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WO2022092352A1 - Procédé pour isoler un exosome spécifique à une maladie - Google Patents

Procédé pour isoler un exosome spécifique à une maladie Download PDF

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Publication number
WO2022092352A1
WO2022092352A1 PCT/KR2020/014890 KR2020014890W WO2022092352A1 WO 2022092352 A1 WO2022092352 A1 WO 2022092352A1 KR 2020014890 W KR2020014890 W KR 2020014890W WO 2022092352 A1 WO2022092352 A1 WO 2022092352A1
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exosomes
magnetic nanoparticles
disease
exosome
bound
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Korean (ko)
Inventor
함승주
문병걸
정혜인
김륜형
강병훈
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University Industry Foundation UIF of Yonsei University
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University Industry Foundation UIF of Yonsei University
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • 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
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/42—Apparatus for the treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • 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
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/06—Tissue, human, animal or plant cell, or virus culture apparatus with filtration, ultrafiltration, inverse osmosis or dialysis means
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present invention relates to a method for isolating disease-specific exosomes from body fluids, and more particularly, to a method for multiple isolation of exosomes overexpressing disease-specific markers from all exosomes at once.
  • Liquid biopsy is an in vitro diagnostic method for diagnosing diseases using circulating tumor cells, cell free circulating tumor DNA, and exosomes present in blood. It is emerging as a next-generation strategy for cancer diagnosis and treatment because it can be used for precise-targeted anti-cancer treatment and can be used for detailed observation of cancer occurrence and metastasis with only the test.
  • Exosomes are spherical vesicles (30-100 nm) that are discharged from cells and contain various proteins, lipids, and genomes that are indicators of cancer progression, metastasis, and drug response. , pleural fluid, saliva, cerebrospinal fluid, breast milk, semen, amniotic fluid, ascites, etc.). In addition, it is attracting attention as the most promising biomarker because it exists at a higher concentration in body fluids than in circulating tumor cells.
  • exosomes derived from disease-related cells such as cancer cells and analyze the genes in the exosomes and disease-specific markers on the surface of the exosomes together.
  • disease-related cells such as cancer cells
  • the number of exosomes overexpressing disease-specific markers is very few, about 1000. Therefore, it is necessary to separate the two types of exosomes so that the signals of exosomes overexpressing disease-specific markers are not obscured by signals of exosomes discharged from normal cells in disease diagnosis and prognosis discrimination.
  • a representative method for isolating exosomes is a method using centrifugation. It takes a long time to separate exosomes by co-precipitating them with PEG or dextran polymers, and each time the exosomes are separated, the sample is lost. .
  • exosomes overexpressing disease-specific markers and non-expressing exosomes cannot be distinguished, in order to separate normal cell-derived exosomes from exosomes overexpressing disease-specific markers, after separating the entire exosomes from body fluids, specific markers should be used to further isolate exosomes overexpressing disease-specific markers. This method has a disadvantage in that the sample loss rate is high and a considerable amount of time (6 hours or more) is required.
  • exosomes to which the magnetic nanoparticles are bound are injected into the microfluidic channel and separated into different recovery paths in the microfluidic channel,
  • the magnetic nanoparticles have a different magnetization from the first magnetic nanoparticles and the first magnetic nanoparticles to which a probe for detecting an exosome common expression marker is bound, and a probe for detecting a disease-specific exosome overexpression marker is bound It provides a method for separating disease-specific exosomes containing two magnetic nanoparticles.
  • the present inventors studied a method for separating disease-specific exosomes present in a small number in body fluids from normal cell-derived exosomes at once. Based on the presence of a large number of , magnetic nanoparticles labeled with a probe for detecting a disease-specific exosome overexpression marker or a probe for detecting a common exosome expression marker were prepared. Relatively few disease-specific markers exist in normal cell-derived exosomes. Therefore, when a sample containing exosomes is brought into contact with the two types of magnetic nanoparticles, both types of magnetic nanoparticles bind to the disease-specific exosomes, or a probe for detecting disease-specific exosome overexpression markers is labeled. A lot of magnetic nanoparticles bind. On the other hand, a lot of magnetic nanoparticles labeled with exosome common expression markers bind to normal cell-derived exosomes.
  • first magnetic nanoparticles and “second magnetic nanoparticles” are for distinguishing nanoparticles having different sizes and labeled probes, and do not limit the scope of magnetic nanoparticles, and may be used interchangeably.
  • the size of the first magnetic nanoparticles may be 100 to 800 nm, and the size of the second magnetic nanoparticles may be 200 to 1000 nm.
  • the size of the first magnetic nanoparticles is 100 to 600 nm
  • the size of the second magnetic nanoparticles may be 200 to 800 nm
  • more preferably the size of the first magnetic nanoparticles is 100 to 500 nm
  • the size of the second magnetic nanoparticles may be 200 to 600 nm.
  • the size of the magnetic nanoparticles is less than 100 nm in diameter, the exosome size is less than 100 nm and the separation ability is lowered, and the magnetic nanoparticles of 1000 nm or more have remarkably reduced dispersibility, making separation impossible.
  • the magnetic nanoparticles may be in the form of magnetic nanocluster.
  • Magnetic nanocluster is a particle synthesized by aggregation of several single magnetic nanoparticles, and can be synthesized in various sizes by controlling the amount of single magnetic nanoparticles. Since the magnetization of the cluster can be increased by the sum of the magnetizations of a single magnetic nanoparticle, the magnetization increases as the size of the magnetic nanocluster increases.
  • the first magnetic nanoparticles and the second magnetic nanoparticles have the same composition so that factors other than the size of the nanoparticles do not affect the magnetic sensitivity or the strength of magnetization.
  • the exosome common expression marker refers to a marker found in all exosomes regardless of the parent cell from which the exosomes are derived, and may be selected from the group consisting of CD9, CD63, CD81 and CD82.
  • CD81 was used as an exosome common expression marker.
  • disease-specific exosome refers to an exosome derived from a disease-associated cell, and a representative disease-associated cell includes cancer cells.
  • the disease-specific exosome expression marker refers to a marker specifically or commonly found in exosomes discharged from disease-associated cells, and may have a specific marker depending on the derived disease-associated cell.
  • the disease-specific exosome expression marker is HER2 (human epidermal growth factor receptor 2), PSA (prostate specific antigen), CEA (carcinoembryonic antigen), AFP (alpha fetoprotein), CA125 (cancer antigen 125), CA 15-3 ( It can be selected from the group consisting of cancer antigen 15-3) and CA 19-9 (cancer antigen 19-9), and HER2 was used in the present invention.
  • the term "probe” refers to a substance capable of specifically binding to a marker expressed in exosomes, monoclonal antibody, polyclonal antibody, chimeric antibody, Fab, F(ab') 2 , Fab' , scFv (single chain fragment variable), single-domain antibody (single-domain antibody), aptamer, it may be selected from the group consisting of a peptide and a peptide nucleic acid (PNA).
  • PNA peptide nucleic acid
  • a HER2 antibody was used as a probe for detecting a disease-specific exosome expression marker
  • a CD9 antibody was used as a probe for detecting a common exosome expression marker.
  • a probe for detecting a common exosome expression marker was coupled to magnetic nanoparticles with a small magnetization (size), and magnetic nanoparticles with a large magnetization (size)
  • a disease-specific exosome expression marker detection probe was bound to the particle.
  • the binding between the magnetic nanoparticles and the probe may be achieved by amide bond, but any binding method may be used as long as it can induce stable binding between the magnetic nanoparticles and the probe.
  • the disease-specific exosome separation method starts with the step of obtaining exosomes to which magnetic nanoparticles are bound by contacting a sample containing exosomes with two or more types of magnetic nanoparticles having different degrees of magnetization.
  • a method of binding exosomes to magnetic nanoparticles may use an antigen-antibody reaction.
  • the exosome and buffer to which the magnetic nanoparticles are bound are injected into the microfluidic channel to which a magnetic field is applied, and the injection rate of the exosome to which the magnetic nanoparticles are bound may vary depending on the size of the microfluidic channel, but 2 ml/hr It is preferable to use from 10 ml/hr. When it is in the above range, the exosomes treated with magnetic nanoparticles can be effectively affected by the magnetic field.
  • the buffer used for a constant laminar flow is preferably injected at a rate of 8 ml/hr to 40 ml/hr according to the injection rate of the sample injection unit and a ratio of 1:4.
  • the injection volume per unit length of the sample injection unit is 1, the injection volume of the buffer injection unit is 4 times larger.
  • the buffer It is desirable to quadruple the injection rate of the injection unit.
  • the injection rate of the buffer injection unit compared to the injection rate of the sample injection unit may be changed according to the design of the microfluidic channel.
  • the flow of the sample and the buffer in the microfluidic channel structure can be controlled using a method well known in the art. These include electroosmotic flow that electrically moves a small amount of liquid sample, a method using a membrane pump, a syringe pump, and the like.
  • the exosomes bound with magnetic nanoparticles are injected into the microfluidic channel through the sample injection unit and pass through the main channel to which an external magnetic field of a certain size is applied. .
  • the first magnetic nanoparticles to which the probe for detecting the common exosome expression marker is bound to the normal cell-derived exosomes, but the first magnetic nanoparticles as well as the first magnetic nanoparticles to the disease-specific exosomes for detecting the disease-specific exosome expression marker Since the second magnetic nanoparticles to which the probe is bound are also bound, there is a large difference in the intensity of magnetic sensitivity or magnetization between the two exosomes.
  • the strength of the magnetic field is preferably set to 500 G to 3000 G (0.05 T to 0.3 T). If the intensity is too weak, the exosomes do not separate from each other, so only disease-specific exosomes cannot be separated.
  • the magnetic device may include applying an external magnetic field from a permanent magnet or an electromagnet.
  • the permanent magnets are produced from nickel, cobalt, iron, alloys thereof and alloys of non-ferromagnetic materials that become ferromagnetic by alloying, ie, alloys known as Heusler alloys (eg, alloys of copper, tin and manganese).
  • Heusler alloys eg, alloys of copper, tin and manganese.
  • suitable alloys for permanent magnets are known and commercially available for making magnets usable in one embodiment of the present invention.
  • a typical such material is a transition metal-semimetal (metalloid) alloy, with a transition metal (usually Fe, Co, or Ni) about 80% and a melting point lowering metalloid component (boron, carbon, silicon, phosphorus or aluminum).
  • Permanent magnets may be crystalline or amorphous.
  • An example of an amorphous alloy is Fe80B20 (Metglas 2605).
  • the external magnetic field is provided by a rectangular (2.5 cm x 2.5 cm x 4.0 cm) neodymium (NdFeB) magnet (K&J Magnetics, Jamison, PA) attached to the upper and lower surfaces of the main channel of the microfluidic channel. do.
  • the method may further include the step of inhaling the exosomes combined with magnetic nanoparticles separated by different recovery paths after step (b) (c), wherein the suction is performed by using a pump connected to the outlet can be done through
  • the inhalation may be made at a rate ranging from 10 ml/hr to 50 ml/hr.
  • disease-specific exosomes can be separated from normal cell-derived exosomes at once, and this method is different from the existing exosome separation method of isolating the disease-related exosomes again after separating the entire exosomes.
  • the process is simple and only disease-related exosomes can be concentrated and isolated, and the isolated disease-specific exosomes can be utilized for disease diagnosis and prognosis prediction.
  • FIG. 1 is a schematic diagram of the principle of multiple separation of exosomes using a microfluidic channel, according to an example of the present invention.
  • Figure 2 shows a schematic diagram of a microfluidic channel for multiple separation of exosomes, according to an example of the present invention.
  • FIG. 3 shows a microfluidic channel structure for multiple separation of exosomes manufactured according to an example of the present invention.
  • Figure 4 is the result of confirming the magnetic nanoparticles of triiron tetraoxide (Fe 3 O 4 ) of various sizes synthesized according to a preparation example of the present invention by a transmission electron microscope: (A) is 100 nm, (B) is 200 nm, (C) is 300 nm and (D) is a 400 nm size of magnetic nanoparticles.
  • VSM vibrating sample magnetometer
  • (A) is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles
  • B is CD9- bound to exosomes derived from HCC_1143 cells Transmission electron microscope image of magnetic nanoparticles
  • C is a scanning electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes
  • D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes
  • E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes.
  • HCC_1954 is an image obtained by confocal microscopy after binding each of magnetic nanoparticles of different sizes (labeled with HER2 antibody or CD9 antibody) with exosomes derived from a HER2-overexpressing cell line (HCC_1954).
  • Figure 10 is the result of confirming the trajectory of the exosomes by optical microscopy after binding each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes with exosomes derived from the HER2-overexpressing cell line (HCC_1954) and injecting them into microfluidic channels: (A-C) Exosome behavior by location in microfluidic channels in the absence of an external magnetic field; (D-F) Exosome behavior by location in microfluidic channels under an external magnetic field; and (G-J) exosome behavior by outlet under an external magnetic field.
  • A-C Exosome behavior by location in microfluidic channels in the absence of an external magnetic field
  • D-F Exosome behavior by location in microfluidic channels under an external magnetic field
  • G-J exosome behavior by outlet under an external magnetic field.
  • each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and these are injected into microfluidic channels at different total flow rates, and then outlet 2 compared to the total flow rate. And it is a result of confirming the ratio of the exosomes recovered in 4.
  • FIG. 12 is a diagram showing each of magnetic nanoparticles (labeled with HER2 antibody or CD9 antibody) of different sizes is combined with exosomes derived from a HER2-overexpressing cell line (HCC_1954), and after injecting them into microfluidic channels, exosomes are recovered from each outlet to indicate disease markers. This is the result of confirming the expression rate of the gene.
  • a pattern using SU-8 photosensitive resin on a silicon wafer was fabricated as shown in FIG. After that, liquid polydimethylsiloxane (PDMS) was solidified in a casting mold to make a chip, and the chip was attached to a slide glass to make a microfluidic channel device.
  • PDMS liquid polydimethylsiloxane
  • the injection hole is divided into a sample inlet (one channel, 1 in FIG. 2), one channel with a buffer inlet (divided into 8 channels after injection, 2 in FIG. 2), and a magnetic field is applied from the outside. It includes an outlet composed of a main channel (4 in FIG. 2) and a total of 4 channels (grouped by 2, 3 in FIG. 2). 3 shows a photograph of the actually fabricated microfluidic channel.
  • the sample inlet was arranged on the side to confirm the behavior of the magnetic nanoparticle-coated exosome sample, and the buffer inlet was increased to eight channels in order to reduce the parabolic fluid flow due to the laminar flow effect. composed.
  • a phosphate buffered saline (PBS) solution mixed with 10% of bovine serum albumin (BSA) was quickly flowed after production. was produced.
  • magnetic nanoclusters having diameters of 200 nm and 400 nm, respectively, were used.
  • ferric chloride FeCl 3
  • sodium acetate sodium citric acid
  • the composition of the magnetic nanoparticles was triiron tetraoxide (Fe 3 O 4 ), and had a surface and various sizes as shown in FIG. 4 .
  • VSM vibrating sample magnetometer
  • an antibody binding to the disease-specific marker and the magnetic nanoparticles synthesized in 2-1 were combined.
  • HER2 antibody EPR19547-12 (ab214275), purchased from Abcam
  • CD9 antibody EPR23105-121 (ab236630), Abcam
  • CD9 antibody binds to CD81 on the surface of exosomes.
  • the antibodies were combined with magnetic nanoparticles of different sizes.
  • amide bonds were induced with 1-ethyl 3-dimethylaminopropyl carbodiimide and hydroxysuccinimide to prepare antibody-bound magnetic nanoparticles: 400 nm magnetic nanoparticles 10 mg + anti-HER2 antibody 10 ⁇ g; and 10 mg of 200 nm magnetic particles + 10 ⁇ g of anti-CD9 antibody.
  • HER2-magnetic nanoparticles 400 nm
  • CD9-magnetic nanoparticles 200 nm
  • exosomes were collected and obtained in a culture medium according to a method known in the art. After culturing HCC_1143 cells, which are HER2 non-expressing cells, exosomes were isolated in the same manner. Each of the isolated exosomes was mixed with 2 mg of HER2-magnetic nanoparticles (400 nm) or 2 mg of CD9-magnetic nanoparticles (200 nm) to prepare a total of four exosome-magnetic nanoparticle complexes.
  • A is a transmission electron microscope image of exosomes not treated with magnetic nanoparticles
  • B is a transmission electron microscope image of CD9-magnetic nanoparticles bound to HCC_1143 cell-derived exosomes
  • C is CD9 bound to HCC_1143 cell-derived exosomes.
  • D is a transmission electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes
  • E is a scanning electron microscope image of HER2-magnetic nanoparticles bound to HCC_1954 cell-derived exosomes.
  • HCC_1954 cells which are HER2-overexpressing cells.
  • FIG. 10 shows an image that appears when a magnet is placed in the lower part of the main channel in the microfluidic channel shown in FIG. 2 and exosomes having magnetic nanoparticles attached thereto are flowed through the microfluidic channel.
  • FIG. 11 shows the fraction of exosomes obtained from outlets 2 and 4 compared to the amount of exosomes injected after performing an exosome separation experiment by changing the total flow rate to 5, 15, 25, 35, and 50 ml/hr shown in a circular table. It was confirmed that the sample could be obtained with high efficiency when the separation experiment was performed at a total flow rate of 20 ml/hr (sample inlet 4 ml/hr).

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Abstract

La présente invention concerne un procédé permettant d'isoler simultanément plusieurs exosomes surexprimant un marqueur spécifique à une maladie à partir de la population totale d'exosomes. Par comparaison avec les procédés existants d'isolement des exosomes, consistant à isoler les exosomes de la population totale d'exosomes, puis à isoler les exosomes liés à la maladie à partir des exosomes isolés de la population totale, le procédé est simple et présente l'avantage de pouvoir concentrer et isoler uniquement les exosomes liés à la maladie, et l'exosome isolé lié à la maladie peut être utilisé pour diagnostiquer et prédire le pronostic des maladies.
PCT/KR2020/014890 2020-10-29 2020-10-29 Procédé pour isoler un exosome spécifique à une maladie Ceased WO2022092352A1 (fr)

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Cited By (1)

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CN117778309A (zh) * 2023-12-26 2024-03-29 广州拾纳客生物科技有限公司 动物干细胞外泌体的分离纯化方法及其应用

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KR20120023684A (ko) * 2009-04-23 2012-03-13 소시에다지 베네피시엔찌 이스라엘리따 브라질레이라 오스삐따우 알버트 아인슈타인 산화철 나노 입자를 이용하여 생물학적 용액으로부터 엑소좀을 분리하는 방법
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KR101892214B1 (ko) * 2017-01-06 2018-08-27 고려대학교 산학협력단 엑소좀을 포함하는 생체분자 연속 분리용 장치 및 이를 이용한 분리방법
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KR102078787B1 (ko) * 2018-02-28 2020-02-19 울산과학기술원 자성 면역 입자 및 그 용도

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117778309A (zh) * 2023-12-26 2024-03-29 广州拾纳客生物科技有限公司 动物干细胞外泌体的分离纯化方法及其应用
CN117778309B (zh) * 2023-12-26 2024-08-09 广州拾纳客生物科技有限公司 动物干细胞外泌体的分离纯化方法及其应用

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