WO2019103548A2 - Procédé d'isolement de vésicules extracellulaires par chromatographie d'exclusion de taille semi-sèche - Google Patents

Procédé d'isolement de vésicules extracellulaires par chromatographie d'exclusion de taille semi-sèche Download PDF

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WO2019103548A2
WO2019103548A2 PCT/KR2018/014600 KR2018014600W WO2019103548A2 WO 2019103548 A2 WO2019103548 A2 WO 2019103548A2 KR 2018014600 W KR2018014600 W KR 2018014600W WO 2019103548 A2 WO2019103548 A2 WO 2019103548A2
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extracellular
endoplasmic reticulum
extracellular endoplasmic
exclusion chromatography
sample
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PCT/KR2018/014600
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English (en)
Korean (ko)
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WO2019103548A3 (fr
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이창진
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㈜로제타엑소좀
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Priority claimed from KR1020180147145A external-priority patent/KR102161280B1/ko
Publication of WO2019103548A2 publication Critical patent/WO2019103548A2/fr
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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
    • B01D15/34—Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation

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  • the present invention relates to a method for separating extracellular endoplasmic reticulum using size exclusion chromatography. More specifically, the present invention relates to a method for separating extracellular endoplasmic reticulum from a fixed bed of semi-annular size exclusion chromatography by preventing adsorption or non- The present invention relates to a method for rapidly and efficiently separating extracellular endoplasmic reticulum from various samples while compensating for the disadvantages in the method of separating extracellular endoplasmic reticulum using semi-dry size exclusion chromatography.
  • 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.
  • the extracellular endoplasmic reticulum is small in size at the nanometer level, and there are many other substances besides the extracellular endoplasmic reticulum in cell culture fluids in body fluids. Therefore, for the analysis of extracellular endoplasmic reticulum, it is important to separate extracellular endoplasmic reticulum from samples such as body fluids and cell culture fluids. Separation of extracellular endoplasmic reticulum is the most important technology in all fields utilizing it.
  • Extracellular ERs have become increasingly popular in size exclusion chromatography purification methods using physical characteristics that their molecular weight is relatively large compared to various materials present in biological samples containing them.
  • the size exclusion chromatography method has an advantage that the purity of the extracellular endoplasmic reticulum can be increased.
  • the principle of size exclusion chromatography has a disadvantage that the final purified material is greatly diluted, and the development time for increasing the purity of the extracellular endoplasmic reticulum There is a problem that there are many difficulties in subsequent analysis or application.
  • Semi-dry size exclusion chromatography is increasingly being used as a technique to overcome the drawbacks of such size exclusion chromatography.
  • Semi-formal size exclusion chromatography is characterized by semi-dry balancing of the stationary phase in the column by either scanning or rotary methods prior to sample loading to minimize the volume of the mobile phase present in the column.
  • the dilution rate of the extracellular endoplasmic reticulum is greatly reduced by eluting the extracellular endoplasmic reticulum after the sample is loaded on the equilibrated column, and the sample can be rapidly separated.
  • It is still another object of the present invention to provide a method for preparing a column comprising the steps of (a) equilibrating a stationary phase filled in a column with a solution containing a blocking substance, (b) loading a sample containing the extracellular endoplasmic reticulum into the column, and and c) eluting the extracellular endoplasmic reticulum from the column.
  • the present invention provides a separation method capable of improving disadvantages of semi-annular size exclusion chromatography, which is a conventional extracellular ER separation method, and improving separation efficiency.
  • the method for separating extracellular fibrils according to the present invention is characterized in that in a method using ordinary semi-dry size exclusion chromatography, a blocking substance that binds non-specifically to a fixed phase is added to extracellular fibrils in the sample to be adsorbed or non-specifically bound to the fixed phase
  • a blocking substance that binds non-specifically to a fixed phase is added to extracellular fibrils in the sample to be adsorbed or non-specifically bound to the fixed phase
  • the efficiency of separation of the extracellular endoplasmic reticulum via semi-dry size exclusion chromatography can be increased.
  • 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, but not limited thereto. It is not.
  • One embodiment of the present invention is a method for preparing a column comprising the steps of (a) balancing a stationary phase packed in a column semitransparently, (b) loading a mixture of a sample containing a blocking substance and an extracellular endoplasmic reticulum onto the stationary phase, and (c) Extracellular < / RTI > A method for separating an extracellular endoplasmic reticulum.
  • FIG. 1 A method for separating the extracellular endoplasmic reticulum according to the present invention is schematically shown in Fig.
  • the method for separating extracellular fibrils according to the present invention includes a step (a) of semi-drying equilibrium of a stationary phase filled in a column after filling a column on a size exclusion chromatography column.
  • column of the present invention is a unit filled with a porous stationary phase used in size exclusion chromatography
  • stationary phase means a porous particle of various sizes for fractionating a substance according to molecular weight.
  • the separation resolution depending on the molecular size of the molecules present in the sample varies depending on the size of the holes existing in the stationary phase. For example, if the pores present in the stationary phase are large, they are efficient for separating relatively large molecules and relatively small molecules are eluted without separation. On the other hand, if the size of the hole existing in the stationary phase is small, the degree of separation of large molecules is low, but it may be efficient to separate molecules having a certain size or smaller.
  • the size of the molecule to be separated and the size of the contaminant in the sample are taken into consideration, and a fixed phase having a hole having a size providing an optimum separation efficiency is selected.
  • the most commonly used stationary phases in size exclusion chromatography are Sepharose (GE Healthcare), Superose (GE Healthcare), Sephadex (Pharmacia), Bio-Gel P (Bio-Rad) and TSKgel® silica-based; Sigma).
  • a Sephacryl S500 stationary phase having pores having a size capable of separating the extracellular endoplasmic reticulum, which is a nanoparticle, from proteins of various sizes, But is not limited thereto.
  • the semi-dry size exclusion chromatography development method of the present invention employs a rotary type, but is not limited thereto.
  • equilibration refers to a process of performing a separation before a sample to be separated is loaded into a column.
  • the sample is loaded into a column in a stationary phase, loaded with a buffer solution, Is minimized.
  • the stationary phase equilibrium step of the present invention may employ a buffer solution used in conventional size exclusion chromatography.
  • the method for separating extracellular fibrils of the present invention includes a step (b) of loading a mixture of a sample containing a blocking substance and an extracellular endoplasmic reticulum into the equilibrium fixed bed.
  • sample includes a biological sample containing cell extracellular medium, a cell culture fluid, a tissue sample, and the like, and specifically includes mammalian cell culture medium, bacterial cell culture medium, yeast culture medium, , Serum, plasma, saliva, tears, sweat, urine, feces, CSF, ascites, amniotic fluid, semen, milk, dust, fresh water, seawater, soil and fermentation One or more of which may be selected from the group consisting of foods.
  • blocking substance refers to a substance that adsorbs or non-specifically binds to a fixed bed of size exclusion chromatography, and particularly acts to compete with extracellular endoplasmic reticulum to prevent the extracellular endoplasmic reticulum from binding to the fixed bed .
  • the blocking material may comprise a protein material that is smaller in size than the extracellular endoplasmic reticulum to be separated.
  • the blocking substance that can be used in the extracellular fibrin separation method of the present invention may be a single substance having the above properties, or a combination of two or more substances.
  • the blocking material refers to a material capable of forming adsorbed or complex nonspecific binding with a stationary phase with a single substance or a combination of various substances capable of adsorbing or non-specific binding with a conventional stationary phase used in size exclusion chromatography.
  • the blocking material of the present invention may be a smaller sized material than the extracellular endoplasmic reticulum.
  • the blocking material is a single protein or protein complex such as Fetal bovine serum (FBS) or Fetal bovine albumin (BSA).
  • FBS Fetal bovine serum
  • BSA Fetal bovine albumin
  • non-specific binding of the present invention means various types of intermolecular noncovalent bonding, specifically hydrogen bonds, ion interactions, hydrophobic interaction, van der Waals force der Waals force). Nonspecific binding between molecules may be due to one type of binding or two or more types of binding may be simultaneously acting.
  • the immobilized phase of size exclusion chromatography is capable of binding or non-specific binding with the extracellular endoplasmic reticulum or blocking substance, and the extracellular endoplasmic reticulum and the blocking substance can competitively bind to the fixed bed and noncovalently.
  • the blocking substance is adsorbed on the fixed bed or forms non-specific binding, thereby preventing the extracellular endoplasmic reticulum contained in the sample from binding to the fixed bed, thereby maximizing the efficiency of separation of the extracellular endoplasmic reticulum.
  • the yield of the extracellular fibrinolysis was improved when the mixture of the blocking substance and the sample was loaded.
  • the bovine serum (FBS) or the bovine serum albumin (BSA) (Fig. 3).
  • the yield of the extracellular endoplasmic reticulum isolated in proportion to the concentration of the blocking substance was increased (Fig. 4), and the blocking effect of the bovine serum albumin was not influenced by the concentration of salt (NaCl) ordinarily used ).
  • the extracellular fibrillar separation method of the present invention includes a step (c) of eluting the extracellular endoplasmic reticulum from the column.
  • extracellular endoplasmic reticulum When a sample containing extracellular endoplasmic reticulum is loaded on a semi-dry-type exclusion chromatography column and a mobile phase is developed by rotation, a large molecule such as extracellular endoplasmic reticulum is selectively eluted and the remaining small-sized substance remains in the column, Large extracellular endoplasmic reticulum can be isolated from the sample.
  • the extracellular endoplasmic reticulum elutes with the mobile phase preferentially in the rotary mobile phase development because the molecular size is over 1,000 kDa and belongs to the larger particle than other impurities.
  • the molecular weight of the eluted substance varies depending on the size and pore size of the porous stationary phase, the length of the column, the flow rate of the mobile phase, and the like.
  • the column (8 x 20 mm) filled with Sephacryl S500 was washed with a rotary (300 xg, 5 min) after adding HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) 2 mg of extracellular extracellular matrix and blocking substances of various concentrations were mixed and loaded onto the semi-permanent stationary bed, and the column was elongated at 700 xg for 5 minutes.
  • a method for preparing a sample comprising the steps of: (a) balancing a stationary phase packed in a column with a solution containing a blocking substance semi-dryly, (b) loading a sample containing an extracellular endoplasmic reticulum into the column And (c) eluting the extracellular endoplasmic reticulum from the column.
  • step (a) of filling a fixed bed in a size exclusion chromatography column and then equilibrating the stationary bed filled with the column using a solution containing a blocking substance, semi-dryly step (a)).
  • a blocking material may be used in combination in both the blocking step (step (a)) and the sample loading step (step (b)).
  • the fixed phase can be blocked in two steps, it was confirmed through the following examples that the extracellular ER separation efficiency can be maximized (FIG. 7).
  • the kind and concentration of the blocking substance and the mixing ratio to the equilibrium buffer solution or the sample may be optimized by the person skilled in the art depending on the properties of the sample to be separated or the extracellular endoplasmic reticulum and the purpose of separation have.
  • the method for separating extracellular ER according to the present invention does not require expensive equipment such as an ultracentrifuge, and since the sample is not exposed to the extreme environment during the separation process, the extracellular ER can be efficiently separated There is an advantage to be able to do.
  • the method of the present invention can be used to effectively isolate extracellular endoplasmic reticulum with high purity by combining with conventional extracellular fibrin separation methods by greatly improving the loss of extracellular endoplasmic reticulum in conventional semi-dry size exclusion chromatography The separation efficiency can be maximized by applying the method before or after the conventional method.
  • the extracellular fibrin separation method of the present invention can effectively separate the extracellular endoplasmic reticulum, and can be quickly applied to clinical diagnosis by applying a small amount of sample to the pretreatment and post-treatment steps.
  • FIG. 1 is a schematic diagram of a method for separating extracellular ER according to the present invention.
  • Fig. 2 shows the result of separation of the extra-cellular endoplasmic reticulum from the colorectal cancer cell line (SW480).
  • Figure 3 shows the results of separation of the extracellular endoplasmic reticulum by semidry size exclusion chromatography in samples in which various blocking materials are mixed.
  • FIG. 4 shows the results of separation of the extracellular endoplasmic reticulum by semi-dry size exclusion chromatography in samples mixed with various concentrations of bovine serum albumin.
  • FIG. 5 shows the results of separation of the extracellular endoplasmic reticulum by semi-dry size exclusion chromatography in a sample in which 10% bovine serum albumin and various concentrations of salt were mixed.
  • FIG. 6 shows the results of separation of the extracellular endoplasmic reticulum by semi-dry size exclusion chromatography in which the fixed phase was blocked with various concentrations of bovine serum albumin.
  • Figure 7 shows the results of separation of extracellular endoplasmic reticulum by semidry size exclusion chromatography with a fixed bed blocked in a sample containing a blocking material.
  • the colon cancer cell line SW480 culture was centrifuged at 500 xg for 10 minutes and at 2,000 xg for 20 minutes to remove the precipitate.
  • the extracellular endoplasmic reticulum was added with polyethylene glycol solution (8.4% Polyethylene Glycol 6000, 250 mM NaCl, 20 mM HEPES, pH 7.4) for the first purification and precipitation of the extracellular endoplasmic reticulum present in the supernatant for 16 hours After refrigerated, the extracellular endoplasmic reticulum was centrifuged at 12,000 xg for 30 minutes and the precipitate was dissolved in HEPES buffered saline (HEPES-buffered saline, 20 mM HEPES, 150 mM NaCl, pH 7.4).
  • HEPES buffered saline HEPES buffered saline
  • HPLC analysis and Western blotting were performed to analyze the purity of the endoplasmic reticulum endoplasmic reticulum endoplasmic reticulum (Fig. 2 (a) and 2 (b)) according to the method of purifying the extracellular endoplasmic reticulum.
  • the specimen extracellular endoplasmic reticulum purified according to the above method was loaded on a TSK gel 6000 column (7.5 x 600 mm) for HPLC, the HEPES buffer solution was flowed at 0.5 ml / min, and the absorbance at 280 nm was traced The purity of the purified sample was verified.
  • the extracellular endoplasmic reticulum isolated from the colorectal cancer cell line had a very high purity, and the mean size was a nanoparticle having an intact double membrane with a diameter of about 160 nm.
  • Example 2 Separation efficiency of extracellular ERs according to the type of blocking substance
  • blocking substances having complex properties including proteins such as bovine serum or bovine serum albumin effectively inhibited the binding between the fixed phase and the extracellular endoplasmic reticulum of semi-dry size exclusion chromatography, thereby remarkably improving the yield of extracellular endoplasmic reticulum .
  • a sample mixed with various concentrations of bovine serum albumin was loaded onto the fixed bed of the semi - dry size exclusion chromatography. Specifically, 2 ⁇ g of the specimen extracellular envelope from purified colon cancer was mixed with 2 ⁇ g of various concentrations of bovine serum albumin (0%, 5%, 10%) and then loaded on a size exclusion chromatography column , Centrifuged at 700 xg for 5 minutes, and subjected to rotary size exclusion chromatography (Fig. 4 (a)).
  • bovine serum albumin was influenced by the concentration of salt (NaCl) added for the purpose of inhibiting hydrogen bond and ion binding in size exclusion chromatography.
  • the extracellular endoplasmic reticulum was isolated by mixing various concentrations (0.15M, 0.5M, 1.0M) of salt into the extracellular ER samples and the 10% BSA mixture from the colon cancer.
  • Western blot and nanoparticle analysis of the extracellular endoplasmic reticulum in the eluate revealed that the blocking effect of the bovine serum albumin was not affected by the salt concentration (FIG. 5).
  • the column was first blocked before loading the sample containing the extracellular endoplasmic reticulum.
  • the immobilized phase was loaded on a prepared size exclusion chromatography column, and the immobilized phase of the size exclusion chromatography column was equilibrated semi-dryly by centrifugation.
  • the column was washed with HBS buffer (20 mM HEPES, 150 mM NaCl, pH 7 .4) 700 ⁇ l, 700 ⁇ l buffer containing 1% bovine serum albumin, or 700 ⁇ l buffer containing 2% bovine serum albumin and then centrifuged to block the column.
  • the buffer solution was loaded on the blocked column and centrifugation was repeated 6 times to wash excess albumin.
  • the final washed column was loaded with 2 ug of colon cancer-derived specimen extracellular endoplasmic reticulum and then the extracellular endoplasmic reticulum was eluted through centrifugation (Fig. 6 (a)).
  • Example 5 Confirmation of separation efficiency of extracellular endoplasmic reticulum upon equilibrium and sample loading of a stationary phase using a blocking substance
  • the size exclusion chromatography column is blocked, a mixture of the sample and the blocking substance is loaded to confirm the extracellular fibrin separation efficiency.
  • the stationary phase was loaded into a prepared size exclusion chromatography column, and the stationary phase of the size exclusion chromatography column was semi-dryly equilibrated by centrifugation.
  • the column was blocked by loading 700 ⁇ l of buffer solution or 700 ⁇ l of 2% bovine serum albumin buffer solution and centrifuging. The blocked column was further loaded with buffer solution and centrifuged 6 times to remove excess albumin.
  • the extracellular endoplasmic reticulum sample, the 10% bovine serum albumin sample, or the extracellular endoplasmic reticulum sample without the addition of the serum albumin were loaded on the final washed column, and then the extracellular endoplasmic reticulum was eluted through centrifugation (Fig. 7 a)), and the yield of the final purified extracellular endoplasmic reticulum was compared by Western blot (Fig. 7 (b)).
  • the extracellular vesicle separation efficiency was the most excellent.

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Abstract

La présente invention concerne un procédé d'isolement de vésicules extracellulaires à l'aide d'une chromatographie d'exclusion de taille et, plus particulièrement, un procédé d'isolement efficace et rapide de vésicules extracellulaires à partir de divers échantillons. L'invention vise à compenser l'inconvénient d'un procédé d'isolement de vésicules extracellulaires utilisant une chromatographie d'exclusion stérique en empêchant la perte de vésicules extracellulaires due à une liaison non spécifique à une phase solide de chromatographie d'exclusion de taille semi-sèche. Le procédé d'isolement de vésicules extracellulaires selon la présente invention ne nécessite pas d'installations coûteuses telles que des ultracentrifugeuses. Étant capable de non seulement isoler des vésicules extracellulaires tandis que les morphologies et les propriétés des vésicules extracellulaires restent intactes, mais également réduire considérablement la perte de vésicules extracellulaires se produisant dans une chromatographie d'exclusion de taille semi-sèche classique, le procédé d'isolement de vésicule extracellulaire selon la présente invention trouve des applications actives dans l'isolement de vésicules extracellulaires de pureté élevée en conjugaison avec un procédé d'isolement de vésicule extracellulaire classique. En outre, le procédé d'isolement de vésicule extracellulaire de la présente invention peut être appliqué à des étapes de pré-traitement et de post-traitement pour une petite quantité d'échantillons et peut ainsi être rapidement utilisé pour un diagnostic clinique.
PCT/KR2018/014600 2017-11-24 2018-11-26 Procédé d'isolement de vésicules extracellulaires par chromatographie d'exclusion de taille semi-sèche WO2019103548A2 (fr)

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KR1020180147145A KR102161280B1 (ko) 2017-11-24 2018-11-26 반건식 크기 배제 크로마토그래피를 이용한 세포밖 소포체의 분리 방법
KR10-2018-0147145 2018-11-26

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CN113092642A (zh) * 2021-03-30 2021-07-09 苏州爱宝德生物科技有限公司 一种用于细胞外囊泡的快速提取装置
US11085089B2 (en) 2019-03-01 2021-08-10 Mercy Bioanalytics, Inc. Systems, compositions, and methods for target entity detection
CN114814020A (zh) * 2022-04-20 2022-07-29 安康市农产品质量安全检验监测中心 一种农产品中残留有机磷农药的分析方法

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JP2001021563A (ja) * 1999-07-06 2001-01-26 Nitto Denko Corp 特異的結合体
KR101761680B1 (ko) * 2015-03-31 2017-08-23 포항공과대학교 산학협력단 수용액 이상계를 이용한 세포 밖 소포체의 분리방법

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11085089B2 (en) 2019-03-01 2021-08-10 Mercy Bioanalytics, Inc. Systems, compositions, and methods for target entity detection
CN113092642A (zh) * 2021-03-30 2021-07-09 苏州爱宝德生物科技有限公司 一种用于细胞外囊泡的快速提取装置
CN114814020A (zh) * 2022-04-20 2022-07-29 安康市农产品质量安全检验监测中心 一种农产品中残留有机磷农药的分析方法
CN114814020B (zh) * 2022-04-20 2023-08-29 安康市农产品质量安全检验监测中心 一种农产品中残留有机磷农药的分析方法

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