Reagent, drug source material, drug, quasi-drug, cosmetic source material, or cosmetic

Abstract

A reagent, a drug source material, a drug, a quasi-drug, a cosmetic source material, or a cosmetic that includes a supernatant of a culture solution of stem cells and is for reducing skin redness or inflammation, inflammation, number of pores, pore size, skin spots, and sebum. A reagent, a drug source material, a drug, a quasi-drug, a cosmetic source material, or a cosmetic that includes a secretion of stem cells and is for reducing skin redness or inflammation, inflammation, number of pores, pore size, skin spots, and sebum. The secretion of the stem cells may be extracellular vesicles. The secretion of the stem cells may be exosomes.

Description

Reagent, pharmaceutical raw material, pharmaceutical product, quasi-drug, cosmetic raw material, or cosmetic

[0001] The present invention relates to a reagent, a pharmaceutical raw material, a pharmaceutical product, a quasi-drug, a cosmetic raw material, or a cosmetic.

[0002] Embryonic stem cells (ES cells) are stem cells established from early embryos of humans and mice. ES cells have pluripotency to differentiate into all cells existing in the living body. Currently, human ES cells can be used for cell transplantation therapy for many diseases such as Parkinson's disease, juvenile diabetes, and leukemia. However, there are also obstacles to the transplantation of ES cells. In particular, the transplantation of ES cells can cause an immune rejection reaction similar to the rejection reaction that occurs following unsuccessful organ transplantation. In addition, there are many critical and opposing opinions from an ethical perspective regarding the use of ES cells established by destroying human embryos.

[0003] Under such background circumstances, Professor Shinya Yamanaka of Kyoto University succeeded in establishing induced pluripotent stem cells (iPS cells) by introducing four genes: Oct3 / 4, Klf4, c-Myc, and Sox2 into somatic cells. As a result, Professor Yamanaka received the Nobel Prize in Physiology or Medicine in 2012 (see, for example, Patent Document 1). iPS cells are ideal pluripotent cells without rejection reactions or ethical problems. Therefore, iPS cells are expected to be used for cell transplantation therapy. On the other hand, there is a report that the medium used for culturing iPS cells was reused in a pharmaceutical composition (see, for example, Patent Document 2). However, as verified by the present inventors, in the culturing method described in Patent Document 2, iPS cells differentiate, so it is considered that, in reality, the medium in which differentiated cells rather than iPS cells were cultured was reused (see, for example, Patent Documents 3 and 4).

[0004] Japanese Patent No. 4183742, Japanese Patent Application Laid-Open No. 2016-128396, Japanese Patent No. 6647545, Japanese Patent No. 7046421

[0005] One object of the present invention is to provide a reagent, a pharmaceutical raw material, a pharmaceutical product, a quasi-drug, a cosmetic raw material, or a cosmetic that effectively utilizes components of stem cells such as iPS cells and a culture solution of stem cells.

[0006] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation may further include an extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation may be administered repeatedly. The pharmaceuticals for reducing skin redness or inflammation may be used by subcutaneous injection.

[0007] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation may further include an extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation may be administered repeatedly. The pharmaceuticals for reducing inflammation may be used by subcutaneous injection.

[0008] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores may be administered repeatedly. The pharmaceuticals for reducing the number of pores may be used by subcutaneous injection.

[0009] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size may be administered repeatedly. The pharmaceuticals for reducing pore size may be used by subcutaneous injection.

[0010] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes may further include an extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes may be administered repeatedly. The pharmaceuticals for reducing skin blemishes may be used by subcutaneous injection.

[0011] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum according to the embodiment include the supernatant of a stem cell culture medium. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum may further include an extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum may be administered repeatedly. The pharmaceuticals for reducing sebum may be used by subcutaneous injection.

[0012] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium in which stem cells were maintained in an undifferentiated state.

[0013] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when stem cells are cultured in two dimensions.

[0014] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when stem cells are cultured in three dimensions.

[0015] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be pluripotent stem cells.

[0016] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0017] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be derived from the subject to which the composition is provided.

[0018] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation may be administered repeatedly. The pharmaceuticals for reducing skin redness or inflammation may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0019] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation may be administered repeatedly. The pharmaceuticals for reducing inflammation may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0020] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores may be administered repeatedly. The pharmaceuticals for reducing the number of pores may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0021] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size may be administered repeatedly. The pharmaceuticals for reducing pore size may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0022] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes may be administered repeatedly. The pharmaceuticals for reducing skin blemishes may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0023] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum according to the embodiment include stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum may further include extracellular matrix. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum may be administered repeatedly. The pharmaceuticals for reducing sebum may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0024] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be pluripotent stem cells.

[0025] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0026] In the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the above embodiment, the stem cells may be derived from the subject to which the composition is provided.

[0027] A therapeutic method for reducing skin redness or inflammation according to an embodiment includes administering a pharmaceutical product for reducing skin redness or inflammation, which contains the supernatant of a stem cell culture medium, to the target. The pharmaceutical product for reducing skin redness or inflammation may further contain an extracellular matrix. The pharmaceutical product for reducing skin redness or inflammation may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing skin redness or inflammation according to an embodiment includes administering a cosmetic product for reducing skin redness or inflammation, which contains the supernatant of a stem cell culture medium, to the target. The cosmetic product for reducing skin redness or inflammation may further contain an extracellular matrix. The cosmetic product for reducing skin redness or inflammation may be administered repeatedly.

[0028] A therapeutic method for reducing inflammation according to an embodiment includes administering a pharmaceutical product for reducing inflammation, which contains the supernatant of a stem cell culture medium, to the target. The pharmaceutical product for reducing inflammation may further contain an extracellular matrix. The pharmaceutical product for reducing inflammation may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing inflammation according to an embodiment includes administering a cosmetic product for reducing inflammation, which contains the supernatant of a stem cell culture medium, to the target. The cosmetic product for reducing inflammation may further contain an extracellular matrix. The cosmetic product for reducing inflammation may be administered repeatedly.

[0029] A therapeutic method for reducing the number of pores according to an embodiment includes administering a pharmaceutical product for reducing the number of pores, which contains the supernatant of a stem cell culture medium, to the target. The pharmaceutical product for reducing the number of pores may further contain extracellular matrix. The pharmaceutical product for reducing the number of pores may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing the number of pores according to an embodiment includes administering a cosmetic product for reducing the number of pores, which contains the supernatant of a stem cell culture medium, to the target. The cosmetic product for reducing the number of pores may further contain extracellular matrix. The cosmetic product for reducing the number of pores may be administered repeatedly.

[0030] A therapeutic method for reducing pore size according to an embodiment includes administering a pore-reducing drug containing the supernatant of a stem cell culture medium to the target. The pore-reducing drug may further contain extracellular matrix. The pore-reducing drug may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing pore size according to an embodiment includes administering a pore-reducing cosmetic containing the supernatant of a stem cell culture medium to the target. The pore-reducing cosmetic may further contain extracellular matrix. The pore-reducing cosmetic may be administered repeatedly.

[0031] A therapeutic method for reducing skin blemishes according to an embodiment includes administering a pharmaceutical product for reducing skin blemishes, which contains the supernatant of a stem cell culture medium, to the target. The pharmaceutical product for reducing skin blemishes may further contain an extracellular matrix. The pharmaceutical product for reducing skin blemishes may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing skin blemishes according to an embodiment includes administering a cosmetic product for reducing skin blemishes, which contains the supernatant of a stem cell culture medium, to the target. The cosmetic product for reducing skin blemishes may further contain an extracellular matrix. The cosmetic product for reducing skin blemishes may be administered repeatedly.

[0032] A therapeutic method for reducing sebum according to an embodiment includes administering a sebum-reducing pharmaceutical containing the supernatant of a stem cell culture medium to the target. The sebum-reducing pharmaceutical may further contain an extracellular matrix. The sebum-reducing pharmaceutical may be administered repeatedly. Administration may be performed by subcutaneous injection. A cosmetic method for reducing sebum according to an embodiment includes administering a sebum-reducing cosmetic containing the supernatant of a stem cell culture medium to the target. The sebum-reducing cosmetic may further contain an extracellular matrix. The sebum-reducing cosmetic may be administered repeatedly.

[0033] In the therapeutic or cosmetic method according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium in which stem cells were maintained and cultured in an undifferentiated state.

[0034] In the therapeutic or cosmetic method according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when stem cells are cultured in two dimensions.

[0035] In the therapeutic or cosmetic method according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when stem cells are cultured in three dimensions.

[0036] In the therapeutic or cosmetic method according to the above embodiment, the stem cells may be pluripotent stem cells.

[0037] In the therapeutic or cosmetic method according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0038] In the therapeutic or cosmetic method according to the above embodiment, the stem cells may be derived from the subject to whom the composition is provided.

[0039] A therapeutic method for reducing skin redness or inflammation according to an embodiment includes administering a pharmaceutical product for reducing skin redness or inflammation that contains stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The pharmaceutical product for reducing skin redness or inflammation may further contain extracellular matrix. The pharmaceutical product for reducing skin redness or inflammation may be administered repeatedly. Administration may be by subcutaneous injection. A cosmetic method for reducing skin redness or inflammation according to an embodiment includes administering a cosmetic product for reducing skin redness or inflammation that contains stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The cosmetic product for reducing skin redness or inflammation may further contain extracellular matrix. The cosmetic product for reducing skin redness or inflammation may be administered repeatedly. The particle size of the extracellular vesicles may be between 70 nm and 600 nm, and the concentration of extracellular vesicles may be between 10,000 vesicles / mL and 100 trillion vesicles / mL.

[0040] A therapeutic method for reducing inflammation according to an embodiment includes administering a drug for reducing inflammation, which contains secretions from stem cells, to a target. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The drug for reducing inflammation may further contain an extracellular matrix. The drug for reducing inflammation may be administered repeatedly. Administration may be by subcutaneous injection. A cosmetic method for reducing inflammation according to an embodiment includes administering a cosmetic for reducing inflammation, which contains secretions from stem cells, to a target. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The cosmetic for reducing inflammation may further contain an extracellular matrix. The cosmetic for reducing inflammation may be administered repeatedly. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0041] A therapeutic method for reducing the number of pores according to an embodiment includes administering a pharmaceutical product for reducing the number of pores, which contains stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The pharmaceutical product for reducing the number of pores may further contain extracellular matrix. The pharmaceutical product for reducing the number of pores may be administered repeatedly. Administration may be by subcutaneous injection. A cosmetic method for reducing the number of pores according to an embodiment includes administering a cosmetic product for reducing the number of pores, which contains stem cell secretions. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The cosmetic product for reducing the number of pores may further contain extracellular matrix. The cosmetic product for reducing the number of pores may be administered repeatedly. The particle size of the extracellular vesicles may be between 70 nm and 600 nm, and the concentration of extracellular vesicles may be between 10,000 vesicles / mL and 100 trillion vesicles / mL.

[0042] A therapeutic method for reducing pore size according to an embodiment includes administering a pore-reducing drug containing stem cell secretions to a target. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The pore-reducing drug may further contain extracellular matrix. The pore-reducing drug may be administered repeatedly. Administration may be by subcutaneous injection. A cosmetic method for reducing pore size according to an embodiment includes administering a pore-reducing cosmetic containing stem cell secretions to a target. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. The pore-reducing cosmetic may further contain extracellular matrix. The pore-reducing cosmetic may be administered repeatedly. The particle size of the extracellular vesicles may be between 70 nm and 600 nm, and the concentration of extracellular vesicles may be between 10,000 vesicles / mL and 100 trillion vesicles / mL.

[0043] The treatment method for reducing skin spots according to the embodiment includes administering a pharmaceutical product for reducing skin spots containing the secretion of stem cells to a subject. The secretion may be extracellular vesicles. The secretion may be exosomes. The secretion may be a protein. The secretion may be an isolated secretion. The secretion may be a concentrated secretion. The pharmaceutical product for reducing skin spots may further contain an extracellular matrix. The pharmaceutical product for reducing skin spots may be administered repeatedly. The administration may be performed by subcutaneous injection. The cosmetic method for reducing skin spots according to the embodiment includes administering a cosmetic product for reducing skin spots containing the secretion of stem cells to a subject. The secretion may be extracellular vesicles. The secretion may be exosomes. The secretion may be a protein. The secretion may be an isolated secretion. The secretion may be a concentrated secretion. The cosmetic product for reducing skin spots may further contain an extracellular matrix. The cosmetic product for reducing skin spots may be administered repeatedly. The particle diameter of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 particles / mL or more and 100 trillion particles / mL or less.

[0044] The treatment method for reducing sebum according to the embodiment includes administering a pharmaceutical product containing the secretion of stem cells to a subject. The secretion may be extracellular vesicles. The secretion may be exosomes. The secretion may be a protein. The secretion may be an isolated secretion. The secretion may be a concentrated secretion. The pharmaceutical product for reducing sebum may further contain an extracellular matrix. The pharmaceutical product for reducing sebum may be administered repeatedly. The administration may be performed by subcutaneous injection. The cosmetic method for reducing sebum according to the embodiment includes administering a cosmetic product containing the secretion of stem cells to a subject. The secretion may be extracellular vesicles. The secretion may be exosomes. The secretion may be a protein. The secretion may be an isolated secretion. The secretion may be a concentrated secretion. The cosmetic product for reducing sebum may further contain an extracellular matrix. The cosmetic product for reducing sebum may be administered repeatedly. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 particles / mL or more and 100 trillion particles / mL or less.

[0045] In the treatment method or cosmetic method according to the above embodiment, the stem cells may be pluripotent stem cells.

[0046] In the treatment method or cosmetic method according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0047] In the treatment method or cosmetic method according to the above embodiment, the stem cells may be derived from the subject to whom the composition is provided.

[0048] The use according to the embodiment is the use of the supernatant of the culture solution of stem cells in the manufacture of a reagent, a pharmaceutical raw material, a pharmaceutical product, a quasi-drug, a cosmetic raw material, or a cosmetic product for reducing skin redness or inflammation. The pharmaceutical product for reducing skin redness or inflammation may be used for subcutaneous injection.

[0049] The embodiment involves the use of the supernatant of stem cell culture medium in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation. Pharmaceuticals for reducing inflammation may be used by subcutaneous injection.

[0050] The embodiment involves the use of the supernatant of stem cell culture medium in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores. Pharmaceuticals for reducing the number of pores may be used by subcutaneous injection.

[0051] The embodiment involves the use of the supernatant of stem cell culture medium in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size. Pharmaceuticals for reducing pore size may be used by subcutaneous injection.

[0052] The embodiment involves the use of the supernatant of stem cell culture medium in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes. Pharmaceuticals for reducing skin blemishes may be used by subcutaneous injection.

[0053] The embodiment involves the use of the supernatant of stem cell culture medium in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum. Pharmaceuticals for reducing sebum may be used by subcutaneous injection.

[0054] In the use according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium in which stem cells were maintained in an undifferentiated state.

[0055] In the use according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when the stem cells were cultured in two dimensions.

[0056] In the use according to the above embodiment, the supernatant of the stem cell culture medium may be the supernatant of the culture medium obtained when the stem cells were cultured in three dimensions.

[0057] In the use according to the above embodiment, the stem cells may be pluripotent stem cells.

[0058] In the use according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0059] In the use according to the above embodiment, the stem cells may be derived from the subject to which the composition is provided.

[0060] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing skin redness or inflammation may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0061] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing inflammation may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm to 600 nm, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL to 100 trillion vesicles / mL.

[0062] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing the number of pores may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0063] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing pore size may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm to 600 nm, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL to 100 trillion vesicles / mL.

[0064] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing skin blemishes may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm to 600 nm, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL to 100 trillion vesicles / mL.

[0065] The embodiment involves the use of stem cell secretions in the manufacture of reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum. The secretions may be extracellular vesicles. The secretions may be exosomes. The secretions may be proteins. The secretions may be isolated secretions. The secretions may be concentrated secretions. Pharmaceuticals for reducing sebum may be used by subcutaneous injection. The particle size of the extracellular vesicles may be 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles may be 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

[0066] In the use according to the above embodiment, the stem cells may be pluripotent stem cells.

[0067] In the use according to the above embodiment, the stem cells may be reprogrammed stem cells.

[0068] In the use according to the above embodiment, the stem cells may be derived from the subject to which the composition is provided.

[0069] According to the present invention, it is possible to provide pharmaceuticals that effectively utilize stem cell components and stem cell culture medium.

[0070] These are photographs and graphs showing the results of injecting the supernatant of the culture medium according to Example 2 into the face of a subject. These are photographs showing the results of injecting the supernatant of the culture medium according to Example 2 into the face of a subject. These are graph3 into the face of a subject. These are graphs showing the results of injecting the supernatant of the culture medium and hyaluronic acid according to Example 4 into the face of a subject. These are graphs showing the results of injecting exosomes according to Example 6 into the face of a subject. These are graphs showing the results of injecting exosomes according to Example 6 into the face of a subject.

[0071] The embodiments of the present invention will be described in detail below. The embodiments shown below are illustrative examples of devices and methods for realizing the technical concept of this invention, and the technical concept of this invention is not limited to the combination of components described below. The technical concept of this invention can be modified in various ways within the scope of the claims.

[0072] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment include the supernatant of a stem cell culture medium and / or stem cell secretions. Examples of the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment include reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin redness or inflammation; reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing inflammation; reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing the number of pores; reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing pore size; reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing skin blemishes; and reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics for reducing sebum. Examples of blemishes include age spots, freckles, melasma, ADM (acquired dermal melanocytosis), hyperpigmentation, dullness, seborrheic keratosis, brown spots, and UV spots.

[0073] The supernatant of the stem cell culture medium is obtained by removing the stem cells by filtration and centrifugation. A filter may be used during filtration. The pore size of the filter is, for example, 0.1 μm to 0.5 μm. The culture medium may be filtered before centrifugation to precipitate the stem cells. The collected supernatant may also be filtered.

[0074] The stem cell culture medium may be the culture medium used during stem cell induction. The culture medium used during stem cell induction is, for example, the culture medium used when somatic cells were reprogrammed into pluripotent stem cells such as iPS cells. iPS cells are induced by introducing factors such as OCT3 / 4, KLF4, c-MYC, and SOX2 into somatic cells, such as differentiated cells including blood cells, fibroblasts, dental pulp stem cells, hair follicle cells, and urine cells. iPS cells may also be induced from CD3-positive T cells, CD14 or CD16-positive monocytes, CD68-positive macrophages, or CD34 or CD43-positive hematopoietic stem cells. Examples of blood cells include umbilical cord blood-derived cells, peripheral blood-derived cells, and mononuclear cells. It is preferable that iPS cells be induced by methods that do not involve gene insertion, such as methods using Sendai virus or RNA lipofection.

[0075] The process of inducing iPS cells is sometimes referred to as reprogramming, reprogramming, transformation, or lineage reprogramming, and cell fate reprogramming. Pluripotent stem cells have a positive rate of at least 30%, 50%, and preferably 80% for any of the following: TRA1-60, OCT3 / 4, SSEA3, SSEA4, TRA1-81, LIN28, and NANOG.

[0076] Pluripotent stem cells may be induced while being cultured in two dimensions, such as adherent culture. Alternatively, pluripotent stem cells may be induced while being cultured in three dimensions, such as suspension culture. In three-dimensional culture, gel culture medium or liquid culture medium may be used. Feeder cells do not need to be used in three-dimensional culture.

[0077] For induction culture, human ES / iPS culture media such as TeSR2 (STEMCELL Technologies) can be used. However, the culture medium is not limited to this, and various stem cell culture media can be used. For example, Primate ES Cell Medium, Reprostem, ReproFF, ReproFF2, ReproXF (Reprocell), mTeSR1, TeSRE8, ReproTeSR (STEMCELL Technologies), Pluristem® Human ES / iPS Medium (Merck), NutriStem® XF / FF Culture Medium for Human iPS and ES Cells, Plurion reprogramming medium (Stemgent), Pluristem®, Stemfit AK02N, Stemfit AK03 (Ajinomoto), ESC-Sure® serum and feeder-free medium for hESC / iPS (Applied StemCell), L7® hPSC Culture System (LONZA), Primate ES Cell Medium (ReproCELL), and Puel (I Peace, Inc.) may also be used. The culture medium does not contain, for example, activin A.

[0078] The gel medium is prepared, for example, by adding gellan gum such as deacylated gellan gum to the above medium so that the final concentration is 0.5% to 0.001% by weight, 0.1% to 0.005% by weight, or 0.05% to 0.01% by weight. In this disclosure, gellan gum includes deacylated gellan gum.

[0079] Alternatively, the stem cell culture medium may be the culture medium used when stem cells are maintained in an undifferentiated state. Stem cells are, for example, pluripotent stem cells such as induced pluripotent stem (iPS) cells and embryonic stem cells (ES cells). Stem cells may be totipotent stem cells or multipotent stem cells. Stem cells may be, for example, reprogrammed cells. Multipotent stem cells may be at least one of the following: CD73-positive, CD90-positive, CD105-positive, CD34-positive, CD45-positive, EPCAM-positive, EGFR-positive, cadherin-positive, CD133 / PROML1-positive, and nestin-positive.

[0080] Whether or not stem cells maintain an undifferentiated state can be confirmed, for example, by checking whether the positive rate of any of TRA1-60, OCT3 / 4, SSEA3, SSEA4, TRA1-81, LIN28, and NANOG is 30% or more, 50% or more, preferably 80% or more.

[0081] For example, human ES / iPS medium such as Puel (I Peace, Inc.) can be used as a culture medium for stem cells. However, the culture medium for stem cells is not limited to this, and various stem cell media can be used. For example, the culture medium for stem cells may be Dulbecco's modified Eagle medium / Ham F-12 (DMEM / F12) supplemented with alternative serum, L-glutamine, non-essential amino acid solution, 2-mercaptoethanol, and penicillin / streptomycin. When maintaining stem cells in an undifferentiated state, the medium contains 10 ng / ml or more of b-FGF, preferably 40 ng / ml or more.

[0082] When maintaining stem cells in an undifferentiated state through culture, the culture may be two-dimensional or three-dimensional. Two-dimensional culture includes adherent culture. Three-dimensional culture includes suspension culture, in which cells can float in the culture medium. In suspension culture, the culture medium may or may not be stirred. Furthermore, three-dimensional culture also includes culture in which cells are fixed in a three-dimensional matrix and do not float. When stem cells are cultured in three dimensions, the culture medium may be a liquid medium or a gel medium. Gel medium can be prepared, for example, by adding gellan gum such as deacylated gellan gum to a stem cell medium to a final concentration of 0.5% to 0.001% by mass, 0.1% to 0.005% by mass, or 0.05% to 0.01% by mass.

[0083] Stem cells may form clamps. When stem cells are cultured in suspension, the culture medium may be agitated at a rate such that 10% or more, 20% to 60%, or 20% to 50% of the cell clamps sink. The density of clamps in the culture medium is, for example, 50 cells / mL to 2500 cells / mL, 300 cells / mL to 1200 cells / mL, 200 cells / mL to 1100 cells / mL, or 100 cells / mL to 1000 cells / mL.

[0084] The temperature used for maintaining stem cell culture is, for example, 37°C. The carbon dioxide concentration used for maintaining stem cell culture is, for example, 5%. The duration of maintaining stem cell culture is, for example, 1 to 90 days, 2 to 60 days, 3 to 40 days, 5 to 30 days, or 7 to 21 days.

[0085] The culture medium may contain at least one high-molecular-weight compound selected from the group consisting of hyaluronic acid, ramsan gum, dieutan gum, xanthan gum, carrageenan, fucoidan, pectin, pectic acid, pectinic acid, heparan sulfate, heparin, heparin sulfate, kerato sulfate, chondroitin sulfate, deltamann sulfate, rhamnan sulfate, and salts thereof. The culture medium may also contain methylcellulose and lipids such as lysophosphatidic acid and sphingosine-1-phosphate. The inclusion of these substances suppresses cell aggregation. The culture medium may also contain natural or artificial extracellular matrix. Examples of extracellular matrix include Matrigel, laminin, and vitronectin and fibronectin. The culture medium may also contain polyethylene glycol (PEG).

[0086] The culture medium may contain a small number of temperature-sensitive gels selected from poly(glycerol monomethacrylate) (PGMA), poly(2-hydroxypropyl methacrylate) (PHPMA), Poly(N-isopropylacrylamide) (PNIPAM), amine-terminated, carboxylic acid-terminated, maleimide-terminated, N-hydroxysuccinimide (NHS) ester-terminated, triethoxysilane-terminated, Poly(N-isopropylacrylamide-co-acrylamide), Poly(N-isopropylacrylamide-co-acrylic acid), Poly(N-isopropylacrylamide-co-butylacrylate), Poly(N-isopropylacrylamide-co-methacrylic acid), Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate), and N-Isopropylacrylamide.

[0087] For example, a ROCK inhibitor may be added to the culture medium at a final concentration of 1000 μmol / L to 0.1 μmol / L, 100 μmol / L to 1 μmol / L, or 5 μmol / L to 20 μmol / L. Adding a ROCK inhibitor to the culture medium promotes colony formation by stem cells.

[0088] The culture medium does not necessarily have to contain growth factors such as bFGF. Alternatively, the culture medium may contain growth factors such as bFGF at low concentrations of 400 μg / L or less, 100 μg / L or less, 40 μg / L or less, or 10 μg / L or less.

[0089] Furthermore, the culture medium may either not contain TGF-β, or it may contain TGF-β at a low concentration of 600 ng / L or less, 300 ng / L or less, or 100 ng / L or less.

[0090] The culture medium for stem cells may be cryopreserved. The supernatant of the culture medium for stem cells contains lipids. Examples of lipids include simple lipids and complex lipids. Examples of simple lipids include triglycerides, cholesterol esters, and waxes. Triglycerides are esters of alcohols and fatty acids. Cholesterol esters are esters of cholesterol and fatty acids. Waxes are esters of higher alcohols and fatty acids. Examples of complex lipids include phospholipids, glycolipids, and derived lipids. Phospholipids are lipids containing phosphoric acid. Examples of phospholipids include diglycerides and triglycerides. Glycolipids are lipids containing sugars. The lipids may also be hydrolysis products of simple lipids or complex lipids, and may be lipid-soluble lipids. Examples of such lipids include fatty acids and cholesterol. When the reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to the embodiment is applied to the skin, the phospholipids contained in the supernatant of the culture medium for stem cells are incorporated into the intercellular lipids and form a lamellar structure. The phospholipids contained in the supernatant of the culture medium for stem cells can be incorporated into liposomes.

[0091] Examples of secretions from stem cells include extracellular vesicles, cytokines, chemokines, and growth factors. Extracellular vesicles are vesicles with a lipid bilayer structure. Extracellular vesicles are also called membrane vesicles. Examples of extracellular vesicles include exosomes and microvesicles. Exosomes are granular substances with a diameter of 30 nm to 150 nm. Exosomes are formed by inward budding of the late endosomal membrane, and then fuse with the cell membrane to form complete particles, which are secreted extracellularly by exocytosis. Exosomes contain lipids and proteins derived from the cell membrane on their surface. Exosomes also contain nucleic acids and proteins derived from the inside of the cell. Exosomes may contain growth factors. Examples of growth factors include IGF, FGF, PDGFB, EGF, PDGFA, FFFR2, PDGFC, IGF, PDGFD, HGF, TGFB3, EGFR, VEGFC, VEGF, and TGF. Examples of IGF include IGF1 and IGF2. Examples of FGF include FGF1, FGF4, FGF10, FGF11, FGF14, FGF16, FGF17, FGF18, FGF22, and FGF23.

[0092] Let's further explain the exosome secretion process. When endocytosis occurs in a cell, early endosomes are formed, followed by late endosomes. At this time, vesicles that form with the endosomal membrane budding inward are called intermembryonic vesicles (ILVs). Endosomes formed by the aggregation of intermembryonic vesicles are called multilocular endosomes (MVBs). When multilocular endosomes fuse with the cell membrane, the intermembryonic vesicles within the multilocular endosome are exocytically secreted outside the cell. These intermembryonic vesicles released outside the cell are exosomes. In this series of secretion processes, the involvement of ceramides, Hrs (a subunit of the endosomal transport sorting complex (ESCR)), and the RAB family, such as RAB11 and RAB35 (low molecular weight GTPases involved in intracellular transport of vesicles), has been reported. Furthermore, the involvement of the membrane-binding protein SNARE has been reported when multilocular endosomes fuse with the cell membrane and exocytosis occurs.

[0093] The method for recovering exosomes from the culture medium is not particularly limited. For example, the culture medium may be centrifuged by ultracentrifugation or density gradient centrifugation to recover exosomes. In ultracentrifugation, the culture medium may be centrifuged at 10,000 g to 100,000 g for 30 minutes or more to isolate exosomes from the culture medium. Exosomes may also be recovered by size exclusion chromatography. Exosomes may also be recovered by co-precipitation with polyethylene glycol (PEG). Exosomes may also be isolated from serum or culture supernatant using immunoprecipitation with antibodies that specifically recognize CD9, CD63, and CD81.

[0094] Exosomes may also be recovered by affinity purification using proteins specifically expressed on exosomes. For example, the phospholipid phosphatidylserine is expressed on the membrane surface of exosomes. Tim4, an exosome receptor expressed on macrophages, binds to phospholipid phosphatidylserine in a calcium-dependent manner. Therefore, by preparing magnetic beads to which the extracellular domain of Tim4 is attached, immobilizing exosomes on the surface of the magnetic beads via the extracellular domain of Tim4, and then eluting the exosomes from the magnetic beads using a chelating agent, it is possible to recover and isolate exosomes from the culture medium.

[0095] Microvesicles have a diameter of 100 nm to 1000 nm and are produced by the budding and separation of the cell membrane. Stem cell secretions may include, for example, ectosomes, secretory microvesicles, oncosomes, and prostasomes. Stem cell secretions may also include, for example, peptides, proteins, growth factors, proliferation factors, cytokines, and hormones. Proteins may have molecular weights of, for example, 3 kDa or more, 10 kDa or more, or 20 kDa or more.

[0096] Stem cell secretions may be contained in the supernatant of the stem cell culture medium. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may contain isolated secretions. Furthermore, reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may contain concentrated secretions. Examples of methods for isolating and concentrating secretions include freeze-drying, ultrafiltration, precipitation and resuspension, and gel filtration. Methods for precipitating proteins include ammonium sulfate precipitation, trichloroacetic acid precipitation, acetone precipitation, and methanol precipitation containing ammonium acetate.

[0097] Examples of marker proteins for extracellular vesicles include the tetraspanin family (CD9, CD63, CD81), the integrin family (ITGA, ITGB), ESCRT complex-binding proteins (TSG101, ALIX), lipid raft markers (Flotillin-1 / 2), and heat shock proteins (HSP70). Therefore, extracellular vesicles may be isolated using antibodies that specifically recognize these marker proteins.

[0098] The secretions may be collected from the supernatant of the stem cell culture medium. The secretions may also be collected from the supernatant of the culture medium while the stem cells are being maintained in culture.

[0099] The exosome concentration in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment is, for example, 10 pg / mL or more and 500,000 pg / mL or less, 15 pg / mL or more and 500,000 pg / mL or less, 20 pg / mL or more and 500,000 pg / mL or less, 50 pg / mL or more and 500,000 pg / mL or less, 500 pg / mL or more and 350,000 pg / mL or less, or 2,000 pg / mL or more and 200,000 pg / mL or less. Alternatively, the exosome concentration in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment is 10,000 or more and 100 trillion or less, 100,000 or more and 50 trillion or less, 1,000,000 or more and 10 trillion or less, 10,000,000 or more and 1 trillion or less, 100,000,000 or more and 500,000,000 or less, or 1,000,000,000 or more and 300,000,000 or less. The particle size of the exosomes in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment is, for example, 50 nm or more and 1,000 nm or less, 60 nm or more and 800 nm or less, or 70 nm or more and 600 nm or less.

[0100] In the supernatant of the culture medium of stem cells contained in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment, the stem cells may be derived from the human subject to whom the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are provided. For example, cells may be collected from the human subject, the collected cells may be reprogrammed to induce pluripotent stem cells, and the supernatant of the culture medium during the induction of pluripotent stem cells, or the supernatant of the culture medium of the induced pluripotent stem cells, may be included in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment, and the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics may be provided to the target.

[0101] In the stem cell-derived secretions contained in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment, the stem cells may be derived from the human subject to whom the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are provided. For example, cells may be collected from the human subject, the collected cells may be reprogrammed to induce pluripotent stem cells, and secretions recovered from the culture medium of the induced pluripotent stem cells may be included in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment, and the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics may be provided to the target.

[0102] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment contain an effective amount of the supernatant of stem cell culture medium and / or stem cell secretions. Here, the effective amount refers to the amount that can exert efficacy as a reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic. The effective amount is appropriately set according to the patient's age, the target disease, the presence or absence of other effective ingredients, and the amount of other formulations.

[0103] The pharmaceutical product according to the embodiment may be an injectable or intravenous solution. The pharmaceutical product according to the embodiment may be used by intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection, and intra-arterial injection. Preferably, the pharmaceutical product according to the embodiment is used for subcutaneous injection. The pharmaceutical product according to the embodiment may be injected into the dermis, epidermis, or from the dermis to the epidermis. The injection is performed using a syringe. A general syringe capable of administering a drug solution through a needle can be used. Examples of needle gauges include 16G to 40G. The needle gauge may be 32G or less. The pharmaceutical product according to the embodiment may further contain at least one selected from water for injection, physiological saline, glucose solution, and amino acid solution. The application site of the reagent, pharmaceutical raw material, pharmaceutical product, quasi-drug, cosmetic raw material, or cosmetic according to the embodiment is not particularly limited, but is the entire body surface, for example, the face and neck. For example, the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are applied to five or more, ten or more, thirty or more, or fifty or more locations on either or both the face and neck.

[0104] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may contain formulation-permissible carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, antiseptics, and physiological saline, etc. Examples of excipients include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrants include carboxymethylcellulose and calcium carbonate. Examples of buffers include phosphates, citrates, and acetates. Examples of emulsifiers include gum arabic, sodium alginate, and tragacanth.

[0105] Examples of suspending agents include glyceryl monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of analgesics include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers include propylene glycol and ascorbic acid. Examples of preservatives include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Examples of antiseptics include benzalkonium chloride, parahydroxybenzoic acid, and chlorobutanol.

[0106] Furthermore, the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment include water, alcohol, surfactants (cationic, anionic, nonionic, and amphoteric surfactants, etc.), humectants (glycerin, 1,3-butylene glycol, propylene glycol, propanediol, pentanediol, polyquaternium, amino acids, urea, pyrrolidone carboxylates, nucleic acids, monosaccharides, oligosaccharides, etc., and their derivatives, etc.), thickeners (polysaccharides, polyacrylates, carboxyvinyl polymers, polyvinylpyrrolidone, polyvinyl alcohol, chitin) Chitosan, alginic acid, carrageenan, xanthan gum, and methylcellulose, etc., and their derivatives, etc.), waxes, petrolatum, hydrocarbon saturated fatty acids, unsaturated fatty acids, and silicone oils, etc., and their derivatives, triglycerides such as caprylic / capric triglyceride and glyceryl trioctanoate, ester oils such as isopropyl stearate, natural oils and fats (olive oil, camellia oil, avocado oil, almond oil, cocoa butter, evening primrose oil, grape seed oil, macadamia nut oil, eucalyptus oil, rosehip oil, squalane, orange roughy - Oils, lanolin, and ceramides, etc.), preservatives (oxybenzoic acid derivatives, dehydroacetates, photosensitizers, sorbic acid, and phenoxyethanol, etc., and their derivatives, etc.), bactericides (sulfur, triclocarbaanilide, salicylic acid, zinc pyrithione, and hinokitiol, etc., and their derivatives, etc.), UV absorbers (para-aminobenzoic acid, methoxycinnamic acid, etc., and their derivatives, etc.), anti-inflammatory agents (allantoin, bisabolol, ε-aminocaproic acid, acetylphanesylcysteine, and glycyrrhizic acid, etc., and their derivatives, etc.), Antioxidants (tocopherol, BHA, BHT, and astaxanthin, etc., and their derivatives, etc.), chelating agents (edetic acid, hydroxyethanediphosphonic acid, etc., and their derivatives, etc.), plant and animal extracts (Angelica keiskei, aloe, rosehip, scutellaria baicalensis, Phellodendron amurense, seaweed, quince, chamomile, licorice, kiwi, cucumber, mulberry, birch, angelica tree, garlic, peony, hops, horse chestnut, lavender, rosemary, eucalyptus, milk, various peptides, placenta, royal jelly, Euglena extract, hydrolyzed Euglena extract, and Euglena oil, etc.)(and their purified or fermented products, etc.), pH adjusters (inorganic acids, inorganic acid salts, organic acids, and organic acid salts, etc., and their derivatives, etc.), vitamins (vitamins A, B, C, D, ubiquinone, and nicotinamide, etc., and their derivatives, etc.), yeast, koji mold and lactic acid bacteria fermentation liquid, galactomyces culture liquid, whitening agents (tranexamic acid, cetyl tranexamate hydrochloride, 4-n-butylresorcinol, arbutin, kojic acid, ellagic acid, licorice flavonoids, niacinamide, and vitamin C) Derivatives, etc.), ceramides and ceramide derivatives, anti-wrinkle agents (retinol and retinal, and their derivatives, nicotinamide and oligopeptides, etc., and their derivatives, natural and synthetic components with neutrophil elastase inhibitory and MMP-1 and MMP-2 inhibitory effects, etc.), titanium dioxide, talc, mica, silica, zinc oxide, iron oxide, silicon, and powders processed from these may be blended within the scope necessary to achieve the purpose of the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment.

[0107] The components that can be added to the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are not limited to those described above, and any components that can be used in ordinary reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics can be freely selected. When the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are used as a poultice, in addition to the above components, a base (such as kaolin and bentonite) and a gelling agent (such as polyacrylate and polyvinyl alcohol) may be added within a range that achieves the purpose. When the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment are used as a bath additive, sulfates, bicarbonates, borates, dyes, and humectants may be added as appropriate within a range that achieves the purpose, and the product may be prepared in powder or liquid form. The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may also be skin application compositions.

[0108] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may contain extracellular matrix. Examples of extracellular matrix include hyaluronic acid, collagen, proteoglycans, and salts thereof. Examples of hyaluronic acid salts include sodium hyaluronate, potassium hyaluronate, magnesium hyaluronate, and calcium hyaluronate. Hyaluronic acid may be extracted from animal tissue, harvested as a product of bacterial fermentation, or produced in commercial quantities by bioprocess technology. The volume concentration of extracellular matrix in the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment is 1% to 80%, 2% to 70%, 5% to 60%, or 10% to 50%.

[0109] The reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may be administered to a subject repeatedly. For example, the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may be administered to a subject every other day, every two days, every three days, every four days, every five days, every six days, every week, every two weeks, or every three weeks. Alternatively, the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may be administered to a subject two or more times a month, or four or more times a month. Alternatively, the reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics according to the embodiment may be administered to a subject once or more times a month for three months or more. Examples of administration methods include subcutaneous administration, intravenous administration, intramuscular administration, intrathecal administration, transdermal administration, and transdermal absorption administration.

[0110] (Example 1: Preparation of supernatant of culture medium used for iPS cell induction) The supernatant of the culture medium used for iPS cell induction was prepared by the following method. Hematopoietic cell medium was prepared by adding recombinant protein FLT3 ligand (PEPROTECH), recombinant human TPO (PEPROTECH), recombinant human IL-6 (PEPROTECH), recombinant human G-CSF (PEPROTECH), and recombinant human SCF (PEPROTECH) to serum-free and animal-derived component-free hematopoietic cell medium (Stemspan ACF, STEMCELL TECHNOLOGIES). 2 × 10⁶ wells were added to each well of a 12-well dish. 5 Human-derived blood cells (peripheral blood mononuclear cells) were seeded. Subsequently, 12 welldishes were placed in a 37°C CO2 incubator, and the cells were cultured in suspension.

[0111] Three days after initiating cell culture, hematopoietic cell culture medium was added to each well as needed. Six days after initiating cell culture, Oct3 / 4, SOX2, KLF4, and c-Myc were introduced into the cells in each well, and then hematopoietic cell culture medium was added to each well. The cells were then cultured on laminin 511 coated dishes, which are a stem cell culture substrate.

[0112] Two days after infection, the culture medium in each well was half replaced with stem cell medium (Puel, I Peace, Inc.) every two days. Fourteen days after infection, it was confirmed that more than 10 iPS cell clusters had been induced. Subsequently, the formed colonies were detached from the wells using triple select (Thermo Fisher), and the colonies were divided so that more than 50% were single cells. Then, the cells were seeded as single cells on laminin 511 coated dishes as described above. After that, the cells were cultured in Puel medium, the culture medium was changed every two days, and subculture was performed when more than 70% were confluent. Subculture was performed more than three times until the introduced factor had been removed from the cells and was no longer detectable. The transgene-free iPS cells were seeded in a culture dish, and the culture supernatant was collected when more than 50% were confluent. The collected culture supernatant was centrifuged with 400g to remove the cells, and filtered through a filter of 0.22 μm or less to obtain the culture supernatant according to Example 1.

[0113] (Example 2: Administration of culture medium supernatant) 20 μL to 200 μL of the culture medium supernatant from Example 1 was injected into more than 50 locations on the entire face and neck of Japanese female subjects using a needle of 32G or less, from the dermis to the epidermis. On the face, injections were particularly given to the nasolabial folds, under the eyes, around the eyes, and the mid-cheek line. The subjects' faces before and two weeks after the injection of the culture medium supernatant were analyzed using a facial skin image analysis counseling system (VISIA, Integral Co., Ltd.). VISIA can calculate scores for wrinkles, redness, and pores by matching them to a large database. As shown in Figure 1, the subjects' faces showed a reduction in wrinkles, redness, and the number and size of pores after injection of the culture medium supernatant.

[0114] Furthermore, as shown in Figures 2, 3A, and 3B, the subjects' faces showed improved skin texture, reduced pore count, decreased UV-induced spots, reduced brown spots, and decreased porphyrin levels (indicating sebum production) after injection of the culture medium supernatant. Here, skin texture refers to the unevenness of the skin surface, composed of furrows and ridges. Shallow furrows and fine ridges result in fine-textured, beautiful skin. The vertical axis in Figures 3A and 3B represents the score calculated by VISIA.

[0115] Furthermore, the subjects' faces were analyzed using VISIA one month after being injected with the culture medium supernatant. The results showed that wrinkles were reduced by 15%, sebum by 11%, and the number of pores by 14% compared to before the injection. In addition, their skin age was reduced by 6 years.

[0116] (Example 3: Repeated administration of culture medium supernatant) Similar to Example 2, the culture medium supernatant from Example 1 was injected into the entire face and neck of a Japanese female subject, from the dermis to the epidermis, three times. The first injection was on July 2, 2024. The second injection was on July 9, 2024. The third injection was on July 23, 2024. On July 2, July 9, July 23, 2024, and July 30, 2024, the number of spots, wrinkles, redness, and pores were evaluated using VISIA, similar to Example 2. As shown in Figure 4, the number of spots, wrinkles, redness, and pores decreased with each injection.

[0117] (Example 4: Administration of culture medium supernatant and hyaluronic acid) The culture medium supernatant from Example 1, a sample prepared by mixing the culture medium supernatant from Example 1 with 1% hyaluronic acid by weight, and a sample prepared by mixing the culture medium supernatant from Example 1 with 50% hyaluronic acid by weight were prepared. The culture medium supernatant from Example 1 and the sample prepared by mixing the culture medium supernatant from Example 1 with 1% hyaluronic acid by weight were injected into half of the face of the first Japanese female subject, from the dermis to the epidermis. The sample prepared by mixing the culture medium supernatant from Example 1 with 50% hyaluronic acid by weight was injected into half of the face of the second Japanese female subject, from the dermis to the epidermis. The faces of the subjects were analyzed using VISIA approximately two weeks later. As a result, as shown in Figure 5, the number of blemishes, wrinkles, redness, sebum, and pores was reduced more in the supernatant of the culture medium with hyaluronic acid added than in the supernatant of the culture medium without hyaluronic acid added.

[0118] (Example 5: Preparation of iPS cell exosomes) Human iPS cells free of inducible factors were cultured in a stem cell culture medium (Puel, I Peace, Inc.) on an adhesion culture dish coated with laminin 511. The human iPS cells were passaged every week. During passage, the human iPS cells were treated with ES cell dissociation solution (TrypLE Select, registered trademark, Thermo Fisher Scientific).

[0119] As described above, human iPS cells maintained in culture were detached from the adherent culture dish using ES cell dissociation solution (TrypLE Select, registered trademark, Thermo Fisher Scientific). The detached human iPS cells were seeded onto a dish coated with laminin (Nippi). Subsequently, the human iPS cells were cultured for one week in stem cell medium (Puel, I Peace, Inc.) supplemented with 10 μmol / L of ROCK inhibitor. The culture medium was changed every two days.

[0120] Cell density is 2 × 10 4 pieces / cm 2After confirming that the cells had reached confluence of 30% or more, the culture medium was replaced with fresh medium, and the supernatant of the stem cell medium was collected two days later. The collected supernatant of the stem cell medium was centrifuged at 400g for 30 minutes, and the supernatant after centrifugation was filtered through a 0.22 μm filter. Furthermore, the iPS cells maintained in culture were confirmed to be positive for the undifferentiated markers alkaline phosphatase, NANOG, OCT3 / 4, SSEA4, SSEA5, and TRA1-60.

[0121] The supernatant of the filtered stem cell medium described above was centrifuged at 400 g for 30 minutes, and the supernatant after centrifugation was filtered through a 0.22 μm filter. Next, the proteins contained in the supernatant of the stem cell medium after centrifugation were concentrated more than 5 times by ultrafiltration using a centrifugal concentration tube (VIVASPIN 20, Funakoshi). Then, using MagCapture Exosome Isolation Kit PS Ver. 2 (Fujifilm Wako Pure Chemical Industries), exosomes were immobilized onto magnetic beads using the affinity method for phosphatidylserine (PS) on the membrane surface, according to the package insert, and the magnetic beads were recovered. Furthermore, exosomes were eluted from the magnetic beads and recovered into fresh stem cell medium (Puel, I Peace, Inc.) to obtain an exosome-containing solution with an exosome concentration of 20 pg / mL. Exosomes may also be recovered and purified using the filter filtration system of the exosome purification kit (ExoIsolator, Dojin Chemical Laboratories).

[0122] (Example 6: Exosome Administration) Similar to Example 2, the exosome-containing solution according to Example 5 was injected into the entire face and neck of a Japanese female subject, from the dermis to the epidermis. The subject's face before injection of the exosome-containing solution and the subject's face two weeks after injection were analyzed using VISIA. As shown in Figure 6, the subject's face showed a reduction in age spots, wrinkles, redness, porphyrin (an indicator of sebum volume), and the number of pores after injection of the exosome-containing solution. Furthermore, as shown in Figure 7, the subject's face showed improved skin texture after injection of the exosome-containing solution.

[0123] (Example 7: Exosome Expression Levels) The supernatant of the culture medium used to induce iPS cells according to Example 1 was prepared. In addition, the supernatant of the culture medium of peripheral blood mononuclear cells (PBMCs) was prepared. RNA sequencing analysis of each supernatant revealed that the expression levels of IGF2, FGF18, PDGFB, FGF23, EGF, PDGFA, FGF22, FFFR2, FGF11, FGF16, FGF4, FGF10, FGF17, PDGFC, FGF1, FGF14, IGF1, PDGFD, HGF, TGFB3, EGFR, and VEGFC were higher in the supernatant of the culture medium used to induce iPS cells than in the supernatant of the PBMC culture medium.

[0124] (Example 8: Exosome particle size and number of particles) Human iPS cells were cultured using stem cell culture medium (Puel, I Peace, Inc.) in a laminin 511-coated flask. After 2 days, when the culture reached 80% confluence, the culture medium was replaced with 5 mL of fresh medium. After 2 days, the culture supernatant was collected, and the exosomes contained in the culture supernatant were purified and concentrated to obtain an exosome-containing solution. Analysis of the exosome-containing solution using a nanoparticle analysis system (NanoSight NS300, blue laser, wavelength 405 nm) revealed particles with particle sizes ranging from 100 nm to 400 nm, with a median particle size of 100 nm to 200 nm. The exosome concentration in the exosome-containing solution ranged from 1,560,000,000 particles / mL to 159,000,000,000 particles / mL.

Claims

1. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing the supernatant of stem cell culture medium for reducing skin redness or inflammation.

2. The pharmaceutical product according to claim 1, which is a pharmaceutical product and is used for subcutaneous injection.

3. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing the supernatant of stem cell culture medium for reducing the number or size of pores.

4. The pharmaceutical product according to claim 3, which is a pharmaceutical product and is used for subcutaneous injection.

5. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing the supernatant of stem cell culture medium for reducing sebum.

6. The pharmaceutical product according to claim 5, which is a pharmaceutical product and is used for subcutaneous injection.

7. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing stem cell secretions for reducing skin redness or inflammation.

8. The reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to claim 7, wherein the secretion is an extracellular vesicle.

9. The pharmaceutical product according to claim 7, which is a pharmaceutical product and is used for subcutaneous injection.

10. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing stem cell secretions for reducing the number or size of pores.

11. The reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to claim 10, wherein the secretion is an extracellular vesicle.

12. The pharmaceutical product according to claim 10, which is a pharmaceutical product and is used for subcutaneous injection.

13. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing stem cell secretions for reducing skin blemishes.

14. The reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to claim 13, wherein the secretion is an extracellular vesicle.

15. The pharmaceutical product according to claim 13, which is a pharmaceutical product and is used for subcutaneous injection.

16. Reagents, pharmaceutical raw materials, pharmaceuticals, quasi-drugs, cosmetic raw materials, or cosmetics containing stem cell secretions for reducing sebum.

17. The reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to claim 16, wherein the secretion is an extracellular vesicle.

18. A reagent, pharmaceutical raw material, pharmaceutical, quasi-drug, cosmetic raw material, or cosmetic according to at least one of claims 8, 11, 14, and 17, wherein the particle size of the extracellular vesicles is 70 nm or more and 600 nm or less, and the concentration of the extracellular vesicles is 10,000 vesicles / mL or more and 100 trillion vesicles / mL or less.

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