JP6471259B2 - Cell preparation for hair regeneration - Google Patents
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- JP6471259B2 JP6471259B2 JP2018121324A JP2018121324A JP6471259B2 JP 6471259 B2 JP6471259 B2 JP 6471259B2 JP 2018121324 A JP2018121324 A JP 2018121324A JP 2018121324 A JP2018121324 A JP 2018121324A JP 6471259 B2 JP6471259 B2 JP 6471259B2
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- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 description 1
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- BTSZTGGZJQFALU-UHFFFAOYSA-N piroctone olamine Chemical compound NCCO.CC(C)(C)CC(C)CC1=CC(C)=CC(=O)N1O BTSZTGGZJQFALU-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
- A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/14—Drugs for dermatological disorders for baldness or alopecia
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/86—Products or compounds obtained by genetic engineering
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
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Description
本発明は、毛髪再生用の細胞製剤に関する。より具体的には、本発明は、再生医療によって毛髪を再生させるための細胞製剤に関する。 The present invention relates to a cell preparation for hair regeneration. More specifically, the present invention relates to a cell preparation for regenerating hair by regenerative medicine.
近年、生活環境の変化やストレス等に起因するホルモン分泌バランスの不均衡等が原因となって、男女を問わず、薄毛や抜け毛で悩む人が多く存在している。そのため、発毛促進、毛髪成長、脱毛抑制等に有効な育毛剤の開発への期待や社会的要求が高まっている。従来、低分子化合物、植物エキス、植物素材の発酵物等を配合した育毛剤が種々報告されている（例えば、特許文献１及び２等参照）。また、近年では、ミノキシジル、塩化カプロニウム、トランス-3,4'-ジメチル-3-ヒドロキシフラバノン、ニコチン酸アミド、ピロクトンオラミン、アデノシン等を配合した育毛剤が実用化されている。しかしながら、従来の育毛剤では、毛乳頭細胞の増殖促進や賦活化を行っているに過ぎず、その効果は個人差に大きく左右され、限界があるといわれている。 In recent years, there are many people who suffer from thinning hair and hair loss regardless of gender due to imbalance in hormone secretion balance caused by changes in living environment and stress. Therefore, the expectation and social demand for the development of a hair growth agent effective for hair growth promotion, hair growth, hair loss suppression and the like are increasing. Conventionally, various hair growth agents containing a low molecular weight compound, a plant extract, a fermented material of a plant material, and the like have been reported (see, for example, Patent Documents 1 and 2). In recent years, hair restorers containing minoxidil, capronium chloride, trans-3,4'-dimethyl-3-hydroxyflavanone, nicotinamide, piroctone olamine, adenosine and the like have been put into practical use. However, the conventional hair restorer only promotes the proliferation and activation of hair papilla cells, and the effect is said to be greatly limited by individual differences and is limited.
一方、近年、多能性幹細胞を利用した再生医療の技術の進歩に伴って、毛髪再生医療の研究も精力的に行われている。例えば、人工多能性幹細胞（iPS細胞）を用い、毛髪を作り出す組織「毛包」を部分的に再生できたことが報告されている。更に、毛包幹細胞から人工的に毛包原器を作製し、これを皮膚内に移植することによって毛包を再生させ得ることも報告されている。 On the other hand, in recent years, research on hair regenerative medicine has been conducted energetically with the advancement of regenerative medicine technology using pluripotent stem cells. For example, it has been reported that tissue “hair follicles” that produce hair can be partially regenerated using artificial pluripotent stem cells (iPS cells). Furthermore, it has been reported that a hair follicle can be regenerated by artificially preparing a hair follicle device from hair follicle stem cells and transplanting it into the skin.
しかしながら、従来報告されている幹細胞を利用した再生医療の技術では、幹細胞を含む細胞製剤を、発毛が望まれる部位に投与することによって毛髪再生させる技術については十分に確立できていないのが現状である。 However, in the regenerative medicine technology using stem cells that has been reported so far, the technology for regenerating hair by administering a cell preparation containing stem cells to a site where hair growth is desired has not been sufficiently established. It is.
本発明は、幹細胞を利用した毛髪再生医療を実現すべく、幹細胞を利用して毛髪再生が可能な細胞製剤を提供することを目的とする。 An object of this invention is to provide the cell formulation which can reproduce hair using a stem cell, in order to implement | achieve the hair regeneration medicine using a stem cell.
本発明者は、前記課題を解決すべく鋭意検討を行ったところ、脂肪組織由来間葉系幹細胞を脂肪細胞と共存させた状態にして、これを発毛が望まれる部位に移植することにより、当該部位において発毛が認められ、毛髪が再生できることを見出した。本発明は、かかる知見に基づいて更に検討することにより完成したものである。 The present inventor made an intensive study to solve the above problems, and made the adipose tissue-derived mesenchymal stem cells coexist with adipocytes, and transplanted it to a site where hair growth is desired. It was found that hair growth was observed at the site, and the hair could be regenerated. The present invention has been completed by further study based on this finding.
即ち、本発明は、下記に掲げる態様の毛髪再生用の細胞製剤を提供する：
項１． 脂肪組織から採取された脂肪組織由来間葉系幹細胞及び脂肪細胞を含有すると共に、
さらに、薬学的に許容される担体、賦形剤、消炎剤、鎮痛剤、免疫抑制剤、コラーゲン、線維芽細胞、成長因子、増殖因子、サイトカイン、ＰＲＰ、又は、組織再生作用を有する他の細胞製剤のいずれか少なくとも１つを含有する、毛髪再生用の細胞製剤。
項２． 脂肪組織から採取された脂肪組織由来間葉系幹細胞及び脂肪細胞に対して、抗原性を除去するための処理を行う、脂肪組織から採取された脂肪組織由来間葉系幹細胞及び脂肪細胞を含有する毛髪再生用の細胞製剤の調製方法。
項３． 前記抗原性を除去するための処理がγ線照射を含む、項２に記載の毛髪再生用の細胞製剤の調製方法。
項４． 採取された脂肪組織から、液体画部及び不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞を含有する脂肪組織を得ることからなる、毛髪再生用の細胞製剤の調製方法。
項５． 前記不純物としては、麻酔薬、老化した脂肪細胞、血液、又は、組織液の中の少なくとも１つが含まれる、項４に記載の毛髪再生用の細胞製剤の調製方法。
項６． 以下の第１〜第３工程を有する、毛髪再生用の細胞製剤の調製方法。
採取された脂肪組織から液体画分を除去し、細胞画分を得る第１工程、
前記第１工程で得られた細胞画分から不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得る第２工程、
前記第２工程で得られた濃縮物の一部から脂肪組織由来間葉系幹細胞と脂肪細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、さらに前記第２工程で得られた濃縮物の一部に混合する第３工程。
項７． 以下の第１〜第３工程を有する、毛髪再生用の細胞製剤の調製方法。
採取された脂肪組織から液体画分を除去し、細胞画分を得る第１工程、
前記第１工程で得られた細胞画分から不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得る第２工程、
前記第２工程で得られた濃縮物中の脂肪細胞を微細化し、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物として分離する第３工程。
項８． 項７に記載の毛髪再生用の細胞製剤の調製方法において、さらに、以下の第４工程を有する、毛髪再生用の細胞製剤の調製方法。
前記第３工程で得られた脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部から脂肪組織由来間葉系幹細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、前記第２工程で得られた濃縮物、又は、前記第３工程で得られた脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部に混合する第４工程。
That is, the present invention provides a cell preparation for hair regeneration having the following aspects:
Item 1. Containing adipose tissue-derived mesenchymal stem cells and adipocytes collected from adipose tissue ;
Furthermore, pharmaceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, collagen, fibroblasts, growth factors, growth factors, cytokines, PRP, or other cells with tissue regeneration activity A cell preparation for hair regeneration containing at least one of the preparations.
Item 2. Adipose tissue- derived mesenchymal stem cells and adipose cells collected from adipose tissue are treated to remove antigenicity, and adipose tissue-derived mesenchymal stem cells and adipocytes collected from adipose tissue are contained. A method for preparing a cell preparation for hair regeneration.
Item 3. Item 3. The method for preparing a cell preparation for hair regeneration according to Item 2, wherein the treatment for removing antigenicity includes γ-ray irradiation.
Item 4. A method for preparing a cell preparation for hair regeneration, comprising removing a liquid fraction and impurities from a collected adipose tissue to obtain an adipose tissue containing adipose tissue-derived mesenchymal stem cells and adipocytes.
Item 5. Item 5. The method for preparing a cell preparation for hair regeneration according to Item 4, wherein the impurities include at least one of an anesthetic, aged fat cells, blood, or tissue fluid.
Item 6. A method for preparing a cell preparation for hair regeneration, comprising the following first to third steps.
A first step of removing a liquid fraction from the collected adipose tissue to obtain a cell fraction;
A second step of removing impurities from the cell fraction obtained in the first step to obtain a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes;
Adipose tissue-derived mesenchymal stem cells and adipocytes are separated from a part of the concentrate obtained in the second step, and the obtained adipose tissue-derived mesenchymal stem cells are further concentrated in the second step. 3rd process which mixes in a part of thing.
Item 7. A method for preparing a cell preparation for hair regeneration, comprising the following first to third steps.
A first step of removing a liquid fraction from the collected adipose tissue to obtain a cell fraction;
A second step of removing impurities from the cell fraction obtained in the first step to obtain a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes;
A third step in which the fat cells in the concentrate obtained in the second step are refined and separated as a mixture of adipose tissue-derived mesenchymal stem cells and refined adipocytes.
Item 8. Item 8. The method for preparing a cell preparation for hair regeneration according to Item 7, further comprising the following fourth step.
The adipose tissue-derived mesenchymal stem cells are separated from a part of the mixture of the adipose tissue-derived mesenchymal stem cells obtained in the third step and the refined adipocytes, and the obtained adipose tissue-derived mesenchymal stem cells are obtained. The 4th process mixed with a part of the mixture of the concentrate obtained by the said 2nd process, or the adipose tissue origin mesenchymal stem cell obtained by the said 3rd process, and the refined fat cell.
本発明によれば、脂肪組織由来間葉系幹細胞（adipose tissue-derived mesenchymal stem cell; AD-MSC, AT-MSC）及び脂肪細胞を含む細胞製剤を、これを発毛が望まれる部位に移植するという簡便な手法によって、当該部位において効率的に毛包を再生させて発毛させることができる。また、本発明は、細胞製剤をそのまま移植に使用でき、一旦、毛包を形成した上での組織移植を要しないため、移植部位に痕跡を殆ど残すことなく毛髪を再生できるという利点もある。 According to the present invention, a cell preparation containing adipose tissue-derived mesenchymal stem cells (AD-MSC, AT-MSC) and adipocytes is transplanted to a site where hair growth is desired. By such a simple method, hair follicles can be efficiently regenerated at the site and hair can be grown. The present invention also has the advantage that the cell preparation can be used as it is for transplantation, and it does not require tissue transplantation once hair follicles have been formed, so that the hair can be regenerated with almost no trace left at the transplant site.
更に、本発明によれば、毛母細胞と共に色素細胞（メラノサイト）を誘導させることができるので、白髪ではなく、黒色の自然な毛髪を再生させることができる。 Furthermore, according to the present invention, since pigment cells (melanocytes) can be induced together with hair matrix cells, natural black hair can be regenerated instead of white hair.
本発明の細胞製剤は、毛髪再生用として使用されるものであって、脂肪組織由来間葉系幹細胞及び脂肪細胞を含有することを特徴とする。以下、本発明の細胞製剤について詳述する。 The cell preparation of the present invention is used for hair regeneration and contains adipose tissue-derived mesenchymal stem cells and adipocytes. Hereinafter, the cell preparation of the present invention will be described in detail.
＜脂肪組織由来間葉系幹細胞及び脂肪細胞＞
脂肪組織由来間葉系幹細胞とは、主に、脂肪組織、筋肉細胞、骨、軟骨、靭帯、皮膚細胞、神経細胞に分化できる幹細胞である。本発明で使用される脂肪組織由来間葉系幹細胞は、生体の脂肪組織から採取されたものであってもよく、また生体の脂肪組織から採取された脂肪組織由来間葉系幹細胞を培養したものであってもよい。本発明で使用される脂肪組織由来間葉系幹細胞として、免疫反応の抑制という観点から、好ましくは生体から採取されたものが挙げられる。特に、皮下脂肪組織内に存在する脂肪組織由来間葉系幹細胞は、比較的低襲撃で容易に採取できる、比較的多量に採取でき培養による増殖の必要が無い、効率的に毛髪を再生できる、脂肪細胞と同時に採取できる、等の利点があり、本発明において好適に使用される。
<Adipose tissue-derived mesenchymal stem cells and adipocytes>
Adipose tissue-derived mesenchymal stem cells are stem cells that can differentiate into adipose tissue, muscle cells, bones, cartilage, ligaments, skin cells, and nerve cells. The adipose tissue-derived mesenchymal stem cells used in the present invention may be collected from living adipose tissue, or cultured adipose tissue-derived mesenchymal stem cells collected from a living adipose tissue It may be. The adipose tissue-derived mesenchymal stem cells used in the present invention are preferably those collected from a living body from the viewpoint of suppression of immune reaction. In particular, the adipose tissue-derived mesenchymal stem cells present in the subcutaneous adipose tissue can be easily collected with relatively low attack, can be collected in a relatively large amount, and does not require growth by culture, and can efficiently regenerate hair. There is an advantage that it can be collected at the same time as fat cells, and it is preferably used in the present invention.
脂肪組織由来間葉系幹細胞の取得方法については、特に制限されないが、好ましくは、後述する「細胞製剤の調製方法」の欄に記載の方法が挙げられる。 The method for obtaining adipose tissue-derived mesenchymal stem cells is not particularly limited, and preferably includes the method described in the section “Method for preparing cell preparation” described later.
脂肪細胞（ｆａｔ ｃｅｌｌ）とは、脂肪を合成、貯蔵、放出する機能を有し、脂肪組織を形成する細胞である。また、脂肪細胞は、完全に分化しているため細胞分裂を起こさない。脂肪細胞には、白色脂肪細胞及び褐色脂肪細胞が存在するが、本発明では、これらのいずれを用いてもよく、両者の混合物を使用してもよい。本発明で使用される脂肪細胞としては、脂肪細胞を単離した状態で使用してもよく、また脂肪細胞周辺に存在する細胞外基質（ｅｘｔｒａｃｅｌｌｕｌａｒ ｍａｔｒｉｘ：ＥＣＭ）と脂肪細胞の混合物を使用してもよい。具体的には、本発明では、脂肪細胞として、採取された脂肪組織をそのまま使用してもよく、また採取された脂肪組織から細胞外基質を取り除いたものを使用してもよい。 A fat cell is a cell that has the function of synthesizing, storing, and releasing fat, and forms adipose tissue. Adipocytes do not undergo cell division because they are completely differentiated. The fat cells include white fat cells and brown fat cells. In the present invention, any of these may be used, or a mixture of both may be used. The adipocytes used in the present invention may be used in a state in which adipocytes are isolated, or by using a mixture of extracellular matrix (ECM) and adipocytes existing around the adipocytes. Also good. Specifically, in the present invention, the collected adipose tissue may be used as it is as the adipocyte, or one obtained by removing the extracellular matrix from the collected adipose tissue may be used.
また、本発明の細胞製剤における脂肪組織由来間葉系幹細胞の濃度については、特に制限されないが、効率的に毛髪再生を促すという観点から、例えば、細胞製剤１ｍＬ当たりに含まれる脂肪組織由来間葉系幹細胞の数として、１万個以上、好ましくは１０万個以上、更に好ましくは１５万個以上、特に好ましくは１５〜２０万個が挙げられる。 Further, the concentration of the adipose tissue-derived mesenchymal stem cells in the cell preparation of the present invention is not particularly limited, but for example, from the viewpoint of efficiently promoting hair regeneration, for example, the adipose tissue-derived mesenchyme contained per mL of cell preparation The number of stem cells is 10,000 or more, preferably 100,000 or more, more preferably 150,000 or more, and particularly preferably 150 to 200,000.
また、本発明の細胞製剤における脂肪細胞の濃度については、特に制限されないが、効率的に毛髪再生を促すという観点から、例えば、細胞製剤１ｍＬ当たりに含まれる脂肪細胞の数として、１，０００個以上、好ましくは５，０００個以上、更に好ましくは１万個以上、特に好ましくは１万〜２万個が挙げられる。 Further, the concentration of adipocytes in the cell preparation of the present invention is not particularly limited, but from the viewpoint of efficiently promoting hair regeneration, for example, the number of adipocytes contained in 1 mL of cell preparation is 1,000. The number is preferably 5,000 or more, more preferably 10,000 or more, and particularly preferably 10,000 to 20,000.
本発明の細胞製剤において、脂肪組織由来間葉系幹細胞及び脂肪細胞の比率については、特に制限されず、脂肪組織由来間葉系幹細胞の含有量が多いほどより一層優れた毛髪再生効果が得られると考えられるが、例えば、脂肪細胞１個に対して脂肪組織由来間葉系幹細胞が、少なくとも１個以上、好ましくは５個以上、より好ましくは１〜１０個、更に好ましくは５〜１０個が挙げられる。 In the cell preparation of the present invention, the ratio between the adipose tissue-derived mesenchymal stem cells and the adipocytes is not particularly limited, and a more excellent hair regeneration effect is obtained as the content of the adipose tissue-derived mesenchymal stem cells is increased. However, for example, at least one adipose tissue-derived mesenchymal stem cell, preferably five or more, more preferably 1 to 10, more preferably 5 to 10, per adipocyte Can be mentioned.
本発明の細胞製剤において、自家移植を目的として、投与される患者由来の脂肪細胞及び脂肪組織由来間葉系幹細胞）を使用することにより、免疫拒絶反応を回避することもできる。但し、本発明の細胞製剤は、投与される患者以外の人由来の脂肪細胞及び／又は脂肪組織由来間葉系幹細胞を用いて調製し、他家移植に使用することを制限するものではない。投与される患者以外の人由来の脂肪細胞及び／又は脂肪組織由来間葉系幹細胞を使用する場合は、移植前に他人から採取した脂肪細胞（脂肪細胞周辺に存在する細胞外基質（ＥＣＭ）を含む）に対し、例えばγ線照射等の抗原性を除去するための処理を行うことが好ましい。また、脂肪組織由来間葉系幹細胞は通常免疫原性を有さない細胞であるが、必要に応じて、前記処理に供したものを使用してもよい。 In the cell preparation of the present invention, immune rejection can be avoided by using patient-derived adipocytes and adipose tissue-derived mesenchymal stem cells to be administered for the purpose of autotransplantation. However, the cell preparation of the present invention is prepared using adipocytes derived from a person other than the patient to be administered and / or adipose tissue-derived mesenchymal stem cells, and is not limited to use in other transplants. When using adipocytes and / or adipose tissue-derived mesenchymal stem cells other than the patient to be administered, adipocytes (extracellular matrix (ECM) present around the adipocytes) collected from others before transplantation are used. For example, it is preferable to perform a treatment for removing antigenicity such as γ-ray irradiation. In addition, adipose tissue-derived mesenchymal stem cells are usually cells that do not have immunogenicity, but those subjected to the treatment may be used as necessary.
また、脂肪組織には、通常、脂肪組織由来間葉系幹細胞と脂肪細胞が含まれているため、本発明の細胞製剤として、採取された脂肪組織をそのまま使用してもよく、また、採取された脂肪組織から不純物（例えば、脂肪採取の際に注入される麻酔液（所謂チュメセント液）、老化した脂肪細胞、血液、組織液等）を除去したもの、採取された脂肪組織から不純物と一部の脂肪細胞を除去したもの等を使用してもよい。当該細胞製剤の調製方法の好適な例としては、後述する方法（ａ）〜（ｃ）が挙げられる。 In addition, since the adipose tissue usually contains adipose tissue-derived mesenchymal stem cells and adipocytes, the collected adipose tissue may be used as it is as the cell preparation of the present invention. Removed impurities from the adipose tissue (for example, anesthetic fluid (so-called tumecent solution), aged adipocytes, blood, tissue fluid, etc. injected during the collection of fat), impurities and some of the collected adipose tissue You may use what removed the fat cell. Preferable examples of the method for preparing the cell preparation include methods (a) to (c) described later.
＜他の含有成分＞
また、本発明の細胞製剤には、必要に応じて従来公知の薬学的に許容される担体、賦形剤、消炎剤、鎮痛剤、免疫抑制剤等を添加することができる。更に、本発明の効果を損なわない範囲でコラーゲン、線維芽細胞、成長因子、増殖因子、サイトカイン等を添加してもよい。
<Other ingredients>
In addition, conventionally known pharmaceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, and the like can be added to the cell preparation of the present invention as necessary. Furthermore, collagen, fibroblasts, growth factors, growth factors, cytokines, etc. may be added as long as the effects of the present invention are not impaired.
また、本発明の効果を損なわない限り、例えば、ＰＲＰ（Ｐｌａｔｅｌｅｔ Ｒｉｃｈ Ｐｌａｓｍａ：多血小板血漿）等の組織再生作用が知られている他の細胞製剤を、本発明の細胞製剤と併用してもよい。但し、毛髪再生効率をより一層高める観点から、トランスフォーミング増殖因子β１（ＴＧＦβ１）を除去して使用することが好ましい。 Moreover, as long as the effects of the present invention are not impaired, for example, other cell preparations known for tissue regeneration such as PRP (Platelet Rich Plasma) may be used in combination with the cell preparations of the present invention. . However, from the viewpoint of further improving the hair regeneration efficiency, it is preferable to use it after removing transforming growth factor β1 (TGFβ1).
また、本発明の細胞製剤には、脂肪幹細胞等の細胞が含まれていてもよい。 The cell preparation of the present invention may contain cells such as adipose stem cells.
＜用途・投与方法＞
本発明の細胞製剤は、毛髪再生が必要とされる部位の真皮や皮下組織に、投与することによって、当該部位において発毛を促し、毛髪を再生させることができる。本発明の細胞製剤は、びまん性脱毛症、分娩後脱毛症、男性型脱毛症、脂漏性脱毛症、老人性脱毛症、円形脱毛症、瘢痕性脱毛症、精神疾患に起因する抜毛症等による薄毛を改善することができる。
<Use and administration method>
When the cell preparation of the present invention is administered to the dermis or subcutaneous tissue of a site where hair regeneration is required, hair growth can be promoted at the site and hair can be regenerated. The cell preparation of the present invention comprises diffuse alopecia, postpartum alopecia, androgenetic alopecia, seborrheic alopecia, senile alopecia, alopecia areata, scarring alopecia, hair loss caused by mental disorders, etc. Can improve thinning hair.
本発明の細胞製剤の投与量については、投与部位の毛髪量、患者の性別、体格等に応じて適宜設定されるが、例えば、投与部位１ｃｍ2当たり、脂肪細胞数に換算して１，０００〜１０万個程度、好ましくは１，０００〜５万個程度、更に好ましくは５，０００〜１万個程度が挙げられる。また、例えば、下記方法（ｃ）で得られる細胞製剤を用いる場合であれば、例えば投与部位１ｃｍ2当たり、０．１〜１．０ｍＬ、好ましくは０．１〜０．５ｍＬ、更に好ましくは０．２〜０．５ｍＬが挙げられる。 The dosage of the cell preparation of the present invention is appropriately set according to the amount of hair at the administration site, the patient's sex, physique, etc. For example, 1,000 cm in terms of the number of fat cells per cm 2 of the administration site. About 100,000, preferably about 1,000 to 50,000, and more preferably about 5,000 to 10,000. Further, for example, in the case of using a cell preparation obtained by the following method (c), for example, 0.1 to 1.0 mL, preferably 0.1 to 0.5 mL, more preferably 0 per 1 cm 2 of the administration site. 2 to 0.5 mL.
本発明の細胞製剤を毛髪再生が必要とされる部位に投与する方法については、特に制限されず、従来公知の投与方法を採用することができ、例えば、シリンジ、カニューレ等で注入する方法が挙げられる。 The method for administering the cell preparation of the present invention to a site where hair regeneration is required is not particularly limited, and a conventionally known administration method can be adopted, for example, a method of injecting with a syringe, cannula or the like. It is done.
本発明の細胞製剤の毛髪再生効果については、個人差があるが、通常、投与後２〜６週間程度で発毛が認められ、当該部位において毛髪が再生される。 Although there are individual differences in the hair regeneration effect of the cell preparation of the present invention, hair growth is usually observed about 2 to 6 weeks after administration, and the hair is regenerated at the site.
＜細胞製剤の調製方法＞
本発明の細胞製剤は、脂肪組織由来間葉系幹細胞と脂肪細胞をそれぞれ採取して混合したものを使用してもよく、また脂肪組織から脂肪組織由来間葉系幹細胞と脂肪細胞の混合物として採取したものを使用してもよい。とりわけ、脂肪組織から液体画分（体液等）を除去して脂肪組織由来間葉系幹細胞と脂肪細胞の濃縮物を得て、本発明の細胞製剤とすることが好ましい。また、より簡便には、本発明の細胞製剤として採取された脂肪組織をそのまま使用してもよい。
<Method for preparing cell preparation>
The cell preparation of the present invention may be obtained by collecting and mixing adipose tissue-derived mesenchymal stem cells and adipocytes, or collected from adipose tissue as a mixture of adipose tissue-derived mesenchymal stem cells and adipocytes. You may use what you did. In particular, it is preferable to obtain a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes by removing a liquid fraction (such as a body fluid) from the adipose tissue to obtain the cell preparation of the present invention. More simply, the adipose tissue collected as the cell preparation of the present invention may be used as it is.
脂肪組織から本発明の細胞製剤を調製する場合、好ましい方法として、下記方法（ａ）〜（ｃ）が挙げられる。当該方法（ａ）〜（ｃ）に従えば、脂肪組織から液体画分（体液等）を除去して脂肪組織由来間葉系幹細胞と脂肪細胞の濃縮物を得ることができる。以下、方法（ａ）〜（ｃ）についてそれぞれ詳述する。 When preparing the cell preparation of the present invention from adipose tissue, preferred methods include the following methods (a) to (c). According to the said method (a)-(c), a liquid fraction (body fluid etc.) can be removed from an adipose tissue, and the concentrate of adipose tissue origin mesenchymal stem cell and an adipocyte can be obtained. Hereinafter, each of the methods (a) to (c) will be described in detail.
方法（ａ）
本発明の細胞製剤を調製する方法（ａ）は、（１ａ）採取された脂肪組織から液体画分を除去し、細胞画分を得る工程を含む。
Method (a)
The method (a) for preparing the cell preparation of the present invention includes the step (1a) of removing a liquid fraction from the collected adipose tissue to obtain a cell fraction.
ここで、採取された脂肪とは、脂肪吸引、脂肪切除、除脂術等により脂肪組織から採取された、脂肪組織由来の細胞画分及び液体画分の混合物を指す。細胞画分には、脂肪細胞、脂肪幹細胞及び不純物（血球、死活細胞、老化細胞等）が含まれる。また、液体画分には、チュメセント液、細胞組織液等が含まれる。 Here, the collected fat refers to a mixture of a cell fraction and a liquid fraction derived from adipose tissue collected from adipose tissue by liposuction, lipectomy, defatting, or the like. The cell fraction contains adipocytes, adipose stem cells, and impurities (blood cells, dead / alive cells, senescent cells, etc.). The liquid fraction includes a tumecent liquid, a cell tissue liquid, and the like.
液体画分と細胞画分の分離は、簡便には、採取された脂肪を静置することによって細胞画分を沈降させて分離することができる。液体画分の除去方法は特に限定されないが、例えば、デカンテーション、吸引等が挙げられる。また、必要に応じて遠心分離、濾過等を行ってもよい。 For separation of the liquid fraction and the cell fraction, the cell fraction can be conveniently separated by allowing the collected fat to stand. The method for removing the liquid fraction is not particularly limited, and examples thereof include decantation and suction. Moreover, you may perform centrifugation, filtration, etc. as needed.
遠心分離及び濾過の条件は、脂肪細胞及び脂肪組織由来間葉系幹細胞を損傷せず、且つ不純物を除去できれば特に限定されないが、例えば、７００〜２５００×ｇで１〜１５分間、好ましくは２０００〜２２００×ｇで５〜１０分間の条件が挙げられる。 Centrifugation and filtration conditions are not particularly limited as long as they do not damage adipocytes and adipose tissue-derived mesenchymal stem cells and can remove impurities, but for example, 700 to 2500 × g for 1 to 15 minutes, preferably 2000 to 2000 A condition of 5 to 10 minutes at 2200 × g can be mentioned.
また、濾過を行う場合であれば、採取された脂肪を、例えば１０〜３００μｍ、好ましくは１０〜１５０μｍ、より好ましくは１０〜１００μｍ、更に好ましくは１５〜２０μｍの穴大きさを有する網目状フィルターを通過させることにより血液、組織液、脂肪を採取する箇所に投与された麻酔薬（チュメセント液）等を除去してフィルター上に脂肪細胞及び脂肪組織由来間葉系幹細胞の混合物を得ることができる。また、このような網目状フィルターを備えた容器に、採取された不純物を含む脂肪を充填し、遠心分離に供してもよい。 In the case of performing filtration, the collected fat is filtered through a mesh filter having a hole size of, for example, 10 to 300 μm, preferably 10 to 150 μm, more preferably 10 to 100 μm, and still more preferably 15 to 20 μm. The mixture of adipocytes and adipose tissue-derived mesenchymal stem cells can be obtained on the filter by removing blood, tissue fluid, anesthetic agent (tumecent solution) and the like administered to the site where the fat is collected. In addition, a container including such a mesh filter may be filled with collected fat containing impurities and subjected to centrifugation.
斯して、液体画分が除去された、脂肪組織由来間葉系幹細胞及び脂肪細胞（細胞外基質を含む）を含む脂肪組織を得ることができ、これを本発明の細胞製剤として使用することができる。 Thus, adipose tissue containing adipose tissue-derived mesenchymal stem cells and adipocytes (including extracellular matrix) from which the liquid fraction has been removed can be obtained, and this can be used as the cell preparation of the present invention. Can do.
方法（ｂ）
本発明の細胞製剤を調製する方法（ｂ）は、下記工程（１ｂ）〜（３ｂ）を含む。
（１ｂ）採取された脂肪組織から液体画分を除去し、細胞画分を得る工程、
（２ｂ）前記工程（１ｂ）で得られた細胞画分の少なくとも一部から脂肪組織由来間葉系幹細胞と脂肪細胞を分離する工程、及び
（３ｂ）前記工程（１ｂ）で得られた細胞画分の一部又は前記工程（２ｂ）で得られた脂肪細胞の一部と、前記工程（２ｂ）で得られた脂肪組織由来間葉系幹細胞とを混合する工程。
Method (b)
The method (b) for preparing the cell preparation of the present invention includes the following steps (1b) to (3b).
(1b) removing the liquid fraction from the collected adipose tissue to obtain a cell fraction;
(2b) a step of separating adipose tissue-derived mesenchymal stem cells and adipocytes from at least a part of the cell fraction obtained in the step (1b), and (3b) a cell fraction obtained in the step (1b). A step of mixing a part of a minute or a part of the adipocyte obtained in the step (2b) and the adipose tissue-derived mesenchymal stem cell obtained in the step (2b).
ここで、工程（１ｂ）は上記方法（１ａ）に記載の通りである。工程（２ｂ）では、工程（１ｂ）で得られた細胞画分の少なくとも一部、好ましくは半量から脂肪組織由来間葉系幹細胞を分離する。そして、工程（３ｂ）において、工程（２ｂ）で得られた脂肪組織由来間葉系幹細胞を、工程（１ｂ）で得られた細胞画分の一部又は前記工程（２ｂ）で得られた脂肪細胞の一部に添加することによって、脂肪組織由来間葉系幹細胞及び脂肪細胞を含む細胞製剤とすることができる。 Here, the step (1b) is as described in the method (1a). In the step (2b), adipose tissue-derived mesenchymal stem cells are separated from at least a part, preferably half, of the cell fraction obtained in the step (1b). In the step (3b), the adipose tissue-derived mesenchymal stem cells obtained in the step (2b) are used as a part of the cell fraction obtained in the step (1b) or the fat obtained in the step (2b). By adding to a part of the cells, a cell preparation containing adipose tissue-derived mesenchymal stem cells and adipocytes can be obtained.
ここで、脂肪組織由来間葉系幹細胞を分離する方法としては、例えば、特表２００５−５１９８８３号公報に記載の方法が挙げられる。より具体的には、細胞間の結合を分解することにより脂肪組織由来間葉系幹細胞と脂肪細胞を分離しやすくするために、細胞画分にタンパク質分解酵素を添加する。タンパク質分解酵素としては、コラゲナーゼ、トリプシン、リパーゼ等が例示され、これらから選択される１種を単独で使用してもよく、２種以上を組合せて使用することもできる。 Here, as a method for separating adipose tissue-derived mesenchymal stem cells, for example, a method described in JP-T-2005-519883 can be mentioned. More specifically, a proteolytic enzyme is added to the cell fraction in order to facilitate separation of adipose tissue-derived mesenchymal stem cells and adipocytes by decomposing bonds between cells. Examples of proteolytic enzymes include collagenase, trypsin, lipase, etc., and one kind selected from these may be used alone, or two or more kinds may be used in combination.
酵素処理された細胞画分から脂肪組織由来間葉系幹細胞と脂肪細胞を分離する方法としては、特に限定されず、従来公知の方法から適宜選択され得るが、例えば、遠心分離、濾過等が挙げられる。遠心分離の条件としては、脂肪組織由来間葉系幹細胞及び脂肪細胞を傷つけることなく分離できるものであれば特に限定されないが、例えば、１００〜５００×ｇで１〜２０分、好ましくは１００〜３００×ｇで５〜１０分間の条件が挙げられる。
このような遠心分離に供することによって、脂肪組織由来間葉系幹細胞が沈殿物として得られ、上清中に脂肪細胞が濃縮される。また、濾過を行う場合であれば、細胞画分又はその懸濁液を１０〜１００μｍ、好ましくは１０〜５０μｍ、更に好ましくは１５〜２０μｍの穴大きさを有する網目状フィルターを通過させることによりフィルター上に脂肪組織由来間葉系幹細胞を得ることができ、濾液中に脂肪細胞を得ることができる。
The method for separating adipose tissue-derived mesenchymal stem cells and adipocytes from the enzyme-treated cell fraction is not particularly limited and may be appropriately selected from conventionally known methods. Examples thereof include centrifugation and filtration. . The conditions for the centrifugation are not particularly limited as long as they can be separated without damaging the adipose tissue-derived mesenchymal stem cells and fat cells, but for example, 100 to 500 × g for 1 to 20 minutes, preferably 100 to 300. The conditions for 5 to 10 minutes are mentioned by xg.
By subjecting to such centrifugation, adipose tissue-derived mesenchymal stem cells are obtained as a precipitate, and adipocytes are concentrated in the supernatant. In the case of filtration, the cell fraction or suspension thereof is passed through a mesh filter having a hole size of 10 to 100 μm, preferably 10 to 50 μm, more preferably 15 to 20 μm. Adipose tissue-derived mesenchymal stem cells can be obtained on top, and adipocytes can be obtained in the filtrate.
酵素処理された脂肪組織由来間葉系幹細胞及び脂肪細胞は、分離後、洗浄処理に供されることが好ましく、洗浄はリン酸緩衝溶液（ＰＢＳ）、生理食塩水、リンゲル液、デキストラン等により行うことができる。洗浄処理は、必要に応じて複数回繰り返してもよく、洗浄後に細胞を回収する際、必要に応じて遠心分離に供してもよい。遠心分離の条件としては前記の酵素処理された細胞画分から脂肪組織由来間葉系幹細胞と脂肪細胞を分離する際に採用される条件に従えばよい。 The enzyme-treated adipose tissue-derived mesenchymal stem cells and adipocytes are preferably subjected to a washing treatment after separation, and washing is performed with a phosphate buffer solution (PBS), physiological saline, Ringer's solution, dextran, or the like. Can do. The washing treatment may be repeated a plurality of times as necessary, and may be subjected to centrifugation as necessary when cells are collected after washing. The conditions for the centrifugation may be the same as those employed when separating the adipose tissue-derived mesenchymal stem cells and adipocytes from the enzyme-treated cell fraction.
このようにして調製される細胞製剤は、脂肪細胞と共に、脂肪組織由来間葉系幹細胞を高濃度で含んでおり、卓越した毛髪再生効果を奏することができる。 The cell preparation thus prepared contains a high percentage of adipose tissue-derived mesenchymal stem cells together with adipocytes, and can exhibit an excellent hair regeneration effect.
方法（ｃ）
本発明の細胞製剤を調製する方法（ｃ）は下記工程（１ｃ）及び（２ｃ）を含む。
（１ｃ）採取された脂肪組織から液体画分を除去し、細胞画分を得る工程；及び
（２ｃ）前記工程（１ｃ）で得られた細胞画分から不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得る工程。
Method (c)
The method (c) for preparing the cell preparation of the present invention comprises the following steps (1c) and (2c).
(1c) removing the liquid fraction from the collected adipose tissue to obtain a cell fraction; and (2c) removing impurities from the cell fraction obtained in the step (1c) to obtain a mesenchymal system derived from adipose tissue Obtaining a concentrate of stem cells and adipocytes.
ここで、工程（１ｃ）については、上記方法（１ａ）に記載の通りである。工程（２ｃ）では、細胞画分から不純物を除去して濃縮する。不純物除去及び濃縮の方法としては特に限定されず、従来公知の方法から適宜選択され得るが、簡便には、例えば特表２００７−５３３３９６号公報に記載されるウェイトフィルターを備えたシリンジを使用することにより、壊れた脂肪細胞から遊離したフリーオイル、死活細胞、老化細胞等の不純物が除去され、健全な脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物が得られる。このようなシリンジは商業的に入手可能であり、具体的にはＬＩＰＯＭＡＸ−ＳＣ用に製造されたシリンジ（Ｍｅｄｉｋａｎ Ｃｏｒｐ．製，韓国 ソウル）が例示される。 Here, the step (1c) is as described in the method (1a). In the step (2c), impurities are removed from the cell fraction and concentrated. The method for removing and concentrating impurities is not particularly limited, and can be appropriately selected from conventionally known methods. For example, a syringe equipped with a weight filter described in JP-T-2007-533396 is used for convenience. Thus, impurities such as free oil, dead and active cells, and senescent cells released from broken fat cells are removed, and a healthy adipose tissue-derived mesenchymal stem cell and fat cell concentrate can be obtained. Such syringes are commercially available, and specific examples include syringes manufactured for LIPOMAX-SC (manufactured by Medikan Corp., Seoul, Korea).
より具体的には、内部にフィルターが備えられたシリンジ中に採取された脂肪を充填し、遠心分離によって脂肪を液体画分と細胞画分に分離し、更にフィルターによって、フリーオイル（排液オイル）が分離される。ここで分離される液体画分は、工程（１ｃ）において完全に分離されずに細胞画分中に混在していたものである。即ち、シリンジ中に、上層にフリーオイル、中間層に脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物ならびに下層に液体画分が分離され、中間層以外を廃棄することによって健全な脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得ることができ、これを本発明の細胞製剤として使用することができる。このようにして得られる細胞製剤は、健全な脂肪組織由来間葉系幹細胞及び脂肪細胞が選択、濃縮されていることから、移植後の壊死、石灰化等を生じにくく、骨間接合部の疾患の症状の緩和及び治療、並びに損傷した筋肉の修復及び筋肉損傷による疼痛の緩和に一層有用である。 More specifically, the collected fat is filled in a syringe equipped with a filter inside, and the fat is separated into a liquid fraction and a cell fraction by centrifugation, and free oil (drained oil) is further separated by a filter. ) Are separated. The liquid fraction separated here is not completely separated in the step (1c) but is mixed in the cell fraction. That is, in the syringe, free oil in the upper layer, adipose tissue-derived mesenchymal stem cells and adipose cell concentrate in the middle layer, and a liquid fraction in the lower layer are separated, and the rest of the middle layer is discarded, resulting in healthy adipose tissue origin A concentrate of mesenchymal stem cells and adipocytes can be obtained, which can be used as the cell preparation of the present invention. Since the cell preparation obtained in this way has selected and concentrated healthy adipose tissue-derived mesenchymal stem cells and adipocytes, it is difficult to cause necrosis, calcification, etc. after transplantation, and diseases of the interosseous junction It is more useful for alleviating and treating the symptoms, and for repairing damaged muscles and alleviating pain due to muscle damage.
前記脂肪を液体画分と細胞画分に分離するための遠心分離の条件としては、脂肪組織由来間葉系幹細胞及び脂肪細胞を損傷することなく、且つ不純物と脂肪組織由来間葉系幹細及び脂肪細胞とが分離され得る条件であれば特に限定されないが、例えば、７００〜２５００×ｇで１〜１５分間、好ましくは２０００〜２２００×ｇで５〜１０分間の条件が挙げられる。 Centrifugation conditions for separating the fat into a liquid fraction and a cell fraction include, without damaging adipose tissue-derived mesenchymal stem cells and adipocytes, and impurities and adipose tissue-derived mesenchymal stem cells and Although it will not specifically limit if it is the conditions from which an adipocyte can be isolate | separated, For example, the conditions of 1 to 15 minutes at 700-2500xg, Preferably it is 5-10 minutes at 2000-2200xg.
また、フィルターの穴大きさは、脂肪組織由来間葉系幹細胞及び脂肪細胞が通過せず、且つ不純物が通過できる程度であれば特に限定されないが、例えば１０〜３００μｍ、好ましくは１０〜１５０μｍ、より好ましくは１０〜１００μｍ、更に好ましくは１５〜２０μｍである。 The pore size of the filter is not particularly limited as long as it does not allow adipose tissue-derived mesenchymal stem cells and adipocytes to pass through and allows impurities to pass through, but is, for example, 10 to 300 μm, preferably 10 to 150 μm. Preferably it is 10-100 micrometers, More preferably, it is 15-20 micrometers.
また、本方法（ｃ）は、工程（２ｃ）で得られた濃縮物の一部から脂肪組織由来間葉系幹細胞と脂肪細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、更に前記工程（２ｃ）で得られた濃縮物の一部に混合する工程（３ｃ'）を含んでもよい。脂肪組織由来間葉系幹細胞の分離の方法は、上記方法（ｂ）の工程（２ｂ）について記載される通りである。本工程（３ｃ'）において、タンパク質分解酵素を用いて脂肪組織由来間葉系幹細胞を分離した場合、工程（２ｃ）で得られた濃縮物１ｃｃあたり通常約７０万個以上、好ましくは約１５０万個以上、更に好ましくは約２００万個以上の脂肪組織由来間葉系幹細胞を得ることができる。本工程（３ｃ'）を経て調製される細胞製剤は、健全な脂肪細胞が選択、濃縮されていることに加え、脂肪組織由来間葉系幹細胞をさらに高濃度で含むことから、卓越した毛髪再生効果を奏することができる。 Moreover, this method (c) separates adipose tissue-derived mesenchymal stem cells and adipocytes from a part of the concentrate obtained in step (2c), and further obtains the obtained adipose tissue-derived mesenchymal stem cells. You may include the process (3c ') mixed with a part of concentrate obtained by the said process (2c). The method for separating the adipose tissue-derived mesenchymal stem cells is as described for the step (2b) of the method (b). In this step (3c ′), when adipose tissue-derived mesenchymal stem cells are separated using a proteolytic enzyme, usually about 700,000 or more, preferably about 1.5 million per cc of the concentrate obtained in step (2c). One or more, more preferably about 2 million or more adipose tissue-derived mesenchymal stem cells can be obtained. The cell preparation prepared through this step (3c ′) contains not only healthy adipocytes, but also a high concentration of adipose tissue-derived mesenchymal stem cells. There is an effect.
また、本方法（ｃ）は、前記工程（２ｃ）の後、得られた濃縮物中の脂肪細胞を微細化し、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物として分離する工程（３ｃ''）を含んでもよい。脂肪細胞を微細化は、当該技術分野において通常採用される方法により行うことができ、特に限定されないが、例えば、必要に応じてドリル等によって脂肪細胞をカッティングした後、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物を分離する。分離方法としては、前記遠心分離の条件を採用することができる。本工程（３ｃ''）を行う際、前記工程（２ｃ）の後、濃縮物をシリンジに充填したまま、脂肪細胞の微細化及び遠心分離に供してもよい。遠心分離後、シリンジ中にフリーオイルと、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物とが分離されるため、フリーオイルを廃棄し、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物を得ることができる。 In the method (c), after the step (2c), the fat cells in the obtained concentrate are refined and separated as a mixture of the adipose tissue-derived mesenchymal stem cells and the refined adipocytes. (3c ″) may be included. Adipocytes can be miniaturized by a method that is usually employed in the art, and is not particularly limited. For example, adipose tissue-derived mesenchymal stem cells are cut after drilling fat cells with a drill or the like as necessary. And separating the micronized adipocyte mixture. As the separation method, the above-mentioned centrifugation conditions can be employed. When performing this process (3c ''), after the said process (2c), you may use for refinement | miniaturization and centrifugation of an adipocyte, with the concentrate filled. After centrifugation, free oil is separated in the syringe from the mixture of adipose tissue-derived mesenchymal stem cells and refined adipocytes, so the free oil is discarded and the adipose tissue-derived mesenchymal stem cells and refined A mixture of adipocytes can be obtained.
このような分離工程を経ることにより、脂肪組織由来間葉系幹細胞と共に、微細化された脂肪細胞を高濃度で含有する細胞製剤を調製することができる。ここで、微細な脂肪細胞とは、２６〜３０ゲージ、好ましくは１８〜３０ゲージの注射針を通過できる程度の大きさを有する脂肪細胞を指す。本工程（３ｃ''）において、脂肪細胞を微細化する処理を少なくとも１回、好ましくは１〜数回、より好ましくは１〜２回行うことができる。脂肪細胞を微細化することによって、細胞製剤の単位容積あたりの脂肪幹細胞の濃度を高くすることができ、毛髪再生果をより一層顕著に奏することができる。 By passing through such a separation step, a cell preparation containing high-concentrated adipocytes together with adipose tissue-derived mesenchymal stem cells can be prepared. Here, the fine fat cells refer to fat cells having a size that can pass through an injection needle of 26 to 30 gauge, preferably 18 to 30 gauge. In this step (3c ″), the treatment for refining fat cells can be performed at least once, preferably 1 to several times, more preferably 1 to 2 times. By making the fat cells finer, the concentration of adipose stem cells per unit volume of the cell preparation can be increased, and the hair regeneration result can be more remarkably exhibited.
また、工程（３ｃ''）で得られた微細化された脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部から脂肪組織由来間葉系幹細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、前記工程（２ｃ）で得られた濃縮物、又は工程（３ｃ''）で得られた微細化された脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部に混合する工程（４ｃ''）を含んでもよい。脂肪幹細胞の分離の方法は、上記方法（ｂ）の工程（２ｂ）について記載される通りである。 Further, the adipose tissue-derived mesenchymal stem cells were separated from a part of the mixture of the refined adipose tissue-derived mesenchymal stem cells obtained in the step (3c ″) and the refined adipocytes, and obtained. The adipose tissue-derived mesenchymal stem cells are the concentrate obtained in the step (2c) or the refined adipose tissue-derived mesenchymal stem cells obtained in the step (3c '') and the refined adipocytes. (4c ″) may be included in a part of the mixture. The method for separating adipose stem cells is as described for step (2b) of method (b) above.
本方法（ｃ）において、特表２００７−５３３３９６号公報に記載されるシリンジを使用することにより、細胞を外気に接触させることなく全ての工程を実施することができる。例えば、特表２００７−５３３３９６号公報に記載されるシリンジにカニューレ等を装
着して脂肪吸引により採取された脂肪を充填し、そのまま不純物の除去及び濃縮処理等に供し、細胞製剤を得ることができる。その後、細胞製剤を投与する際も、シリンジから直接、細胞製剤を注入するためのシリンジに移し、患部に投与することができるため、汚染等の問題のない安全性の高い細胞製剤とすることができる。簡便には、本方法（ｃ）を商業的に入手可能なキットを使用して行うこともでき、そのようなキットとしては、具体的には、ＬＩＰＯＭＡＸ−ＳＣ（ＭＥＤＩＫＡＮ Ｃｏｒｐ．製）等が例示される。
In this method (c), by using the syringe described in JP-T-2007-533396, all steps can be performed without bringing the cells into contact with the outside air. For example, a syringe described in JP-T-2007-533396 can be equipped with a cannula or the like, filled with fat collected by liposuction, and directly subjected to removal of impurities, concentration treatment, and the like to obtain a cell preparation. . After that, when administering the cell preparation, it can be transferred directly from the syringe to the syringe for injecting the cell preparation and administered to the affected area, so that the cell preparation should be highly safe without problems such as contamination. it can. For convenience, this method (c) can also be carried out using a commercially available kit. Specific examples of such a kit include LIPOMAX-SC (manufactured by MEDIKAN Corp.) and the like. Is done.
以上のいずれの方法によっても、本発明の細胞製剤を得ることができるが、好ましくは方法（ｂ）又は（ｃ）で得られる細胞製剤、更に好ましくは方法（ｃ）で得られる細胞製剤、より好ましくは工程（３ｃ''）を含む方法（ｃ）で得られる細胞製剤、特に好ましくは工程（３ｃ''）及び工程（４ｃ''）を含む方法（ｃ）で得られる細胞製剤が挙げられる。工程（３ｃ''）、又は工程（３ｃ''）及び工程（４ｃ''）を含む方法（ｃ）を経て得られる細胞製剤は、不純物が除去され、脂肪組織由来間葉系幹細胞と共に、微細化された健全な脂肪細胞を高濃度で含有するものであり、顕著な毛髪再生効果を奏することができる。 The cell preparation of the present invention can be obtained by any of the above methods, but is preferably a cell preparation obtained by the method (b) or (c), more preferably a cell preparation obtained by the method (c), Preferably, the cell preparation obtained by the method (c) including the step (3c ″), particularly preferably the cell preparation obtained by the method (c) including the step (3c ″) and the step (4c ″). . The cell preparation obtained through the step (3c ″) or the method (c) including the step (3c ″) and the step (4c ″) is finely mixed with adipose tissue-derived mesenchymal stem cells. It contains high-concentrated healthy fat cells and can exhibit a remarkable hair regeneration effect.
以下、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例に限定されて解釈されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is limited to these Examples and is not interpreted.
（１）細胞製剤の調製
細胞製剤の調製には、ＬＩＰＯＭＡＸ−ＳＣ（Ｍｅｄｉｋａｎ Ｃｏｒｐ．製，韓国 ソウル）キットを使用した。本細胞製剤の調製工程においては、細胞を全く外気に触れさせることなく行うことが可能であった。具体的な方法は以下の通りである。
(1) Preparation of cell preparation A LIPOMAX-SC (Medikan Corp., Seoul, Korea) kit was used for preparation of a cell preparation. In the preparation process of the present cell preparation, it was possible to carry out the cells without exposing them to the open air. A specific method is as follows.
シリンジ（ＬＩＰＯＭＡＸ−ＳＣ用に製造されたシリンジ：Ｍｅｄｉｋａｎ Ｃｏｒｐ．製，韓国 ソウル）を用いて、各被験者から脂肪吸引を行った。採取された脂肪を含むシリンジを約１０分間室温（約２５℃）で静置して細胞画分と液体画分に分離し、チュメセント液が含まれる液体画分を廃液した。その後、シリンジを２２００×ｇで８分間遠心分離にかけた。遠心分離により、上層（フリーオイル）、中間層（脂肪細胞及び脂肪組織由来間葉系幹細胞）、下層（細胞組織液等の液体画分）の三層に分離した。その後、中間層のみをシリンジ内に残して上下層を廃棄することにより、脂肪組織由来間葉系幹細胞と脂肪細胞の濃縮物を得た。この濃縮物に対してジェリリング（Ｇｅｌｌｉｎｇ）処理を行い、比較的大きな脂肪細胞の微細化を行った。ジェリリング処理は、１〜３回行った。ジェリリングには、ドリル（ＦｉｌｌｅｒＧｅｌｌｅｒカッター：ＭＥＤＩＫＡＮ社製）を用い、脂肪細胞をカッティングして微細化し、更に２２００×ｇで５分間遠心分離することにより、脂肪組織由来間葉系幹細胞と微細化された脂肪細胞を含む細胞製剤を得た。 Liposuction was performed from each subject using a syringe (syringe manufactured for LIPOMAX-SC: manufactured by Medikan Corp., Seoul, Korea). The collected fat-containing syringe was allowed to stand at room temperature (about 25 ° C.) for about 10 minutes to separate the cell fraction and the liquid fraction, and the liquid fraction containing the tumecent liquid was discarded. The syringe was then centrifuged at 2200 × g for 8 minutes. The mixture was separated into three layers by centrifugation, an upper layer (free oil), an intermediate layer (adipocytes and adipose tissue-derived mesenchymal stem cells), and a lower layer (liquid fraction such as cell tissue fluid). Thereafter, the concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes was obtained by leaving only the intermediate layer in the syringe and discarding the upper and lower layers. A gelling process was performed on the concentrate to refine relatively large adipocytes. The jelliing process was performed 1 to 3 times. For the jelliing, using a drill (FillerGeller cutter: manufactured by MEDIKAN), the fat cells are cut and refined, and further centrifuged at 2200 × g for 5 minutes to refine the adipose tissue-derived mesenchymal stem cells. A cell preparation containing adipocytes was obtained.
なお、脂肪細胞及び脂肪幹細胞は各患者由来のものを使用したため、上述の操作を各患者について行った。得られた細胞製剤中の脂肪組織由来間葉系幹細胞と脂肪細胞の細胞数は、患者によって異なるが、細胞製剤１ｍｌ当たり脂肪組織由来間葉系幹細胞が約１５〜２０万個、脂肪細胞が約１万〜２万個含まれていた。 In addition, since the fat cell and the fat stem cell used the thing derived from each patient, the above-mentioned operation was performed about each patient. The number of adipose tissue-derived mesenchymal stem cells and adipocytes in the obtained cell preparation varies depending on the patient, but about 1 to 200,000 adipose tissue-derived mesenchymal stem cells per ml of cell preparation and about adipocytes 10,000 to 20,000 were included.
（２）毛髪再生効果の確認
表１に示す、脱毛症等で前頭部が薄毛になっている被験者５名に対して、前記で得られた細胞製剤２．５ｍｌを、薄毛になっている右前頭部（約１２．５ｃｍ2）の皮下にシリンジを用いて注入した。
(2) Confirmation of hair regeneration effect As shown in Table 1, 2.5 ml of the cell preparation obtained above was thinned for five subjects with thinning frontal head due to alopecia etc. A syringe was used to inject the right frontal region (about 12.5 cm 2 ) subcutaneously.
細胞製剤投与から１〜２ヵ月後に投与部位での発毛状況を観察した結果を表１に示す。また、被験者Ｂ、Ｃ、及びＥについて、投与前後における投与部位の発毛状況を観察した結果を、それぞれ図１〜３に示す。この結果から、脂肪組織由来間葉系幹細胞と脂肪細胞を含む細胞製剤には、薄毛部位において発毛を促して毛髪を再生させる作用があることが確認された。 Table 1 shows the results of observation of hair growth at the administration site 1 to 2 months after administration of the cell preparation. Moreover, about subject B, C, and E, the result of having observed the hair growth condition of the administration site | part before and behind administration is shown to FIGS. 1-3, respectively. From this result, it was confirmed that the cell preparation containing adipose tissue-derived mesenchymal stem cells and adipocytes has an action of promoting hair growth and regenerating hair in the thin hair region.
Claims (8)
さらに、薬学的に許容される担体、賦形剤、消炎剤、鎮痛剤、免疫抑制剤、コラーゲン、線維芽細胞、成長因子、増殖因子、サイトカイン、ＰＲＰ、又は、組織再生作用を有する他の細胞製剤のいずれか少なくとも１つを含有する、毛髪再生用の細胞製剤。 Containing adipose tissue-derived mesenchymal stem cells and adipocytes collected from adipose tissue ;
Furthermore, pharmaceutically acceptable carriers, excipients, anti-inflammatory agents, analgesics, immunosuppressants, collagen, fibroblasts, growth factors, growth factors, cytokines, PRP, or other cells with tissue regeneration activity A cell preparation for hair regeneration containing at least one of the preparations.
採取された脂肪組織から液体画分を除去し、細胞画分を得る第１工程、
前記第１工程で得られた細胞画分から不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得る第２工程、
前記第２工程で得られた濃縮物の一部から脂肪組織由来間葉系幹細胞と脂肪細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、さらに前記第２工程で得られた濃縮物の一部に混合する第３工程。 A method for preparing a cell preparation for hair regeneration, comprising the following first to third steps.
A first step of removing a liquid fraction from the collected adipose tissue to obtain a cell fraction;
A second step of removing impurities from the cell fraction obtained in the first step to obtain a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes;
Adipose tissue-derived mesenchymal stem cells and adipocytes are separated from a part of the concentrate obtained in the second step, and the obtained adipose tissue-derived mesenchymal stem cells are further concentrated in the second step. 3rd process which mixes in a part of thing.
採取された脂肪組織から液体画分を除去し、細胞画分を得る第１工程、
前記第１工程で得られた細胞画分から不純物を除去し、脂肪組織由来間葉系幹細胞及び脂肪細胞の濃縮物を得る第２工程、
前記第２工程で得られた濃縮物中の脂肪細胞を微細化し、脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物として分離する第３工程。 A method for preparing a cell preparation for hair regeneration, comprising the following first to third steps.
A first step of removing a liquid fraction from the collected adipose tissue to obtain a cell fraction;
A second step of removing impurities from the cell fraction obtained in the first step to obtain a concentrate of adipose tissue-derived mesenchymal stem cells and adipocytes;
A third step in which the fat cells in the concentrate obtained in the second step are refined and separated as a mixture of adipose tissue-derived mesenchymal stem cells and refined adipocytes.
前記第３工程で得られた脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部から脂肪組織由来間葉系幹細胞を分離し、得られた脂肪組織由来間葉系幹細胞を、前記第２工程で得られた濃縮物、又は、前記第３工程で得られた脂肪組織由来間葉系幹細胞及び微細化された脂肪細胞の混合物の一部に混合する第４工程。 The method for preparing a cell preparation for hair regeneration according to claim 7, further comprising the following fourth step.
The adipose tissue-derived mesenchymal stem cells are separated from a part of the mixture of the adipose tissue-derived mesenchymal stem cells obtained in the third step and the refined adipocytes, and the obtained adipose tissue-derived mesenchymal stem cells are obtained. The 4th process mixed with a part of the mixture of the concentrate obtained by the said 2nd process, or the adipose tissue origin mesenchymal stem cell obtained by the said 3rd process, and the refined fat cell.
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