WO2017175736A1 - Procédé de préparation de vésicules membranaires convenant à l'analyse biophysique - Google Patents

Procédé de préparation de vésicules membranaires convenant à l'analyse biophysique Download PDF

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WO2017175736A1
WO2017175736A1 PCT/JP2017/013999 JP2017013999W WO2017175736A1 WO 2017175736 A1 WO2017175736 A1 WO 2017175736A1 JP 2017013999 W JP2017013999 W JP 2017013999W WO 2017175736 A1 WO2017175736 A1 WO 2017175736A1
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buffer solution
membrane
membrane vesicles
solution containing
membrane vesicle
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PCT/JP2017/013999
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則行 石井
光志 池本
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国立研究開発法人産業技術総合研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor

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  • the present invention relates to a method for preparing membrane vesicles suitable for biophysical analysis.
  • Membrane vesicles secreted from cells such as exosomes contain various biomolecules such as proteins and miRNA on the surface and inside, and are stably present in body fluids such as blood, urine, saliva and the like. It has also been clarified that exosomes play an important role as an intercellular communication tool. For this reason, membrane vesicles such as exosomes are attracting attention as biomarker resources, and active research has been conducted to diagnose diseases such as cancer by analyzing biomolecular information contained in membrane vesicles. (Non-Patent Document 1). In addition, since membrane vesicles are composed of biological materials, they are non-cytotoxic and have a stable structure, so a drug delivery system (DDS) for specifically delivering drugs to target tissues and diseased sites. ) Is also expected.
  • DDS drug delivery system
  • Exosomes isolated and purified by any of the above conventional methods are distributed in a single peak around 100 nm.
  • recent studies have confirmed by electron microscopic analysis that exosomes immediately before or after being secreted from cells have a particle size of about 50 nm (Non-patent Documents 5 and 6). Therefore, this report suggests that the membrane vesicles isolated and purified by the conventional method may not maintain the state immediately after being secreted from the cells, that is, may not be intact.
  • Membrane vesicles have not only properties as biological lipid membranes but also properties as nanocolloid particles having physicochemical properties such as inherent charge and particle size.
  • Conventional research has been aimed mainly at elucidating the constituents of membrane vesicles, and therefore purification and isolation of membrane vesicles has been carried out without considering the characteristics of membrane vesicles as nanocolloid particles. I have been. For this reason, in previous research, it was imagined that useful and enormous physicochemical information possessed by membrane vesicles, which contributes to the search for new highly sensitive disease markers and the development of early disease diagnosis methods, was lost. Not difficult.
  • An object of the present invention is to provide a method for preparing membrane vesicles for separating and purifying intact membrane vesicles maintaining physicochemical properties.
  • the present inventors paid attention to the characteristics of membrane vesicles as nanocolloid particles, and as a result of intensive studies, the separation of intact membrane vesicles by density gradient centrifugation using a buffer solution that does not contain colloidal flocculants. We found for the first time that purification is possible.
  • the present invention provides the following method: [1] (1) a step of ultrafiltration of a sample solution containing membrane vesicles; (2) replacing the sample solution obtained by the step (1) with a buffer solution containing no colloidal flocculant in order to prepare a buffer solution containing membrane vesicles; (3) A method for preparing a membrane vesicle population, comprising the step of separating membrane vesicles contained in the buffer solution obtained by the step (2) by density gradient centrifugation. [2] The method according to [1] above, wherein the sample solution containing the membrane vesicle is a biological fluid sample or a culture solution.
  • the present invention also provides the following membrane vesicle population according to one embodiment: [8] A membrane vesicle population separated and purified from a sample solution containing membrane vesicles, wherein the membrane vesicles contained in the membrane vesicle population are dispersed, and (I) The membrane vesicles contained in the membrane vesicle population maintain the surface charge when present in the sample solution before separation and purification. (Ii) The membrane vesicles contained in the membrane vesicle population maintain the form when they existed in the sample solution before separation and purification, and / or (iii) the membrane vesicle population is separated Maintaining the particle size distribution pattern when present in the sample solution before purification, Membrane vesicle population.
  • the present invention also provides the following composition according to one embodiment: [12] A composition comprising the membrane vesicle population according to any one of [8] to [11] above in a buffer solution containing no colloidal flocculant. [13] The composition according to [12] above, wherein the buffer solution containing no colloidal flocculant is a buffer solution substantially free of salt. [14] The composition described in [12] or [13] above, wherein the buffer solution not containing the colloidal flocculant has an ionic strength of 0 to 135 mM. [15] The composition according to any one of [12] to [14] above, wherein the buffer solution not containing the colloidal flocculant has a buffering action in a pH range of 5.5 to 11.1.
  • the buffer solution containing no colloidal flocculant further contains an amino acid.
  • the method for preparing a membrane vesicle population according to the present invention is superior to the conventional membrane vesicle purification method in the following points and useful.
  • Intact membrane vesicles for example, exosomes
  • the biophysical properties charge, morphology, particle size distribution pattern, etc.
  • the molecular biological and biochemical characteristics of intact membrane vesicles can be analyzed.
  • Membrane vesicles eg, exosomes
  • Membrane vesicles can be separated and purified simply, quickly and inexpensively.
  • Membrane vesicles (for example, exosomes) that can be used as they are for various purposes without additional treatment can be prepared.
  • the separated and purified membrane vesicle population according to the present invention is superior and useful in the following respects compared to the membrane vesicle population prepared by the conventional method.
  • (1) It is intact both in molecular biology, biochemistry and biophysics.
  • (2) Enables integrated profiling analysis of membrane vesicles by combining molecular biological / biochemical indices and biophysical chemical indices.
  • (3) Enables the development of early disease diagnosis methods.
  • It It enables identification and development of highly sensitive novel disease molecular markers.
  • It It enables development of disease treatment methods.
  • (6) Enables the development of improved DDS.
  • composition containing the membrane vesicle population according to the present invention can stably maintain the intact state of the membrane vesicle population over a long period of time.
  • FIG. 1 is a schematic diagram showing an embodiment of the method of the present invention.
  • FIG. 2 shows the results of Dot Blot analysis using antibodies against various exosome molecular markers after separating and purifying membrane vesicles derived from HEK293 cells into 12 fractions by the method of the present invention.
  • FIG. 3 shows the results of Dot Blot analysis using an antibody against CD63, an exosome molecular marker, after separating and purifying membrane vesicles derived from a plurality of cell types into 12 fractions by the method of the present invention.
  • FIG. 4 shows imaging of membrane vesicles obtained as Fraction # 9 and Fraction # 10 after separation and purification of membrane vesicles derived from HEK293 cells into 12 fractions by the method of the present invention under a transmission electron microscope (TEM). The image is shown.
  • FIG. 5 shows membrane vesicles derived from HEK293 cells by the density gradient centrifugation method according to the method of the present invention, and the fractions containing membrane vesicles were measured using the nanoparticle size measurement system DelsaMax CORE. It is a graph which shows the result of having analyzed the diameter and the particle size distribution pattern.
  • FIG. 5 shows membrane vesicles derived from HEK293 cells by the density gradient centrifugation method according to the method of the present invention, and the fractions containing membrane vesicles were measured using the nanoparticle size measurement system DelsaMax CORE. It is a graph which shows the result of having analyzed the diameter and the particle size distribution pattern.
  • FIG. 6 shows the results of immunoelectron microscopic analysis using an anti-CD63 antibody on the fraction containing membrane vesicles after fractionation of membrane vesicles derived from HEK293 cells by density gradient centrifugation using the method of the present invention. It is an image.
  • FIG. 7 shows the result of measuring the membrane vesicle particle size distribution using an electron microscope after fractionating membrane vesicles derived from HEK293 cells by the density gradient centrifugation method according to the method of the present invention. It is a graph which shows.
  • FIG. 8 shows density centrifugation of membrane vesicles derived from HEK293 cells using 25 mM Bis-Tris (pH ⁇ 6.5), 25 mM Bis-Tris (pH 6.5) containing 140 mM NaCl, or PBS (-). It is a graph which shows the result of having analyzed the particle size and particle size distribution pattern of the membrane vesicle at the time of fractionating by the isolation
  • FIG. 9 shows the result of Dot Blot analysis using an anti-CD63 antibody for the fraction obtained by density gradient centrifugation using 25 mM Bis-Tris (pH 6.5) containing / without amino acid.
  • the present invention provides (1) a step of ultrafiltration of a sample solution containing membrane vesicles, and (2) a preparation of a buffer solution containing membrane vesicles, The step of replacing the sample solution obtained by the step 1) with a buffer solution containing no colloidal flocculant, and (3) the density of the membrane vesicles contained in the buffer solution obtained by the step (2)
  • a method of preparing a membrane vesicle population comprising a step of separating by gradient centrifugation.
  • membrane vesicle means a lipid bilayer vesicle secreted from a cell or tissue.
  • the membrane vesicle in the present specification is not limited to the following, but for example, a membrane vesicle having a particle size of about 20 to 200 ⁇ m, which is conventionally called exosome / exosome, or a particle size of about 10 to 1,000 ⁇ m And microvesicles having apoptotic and apoptotic vesicles.
  • the cell or tissue that secretes the membrane vesicle may be from any species.
  • Examples of organisms from which cells or tissues that secrete membrane vesicles are derived include, but are not limited to, microorganisms, protozoa, nematodes, insects, fish, reptiles, amphibians, birds, mammals, plants, and the like.
  • Examples of mammals include primates such as humans, monkeys, orangutans, chimpanzees, rodents such as mice, rats, hamsters, guinea pigs, pigs, cows, goats, horses, sheep, rabbits, dogs, cats, etc. It is done.
  • Examples of cells that secrete membrane vesicles include nerve cells, immune cells, epithelial cells, cardiomyocytes, skeletal muscle cells, connective tissue cells, stem cells, iPS cells, ES cells, tumor cells and other disease cells, primary cultured cells (Derived from various organs), established cultured cells (derived from various organs) and the like.
  • Examples of tissues that secrete membrane vesicles include brain, heart, liver, kidney, pancreas, spleen, stomach, small intestine, and large intestine.
  • sample solution containing membrane vesicles is not particularly limited as long as it is a liquid sample containing membrane vesicles, and may be, for example, a culture solution obtained by culturing cells or tissues. It may also be a biological fluid sample such as blood, serum, plasma, lymph, urine, saliva, breast milk, amniotic fluid, malignant ascites, cerebrospinal fluid, and ventricular fluid.
  • a sample solution containing membrane vesicles is subjected to an ultrafiltration treatment (step (1)).
  • impurities can be removed from the sample solution, and at the same time, the sample solution can be concentrated.
  • Contaminants mean cell debris, lipids, proteins, etc. that can interfere with the analysis of membrane vesicles.
  • impurities are completely removed by this step, some impurities may remain as long as they do not hinder the analysis of the finally obtained membrane vesicles.
  • the ultrafiltration treatment in the present embodiment does not pass through membrane vesicles to be separated and purified, and is smaller in size than the membrane vesicles, such as sugars, proteins, nucleic acids, protein nucleic acid complexes, proteinaceous aggregates, etc. It can carry out using the ultrafiltration membrane which has a molecular weight cut off so that a low molecule may pass.
  • an ultrafiltration membrane having a smaller fractional molecular weight.
  • the separation performance of the ultrafiltration membrane that can be used in this embodiment is preferably a fractional molecular weight of 3 to 100 kDa, and particularly preferably a fractional molecular weight of 10 kDa.
  • a commercially available product can be used. For example, Amicon (trademark) Ultra Centrifugal Filters (NMWL: 10K) (Millipore) can be used.
  • the ultrafiltration treatment can be performed by a known method. Specifically, for example, 5 ⁇ ml of a sample solution containing membrane vesicles can be added to an ultrafiltration filter unit, and ultrafiltration treatment can be performed by centrifuging at 6,000 ⁇ rpm for 30 minutes. And the volume of the sample solution containing membrane vesicles can be concentrated to about 300 ⁇ l.
  • a sample solution it is preferable to perform the pretreatment according to the component contained in a sample solution suitably.
  • the sample solution is a culture solution
  • the sample solution is a biological fluid sample containing a large amount of lipid components such as breast milk
  • the sample solution is blood
  • blood albumin is preferably removed as a pretreatment.
  • those skilled in the art can appropriately select and implement a preferred pretreatment for each component contained in the sample.
  • step (2) the sample solution after the ultrafiltration treatment is replaced with a buffer solution containing no colloidal flocculant, thereby preparing a buffer solution containing membrane vesicles.
  • Membrane vesicles have a specific surface charge (plus or minus charge) depending on the cell or tissue from which they are derived, and therefore have properties as colloidal particles and are dispersed in the sample solution. . Therefore, by using a buffer solution that does not contain a colloidal flocculant, it is possible to prepare a buffer solution containing membrane vesicles in which a dispersed state is maintained.
  • the “colloid flocculant” means a compound that weakens the repulsive force between the membrane vesicles and neutralizes the membrane vesicles by neutralizing the surface charge of the membrane vesicles.
  • the colloid flocculant in this embodiment includes both inorganic flocculants and organic flocculants.
  • Examples of the inorganic flocculant include, but are not limited to, metal salts such as sodium chloride, potassium chloride, magnesium chloride, calcium chloride, and aluminum sulfate; phosphates; onium salts such as ammonium salts, oxonium salts, and sulfonium salts; Examples thereof include salts such as amine salts such as triethanolamine salt and hydroxylamine salt.
  • Examples of organic flocculants include, but are not limited to, synthetic polymer flocculants such as polyamines, polyamides, and acrylamides, and natural polymer flocculants such as chitin / chitosan and cellulose.
  • the “buffer solution containing no colloid flocculant” that can be used in the present embodiment may be any buffer solution that does not contain colloid flocculant or substantially does not contain colloid flocculant.
  • the buffer solution containing no colloidal flocculant in the present embodiment is preferably a buffer solution substantially free of salt.
  • substantially free of the colloidal flocculant means that the concentration of the colloidal flocculant contained in the buffer solution is less than the concentration causing aggregation of membrane vesicles. That is, the buffer solution containing no colloidal flocculant in this embodiment preferably has an ionic strength of 0 to 135 ⁇ mM, more preferably 0 to 65 ⁇ mM, and particularly preferably 0 to 30 ⁇ mM.
  • the buffer solution containing no colloidal flocculant that can be used in the present embodiment preferably has a buffering action in the pH range of 5.5 to 11.1, and more preferably has a buffering action in the pH range of 6 to 8.
  • Such a buffer solution containing no colloidal flocculant is not limited to the following, but, for example, a Good buffer solution such as Bis-Tris buffer solution, MES buffer solution, PIPES buffer solution, etc. can be used, most preferably Bis. -Tris buffer can be used.
  • the Good buffer solution is (1) well dissolved in water and capable of producing a high concentration buffer solution, (2) chemically stable, and highly purified by recrystallization, (3 )
  • the target component is easy to detect because it has no visible or ultraviolet absorption, (4) has little salt effect on biological systems, (5) hardly penetrates biological membranes and cell membranes, (6) It has many advantages such as acid dissociation constant is less affected by concentration, temperature, and ion composition, and (7) low ability to form complex with metal ions (Norman E. Good, G. Douglas Winget, Wilhelmina Winter, Thomas N. Connolly, Seikichi Izawa, and Raizada M. M. Singh, "Hydrogen Ion Buffersyfor Biological Research", Biochemistry, Vol. 5, pp. 467-477, 1966).
  • the membrane vesicles contained in the buffer solution obtained as described above are separated by density gradient centrifugation (step (3)).
  • the density gradient centrifugation can be performed by a known method.
  • the solute used to prepare the density gradient solution is a nonionic substance and may be any one as long as it can hold the membrane vesicle in a dispersed state, and is not limited to the following. Sucrose, glycerol, polysaccharides, Ficoll (GE Healthcare Bioscience), Nycodenz (AXS), OptiPrep (AXS) and the like can be used. Those skilled in the art can appropriately select preferable density gradient conditions and centrifugal conditions according to the solute used.
  • the density gradient can be set to 10 to 60%, and the centrifugation condition can be 21,000 rpm (75,600 ⁇ g) for 5 hours.
  • sucrose when sucrose is used, a sample separated by density gradient centrifugation can be directly used as a sample for analysis of membrane vesicles, which is preferable.
  • FIG. 1 The outline of the method of this embodiment is shown in FIG.
  • the sample solution is replaced with Bis-Tris buffer as a buffer solution containing no colloidal flocculant (FIG. 1 left), and then by sucrose density gradient ultracentrifugation.
  • Membrane vesicles are separated (middle of FIG. 1).
  • the separated membrane vesicle sample can be used as it is as a sample for various analyzes (right in FIG. 1).
  • the above steps can be completed in 2 to 3 days.
  • the “membrane vesicle population” in the present embodiment means a population containing two or more membrane vesicles substantially separated and purified. “Substantially separated and purified” means a state in which impurities are removed to such an extent that the analysis or use of membrane vesicles is not affected.
  • the membrane vesicle population in the present embodiment may include only a single type of membrane vesicle or may include a plurality of types of membrane vesicles.
  • intact state means not only biochemical properties such as constituents and ratios of membrane vesicles such as proteins and lipids, but also forms such as surface charge, size and shape,
  • biophysical properties of membrane vesicles such as particle size distribution pattern, dispersion state, etc. were also maintained as they were in the sample solution as they were released from the cell or tissue. This means that all the characteristics of the membrane vesicles are preserved.
  • the present invention is a membrane vesicle population separated and purified from a sample solution containing membrane vesicles according to the second embodiment, wherein the membrane vesicles contained in the membrane vesicle population are dispersed. And (i) the membrane vesicles contained in the membrane vesicle population maintain the surface charge when present in the sample solution before separation and purification, (ii) the membrane vesicle population The contained membrane vesicles maintain the form as they existed in the sample solution before separation and purification, and / or (iii) the membrane vesicle population is present in the sample solution before separation and purification It is a membrane vesicle population that maintains the particle size distribution pattern.
  • membrane vesicle The “membrane vesicle”, “membrane vesicle population”, and “sample solution” in this embodiment are the same as those defined in the first embodiment.
  • the membrane vesicle population of this embodiment can be prepared by the method of the first embodiment.
  • Dispersed means a state in which a plurality of membrane vesicles contained in a membrane vesicle population are separated without interfering with each other.
  • interference means that biochemical properties and / or biophysical properties of membrane vesicles are irreversibly changed by the interaction between membrane vesicles. Therefore, “separated without interference” means that a plurality of membrane vesicles contained in the membrane vesicle population are not in contact with each other at all or even if they are in contact with each other instantaneously.
  • the membrane vesicle population in this embodiment is preferably completely dispersed.
  • the membrane vesicle population is dispersed as long as it does not interfere with the analysis and use of the finally obtained membrane vesicle population. May contain no membrane vesicles. That is, at least 90% or more of the membrane vesicles included in the membrane vesicle population in this embodiment may be dispersed.
  • “Maintaining the surface charge when present in the sample solution before separation and purification” means a membrane vesicle when secreted into the culture solution or a membrane when secreted into the biological fluid It means that the surface charge is maintained to such an extent that the characteristics of the vesicle as colloidal particles can be maintained.
  • the surface charge can be positive or negative depending on the cell or tissue from which the membrane vesicle is derived.
  • “Maintaining the form when it was present in the sample solution before separation and purification” means that the membrane vesicle at the time of secretion into the culture medium or the membrane vesicle at the time of secretion into the biological fluid This means that the size and shape of the vesicles are maintained.
  • normal membrane vesicles derived from normal cells are considered to be basically spherical, whereas membrane vesicles isolated from abnormal cells such as disease cells may not be spherical. ing.
  • the membrane vesicle population of the present embodiment may include a membrane vesicle having a shape other than a spherical shape.
  • “Maintaining the particle size distribution pattern when present in the sample solution before separation and purification” means that both the range and the ratio of the particle size distribution of the membrane vesicle when present in the sample solution are maintained.
  • the membrane vesicle population of this embodiment preferably contains at least 5%, and particularly preferably contains at least 10% of membrane vesicles having a particle size of 60 nm or less.
  • the membrane vesicle population in this embodiment maintains an intact state both biochemically and physicochemically. Therefore, it is useful for the search for a novel highly sensitive disease marker and the development of an early disease diagnosis method, and for the development of an improved DDS.
  • the present invention is a composition comprising the membrane vesicle population described above in a buffer solution that does not contain a colloidal flocculant.
  • the “colloid flocculant” and “buffer solution not containing colloid flocculant” in the present embodiment are the same as those defined in the first embodiment.
  • the buffer solution not containing the colloidal flocculant preferably further contains an amino acid.
  • an amino acid any natural amino acid can be used as the amino acid in the present embodiment, a hydrophobic amino acid such as phenylalanine or a polar uncharged amino acid such as threonine can be preferably used.
  • the buffer solution containing no colloidal flocculant in the present embodiment may contain one type of amino acid or two or more types of amino acids.
  • composition in the present embodiment is useful because the membrane vesicle population separated and purified from the sample solution can be maintained in an intact state for a long time.
  • a membrane vesicle analysis method comprising a step of analyzing a membrane vesicle obtained by the separation and purification method of the present invention by a physicochemical or biochemical technique. is there.
  • the method for analyzing membrane vesicles obtained by the separation and purification method of the present invention is not particularly limited, and a known physicochemical or biochemical method can be used. Specifically, but not limited to: i) microarray analysis, nucleic acid factors such as miRNA, mRNA, free circulating RNA, Non-coding RNA, Long non-coding RNA, Genomic DNA, next-generation sequencing analysis, PCR Analysis, transcriptome analysis, ii) proteome analysis, proteinome analysis, protein array analysis, antibody array analysis, Western Blot analysis, Dot Blot analysis, mass spectrometry (MALDI-MS), iii) Particle size distribution analysis such as dynamic / static light scattering analysis, nanoparticle tracking (trajectory) analysis, charge distribution analysis such as light scattering electrophoresis analysis, single particle structure analysis, cryo electron microscope analysis, transmission electron microscope analysis, etc. Analysis of biophysical properties of membrane vesicles, including analysis of membrane lipid components such as structure / morphological analysis, mass spect
  • the method according to the present embodiment is useful because the biophysical characteristics of intact membrane vesicles can be analyzed by using the membrane vesicles obtained by the separation and purification method of the present invention.
  • the present invention provides a separation and purification solvent for separating membrane vesicles dispersed in a sample solution, the separation and purification solvent comprising a buffer solution containing no colloidal flocculant. is there.
  • the present invention is a membrane vesicle separation and purification kit comprising a separation and purification solvent comprising a buffer solution that does not contain a colloidal flocculant.
  • the kit can contain known reagents and equipment necessary for separating and purifying membrane vesicles from the sample solution. Examples of such reagents and equipment include ultrafiltration membranes, filtration membranes, culture solutions for culturing cells, culture dishes, centrifuge tubes, reagents for density gradient centrifugation (eg, sucrose), etc. Can be mentioned.
  • the separation and purification solvent in the fifth embodiment and the membrane vesicle separation and purification kit in the sixth embodiment are useful because they can separate and purify intact membrane vesicles.
  • Each of the above cells is suspended in 10 ml of Dulbecco's modified Eagle medium (Sigma-Aldrich) (hereinafter referred to as 10% FCS-DMEM medium) containing 10% fetal calf serum (Gibco), and a 9 cm diameter plastic dish (Greiner Bio-One)
  • 10% FCS-DMEM medium containing 10% fetal calf serum (Gibco)
  • a 9 cm diameter plastic dish (Greiner Bio-One)
  • the cells were cultured in a 37 ° C., 100% humidity, 5% CO 2 atmosphere until the cell density reached about 90%.
  • 10% FCS-DMEM medium was completely removed by aspiration, and then 5 ml of PBS (+) solution was added to wash the cells. This process was repeated 5 times to almost completely remove FCS-derived membrane vesicles.
  • DMEM medium serum-free Dulbecco's modified Eagle's medium
  • Sucrose density gradient ultracentrifugation method Prepare 6 types of sucrose solutions of 10%, 20%, 30%, 40%, 50% or 60% sucrose / 25 mM Bis-Tris (pH 6.5). In a 12.5 ml Open Top ultracentrifuge tube (Beckman Coulter), 2 ml each was gently piled to form a 10-60% sucrose density gradient, and allowed to stand at 4 ° C. for 1 hour or longer. Thereafter, the sample (about 300 ⁇ l) prepared in (2) above was gently layered, and 21,000 rpm (75,600 ⁇ g) using an ultracentrifuge Optima L-100 XP (SW41-Ti rotor) (Beckman Coulter).
  • sucrose density measurement For sucrose density, portable sugar refractometer (high contrast type) No. 1 (analytical sugar degree 0-32%) and No. 2 (analytical sugar degree 28-62%) (Pika Seiko Co., Ltd.) And measured.
  • the nitrocellulose membrane was washed with 50 ml of a washing solution (0.1% Tween20 / PBS (-)) three times, and then reacted with a secondary antibody labeled with Horse Radish Peroxidase (HRP) at room temperature for 2 hours.
  • the nitrocellulose membrane was washed 3 times with 50 ml of washing solution, and then chemiluminescence detection was performed using Clarity (trademark) Western ECL Substrate (Bio Rad). LAS3000 (Fuji Film) was used for detection of the chemiluminescence signal.
  • the types and dilution ratios of the primary antibody and HRP-labeled secondary antibody used in this analysis are as follows. Exosome molecular marker primary antibodies: Mouse anti-CD63 IgG (Santa Cruz) (1: 100 dilution), Mouse anti-CD9 IgG (Santa Cruz) (1: 100 dilution), Mouse anti-TSG101 IgG (Santa Cruz) (1 : 100 dilution).
  • HRP-labeled secondary antibody Rabbit anti-Mouse IgG HRP (Chemicon) (1: 1,500 dilution).
  • the particle size (particle size) measurement and distribution mode of membrane vesicles were analyzed by a nano particle size measurement system DelsaMax CORE (Beckman Coulter).
  • the measurement system is based on two different analysis methods: dynamic light scattering (DLS) and static light scattering (SLS).
  • DLS is generally used as a technique for evaluating the characteristics of nanoparticles present in a liquid phase, and can obtain hydrodynamic characteristic values (variables) such as particle size.
  • the particles dispersed in the solution have a Brownian motion, and the movement is slower for larger particles and faster for smaller particles.
  • exosome positive fraction for exosome positive fraction (CD63 positive fraction), 4 ⁇ l of sample is taken in a disposable cell for measurement, and laser light with a wavelength of 660.9 nm is used at 4 ° C. or 20 ° C. for 5 seconds. Measurement was performed three times.
  • % Intensity Relative ratio of scattering intensity.
  • Each histogram bin (bin, binning) in the particle size histogram is analyzed from the fluctuation of scattered light intensity from a group of particles with different particle size present in the sample, and then analyzed by autocorrelation function. The value (relatively allocated for each particle size bin) was calculated so that the total (frequency) of the scattering intensity was 100%.
  • Electron microscope analysis The shape and particle size of the membrane vesicles were observed with a transmission electron microscope.
  • a transmission electron microscope Tecnai F20 (FEI) was operated at an acceleration voltage of 120 kV, and images were taken at a magnification of 5,000, 11,500, 25,000, and 50,000 times in a low electron dose irradiation mode. Acquisition of image data was performed by DigitalMicrograph (registered trademark) (Gatan) using a slow scan CCD camera (Gatan) mounted on a microscope.
  • the sample of the exosome positive fraction was negatively stained with uranyl acetate (1-2%), and a plurality of electron microscope images were obtained.
  • ImageJ 1.48v NIR
  • the particle size and number of membrane vesicles in the image were measured and the particle size distribution analysis was performed by a method that excluded subjectivity.
  • the frequency distribution calculation uses a function provided in Excel (Microsoft), and the graph is drawn with KaleidaGraph IV 4.5 (HULINKS).
  • FIG. 2 shows the results of Dot Blot analysis using various exosome molecular marker antibodies on the purified fraction derived from HEK293 cells.
  • the results of FIG. 2 showed that the exosomes secreted into the culture medium were purified, and that there were differences in the fraction distribution pattern and expression level for each type of exosome molecular marker.
  • FIG. 3 shows the results of Dot Blot analysis using various anti-CD63 antibodies for purified fractions derived from various cells. From the results shown in FIG. 3, it was revealed that exosomes could be purified from any cell-derived sample and that a unique and characteristic exosome distribution pattern was exhibited depending on the cell type.

Abstract

La présente invention concerne un procédé de préparation d'une population de vésicules membranaires, le procédé comprenant : (1) une étape de traitement par ultrafiltration d'une solution échantillon comprenant des vésicules membranaires ; (2) une étape de substitution de la solution échantillon obtenue à partir de l'étape (1) par une solution tampon sans floculant colloïdal, ce qui permet de préparer une solution tampon comprenant des vésicules membranaires ; et (3) une étape de séparation des vésicules membranaires comprises dans la solution tampon obtenue à partir de l'étape (2) par centrifugation par gradient de densité. L'invention concerne également une population de vésicules membranaires séparée et purifiée d'une solution échantillon, dans laquelle : les vésicules membranaires comprises dans la population de vésicules membranaires sont dispersées ; et (i) les vésicules membranaires comprises dans la population de vésicules membranaires conservent une charge de surface lorsque présentes dans la solution échantillon avant la séparation et la purification, (ii) les vésicules membranaires comprises dans la population de vésicules membranaires conservent leur forme lorsqu'elles sont présentes dans la solution échantillon avant la séparation et la purification, et/ou (iii) la population de vésicules membranaires conserve un profil de distribution granulométrique lorsque présente dans la solution échantillon avant la séparation et la purification.
PCT/JP2017/013999 2016-04-04 2017-04-03 Procédé de préparation de vésicules membranaires convenant à l'analyse biophysique WO2017175736A1 (fr)

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