WO2019168184A1 - Population de vésicules extracellulaires et sa méthode de fabrication - Google Patents

Population de vésicules extracellulaires et sa méthode de fabrication Download PDF

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Publication number
WO2019168184A1
WO2019168184A1 PCT/JP2019/008235 JP2019008235W WO2019168184A1 WO 2019168184 A1 WO2019168184 A1 WO 2019168184A1 JP 2019008235 W JP2019008235 W JP 2019008235W WO 2019168184 A1 WO2019168184 A1 WO 2019168184A1
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extracellular vesicles
population
cells
extracellular
zeta potential
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PCT/JP2019/008235
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English (en)
Japanese (ja)
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一木　隆範
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公益財団法人川崎市産業振興財団
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Priority to JP2020503662A priority Critical patent/JP7296937B2/ja
Publication of WO2019168184A1 publication Critical patent/WO2019168184A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/20—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents
    • A23L29/275—Foods or foodstuffs containing additives; Preparation or treatment thereof containing gelling or thickening agents of animal origin, e.g. chitin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q90/00—Cosmetics or similar toiletry preparations for specific uses not provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions

Definitions

  • the present invention relates to a population of extracellular vesicles and a method for producing the same. Furthermore, the present invention relates to a method for evaluating the quality of a population of extracellular vesicles, a composition containing extracellular vesicles, and the like.
  • Exosome is a kind of extracellular vesicle secreted by cells. Exosomes are attracting attention as candidate disease markers that can be used for early detection of intractable diseases including cancer and determination of therapeutic effects. Exosomes are also expected to be used as carriers for drug delivery systems (DDS) and therapeutic applications such as regenerative medicine using mesenchymal stem cell-derived exosomes.
  • DDS drug delivery systems
  • therapeutic applications such as regenerative medicine using mesenchymal stem cell-derived exosomes.
  • it is difficult to analyze and identify a heterogeneous particle population having a diameter of several tens to 100 nm and a heterogeneity.
  • the present invention has been made in view of the above circumstances, and a method for producing such extracellular vesicles, such as a group of extracellular vesicles of uniform quality and a composition containing such extracellular vesicles, and cells It is an object to provide a method for evaluating the quality of outer vesicles.
  • the present invention includes the following aspects.
  • [1] A population of extracellular vesicles having a standard deviation of zeta potential of extracellular vesicles of 5 mV or less.
  • [2] The population of extracellular vesicles according to [1], wherein the standard deviation of the zeta potential is 4.5 mV or less.
  • [3] The population of extracellular vesicles according to [2], wherein the standard deviation of the zeta potential is 4 mV or less.
  • [4] The population of extracellular vesicles according to [3], wherein the standard deviation of the zeta potential is 3.5 mV or less.
  • [5] The population of extracellular vesicles according to [4], wherein the standard deviation of the zeta potential is 3 mV or less.
  • [6] The population of extracellular vesicles according to any one of [1] to [5], wherein the extracellular vesicle is an exosome.
  • [7] (a) A step of synchronizing the cell cycle of a plurality of cells; (b) After the step (a), the medium of the plurality of cells is replaced with a medium substantially free of extracellular vesicles.
  • Process (C) culturing the plurality of cells in the medium exchanged, (d) recovering a population of extracellular vesicles from the medium after step (c);
  • a method for producing a population of extracellular vesicles comprising: [8] The cell according to [7], wherein the population of extracellular vesicles obtained after the step (d) is the population of extracellular vesicles according to any one of [1] to [6].
  • a method for producing a population of outer vesicles [9] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a medium containing a cell cycle synchronizer.
  • [10] The method for producing a population of extracellular vesicles according to [7] or [8], wherein the step (a) is performed by culturing the plurality of cells in a confluent state.
  • the method further includes (e) a step of measuring the zeta potential of the extracellular vesicles contained in the collected extracellular vesicle population.
  • [7] to [11] A method for producing a population of extracellular vesicles according to any one of the above.
  • [13] A population of extracellular vesicles produced by the method for producing a population of extracellular vesicles according to any one of [7] to [12].
  • [14] A method for evaluating the quality of a population of extracellular vesicles, wherein (a) measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles; Calculating the standard deviation of the zeta potential measured in step (a), and (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b).
  • step (c) when the standard deviation is 5 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
  • step (c) when the standard deviation is 4.5 mV or less, it is determined that the uniformity of the population of the extracellular vesicles is high. A method of assessing the quality of a population.
  • step (c) when the standard deviation is 4 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality.
  • step (c) when the standard deviation is 3.5 mV or less, it is determined that the uniformity of the population of extracellular vesicles is high. A method of assessing the quality of a population. [19] In the step (c), when the standard deviation is 3 mV or less, it is determined that the uniformity of the extracellular vesicle population is high. How to evaluate quality. [20] A composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less. [21] The composition according to [20], wherein the standard deviation of the zeta potential is 4.5 mV or less.
  • a pharmaceutical composition comprising the population of extracellular vesicles according to any one of [1] to [6].
  • a cosmetic comprising the population of extracellular vesicles according to any one of [1] to [6].
  • a food comprising the population of extracellular vesicles according to any one of [1] to [6].
  • the food according to [30] which is a health food or a functional food
  • a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle.
  • FIG. 3 is a partial cross-sectional view of a fluid device partially cut along a yz plane.
  • FIG. 7 is a cross-sectional view taken along line AA in FIG. It is a figure which shows schematic structure of the irradiation part and adjustment part of a fluid device. It is a partial detail drawing of the adjustment part of a fluid device, and a fluid device. It is a figure which shows typically the optical path through which illumination light passes the end surface of a reservoir member, and the side surface of a flow path. It is a figure which shows schematic structure of the control apparatus of a fluid device. It is a figure which shows an example of the particle
  • FIG. 2 is a graph showing the measurement results of the particle size and zeta potential of extracellular vesicles collected in Example 1.
  • the present invention provides a population of extracellular vesicles wherein the standard deviation of the zeta potential of the extracellular vesicle is 5 mV or less.
  • Extracellular vesicles are vesicles released by cells.
  • the size of the extracellular vesicle is about 30 nm to 1 ⁇ m in diameter.
  • An extracellular vesicle is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface.
  • Examples of extracellular vesicles include exosomes, apoptotic bodies, microvesicles and the like.
  • a typical example of an extracellular vesicle is exosome. Exosomes are lipid vesicles with a diameter of about 30 to 200 nm.
  • various cells such as tumor cells, dendritic cells, T cells, and B cells can be used for blood, urine, saliva, etc. Secreted into body fluids.
  • Abnormal cells such as cancer cells existing in the body express a protein specific to the cell membrane.
  • An exosome is a secreted product of a cell and expresses a cell-derived protein as a secretory source on its surface.
  • the surface of the exosome is a membrane surface of a lipid vesicle secreted from a cell, and refers to a portion where the secreted exosome is in contact with the environment in the living body.
  • Extracellular vesicles may be processed extracellular vesicles released by cells.
  • the method for processing the extracellular vesicle is not particularly limited as long as the processed extracellular vesicle maintains the vesicle structure.
  • Examples of extracellular vesicle processing methods include modification of the membrane surface of the extracellular vesicle (for example, modification with a peptide, sugar chain, etc.), encapsulation of a drug in the extracellular vesicle, and the like.
  • a group of extracellular vesicles refers to two or more extracellular vesicles, for example, 10 3 or more, 10 4 or more, 10 5 or more, 10 6 or more extracellular vesicles Is done.
  • the upper limit of the number of extracellular vesicles contained in the population of extracellular vesicles is not particularly limited. For example, 10 15 or less, 10 14 or less, 10 13 or less, 10 12 or less, 10 11 or less, Or 10 10 or less are illustrated.
  • the population of extracellular vesicles may be, for example, 10 3 to 10 15 , 10 4 to 10 12 or more, or 10 5 to 10 10 extracellular vesicles.
  • the population of extracellular vesicles of this embodiment is preferably a population of extracellular vesicles obtained by culturing the same type of cells. That is, it is preferable that all the extracellular vesicles constituting the population of extracellular vesicles of this embodiment are extracellular vesicles released from the same type of cells.
  • the cells are not particularly limited as long as they have the ability to release extracellular vesicles. Examples of such cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells, and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells, hematopoietic stem cells, and the like.
  • Somatic stem cells pluripotent stem cells such as ES cells and iPS cells; germ cells and the like, but not limited thereto.
  • the biological species from which the cells are derived is not particularly limited.
  • the cell may be a human cell.
  • cells other than mammals other than humans for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc. may be used.
  • the extracellular vesicle population includes, for example, various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; adipocytes; mesenchymal stem cells and hematopoietic stem cells.
  • Pluripotent stem cells such as somatic stem cells such as ES cells, iPS cells, etc .; a population of extracellular vesicles released from various cells such as germ cells.
  • the population of extracellular vesicles of this embodiment has a standard deviation of the zeta potential of the extracellular vesicles of 5 mV or less.
  • the standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • the standard deviation of the zeta potential can be obtained by measuring the zeta potential of individual extracellular vesicles constituting the population of extracellular vesicles and calculating the standard deviation from the measured values.
  • the number of extracellular vesicles for measuring the zeta potential is sufficient if it is sufficient to calculate the standard deviation, and is appropriately selected according to the size of the extracellular vesicle population (number of extracellular vesicles). That's fine.
  • the number of extracellular vesicles for measuring zeta potential can be 100 or more, 300 or more, 500 or more, 600 or more, 700 or more, or 800 or more.
  • the standard deviation of the zeta potential of a population of extracellular vesicles can be calculated by the following formula (s).
  • the population of extracellular vesicles of this embodiment can be produced by a method for producing a population of extracellular vesicles described later.
  • the population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and uniform quality. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods. Since the population of extracellular vesicles of this embodiment has uniform quality, it can be suitably used as a drug delivery system (DDS) carrier or a pharmaceutical product such as regenerative medicine.
  • DDS drug delivery system
  • the zeta potential of individual extracellular vesicles constituting the extracellular vesicle population can be measured by a known method, apparatus, or system. Examples of such a method, apparatus, or system include the method, apparatus, or system described in International Publication No. 2016/171198, International Publication No. 2016/063912, International Publication No. 2014/030590, and the like. Is done. Below, an example of the zeta potential measuring device of an extracellular vesicle is described.
  • the zeta potential is measured by binding a specific binding substance to the extracellular vesicle, but the zeta potential of the extracellular vesicle is measured without binding the specific binding substance. Is also possible.
  • the zeta potential of the extracellular vesicle population of this embodiment is preferably measured without binding a specific binding substance to the extracellular vesicle. That is, in the explanation illustrated below, it is preferable not to perform the binding reaction between the specific binding substance and the extracellular vesicle.
  • the zeta potential of the extracellular vesicle may be measured using a specific binding substance for the protein.
  • the zeta potential of the extracellular vesicle is measured.
  • a population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles having high uniformity with respect to the expression level of a specific membrane protein.
  • the zeta potential of the extracellular vesicle may be measured by binding a specific binding substance specific to each membrane protein to a plurality of types of membrane proteins.
  • a population of extracellular vesicles having a standard deviation of zeta potential measured by this method of 5 mV or less is a population of extracellular vesicles with high uniformity in the expression levels of the plurality of types of membrane proteins.
  • extracellular vesicle zeta potential refers to the zeta potential measured without binding a specific binding substance to the extracellular vesicle, specific to a specific membrane protein in the extracellular vesicle.
  • zeta potential specific binding substance-data potential of extracellular vesicle complex
  • Zeta potentials data potentials of multiple specific binding substances-extracellular vesicle complexes.
  • the plurality of types of membrane proteins are not particularly limited as long as they are two or more types, and examples thereof include 2 to 50 types, 2 to 30 types, 2 to 20 types, and 2 to 10 types.
  • the “specific binding substance” means a substance having an ability to specifically bind to a specific molecule (for example, a protein).
  • the phrase “specifically binds to a specific molecule” means that it has a high binding affinity for the specific molecule and a low binding affinity for other molecules.
  • the specific binding substance varies depending on the specific molecule to be bound, and can be variously selected according to the type of the specific molecule.
  • Specific molecules to be bound by specific binding substances include molecules present on the surface of extracellular vesicles, and examples include antigens, membrane proteins, nucleic acids, sugar chains, glycolipids, and the like.
  • Specific binding substances for proteins include, for example, antibodies (including chimeric antibodies, humanized antibodies, modified antibodies, multivalent antibodies, multispecific antibodies, modified antibodies such as antibody fragments), aptamers (nucleic acid aptamers, peptide aptamers, etc.) And ligand molecules.
  • the class of the antibody as a specific binding substance is not particularly limited, and may be any antibody class such as IgG, IgA, IgD, IgE, and IgM.
  • Examples of IgG include IgG1, IgG2, IgG3, and IgG4.
  • Examples of IgA include IgA1 and IgA2.
  • IgM include IgM1 and IgM2.
  • Examples of antibody fragments include scFv, Fab, F (ab ') 2, Fv and the like.
  • the ligand molecule include a ligand of the receptor protein when the specific molecule to be bound is a receptor protein.
  • examples of the ligand molecule include G protein.
  • the specific binding substance may be labeled with a labeling substance.
  • labeling substances include biotin, avidin, streptavidin, neutravidin, glutathione-S-transferase, glutathione, fluorescent dyes, polyethylene glycol, charged molecules such as melittic acid, and the like.
  • FIG. 1 is a schematic plan view of a particle detection apparatus 100 that can be used for zeta potential measurement.
  • FIG. 2 is a schematic front view of the particle detection apparatus 100.
  • the particle detection apparatus 100 detects information related to particles in the fluid device C by irradiating the fluid device C with the illumination light L1 and observing the scattered light L2 from the fluid device C with the fluid device C as a detection target.
  • the particle detection apparatus 100 includes a light source unit LS, an irradiation unit 20, an adjustment unit CL, a stage unit ST, a detection unit 30, a transmission unit 40, and a control device 5.
  • a particle detection system 1 is configured by the particle detection apparatus 100 and the fluid device C.
  • the direction orthogonal to the orthogonal surface (not shown) orthogonal to the installation surface STa of the stage part ST is the x direction (x axis; third direction), and the direction parallel to the installation surface STa and orthogonal to the x direction.
  • the y direction (y axis) and the vertical direction perpendicular to the x direction and the y direction are appropriately described as the z direction (z axis; second direction).
  • the fluid device C that is a detection target will be described.
  • the fluid device C in this embodiment is an electrophoresis analysis chip used when analyzing an extracellular vesicle as an example.
  • an extracellular vesicle analysis chip (electrophoresis analysis chip) will be described by taking an example of analyzing exosomes as extracellular vesicles.
  • analysis of exosomes using an extracellular vesicle analysis chip can be performed as follows. First, the exosome to be detected is purified. Next, the exosome is brought into contact with the specific binding substance. As the specific binding substance, a substance that can specifically bind to a molecule present on the surface of the exosome is selected. Next, the zeta potential of exosome is measured and analyzed using an extracellular vesicle analysis chip. This analysis can be applied not only to exosomes but also to analysis of extracellular vesicles in general. In addition, the exosome may be subjected to analysis without contact with a specific binding substance.
  • exosomes are purified from a sample containing exosomes.
  • the sample include a cell culture solution.
  • Examples of the method for purifying exosomes include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography, and a method using a ⁇ -TAS (Micro-Total Analysis Systems) device.
  • ⁇ -TAS Micro-Total Analysis Systems
  • the exosome is brought into contact with the specific binding substance.
  • a specific binding substance-exosome complex is formed.
  • an abnormality associated with a disease such as cancer, obesity, diabetes, or neurodegenerative disease can be detected.
  • the expression level of the molecule to be detected on the exosome surface can be analyzed. For example, it is possible to evaluate exosomes with altered functions, such as using a specific binding substance that specifically binds to the peptide or protein of the exosome that artificially expresses the peptide or protein on the membrane surface. it can.
  • the zeta potential of the exosome reacted with the antibody is measured.
  • the zeta potential is the surface charge of the fine particles in the solution.
  • exosomes are negatively charged while antibodies are positively charged.
  • the zeta potential of the antibody-exosome complex is shifted positively compared to the zeta potential of the exosome alone. Therefore, by measuring the zeta potential of the exosome reacted with the antibody, the expression of the antigen on the exosome membrane surface can be detected. This is true not only for antibodies but also for other positively charged specific binding substances.
  • the exosome zeta potential ⁇ is obtained by performing exosome electrophoresis in the microchannel of an extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the measured exosome electrophoresis. Based on the speed S, it can be calculated using the Smolkovsky equation shown in the following equation (1).
  • Equation (1) U is the electrophoretic mobility of the exosome to be measured, and ⁇ and ⁇ are the dielectric constant and viscosity coefficient of the sample solution, respectively.
  • the electrophoretic mobility U can be calculated by dividing the electrophoretic velocity S by the electric field strength in the microchannel.
  • the exosome electrophoresis speed S is obtained by electrophoresing an exosome in a microchannel of an extracellular vesicle analysis chip, and as an example, irradiating the exosome flowing in the microchannel with a Rayleigh Measurement can be performed by obtaining a particle image by scattered light.
  • the laser beam one having a wavelength of 405 nm and an intensity of 150 mW can be given.
  • the particle diameter d of the exosome is obtained by performing exosome electrophoresis in the microchannel of the extracellular vesicle analysis chip, optically measuring the exosome electrophoresis speed S, and measuring the exosome electrophoresis. Based on the speed S, it can be calculated using the Einstein-Stokes equation shown in the following equation (2).
  • d is the particle diameter of exosome
  • k is Boltzmann constant
  • T absolute temperature
  • ⁇ is the viscosity coefficient of the sample solution
  • D is the diffusion coefficient of the fine particles. That is, the particle diameter d of the exosome can be calculated based on the Brownian motion state of the exosome to be measured.
  • FIG. 3 is a perspective view showing the basic structure of the extracellular vesicle analysis chip.
  • 4 is a cross-sectional view taken along line II-II in FIG.
  • the extracellular vesicle analysis chip CH includes a first reservoir 110, a second reservoir 120, an electrophoresis channel 150 that connects the first reservoir 110 and the second reservoir 120, and a base material 160.
  • the migration channel 150 is, for example, a millimeter channel or a micro channel.
  • the migration channel 150 has a width of about 200 ⁇ m, a height of 400 ⁇ m, and a length of about 10 mm.
  • the electrophoresis channel 150 is a specific binding formed by interaction between an extracellular vesicle or a specific binding substance that specifically binds to a biomolecule existing on the surface of the extracellular vesicle and the extracellular vesicle.
  • the substance-extracellular vesicle complex (for example, antibody-exosome complex) is electrophoresed.
  • the electrophoresis channel 150 has one end connected to the first reservoir 110 and the other end connected to the second reservoir 120.
  • the first reservoir 110 and the second reservoir 120 are provided on the base material 160 and have an electrode 130 and an electrode 140, respectively.
  • the electrode 130 is provided at the bottom of the first reservoir 110
  • the electrode 140 is provided at the bottom of the second reservoir 120.
  • the electrode 130 and the electrode 140 are each provided in the vicinity of the end of the migration channel 150.
  • a sample eg, exosome to be analyzed
  • a buffer solution is introduced into the second reservoir 120. Note that the buffer solution may be introduced into the first reservoir 110.
  • the extracellular vesicle analysis chip CH is suitable for measuring the zeta potential of extracellular vesicles.
  • a method for measuring the zeta potential of exosomes using the extracellular vesicle analysis chip CH will be described, taking as an example the case of analyzing exosomes as extracellular vesicles.
  • a sample solution containing exosomes to be analyzed is introduced into the first reservoir 110.
  • the exosome to be analyzed may have been reacted with a specific binding substance.
  • the exosome is obtained from, for example, the culture supernatant, and the sample solution is an exosome suspension in which the exosome is suspended in a buffer solution such as a phosphate buffer (Phosphate Buffered Saline, PBS).
  • a sample solution containing exosomes is introduced into the migration channel 150.
  • the exosome can be introduced into the electrophoresis channel 150 by connecting a syringe to the second reservoir 120 and sucking the sample solution.
  • the buffer solution is put into the first reservoir 110 and the second reservoir 120.
  • liquid level (liquid level height) between the first reservoir 110 and the second reservoir 120 By adjusting the liquid level (liquid level height) between the first reservoir 110 and the second reservoir 120 by using a liquid level adjusting means, which will be described later, it is possible to prevent the generation of a hydrostatic pressure flow that occurs in the migration channel 150 and to measure the zeta potential. The accuracy can be improved.
  • a voltage is applied between the electrodes 130 and 140 by a control unit (for example, a control device 5 described later or a computer), and the exosome is electrophoresed.
  • the control unit applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds.
  • the electrophoresis channel 150 is irradiated with laser light, and the scattered light passing through the exosome, which is emitted from the electrophoresis channel 150, is collected using an objective lens or the like, and a light receiving sensor (eg, high A sensitivity camera is used to image exosomes or specific binding substance-exosome complexes.
  • the magnification of the objective lens is about 60 times as an example.
  • the wavelength and intensity of the laser are, for example, a wavelength of 405 nm and an intensity of 150 mW.
  • the extracellular vesicle analysis chip CH By using the extracellular vesicle analysis chip CH, not only the average value of the zeta potential of the exosome or specific binding substance-exosome complex, but also the zeta potential of the exosome or specific binding substance-exosome complex at the level of one particle. It can be measured. Therefore, the standard deviation of the zeta potential in a population of exosomes can be determined from the zeta potential of individual exosomes or specific binding substance-exosome complexes.
  • the exosome or the specific binding substance-exosome complex may be simply referred to as “exosome”.
  • FIG. 5 is a plan view in which the fluid device C is installed on the installation surface STa of the stage unit ST.
  • FIG. 6 is a partial cross-sectional view in which the fluid device C is partially cut in the yz plane.
  • 7 is a cross-sectional view taken along line AA in FIG.
  • the fluid device C is formed in a rectangular shape in plan view.
  • the fluid device C includes a reservoir member (first base material) 10 and a bottom plate (second base material) 11 that are sequentially stacked in the z direction.
  • the fluid device C has a laminated structure (laminated body) composed of at least the reservoir member 10 and the bottom plate 11.
  • the laminated structure of the fluid device C has a two-layer structure.
  • such a laminated structure of the fluid device C is formed by bonding the reservoir member 10 and the bottom plate 11 to each other.
  • the reservoir member 10 is formed of a material that can be elastically deformed in at least one direction by an external force or the like.
  • Examples of the material of the reservoir member 10 include elastomers such as silicone rubber and PDMS (polydimethylsiloxane).
  • the bottom plate 12 is made of a material through which scattered light L2 generated by irradiation with the illumination light L1 is transmitted.
  • the bottom plate 12 is formed of a glass material as an example.
  • the fluidic device C includes a plurality of (three in FIG. 5) lanes 2 arranged in the length direction (y direction).
  • Each lane 2 includes a first reservoir 12A, a second reservoir 12B, a flow path 13, and electrodes 18A and 18B.
  • the first reservoir 12A and the second reservoir 12B are arranged at an interval in the y direction.
  • the first reservoir 12 ⁇ / b> A and the second reservoir 12 ⁇ / b> B are arranged at an interval in the flow path direction of the flow path 13.
  • the plurality of lanes are arranged in the flow path direction (in series), so that it is easy to irradiate light from the side.
  • a plurality of lanes may be analyzed in order for each lane, or may be analyzed simultaneously by a plurality of detection systems.
  • the plurality of lanes 2 may be arranged in the height direction (z direction).
  • the solution may be injected from the length direction (x direction) or from the y direction.
  • there are a plurality of irradiation light sources and each of the light sources irradiates fine particles flowing through the lane 2 having a corresponding height. Further, the fine particles flowing in the lane 2 may be irradiated by changing the irradiation direction from at least one irradiation light source.
  • the illumination light applied to each lane 2 may be adjusted by adjusting the shape of the illumination light by moving the objective lens. Further, when there are a plurality of lanes 2, a configuration in which the measurement target lane 2 is selected (switched) from among the plurality of lanes 2 by moving a stage on which the fluid device C is placed may be employed.
  • the first reservoir 12A has a holding space 14A that has a circular cross section in a plane parallel to the xy plane and extends in the z direction, and a funnel shape that gradually increases in diameter from the + z side end of the holding space 14A toward the + z direction.
  • the introduction part 15A is provided.
  • the holding space 14A opens at the ⁇ z side end facing the bottom plate 11.
  • the holding space 14 ⁇ / b> A is connected to the flow path 13.
  • the second reservoir 12B has a holding space 14B having a circular cross section in a plane parallel to the xy plane and extending in the z direction, and a funnel shape gradually increasing in diameter from the + z side end of the holding space 14B toward the + z direction.
  • the introduction part 15B is provided.
  • the holding space 14B has an end on the ⁇ z side facing the bottom plate 11 and opening.
  • the holding space 14 ⁇ / b> B is connected to the flow path 13.
  • the flow path 13 is a flow path for electrophoresis (flow path for electrophoresis).
  • the flow path 13 extends in the y direction, which is the length direction of the fluid device C.
  • the channel 13 is provided on the surface facing the bottom plate 11 so as to connect the holding space 14A and the holding space 14B.
  • the flow path 13 is formed in a rectangular cross section surrounded by the groove 10 ⁇ / b> A formed in the reservoir member 10 and the surface (second surface) 11 a of the bottom plate 11.
  • the groove portion 10A is formed to be surrounded by side surfaces (first surfaces) 16a and 16b facing in the x direction and a bottom surface (second surface) 16c facing the surface 11a of the bottom plate 11 in the z direction.
  • the side surfaces 16a, 16b, the bottom surface 16c, and the surface 11a constituting the groove 10A are mirror-finished.
  • the first surface includes a side surface 16a that is a first side surface and a side surface 16b that is a second side surface.
  • the side surface 16a and the side surface 16b face each other and are separated from each other in the x direction which is the first direction.
  • the lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the + x side than the center with respect to the optical axis direction (incident direction) of the illumination light L1 that is the width direction of the fluid device C.
  • the lane 2 is arranged so as to be biased toward the side closer to the end surface 17 on the incident side of the illumination light L1 than the center in the width direction (the x direction in FIG. 5) of the fluid device C that is the optical axis direction of the incident illumination light L1.
  • the end face 17 is mirror-finished in a range where at least the lane 2 is provided in the y direction.
  • the channel 13 is formed in a size of about 200 ⁇ m in width, about 400 ⁇ m in height (depth of the groove 10A), and about 10 mm in length.
  • An electrode 18A is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14A.
  • An electrode 18B is provided on the surface 11a of the bottom plate 11 so as to face the holding space 14B.
  • Examples of the material for the electrode 18A and the electrode 18B include gold, platinum, and carbon.
  • the end surface (second end surface) 19 located on the incident side of the illumination light L1 in the bottom plate 11 is closer to the incident side of the illumination light L1 than the position of the end surface 17 of the reservoir member 10 in the x direction. Are spaced apart on the opposite side, -x side.
  • the light source unit LS has a wavelength that does not adversely affect the particles.
  • the light source unit LS has a wavelength of 405 nm and an intensity of 150 mW.
  • Laser light having a deflection direction in the z direction at 0.8 mm is emitted as illumination light L1.
  • the illumination light L1 may be polarized light (for example, linearly polarized light) or non-polarized light. However, in the present embodiment, vertical polarization is used and there is no directivity of Rayleigh scattering.
  • the illumination light L1 is applied to the fluid device C along an optical axis extending in a direction intersecting the orthogonal plane described above. In the present embodiment, the optical axis of the illumination light L1 is parallel to the x direction.
  • the illumination light L1 of the present embodiment is applied to the fluid device C along the optical axis extending in the x direction.
  • FIG. 8 is a diagram illustrating a schematic configuration of the irradiation unit 20 and the adjustment unit CL.
  • the irradiation unit 20 includes a ⁇ / 2 plate 21 and an expander lens 22 that are sequentially arranged along the optical axis of the illumination light L1.
  • the optical axis of the illumination light L1 extends in the y direction, but the illumination light L1 that finally irradiates the fluid device C (channel 13) is x. Since the optical axis is along the direction, the illumination light L1 shown in FIG. 8 is illustrated on the assumption that the optical axis is along the x direction.
  • the illumination light L1 emitted from the light source unit LS is transmitted through the ⁇ / 2 plate 21 so that the polarization direction is rotated in the y direction.
  • the ⁇ / 2 plate 21 is not necessary when the light source unit LS emits the illumination light L1 having the deflection direction in the y direction.
  • the expander lens 22 includes cylindrical lenses 22A and 22B facing each other. Since the cylindrical lenses 22A and 22B have no power in the y direction, the illumination light L1 has a constant width in the y direction. The width of the illumination light L1 in the z direction is enlarged or reduced according to the distance in the optical axis direction of the cylindrical lenses 22A and 22B. The expander lens 22 enlarges the width of the illumination light L1 in the z direction as an example by a factor of two.
  • the adjustment unit CL adjusts the incident illumination light L ⁇ b> 1 that has been expanded by the expander lens 22 in the width in the z direction.
  • the adjustment unit CL is disposed in the optical path between the light source unit LS and the objective lens 31.
  • the adjustment unit CL is disposed in the optical path between the ⁇ / 2 plate 21 or the expander lens 22 and the objective lens 31.
  • the adjustment unit CL may include a drive mechanism, and the light collection point may be adjusted by the movement of the adjustment unit CL.
  • the adjustment unit CL can be driven in the x direction, for example. In this case, even when a chip having a different position of the flow path 13 is used, it is possible to adjust so that the light collecting point is located in the flow path 13.
  • FIG. 9 is a partial detailed view of the adjustment unit CL and the fluid device C according to the embodiment.
  • the adjustment unit CL is configured by a cylindrical lens.
  • the adjustment portion CL has a minimum width in the z-direction of the illumination light L1 inside the flow path 13, and the passage region of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a.
  • the convergence angle is adjusted so as to be limited.
  • the adjustment portion CL has a minimum width in the z direction of the illumination light L1 inside the flow path 13, and the irradiation area of the illumination light L1 at the position of the side face 16a on the irradiation light incident side of the flow path 13 is within the side face 16a.
  • the illumination light L1 is adjusted to a convergence angle that condenses light.
  • the adjustment part CL is adjusted to the convergence angle which converges so that the passage area
  • the adjusting unit CL adjusts the illumination light L1 to a convergence angle such that the irradiation region of the illumination light L1 (irradiation light beam) at the position of the side surface 16b on the irradiation light emission side of the flow path 13 is condensed in the side surface 16b. . Further, the adjustment unit CL adjusts the convergence angle so that the irradiation region of the illumination light L1 at the position of the end surface 17 of the reservoir member 10 converges in the end surface 17. Further, the adjustment unit CL adjusts the convergence angle so that the illumination light L1 has a convergence point in the detection region in the flow path 13.
  • the illumination light beam of the illumination light L1 outside the focal depth of the detection unit 30 has a convergence angle that is smaller than the illumination light beam within the focal depth.
  • the above-described orthogonal surface includes the end surface 17 of the reservoir member 10, the side surface 16a on the irradiation light incident side of the flow path 13, or the side surface 16b on the irradiation light emission side of the flow path 13.
  • the convergence angle in the medium is ⁇
  • the wavelength of the illumination light L1 is ⁇
  • the beam width in the z direction at the position x and the convergence angle ⁇ is ⁇ (x, ⁇ )
  • the beam profile factor of the illumination light L1 is M2
  • the minimum width ⁇ 0 Assuming that the distance from the position in the x direction to the side surface 16a is xL, the following formula (3) and formula (4) must satisfy formula (5).
  • the beam width ⁇ (x, ⁇ ) included in the above equations (3) to (5) is 1 / e2 with respect to the peak value of the intensity of the illumination light L1. It is specified by the width. Even when the convergence angle ⁇ satisfies the expressions (1) to (3), the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is outside the beam width ⁇ (xL, ⁇ ). Therefore, when the convergence angle ⁇ is set, the beam width of the illumination light L1 having an intensity that is 1 / e2 or less with respect to the peak value is also taken into consideration.
  • FIG. 10 is a diagram schematically showing an optical path through which the illumination light L1 passes through the end face 17 of the reservoir member 10 and the side face 16a of the flow path 13.
  • the angle ⁇ 3 is the elevation angle of the illumination optical axis viewed from the focal plane F, and the counterclockwise direction from the focal plane F is the positive direction.
  • the incident angle and the emission angle at the interface, the inclination angle of the end surface 17 of the reservoir member 10 and the side surface 16a of the flow path 13 with respect to the yz plane, the illumination light flux in the material of the air / flow path device C and in the flow path The following relationship is established between the elevation angle with respect to the focal plane F, the medium outside the channel device C, the material of the channel device C, and the refractive index of the medium in the channel 13.
  • Incident angle / outgoing angle angle from the perpendicular to the end face 17 and the wall surface 16a Tilt angle: angle
  • the elevation angle ⁇ 3 of the illumination light L1 in the flow path 13 is expressed by the following equation (7).
  • the inclination angle of the end face 17 of the reservoir member 10 and the wall surface 16a of the flow path 13 and the elevation angle ⁇ 3 of the illumination light L1 are the refractive index n1 of the free space medium, the refractive index n2 of the material of the reservoir member 10 and the flow path 13
  • the refractive index n3 of the medium it is necessary to be selected, manufactured and adjusted so as to satisfy Expression (8).
  • the stage unit ST moves in the x direction, the y direction, and the z direction by driving the stage driving unit 60 shown in FIG.
  • the driving of the stage driving unit 60 is controlled by the control device 5.
  • the stage unit ST includes an installation surface STa on which the fluid device C is installed.
  • the installation surface STa is a surface parallel to the xy plane.
  • the installation surfaces STa are arranged at intervals in the y direction.
  • the installation surface STa supports both ends in the y direction where the lane 2 of the flow channel device C is not provided from the ⁇ Z side.
  • the region where the lane 2 is arranged is supported on the installation surface STa without hindering the observation from the ⁇ Z side by the detection unit 30.
  • stage part ST does not exist in the optical path of the illumination light L1 until the lane 2 in the fluid device C is irradiated, a part of the illumination light L1 incident on the fluid device C enters the stage part ST, which will be described later. Adversely affecting the particle detection.
  • the fixing pin 51 protrudes from the installation surface STa.
  • the fixing pin 51 includes two fixing pins 51 a that contact the long side of the fluid device C and one fixing pin 51 b that contacts the short side of the fluid device C.
  • the fixing pins 51a are arranged in the vicinity of both sides of the fluid device C in the y direction.
  • the fixing pin 51b contacts the short side located on the + y side.
  • a pressing piece 52 is provided at a corner located opposite to the corner where the fixing pin 51a and the fixing pin 51b located on the + y side are arranged.
  • the pressing piece 52 presses the fluid device C diagonally against the stage part ST.
  • the pressed fluid device C is fixed in a state where the fluid device C is positioned on the stage portion ST in the xy direction so that the flow path 13 (lane 2) is parallel to the y direction by contacting the fixing pins 51a and 51b.
  • the detection unit 30 includes an objective lens 31 and an imaging unit 32.
  • the objective lens 31 is disposed on the ⁇ Z side of the stage unit ST and the fluid device C. As shown in FIG. 9, the objective lens 31 is disposed at a position where the detection axis 31a passes through the center of the flow path 13 in the x direction.
  • the detection axis 31a is orthogonal to the optical axis of the illumination light L1.
  • the imaging unit 32 includes an EMCD (Electron Multiplying Charge Coupled Device) camera as an example, and captures an image of incident light.
  • the imaging unit 32 acquires image information of side scattered light that enters through the objective lens 31.
  • the transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
  • the operation of the particle detection apparatus 100 includes an installation process, an introduction process, an irradiation process, and a detection process.
  • the installation process is a process of installing the fluid device C on the installation surface STa of the stage part ST. Specifically, as shown in FIG. 5, by pressing the fluid device C diagonally with the pressing piece 52, the fluid device C is pressed against the fixing pins 51a and 51b, and the flow path 13 (lane 2). Is placed on the installation surface STa in a state of being positioned on the stage portion ST so as to be parallel to the y direction.
  • the introducing step is a step of introducing a sample containing particles into the holding spaces 14A and 14B and the flow path 13 of the fluid device C.
  • a sample containing particles such as a sample containing particles
  • an exosome suspension in which exosomes are suspended in a buffer solution (medium) such as a phosphate buffer can be used as the sample.
  • the control device 5 drives the stage drive unit 60 so that the lane 2 to be detected is positioned on the optical path of the illumination light L 1 and the detection axis 31 a of the detection unit 30.
  • the control device 5 controls the power supply unit BT to apply an electric field to the electrodes 18A and 18B, and applies a force for causing the exosome to electrophores along the flow path 13. To do.
  • the control device 5 applies a voltage having an electric field strength of about 50 V / cm for about 10 seconds.
  • the moving direction of the exosome is parallel to the y direction.
  • the irradiation process is a process of irradiating the flow path 13 of the flow path device C with the illumination light L1 parallel to the x direction.
  • the irradiation unit 20 and the adjustment unit CL that irradiate the illumination light L1 have a constant width in the y direction, and have a sheet beam shape that converges in the z direction at a convergence angle ⁇ that satisfies the above-described equations (3) to (8).
  • Irradiation light L1 is irradiated.
  • the minimum beam thickness (beam width in the z direction) of the illumination light L1 is 10 ⁇ m.
  • the minimum beam thickness (beam width in the z direction) direction of the illumination light L1 is the z direction in FIGS.
  • the direction of the minimum beam thickness (beam width in the z direction) of the illumination light L1 is different from the optical axis direction and the flow path direction of the illumination light L1 on the incident surface (end surface 17 and side surface 16a). It is a direction orthogonal to the flow path direction.
  • the channel direction is a direction in which the channel 13 extends.
  • the flow path direction is a direction in which fluid flows through the flow path 13.
  • the irradiated illumination light L1 is one end surface (illumination light incident side end surface) 17 of the fluid device C, the side surface (illumination light incident side side surface) 16a of the flow channel 13, the inside of the flow channel 13, the side surface of the flow channel 13 ( The light passes through the illumination light emission side surface 16b and the other end surface (illumination light emission side end surface) 27 (see FIG. 5) of the fluid device C sequentially.
  • the illumination light L1 is irradiated in a direction orthogonal to the moving direction of the exosome. As shown in FIG.
  • the irradiated illumination light L1 converges so that the width in the z direction is minimized inside the flow path 13, and the irradiation light flux passage region at the position of the side surface 16 a of the flow path 13. Converges to be confined within the side surface 16a. Furthermore, the irradiated illumination light L1 converges so that the passage region of the irradiated light beam at the position of the side surface 16b on the illumination light exit side of the flow path 13 is limited to the side surface 16a.
  • the illumination light L1 is adjusted to a convergence angle such that the irradiation region at the position of the side surface 16a is condensed in the side surface 16a and the irradiation region at the position of the side surface 16b is condensed in the side surface 16b. Further, the irradiated illumination light L1 has a convergence point in the detection region of the detection unit 30 in the flow path 13.
  • the detection unit 30 observes (images) and detects the scattered light generated from the particles in the flow path 13 by irradiation of the illumination light L1 in parallel with the x direction. Since the detection axis 31a of the objective lens 31 in the detection unit 30 is orthogonal to the optical axis of the illumination light L1, the detection unit 30 detects side scattered light generated from the particles. The detection unit 30 detects light scattered toward the z direction perpendicular to the x direction by irradiation of the illumination light L1 irradiated in parallel with the x direction. The image of the particles in which the scattered light is observed is picked up by the image pickup unit 32. The transmission unit 40 transmits the image information captured by the imaging unit 32 to the control device 5.
  • the control device 5 comprehensively controls the particle detection system 1.
  • the control device 5 controls the movement of the stage unit ST and the fluid device C via the stage driving unit 60.
  • the control device 5 controls the power supply unit (application unit) BT to apply an electric field in the direction along the flow path 13 to the electrodes 18A and 18B.
  • the control apparatus 5 performs various determinations by processing the image captured by the particle detection apparatus 100. Details of the configuration of the control device 5 will be described with reference to FIGS. 11 to 16.
  • FIG. 11 is a diagram showing a schematic configuration of the control device 5 of the present embodiment.
  • the control device 5 includes a calculation unit 500 and a storage unit 520.
  • the storage unit 520 includes storage devices such as a flash memory, an HDD (Hard Disk Drive), a RAM (Random Access Memory), a ROM (Read Only Memory), and a register.
  • the storage unit 520 stores a program (firmware) executed by the calculation unit 500 in advance.
  • the storage unit 520 stores a calculation result obtained by performing the calculation process by the calculation unit 500.
  • the calculation unit 500 includes a CPU (Central Processing Unit) and performs various calculations.
  • the calculation unit 500 includes, as its functional units, an acquisition unit 501, an identification unit 502, a zeta potential determination unit 503, a particle diameter determination unit 504, a correlation unit 505, a state determination unit 506, and an evaluation unit 507. It has.
  • the acquisition unit 501 acquires an image captured by the particle detection device 100. Specifically, as described above, the imaging unit 32 of the particle detection apparatus 100 captures an image of side scattered light that is incident through the objective lens 31 and transmits image information of the captured image to the transmission unit 40. Output to. The acquisition unit 501 acquires the image information of the side scattered light image captured by the imaging unit 32 via the transmission unit 40. The acquisition unit 501 outputs the acquired image to the identification unit 502.
  • the identification unit 502 extracts a fine particle image from the image captured by the particle detection device 100. For example, the identification unit 502 extracts a fine particle image by performing known filter processing and pattern matching processing on the image supplied from the acquisition unit 501. At this time, the identification unit 502 may assign a particle number for each fine particle to the extracted fine particle image. When the identification target microparticle is an extracellular vesicle, the particle number may be an extracellular vesicle identifier. That is, the identification unit 502 may label the fine particles. This facilitates the correlation between the zeta potential ⁇ of the fine particles and the particle diameter d of the fine particles in the correlation section described later.
  • the identification unit 502 performs tracking on the labeled fine particle based on the difference between frames of the image captured by the particle detection device 100.
  • tracking refers to tracking changes with time in the coordinates of particles in an image. An example of the result of the identification unit 502 tracking fine particles is shown in FIG.
  • FIG. 12 is a diagram illustrating an example of the particle list LS1 stored in the storage unit 520.
  • the particle list LS1 stores the coordinates (X, Y) of the image of each fine particle at each time, with the row direction as the labeled particle number and the column direction as the imaging time.
  • the coordinates of each fine particle from the fine particle P1 to the fine particle Pn at each time from the time t0 to the time t50 are stored in the particle list LS1.
  • the zeta potential determination unit 503 determines the zeta potential ⁇ for each fine particle based on the result tracked by the identification unit 502. For example, the zeta potential determination unit 503 determines the zeta potential ⁇ 1 of the fine particle P1 based on the moving speed v1 of the fine particle P1 from the time t0 to the time t1 in the tracking result of the fine particle P1 performed by the identification unit 502. To do. The zeta potential determination unit 503 determines the zeta potential ⁇ based on the above equation (1). In this example, the dielectric constant ⁇ of the sample solution and the viscosity coefficient ⁇ of the sample solution are stored in the storage unit 520 in advance.
  • the zeta potential determination unit 503 is configured based on the permittivity ⁇ of the sample solution and the viscosity coefficient ⁇ of the sample solution stored in the storage unit 520 and the moving speed of the particles obtained from the tracking result by the identification unit 502. The zeta potential ⁇ is determined.
  • the particle diameter determination unit 504 determines the diameter of the fine particles based on the amount of movement of the fine particles due to Brownian motion in the sample solution and the above equation (2).
  • the particle size determination unit 504 determines the particle size of the fine particles P1.
  • the Boltzmann constant k and the absolute temperature T of the sample solution are stored in the storage unit 520 in advance.
  • the particle diameter determination unit 504 calculates the movement amount of the fine particles P1 based on the result tracked by the identification unit 502.
  • the particle size determination unit 504 determines the particles of the fine particles P1 based on the calculated movement amount of the fine particles P1, the Boltzmann constant k and the absolute temperature T stored in the storage unit 520, and the above equation (2).
  • the diameter d1 is determined.
  • the correlation unit 505 associates the zeta potential ⁇ of the fine particles determined by the zeta potential determination unit 503 with the particle size d of the fine particles determined by the particle size determination unit 504. Specifically, the first zeta potential ⁇ 1 determined for the first fine particles in the zeta potential determination unit 503, and the first particle diameter d1 determined for the first fine particles in the particle size determination unit 504 Are correlated with each other as data on the first fine particles in the correlation unit 505.
  • FIG. 13 shows an example of the particle correlation list LS2 that is a result associated with the correlation unit 505.
  • FIG. 13 is a diagram illustrating an example of the particle correlation list LS2 stored in the storage unit 520.
  • the particle diameter d and the zeta potential ⁇ are associated with each particle number assigned by the identification unit 502.
  • the correlation unit 505 associates the particle diameter d1 of the fine particle P1 with the zeta potential ⁇ 1 of the fine particle P1 and stores it as particle correlation information PC1 (d1, ⁇ 1) in the particle correlation list LS2 for the fine particle P1. Further, the correlation unit 505 associates the particle diameter d2 of the fine particle P2 with the zeta potential ⁇ 2 of the fine particle P2 and stores the particle P2 in the particle correlation list LS2 as particle correlation information PC2 (d2, ⁇ 2). In this way, the correlation of the state of the fine particles existing in the medium can be determined.
  • the state determination unit 506 determines the state of the fine particles based on the particle correlation list LS2 generated by the correlation unit 505.
  • the storage unit 520 stores reference range information indicating the reference range of the particle diameter d and the reference range of the zeta potential ⁇ .
  • state determination by the state determination unit 506 a case will be described in which it is determined whether or not the microparticles identified by the identification unit 502 are exosomes in a sample containing particles other than exosomes.
  • the characteristics of exosomes are microparticles with a particle size of about 30 to 200 nm, and the presence of chaperone molecules Hsc70, Hsc90 and tetraspanins (CD9, CD63, CD81) as constituent factors.
  • the storage unit 520 stores a threshold value Thd of particle diameter as reference range information.
  • the storage unit 520 stores a threshold value Th ⁇ of zeta potential as reference range information.
  • the storage unit 520 may be rephrased as a reference storage unit. An example of the threshold value Thd and the threshold value Th ⁇ is shown in FIG.
  • FIG. 14 is a diagram illustrating an example of threshold values stored in the storage unit 520 of the present embodiment.
  • the particle diameter of exosome is about 30 to 100 nm in diameter, and among the particles to be determined, the particle diameter of particles other than exosome exceeds 100 nm in diameter.
  • the zeta potential ⁇ of the exosome is equal to or lower than the threshold Th ⁇ and the zeta potential ⁇ of fine particles other than the exosome exceeds the threshold Th ⁇ .
  • the state determination unit 506 can determine the particle based on the particle diameter of the particle and the zeta potential ⁇ of the particle.
  • the determination of fine particles performed by the state determination unit 506 may be paraphrased as identification of fine particles.
  • the storage unit 520 stores 100 nm as the particle size threshold Thd.
  • the storage unit 520 stores ⁇ 6 mV as the threshold value Th ⁇ of the zeta potential.
  • the state determination unit 506 is a particle whose particle diameter d is equal to or less than the threshold Thd in the particle correlation information PC stored in the particle correlation list LS2, and the zeta potential ⁇ is equal to or less than the threshold Th ⁇ .
  • a microparticle is determined to be an exosome.
  • the state determination unit 506 among the particle correlation information PC stored in the particle correlation list LS2, fine particles having a particle diameter d exceeding the threshold value Thd, or fine particles having a zeta potential ⁇ exceeding the threshold value Th ⁇ . Is determined not to be an exosome.
  • the particle diameter of exosome is about 30 to 100 nm, and among the fine particles to be determined, the particle diameter of fine particles other than exosome may exceed 200 nm.
  • the state determination unit 506 can determine the state of the fine particles based only on the particle diameter.
  • fine particles other than exosomes may be included in the diameter range of 100 to 200 nm.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the fine particle is an exosome.
  • a single exosome may not be contained in the range whose diameter is larger than 200 nm.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether or not the microparticle is a single exosome.
  • a range in which the diameter is larger than 200 nm may include fine particles in which a plurality of single exosomes are aggregated.
  • the threshold value Thd (200 nm) of the particle diameter can be used as one element for determining whether the microparticle is a single exosome or an aggregated exosome.
  • the threshold value serving as the reference value stored in the reference storage unit can be used as a factor for determining the state of the fine particles.
  • the state determination unit 506 determines whether or not the exosome has reacted with the antibody when the microparticles identified by the identification unit 502 are exosomes.
  • the storage unit 520 stores a threshold value Th ⁇ of zeta potential as reference range information. As described above, the zeta potential of the antibody-exosome complex is positively shifted compared to the zeta potential of exosome alone. In this case, the storage unit 520 stores a zeta potential (eg, ⁇ 6 mv) between the zeta potential of the exosome alone and the zeta potential of the antibody-exosome complex as the zeta potential threshold Th ⁇ . .
  • the state determination unit 506 determines that, among the particle correlation information PC stored in the particle correlation list LS2, the fine particles whose zeta potential of the fine particles is less than the threshold Th ⁇ is a single exosome that has not reacted with the antibody. judge. On the other hand, in the particle correlation information PC stored in the particle correlation list LS2, the state determination unit 506 determines that the microparticles whose microparticle zeta potential is greater than or equal to the threshold Th ⁇ are antibody-exosome complexes. .
  • the threshold value Th ⁇ for example, ⁇ 6 mV
  • the threshold value Th ⁇ ( ⁇ 6 mV) of the zeta potential can be used as one element for determining whether the microparticle is a single exosome.
  • the state determination unit 506 can also determine the state of the fine particles by combining the threshold value of the particle diameter d and the threshold value of the zeta potential ⁇ .
  • the antibody-exosome complex has a low zeta potential compared to a single exosome. For this reason, the Coulomb force acting between the microparticles is weaker in the antibody-exosome complex than in the single exosome.
  • the Coulomb force acting between the fine particles acts as a repulsive force that keeps the fine particles apart. That is, the antibody-exosome complex has less repulsive force acting between the microparticles than the single exosome. For this reason, the antibody-exosome complex tends to aggregate more easily than a single exosome.
  • the particle diameter determination unit 504 shifts the particle diameter d to be larger than that in the case where the particles are not aggregated by determining a plurality of aggregated fine particles as one fine particle.
  • a case where a fine particle having a particle diameter d of 200 nm or less is determined to be an exosome will be described as an example.
  • the particle size determination unit 504 may determine that the antibody-exosome complex is a fine particle having a diameter exceeding 200 nm.
  • the state determination unit 506 determines only by the particle diameter d, the particle diameter d of the antibody-exosome complex exceeds the threshold value Thd of the particle diameter d whether or not the antibody is an exosome. -It may be determined that the exosome complex is not an exosome. Therefore, the state determination unit 506 determines that a fine particle having a particle diameter d of 200 nm or less is an exosome, and the zeta potential ⁇ is equal to or less than a threshold Th ⁇ even if the particle diameter d is greater than 200 nm. Is determined to be an exosome. That is, the state determination unit 506 determines whether or not the fine particle is an exosome by combining the threshold value Thd of the particle diameter d and the threshold value Th ⁇ of the zeta potential ⁇ .
  • an antibody that specifically binds to the exosome such as tetraspanin (CD9, CD81, etc.) can be used. That is, it is possible to determine whether or not the microparticle is an exosome based on the respective changes in the zeta potential ⁇ and the particle diameter d caused by causing the antibody to act on the exosome.
  • the particle detection system 1 has an advantage that fine particles can be evaluated by variously combining the above-described evaluation conditions based on the zeta potential ⁇ and the particle diameter d.
  • the state determination unit 506 can determine the state of the fine particles based on the result of tracking by the identification unit 502 after combining the threshold value Thd of the particle diameter d and the threshold value Th ⁇ of the zeta potential ⁇ . . Specifically, the state determination unit 506 tracks the progress until the antibody reacts with a single exosome and further aggregates the antibody-exosome complexes based on the result of tracking by the identification unit 502. Specifically, the state determination unit 506 moves the particle diameter d and the zeta potential ⁇ of each fine particle from any of the regions DM1 to DM4 shown in FIG.
  • the state of the fine particles is determined depending on whether or not As an example, the state determination unit 506 determines that when a microparticle (for example, a single exosome) present in the region DM3 moves to the region DM2, the exosome reacts with the antibody and changes to an antibody-exosome complex. judge. Further, when the exosome moves from the region DM2 to the region DM1, the state determination unit 506 determines that the antibody-exosome complexes are aggregated.
  • a microparticle for example, a single exosome
  • Evaluation unit 507 evaluates the quality of the fine particles.
  • the evaluation unit 507 ranks the state of the fine particles into A rank, B rank, and C rank based on the state of the fine particles determined by the state determination unit 506.
  • the A rank is a case where both the particle diameter d and the zeta potential ⁇ of the fine particles are included in the reference range.
  • the rank B is a case where either one of the particle diameter d and the zeta potential ⁇ of the fine particles is not included in the reference range.
  • the C rank is a case where neither the particle diameter d of the fine particles nor the zeta potential ⁇ is included in the reference range.
  • the evaluation unit 507 evaluates whether or not the microparticle is a single exosome.
  • the evaluation unit 507 determines that the rank of the fine particles is rank A when the fine particles are present in the region DM3. Further, the evaluation unit 507 determines that the rank of the fine particles is rank B when the fine particles are present in the region DM2 or the region DM4. The evaluation unit 507 determines that the rank of the fine particles is rank C when the fine particles are present in the region DM1.
  • FIG. 15 is a diagram illustrating an example of the operation of the control device 5.
  • the particle detection apparatus 100 captures an image of side scattered light at a predetermined time interval.
  • the acquisition unit 501 acquires images captured by the imaging unit 32 of the particle detection device 100 one by one from the particle detection device 100 (step S10).
  • This image includes an image of fine particles that are electrophoresed in the migration channel 150.
  • the fine particle image includes an exosome image.
  • the identification unit 502 extracts a fine particle image from the image acquired in step S10, and assigns a unique particle number to each fine particle. That is, the identification unit 502 labels the fine particles (step S20).
  • the identification unit 502 determines whether or not labeling has been completed for all captured images (step S30). If it is determined that the labeling has not been completed for all captured images (step S30; NO), the identification unit 502 returns the process to step S10 and performs the labeling for the next image. If it is determined that the labeling has been completed for all captured images (step S30; YES), the identification unit 502 advances the process to step S40 and performs tracking for the identified fine particles.
  • the zeta potential determination unit 503 determines the zeta potential ⁇ for each fine particle based on the result tracked by the identification unit 502 (step S50). Further, the particle size determination unit 504 determines the particle size for each fine particle based on the result of tracking by the identification unit 502 (step S60). Note that the order of step S50 and step S60 may be reversed or may be executed in parallel.
  • correlation section 505 associates the zeta potential ⁇ of the fine particles determined by zeta potential determination section 503 with the particle diameter d of the fine particles determined by particle diameter determination section 504 (step SS70).
  • the correlation unit 505 generates a particle correlation list LS2 indicating the associated result, and stores the generated particle correlation list LS2 in the storage unit 520.
  • the correlation unit 505 determines that the association has not been completed for all the fine particles (step S80; NO)
  • the correlation unit 505 returns the process to step S40.
  • the correlation unit 505 determines that the association has been completed for all the fine particles (step S80; YES)
  • the correlation unit 505 advances the process to step S90.
  • the state determination unit 506 and the evaluation unit 507 perform particle state determination and evaluation based on the particle correlation list LS2 generated in step S70.
  • the particle detection apparatus 100 is an example of an apparatus for measuring the zeta potential of extracellular vesicles, and can be a device capable of measuring the zeta potential of individual extracellular vesicles in a population of extracellular vesicles. Can be used without any particular restrictions.
  • the particle detector configured as described above, or other zeta potential measuring device the zeta potential of individual extracellular vesicles in a population of extracellular vesicles is measured, and the standard deviation of those zeta potentials is calculated. It can be calculated as the standard deviation of the zeta potential of the population of extracellular vesicles.
  • the standard deviation of the zeta potential of the extracellular vesicle calculated as described above is 5 mV or less, it is determined that the group is the extracellular vesicle group of the present embodiment.
  • the population of extracellular vesicles of this embodiment is a population of extracellular vesicles having a standard deviation of zeta potential of 5 mV or less and high homogeneity with respect to the state of extracellular vesicles, particularly the surface state of extracellular vesicles. is there. Therefore, it can be used for various uses such as pharmaceuticals, cosmetics, and foods.
  • the extracellular vesicle population of this embodiment can be suitably used as a pharmaceutical product that requires particularly high homogeneity because the quality of the extracellular vesicles constituting the population is uniform.
  • Examples of the pharmaceutical use include, but are not limited to, use as a carrier encapsulating a drug and use of a specific cell-derived exosome such as a mesenchymal stem cell as a drug.
  • a specific cell-derived exosome such as a mesenchymal stem cell as a drug.
  • the zeta potential is measured in a state in which a specific binding substance is bound to an extracellular vesicle, the extracellular smallness with high homogeneity particularly with respect to the expression state of the molecule to which the specific binding substance is bound.
  • a population of cells can be obtained.
  • the membrane surface of an extracellular vesicle is modified with a molecule that targets a disease site or the like (for example, an antibody against a cancer cell membrane surface antigen)
  • the zeta potential can be reduced using a specific binding substance for the molecule.
  • Measurement may be performed to obtain a population of extracellular vesicles having a standard deviation of the zeta potential of 5 mV or less.
  • Extracellular vesicles constituting such a population of extracellular vesicles are particularly homogeneous with respect to the state of modification of the membrane surface by the molecule. Therefore, it can be suitably used as a carrier for DDS to the disease site.
  • the present invention provides a method for producing a population of extracellular vesicles.
  • the method of the present embodiment includes (a) a step of synchronizing the cell cycle of a plurality of cells, and (b) a medium of the plurality of cells substantially not including extracellular vesicles after the step (a). Replacing the medium; (c) culturing the plurality of cells in the medium replaced; and (d) recovering a population of extracellular vesicles from the medium after the step (c). And including.
  • Step (a) is a step of synchronizing the cell cycle of a plurality of cells.
  • the plurality of cells are preferably the same type of cells. By culturing the same type of cells, extracellular vesicles of uniform quality can be obtained.
  • the cell type is not particularly limited as long as it releases extracellular vesicles.
  • Examples of cells include various disease cells such as tumor cells; immune cells such as dendritic cells, T cells and B cells; various tissue cells such as nerve cells; fat cells; mesenchymal stem cells and hematopoietic stem cells. Examples include, but are not limited to, stem cells; pluripotent stem cells such as ES cells and iPS cells; germ cells and the like.
  • the biological species from which the cells are derived is not particularly limited.
  • the biological species from which the cells are derived is not particularly limited, and examples thereof include cells of humans and mammals other than humans (for example, mice, guinea pigs, monkeys, dogs, cats, cows, horses, pigs, etc.).
  • the cells may be appropriately selected according to the use of the exosome to be produced.
  • human mesenchymal stem cells and the like can be selected.
  • “More cells” if two or more cells is not particularly limited, for example, 10 2 or more, 10 3 or more, may be a cell, such as a 10 4 or more.
  • the plurality of cells may be, for example, 10 2 to 10 15 cells, 10 3 to 10 12 cells, or 10 4 to 10 10 cells.
  • the cell cycle is divided into an interphase and an M phase, and the interphase is further divided into a G1 phase, an S phase, and a G2 phase (see FIG. 16).
  • the cell cycle for synchronizing a plurality of cells is not particularly limited, and may be any of G1 phase, S phase, G2 phase, and M phase. Further, it may be synchronized with the boundary of two periods such as the boundary of G1 period / S period.
  • the method for synchronizing the cell cycle of the plurality of cells is not particularly limited, and a known method can be used. Examples of such a method include a method of culturing in a medium containing a cell cycle synchronizer, a method of culturing cells in a confluent state, a method of culturing cells in a serum-starved state, and a thymidine block method.
  • the “cell cycle synchronizer” is an agent having an action of synchronizing the cell cycle of a plurality of cells.
  • the cell cycle synchronizer include a drug having an action of stopping the progression of the cell cycle in a specific cell cycle.
  • a well-known thing can be especially used for a cell cycle synchronizing agent without a restriction
  • leptomycin A, leptomycin B, etc. are mentioned as a cell cycle synchronizer which synchronizes a cell to G1 phase.
  • a cell cycle synchronizer is added to a culture medium, and a plurality of cells are cultured. What is necessary is just to select a culture medium suitably according to the kind of cell.
  • a known human cell culture medium can be used without any particular limitation.
  • An example of the human cell culture medium is RPMI medium to which fetal bovine serum (FBS) or the like is added.
  • the culture time in a medium containing a cell cycle synchronizer may be appropriately selected according to the cell type. It is preferable to culture for a period of time longer than the cell cycle proceeds for one cycle. Examples of the culture time include 10 hours or more, 15 hours or more, or 20 hours or more.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 5 cycles, 4 cycles, 3 cycles, or 2 cycles, etc. Is mentioned. Specific examples of the upper limit of the culture time include, for example, within 50 hours, within 40 hours, or within 30 hours. As an example, the culture time can be 24 hours.
  • the cell concentration at the start of the culture is not particularly limited, and may be, for example, 10 3 to 10 10 cells / mL, 10 3 to 10 9 cells / mL, 10 4 to 10 9 cells / mL, and the like.
  • the cell culturing conditions are not particularly limited, and conditions generally used for culturing the cells may be used according to the cell type.
  • the temperature conditions include 25 to 40 ° C., 30 to 37 ° C. and the like.
  • the “confluent state” refers to a state in which cells reach a concentration capable of growing in a culture container and cell growth is almost stopped. By culturing cells in a confluent state, the cell cycle can be synchronized with the G1 phase.
  • the cells when cells are cultured in a culture container such as a petri dish, the cells proliferate and reach a confluent state.
  • a confluent state For example, when culturing in a petri dish or the like, it can be determined that a confluent state has been reached when cells have spread throughout the petri dish. In this state, the cell cycle can be synchronized by continuing the culture.
  • the culture time after reaching the confluent state is preferably cultivated for at least the time required for one cycle of the cell cycle, as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized.
  • the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. .
  • Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours.
  • the culture time can be 24 hours.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing cell cycle synchronizer is used.
  • the culture in the serum starvation state may be performed by a known method by culturing cells in a medium not containing serum.
  • a medium not containing serum can be prepared, for example, by adding no serum in the composition of the medium appropriately selected according to the cell type.
  • the cell cycle can be synchronized with the G1 phase.
  • the culture time in the serum starvation state is preferably cultivated for a time longer than the time required for one cycle of the cell cycle as described above. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium not containing serum is used.
  • the thymidine block may be performed by a known method by culturing cells in a medium containing an excessive amount of thymidine. By performing thymidine blocking, the cell cycle can be synchronized to the S phase. Examples of the concentration of thymidine in the medium include 1 to 5 mM, 1.5 to 4 mM, and 2 to 3 mM. The medium may be appropriately selected according to the cell type, except that thymidine is added in excess. As in the above, the culture time in the medium containing excess thymidine is preferably cultivated for at least the time required for one cycle of the cell cycle. Examples of the culture time include 10 hours or more, 15 hours or more, 20 hours or more, and the like.
  • the upper limit of the culture time is not particularly limited, but it is not necessary to culture for a long time after the cell cycle is synchronized. For example, the time is shorter than when the cell cycle proceeds for 4 cycles, 3 cycles, or 2 cycles, etc. . Specific examples of the upper limit of the culture time can be, for example, within 50 hours, within 40 hours, and within 30 hours. As an example, the culture time can be 24 hours.
  • the cell cycle may be synchronized using the double thymidine block method.
  • the double thymidine block method can also be performed by a known method.
  • thymidine block method thymidine block is performed twice. For example, after culturing in a medium containing excess thymidine for a certain period (for example, about 10 to 30 hours), the medium is changed, and culturing is performed for a certain period (for example, about 6 to 20 hours) in a medium not containing thymidine. Thereafter, the cell cycle can be synchronized with the boundary between G1 phase / S phase by culturing again in a medium containing excess thymidine.
  • the culture method may be performed in the same manner as described above (method using cell cycle synchronizer) except that a medium containing excess thymidine is used.
  • Step (b) is a step of replacing the medium of the plurality of cells with a medium substantially free of extracellular vesicles after the step (a).
  • the medium can be exchanged, for example, by removing the culture supernatant from the culture vessel in step (a) and adding a medium substantially free of extracellular vesicles. Further, after removing the medium supernatant, the cells may be washed about 1 to 3 times with a medium substantially free of extracellular vesicles. When the cells are floating cells or the like, the cells may be collected by centrifugation, filter filtration, etc., washed appropriately, and seeded in a medium substantially free of extracellular vesicles. .
  • the “medium substantially free of extracellular vesicles” is a medium substantially free of extracellular vesicles in the medium components. “Substantially free of extracellular vesicles” means that the content is negligible even if extracellular vesicles are contained, or no extracellular vesicles.
  • the concentration of extracellular vesicles contained in the medium is, for example, about 0 to 10 cells / mL, preferably about 0 to 5 cells / mL, more preferably Examples of the medium include about 0 to 3 / mL, more preferably about 0 to 1 / mL, and particularly preferably about 0 to 0.5 / mL.
  • the culture medium contains both extracellular vesicles released from the cells and extracellular vesicles originally contained in the medium. It is not possible to obtain extracellular vesicles with uniform size. Therefore, in this step, the cell culture medium is replaced with a medium that is substantially free of extracellular vesicles. By this step, it is possible to remove extracellular vesicles released before the cell cycle is synchronized, and to eliminate the introduction of extracellular vesicles from the medium.
  • the method for producing a medium substantially free of extracellular vesicles is not particularly limited, and known methods for recovering extracellular vesicles can be applied.
  • extracellular vesicles in the medium can be removed by performing ultracentrifugation, ultrafiltration, or the like. Therefore, it can also be said that the medium substantially free of extracellular vesicles is a medium that has been subjected to a treatment for removing extracellular vesicles in the medium after the medium has been adjusted.
  • Step (c) is a step of culturing the plurality of cells in the medium exchanged.
  • the cells can be cultured in the same manner as described in the above step (a) except that the cells are cultured in a medium not containing extracellular vesicles.
  • the culture time in this step may be a time sufficient for the cells to release extracellular vesicles. For example, it can be 30 minutes or longer, 1 hour or longer, 1.5 hours or longer. Further, for example, it may be 30 minutes to 5 hours, 1 to 4 hours, 1.5 to 3 hours, and the like. For example, the culture time can be 2 hours.
  • Step (d) is a step of collecting a population of extracellular vesicles from the medium after step (c).
  • Extracellular vesicles can be recovered, for example, from the culture supernatant after step (c).
  • the cells and the culture supernatant can be separated by centrifuging the culture solution or filtering the filter.
  • the method for recovering extracellular vesicles from the culture supernatant is not particularly limited, and a known method can be used.
  • methods for recovering extracellular vesicles include ultracentrifugation, ultrafiltration, continuous flow electrophoresis, chromatography and the like. In this way, a population of extracellular vesicles can be obtained.
  • the population of extracellular vesicles obtained by the production method of the present embodiment is a population of extracellular vesicles released from cells synchronized in cell cycle and of uniform quality.
  • the group of extracellular vesicles having uniform quality is, for example, a group of extracellular vesicles in which the standard deviation of the zeta potential of the extracellular vesicles is an arbitrary threshold value or less.
  • the extracellular vesicles whose standard deviation of zeta potential of extracellular vesicles is 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, further preferably 3.5 mV or less, particularly preferably 3 mV or less. It is a group of.
  • the population of extracellular vesicles obtained by the production method of the present embodiment has the same quality as described above, it is suitable for use as a drug for DDS carriers and regenerative medicine.
  • the manufacturing method of this embodiment may include an optional step in addition to the steps (a) to (d).
  • an arbitrary process is not specifically limited, For example, (e) The process of measuring the zeta potential of the extracellular vesicle contained in the collect
  • the production method of this embodiment may include a step of measuring the zeta potential of extracellular vesicles contained in the population of extracellular vesicles collected in step (d).
  • the zeta potential of the extracellular vesicle can be measured by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “ ⁇ Extracellular vesicle population>”.
  • the manufacturing method of the present embodiment may further include a step of (f) calculating a standard deviation of the zeta potential obtained by the step (e). Further, (g) a step of selecting a population of extracellular vesicles in which the standard deviation is not more than a predetermined threshold value may be included.
  • the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • a population of extracellular vesicles having uniform quality can be obtained by steps (a) to (d).
  • the quality can be further improved by carrying out steps (e) to (g).
  • a group of extracellular vesicles with uniform thickness can be obtained.
  • the present invention provides a method for assessing the quality of a population of extracellular vesicles.
  • the method of this embodiment includes (a) a step of measuring zeta potential of a plurality of extracellular vesicles contained in a population of extracellular vesicles, and (b) a standard of zeta potential measured in the step (a). And (c) evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b).
  • Step (a) is a step of measuring the zeta potential of a plurality of extracellular vesicles contained in the population of extracellular vesicles. Measurement of the zeta potential of individual extracellular vesicles in a population of extracellular vesicles can be performed by a known method. Examples of the method for measuring the zeta potential of extracellular vesicles include the method described in the above “ ⁇ Extracellular vesicle population>”.
  • Step (b) is a step of calculating the standard deviation of the zeta potential measured in step (a).
  • the standard deviation of the zeta potential can be calculated by the method described above in “ ⁇ population of extracellular vesicles>”.
  • Step (c) is a step of evaluating the quality of the population of extracellular vesicles based on the standard deviation calculated in step (b). For example, when the standard deviation threshold is set based on the type of cell from which the extracellular vesicle is derived, the use of the extracellular vesicle, etc., and the standard deviation calculated in step (b) is equal to or less than the threshold It can be evaluated that the uniformity of the extracellular vesicle population is high. That is, it can be determined that the quality of the population of extracellular vesicles is high (the quality is uniform). Examples of the standard deviation threshold include 5 mV or less, preferably 4.5 mV or less, more preferably 4 mV or less, still more preferably 3.5 mV or less, and particularly preferably 3 mV or less.
  • the method of the present embodiment may further include a step of selecting a population of extracellular vesicles evaluated as having high uniformity by the step (c).
  • the method may include a step of discarding a population of extracellular vesicles evaluated as having low uniformity by the step (c).
  • the quality of the extracellular vesicle population can be managed, and the extracellular vesicle population with uniform quality can be maintained.
  • the present invention provides a composition comprising a plurality of extracellular vesicles, wherein the standard deviation of the zeta potential of the extracellular vesicles contained in the composition is 5 mV or less. To do.
  • the standard deviation of the zeta potential of the extracellular vesicles contained in the composition of this embodiment is 5 mV or less.
  • the standard deviation of the zeta potential is preferably 4.5 mV or less, more preferably 4 mV or less, preferably 3.5 mV or less, and more preferably 3 mV or less.
  • the plurality of extracellular vesicles may be two or more, and is not particularly limited.
  • the number of extracellular vesicles is 10 3 to 10 15 , 10 4 to 10 12 or more, 10 5 to 10 10 extracellular vesicles. May be.
  • the plurality of extracellular vesicles contained in the composition of the present embodiment is the population of extracellular vesicles of the above-described embodiment described in the above “ ⁇ population of extracellular vesicles>”.
  • composition of the present embodiment may contain an arbitrary component in addition to a plurality of extracellular vesicles.
  • arbitrary components are not specifically limited, For example, various buffer solutions (physiological saline, a phosphate buffer, a HEPES buffer etc.), a cell culture solution, etc. are mentioned.
  • composition of the present embodiment may be a pharmaceutical composition, cosmetics, food (including functional food, health food, etc.) and the like.
  • composition of this embodiment is a pharmaceutical composition, cosmetics, food, or the like, various components described later may be included depending on the application.
  • the present invention provides a pharmaceutical composition comprising the population of extracellular vesicles of the above embodiment.
  • the population of extracellular vesicles of the above embodiment, or the population of extracellular vesicles produced by the production method of the above embodiment (hereinafter sometimes collectively referred to as “the population of extracellular vesicles”). Due to its high uniformity, it can be contained in a pharmaceutical composition.
  • the population of extracellular vesicles of the above-described embodiment can be used, for example, for a carrier containing a drug (for example, a carrier of DDS) or regenerative medicine.
  • the pharmaceutical composition of this embodiment may contain other components in addition to the population of the present extracellular vesicles.
  • it may contain at least one pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” means a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the administration subject. By “not exhibiting substantial toxicity” is meant that the ingredient is not toxic to the administered subject at the doses normally used.
  • Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredients that are typically considered inactive ingredients.
  • the pharmaceutically acceptable carrier is not particularly limited, and examples thereof include solvents, diluents, vehicles, excipients, glidants, binders, granulating agents, dispersing agents, suspending agents, wetting agents, Lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, preservatives (for example, antioxidants), chelating agents, flavoring agents, sweeteners, thickeners, buffers, colorants, etc. Can be mentioned.
  • the solvent include water, physiological saline, phosphate buffer, HEPES buffer, cell culture medium, DMSO, dimethylacetamide, ethanol, glycerol, mineral oil, and the like.
  • a pharmaceutically acceptable carrier may be used alone or in combination of two or more.
  • components commonly used in the pharmaceutical field can be used without particular limitation.
  • the pharmaceutical composition of the present embodiment includes, for example, a solubilizing agent, a suspending agent, an isotonic agent, a buffer, a pH adjusting agent, an excipient, a stabilizer, an antioxidant, an osmotic pressure adjusting agent, and an antiseptic.
  • Coloring agents, fragrances and the like may be included. These may be used alone or in combination of two or more.
  • the pharmaceutical composition of this embodiment may contain the chemical
  • medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the pharmaceutical composition of this embodiment.
  • Examples of the drug include anticancer agents, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, blood circulation promoters, stimulants, hormones, stimulus relieving agents, analgesics, cell activators, plants and animals. ⁇ Microbial extract, antipruritics, anti-inflammatory analgesics, antifungals, antihistamines, hypnotic sedatives, tranquilizers, antihypertensives, antihypertensive diuretics, antibiotics, anesthetics, antibacterial substances, antiepileptics, coronary vasodilation Examples include, but are not limited to, agents, herbal medicines, antipruritic agents, and keratin softening release agents. A medicine may be used individually by 1 type and may use 2 or more types together. The drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
  • the dosage form of the pharmaceutical composition of the present embodiment is not particularly limited, and can be a dosage form generally used as a pharmaceutical preparation.
  • Examples of the dosage form of the pharmaceutical composition of the present embodiment include orally administered dosage forms such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, and emulsions, or Examples include dosage forms administered parenterally such as injections, suppositories, and external preparations for skin.
  • the pharmaceutical composition of these dosage forms can be formulated according to a conventional method (for example, a method described in the Japanese Pharmacopoeia).
  • the administration route of the pharmaceutical composition of this embodiment is not particularly limited, and can be administered by oral or parenteral routes.
  • the parenteral route includes all routes other than oral administration such as intravenous, intramuscular, subcutaneous, intranasal, intradermal, ophthalmic, intracerebral, intrarectal, intravaginal, intraperitoneal, etc. To do. Administration may be local or systemic.
  • the pharmaceutical composition of the present embodiment can be administered in a single dose or multiple doses, and the administration period and interval thereof include the type of drug, the type and condition of the disease, the administration route, the age of the administration subject, It can be appropriately selected depending on body weight and sex.
  • the administration interval can be, for example, 1 to 3 times a day, every 3 days, every week, etc.
  • the dosage of the pharmaceutical composition of the present embodiment can be appropriately selected depending on the type of drug, the type and condition of the disease, the administration route, the age, weight and sex of the administration subject.
  • the dosage of the pharmaceutical composition of the present embodiment can be a therapeutically effective amount of the drug contained in the pharmaceutical composition, for example, about 0.01 to 1000 mg per kg body weight at a time, 0.1 to 500 mg. About 0.1 to 100 mg.
  • the present invention provides a cosmetic product comprising the population of extracellular vesicles of the above embodiment.
  • the cosmetic product of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles.
  • the cosmetic of this embodiment can be manufactured according to a known method according to the type of cosmetic.
  • Other components are not particularly limited, and examples thereof include pharmaceutically acceptable carriers. Examples of the pharmaceutically acceptable carrier are the same as those exemplified in the above “ ⁇ Pharmaceutical composition>”.
  • the cosmetics of this embodiment may use materials known as cosmetic additives as other components. Other components may be used alone or in combination of two or more.
  • the cosmetic product of this embodiment may contain a drug (active ingredient) having a cosmetic effect or the like.
  • medical agent is not specifically limited, What is necessary is just to select suitably according to the use of the cosmetics of this embodiment.
  • the agent include whitening materials, ultraviolet absorbers, hair-growth agents, astringents, anti-wrinkle agents, anti-aging agents, tanning agents, antiperspirants, moisturizers, vitamins and derivatives thereof, anti-inflammatory agents, anti-inflammatory agents, Examples include, but are not limited to, inflammatory agents, blood circulation promoters, stimulants, hormones, stimulant mitigators, cell activators, plant / animal / microbe extracts, herbal medicines, antipruritic agents, keratin softening release agents, and the like.
  • a medicine may be used individually by 1 type and may use 2 or more types together.
  • the drug may be encapsulated in, for example, extracellular vesicles that constitute the population of the present extracellular vesicles.
  • the type of cosmetic product is not particularly limited.
  • cosmetics include basic cosmetics such as lotions, emulsions, lotions, creams, gels, sunscreens, packs, masks, and cosmetics; makeup cosmetics such as foundations, makeup bases, lipsticks, lip glosses, and blushers Cleaning agents such as facial cleansers, body shampoos and cleansing agents; hair cosmetics such as shampoos, rinses, hair conditioners, treatments, hair styling agents; and body cosmetics such as body powders and body lotions, but are not limited to these Not.
  • the cosmetic product of the present embodiment can be used in the same manner as a normal cosmetic product, depending on the use of the cosmetic product.
  • the present invention provides a food product comprising the population of extracellular vesicles of the above embodiment.
  • the food of this embodiment may contain other components as appropriate in addition to the population of the present extracellular vesicles.
  • the food of this embodiment can be produced according to a known method according to the type of food.
  • Other components are not particularly limited, and for example, materials known as food additives may be used as other components.
  • Other components may be used alone or in combination of two or more.
  • the type of food is not particularly limited.
  • Examples of food include various types of noodles such as buckwheat, udon, harusame, Chinese noodles, instant noodles, cup noodles; carbohydrates such as bread, flour, rice flour, hot cakes, mashed potatoes; green juices, soft drinks, carbonated drinks, Beverages such as nutritional drinks, fruit drinks, vegetable drinks, lactic acid drinks, milk drinks, sports drinks, tea and coffee; bean products such as tofu, okara and natto; various soups such as curry roux, stew roux and instant soup; ice Frozen confectionery such as cream, ice sherbet, shaved ice; sweets such as candy, cookies, candy, gum, chocolate, tablet confectionery, snack confectionery, biscuits, jelly, jam, cream, and other baked confectionery; kamaboko, hampen, ham, sausage Processed fishery and livestock products such as processed milk, fermented milk, butter, cheese, yogurt and other milk Products; salad oil, tempura oil, margar
  • the food of this embodiment may be health food, functional food, or the like. In this case, it may be formulated into a dry powder, granule, tablet, jelly, drink or the like by a known formulation method.
  • the food of this embodiment may be taken in the same way as normal food.
  • HL60 cells which are human acute myeloid leukemia cells, were cultured in Roswell Park Memorial Institute (RPMI) medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS).
  • RPMI Roswell Park Memorial Institute
  • FBS fetal bovine serum
  • EV removal medium in which EV in FBS was removed by ultracentrifugation was used.
  • EV purification from the collected culture supernatant was performed according to the following procedure. First, to remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 ⁇ g for 10 minutes, 2,000 ⁇ g for 20 minutes, and 10,000 ⁇ g for 100 minutes.
  • the supernatant was centrifuged at 100,000 ⁇ g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer to obtain an EV sample.
  • the EV sample was introduced into the micro-channel of the analysis chip in which the channel for adjusting the liquid level was arranged in parallel, and the barycentric position of each particle was visualized and tracked by scattered light imaging.
  • the particle size was estimated by Brownian motion analysis, and the zeta potential was estimated by electrophoresis analysis.
  • Example 1 The number of HL60 cells, which are human acute myeloid leukemia, was counted using a blood count plate, and the initial number of cells was adjusted to 1 ⁇ 10 7 .
  • a medium for cell culture a RPMI medium (normal medium) supplemented with 10% fetal bovine serum (FBS) was used.
  • FBS fetal bovine serum
  • LMB-added medium obtained by adding 100 nM Leptomycin B (LMB), which is an agent for synchronizing cells to the G1 phase, in a normal medium was used as a medium for cell cycle synchronization.
  • a PRMI medium supplemented with 10% FBS from which EV was removed by ultracentrifugation was used as a medium that does not contain EV (EV removal medium).
  • HL60 cells were cultured in a normal medium or a medium supplemented with LMB for 24 hours, then replaced with an EV removal medium, and cultured for 2 hours. Thereafter, cells and cell supernatant were collected. For the collected cells, the cell cycle was examined by the amount of intracellular DNA. Specifically, the cells were fixed with ethanol, stored at 4 ° C. for 24 hours or more, and then washed with PBS. Thereafter, RNase A was added to degrade RNA and incubated at 37 ° C.
  • EV was purified from the collected culture supernatant by the following procedure. To remove large particles such as cell debris and microvesicles, centrifugation was performed at 300 ⁇ g for 10 minutes, 2,000 ⁇ g for 20 minutes, and 10,000 ⁇ g for 100 minutes. Next, the supernatant was centrifuged at 100,000 ⁇ g for 200 minutes, and the precipitate was suspended in 10 mM HEPES buffer.
  • the prepared EV sample was introduced into the flow path of the liquid level difference compensation type analysis chip, set in a high-precision single nanoparticle measurement system (see FIG. 19), and the particle size and zeta potential were measured (see FIG. 20). .
  • An outline of the procedure from cell culture to EV recovery is shown in FIG.
  • An outline of the procedure of the method for purifying EV from cells is shown in FIG.
  • FIG. 23 shows the result of FACS measurement of the fluorescence intensity of DNA stained with PI.
  • the cells cultured in the LMB-added medium are 36.4% to 14.5% in the S phase cells and 22.2 in the G2 / M phase, compared to the cells cultured in the normal medium (without the LMB medium). From 1% to 14.5%.
  • G1 phase cells increased from 41.4% to 69.7%.
  • LMB inhibited the transition from the G1 phase to the S phase, confirming that the cell cycle was synchronized with the G1 phase (FIG. 23).
  • the measurement results of EV particle size and zeta potential are shown in FIG.
  • the average particle size and standard deviation of EV particle size and zeta potential derived from cells cultured in normal medium were 129 ⁇ 80.3 nm and ⁇ 12.2 ⁇ 5.73 mV, respectively.
  • the average value and standard deviation of the particle size and zeta potential of EVs derived from cells cultured in the LMB-added medium were 193 ⁇ 115 nm and ⁇ 13.4 ⁇ 2.93 mV, respectively.
  • EV-derived cells cultured in a medium supplemented with LMB had a smaller zeta potential distribution range.
  • the distribution range (standard deviation) of the zeta potential of the EV secreted from the cells was reduced, and it was found that the EV secreted for each cell cycle was different. .
  • the standard deviation of the zeta potential is 2.93 mV, and the standard deviation of EV secreted from cells not synchronized in the cell cycle (5.73 mV) The standard deviation value was smaller. From this result, it was confirmed that the standard deviation of the zeta potential of EV can be reduced by synchronizing the cell cycle.
  • a method for producing an extracellular vesicle and a method for evaluating the quality of the extracellular vesicle such as a group of extracellular vesicles having a uniform quality and a composition containing the extracellular vesicle.
  • the extracellular vesicles of the present invention can be used for various uses such as pharmaceuticals, cosmetics, and foods.

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Abstract

L'invention concerne une population de vésicules extracellulaires dans laquelle l'écart-type du potentiel zêta des vésicules extracellulaires est égal ou inférieur à 5 mV. La présente invention concerne également une composition contenant la population de vésicules extracellulaires. De plus, la présente invention concerne une méthode de fabrication d'une population de vésicules extracellulaires, la méthode comprenant : (a) une étape de synchronisation de cycles cellulaires d'une pluralité de cellules ; (b) une étape d'échange, après l'étape (a), du milieu pour la pluralité de cellules avec un milieu qui ne contient sensiblement pas de vésicules extracellulaires ; (c) une étape de culture de la pluralité de cellules dans le milieu qui a été échangé ; et (d) une étape de récupération de la population de vésicules extracellulaires à partir du milieu après l'étape (c).
PCT/JP2019/008235 2018-03-02 2019-03-01 Population de vésicules extracellulaires et sa méthode de fabrication WO2019168184A1 (fr)

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JP7371975B1 (ja) 2022-09-27 2023-10-31 セルソース株式会社 細胞外小胞の製造方法及び細胞外小胞含有組成物
JP7423016B1 (ja) 2022-09-27 2024-01-29 セルソース株式会社 細胞外小胞分泌用培地

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JP7372294B2 (ja) 2021-09-27 2023-10-31 矢崎総業株式会社 端子挿入装置

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WO2014030590A1 (fr) * 2012-08-24 2014-02-27 国立大学法人東京大学 Méthode d'analyse d'un exosome, appareil d'analyse d'un exosome, complexe anticorps-exosome et puce pour l'électrophorèse d'un exosome
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7371975B1 (ja) 2022-09-27 2023-10-31 セルソース株式会社 細胞外小胞の製造方法及び細胞外小胞含有組成物
JP7423016B1 (ja) 2022-09-27 2024-01-29 セルソース株式会社 細胞外小胞分泌用培地

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