or adult bovine serum.
  • PEG-10,000 e.g., 50% by weight in PBS
  • FBS fetal bovine serum
  • Exo-FBSTM fetal bovine serum
  • Exo-FBSTM is devoid of bovine CD63 positive exosomes and does not have any measurable bovine microRNAs.
  • Exo-FBSTM supports cell growth equivalent to untreated FBS, and in many types of cells in culture.
  • Lentivirus particles have an average diameter of about 80nm, a size that is within the estimated range of exosome size.
  • Formulations of polyethylene glycol (PEG-6000) had been previously used to precipitate and concentrate lentiviral particles from conditioned cell culture medium (Lewis and Metcalf, Applied and Environmental Microbiology, Vol. 54, No. 8 (1988).
  • exosomes have similar diameters to a lentiviral particle, it was hypothesized that PEG formulations might be used to isolate exosomes from conditioned cell culture media that had been used to culture exosome-producing cell lines in vitro, and additionally, may also be used to isolate exosomes from other liquids such as biofluids.
  • Any PEG polymer species defined by a particular molecular weight is in fact a mixed population of PEG molecules of varying lengths, but where the molecule population is characterized by its average molecular weight. It is understood that when a reagent such as PEG-8,000 is specified, the vast preponderance of molecules in that preparation will have a molecular weight of about 8,000 kilodaltons (within the accuracy range of experimental determination), but the population of molecules will also contain some small fraction of molecules that are slightly smaller and slightly larger than 8,000 kilodaltons.
  • a matrix of varying PEG polymer sizes was used to test whether exosomes could be recovered from culture medium that has been conditioned using exosome-producing cells in serum- free conditions.
  • Conditioned media was obtained from the culture of human embryonic kidney (HEK)-293 cells in 10 mL media in serum-free conditions. Fresh serum-free media was applied to an established HEK-293 cell culture, incubated for 2 days, and then collected. The conditioned media samples were then centrifuged at 1500 x g for 30 minutes to remove whole cells or cell debris.
  • a precipitation solution was prepared that contained the various PEG species (e.g., the PEG-8,000 ExoQuick-TCTM reagent).
  • PEG-8,000 ExoQuick-TCTM reagent
  • that PEG was tested using a broad range of molar concentrations from about 0.06 mol/L to 1.25 mol/L. From this range, optimal conditions were identified using between about 300 milligrams per milliliter (mg/mL) to about 500 mg/mL of the PEG polymer.
  • the PEG in the precipitation solution was most frequently in a concentration of about 500 mg/mL.
  • the PEG precipitation was typically prepared in a standard phosphate buffered saline (PBS) solution. This PEG stock solution was a clear, slightly viscous liquid that was stored at 5°C.
  • PBS phosphate buffered saline
  • the resuspension used was either water or phosphate buffered saline (PBS).
  • PBS phosphate buffered saline
  • the exosome suspension derived from urine was also used in a Western blot to detect the known exosome protein marker CD9 using the same set of antibodies as used in the ELISA.
  • Samples were separated on gradient 4-15% PAGE gels, transferred to PVDF membranes and probed with anti-CD9 primary antibody, then probed with the secondary antibody, and subjected to color development.
  • the results are shown in FIG. 2 in the insert.
  • This Western blot reveals the abundant presence of an anti-CD9-reactive protein having the 22 kilodalton predicted molecular weight of the CD9 marker.
  • Formvar/carbon-coated nickel grids were incubated on drops of 0.1% poly-L-lysine for 5 minutes, rinsed by water and dried. Freshly prepared grids were placed on top of exosome drops and incubated for 10 minutes to adsorb exosomes. The grids were washed on several drops of PBS and fixed with 2.5% glutaraldehyde in 0.1 M cacodylate buffer for 15 minutes.
  • exosome microvesicles were visualized and photographed, a representative field is shown in FIG. 3. Arrows pointing to a few exosomes are indicated for reference.
  • the exosomes isolated using the PEG-8,000 precipitation solution show the expected size distribution of 40-150nm in diameter. Careful examination also shows the anticipated double-membrane structures of the exosome microvesicles.
  • the size bar in the lower left corner designates 100 nm length.
  • PC-3 prostate cancer cells were grown in a T75 flask in 20ml DMEM supplemented with 10% fetal bovine serum (FBS), where the FBS supplement was previously depleted of exosomes (see Example 11). The cells were cultured for seven (7) days.
  • FBS fetal bovine serum
  • Exosome material was isolated from the conditioned cell culture media alternatively with either the LifeTechnologiesTM kit for total exosome isolation from conditioned media
  • exosomes were isolated.
  • the number of exosomes isolated was measured by ELISA using an antibody specific for the CD63 protein marker (System Biosciences, Inc., ELISA Kit Catalog No. EXOEL-CD63A-1).
  • the PEG-8,000 precipitation reagent precipitated significantly more exosomes compared to the LifeTechnologiesTM product.
  • NanoSight LM10 instrument is based on a conventional optical microscope and uses a laser light source to illuminate nano-scale particles within a 0.3 ml sample introduced to the viewing unit with a disposable syringe. Particles appear individually as point-scatterers moving under Brownian motion.
  • FIG. 5A shows that PEG-8,000 precipitation solution isolated exosomes having an average diameter of approximately 133 nm, with a recovery of 1.74 x 10 9 particles/ml.
  • FIG. 5B shows a screen shot of NanoSight exosome NTA tracking data collection using the NanoSight LM10 instrument.
  • HEK Human embryonic kidney
  • conditioned media serum-free
  • Ten milliliters of the media was combined with 2ml PEG-8,000 precipitation solution to pellet to pellet the exosomes overnight.
  • the exosome pellet was resuspended in 1ml PBS and visualized on the NanoSight LM10 instrument undiluted.
  • the analysis in FIG. 6A shows that PEG- 8,000 precipitation solution to pellet isolated 137 nm exosomes with a recovery of 3.33 x 10 8 particles/ml.
  • FIG. 6B shows a screenshot capture of the exosome particles observed during the NTA analysis.
  • FIG. 7A The analysis shows that PEG-8,000 precipitation solution isolated exosomes having an average size of 107 nm in diameter and with a recovery of 4.80 x 10 9 particles/ml.
  • FIG. 7B shows a screenshot capture of the exosome particles observed during the NTA analysis.
  • MDM Monocyte-derived macrophage
  • a filter membrane prior to and after migration toward the enriched vesicles in a Boyden chemotaxis chamber (scale bar, 1 urn; expanded box, 10 um) and then imaged using scanning electron microscopy.
  • Exosome membranes demonstrated a high affinity for the surface of the target MDM cells, and their adhesion to the plasma membrane was resistant to washes.
  • This Western blot is shown in FIG. 14A. This blot verifies that the pellet material following centrifugation contains exosome-specific markers CD63 and CD9, but those same markers are absent from the serum supernatant.
  • FIG. 14B provides Western blot data verifying the presence of known exosome markers CD9, CD63, CD81 and Hsp70 in exosome samples isolated from normal human serum using System Biosciences, Inc., ExoQuickTM (comprising PEG 10,000).
  • the exosome markers were detected using four primary antibodies and reagents from the System Biosciences, Inc. Exosome Sampler Kit (catalog no. EXOAB-KIT-1) and visualized with goat anti-rabbit HRP secondary antibody (THERMO FISHER SCIENTIFICTM catalog no. 31460, goat anti-rabbit IgG (H+L), peroxidase conjugated secondary antibody) at a 1:20,000 dilution.
  • the signals were developed using SuperSignal West Femto substrate kit (THERMO FISHER SCIENTIFICTM catalog no. 34094).
  • exosomes can be isolated using polyethylene glycol (PEG) polymer formulations, as demonstrated by known exosome markers in Western blots, ELISAs, physical dimensions such as diameters measured using electron microscopy and NanoSight particle analysis methods. Exosomes isolated by the protocols descrtibed herin also contain exosome RNA cargo.
  • PEG polyethylene glycol
  • exosome microvesicles contain small RNAs encapsulated in the vesicle.
  • Exosomes isolated from human urine as described in Example 2 were lysed using Trizol reagents and the RNA extracted using standard methods. The RNA was separated on a 15% TBE-Urea gel and visualized using SYBR green staining for RNA.
  • RNA enrichment was compared between the supernatant and the exosome pellet.
  • the data shown in FIG. 9 reveals a nearly quantitative enrichment of small RNAs from the exosomes isolated using the PEG-8,000 precipitation method of the invention.
  • Standard FBS was thawed from frozen and mixed by inversion. 200ml of the FBS was transferred to a sterile 250 mL screw cap conical bottom centrifuge tube (Corning, catalog no. 430776).
  • Example 11 The exosome-depleted serum described in Example 11 was analyzed for the presence of exosome particles using NanoSight particle analysis. The original untreated serum was also analyzed in parallel.
  • bottom panel is characterized by a vast reduction in the number of particles compared to the untreated serum (top panel), as seen in both the particle sorting and microscope field of view.
  • Exosome-Depleted Serum is Devoid of Detectable Bovine CD63 Protein
  • Example 11 The exosome-depleted serum described in Example 11 was analyzed for the presence of bovine CD63 protein, as measured by ELISA. The original untreated serum was also analyzed in parallel.
  • Example 11 The exosome-depleted serum (Exo-FBS) described in Example 11 was analyzed for its ability to support cell growth in culture, as compared to untreated fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • DMEM media supplemented with either 10% standard FBS or with 10% exosome-depleted FBS.
  • HT1080 fibrosarcoma cells and HEK293 cells were cultured under standard conditions at 37°C with 5% C0 2 for 5 days in the two different DMEM media indicated.
  • Example 11 The exosome-depleted serum described in Example 11 was analyzed for its RNA content. Standard FBS and exosome-depleted-FBS media supplements (4 mL each) were treated with Trizol extraction methods to recover exosome RNAs. The extracted RNA was resuspended in 20 ⁇ _ water.

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Abstract

La présente invention concerne des compositions et des procédés d'isolement de microvésicules produites par des cellules mammifères. Ces microvésicules, connues en tant que microvésicules extracellulaires ou microvésicules circulantes, sont isolées à partir d'échantillons, tels que des fluides corporels, ou d'un milieu de culture cellulaire utilisé pour cultiver ou conserver des cellules mammifères in vitro. L'isolement de microvésicules tel que décrit ici entraîne la purification et la concentration desdites microvésicules. L'invention porte également sur des procédés afférents de production de sérum sanguin et/ou de plasma sanguin exempt de microvésicules détectables, considérablement appauvri en microvésicules, ou qui présente une concentration réduite en microvésicules par rapport au matériau de départ de sérum sanguin ou de plasma sanguin (collectivement qualifiés de « appauvri en microvésicules »). La production de plasma ou de sérum sanguin appauvri en microvésicules est essentielle pour permettre une utilisation dans des systèmes expérimentaux pour lesquels il est souhaitable d'utiliser un milieu de culture cellulaire ne contenant pas de microvésicule endogène, ou qui a été appauvri en microvésicules endogènes, à partir du matériau source.
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JP2016518109A (ja) * 2013-03-13 2016-06-23 ユニバーシティ・オブ・マイアミＵｎｉｖｅｒｓｉｔｙ Ｏｆ Ｍｉａｍｉ 細胞培養上清および生体液からの微小胞の単離および精製のための方法
EP2972193A4 (fr) * 2013-03-13 2016-09-07 Univ Miami Procédé pour isoler et purifier les microvésicules de surnageants de culture cellulaire et fluides biologiques
US10500231B2 (en) 2013-03-13 2019-12-10 University Of Miami Method for isolation and purification of microvesicles from cell culture supernatants and biological fluids
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EP2972193A1 (fr) 2013-03-13 2016-01-20 University Of Miami Procédé pour isoler et purifier les microvésicules de surnageants de culture cellulaire et fluides biologiques
WO2014159662A1 (fr) 2013-03-13 2014-10-02 University Of Miami Procédé pour isoler et purifier les microvésicules de surnageants de culture cellulaire et fluides biologiques
CN106399250A (zh) * 2015-07-31 2017-02-15 广州市锐博生物科技有限公司 一种分离外泌体的方法及其试剂盒
WO2017178472A1 (fr) 2016-04-12 2017-10-19 Unicyte Ev Ag Isolement de vésicules extracellulaires (ve) à partir d'échantillons de fluide biologique
US11484816B2 (en) 2016-04-12 2022-11-01 Unicyte Ev Ag Isolation of extracellular vesicles (EVs) from biological fluid samples
EP3602055B1 (fr) * 2017-03-24 2021-01-13 Pfaffl, Michael W. Procédés et trousses d'isolement et de quantification d'exosomes
US11079374B2 (en) * 2017-03-24 2021-08-03 Dapi Meng Lin CHIANG Methods and kits for exosome isolation and quantification
WO2019229271A1 (fr) 2018-06-01 2019-12-05 Alexander May Microvésicules dérivées de produits à base de plantes fermentées, leur procédé de préparation et d'utilisation
CN111117949A (zh) * 2020-01-19 2020-05-08 承启医学（深圳）科技有限公司 一种基于改良聚乙二醇沉淀法分离外泌体的方法
CN111117949B (zh) * 2020-01-19 2023-08-22 承启医学（深圳）科技有限公司 一种基于改良聚乙二醇沉淀法分离外泌体的方法
CN114323851A (zh) * 2021-12-24 2022-04-12 多莱泌生物科技(武汉)有限公司 一种基于超滤和亲和层析技术分离血清血浆中外泌体方法

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