JP7520397B2 - Stem cell culture supernatant and its manufacturing method - Google Patents
Stem cell culture supernatant and its manufacturing method Download PDFInfo
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- JP7520397B2 JP7520397B2 JP2022130872A JP2022130872A JP7520397B2 JP 7520397 B2 JP7520397 B2 JP 7520397B2 JP 2022130872 A JP2022130872 A JP 2022130872A JP 2022130872 A JP2022130872 A JP 2022130872A JP 7520397 B2 JP7520397 B2 JP 7520397B2
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- mesenchymal stem
- culture supernatant
- stem cells
- cell culture
- cells
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Description
本発明は、幹細胞培養上清液およびその製造方法に関する。より詳細には、複数の組織に由来する間葉系幹細胞を共培養することで製造される間葉系幹細胞培養上清液とその製造方法に関する。また、本発明は、間葉系幹細胞培養上清液またはその乾燥粉末を含む医薬組成物または化粧用組成物にも関する。 The present invention relates to a stem cell culture supernatant and a method for producing the same. More specifically, the present invention relates to a mesenchymal stem cell culture supernatant produced by co-culturing mesenchymal stem cells derived from multiple tissues and a method for producing the same. The present invention also relates to a pharmaceutical composition or cosmetic composition containing the mesenchymal stem cell culture supernatant or a dried powder thereof.
間葉系幹細胞は、骨髄や滑膜、脂肪、臍帯の中に存在する体性幹細胞であり、軟骨や骨、脂肪、神経細胞への分化能を有することが報告されている（非特許文献1）。成人の組織からも分離できることから、半月板や軟骨の損傷部の再生治療に用いられている（非特許文献2）。また、間葉系幹細胞は抗炎症作用や免疫調節等の作用も有することから、肝疾患や移植片対宿主病、自己免疫疾患など、既に様々な疾患に対して幅広く利用されていることが知られている。 Mesenchymal stem cells are somatic stem cells found in bone marrow, synovium, fat, and umbilical cord, and have been reported to have the ability to differentiate into cartilage, bone, fat, and nerve cells (Non-Patent Document 1). Because they can be isolated from adult tissues, they are used in regenerative treatments for damaged areas of the meniscus and cartilage (Non-Patent Document 2). In addition, mesenchymal stem cells also have anti-inflammatory and immunoregulatory effects, and are known to have been widely used for a variety of diseases, including liver disease, graft-versus-host disease, and autoimmune diseases.
こうした中、細胞移植治療において生体内に移植されたこれらの間葉系幹細胞の働きには、間葉系細胞から分泌される種々のサイトカイン等の生理活性物質が大きく寄与していることが明らかにされてきた。 In this context, it has become clear that the function of mesenchymal stem cells transplanted into the body in cell transplantation therapy is greatly influenced by various physiologically active substances such as cytokines secreted by mesenchymal cells.
間葉系幹細胞をインビトロで培養した際には、培養液中にこうした生理活性物質が放出されることになる。そこで、間葉系幹細胞の培養に使用した培養液を回収し、細胞から放出される物質を多く含むこの培養液を利用して、組織を再生すること等に成功した例が報告されている（非特許文献3、非特許文献4、非特許文献5）。 When mesenchymal stem cells are cultured in vitro, these physiologically active substances are released into the culture medium. There have been reports of successful cases in which the culture medium used to culture mesenchymal stem cells has been recovered and used to regenerate tissues, as this culture medium contains a large amount of substances released from the cells (Non-Patent Documents 3, 4, and 5).
このように、間葉系幹細胞の培養過程で生成される培養上清液は、エクソソームを始めとする幹細胞から分泌した成長因子や免疫調節因子（サイトカイン）など、細胞活性に重要な働きをする情報伝達物質を豊富に含んでおり、体内の損傷を受けた組織や細胞の機能回復に役立つと考えられている。上清液に豊富に含まれるエクソソーム・成長因子・サイトカインなどが周囲に存在している幹細胞に作用し、老化や損傷などによって機能が低下した箇所に細胞を集めることによって、組織再生や免疫力向上などの機能回復効果が期待されている。 In this way, the culture supernatant produced during the mesenchymal stem cell culture process is rich in signaling substances that play an important role in cell activity, such as exosomes and other growth factors and immune regulatory factors (cytokines) secreted by stem cells, and is thought to be useful in restoring the function of damaged tissues and cells in the body. The exosomes, growth factors, and cytokines contained in the supernatant act on surrounding stem cells, and by collecting cells in areas where function has been reduced due to aging or damage, it is expected to have a functional recovery effect, such as tissue regeneration and improved immunity.
本開示は、従来とは異なる活性を有する幹細胞培養上清液を提供することを目的の1つとする。また、本開示は、従来とは異なる活性を有する幹細胞培養上清液に基づく医薬品および／または化粧品を提供することも目的の1つとする。 One of the objectives of the present disclosure is to provide a stem cell culture supernatant having an activity different from that of conventional products. Another objective of the present disclosure is to provide a pharmaceutical and/or cosmetic product based on a stem cell culture supernatant having an activity different from that of conventional products.
本発明者らは、2種類以上の間葉系幹細胞を共培養することにより、予想外の成分を含み、優れた活性を有する幹細胞培養上清液が得られることを見出した。 The inventors have discovered that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant containing unexpected components and having excellent activity can be obtained.
本開示はこのような知見に基づくものであり、以下の態様を包含する：
（実施態様１）
間葉系幹細胞培養上清液の製造方法であって、
i）2種類以上の間葉系幹細胞を共培養すること、および
ii）培養上清液を回収すること
を含む、方法。
（実施態様２）
共培養に先立ち、前記2種類以上の間葉系幹細胞を個別に培養することをさらに含む、実施態様1記載の方法。
（実施態様３）
前記間葉系幹細胞が、脂肪、臍帯、羊膜、羊水、歯髄、ワルトン膠様質（Wharon’s jelly）、CPJ（Cord Placenta Junction）、絨毛膜、肝臓、肺、脊椎、臍帯血、胎盤、末梢血、真皮、子宮内膜、母乳、および毛包から成る群から選択される組織由来の細胞である、実施態様1記載の方法。
（実施態様４）
2種類の間葉系幹細胞が用いられる、実施態様1記載の方法。
（実施態様５）
ヒト脂肪幹細胞とヒト臍帯幹細胞、ヒト脂肪幹細胞とヒト羊膜幹細胞、またはヒト臍帯幹細胞とヒト羊膜幹細胞が用いられる、実施態様1記載の方法。
（実施態様６）
間葉系幹細胞培養上清液中に、前記2種類以上の間葉系幹細胞を個別に培養した場合には存在しない成分が含まれている、実施態様1記載の方法。
（実施態様７）
間葉系幹細胞培養上清液中に、Double-strand-break repair protein rad21 homolog（RAD21）、Glutamate-cysteine ligase catalytic subunit (GCLC)、およびRho-related GTP-binding protein RhoE（RND3）から成る群から選択される成分が含まれている、実施態様1記載の方法。
（実施態様８）
間葉系幹細胞培養上清液中に、前記2種類以上の間葉系幹細胞を個別に培養した場合に含まれる量に比べて増加した量のDouble-strand-break repair protein rad21 homolog（RAD21）、Glutamate-cysteine ligase catalytic subunit (GCLC)、およびRho-related GTP-binding protein RhoE（RND3）から成る群から選択される成分が含まれている、実施態様1記載の方法。
（実施態様９）
MSCの未分化性を維持する無血清培地、基本培地に血清を添加した培地、ヒト間葉系幹細胞用培養培地、間葉系幹細胞用フィーダーレス培地、および／または化学的に定義された無血清培地中で細胞を培養することを含む、実施態様1記載の方法。
（実施態様１０）
実施態様1～9のいずれか1項記載の方法により得られる細胞培養上清液。
（実施態様１１）
実施態様1～9のいずれか1項記載の方法により得られる細胞培養上清液を含む医薬組成物。
（実施態様１２）
実施態様1～9のいずれか1項記載の方法により得られる細胞培養上清液を含む化粧用組成物 。
（実施態様１３）
実施態様1～9のいずれか1項記載の方法により得られる細胞培養上清液を凍結乾燥させることを含む、間葉系幹細胞培養上清粉末の製造方法。
（実施態様１４）
実施態様13記載の方法により得られる間葉系幹細胞培養上清粉末。
（実施態様１５）
実施態様13記載の方法により得られる間葉系幹細胞培養上清粉末を含む医薬組成物。
（実施態様１６）
実施態様13記載の方法により得られる間葉系幹細胞培養上清粉末を含む化粧用組成物。
（実施態様１７）
眼疾患、眼障害、がん、および脱髄性疾患から成る群から選択される疾患または障害の治療、予防若しくは改善に用いるための、実施態様11に記載の医薬組成物。
（実施態様１８）
眼疾患、眼障害、がん、および脱髄性疾患から成る群から選択される疾患または障害の治療、予防若しくは改善に用いるための、実施態様15に記載の医薬組成物。
The present disclosure is based on these findings and includes the following aspects:
(Embodiment 1)
A method for producing a mesenchymal stem cell culture supernatant, comprising:
i) co-culturing two or more types of mesenchymal stem cells; and
ii) recovering the culture supernatant.
(Embodiment 2)
The method of embodiment 1, further comprising separately culturing said two or more types of mesenchymal stem cells prior to co-culturing.
(Embodiment 3)
The method of embodiment 1, wherein the mesenchymal stem cells are cells derived from a tissue selected from the group consisting of adipose, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, and hair follicles.
(Embodiment 4)
The method of embodiment 1, wherein two types of mesenchymal stem cells are used.
(Embodiment 5)
The method of embodiment 1, wherein human adipose stem cells and human umbilical cord stem cells, human adipose stem cells and human amniotic stem cells, or human umbilical cord stem cells and human amniotic stem cells are used.
(Embodiment 6)
The method according to embodiment 1, wherein the mesenchymal stem cell culture supernatant contains a component that is not present when the two or more types of mesenchymal stem cells are cultured individually.
(Embodiment 7)
The method of embodiment 1, wherein the mesenchymal stem cell culture supernatant contains a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), Glutamate-cysteine ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3).
(Embodiment 8)
The method of embodiment 1, wherein the mesenchymal stem cell culture supernatant contains an increased amount of a component selected from the group consisting of double-strand-break repair protein rad21 homolog (RAD21), Glutamate-cysteine ligase catalytic subunit (GCLC), and Rho-related GTP-binding protein RhoE (RND3) compared to the amount contained when the two or more types of mesenchymal stem cells are cultured individually.
(Embodiment 9)
The method of embodiment 1, comprising culturing the cells in a serum-free medium that maintains the undifferentiated state of MSCs, a medium containing serum added to a basal medium, a culture medium for human mesenchymal stem cells, a feederless medium for mesenchymal stem cells, and/or a chemically defined serum-free medium.
(Embodiment 10)
A cell culture supernatant obtainable by the method according to any one of embodiments 1 to 9.
(Embodiment 11)
A pharmaceutical composition comprising a cell culture supernatant obtainable by the method according to any one of embodiments 1 to 9.
(Embodiment 12)
A cosmetic composition comprising a cell culture supernatant obtainable by the method according to any one of embodiments 1 to 9.
(Embodiment 13)
A method for producing a mesenchymal stem cell culture supernatant powder, comprising freeze-drying a cell culture supernatant obtained by the method according to any one of embodiments 1 to 9.
(Embodiment 14)
Mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13.
(Embodiment 15)
A pharmaceutical composition comprising mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13.
(Embodiment 16)
A cosmetic composition comprising mesenchymal stem cell culture supernatant powder obtained by the method described in embodiment 13.
(Embodiment 17)
12. The pharmaceutical composition of embodiment 11 for use in the treatment, prevention or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases.
(Embodiment 18)
16. The pharmaceutical composition of embodiment 15 for use in the treatment, prevention or amelioration of a disease or disorder selected from the group consisting of eye diseases, eye disorders, cancer, and demyelinating diseases.
上述のとおり、本発明者らは、2種類以上の間葉系幹細胞を共培養することにより、予想外の優れた活性を有する幹細胞培養上清液が得られることを見出した。以下に、本発明を詳細に説明する。 As described above, the present inventors have discovered that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant with unexpectedly excellent activity can be obtained. The present invention is described in detail below.
（MSCの共培養）
本開示の1つの態様は、間葉系幹細胞培養上清液の製造方法に関し、より具体的には、例えば、i）2種類以上の間葉系幹細胞を共培養すること、およびii）培養上清液を回収することを含む、方法に関する。また、本開示の別の態様は、上記の製造方法により得られる細胞培養上清液に関する。
(MSC co-culture)
One embodiment of the present disclosure relates to a method for producing a mesenchymal stem cell culture supernatant, more specifically, the method includes, for example, i) co-culturing two or more types of mesenchymal stem cells, and ii) recovering the culture supernatant. Another embodiment of the present disclosure relates to a cell culture supernatant obtained by the above-mentioned production method.
間葉系幹細胞（MSC）は、臍帯、骨髄、脂肪組織などの複数の組織に存在する多能性の成体幹細胞である。間葉系幹細胞（MSC）は稀な細胞であるが、骨髄には1万から10万個の有核骨髄細胞につき1個の割合で存在すると推定されている。間葉系幹細胞（MSC）は自己増殖能を有し、骨芽細胞（骨細胞）、軟骨細胞、筋細胞、脂肪細胞などの様々な細胞種に分化することができる。 Mesenchymal stem cells (MSCs) are multipotent adult stem cells present in multiple tissues, including the umbilical cord, bone marrow, and adipose tissue. Although MSCs are rare cells, they are estimated to be present in bone marrow at a ratio of 1 per 10,000 to 100,000 nucleated bone marrow cells. MSCs have the ability to self-renew and can differentiate into various cell types, including osteoblasts (bone cells), chondrocytes, muscle cells, and adipocytes.
いくつかの実施形態において、本開示に係る方法で用いられる間葉系幹細胞には、ヒトの組織、例えば、脂肪、臍帯、羊膜、羊水、歯髄、ワルトン膠様質（Wharon’s jelly）、CPJ（Cord Placenta Junction）、絨毛膜、肝臓、肺、脊椎、臍帯血、胎盤、末梢血、真皮、子宮内膜、母乳、または毛包から単離された細胞が含まれるが、これらに限定はされない。 In some embodiments, mesenchymal stem cells used in the methods of the present disclosure include, but are not limited to, cells isolated from human tissues, such as adipose, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, or hair follicles.
間葉系幹細胞（MSC）は、Dominici et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement., Cytotherapy. 2006;8(4):315-7による定義によれば、（1）プラスチック培養容器に接着し、（2）CD105（エンドグリン）、CD73（エクト5’－ヌクレオチダーゼ）、CD90（Thy－1）を陽性マーカー、CD45、CD34、CD14、CD11b、CD79α、CD19、HLA－ClassII（DR）を陰性マーカーとし、（3）骨、脂肪、軟骨への分化能を有する細胞であるとされている。従来的な間葉系幹細胞の単離方法では、プラスチック培養容器に接着して増殖するというMSCの性質が利用されるが、細胞表面マーカーに基づき、FACS（fluorescence activated cell sorting）などを用いてMSCを単離することもできる。FACSでは、蛍光抗体で細胞を染色し、個々の細胞が発する蛍光を測定して分析することによって、特定の細胞を分取（ソーティング）する。FACSシステムとしては、例えば、BDバイオサイエンス社のBD FACSAria（商標）セルソーターを使用することができる。 According to the definition by Dominici et al., Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement., Cytotherapy. 2006;8(4):315-7, mesenchymal stem cells (MSCs) are (1) cells that adhere to plastic culture vessels, (2) cells with positive markers of CD105 (endoglin), CD73 (ecto-5'-nucleotidase), and CD90 (Thy-1), and negative markers of CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-Class II (DR), and (3) cells that have the ability to differentiate into bone, fat, and cartilage. Conventional methods for isolating mesenchymal stem cells utilize the property of MSCs that they adhere to plastic culture vessels and proliferate, but MSCs can also be isolated based on cell surface markers using FACS (fluorescence activated cell sorting) and other methods. In FACS, cells are stained with fluorescent antibodies, and specific cells are sorted by measuring and analyzing the fluorescence emitted by individual cells. For example, a BD FACSAria (trademark) cell sorter from BD Biosciences can be used as a FACS system.
上述のように、間葉系幹細胞は多くの場合、骨髄等の組織から得た細胞を長期間培養した後に、培養皿に付着した細胞を増殖させることによって単離されるが、表面抗原マーカーを用いて間葉系幹細胞を単離する技術も開発されており、例えば、Ogataらは細胞表面マーカーとしてLNGFRとTHY－1の2つを用いてヒトMSCを純化できることを示している（Ogata et al., PLoS One. 2015 Jun 8;10(6):e0129096.）。 As mentioned above, mesenchymal stem cells are often isolated by culturing cells obtained from tissues such as bone marrow for a long period of time and then proliferating the cells that adhere to the culture dish. However, techniques have also been developed to isolate mesenchymal stem cells using surface antigen markers. For example, Ogata et al. have shown that human MSCs can be purified using two cell surface markers, LNGFR and THY-1 (Ogata et al., PLoS One. 2015 Jun 8;10(6):e0129096.).
MSCとして同定されるためには、培養された細胞集団の95％よりも多くがCD73、CD90およびCD105を発現しなければならず、そして、CD11bまたはCD14、CD34、CD45、CD19またはCD79α、およびHLA－DRに関して陰性（≦2％の陽性細胞）でなければならない。 To be identified as MSCs, greater than 95% of the cultured cell population must express CD73, CD90, and CD105, and be negative (≤2% positive cells) for CD11b or CD14, CD34, CD45, CD19 or CD79α, and HLA-DR.
いくつかの実施形態において、本開示に係る方法は、2種類以上の間葉系幹細胞を共培養するステップを含む。例えば、脂肪、臍帯、羊膜、羊水、歯髄、ワルトン膠様質（Wharon’s jelly）、CPJ（Cord Placenta Junction）、絨毛膜、肝臓、肺、脊椎、臍帯血、胎盤、末梢血、真皮、子宮内膜、母乳、または毛包に由来するMSCのうち、任意の2種類以上（例えば、2種類、3種類、4種類、5種類、またはそれ以上）の組み合わせでの共培養を行うことができる。例えば、共培養のために、脂肪幹細胞、臍帯幹細胞、および羊膜幹細胞のうちの2種類、または3種類すべてを選択することができ、例えば、ヒト脂肪幹細胞とヒト臍帯幹細胞の組み合わせ、ヒト脂肪幹細胞とヒト羊膜幹細胞の組み合わせ、またはヒト臍帯幹細胞とヒト羊膜幹細胞の組み合わせが使用されうるが、これらに限定はされない。 In some embodiments, the method of the present disclosure includes a step of co-culturing two or more types of mesenchymal stem cells. For example, any two or more types (e.g., two, three, four, five, or more types) of MSCs derived from fat, umbilical cord, amniotic membrane, amniotic fluid, dental pulp, Wharton's jelly, CPJ (Cord Placenta Junction), chorion, liver, lung, spine, umbilical cord blood, placenta, peripheral blood, dermis, endometrium, breast milk, or hair follicles can be co-cultured. For example, two or all three types of adipose stem cells, umbilical cord stem cells, and amniotic stem cells can be selected for co-culture, and for example, a combination of human adipose stem cells and human umbilical cord stem cells, a combination of human adipose stem cells and human amniotic stem cells, or a combination of human umbilical cord stem cells and human amniotic stem cells can be used, but are not limited thereto.
いくつかの実施形態において、本開示に係る方法は、共培養に先立ち、2種類以上の間葉系幹細胞を個別に培養するステップを含んでいてもよい。 In some embodiments, the method of the present disclosure may include a step of separately culturing two or more types of mesenchymal stem cells prior to co-culturing.
細胞の培養には、接着培養法、浮遊培養法、旋回培養法、ハンギングドロップ培養法、不織布培養法、またはチューブ培養法が用いられうるが、これらに限定はされない。 Cell culture may be performed using, but is not limited to, adhesion culture, suspension culture, rotation culture, hanging drop culture, nonwoven fabric culture, or tube culture.
細胞の培養には、培養用容器として、例えば、接着培養用の培養容器、例えば、コーニング社から入手可能なCellBIND、コーニング社から入手可能な組織培養用ディッシュ、Falcon社から入手可能なセルカルチャーディッシュ、IWAKI社から入手可能な組織培養用ディッシュ、Greinar社から入手可能なCELLSTAR Advancedシャーレなどを使用することができるが、これらに限定はされない。 For cell culture, a culture vessel for adhesion culture, such as CellBIND available from Corning, tissue culture dishes available from Corning, cell culture dishes available from Falcon, tissue culture dishes available from IWAKI, CELLSTAR Advanced petri dishes available from Greinar, etc., can be used, but are not limited to these.
細胞の播種は、例えば、1,000～25,000 cells/cm2の細胞密度、例えば、約1,000 cells/cm2、約2,000 cells/cm2、約3,000 cells/cm2、約4,000 cells/cm2、約5,000 cells/cm2、約6,000 cells/cm2、約7,000 cells/cm2、約8,000 cells/cm2、約9,000 cells/cm2、または約10,000 cells/cm2の細胞密度で行うことができる。なお、本明細書で使用される「約」という用語は、記載されている値の＋／－10％を意味するものとする。つまり、本明細書において、「約1,000」という表現は、900～1,100の範囲に含まれる任意の数値を意味しうる。 The cells can be seeded at a cell density of, for example, 1,000 to 25,000 cells/ cm2 , such as about 1,000 cells/ cm2 , about 2,000 cells/ cm2 , about 3,000 cells/ cm2 , about 4,000 cells/ cm2 , about 5,000 cells/ cm2 , about 6,000 cells/ cm2 , about 7,000 cells/ cm2 , about 8,000 cells/ cm2 , about 9,000 cells/ cm2 , or about 10,000 cells/ cm2 . As used herein, the term "about" refers to +/- 10% of the stated value. In other words, the expression "about 1,000" can refer to any number in the range of 900 to 1,100.
細胞の培養には、培養培地として、例えば、MSCの未分化性を維持する無血清培地、基本培地に血清を添加した培地、ヒト間葉系幹細胞用培養培地、間葉系幹細胞用フィーダーレス培地、化学的に定義された無血清培地、例えば、ニプロ社から入手可能なヒト間葉系幹細胞用培養液（#87-072）、味の素社から入手可能なStemFit For Mesenchymal Stem Cell、コージンバイオ社から入手可能なKBM ADSC-4培地（#16030044）、ロート製薬社から入手可能なR:STEM Medium for hMSC High Growthなどを使用することができるが、これらに限定はされない。 For cell culture, a culture medium that can be used is, for example, a serum-free medium that maintains the undifferentiated state of MSCs, a medium containing serum added to a basal medium, a culture medium for human mesenchymal stem cells, a feederless medium for mesenchymal stem cells, or a chemically defined serum-free medium, such as a culture medium for human mesenchymal stem cells available from Nipro (#87-072), StemFit For Mesenchymal Stem Cell available from Ajinomoto Co., KBM ADSC-4 medium (#16030044) available from Kohjin Bio Co., Ltd., or R:STEM Medium for hMSC High Growth available from Rohto Pharmaceutical Co., Ltd., but is not limited to these.
典型的には、細胞は37℃および5%CO2で培養されるが、条件は適宜調整されうる。細胞は低酸素または高酸素条件で培養されてもよく、また接着培養や浮遊培養、マイクロビーズや不織布を使った大量培養法でも構わない。 Typically, cells are cultured at 37°C and 5% CO2 , but conditions can be adjusted accordingly. Cells may be cultured under hypoxic or hyperoxic conditions, and may be cultured in adherent or suspension culture, or in large-scale culture using microbeads or nonwoven fabrics.
培地交換頻度は、例えば、1日1回、2日に1回、3日に1回、4日に1回、5日に1回、または1週間に1回の頻度でありうる。 The frequency of medium change can be, for example, once a day, once every two days, once every three days, once every four days, once every five days, or once a week.
接着培養によりMSCを単離する際には、組織由来の細胞の培養を少なくとも1回、継代して培養することができる。MSCの単離のために行われる継代の回数は、例えば、1回、2回、3回、4回、5回、6回、7回、8回、9回、または10回でありうるが、これらに限定はされない。 When isolating MSCs by adherent culture, the tissue-derived cells can be passaged at least once. The number of passages performed to isolate MSCs can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, but is not limited to these.
2種類以上の異なる組織に由来するMSCを個別に培養した後、2種類以上の異なる組織に由来するMSCを共培養に付すことができる。共培養の際、各MSCはそれぞれ等しい細胞数（または比率）で播種されてもよいし、異なる細胞数（または比率）で播種されてもよい。2種類の細胞が用いられる場合、比率は、例えば、50：50、40：60、30：70、20：80、または10：90でありうるが、これらに限定はれない。2種類の細胞が用いられる場合、比率は、例えば、約50：50でありうる。3種類以上の細胞が用いられる場合も、任意の比率が用いられうる。 After MSCs derived from two or more different tissues are cultured separately, the MSCs derived from two or more different tissues can be co-cultured. During co-culture, the MSCs may be seeded at equal cell numbers (or ratios) or at different cell numbers (or ratios). When two types of cells are used, the ratio may be, for example, but is not limited to, 50:50, 40:60, 30:70, 20:80, or 10:90. When three or more types of cells are used, any ratio may be used.
いくつかの実施形態において、培養上清液の回収は、共培養を行った細胞がコンフルエントに達した後に行われうる。培養上清液とは、細胞培養の過程で採取される上澄み液のことをいう。培養上清液には、タンパク質の一種であるサイトカインや成長因子が数十から数百種類も含まれており、多岐にわたる治療効果が期待できる。また、遺伝情報（細胞成分やDNA）を含まず、投与に伴う拒絶反応や異物反応が生じにくく、汎用性が高い。いくつかの実施形態において、培養上清液の回収は、共培養を行った細胞がコンフルエントに達した後、培地の交換を行い、さらに培養を行った後（例えば、12時間、1日、2日、3日、4日、または5日間の培養後）に行われうる。 In some embodiments, the culture supernatant may be collected after the co-cultured cells reach confluence. The culture supernatant refers to the supernatant collected during the cell culture process. The culture supernatant contains tens to hundreds of types of cytokines and growth factors, which are a type of protein, and is expected to have a wide range of therapeutic effects. In addition, the culture supernatant does not contain genetic information (cell components and DNA), and is less likely to cause rejection or foreign body reactions upon administration, making it highly versatile. In some embodiments, the culture supernatant may be collected after the co-cultured cells reach confluence, after the medium is replaced, and after further culturing (e.g., after culturing for 12 hours, 1 day, 2 days, 3 days, 4 days, or 5 days).
いくつかの実施形態では、回収した上清液には、滅菌処理（例えば、フィルター滅菌）が行われうる。いくつかの実施形態では、回収した上清液は、凍結保存されうる（例えば、－80℃での凍結保存）。いくつかの実施形態では、回収した上清液は、凍結乾燥されうる。いくつかの実施形態では、凍結乾燥された上清は、粉末化されうる。いくつかの実施形態では、粉末化された上清は、さらに錠剤化されうる。 In some embodiments, the recovered supernatant may be sterilized (e.g., filter sterilized). In some embodiments, the recovered supernatant may be frozen (e.g., frozen at -80°C). In some embodiments, the recovered supernatant may be lyophilized. In some embodiments, the lyophilized supernatant may be powdered. In some embodiments, the powdered supernatant may be further tableted.
本開示の1つの態様は、間葉系幹細胞培養上清粉末の製造方法であって、i）2種類以上の間葉系幹細胞を共培養すること、ii）培養上清液を回収すること、およびiii）回収した培養上清液を凍結乾燥させることを含む、製造方法に関する。また、本開示の別の態様は、上記の製造方法により得られる細胞培養上清粉末に関する。 One aspect of the present disclosure relates to a method for producing a mesenchymal stem cell culture supernatant powder, the method comprising: i) co-culturing two or more types of mesenchymal stem cells; ii) recovering the culture supernatant; and iii) freeze-drying the recovered culture supernatant. Another aspect of the present disclosure relates to a cell culture supernatant powder obtained by the above-mentioned production method.
いくつかの実施形態では、本開示に係る共培養法により得られる細胞培養上清液には、2種類以上の間葉系幹細胞を個別に培養した場合には存在しない成分が含まれている。細胞培養上清液中に含まれる成分は、例えば、プロテオーム解析により調べることができる。表1に本開示に係る共培養法により得られる細胞培養上清液に含まれるが、各間葉系幹細胞を個別に培養した場合には存在しない成分をまとめた。 In some embodiments, the cell culture supernatant obtained by the co-culture method according to the present disclosure contains components that are not present when two or more types of mesenchymal stem cells are cultured individually. The components contained in the cell culture supernatant can be examined, for example, by proteomic analysis. Table 1 lists the components that are contained in the cell culture supernatant obtained by the co-culture method according to the present disclosure but are not present when each type of mesenchymal stem cell is cultured individually.
いくつかの実施形態では、間葉系幹細胞培養上清液中に、Glutamate-cysteine ligase catalytic subunit (GCLC)、Rho-related GTP-binding protein RhoE（RND3）、およびDouble-strand-break repair protein rad21 homolog（RAD21）から成る群から選択される成分が含まれている。これらの成分は、脂肪、臍帯、羊膜由来のMSCをそれぞれ個別に培養した場合の細胞培養上清液には含まれていないが、脂肪MSC、臍帯MSC、羊膜MSCのうちの2つを組み合わせて共培養した細胞培養上清液中には存在している。 In some embodiments, the mesenchymal stem cell culture supernatant contains a component selected from the group consisting of Glutamate-cysteine ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21). These components are not present in the cell culture supernatant when adipose-, umbilical cord-, or amniotic membrane-derived MSCs are cultured individually, but are present in the cell culture supernatant when two of adipose MSCs, umbilical cord MSCs, or amniotic membrane MSCs are co-cultured in combination.
よって、本開示の別の態様は、Glutamate-cysteine ligase catalytic subunit (GCLC)、Rho-related GTP-binding protein RhoE（RND3）、およびDouble-strand-break repair protein rad21 homolog（RAD21）から成る群から選択される成分を含む、間葉系幹細胞培養上清液に関する。さらに、本開示の別の態様は、Glutamate-cysteine ligase catalytic subunit (GCLC)、Rho-related GTP-binding protein RhoE（RND3）、およびDouble-strand-break repair protein rad21 homolog（RAD21）から成る群から選択される成分を含む、間葉系幹細胞培養上清粉末に関する。 Therefore, another aspect of the present disclosure relates to a mesenchymal stem cell culture supernatant liquid containing an ingredient selected from the group consisting of Glutamate-cysteine ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21). Furthermore, another aspect of the present disclosure relates to a mesenchymal stem cell culture supernatant powder containing an ingredient selected from the group consisting of Glutamate-cysteine ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21).
また、本開示の別の態様は、Glutamate-cysteine ligase catalytic subunit (GCLC)、Rho-related GTP-binding protein RhoE（RND3）、およびDouble-strand-break repair protein rad21 homolog（RAD21）から成る群から選択されるタンパク質の製造方法に関する。 Another aspect of the present disclosure relates to a method for producing a protein selected from the group consisting of Glutamate-cysteine ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21).
いくつかの実施形態では、本開示に係る共培養法により得られる細胞培養上清液には、2種類以上の間葉系幹細胞を個別に培養した場合に含まれる量に比べて増加した量のGlutamate-cysteine ligase catalytic subunit (GCLC)、Rho-related GTP-binding protein RhoE（RND3）、およびDouble-strand-break repair protein rad21 homolog（RAD21）から成る群から選択される成分が含まれている。 In some embodiments, the cell culture supernatant obtained by the co-culture method of the present disclosure contains an increased amount of a component selected from the group consisting of Glutamate-cysteine ligase catalytic subunit (GCLC), Rho-related GTP-binding protein RhoE (RND3), and double-strand-break repair protein rad21 homolog (RAD21) compared to the amount contained when two or more types of mesenchymal stem cells are cultured individually.
（医薬組成物）
本開示の1つの態様は、上記の細胞培養上清液、または上記の細胞培養上清粉末を含む医薬組成物に関する。
Pharmaceutical Compositions
One embodiment of the present disclosure relates to a pharmaceutical composition comprising the above-mentioned cell culture supernatant liquid or the above-mentioned cell culture supernatant powder.
一般的に、MSC培養上清液には、皮膚などの組織の再生・修復効果、創傷治癒効果、血管新生効果などがあると考えられている。また、表２に示されているように、例えば、Glutamate-cysteine ligase catalytic subunit (GCLC)は、がん（例えば、結腸がん）に対して抑制的に働くことから、GCLCを含む本開示に係るMSC培養上清液は、抗がん剤として、がんの治療／予防に利用することができる。また、Rho-related GTP-binding protein RhoE（RND3）は、オリゴデンドロサイトの形成とミエリン化に必要であり、RND3を含む本開示に係るMSC培養上清液は、多発性硬化症などの脱髄性疾患の治療／予防に利用されうる。さらに、Double-strand-break repair protein rad21 homolog（RAD21）は、角膜基質の発生において重要な働きを担うことが知られており、RAD21を含む本開示に係るMSC培養上清液は、眼疾患および眼障害（例えば、強膜化角膜を含む）の薬剤として有用となりうる。さらに、これらいずれの因子もUV刺激による細胞死及び傷害に関与しており、化粧品（日焼け止めクリームなど）としての有用性が想定される。 In general, MSC culture supernatant is believed to have effects such as regeneration and repair of tissues such as skin, wound healing, and angiogenesis. As shown in Table 2, for example, Glutamate-cysteine ligase catalytic subunit (GCLC) acts to suppress cancer (e.g., colon cancer), and therefore the MSC culture supernatant according to the present disclosure containing GCLC can be used as an anticancer drug for the treatment/prevention of cancer. In addition, Rho-related GTP-binding protein RhoE (RND3) is necessary for the formation and myelination of oligodendrocytes, and the MSC culture supernatant according to the present disclosure containing RND3 can be used for the treatment/prevention of demyelinating diseases such as multiple sclerosis. Furthermore, double-strand-break repair protein rad21 homolog (RAD21) is known to play an important role in the development of the corneal stroma, and the MSC culture supernatant of the present disclosure, which contains RAD21, may be useful as a drug for eye diseases and disorders (e.g., including scleralized cornea). Furthermore, all of these factors are involved in cell death and damage caused by UV stimulation, and are expected to be useful as cosmetics (such as sunscreen creams).
いくつかの実施形態は、がん、脱髄性疾患、眼疾患および眼障害から成る群から選択される疾患または障害の治療、予防若しくは改善に用いるための医薬組成物に関する。 Some embodiments relate to a pharmaceutical composition for use in treating, preventing, or ameliorating a disease or disorder selected from the group consisting of cancer, a demyelinating disease, an eye disease, and an eye disorder.
本開示に係る本開示に係る医薬組成物は、錠剤、粉末、液体、半固体などの任意の形態をとることができる。本開示に係る医薬組成物は、皮膚を通して、あるいは皮膚病巣に直接加える局所治療に用いる塗布剤のような外用薬であってもよく、基剤に各種の主剤を配合して調製されうる。本開示に係る医薬組成物は、上記の有効成分に加えて、薬学的に許容可能な賦形剤、添加剤、緩衝剤、等張調節のための塩類、抗酸化剤、保存剤、薬剤安定剤等を含みうる。賦形剤としては、例えば、水、精製水、アルコール、グリセリン、乳糖、デンプン、デキストリン、白糖、沈降シリカ、蜂蜜、コメデンプン、トラガントが挙げられるが、これらに限定はされない。また、本開示に係る医薬組成物には、他の活性成分が配合されていてもよい。各成分の配合量は、医薬として許容される範囲で適宜決定することができる。また、組成物の投与量は、使用する薬剤の種類、投与する対象に応じて、適宜決定することができる。例えば、有効成分は、0．01～15重量％、例えば、0．1～5重量％とすることもできる。 The pharmaceutical composition according to the present disclosure may take any form, such as a tablet, powder, liquid, or semisolid. The pharmaceutical composition according to the present disclosure may be an external medicine, such as a liniment for topical treatment applied through the skin or directly to a skin lesion, and may be prepared by blending various main agents with a base. In addition to the above-mentioned active ingredients, the pharmaceutical composition according to the present disclosure may contain pharma- ceutical acceptable excipients, additives, buffers, salts for adjusting isotonicity, antioxidants, preservatives, drug stabilizers, and the like. Examples of excipients include, but are not limited to, water, purified water, alcohol, glycerin, lactose, starch, dextrin, sucrose, precipitated silica, honey, rice starch, and tragacanth. The pharmaceutical composition according to the present disclosure may also contain other active ingredients. The amount of each ingredient may be appropriately determined within a medicament-acceptable range. The dosage of the composition may be appropriately determined depending on the type of drug used and the subject to which it is administered. For example, the active ingredient may be 0.01 to 15% by weight, e.g., 0.1 to 5% by weight.
投与経路についても、使用する薬剤の種類、投与する対象に応じて、適宜決定することができる。本開示に係る医薬組成物の投与方法としては特に制限されないが、血管内投与（好ましくは静脈内投与）、腹腔内投与、腸管内投与、皮下投与、皮内投与、筋肉内投与、点眼等を好適に例示することができ、中でも、血管内投与をより好適に例示することができる。本開示に係る医薬組成物は、ドレッシング材のような創傷被覆材に含まれていてもよい。ドレッシング材は、湿潤環境を維持して創傷治癒に最適な環境を提供することのできる医療材料である。 The route of administration can also be appropriately determined depending on the type of drug used and the subject to which it is administered. The method of administration of the pharmaceutical composition according to the present disclosure is not particularly limited, but suitable examples include intravascular administration (preferably intravenous administration), intraperitoneal administration, intraintestinal administration, subcutaneous administration, intradermal administration, intramuscular administration, eye drops, etc., and among these, intravascular administration is more suitable. The pharmaceutical composition according to the present disclosure may be contained in a wound covering material such as a dressing material. A dressing material is a medical material that can maintain a moist environment and provide an optimal environment for wound healing.
哺乳動物としては、例えば、ヒト、ならびにサル、マウス、ラット、ウサギ、イヌ、ネコ、ヒツジ、ヤギ、アルパカ、ウマ、ウシ、ブタ、ミンク、キツネ、テン、タヌキ、チンチラ、ラッコ、カワウソ、ビーバー、アザラシなどの非ヒト哺乳動物が含まれる。投与量は、使用する薬剤の種類、投与する対象に応じて、適宜決定することができる。投与経路についても、使用する薬剤の種類、投与する対象に応じて、適宜決定することができる。好ましい投与経路としては、例えば、液剤、ローション剤、クリーム剤の皮膚への塗布または噴霧、あるいは、液剤の皮下注射、静脈内注射、髄腔内注射、点眼、固形剤、液剤の経口投与を挙げることができる。本開示に係る組成物を含有する貼付剤を調製して皮膚に適用してもよい。 Mammals include, for example, humans, as well as non-human mammals such as monkeys, mice, rats, rabbits, dogs, cats, sheep, goats, alpacas, horses, cattle, pigs, minks, foxes, martens, raccoon dogs, chinchillas, sea otters, otters, beavers, and seals. The dosage can be appropriately determined depending on the type of drug used and the recipient. The route of administration can also be appropriately determined depending on the type of drug used and the recipient. Preferred routes of administration include, for example, application or spraying of a liquid, lotion, or cream to the skin, or subcutaneous injection, intravenous injection, intrathecal injection, eye drops, and oral administration of a solid or liquid agent. A patch containing the composition according to the present disclosure may be prepared and applied to the skin.
また、本開示の1つの態様は、処置を必要とする対象に対して、治療有効量の本開示に係る医薬組成物を投与することを含む、疾患または障害の治療および／または予防するための方法に関し、ここで、疾患または障害は、がん、脱髄性疾患、眼疾患および眼障害から成る群から選択されうる。さらに、本開示の別の態様は、疾患または障害の治療および／または予防に用いるための医薬品の製造における、本開示に係る細胞培養上清液の使用に関し、ここで、疾患または障害は、がん、脱髄性疾患、眼疾患および眼障害から成る群から選択されうる。 Also, one aspect of the present disclosure relates to a method for treating and/or preventing a disease or disorder, comprising administering to a subject in need of treatment a therapeutically effective amount of a pharmaceutical composition according to the present disclosure, wherein the disease or disorder may be selected from the group consisting of cancer, a demyelinating disease, an eye disease, and an eye disorder. Still further, another aspect of the present disclosure relates to the use of a cell culture supernatant according to the present disclosure in the manufacture of a medicament for use in treating and/or preventing a disease or disorder, wherein the disease or disorder may be selected from the group consisting of cancer, a demyelinating disease, an eye disease, and an eye disorder.
（化粧用組成物）
本開示の1つの態様は、上記の細胞培養上清液、または上記の細胞培養上清粉末を含む化粧用組成物に関する。表３に示されているように、本開示に係る共培養によって得られる細胞培養上清液には、皮膚にとって有益な成分が多数含まれていると考えられる。
(Cosmetic Composition)
One aspect of the present disclosure relates to a cosmetic composition containing the above cell culture supernatant or the above cell culture supernatant powder. As shown in Table 3, the cell culture supernatant obtained by the co-culture according to the present disclosure is believed to contain many components that are beneficial to the skin.
実施形態の1つにおいて、本開示に係る化粧用組成物は、スキンケアに用いるための組成物である。いくつかの実施形態において、本開示に係る化粧用組成物は、例えば、皮膚のシワ、シミ、および／またはハリの改善のために、または皮膚の再生の促進のために用いられうる。実施形態の1つにおいて、本開示に係る化粧用組成物は、皮膚表面への適用のための組成物に関する。本組成物は、限定はされないが、溶液、懸濁液、ローション、クリーム、ゲル、化粧水、スティック、ペンシル、スプレー、エアゾール、軟膏、クレンジング洗剤液およびクレンジング棒状固形物、シャンプーおよびヘアコンディショナー、パスタ、フォーム、パウダー、ムース、髭剃りクリーム、ワイプ、ストリップ、パッチ、電動パッチ、創傷被覆材および粘着性包帯、ヒドロゲル、フィルム形成製品、顔および皮膚マスク（不溶性シートを有するおよび有さない）、ファンデーション、アイライナーおよびアイシャドウなどのメイクアップ用品などの様々な製品形態をとり得る。 In one embodiment, the cosmetic composition according to the present disclosure is a composition for use in skin care. In some embodiments, the cosmetic composition according to the present disclosure may be used, for example, to improve wrinkles, age spots, and/or firmness of the skin, or to promote skin regeneration. In one embodiment, the cosmetic composition according to the present disclosure relates to a composition for application to a skin surface. The composition may take a variety of product forms, including, but not limited to, solutions, suspensions, lotions, creams, gels, toners, sticks, pencils, sprays, aerosols, ointments, cleansing liquids and bars, shampoos and hair conditioners, pastes, foams, powders, mousses, shaving creams, wipes, strips, patches, power patches, wound dressings and adhesive bandages, hydrogels, film-forming products, face and skin masks (with and without insoluble sheets), makeup products such as foundations, eyeliners and eyeshadows.
本開示に係る化粧用組成物は、皮膚抗老化剤、皮膚色調剤、抗炎症剤、日焼け止め剤のうちの少なくとも1つを更に含んでいてもよい。皮膚抗老化剤としては、例えば、抗老化作用を有するペプチド（WO2014157485A1）が挙げられるが、これに限定はされない。好適な皮膚色調剤としては、糖アミン、アルブチン、デオキシアルブチン、ヘキシルレゾルシノール、コウジ酸、ヘキサミジン化合物、サリチル酸、並びにプロピオン酸レチノールおよびプロピオン酸レチニルを含むレチノイドが挙げられるが、これらに限定されない。 The cosmetic composition according to the present disclosure may further comprise at least one of a skin anti-aging agent, a skin toning agent, an anti-inflammatory agent, and a sunscreen agent. Examples of skin anti-aging agents include, but are not limited to, peptides having anti-aging activity (WO2014157485A1). Suitable skin toning agents include, but are not limited to, sugar amines, arbutin, deoxyarbutin, hexylresorcinol, kojic acid, hexamidine compounds, salicylic acid, and retinoids including retinol propionate and retinyl propionate.
好適な抗炎症剤としては、非ステロイド系抗炎症剤（イブプロフェン、ナプロキセンなどのNSAIDS）、グリチルリチン酸（グリチルレチン酸グリコシドとしても既知である）、およびグリチルリチン酸ジカリウムなどの塩、グリシルレテン酸（glycyrrhetenic acid）、甘草抽出物、ビサボロール（例えば、アルファビサボロール）、マンジスタ（アカネ属の植物、特にインドアカネ（Rubia cordifolia）から抽出される）、およびガッグル（guggal）（コンミフォラ属の植物、特にグッグル（Commiphora mukul）から抽出される）、コーラノキ抽出物、カミツレ、ムラサキツメクサ抽出物、およびムチサンゴ抽出物（ヤギ目の植物からの抽出物）、上記のいずれかの誘導体、およびこれらの混合物が挙げられるがこれらに限定されない。 Suitable anti-inflammatory agents include, but are not limited to, nonsteroidal anti-inflammatory agents (NSAIDS, such as ibuprofen, naproxen, etc.), glycyrrhizic acid (also known as glycyrrhetinic acid glycoside) and salts such as dipotassium glycyrrhizinate, glycyrrhetenic acid, licorice extract, bisabolol (e.g., alpha bisabolol), manjistha (extracted from plants of the genus Rubia, particularly Rubia cordifolia), and guggal (extracted from plants of the genus Commiphora, particularly Commiphora mukul), kola extract, chamomile, red clover extract, and whip coral extract (extract from plants of the order Gorgonian), derivatives of any of the above, and mixtures thereof.
好適な日焼け止め剤は、2－エチルヘキシル－p－メトキシシンナメート、4，4'－t－ブチルメトキシジベンゾイルメタン、2－ヒドロキシ－4－メトキシベンゾフェノン、オクチルジメチル－p－アミノ安息香酸、ジガロイルトリオレエート、2，2－ジヒドロキシ－4－メトキシベンゾフェノン、エチル－4－（ビス（ヒドロキシプロピル）アミノベンゾエート、2－エチルヘキシル－2－シアノ－3，3－ジフェニルアクリレート、2－エチルヘキシルサリチレート、グリセリル－p－アミノベンゾエート、3，3，5－トリ－メチルシクロヘキシルサリチレート、アントラニル酸メチル、p－ジメチル－アミノ安息香酸またはアミノベンゾエート、2－エチルヘキシル－p－ジメチルアミノベンゾエート、2－フェニルベンゾイミダゾール－5－スルホン酸、2－（p－ジメチルアミノフェニル）－5－スルホンベンゾオキサゾイン酸、オクトクリレン、酸化亜鉛、ベンジリデンカンファーおよびその誘導体、二酸化チタン、並びにこれらの混合物が挙げられるが、これらに限定はされない。 Suitable sunscreens include 2-ethylhexyl-p-methoxycinnamate, 4,4'-t-butylmethoxydibenzoylmethane, 2-hydroxy-4-methoxybenzophenone, octyldimethyl-p-aminobenzoic acid, digalloyl trioleate, 2,2-dihydroxy-4-methoxybenzophenone, ethyl-4-(bis(hydroxypropyl)aminobenzoate, 2-ethylhexyl-2-cyano-3,3-diphenylacrylate, 2-ethylhexyl salicylate, glyceryl-p-amino These include, but are not limited to, benzoate, 3,3,5-tri-methylcyclohexyl salicylate, methyl anthranilate, p-dimethyl-aminobenzoic acid or aminobenzoate, 2-ethylhexyl-p-dimethylaminobenzoate, 2-phenylbenzimidazole-5-sulfonic acid, 2-(p-dimethylaminophenyl)-5-sulfonebenzoxazoic acid, octocrylene, zinc oxide, benzylidene camphor and its derivatives, titanium dioxide, and mixtures thereof.
本開示係る化粧用組成物は、化粧品分野において従来から使用され、本開示に係る組成物の特性には影響を及ぼさない、少なくとも1種の添加剤、例えば、増粘剤、香料、真珠光沢剤、保存料、日焼け防止剤、陰イオンもしくは非イオンもしくは陽イオンあるいは両性ポリマー、タンパク質、タンパク質加水分解物、例えば18－メチルエイコサン酸、ビタミン、パンテノール、シリコン、植物油、動物油、鉱物油または合成油等の脂肪酸、ゲル化剤、酸化防止剤、溶媒、フィルター、スクリーニング剤、臭気吸収剤、着色料、研磨剤、吸収剤、芳香剤などの美容成分、色素、顔料／着色剤、精油、アンチケーキング剤、消泡剤、抗菌剤、結合剤、生物学的添加剤、緩衝剤、充填剤、キレート化剤、化学添加剤、美容収斂剤、美容殺生物剤、変性剤、薬物収斂剤、皮膚軟化剤、外用鎮痛剤、フィルム形成剤または材料、乳白剤、pH調整剤、保存剤、噴霧剤、還元剤、捕捉剤、皮膚冷却剤、皮膚保護剤、増粘剤粘度調節剤、ビタミン、およびこれらの組み合わせも含んでもよい。これらの添加剤は、組成物の総重量に関し、限定されないが、好ましくはまたは有利には0から50重量％、5から40重量％または30から50重量％の範囲に収まる割合で、本開示に係る組成物中に存在し得る。 The cosmetic composition according to the present disclosure may contain at least one additive that has been conventionally used in the cosmetic field and does not affect the properties of the composition according to the present disclosure, such as a thickener, a fragrance, a pearlescent agent, a preservative, a sunscreen, an anionic, nonionic, cationic or amphoteric polymer, a protein, a protein hydrolysate, such as 18-methyl eicosanoic acid, a vitamin, panthenol, silicone, a fatty acid such as a vegetable oil, an animal oil, a mineral oil or a synthetic oil, a gelling agent, an antioxidant, a solvent, a filter, a scrub, etc. The cosmetic ingredients may also include cosmetic ingredients such as cleaning agents, odor absorbers, colorants, abrasives, absorbents, fragrances, dyes, pigments/colorants, essential oils, anti-caking agents, defoamers, antimicrobial agents, binders, biological additives, buffers, fillers, chelating agents, chemical additives, cosmetic astringents, cosmetic biocides, denaturants, drug astringents, emollients, topical analgesics, film formers or materials, opacifiers, pH adjusters, preservatives, propellants, reducing agents, scavengers, skin cooling agents, skin protectants, thickeners, viscosity regulators, vitamins, and combinations thereof. These additives may be present in the compositions according to the present disclosure in proportions that are not limited to, but are preferably or advantageously in the range of 0 to 50% by weight, 5 to 40% by weight, or 30 to 50% by weight, based on the total weight of the composition.
本開示係る化粧用組成物は、皮膚への局所適用のために、特に、水性または油性溶液、ローションまたはセラム型の分散物、水相中への脂肪相の分散（O／W）またはその逆（W／O）によって得られる、ミルク型の液体または半液体の稠度を有する乳液、水性もしくは無水ゲルまたはクリーム型の軟稠度を有する懸濁液または乳液、他にはマイクロカプセルもしくは微粒子、イオンおよび／または非イオン型の小胞分散物、或いは泡状の形態を有し得る。これらの組成物は、通常の方法に従って調製される。組成物は、5から99．5％の間の水相を有することが好ましい。 The cosmetic compositions according to the present disclosure may be, for topical application to the skin, in particular in the form of aqueous or oily solutions, lotion or serum type dispersions, emulsions with a liquid or semi-liquid consistency of the milk type obtained by dispersion of a fatty phase in an aqueous phase (O/W) or vice versa (W/O), suspensions or emulsions with a soft consistency of the aqueous or anhydrous gel or cream type, or else in the form of microcapsules or microparticles, vesicular dispersions of ionic and/or non-ionic type, or foams. These compositions are prepared according to the usual methods. The compositions preferably have an aqueous phase between 5 and 99.5%.
本開示に係る化粧用組成物のpHは、限定されないが、一般的には2から12の間であり、好ましくは3から9の間である。pHは、例えば、アンモニア、水酸化ナトリウム、水酸化カリウム、またはモノエタノールアミン、ジエタノールアミン、トリエタノールアミン、イソプロパノールアミンもしくは1，3－プロパンジアミン等の第1級、第2級もしくは第3級（ポリ）アミンの塩基（有機または無機）を組成物に添加することにより、またはリジンもしくはアルギニンのような塩基性アミノ酸もしくはポリアミノ酸を伴って、あるいは、無機酸もしくは有機酸、好ましくは、例えばクエン酸等のカルボン酸を添加することにより、目標値に調製され得る。 The pH of the cosmetic composition according to the present disclosure is not limited, but is generally between 2 and 12, preferably between 3 and 9. The pH can be adjusted to the target value by adding a base (organic or inorganic) to the composition, for example, ammonia, sodium hydroxide, potassium hydroxide, or a primary, secondary or tertiary (poly)amine, such as monoethanolamine, diethanolamine, triethanolamine, isopropanolamine or 1,3-propanediamine, or with a basic amino acid or polyamino acid, such as lysine or arginine, or by adding an inorganic or organic acid, preferably a carboxylic acid, such as, for example, citric acid.
本開示に係る化粧用組成物は、特に、顔、手、足、主要な身体構造上のひだもしくは体を、洗う、保護する、処理するもしくはケアするためのクリーム（例えば、デイクリーム、ナイトクリーム、アンチエイジングクリーム、保湿クリーム、メイクアップ除去用クリーム、ファンデーションクリーム、またはサンクリーム）、リキッドファンデーション、メイクアップ除去用ミルク、保護用もしくはケア用ボディミルク、サンミルク、ローション、皮膚をケアするためのゲルまたはフォーム、例えばクレンジングローション、サンローション、人工日焼けローション、入浴用組成物、殺菌剤を含む脱臭組成物、アフターシェーブゲルまたはローション、脱毛クリーム、虫刺されに対応するための組成物を構成する。 The cosmetic compositions according to the present disclosure comprise, in particular, creams for washing, protecting, treating or caring for the face, hands, feet, major anatomical folds or body (e.g. day creams, night creams, anti-aging creams, moisturizing creams, make-up removing creams, foundation creams or sun creams), liquid foundations, make-up removing milks, protective or caring body milks, sun milks, lotions, gels or foams for caring for the skin, e.g. cleansing lotions, sun lotions, artificial tanning lotions, bath compositions, deodorant compositions containing a germicide, aftershave gels or lotions, depilatory creams, compositions for treating insect bites.
本開示に係る化粧用組成物は、処置期間中、少なくとも1日1回、1日2回、または1日により頻繁に適用され得る。1日2回適用する場合、1回目と2回目の適用には、例えば、少なくとも1～12時間の間隔をあける。典型的には、組成物は、朝および／または就寝前の夜に適用され得る。 The cosmetic compositions of the present disclosure may be applied at least once a day, twice a day, or more frequently per day during the treatment period. If applied twice a day, the first and second applications may be separated, for example, by at least 1-12 hours. Typically, the compositions may be applied in the morning and/or in the evening before bedtime.
また、本開示の1つの態様は、処置を必要とする対象に対して、有効量の本開示に係る化粧用組成物を投与または塗布することを含む、美容方法に関する。さらに、本開示の1つの態様は、化粧品の製造における、本開示に係る細胞培養上清液の使用に関する。 An embodiment of the present disclosure also relates to a cosmetic method comprising administering or applying an effective amount of a cosmetic composition according to the present disclosure to a subject in need of treatment. Furthermore, an embodiment of the present disclosure relates to the use of a cell culture supernatant according to the present disclosure in the manufacture of a cosmetic product.
以下に、実施例を示して本発明を具体的に説明するが、これらにより本発明は何ら制限を受けるものではない。 The present invention will be specifically explained below with reference to examples, but the present invention is not limited in any way by these examples.
＜例1：MSCの共培養＞
ヒト脂肪由来MSC細胞、ヒト臍帯由来MSC細胞、ヒト羊膜由来MSC細胞をそれぞれ以下の方法で培養した。細胞培養プレート上に細胞密度5,000 cells/cm2で細胞を播種し、MSC培養培地中で培養した。培地交換は2～3日に1度の頻度で行った。脂肪由来MSC、臍帯由来MSC、羊膜由来MSCを複数回継代した後、酵素処理により細胞剥離を行い、その後、下記の6条件で細胞播種を行った。
<Example 1: Co-culture of MSCs>
Human adipose-derived MSC cells, human umbilical cord-derived MSC cells, and human amnion-derived MSC cells were cultured using the following method. Cells were seeded on cell culture plates at a cell density of 5,000 cells/ cm2 and cultured in MSC culture medium. Medium was changed every 2-3 days. Adipose-derived MSCs, umbilical cord-derived MSCs, and amnion-derived MSCs were passaged multiple times, after which the cells were detached by enzyme treatment and seeded under the following 6 conditions.
1）ヒト脂肪由来MSC細胞を5,000 cells/cm2の細胞密度で播種する群
2）ヒト臍帯由来MSC細胞を5,000 cells/cm2の細胞密度で播種する群
3）ヒト羊膜由来MSC細胞を5,000 cells/cm2の細胞密度で播種する群
4）脂肪MSCと臍帯MSCを1:1の割合で合計5,000 cells/cm2の細胞密度で播種する群
5）臍帯MSCと羊膜MSCを1:1の割合で合計5,000 cells/cm2の細胞密度で播種する群
6）脂肪MSCと羊膜MSCを1:1の割合で合計5,000 cells/cm2の細胞密度で播種する群
1) Human adipose-derived MSC cells were seeded at a cell density of 5,000 cells/ cm2
2) Human umbilical cord-derived MSC cells were seeded at a cell density of 5,000 cells/ cm2
3) Human amnion-derived MSC cells were seeded at a cell density of 5,000 cells/ cm2.
4) Adipose MSCs and umbilical cord MSCs were seeded at a 1:1 ratio at a total cell density of 5,000 cells/ cm2.
5) A group in which umbilical cord MSCs and amniotic membrane MSCs were seeded in a 1:1 ratio at a total cell density of 5,000 cells/ cm2
6) Adipose MSCs and amniotic MSCs were seeded at a 1:1 ratio at a total cell density of 5,000 cells/ cm2.
これらの細胞を播種し、コンフルエントに達した段階で培地を取り除き、PBSによる洗浄を2回繰り返した後に、基本培地を加えた。基本培地を加えて培養を行った後、培地を回収し、細胞培養上清液を取得した。上清液を0.22μmフィルターでろ過滅菌し、その後、－80℃で保管した。 These cells were seeded, and when they reached confluence, the medium was removed, and the cells were washed twice with PBS, after which basal medium was added. After culturing with the addition of basal medium, the medium was collected and the cell culture supernatant was obtained. The supernatant was sterilized by filtration using a 0.22 μm filter, and then stored at -80°C.
＜例2：上清液のプロテオーム解析＞
回収した上清液中の成分のプロテオーム解析はプロメガ株式会社（東京）に依頼して行った。その結果、表1に示すように、脂肪由来MSC、臍帯由来MSC、羊膜由来MSCをそれぞれ個別に培養した場合の細胞培養上清液中には存在せず、共培養を行った場合にのみ存在が確認される複数の成分が同定された。表1には、脂肪由来MSC、臍帯由来MSC、羊膜由来MSCをそれぞれ個別に培養した場合、および脂肪×臍帯、臍帯×羊膜、脂肪×羊膜の組み合わせで共培養した場合に細胞培養上清液中に存在する各タンパク質の定量値がまとめられている。この結果は、共培養により得られる細胞培養上清液が、従来のような単独培養で得られる細胞培養上清液とは異なる活性を有しており、医薬品または化粧品として有用となることを示している。表2および表3に、これらの成分の一部（GCLC、RAD21、RND3）の薬理効果および美容効果をまとめた。
<Example 2: Proteome analysis of supernatant>
Promega Corporation (Tokyo) was commissioned to perform proteome analysis of the components in the collected supernatant. As a result, as shown in Table 1, several components were identified that were not present in the cell culture supernatant when adipose-derived MSCs, umbilical cord-derived MSCs, and amniotic membrane-derived MSCs were cultured individually, but were present only when co-cultured. Table 1 summarizes the quantitative values of each protein present in the cell culture supernatant when adipose-derived MSCs, umbilical cord-derived MSCs, and amniotic membrane-derived MSCs were cultured individually, and when co-cultured in the combinations of adipose x umbilical cord, umbilical cord x amniotic membrane, and adipose x amniotic membrane. This result indicates that the cell culture supernatant obtained by co-culture has different activities from the cell culture supernatant obtained by conventional single culture, and is useful as a pharmaceutical or cosmetic. Tables 2 and 3 summarize the pharmacological and cosmetic effects of some of these components (GCLC, RAD21, RND3).
本明細書には、本発明の好ましい実施態様を示してあるが、そのような実施態様が単に例示の目的で提供されていることは、当業者には明らかであり、当業者であれば、本発明から逸脱することなく、様々な変形、変更、置換を加えることが可能であろう。本明細書に記載されている発明の様々な代替的実施形態が、本発明を実施する際に使用されうることが理解されるべきである。また、本明細書中において参照している特許および特許出願書類を含む、全ての刊行物に記載の内容は、その引用によって、本明細書中に明記された内容と同様に取り込まれていると解釈すべきである。 While the present specification illustrates preferred embodiments of the present invention, it will be apparent to those skilled in the art that such embodiments are provided by way of example only, and that various modifications, changes, and substitutions may be made by those skilled in the art without departing from the present invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the present invention. In addition, the contents of all publications, including patents and patent applications, referenced in this specification should be construed as being incorporated by reference as if set forth herein.
本発明者らは、2種類以上の間葉系幹細胞を共培養することにより、予想外の優れた活性を有する幹細胞培養上清液が得られることを見出した。例えば、脂肪、臍帯、羊膜由来のMSCのうち2つを組み合わせて共培養することによって、各MSC単独の培養上清液に含まれない成分を有する上清液を得ることができる。共培養によって得られる成分には、医薬品または化粧品として有用な効能を有するものが含まれており、産業上の利用が可能である。
The present inventors have found that by co-culturing two or more types of mesenchymal stem cells, a stem cell culture supernatant with unexpectedly excellent activity can be obtained. For example, by co-culturing two of MSCs derived from fat, umbilical cord, or amniotic membrane, a supernatant containing components not contained in the culture supernatant of each MSC alone can be obtained. The components obtained by co-culturing include those that have useful efficacy as pharmaceuticals or cosmetics, and can be used industrially.
Claims (13)
i）2種類以上の間葉系幹細胞を共培養すること、および
ii）培養上清液を回収すること
を含み、
脂肪幹細胞と臍帯幹細胞が用いられる、方法。 A method for producing a mesenchymal stem cell culture supernatant , the method being for producing a pharmaceutical composition or for producing a cosmetic composition, comprising:
i) co-culturing two or more types of mesenchymal stem cells; and
ii) recovering the culture supernatant,
A method in which adipose stem cells and umbilical cord stem cells are used.
i）2種類以上の間葉系幹細胞を共培養すること、および
ii）培養上清液を回収すること
を含み、
脂肪幹細胞と臍帯幹細胞が用いられ、
細胞培養上清液を凍結乾燥させることを含む、方法。 A method for producing a mesenchymal stem cell culture supernatant powder , the method being for producing a pharmaceutical composition or for producing a cosmetic composition, comprising:
i) co-culturing two or more types of mesenchymal stem cells; and
ii) recovering the culture supernatant,
Adipose stem cells and umbilical cord stem cells are used,
A method comprising freeze-drying a cell culture supernatant.
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| WO2015170347A2 (en) | 2014-05-09 | 2015-11-12 | Reelabs Private Limited | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders |
| JP2020164473A (en) | 2019-03-29 | 2020-10-08 | 株式会社再生医学研究所 | Hair restorer containing dental pulp stem cell culture supernatant and dermal papilla stem cell culture supernatant and method for producing the same |
| CN113444689A (en) | 2021-07-21 | 2021-09-28 | 东莞十度生物科技有限公司 | Method for inducing and differentiating human-derived somatic adipose-derived stem cells and umbilical cord mesenchymal stem cells into neural-like stem cells through co-culture |
| CN114191342A (en) | 2021-11-23 | 2022-03-18 | 广州国色天香生物科技有限公司 | Skin care lotion for promoting skin metabolism and preparation method thereof |
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| WO2015170347A2 (en) | 2014-05-09 | 2015-11-12 | Reelabs Private Limited | Foetal polymix of mesenchymal stem cells under hypoxic conditions for the treatment of clinical disorders |
| JP2020164473A (en) | 2019-03-29 | 2020-10-08 | 株式会社再生医学研究所 | Hair restorer containing dental pulp stem cell culture supernatant and dermal papilla stem cell culture supernatant and method for producing the same |
| CN113444689A (en) | 2021-07-21 | 2021-09-28 | 东莞十度生物科技有限公司 | Method for inducing and differentiating human-derived somatic adipose-derived stem cells and umbilical cord mesenchymal stem cells into neural-like stem cells through co-culture |
| CN114191342A (en) | 2021-11-23 | 2022-03-18 | 广州国色天香生物科技有限公司 | Skin care lotion for promoting skin metabolism and preparation method thereof |
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