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WO2019022542A2 - Procédé d'isolement de vésicules extracellulaires à l'aide de cations - Google Patents

Procédé d'isolement de vésicules extracellulaires à l'aide de cations Download PDF

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WO2019022542A2
WO2019022542A2 PCT/KR2018/008485 KR2018008485W WO2019022542A2 WO 2019022542 A2 WO2019022542 A2 WO 2019022542A2 KR 2018008485 W KR2018008485 W KR 2018008485W WO 2019022542 A2 WO2019022542 A2 WO 2019022542A2
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endoplasmic reticulum
cation
extracellular
extracellular endoplasmic
present
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Korean (ko)
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WO2019022542A3 (fr
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고용송
이창진
김지현
송성현
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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POSTECH Academy Industry Foundation
Rosetta Exosome Co Ltd
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Priority to US16/634,086 priority Critical patent/US11904259B2/en
Priority to JP2020527719A priority patent/JP2020528766A/ja
Priority to CN201880062590.7A priority patent/CN111148828A/zh
Priority to EP18837656.0A priority patent/EP3660142A4/fr
Priority claimed from KR1020180087354A external-priority patent/KR102107844B1/ko
Publication of WO2019022542A2 publication Critical patent/WO2019022542A2/fr
Publication of WO2019022542A3 publication Critical patent/WO2019022542A3/fr
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

Definitions

  • the present invention relates to a method for separating extracellular endoplasmic reticulum from various samples using the affinity of extracellular endoplasmic reticulum for various cations.
  • Extracellular vesicles are universal cellular mechanisms, nano-sized vesicles that are naturally secreted by all living things or cells, from humans to bacteria.
  • the extracellular endoplasmic reticulum derived from eukaryotic cells is involved in the erythropoietic differentiation and regulation of the immune response. Especially, important functions of cancer progression, metastasis and angiogenesis are revealed in the cancer cell microenvironment, And has received high interest in the utilization of
  • the extracellular endoplasmic reticulum secreted from prokaryotic cells contains prokaryotic components similar to extracellular endoplasmic reticulum.
  • acute pulmonary inflammatory diseases are induced according to the pathway including systemic inflammation.
  • chronic inflammation of the local skin tissues can be induced to cause atopic dermatitis, one of the representative diseases of modern people.
  • the extracellular endoplasmic reticulum from prokaryotic cells has also attracted a great deal of attention due to the emergence of bacterial-derived extracellular endoplasmic reticulum in the human body and cancer incidence.
  • extracellular endoplasmic reticulum The function of extracellular endoplasmic reticulum is most important because they are an important component of the intercellular information exchange mechanism. Therefore, the constituents of extracellular endoplasmic reticulum have also received high interest in the basic and medical fields.
  • the extracellular endoplasmic reticulum is a bio-nanoparticle secreted from various kinds of cells in vivo or in vitro. It is present in body fluid such as blood, urine, saliva, tears, etc. and contains a cell-derived lipid bilayer. It is a vesicle of membrane structure with various sizes.
  • extracellular endoplasmic reticulum binds to other cells and tissues and acts as a transporter that transports intracellular substances such as membrane components, proteins, and RNAs, the proteins, lipids, amino acids, RNA, and so on, thus providing an important basis for understanding the physiological and pathological characteristics of the parent cells.
  • nucleic acid, growth hormone, and protein contained in the extracellular endoplasmic reticulum are protected by a phospholipid in the form of a cell membrane and can perform more stable functions than a soluble growth factor and cytokine. It is expected to be used for various purposes including diagnosis and treatment of diseases by analyzing substances contained in extracellular endoplasmic reticulum.
  • extracellular endoplasmic reticulum is small in nanometer level and numerous substances exist in body fluid and cell culture fluid in addition to extracellular endoplasmic reticulum, extracellular endoplasmic reticulum from samples such as body fluids and cell culture fluids for extracellular endoplasmic reticulum analysis It is important to separate, and it is the most important technology in all areas that utilize extracellular endoplasmic reticulum.
  • the most efficient method of material separation is to sequentially remove contaminants from the complex environment using selective binding to the target material without losing the material during the separation process.
  • the substance having such selective binding properties is limited to some antibodies or protein ligands, and separation of the extracellular endoplasmic reticulum using such an antibody or protein is not only inefficient, Ligand development is difficult, and high cost is required, which is very limited.
  • the present invention provides a method for separating extracellular endoplasmic reticulum easily and efficiently with affinity of a cation and an extracellular endoplasmic reticulum.
  • the extracellular endoplasmic reticulum and cations bind to each other in the sample to form an insoluble complex.
  • the extracellular endoplasmic reticulum-cation complex can be separated by various methods such as centrifugation, ultrafiltration, precipitation by gravity, and then the extracellular endoplasmic reticulum can be separated by desorbing the cation from the complex. This makes it possible to quickly and easily separate the extracellular endoplasmic reticulum from a variety of samples without physical and chemical modification.
  • the extracellular endoplasmic reticulum can be used for diagnosis, treatment, multi-omics studies, It is easy.
  • " extracellular < / RTI > endoplasmic reticulum " of the present invention collectively refers to a living body nanoparticle derived from cells of Archaea, Prokarya or Eukarya and includes extracellular endoplasmic reticulum (exosome), argosomes , Dexosomes, ectosomes, exovesicles, oncosomes, prominosomes, prostasomes, tolerosomes, microparticles (e. G.
  • microvesicles microparticles, microvesicles, nanovesicles, blebbing vesicles, budding vesicles, exosome-like vesicles, matrix vesicles, , Membrane vesicles, shedding vesicles, membrane particles, shedding microvesicles, membrane blebs, epididymosomes, promininosome, texosome, or archeosome, Information that is not.
  • the present invention relates to a method for preparing a biological sample, comprising the steps of (a) adding a cation to a biological sample, (b) reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex, (c) And (d) separating the cation from the complex to purify the extracellular endoplasmic reticulum.
  • FIG. 1 A method of separating the extracellular endoplasmic reticulum according to an embodiment of the present invention is schematically shown in FIG.
  • the method for separating extracellular ER of the present invention comprises the steps of adding a cation to a biological sample (step (a)) and reacting the extracellular endoplasmic reticulum contained in the biological sample with a cation to form a complex (Step (b)).
  • biological sample or " sample” of the present invention includes biological samples or cell culture fluids, tissue samples and the like, including extracellular endoplasmic reticulum, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, blood, urine, Seawater, soil, and fermented food.
  • mammalian cell culture medium bacterial cell culture medium, yeast culture medium, (CSF), cerebrospinal fluid (CSF), ascites, amniotic fluid, semen, milk, dust, fresh water, blood, urine, Seawater, soil, and fermented food.
  • CSF mammalian cell culture medium
  • CSF yeast culture medium
  • CSF cerebrospinal fluid
  • cation " of the present invention is electrically positively charged and has a specific affinity for the extracellular endoplasmic reticulum and can bind to the extracellular endoplasmic reticulum in the sample, preferably a metal cation.
  • metal cation " of the present invention may include alkali metal ions, alkaline earth metal ions, transition metal ions, and transition metal ions.
  • the cation or metal cation of the present invention may preferably be a transition metal ion or an alkaline earth metal, but is not limited thereto, so long as it is a cation having a specific affinity for an extracellular endoplasmic reticulum to be separated.
  • Alkali metal is a group of chemical elements other than hydrogen among the group 1 of the periodic table and includes lithium (Li), sodium (Na), potassium (K), rubidium (Rb), cesium (Cs) Fr).
  • the alkaline earth metal is a Group 2 element of the periodic table and includes beryllium (Be), magnesium (Mg), calcium (Ca), strontium (Sr), barium (Ba) and radium (Ra).
  • the transition metal contains elements 4 to 7 and 3 to 12 of the Periodic Table of the Chemical Periods. It forms an ion-binding compound together with a nonmetal and exists in complex ion form.
  • the transition metal of the present invention is a transition metal selected from the group consisting of Sc, Y, Ti, Zr, Hf, Rf, V, Nb, Tantalum (Ta), dibonium (Db), chromium (Cr), molybdenum (Mo), tungsten (W), sb, manganese (Mn), technetium (Tc), rhenium (Re) ), Iron (Fe), ruthenium (Ru), Os, Os, cobalt, rhodium, iridium, (Pd), Pt, Ds, Cu, Ag, Au, Rg, Zn, Cd, Hg) and copper (Cn).
  • Post-transition metal means a metal element in the p-zone of the periodic table and is a metal element of aluminum (Al), gallium (Ga), indium (In), thallium (Tl), tin (Sn) Pb), bismuth (Bi), and polonium (Po).
  • the method of adding the cation of the present invention includes a method in which a solution containing a cation is added to a sample and a method in which the solution is added in a solid form to dissolve it.
  • a form capable of reacting with an extracellular endoplasmic reticulum in a sample in a cationic state It is not limited.
  • the present invention relates to a method for separating an extracellular endoplasmic reticulum associated with a cation from a sample using a property of specifically binding an extracellular endoplasmic reticulum to a cation to be separated.
  • the extracellular endoplasmic reticulum and the cation are specifically bound in the sample, and an insoluble extracellular endoplasmic reticulum-cation complex is formed.
  • insoluble complexes were formed by adding calcium ion, manganese ion, cobalt ion, copper ion or zinc ion to a culture medium sample or urine sample containing an extracellular endoplasmic reticulum. Also, it was confirmed that the insoluble complex can be easily separated because it is submerged by gravity.
  • the method for separating extracellular fibrils includes separating the extracellular endoplasmic reticulum and the cation complex from the sample (step (c)).
  • the step of separating the extracellular endoplasmic reticulum and the cation complex of the present invention is a step of separating the insoluble complex formed in the step from the sample containing various water-soluble substances, and the step of centrifuging, ultracentrifugation, filtration, ultrafiltration, gravity,
  • One or more methods may be selected from, but are not limited to, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography, polymer-based precipitation or organic solvent precipitation.
  • the method for separating extracellular fibrils comprises separating cations from the complex to purify the extracellular fibrils (step (d)).
  • the step of purifying the extracellular endoplasmic reticulum of the present invention can separate only the extracellular endoplasmic reticulum from the complex by eliminating the specific binding state between the extracellular endoplasmic reticulum and the cations, and it is possible to use various methods or conditions which those skilled in the art can understand Can be applied.
  • the step may comprise adding a chelating agent to the separated extracellular endoplasmic reticulum and cation complex.
  • chelate agents or " chelating ligands” of the present invention means an ion, molecule or atomic group containing two or more coordinating atoms forming a chelate complex stable to metal ions, Depending on the number of atoms, tridentate ligand, tetradentrate ligand, pentadentrate ligand, and hexadentrate ligand are also called.
  • the chelate ligand of the present invention may be selected from the group consisting of iminodiacetic acid (IDA), nitrilotriacetic acid (NTA), tris (carboxymethyl) ethylenediamine (TED), ethylenediamine, Ethylenediamine tetraacetate (EDTA), alkylenediamine triacetic acid, diethylenetriaminepentaacetic acid (DTPA), ethylene glycol bis (beta-aminoethyl ether) -N, N, N, N ', N'-tetraacetic acid (EGTA), phosphoserine and 1,4,7-triazo (TACN, 1,4,7-triazocyclononane).
  • IDA iminodiacetic acid
  • NTA nitrilotriacetic acid
  • TED tris (carboxymethyl) ethylenediamine
  • EDTA Ethylenediamine tetraacetate
  • DTPA diethylenetriaminepentaacetic acid
  • the above step may use a method of changing the pH value of the solution containing the separated extracellular endoplasmic reticulum and the cation complex.
  • the above step further comprises the step of adding an imidazole, histidine, ethylenediamine tetraacetate (EDTA), or salt (s) to a solution containing a separated extracellular endoplasmic reticulum and a cation complex methods of purifying the extracellular endoplasmic reticulum from the complex by varying the concentration of the salts.
  • an imidazole, histidine, ethylenediamine tetraacetate (EDTA), or salt (s) to a solution containing a separated extracellular endoplasmic reticulum and a cation complex methods of purifying the extracellular endoplasmic reticulum from the complex by varying the concentration of the salts.
  • the step of purifying the extracellular endoplasmic reticulum of the present invention can be carried out by a complex selection of one or more of the above methods.
  • the purification conditions of the present invention include, but are not limited to, buffers of pH 10 or less, 0-5 M NaCl, 0-2 M imidazole, 0-2 M metal chelating agents, or combinations of the above conditions .
  • the method for separating extracellular fibrils may further comprise pretreating the sample before adding the cation to the sample.
  • the pretreatment step of the present invention is a step of partially purifying an untreated sample, which comprises centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity chromatography , Polymer-based precipitation, or organic solvent precipitation, but is not limited thereto.
  • it may further comprise post-treating the extracellular endoplasmic reticulum isolated according to the extracellular endoplasmic reticulum isolation method of the present invention.
  • the post-treatment step of the present invention is a step of purifying the separated extracellular endoplasmic reticulum by centrifugation, ultracentrifugation, filtration, ultrafiltration, sonication, density gradient ultracentrifugation, size exclusion chromatography, ion exchange chromatography, affinity
  • One or more methods may be selected from, but are not limited to, chromatography, polymer-based precipitation or organic solvent precipitation.
  • the method for separating extracellular fibrils may further include adding a polymer or salting-out ion in the step of adding a cation to the sample.
  • the rate of insoluble complex formation can be remarkably increased by adding a polymer or salting-out ion together with cations, and the efficiency and time for separation of extracellular fibrils can be remarkably improved .
  • the polymer or salting-out ion can be added to the sample simultaneously with the cation.
  • polymer or salting-out ion may be added before the cation is added to the sample.
  • polymer or salting-out ion may be added after adding the cation to the sample.
  • the polymer may be polyethylene glycol (PEG) or polyoxazoline
  • the polyoxazoline may be selected from the group consisting of polymethyloxazoline (PMOZ , poly (2-methyl-2-oxazoline), poly (2-ethyl-2-oxazoline) or poly (2-propyl-2-oxazoline)
  • PMOZ polymethyloxazoline
  • the polymer may be polyethylene glycol (PEG), poly (2-ethyl-2-oxazoline), or polyethylene oxide (PEOZ).
  • salting-out ion of the present invention refers to a kosmotropic salt for stabilizing a water structure for reducing the solubility of water in a solution to increase the strength of the hydrophobic interaction.
  • These cosmotropic salts are represented by the Hofmeister series according to their ability to affect the solubility of soluble materials in solution and the anion series are: SO 4 2- ⁇ HPO 4 2- OH - ⁇ F - ⁇ HCOO - ⁇ CH 3 COO - ⁇ Cl - ⁇ Br - ⁇ NO 3 - ⁇ I - ⁇ SCN - ⁇ ClO 4 - .
  • the cation series are: NH 4 + , Rb + , K + , Na + , Cs + , Li + , Ca 2+ , Mg 2+ , and Ba 2+ .
  • Cosmotropic salts act as salting ions for hydrophobic particles according to the Hope Meister series.
  • the salting-out ion of the present invention may be an anion stabilizing the water structure in the Hofmeister system and a cosmotropic salt of a counter cation thereof.
  • the method of separating extracellular ER according to the present invention does not require expensive equipments such as centrifugal separator and the sample is not exposed to the extreme environment during the separation process, the extracellular ER can be efficiently separated while preserving the shape or properties of extracellular ER .
  • the method of the present invention can be applied in combination with a conventional extracellular ER separation method, and can be maximized by applying the method before or after the conventional method.
  • the extracellular fibrin separation method of the present invention can separate the extracellular endoplasmic reticulum easily and effectively, it can be utilized as an important factor in the mass purification of the extracellular endoplasmic reticulum, as well as the pre- To be used for clinical diagnosis.
  • the method of separating extracellular endoplasmic reticulum of the present invention can fractionate a subset of extracellular endoplasmic reticulum with various cations using different properties of affinity for specific cations according to the type of extracellular endoplasmic reticulum.
  • the extracellular extracellular subsets can be used to diagnose a multidimensional disease.
  • FIG. 1 is a schematic diagram of a method for separating extracellular ER according to an embodiment of the present invention.
  • FIG. 2 shows the results of the separation and characterization of the extracellular endoplasmic reticulum according to an embodiment of the present invention.
  • FIG. 3 is a graph showing the results of HPLC analysis showing that extracellular endoplasmic reticulum can be isolated from cell culture medium by adding various cations (Ca 2+ , Cu 2+ , Zn 2+ ) at various concentrations according to an embodiment of the present invention.
  • FIG. 4 shows that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of copper cation (copper (II) chloride) according to an embodiment of the present invention. to be.
  • FIG. 5 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of copper cations (copper sulfate (II)) according to an embodiment of the present invention .
  • FIG. 6 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by adding cobalt cations (cobalt chloride) at various concentrations according to an embodiment of the present invention.
  • FIG. 7 is a graph showing the results of nano particle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum is separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese chloride (II)) according to an embodiment of the present invention to be.
  • manganese cations manganese chloride (II)
  • FIG. 8 shows that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of manganese cations (manganese sulfide (II)) according to an embodiment of the present invention. to be.
  • manganese cations manganese cations
  • FIG. 9 shows the results of the nanoparticle analysis (a) and Western blot (b) showing that the extracellular endoplasmic reticulum was separated from the cell culture medium by the addition of various concentrations of calcium cation (calcium chloride) according to an embodiment of the present invention.
  • FIG. 10 shows the results of the nanoparticle analysis (a) and the Western blot (b) that the extracellular endoplasmic reticulum was separated from the cell culture medium by addition of various concentrations of zinc cation (zinc chloride) according to an embodiment of the present invention.
  • Figure 11 shows the results of nano particle analysis (a) and western blot (b) showing that the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of calcium cation (calcium chloride) according to one embodiment of the present invention.
  • Figure 12 shows that extracellular endoplasmic reticulum is isolated in human urine by the addition of various concentrations of manganese cations (manganese sulfate (II)) according to one embodiment of the present invention, as confirmed by nanoparticle analysis (a) and Western blot (b) to be.
  • manganese cations manganese cations
  • Figure 13 shows the results of the nanoparticle analysis (a) and western blot (b) in which the extracellular endoplasmic reticulum was isolated in human urine by the addition of various concentrations of zinc cation (zinc chloride) according to one embodiment of the present invention.
  • FIG. 14 is a graph showing the results of nanoparticle analysis in which extracellular endoplasmic reticulum is separated from a cell culture medium by adding various cations (copper (II) chloride, manganese (II) sulfate) and polymers (PEG and PEOZ) together according to an embodiment of the present invention .
  • FIG. 15 is a result of western blot analysis to confirm that extracellular endoplasmic reticulum is isolated in a cell culture solution by adding copper cation (copper sulfate (II)) and polymer (PEOZ) together according to an embodiment of the present invention.
  • FIG. 16 is a result of confirming the separation of extracellular endoplasmic reticulum by combining a conventional salting-out method (ammonium sulfide) precipitation method with a copper cation (copper sulfate (II)) method according to the present invention.
  • a conventional salting-out method ammonium sulfide
  • copper cation copper sulfate (II)
  • Figure 17 compares the separation of extracellular endoplasmic reticulum (a) with the conventional polyethylene glycol (PEG) according to one embodiment of the present invention and the result of separation (b) of extracellular endoplasmic reticulum using the method of the present invention.
  • PEG polyethylene glycol
  • Colorectal cancer cells SW480 culture was centrifuged at 500xg for 10 minutes (2 times in total) to remove remaining cells and sediments. The supernatant was centrifuged again (2 times in total) at 2,000 x g for 20 minutes to remove precipitates.
  • the sample was mixed with Optiprep (final concentration 30%) and placed in the lowest layer of the ultracentrifuge, and then 20% Optiprap, 5 The layers were stacked in order of% optimaprap.
  • OptiPrap buoyancy density gradient ultracentrifugation (30%, 20%, 5% OptiPrap triple layer) was carried out at 200,000 x g for 2 hours and the supernatant after the ultracentrifugation was subjected to an equal density of 1.08 to 1.12 g / ml ) Area were harvested.
  • FIG. 2 (a) shows the process of separating the extracellular ER from the present specimen.
  • the extracellular endoplasmic reticulum from the purified colon cancer cells was analyzed by HPLC chromatogram. As a result, molecular size-specific chromatography showed an absorption band of 280 nm at 3.6 min (FIG. 2 (b)). As a result of the nanoparticle tracking analysis (NTA) of each fraction by chromatography, it was possible to detect high nanoparticle signals in samples eluted between 3.01 and 4.5 minutes, confirming that this signal is consistent with the extinction band at 280 nm , It was found that the extracellular endoplasmic reticulum was a band detected at 3.6 minutes in the HPLC analysis (Fig. 2 (b)).
  • the extracellular endoplasmic reticulum from the colorectal cancer cell line (SW480) was confirmed by electron microscopy. As a result, as shown in Fig. 2 (d), it was confirmed that the extracellular endoplasmic reticulum derived from SW480 colon cancer cells was about 50 to 200 nm in size.
  • TSG101 and CD9 which are markers of the extracellular endoplasmic reticulum, were identified through western blotting and are shown in Fig. 2 (e).
  • the absorption band at 280 nm which was detected at 3.6 min, increased in proportion to the concentration of the cation, depending on the treatment concentration of the calcium cation, the copper cation and the zinc cation added to the culture medium for colon cancer cells, (d).
  • the extinction bands and extracellular extracellular extinction band showed the same detection time, and the yield of extracellular extracellular cells isolated from the cell culture was increased with the concentration of cation added.
  • FIG. 5 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of copper cation Is shown in Fig. 5 (b).
  • Example 5 Isolation of extracellular endoplasmic reticulum using cobalt cation (cobalt chloride)
  • Example 8 Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride)
  • Example 9 Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride)
  • Example 10 Isolation of extracellular endoplasmic reticulum using calcium cation (calcium chloride) in human urine
  • FIG. 11 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the calcium cation concentration Is shown in Fig. 11 (b).
  • Example 11 Isolation of extracellular endoplasmic reticulum using manganese cation (manganese sulfide (II)) in human urine
  • FIG. 12 (a) shows that the signal for CD9, a typical extracellular ER marker, increases with the concentration of calcium cation Is shown in Fig. 12 (b).
  • Example 12 Isolation of extracellular endoplasmic reticulum using zinc cation (zinc chloride) in human urine
  • Human urine was centrifuged at 2,000 x g for 15 min (total 2 replicates) to remove any remaining precipitate.
  • Various concentrations of zinc cations were added to the prepared human urine, and the mixture was centrifuged at 3,000 ⁇ g for 10 minutes.
  • the precipitates were harvested and dissolved in HEPES buffer containing 50 mM EDTA.
  • the extracellular endoplasmic reticulum isolated using the above method was confirmed by nanoparticle analysis and Western blot analysis.
  • FIG. 13 (a) shows that the signal for the normal extracellular ER marker CD9 increases with the zinc cation concentration Is shown in Fig. 13 (b).
  • Example 13 Increased efficiency of separation of extracellular endoplasmic reticulum through combination of various cations (copper (II) chloride, manganese (II) sulphide (II)) and polymer
  • extracellular endoplasmic reticulum was isolated by adding cation alone, polymer alone, or cation and polymer together.
  • polyethylene glycol (PEG) or polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was used as a polymer for extracellular ER separation, Only polyethylene glycol (PEG) was added so that the concentration was 8.3% or only polyethyl oxazoline (PEOZ, Poly (2-ethyl-2-oxazoline)) was added so that the final concentration was 10%.
  • PEG polyethylene glycol
  • PEOZ Poly (2-ethyl-2-oxazoline)
  • Nanoparticle analysis was performed to compare the yield of the extracellular endoplasmic reticulum harvested under the conditions of polymer alone, cation alone, or cation-polymer blend.
  • the yield of the extracellular endoplasmic reticulum under the conditions of culturing the polymer alone for 16 hours was 10 to 20 minutes
  • the yield of extracellular ER was further increased when the reaction was performed for 10 minutes under the condition of mixed cation and polymer. From these results, it was found that the efficiency of polymer alone was very low. However, it was found that the addition of polymer in the presence of cations further increased the extracellular precipitation efficiency of cations.
  • Example 14 CD9 analysis of extracellular endoplasmic reticulum with polymer and copper cation (copper (II) sulfide) concentration
  • Example 15 Isolation of extracellular endoplasmic reticulum by combination of ammonium sulphide and copper cation (copper (II) sulfide)
  • the yield of the extracellular endoplasmic reticulum under the condition of ammonium sulphate alone for 30 minutes was extremely low, while the yield of the high extracellular endoplasmic reticulum was maintained under the conditions of only 10 mM of copper cation for 30 minutes
  • the yield of the extracellular vesicles in the sample was increased in proportion to the concentration of the copper cation in the condition of 30 minutes of reaction with the copper cation and ammonium sulphide.
  • the precipitation efficiency of the extracellular vesicle is very low in the case of ammonium sulphate alone but in the case of the copper cation alone. It was also found that when the copper cation and ammonium sulfide were added together to precipitate the extracellular endoplasmic reticulum, the extracellular ER precipitation efficiency of the copper cation was maximized.
  • Example 16 Comparison of the extracellular endoplasmic reticulum and the extracellular endoplasmic reticulum using the polymer
  • polyethylene glycol PEG, polyethylene glycol
  • HEPES buffer 20 mM HEPES, pH 7.2, 150 mM NaCl
  • copper cations were added to the same volume of cell culture medium, followed by incubation for 10 minutes.
  • the precipitates were harvested by centrifugation at 3,000 ⁇ g for 10 minutes and dissolved in HEPES buffer containing 50 mM EDTA. Samples containing extracellular endoplasmic reticulum isolated using the above method were further separated by size-exclusion chromatography and analyzed by size exclusion chromatography using an HPLC system.
  • the yield and purity of the extracellular endoplasmic reticulum were significantly improved by using the cation as compared with the conventional method of separating the extracellular endoplasmic reticulum using the polymer.

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Abstract

La présente invention concerne un procédé d'isolement de vésicules extracellulaires à l'aide de cations, et plus particulièrement, un procédé d'isolement de vésicules extracellulaires à partir de divers échantillons en utilisant l'affinité entre les vésicules extracellulaires et les cations. Un procédé d'isolement de vésicules extracellulaires selon la présente invention ne nécessite pas d'équipement coûteux, peut être appliqué quelle que soit la quantité d'échantillon, et présente l'avantage d'être capable d'isoler efficacement les vésicules extracellulaires tout en préservant leur forme ou leurs caractéristiques. De plus, le procédé selon la présente invention peut être combiné à des procédés d'isolement existants pour augmenter au maximum l'efficacité d'isolement des vésicules extracellulaires, et peut être appliqué au diagnostic de maladies, au traitement de maladies, et à la recherche multiomique utilisant des vésicules extracellulaires isolées, ainsi qu'à la recherche sur les propriétés des vésicules extracellulaires.
PCT/KR2018/008485 2017-07-26 2018-07-26 Procédé d'isolement de vésicules extracellulaires à l'aide de cations Ceased WO2019022542A2 (fr)

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