JP2023150539A - Composition including isolated exosome and method for producing the same - Google Patents

Abstract

To provide a composition including a novel type of functional exosome.SOLUTION: A composition includes an isolated exosome with an electric conductivity of 70-120 mS/cm when measured under the following conditions. Temperature: 25°C. Solvent: 50 mM HEPES (pH 7.0) buffer+NaCl (with a concentration gradient from 0.02M to 2M). Column: polymer anion-exchange column chromatography.SELECTED DRAWING: Figure 1

Description

The present disclosure relates to compositions containing isolated exosomes and methods for producing the same.

In recent years, therapeutic research using exosomes has been gradually conducted. For example, the advantages of exosome therapy over cell therapy include the following: (1) Large amounts of exosomes can be obtained by maintaining and culturing producing cells for a long period of time (manufacturing costs can be suppressed); (2) It is not eliminated by the body's immune system (allogeneic treatment is possible), (3) it is not a cell itself, so there is no risk of abnormal growth (tumorization) within the body, and (4) it is effective even in the environment of the affected area where stem cells have difficulty surviving. (5) Large amounts of exosomes can be administered as they do not clog the capillaries due to intravenous administration (can be treated on an outpatient basis, no need for hospitalization).

Methods for effectively purifying such functional and industrially valuable exosomes include ultracentrifugation and affinity methods, as well as anion exchange chromatography, cation exchange chromatography, and their Combinations and the like have been proposed (Patent Documents 1 to 3).

International Publication No. 2020/027185 International Publication No. 2021/163696 International Publication No. 2020/191369

Elucidation of the functions of exosomes and their utilization have not yet been fully elucidated, and further utilization is expected.
An object of the present disclosure is to provide a composition containing a new type of functional exosome, a method for producing the same, and a use thereof.

The present disclosure is as follows.
[1] A composition comprising isolated exosomes having an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography [2] The composition according to [1] for suppressing fibrosis in at least one selected from the group consisting of tissues and organs.
[3] A composition comprising isolated exosomes having an electrical conductivity of 15 to 55 mS/cm when measured under the following conditions.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography [4] Isolated exosomes with an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions, and isolated exosomes with an electrical conductivity of 15 to 120 mS/cm when measured under the following conditions. 55 mS/cm.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography [5] The isolated exosome according to any one of [1] to [4], wherein at least one surface marker selected from the group consisting of CD9 and CD63 is positive. Composition.
[6] The composition according to any one of [1] to [5], wherein the isolated exosome is derived from mesenchymal stem cells.
[7] A method for producing a composition, comprising:
Including the following step (1),
An isolated exosome in which the composition has an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions, and an isolated exosome whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. The production method, which is a composition comprising at least one isolated exosome selected from the group consisting of:
Step (1) Selected from the group consisting of exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the following conditions and exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. isolating at least one exosome from a liquid sample containing exosomes.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography [8] The production method according to [7], wherein the step (1) is performed by subjecting the liquid sample containing the exosome to anion exchange chromatography. .
[9] The manufacturing method according to [7] or [8], wherein the step (1) further includes at least one step selected from the group consisting of the following steps (a1) to (a3):
Step (a1): performing cation exchange chromatography,
Step (a2): performing ultrafiltration,
Step (a3): A step of performing size exclusion chromatography.
[10] In step (1), a liquid sample containing exosomes is subjected to at least one selected from the group consisting of cation exchange chromatography and ultrafiltration to obtain a mixture, and the mixture is further subjected to anion exchange chromatography. The manufacturing method according to [7], which is carried out by subjecting the method to lithography.
[11] The production method according to any one of [7] to [10], further comprising the following step (2): Step (2): Formulating the isolated exosome as an active ingredient.
[12] The production method according to any one of [7] to [10], wherein the liquid sample containing exosomes is a culture supernatant of mesenchymal stem cells.

According to the present disclosure, a composition containing a new type of functional exosome, a method for producing the same, and a use thereof are provided.

It is a figure showing the measurement results of surface antigen constant concentration in each elution fraction and the absorbance at each wavelength of 260 nm and 280 nm. FIG. 3 is a diagram showing the results of a fibrosis inhibition test for each solution.

<Composition containing isolated exosomes with electrical conductivity of 70 to 120 mS/cm>
One embodiment of the present disclosure is a composition comprising isolated exosomes having an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography

A composition according to the present disclosure (composition 1) includes isolated exosomes having a specific range of electrical conductivity. Therefore, the present composition is expected to exhibit functions based on specific isolated exosomes. To explain this further, it has been found that certain types of exosomes can be identified as isolated exosomes by using an indicator called electrical conductivity. The composition can have functionality based on this identified isolated exosome.

Exosomes in the present disclosure are known as extracellular vesicles and can be understood as cell-derived components containing a lipid bilayer. The diameter of an exosome is, for example, 50 nm to 1000 nm, 50 nm to 300 nm, or 50 nm to 200 nm. The diameter of exosomes can be measured by nanoparticle tracking analysis (NTA), electron microscopy, or the like.
"Isolated exosome" means an exosome that has been separated and purified as an exosome having electrical conductivity within a specific range. The method of separation and purification is not particularly limited, and examples thereof include the method described in the isolation step in the production method described below.

In the present disclosure, methods for measuring the electrical conductivity of isolated exosomes can be performed using devices and methods known to those skilled in the art. Electrical conductivity can be measured, for example, using a device such as AKTA (registered trademark) purifier manufactured by Cytiva.For specific device operations, measurements of UV and electrical conductivity, etc., refer to the manufacturer's manual. This can be done according to the following. Electrical conductivity can also be measured when exosomes are isolated by anion exchange column chromatography, particularly polymer anion exchange column chromatography. Examples of the anion exchange column chromatography, polymer, and polymer include anion exchange column chromatography and polymer anion exchange column chromatography, respectively, which will be described below.

The conditions for measuring electrical conductivity can be as follows.
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography where HEPES stands for 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. Polymer anion exchange column chromatography is column chromatography that uses a polymer such as polyacrylic acid or polymethacrylic acid as a carrier and has an anion exchange group on the surface of the carrier. Examples of the anion exchange group include functional groups such as quaternary ammonium (QA), diethylaminoethyl (DEAE), and ethylenediamine (EDA). As the electrical conductivity measured under these conditions, the electrical conductivity from the start of fractionation to the end of fractionation is employed.

Conditions other than the above-mentioned temperature, solvent, and column for measuring electrical conductivity are not particularly limited, and regarding other specific conditions for measuring electrical conductivity, please refer to <Including isolated exosomes described below. Each description of steps 1 to 4 of step (1) in the section ``Method for producing a composition'' can be cited.

The electrical conductivity of isolated exosomes contained in the composition of this embodiment can be confirmed not only as an electrical conductivity of a specific value but also as an electrical conductivity that has a certain range. Here, "electrical conductivity having a certain range" refers to the electrical conductivity from the start of acquisition (i.e., the beginning of fractionation) to the end of acquisition (i.e., the end of fractionation) in acquiring the elution fraction. . That is, due to the fact that the concentration of NaCl in the solvent (i.e., the eluent) can change depending on the concentration gradient when measured according to the above conditions, and the fact that it takes a certain amount of time to obtain the elution fraction, Electrical conductivity is expressed as a range.
The electrical conductivity when measured under the above conditions may be in the range of 70 to 120 mS/cm, and from the viewpoint of homogeneity, it may be in the range of 75 to 110 mS/cm. It may well be between 80 and 100 mS/cm. The electrical conductivity of such isolated exosomes is not particularly limited as long as it falls within the above range, and is 70 mS/cm or more, 76 mS/cm or more, 77 mS/cm or more, 83 mS/cm or more, 85 mS/cm. It may be 91 mS/cm or more, or 98 mS/cm or more, and 111 mS/cm or less, 105 mS/cm or less, or 104 mS/cm or less. The difference between the electrical conductivity at the beginning of fractionation and the electrical conductivity at the end of fractionation in isolated exosomes is not particularly limited as long as the electrical conductivity is within the above range, and is within 50 mS/cm, It can be within 35 mS/cm, or within 20 mS/cm, and it only needs to be above the detection limit, that is, above 0 mS/cm, and may be 1 mS/cm or more, 5 mS/cm or more, or 10 mS/cm or more. can do.
Isolated exosomes having electrical conductivity in these ranges can be expected to be highly effective as more homogeneous isolated exosomes.

The composition of this embodiment is a composition having a specific function, and may be a composition for suppressing fibrosis of at least one selected from the group consisting of tissues and organs, for example. Fibrosis is a phenomenon that occurs in various tissues and organs, and examples of tissues or organs where fibrosis occurs include the liver, lungs, kidneys, pancreas, heart, and blood vessels (eg, coronary arteries).
In this embodiment, suppressing fibrosis means that when the composition is administered to a subject, no fibrosis occurs at all compared to a control in which the composition is not administered to the subject, or fibrosis does not occur when the composition is administered to a subject. Even if fibrosis occurs in a smaller amount or area than a control, it means that fibrosis occurs only in a smaller amount or area than a control.
The composition of this embodiment may be a pharmaceutical composition, and can be used to treat or prevent fibrosis in at least one selected from the group consisting of tissues and organs.

Suppression of fibrosis can be confirmed by methods known in the art. Examples of a method for confirming such inhibition of fibrosis include a method using measurement of the expression level of actin α2 (ACTA2).

The composition of this embodiment may have other functions than the fibrosis-inhibiting function. Functions other than the fibrosis-inhibiting function include, for example, anti-inflammatory effect, angiogenic effect, anti-aging effect, cell proliferation promoting effect, cell migration promoting effect, and promoting effect on the production of extracellular matrix proteins such as collagen. . The composition of this embodiment may be a pharmaceutical composition that can be used to treat or prevent symptoms or diseases based on functions other than these fibrosis-inhibiting functions.

The isolated exosomes included in the composition of the present embodiment can usually express surface molecules known to be expressed by exosomes. Surface molecules known to be expressed by exosomes and unique to the surface of exosomes include C
D9, CD63, CD81, etc. can be mentioned. The isolated exosome is, for example, positive for at least one surface marker selected from the group consisting of CD9 and CD63. Furthermore, the isolated exosomes contained in the composition of this embodiment may be positive not only for surface markers such as CD9 and CD63, but also for any other surface marker.

The origin of the isolated exosomes contained in the composition of this embodiment is not particularly limited as long as they have a specific electrical conductivity, and examples include mesenchymal stem cells, hematopoietic stem cells, nervous system stem cells, bone marrow stem cells, etc. The cells may be derived from mesenchymal stem cells such as , fat, bone marrow, umbilical cord blood, placenta, etc., or may be adipose-derived stem cells.

In this embodiment, the composition may be a single component containing only the above-mentioned isolated exosomes as its component, or a multi-component composition containing isolated exosomes and any other components. Good too. The form of the composition may be liquid, gel, or solid. Examples of the solid form include a frozen state, a freeze-dried state, and the like.
Here, other optional components may be pharmaceutically acceptable carriers, solvents, additives, etc., such as diluents, excipients, binders, stabilizers, pH adjusters, thickeners, Antioxidants, tonicity agents, buffers, solubilizing agents, suspending agents, preservatives, antifreeze agents, cryoprotectants, lyoprotectants, bacteriostatic agents, and the like can be mentioned.

In this embodiment, the method of administering the composition to a subject, the content of isolated exosomes contained in the composition, the amount of the composition to be administered to a subject, etc. It is not particularly limited as long as it can exhibit a specific function or activity such as suppressive ability, and can be determined as appropriate depending on the type of disease, degree of disease, symptoms, age, body weight, etc. of the subject to be administered.

The dosage form of the composition of this embodiment can be appropriately selected depending on the route of administration and the like. For example, in the case of oral administration, it can be formulated into solid preparations such as tablets, granules, powders, and capsules; liquid preparations such as solutions, syrups, suspensions, and emulsions. In the case of parenteral administration, the composition can be formulated into injections, solutions, suspensions, etc., and the compositions in these dosage forms can be administered intraarterially, intravenously, intrathecally, intrathecally, in the intestine, etc. It can be administered to a subject via the same route of administration.

As long as the composition of this embodiment can exhibit a function or activity such as a fibrosis-inhibiting effect, the content of isolated exosomes in the composition is not particularly limited.
For example, in the case of a solid preparation, when expressed as the number of exosomes per 1 g of the composition, the lower limit is 1.0 x 10 ⁵ or more / g, 1.0 x 10 ⁶ or more / g, or 1.0 x 10 ⁷ /g or more, 1.0×10 ⁸ pieces/g or more, 1.0×10 ⁹ pieces/g or more, 1.0×10 ¹⁰ pieces/g or more, 1.0×10 ¹¹ pieces/g or more and the upper limit is 1.0×10 ⁶ pieces/g or less, 1.0×10 ⁷ pieces/g or less, 1.0×10 ⁸ pieces/g or less, 1.0×10 ⁹ pieces/g or less, The number can be 1.0×10 ¹⁰ pieces/g or less, 1.0×10 ¹¹ pieces/g or less, and 1.0×10 ¹² pieces/g or less. Further, these lower and upper limits may be arbitrarily combined to form a certain range, for example, 1.0×10 ⁵ pieces/g or more and 1.0×10 ¹² pieces/g or less, 1.0× The number may be 10 ⁸ pieces/g or more and 1.0×10 ¹⁰ pieces/g or less. Further, the amount may be 1 ng to 1000 mg, 1 μg to 1000 mg, or 1 mg to 1000 mg per gram of the composition.

In addition, in the case of liquid preparations, when expressed as the number of exosomes per mL of the composition, the lower limit is 1.0 x 10 ⁵ cells/mL or more, 1.0 x 10 ⁶ cells/mL or more, and 1.0 x 10 ⁷ cells/mL. or more, 1.0 x 10 ⁸ pieces/mL or more, 1.0 x 10 ⁹ pieces/mL or more, 1.0 x 10 ¹⁰ pieces/mL or more, 1.0 x 10 ¹¹ pieces/mL or more. , and the upper limit is 1.0 x 10 ⁶ cells/mL or less, 1.0 x 10 ⁷ cells/mL or less, 1.0 x 10 ⁸ cells/mL or less, 1.0 x 10 ⁹ cells/mL or less, 1. It can be set to 0×10 ¹⁰ pieces/mL or less, 1.0×10 ¹¹ pieces/mL or less, and 1.0×10 ¹² pieces/mL or less. Further, these lower and upper limits may be arbitrarily combined to form a certain range, for example, 1.0×10 ⁵ cells/mL or more and 1.0×10 ¹² cells/mL or less, 1.0× It may be 10 ⁸ pieces/mL or more and 1.0×10 ¹⁰ pieces/mL or less. Further, the amount may be 1 ng/mL to 1000 mg/mL, 1 μg/mL to 1000 mg/mL, or 1 mg/mL to 1000 mg/mL per mL of the composition.

The dosage of the composition of this embodiment is not limited as long as it can exhibit the function or activity such as the fibrosis-inhibiting effect, but for example, the dosage per day when converted to the amount of isolated exosomes in a 60 kg human. At ^{a dosage of 8} pieces/g or more, 1.0×10 ⁹ pieces/g or more, 1.0×10 ¹⁰ pieces/g or more, 1.0×10 ¹¹ pieces/g or more, and the upper limit is 1.0 ×10 ⁶ pieces/g or less, 1.0×10 ⁷ pieces/g or less, 1.0×10 ⁸ pieces/g or less, 1.0×10 ⁹ pieces/g or less, 1.0×10 ¹⁰ pieces/g Hereinafter, the number can be set to 1.0×10 ¹¹ pieces/g or less, and 1.0×10 ¹² pieces/g or less. Further, these lower and upper limits may be arbitrarily combined to form a certain range, for example, 1.0×10 ⁵ pieces/g or more and 1.0×10 ¹² pieces/g or less, 1.0× The number may be 10 ⁸ pieces/g or more and 1.0×10 ¹⁰ pieces/g or less. Further, the amount may be 1 ng to 1000 mg, 1 μg to 1000 mg, or 1 mg to 1000 mg per gram of the composition.

In addition, in the case of a liquid drug, the lower limit is 1.0 x 10 ⁵ cells/mL or more, 1.0 x 10 ⁶ cells/mL or more in terms of the daily dose converted to the amount of isolated exosomes for a 60 kg human. 1.0×10 ⁷ cells/mL or more, 1.0×10 ⁸ cells/mL or more, 1.0×10 ⁹ cells/mL or more, 1.0×10 ¹⁰ cells/mL or more, 1.0×10 ¹¹ The upper limit can be 1.0×10 ⁶ cells/mL or less, 1.0×10 ⁷ cells/mL or less, 1.0×10 ⁸ cells/mL or less, 1.0× It can be ¹⁰⁹ pieces/mL or less, 1.0× ¹⁰¹⁰ pieces/mL or less, 1.0× ¹⁰¹¹ pieces/mL or less, and 1.0× ¹⁰¹² pieces/mL or less. Further, these lower and upper limits may be arbitrarily combined to form a certain range, for example, 1.0×10 ⁵ cells/mL or more and 1.0×10 ¹² cells/mL or less, 1.0× It may be 10 ⁸ pieces/mL or more and 1.0×10 ¹⁰ pieces/mL or less. Further, the amount may be 1 ng/mL to 1000 mg/mL, 1 μg/mL to 1000 mg/mL, or 1 mg/mL to 1000 mg/mL per mL of the composition.

When using the composition of this embodiment, the solvent can be replaced as appropriate by a method known to those skilled in the art. For example, when the solvent is replaced with Dulbecco's phosphate buffered saline (DPBS), the temperature: 25 ° C. Solvent: DPBS, Column: Polymer It may be prepared so that the electrical conductivity when measured under the conditions of anion exchange column chromatography is 20 mS/cm or less.

For the method for producing a composition containing isolated exosomes of the present embodiment, the description in the section <Method for producing a composition containing isolated exosomes> below can be referred to.

<Composition containing isolated exosomes with electrical conductivity of 15 to 55 mS/cm>
Another embodiment of the present disclosure is a composition comprising isolated exosomes having an electrical conductivity of 15-55 mS/cm when measured under the conditions described below.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography where HEPES stands for 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. Polymer anion exchange column chromatography is column chromatography that uses a polymer such as polyacrylic acid or polymethacrylic acid as a carrier and has an anion exchange group on the surface of the carrier. Examples of the anion exchange group include functional groups such as quaternary ammonium (QA), diethylaminoethyl (DEAE), and ethylenediamine (EDA). As the electrical conductivity measured under these conditions, the electrical conductivity from the start of fractionation to the end of fractionation is employed.

Another composition according to the present disclosure (Composition 2) includes other isolated exosomes with a specific range of electrical conductivity. Therefore, the present composition is expected to exhibit functions based on specific isolated exosomes. To explain this further, it has been found that certain types of exosomes can be identified as isolated exosomes by using an indicator called electrical conductivity. The present composition (composition 2) containing isolated exosomes with an electrical conductivity of 15 to 55 mS/cm has an electrical conductivity of 70 to 120 mS/cm, as described above, as a function based on this identified isolated exosome. The composition containing isolated exosomes (composition 1) may have the same or different function.

The electrical conductivity of isolated exosomes contained in the composition of this embodiment can be confirmed not only as an electrical conductivity of a specific value but also as an electrical conductivity that has a certain range. Here, "electrical conductivity having a certain range" refers to the electrical conductivity from the start of acquisition (i.e., the beginning of fractionation) to the end of acquisition (i.e., the end of fractionation) in acquiring the elution fraction. . That is, due to the fact that the concentration of NaCl in the solvent (i.e., the eluent) can change depending on the concentration gradient when measured according to the above conditions, and the fact that it takes a certain amount of time to obtain the elution fraction, Electrical conductivity is expressed as a range.
The electrical conductivity when measured under the above conditions may be in the range of 15 to 55 mS/cm, and from the viewpoint of homogeneity, it may be in the range of 25 to 50 mS/cm. Often it may be 35-45 mS/cm. The electrical conductivity of such isolated exosomes is not particularly limited as long as it is within the above range, and is 17 mS/cm or more, 18 mS/cm or more, 24 mS/cm or more, 27 mS/cm or more, 32 mS/cm. It may be 36 mS/cm or more, 39 mS/cm or more, 45 mS/cm or more, or 47 mS/cm or more, and 55 mS/cm or less, or 53 mS/cm or less. The difference between the electrical conductivity at the beginning of fractionation and the electrical conductivity at the end of fractionation in isolated exosomes is not particularly limited as long as the electrical conductivity is within the above range, and is within 40 mS/cm, It may be within 25 mS/cm, or within 10 mS/cm, and may be greater than or equal to the detection limit, that is, greater than 0 mS/cm, and may be greater than or equal to 1 mS/cm, or greater than or equal to 5 mS/cm.
Isolated exosomes having electrical conductivity in these ranges can be expected to be highly effective as more homogeneous isolated exosomes.

The composition of the present embodiment exhibits functions such as anti-inflammatory effect, angiogenic effect, anti-aging effect, cell proliferation promoting effect, cell migration promoting effect, and promoting effect on the production of extracellular matrix proteins such as collagen. You can.

In this embodiment, methods for measuring electrical conductivity, conditions for measuring electrical conductivity, surface markers of isolated exosomes, origins of isolated exosomes, components contained in the composition, dosage form, administration method, and components contained in the composition are described. For each description of the content of isolated exosomes, the amount of administration of the composition to the subject, etc., the description in the section <Composition containing isolated exosomes with electrical conductivity of 70 to 120 mS/cm> may be referred to. can.

<Composition containing isolated exosomes with electrical conductivity of 70 to 120 mS/cm and isolated exosomes with electrical conductivity of 15 to 55 mS/cm>
Other embodiments of the present disclosure provide isolated exosomes with an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions, and an isolated exosome with an electrical conductivity of 15 to 55 mS/cm when measured under the following conditions. A composition comprising isolated exosomes.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography where HEPES stands for 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. Polymer anion exchange column chromatography is column chromatography that uses a polymer such as polyacrylic acid or polymethacrylic acid as a carrier and has an anion exchange group on the surface of the carrier. Examples of the anion exchange group include functional groups such as quaternary ammonium (QA), diethylaminoethyl (DEAE), and ethylenediamine (EDA). As the electrical conductivity measured under these conditions, the electrical conductivity from the start of fractionation to the end of fractionation is employed.

Another composition according to the present disclosure (Composition 3) comprises a plurality of isolated exosomes having a specific range of electrical conductivity. Therefore, the present composition is expected to exhibit multiple functions based on specific isolated exosomes. To explain this further, it has been found that by using an index called electrical conductivity, certain combinations of exosomes can be identified as isolated exosomes. The present composition (composition 3), which includes isolated exosomes having an electrical conductivity of 70 to 120 mS/cm and isolated exosomes having an electrical conductivity of 15 to 55 mS/cm, has the above-mentioned electrical conductivity. It has both the function of a composition (composition 1) containing isolated exosomes with an electrical conductivity of 70 to 120 mS/cm and the function of a composition (composition 2) containing an electrical conductivity of 15 to 55 mS/cm. In addition, composition 3 can have a unique function that composition 1 and composition 2 do not have.

The electrical conductivity of isolated exosomes contained in the composition of this embodiment can be confirmed not only as an electrical conductivity of a specific value but also as an electrical conductivity that has a certain range. Here, "electrical conductivity having a certain range" refers to the electrical conductivity from the start of acquisition (i.e., the beginning of fractionation) to the end of acquisition (i.e., the end of fractionation) in acquiring the elution fraction. . That is, due to the fact that the concentration of NaCl in the solvent (i.e., the eluent) can change depending on the concentration gradient when measured according to the above conditions, and the fact that it takes a certain amount of time to obtain the elution fraction, Electrical conductivity is expressed as a range.

For isolated exosomes with an electrical conductivity of 70 to 120 mS/cm, the electrical conductivity when measured under the conditions described above may be in the range of 70 to 120 mS/cm, depending on the homogeneity. From this point of view, it may be in the range of 75 to 110 mS/cm, and may be 80 to 100 mS/cm. The electrical conductivity of such isolated exosomes is not particularly limited as long as it falls within the above range, and is 70 mS/cm or more, 76 mS/cm or more, 77 mS/cm or more, 83 mS/cm or more, 85 mS/cm. It may be 91 mS/cm or more, or 98 mS/cm or more, and 111 mS/cm or less, 105 mS/cm or less, or 104 mS/cm or less. The difference between the electrical conductivity at the beginning of fractionation and the electrical conductivity at the end of fractionation in isolated exosomes is not particularly limited as long as the electrical conductivity is within the above range, and is within 50 mS/cm, It can be within 35 mS/cm, or within 20 mS/cm, and it only needs to be above the detection limit, that is, above 0 mS/cm, and may be 1 mS/cm or more, 5 mS/cm or more, or 10 mS/cm or more. can do.

For isolated exosomes with an electrical conductivity of 15 to 55 mS/cm, the electrical conductivity when measured under the above conditions may be in the range of 15 to 55 mS/cm, depending on the homogeneity. From this point of view, it may be in the range of 25 to 50 mS/cm, and may be 35 to 45 mS/cm. The electrical conductivity of such isolated exosomes is not particularly limited as long as it is within the above range, and is 17 mS/cm or more, 18 mS/cm or more, 24 mS/cm or more, 27 mS/cm or more, 32 mS/cm. It may be 36 mS/cm or more, 39 mS/cm or more, 45 mS/cm or more, or 47 mS/cm or more, and 55 mS/cm or less, or 53 mS/cm or less. The difference between the electrical conductivity at the beginning of fractionation and the electrical conductivity at the end of fractionation in isolated exosomes is not particularly limited as long as the electrical conductivity is within the above range, and is within 40 mS/cm, It may be within 25 mS/cm, or within 10 mS/cm, and may be greater than or equal to the detection limit, that is, greater than 0 mS/cm, and may be greater than or equal to 1 mS/cm, or greater than or equal to 5 mS/cm.
Isolated exosomes having electrical conductivity in these ranges can be expected to be highly effective as more homogeneous isolated exosomes.

Examples of the composition of this embodiment include the following compositions.
・Isolated exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 25 to 50 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 35 to 45 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 75 to 110 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 75 to 110 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 25 to 50 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 75 to 110 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 35 to 45 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 80 to 100 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 80 to 100 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 25 to 50 mS/cm when measured under the above conditions. A composition comprising;
・Isolated exosomes whose electrical conductivity is 80 to 100 mS/cm when measured under the above conditions, and isolated exosomes whose electrical conductivity is 35 to 45 mS/cm when measured under the above conditions. A composition comprising.

By containing a combination of these two types of isolated exosomes having specific electrical conductivity, the composition of the present embodiment exhibits the functions of each of these two types of isolated exosomes having specific electrical conductivity. It may be a substance that has the effect of mutually enhancing the functions of each isolated exosome, or it may be a substance that produces a new effect by combining the respective isolated exosomes. Good too.

Further, for example, when the electrical conductivity of the object is measured under the above-mentioned conditions, if the electrical conductivity of the object is greater than 55 mS/cm and less than 70 mS/cm, a large amount of nucleic acid as an impurity may be detected in the object. The composition of this embodiment does not contain such impurities, that is, it contains specific exosomes with high purity.

In this embodiment, methods for measuring electrical conductivity, conditions for measuring electrical conductivity, surface markers of isolated exosomes, origins of isolated exosomes, components contained in the composition, dosage form, administration method, and components contained in the composition are described. Each description of the content of isolated exosomes, the amount of administration of the composition to the subject, etc. is as described in the section <Composition containing isolated exosomes with electrical conductivity of 70 to 120 mS/cm> and <The electrical conductivity is The description in the section ``Composition comprising isolated exosomes with a velocity of 15 to 55 mS/cm'' can be referred to.

<Method for producing a composition containing isolated exosomes>
Another embodiment of the present disclosure is a method of manufacturing a composition, the method comprising:
Including the following step (1),
An isolated exosome in which the composition has an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions, and an isolated exosome whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. The production method, which is a composition comprising at least one isolated exosome selected from the group consisting of:
Step (1) Selected from the group consisting of exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the following conditions and exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. isolating at least one exosome from a liquid sample containing exosomes.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography where HEPES stands for 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid. Polymer anion exchange column chromatography is column chromatography that uses a polymer such as polyacrylic acid or polymethacrylic acid as a carrier and has an anion exchange group on the surface of the carrier. Examples of the anion exchange group include functional groups such as quaternary ammonium (QA), diethylaminoethyl (DEAE), and ethylenediamine (EDA). As the electrical conductivity measured under these conditions, the electrical conductivity from the start of fractionation to the end of fractionation is employed.

According to the production method according to the present disclosure, a composition containing one or two specific isolated exosomes according to the present disclosure can be efficiently obtained.

The isolation step of step (1) may be performed by subjecting a liquid sample containing exosomes to ion exchange chromatography, and is not particularly limited as long as specific exosomes can be isolated. 4.

Step 1. Equilibration of Column As a column, for example, an anion exchange column, CIMmultus (registered trademark) EV 1 mL Monolithic Column (2 μm channels) manufactured by BIA Separations, can be used. The isolation step of step (1) may be performed by subjecting a liquid sample containing exosomes to anion exchange chromatography, in which case the column contains quaternary ammonium (QA) as a surface functional group. ), diethylaminoethyl (DEAE), ethylenediamine (EDA), etc. Alternatively, the isolation step in step (1) may be performed by subjecting a liquid sample containing exosomes to cation exchange chromatography, in which case the column contains sulfonic acid as a surface functional group. (-SO ₃ H) or the like. Further, the column may have a monolith structure, and since the monolith structure is an integrated porous body with a characteristic structure in which network-like skeletons are connected, when a monolith column is used, it is usually Compared to particle-packed columns, it is possible to perform chromatography with high flow rates and high resolution, and as a result, it is possible to process large quantities.
An apparatus for performing ion exchange chromatography can be, for example, AKTA (registered trademark) purifier manufactured by Cytiva. Regarding the specific operation of the device and the measurement of UV and electrical conductivity, etc., it can be performed according to the manufacturer's manual.

As the eluent used for ion exchange chromatography, for example, 50 mM HEPES buffer (pH 7.0) (solution A) and 50 mM HEPES buffer (pH 7.0) containing 2M NaCl (solution B) can be used. Alternatively, in the isolation step, KCl may be used instead of NaCl. Alternatively, in the isolation step, the pH of the eluent may be around neutral, and may be about pH 6.8 to 7.2.
Equilibration of the column can be performed by passing 20 mL of a mixture of 99% liquid A and 1% liquid B (NaCl concentration 20 mM) through the column.

Step 2. Adsorption of Exosomes onto a Column Exosomes can be adsorbed onto the column by passing a solution containing exosomes through the equilibrated column at a flow rate of, for example, 5 mL/min.

Step 3. Elution of non-adsorbed components For example, 20 mL of a mixture of 99% solution A and 1% solution B (NaCl concentration 20 mM) is passed through the column that has adsorbed exosomes, and the components not adsorbed to the column can be eluted. . The flow rate at this time may be, for example, 5 mL/min.

Step 4. Elution of exosomes Apply a linear gradient or step gradient eluent (NaCl concentration from 20mM to 2M) from 99% of solution A and 1% of solution B to 0% of solution A and 100% of solution B (NaCl concentration 20mM to 2M) to the column adsorbed with exosomes. 20 mL) to elute the exosomes adsorbed to the column. The flow rate of the eluent containing NaCl may be, for example, 1 mL/min. The temperature of the column, culture supernatant, and mobile phase may be 25°C. The width of the NaCl concentration gradient in the eluent may be narrowed or widened as necessary.

Moreover, the isolation step of step (1) can also be performed on a liquid sample containing exosomes by a method other than ion exchange chromatography. For example, by contacting a solution containing exosomes with a carrier that has a surface that can bind anions through electrical bonding, the exosomes are adsorbed onto the carrier, and then by contacting a solution that separates the exosomes from the carrier. A mode of isolation is possible. The shape of the carrier may be any shape, for example, spherical or flat.
An operation can be performed to efficiently recover a carrier adsorbing exosomes from a solution containing exosomes. Examples of the recovery operation include, for example, when the carrier is spherical, a method of separating the carrier adsorbed with exosomes from a solution containing exosomes using a membrane having pores smaller than the long axis of the carrier. Furthermore, if the carrier is magnetic, it is possible to separate the carrier adsorbed with exosomes from a solution containing exosomes using a magnet.

Step (1) may further include at least one step selected from the group consisting of steps (a1) to (a3) below.
Step (a1): performing cation exchange chromatography,
Step (a2): performing ultrafiltration,
Step (a3): A step of performing size exclusion chromatography.

For the description of cation exchange chromatography in step (a1), the description of step (1) above can be used. The degree of purification of exosomes can be increased by subjecting them to cation exchange chromatography. When anion exchange chromatography is used in combination with cation exchange chromatography, cation exchange chromatography may be performed after anion exchange chromatography, or anion exchange chromatography may be performed after cation exchange chromatography. Good too. Moreover, each means of anion exchange chromatography and cation exchange chromatography may be performed one or more times.

The ultrafiltration in step (a2) involves flowing a liquid in parallel along the surface of the filter. An example of ultrafiltration is tangential flow filtration.
Combining ion exchange chromatography and ultrafiltration is useful not only from the viewpoint of increasing the degree of purification but also from the viewpoint of mass productivity or cost.

In ultrafiltration, the device used, the molecular weight cutoff (NMWC) of the filter used, the flow rate, the replacement solvent, etc. are not particularly limited as long as impurities can be removed from the liquid to be filtered by ultrafiltration.
For example, the KR2i system (manufactured by Repligen) can be used as the device. Further, a tangential flow filter used for tangential flow filtration may have a molecular weight cutoff (NMWC) of, for example, 500 kDa, and a commercially available filter may be used. Further, the flow rate when performing tangential filtration may be, for example, 50 mL/min, and as a replacement solvent for purification, for example, 50 mM Tris buffer (pH 8.0) may be used. The replacement solvent for purification may further contain 0.1 wt% nonionic surfactant and may contain 500 mM NaCl. This purification treatment may be performed one or more times. Examples of the nonionic surfactant include polyoxyethylene-polyoxypropylene copolymer.

Furthermore, nucleic acid decomposition treatment may be performed. Thereby, nucleic acids as impurities in the liquid sample containing exosomes can be removed. The nucleic acid decomposition treatment may be performed before ultrafiltration, after ultrafiltration, or both before and after ultrafiltration. When carrying out the nucleic acid decomposition treatment before ultrafiltration, for example, a nucleic acid degrading enzyme or the like may be added to the solvent and the enzymatic reaction may be carried out for a certain period of time. When the nucleic acid decomposition treatment is performed after ultrafiltration, for example, a nucleic acid degrading enzyme or the like may be added to the filtrate obtained by ultrafiltration, and an enzyme reaction may be performed for a certain period of time. When performing nucleic acid decomposition treatment both before and after ultrafiltration, for example, a nucleic acid decomposition enzyme is added to the solvent, the enzyme reaction is carried out for a certain period of time, and the filtrate obtained by ultrafiltration is subjected to nucleic acid decomposition treatment. An enzyme or the like may be added to carry out an enzymatic reaction for a certain period of time. During the nucleic acid decomposition treatment, the nuclease may be activated using a nuclease activator together with the nuclease. After the nucleic acid decomposition treatment, the nuclease-containing solvent may be replaced with a nuclease-free solvent.
When anion exchange chromatography is used in combination with ultrafiltration, anion exchange chromatography may be followed by ultrafiltration, or ultrafiltration may be followed by anion exchange chromatography. Moreover, each means of anion exchange chromatography and ultrafiltration may be performed one or more times.

As mentioned above, the nucleic acid degradation treatment may be performed before ultrafiltration, after ultrafiltration, or both before and after ultrafiltration. Therefore, when anion exchange chromatography is used in combination with ultrafiltration and nucleic acid decomposition treatment, it can be performed, for example, in the following order.
・Nucleic acid degradation treatment, ultrafiltration, anion exchange chromatography in order;
・Nucleic acid decomposition treatment, anion exchange chromatography, ultrafiltration in order;
・The order of ultrafiltration, nucleic acid decomposition treatment, and anion exchange chromatography;
・The order of ultrafiltration, anion exchange chromatography, and nucleic acid decomposition treatment;
・Anion exchange chromatography, nucleic acid decomposition treatment, ultrafiltration in order;
・The order of anion exchange chromatography, ultrafiltration, and nucleic acid decomposition treatment;
・Nucleic acid decomposition treatment, ultrafiltration, nucleic acid decomposition treatment, anion exchange chromatography in order;
・Nucleic acid decomposition treatment, ultrafiltration, anion exchange chromatography, and nucleic acid decomposition treatment;
・Nucleic acid decomposition treatment, anion exchange chromatography, ultrafiltration, and nucleic acid decomposition treatment;
・Nucleic acid decomposition treatment, anion exchange chromatography, nucleic acid decomposition treatment, ultrafiltration in order;
・The order of ultrafiltration, nucleic acid decomposition treatment, anion exchange chromatography, and nucleic acid decomposition treatment;
・Anion exchange chromatography, nucleic acid decomposition treatment, ultrafiltration, and nucleic acid decomposition treatment in this order.
Furthermore, in the method for producing exosomes of the present disclosure, each of anion exchange chromatography, ultrafiltration, and nucleic acid decomposition treatment may be used one or more times.

Size exclusion chromatography in step (a3) is a method of separating molecules based on differences in molecular size. Size exclusion chromatography is not particularly limited as long as it improves the degree of purification, and can be performed by methods known to those skilled in the art, such as gel filtration chromatography, gel permeation chromatography, etc. It may be gel filtration chromatography.
When anion exchange chromatography is used in combination with size exclusion chromatography, the anion exchange chromatography may be followed by the size exclusion chromatography, or the size exclusion chromatography may be followed by the anion exchange chromatography. Moreover, each chromatography of anion exchange chromatography and size exclusion chromatography may be performed one or more times.

In the manufacturing method of the present disclosure, anion exchange chromatography can be used in combination with at least one selected from the group consisting of cation exchange chromatography, ultrafiltration, and size exclusion chromatography. Therefore, in this embodiment, for example, a combination of the following methods can be used.
- Combination of anion exchange chromatography and cation exchange chromatography; - Combination of anion exchange chromatography and ultrafiltration;
- Combination of anion exchange chromatography and size exclusion chromatography;
- Combination of anion exchange chromatography, cation exchange chromatography, and ultrafiltration;
- combination of anion exchange chromatography, cation exchange chromatography, and size exclusion chromatography;
- combination of anion exchange chromatography, ultrafiltration, and size exclusion chromatography;
- Combination of anion exchange chromatography, cation exchange chromatography, ultrafiltration, and size exclusion chromatography.
Furthermore, in the combination using ultrafiltration, nucleic acid decomposition treatment can be further combined as described above.

In this embodiment, the particle concentration in the liquid can be measured in each step by a method known to those skilled in the art, for example, using the nanoparticle tracking device Zeta View (manufactured by Particle Metrics) and using the EV measurement method included in the software. It can be measured using a 488 nm laser.

Step (1) involves subjecting a liquid sample containing exosomes to at least one selected from the group consisting of cation exchange chromatography and tangential flow filtration to obtain a mixture, and further subjecting the mixture to anion exchange chromatography. It may also be carried out by subjecting it to. In this way, by using a combination of at least one selected from the group consisting of cation exchange chromatography and tangential flow filtration and anion exchange chromatography, exosomes with an even higher degree of purification can be obtained. be able to. Exosomes with a further improved degree of purification are suitable as raw materials for medical preparations.

Furthermore, it may include step (2): a step of formulating the isolated exosome as an active ingredient. The formulation method is not particularly limited, and can be carried out by methods known to those skilled in the art.
For example, it can be formulated to contain the ingredients described in the above section <Composition containing isolated exosomes with electrical conductivity of 70 to 120 mS/cm>, and the dosage form and content are also as described above. It is as follows.

The liquid sample containing exosomes may be, for example, a biological sample such as blood or tissue fluid, or a culture supernatant of cells that produce exosomes. The culture supernatant of cells that produce exosomes may be the culture supernatant obtained when culturing cells from which exosomes are derived, and may be the culture supernatant of mesenchymal stem cells, or the culture supernatant of adipose-derived hepatocytes. It may be a culture supernatant.

A liquid sample containing exosomes may be pretreated to increase separation and purification efficiency. Examples of the pretreatment include centrifugation, filter filtration, a combination of centrifugation and filter filtration, and the like. The conditions for centrifugation are not particularly limited as long as impurities can be removed from the liquid sample, and the centrifugal acceleration, temperature, and centrifugation time can be determined as appropriate. For example, centrifugation may be performed at 1500G and 4°C for 10 minutes, or centrifugation may be performed at 10000G and 4°C for 30 minutes. Further, filtration may be performed using a 0.22 μm filter or the like. Centrifugation and filter filtration may be performed one or more times each, or centrifugation and filter filtration may be performed in combination.

<Exosome isolation method>
Another embodiment of the present disclosure is a method of isolating exosomes, the method comprising:
At least one selected from the group consisting of exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the following conditions and exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. A method for isolating exosomes from a liquid sample containing exosomes.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography

According to the isolation method according to the present disclosure, one or two specific exosomes according to the present disclosure can be efficiently isolated.
In this embodiment, each description in <Method for producing a composition containing isolated exosomes> can be cited.

In this specification, a numerical range indicated using "~" indicates a range that includes the numerical values written before and after "~" as the minimum and maximum values, respectively.

The present disclosure will be described in more detail with reference to Examples below, but the present disclosure is not limited to these Examples.

<Example 1: Preparation of culture supernatant>
Adipose-derived stem cells (manufactured by Lonza) were suspended in PRIME-XV MDF-1 medium (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), and the resulting cell suspension was placed in CellBIND surface-treated T175 flasks (manufactured by Corning) per flask. After adding 2.0×10 ⁵ cells, the cells were cultured at 37° C. and 5% CO ₂ for 72 hours. After culturing, the culture supernatant was removed and fibroblast growth factor-2 (FGF-2; manufactured by R&D Technology) and insulin-transferrin-selenium solution (ITS) were added to final concentrations of 10 ng/mL and 1%, respectively. 25 mL of DMEM/F12 medium (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.) to which a solution (manufactured by Ventria Biosciences) was added was added, and cultured at 37° C. and 5% CO ₂ for 48 hours. After culturing, the collected culture supernatant was centrifuged at 1500 G and 4° C. for 10 minutes, and then the supernatant was filtered through a 0.22 μm filter (manufactured by Merck) to obtain a culture supernatant.

<Example 2: Purification of culture supernatant by tangential flow filtration (TFF)>
An ultrafiltration filter (D02-E500-05N) was attached to the KR2i system (manufactured by Repligen), and filtration was performed while circulating 200 mL of the culture supernatant obtained in Example 1 at a flow rate of 50 mL/min. Concentrated. 15 volumes of 50 mL of the obtained concentrated solution were replaced with 50 mM HEPES buffer (pH 7.0) containing 20 mM NaCl to obtain a purified solution.

<Example 3: Measurement of particle concentration>
The particle concentration of the purified liquid prepared in Example 2 was measured using a nanoparticle tracking device Zeta View (manufactured by Particle Metrics) using a 488 nm laser and an EV measurement method attached to the software. As a result of measurement under conditions where Sensitivity was set to 82 and Shutter was set to 100, the particle concentration in the purified liquid was 5.8 x 10 ⁹ particles/mL.

<Example 4: Purification of exosomes by column>
(1) Column equilibration The column was an anion exchange column, CIMmultus (registered trademark) EV 1 mL Monolithic Column (2 μm) manufactured by BIA Separations.
channels) was used. Using AKTA (registered trademark) purifier manufactured by Cytiva, absorbance at 260 nm (UV 260 nm), absorbance at 280 nm (UV 280 nm), and electrical conductivity were measured according to the manufacturer's manual. As eluents, 50 mM HEPES buffer (pH 7.0) (solution A) and 50 mM HEPES buffer (pH 7.0) containing 2M NaCl (solution B) were prepared and used. 20 mL of a mixture of 99% liquid A and 1% liquid B (NaCl concentration 20 mM) was passed through the column to equilibrate the column.
(2) Adsorption of exosomes to the column 13.5 mL of the purified solution prepared in Example 2 (5.8 × 10 ⁹ cells/mL) was applied to the column equilibrated in (1) above, and the culture supernatant was The contained exosomes were adsorbed onto the column. The flow rate of the culture supernatant during adsorption onto the column was 5 mL/min.

(3) Elution of non-adsorbed components 20 mL of a mixed solution (NaCl concentration 20 mM) of 99% of liquid A and 1% of liquid B was passed through the column to which the sample had been adsorbed, and the components not adsorbed to the column were eluted. The flow rate during elution was 5 mL/min.
(4) Elution of exosomes Apply a linear gradient of eluent (20 mL) from 99% of solution A and 1% of solution B to 0% of solution A and 100% of solution B (NaCl concentration 20mM to 2M) to the column adsorbed with exosomes. ) was passed through the column, and the exosomes adsorbed to the column were eluted. The elution fraction was collected in 1 mL portions. The flow rate of the buffer containing NaCl was 1 mL/min. Note that the temperature of the column, culture supernatant, and mobile phase was 25°C.

<Example 5: Quantification of surface antigen of collected fraction>
The surface antigen concentration in each elution fraction obtained in Example 4 (4) was
Perform enzyme-linked immunosorbent assay (ELISA) using Exosome ELISA Kit, Human (manufactured by Cosmo Bio), CD9/CD9 Exosome ELISA Kit, CD63/CD63 Exosome ELISA Kit (manufactured by Hakarel) according to the manual provided by the manufacturer. It was quantified by Each elution fraction was diluted with the assay buffer attached to each ELISA kit at the dilution rate shown in the table below, and the prepared diluted fractions were subjected to measurement. The measurement results of each ELISA were multiplied by the dilution rate to determine the concentration of each surface antigen. In each ELISA, the fraction showing the maximum value was set as 1, and the relative value of each fraction was calculated.

The above results are shown in Tables 1 to 3 below. In addition, the ultraviolet absorption spectrum (UV) and electrical conductivity of each elution fraction detected by high-performance protein liquid chromatography (FPLC) using AKTA (registered trademark) purifier (manufactured by Cytiva), and the surface antigen measured by ELISA. The relative values (also referred to as relative ratios) of are shown in FIG. UV showed absorbance at 260 nm (UV260 nm) and absorbance at 280 nm (UV280 nm). In addition, the relative value of surface antigen indicates the relative value when the amount of surface antigen (CD9/CD63, CD9/CD9, CD63/CD63) in each elution fraction is set to 1 in the elution fraction that showed the maximum value. Ta. The bar graph representing the relative values of surface antigens represents the measurement results of CD9/CD63, CD9/CD9, and CD63/CD63 from the left for each fraction.

The electrical conductivity from the start of fractionation to the end of fractionation was defined as the electrical conductivity of this exosome. It was shown that there are two peaks in the expression levels of CD9 and CD63, which are surface markers expressed on exosomes, near fraction 4 and near fraction 11. Therefore, fractions 3, 4, 5, 10, 11, and 12, which are fractions near the peak, were used in the following examples. Note that n.d. indicates that it could not be detected (the same applies below).

<Example 6: Measurement of particle concentration of purified fraction>
Among the fractions collected in Example 4, the particle concentrations in each of fractions 3, 4, 5, 10, 11, and 12, which had high expression levels of CD9 and CD63, were measured according to Example 3. The results are shown in Table 4.

<Example 7: Preparation of exosome sample for fibrosis inhibition test>
An ultrafiltration filter (D02-E500-05N) was attached to the KR2i system (manufactured by Repligen), and filtration was performed while circulating 200 mL of the culture supernatant obtained in Example 1 at a flow rate of 50 mL/min. Concentrated. 50 mL of the obtained concentrate was mixed with 6 volumes of 50 mM Tris buffer (pH 8.0) containing 500 mM NaCl and containing 0.1 wt% nonionic surfactant (polyoxyethylene-polyoxypropylene copolymer). Replacement was made. To the obtained purified solution, add one vial each of a nucleolytic enzyme (Kryptonase) attached to the BIAseparations CORNERSTONE Exosome Process Development Pack and an activator for the nucleolytic enzyme, and filter the resulting mixture. It was circulated for 2 hours using the KR2i system. Thereafter, 12 volumes of 50mM HEPES buffer (pH 7.0) containing 20mM NaCl containing 0.1wt% nonionic surfactant (polyoxyethylene-polyoxypropylene copolymer) were substituted to remove the purified solution. (5.4×10 ⁹ cells/mL) was obtained. 45.9 mL of the obtained purified liquid was subjected to the method of Example 4 to elute exosomes to obtain a fraction containing exosomes. The electrical conductivity of each obtained fraction was measured using this system, and the particle concentration in each fraction was also measured by the method of Example 3. Table 5 shows the electrical conductivity and particle concentration of each fraction from the beginning of the fraction to the end of the fraction.

<Example 8: Preparation of Mag Exo solution>
The culture supernatant (20 mL) prepared in Example 1 was added to a centrifugal filtration tube (manufactured by Sartorius) with a membrane molecular weight cutoff (MWCO) of 100 kDa, and concentrated at 3000 G and 4 ° C. until it became 1 mL or less. . From the obtained concentrate, Mag Capture
(Registered Trademark) Using Exosome Isolation Kit PS (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a purified exosome solution was prepared according to the protocol attached to the kit, and was designated as "Mag Exo solution".

<Example 9: Fibrosis inhibition test>
(1) Concentration of sample and substitution with PBS buffer From the fractions obtained in Example 7, exosome solution A (electrical conductivity: 18-53 mS/cm; fractions 1-4), exosome solution B (electrical conductivity: 53 ~76 mS/cm; fractions 5 to 7), exosome solution C (electrical conductivity: 83 to 104 mS/cm; fractions 9 to 11), and Mag Exo solution, respectively, were filtered through centrifugal filters (manufactured by Merck). ) for 2 minutes at 14,000 G and 25°C. After concentration, Dulbecco's phosphate buffered saline (DPBS) (manufactured by Gibco) was added to the resulting concentrated solution, and the DPBS of each exosome was A solution (500 μL) was prepared. When adding the exosome solution to the fibroblast wells described below, the concentration operation by centrifugation and the addition of DPBS were repeated so that the NaCl concentration between the wells was approximately the same. By repeating this operation, the electrical conductivity of each exosome solution was kept below the electrical conductivity of DPBS (20 mS/cm), and each exosome solution was finally concentrated to 100 μL or less.

(2) Conducting fibrosis inhibition test Lung-derived fibroblasts (manufactured by Promocell) were mixed with fetal bovine serum (FBS) (manufactured by Gibco) and gentamicin sulfate at final concentrations of 10% and 0.1%, respectively. A cell suspension was prepared by suspending the cells in DMEM (manufactured by Gibco) to which an amphotericin solution (manufactured by Lonza) was added. Next, the obtained cell suspension (500 μL) was added to each well of a 24-well plate at 4000 lung-derived fibroblasts per well, and incubated at 37° C. and 5% CO ₂ for 24 hours. Cultured. After culturing, after removing the culture supernatant, add DMEM medium to which gentamicin sulfate-amphotericin solution (manufactured by Lonza) was added so that the final concentration was 0.1%, and the final concentration was 0.1% and 10 ng, respectively. Add 500 μL of each DMEM medium supplemented with gentamicin sulfate-amphotericin solution and transforming growth factor-β (TGF-β; manufactured by Peprotech) so that the amount of the solution was 500 μL, and incubate at 37° C. with 5% CO ₂ . The cells were cultured for 24 hours. After culturing, exosome solution A (final concentration 2.0 × 10 ⁹ cells/mL; DPBS solution) and exosome solution B (final concentration 1.0 × 10 ⁹ cells/mL; DPBS solution) were added to the well containing TGF-β. solution), exosome solution C (final concentration 1.0 × 10 ⁹ cells/mL; DPBS solution), and Mag Exo solution (final concentration 1.0 × 10 ⁹ cells/mL; DPBS solution) were added, and 5% The cells were cultured for 48 hours at 37° C. and CO ₂ . After culturing, the culture supernatant was removed, and then washed with 500 μL of DPBS. After washing, detach the cells with 200 μL of Accutase (manufactured by Nacalai Tesque), add 400 μL of DPBS, collect the cells in a 1.5 mL tube (manufactured by Sumitomo Bakelite), and centrifuge at 300 G and 25° C. for 3 minutes. and the supernatant was removed.

RNA was extracted from the collected cells using PureLink (registered trademark) RNA Mini kit (manufactured by Thermo Fisher Scientific) according to the protocol of the kit. A cDNA solution was prepared for the RNA using a High Capacity cDNA Reverse Transcription Kit (manufactured by Thermo Fisher Scientific) according to the protocol of the kit. Quantitative PCR was performed on the cDNA using TaqMan Fast Advanced Master Mix (manufactured by Thermo Fisher Scientific) according to the protocol of the reagent.

As a probe for quantitative PCR, TaqMan Gene Expression Assay (FAM) actin α2 probe (ACTA2: Hs00426835_g1) or glyceraldehyde-3-phosphate dehydrogenase probe (GAPDH: Hs02786624_g1) was used. The expression level of ACTA2 and the expression level of GAPDH, which is an endogenous control, were determined by the ΔΔCt method. The relative expression level of ACTA2 to the GAPDH expression level was calculated. The relative expression level of ACTA2 in each sample was calculated assuming that the relative expression level of ACTA2 in cells without TGF-β addition was 1.0, and the results are shown in FIG.

In Figure 2, TGF- indicates the relative expression level of ACTA2 in cells without the addition of TGF-β, TGF+ indicates the relative expression level of ACTA2 in the case without the addition of exosomes, and exosome solution A has an electrical conductivity of 18 to The relative expression level of ACTA2 is shown when a DPBS solution of exosomes with an electrical conductivity of 53 mS/cm is added to cells. Exosome solution C shows the relative expression amount of ACTA2 when a DPBS solution of exosomes with an electrical conductivity of 83 to 104 mS/cm is added to cells, and the Exo Mag solution shows the relative expression amount of ACTA2 in Example 8. The relative expression level of ACTA2 is shown when the prepared exosome solution (“Exo Mag solution”) is added to cells. * indicates P<0.01.

There was a significant difference in the relative expression level of ACTA2 when exosome solution C was added compared to the relative expression level of ACTA2 when exosomes were not added (i.e., the relative expression level of ACTA2 with TGF+) (P<0.01 ), exosome solution C was found to exhibit fibrosis-inhibiting effects.
On the other hand, no difference was observed in the relative expression level of ACTA2 when exosome solution A and exosome solution B were added compared to the relative expression level of ACTA2 when exosomes were not added. Furthermore, Exosome Solution C exhibited a stronger fibrosis-inhibiting effect compared to Mag Exo Solution under the same exosome concentration conditions (Figure 2).
From this, it can be seen that the composition containing the isolated exosomes having an electrical conductivity of 83 to 104 mS/cm obtained in Example 7 has a fibrosis-inhibiting effect, and therefore, exosomes having a strong fibrosis-inhibiting effect It turns out that it is possible to produce a solution.

Claims (12)

A composition comprising isolated exosomes having an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography The composition according to claim 1, for inhibiting fibrosis in at least one selected from the group consisting of tissues and organs. A composition comprising isolated exosomes having an electrical conductivity of 15 to 55 mS/cm when measured under the following conditions.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography A composition comprising isolated exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the following conditions, and isolated exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. thing.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography The composition according to any one of claims 1 to 4, wherein the isolated exosome is positive for at least one surface marker selected from the group consisting of CD9 and CD63. The composition according to any one of claims 1 to 5, wherein the isolated exosomes are derived from mesenchymal stem cells. A method for producing a composition, the method comprising:
Including the following step (1),
An isolated exosome in which the composition has an electrical conductivity of 70 to 120 mS/cm when measured under the following conditions, and an isolated exosome whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. The production method, which is a composition comprising at least one isolated exosome selected from the group consisting of:
Step (1) Selected from the group consisting of exosomes whose electrical conductivity is 70 to 120 mS/cm when measured under the following conditions and exosomes whose electrical conductivity is 15 to 55 mS/cm when measured under the following conditions. isolating at least one exosome from a liquid sample containing exosomes.
conditions:
Temperature: 25℃
Solvent: 50mM HEPES (pH 7.0) buffer + NaCl (concentration gradient from 0.02M to 2M)
Column: Polymer anion exchange column chromatography The manufacturing method according to claim 7, wherein the step (1) is performed by subjecting the liquid sample containing the exosome to anion exchange chromatography. The manufacturing method according to claim 7 or 8, wherein the step (1) further includes at least one step selected from the group consisting of the following steps (a1) to (a3):
Step (a1): performing cation exchange chromatography,
Step (a2): performing ultrafiltration,
Step (a3): A step of performing size exclusion chromatography. In step (1), a liquid sample containing exosomes is subjected to at least one selected from the group consisting of cation exchange chromatography and ultrafiltration to obtain a mixture, and the mixture is further subjected to anion exchange chromatography. The manufacturing method according to claim 7, which is carried out by. The manufacturing method according to any one of claims 7 to 10, further comprising the following step (2):
Step (2): A step of formulating the isolated exosome as an active ingredient. The manufacturing method according to any one of claims 7 to 11, wherein the liquid sample containing exosomes is a culture supernatant of mesenchymal stem cells.