JPH0499482A - Method fro culturing hair mother cell - Google Patents
Method fro culturing hair mother cellInfo
- Publication number
- JPH0499482A JPH0499482A JP2214181A JP21418190A JPH0499482A JP H0499482 A JPH0499482 A JP H0499482A JP 2214181 A JP2214181 A JP 2214181A JP 21418190 A JP21418190 A JP 21418190A JP H0499482 A JPH0499482 A JP H0499482A
- Authority
- JP
- Japan
- Prior art keywords
- cells
- hair
- culture
- hair matrix
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 210000004209 hair Anatomy 0.000 title claims abstract description 64
- 238000000034 method Methods 0.000 title claims description 25
- 238000012258 culturing Methods 0.000 title claims description 14
- 210000000130 stem cell Anatomy 0.000 title abstract description 8
- 210000004027 cell Anatomy 0.000 claims abstract description 88
- 210000000170 cell membrane Anatomy 0.000 claims abstract description 8
- 239000011159 matrix material Substances 0.000 claims description 47
- 230000002500 effect on skin Effects 0.000 claims description 32
- 238000012136 culture method Methods 0.000 abstract description 14
- 230000035790 physiological processes and functions Effects 0.000 abstract description 5
- 241000283984 Rodentia Species 0.000 abstract description 2
- 102000004169 proteins and genes Human genes 0.000 abstract description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 210000001519 tissue Anatomy 0.000 description 14
- 239000000243 solution Substances 0.000 description 7
- 230000035755 proliferation Effects 0.000 description 6
- 239000000725 suspension Substances 0.000 description 6
- 230000004663 cell proliferation Effects 0.000 description 5
- 210000003780 hair follicle Anatomy 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 201000004384 Alopecia Diseases 0.000 description 4
- 229920001917 Ficoll Polymers 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 239000006285 cell suspension Substances 0.000 description 4
- 210000004207 dermis Anatomy 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 210000003491 skin Anatomy 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 238000000432 density-gradient centrifugation Methods 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000000512 collagen gel Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000003779 hair growth Effects 0.000 description 2
- 230000003676 hair loss Effects 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 206010033675 panniculitis Diseases 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 210000004304 subcutaneous tissue Anatomy 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- DWRXFEITVBNRMK-UHFFFAOYSA-N Beta-D-1-Arabinofuranosylthymine Natural products O=C1NC(=O)C(C)=CN1C1C(O)C(O)C(CO)O1 DWRXFEITVBNRMK-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- 206010068168 androgenetic alopecia Diseases 0.000 description 1
- 201000002996 androgenic alopecia Diseases 0.000 description 1
- IQFYYKKMVGJFEH-UHFFFAOYSA-N beta-L-thymidine Natural products O=C1NC(=O)C(C)=CN1C1OC(CO)C(O)C1 IQFYYKKMVGJFEH-UHFFFAOYSA-N 0.000 description 1
- JAONZGLTYYUPCT-UHFFFAOYSA-K bismuth subgallate Chemical compound OC(=O)C1=CC(O)=C2O[Bi](O)OC2=C1 JAONZGLTYYUPCT-UHFFFAOYSA-K 0.000 description 1
- 208000037516 chromosome inversion disease Diseases 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 208000024963 hair loss Diseases 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001293 nucleolytic effect Effects 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- -1 stir well Substances 0.000 description 1
- 229940104230 thymidine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、毛母細胞培養法、詳しくは、生理的な状態の
増殖・分化能力を有した毛母細胞を定常的に培養し得る
毛母細胞培養法に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a method for culturing hair matrix cells, and more particularly, to a method for culturing hair matrix cells that can constantly culture hair matrix cells that have the ability to proliferate and differentiate under physiological conditions. Regarding mother cell culture methods.
毛母細胞は毛の成長を支える基となる細胞であることか
ら、男性型脱毛症いわゆる若禿をはしめ種々の脱毛症の
改善を目的に、毛母細胞増殖促進物質の探索が広く行わ
れているが、本発明はこの探索に利用し得る。Since hair matrix cells are the fundamental cells that support hair growth, the search for hair matrix growth-promoting substances has been widely carried out with the aim of improving various types of alopecia, including androgenetic alopecia (premature baldness). However, the present invention can be used for this search.
〔従来の技術］
従来、毛包組織中で毛母細胞は***速度が著しく高いこ
とから、毛母細胞の培養法としては、毛包組織を培養す
ることによって毛母細胞を培養する方法（器官培養法）
が用いられている。このような器官培養法としては、例
えば、Ｂｕｈｌ、Ａ、Ｅ等の方法（Ｊ、Ｉｎｖｅｓｔ、
Ｄｅｒ＋ｍａｔｏ１．９２，３１５．１９８９ ）など
が知られている。[Prior Art] Conventionally, since hair matrix cells in hair follicle tissues have a significantly high division rate, methods of culturing hair matrix cells by culturing hair follicle tissues have been used. culture method)
is used. As such an organ culture method, for example, the method of Buhl, A, E, etc. (J, Invest,
Der+mato1.92, 315.1989) and others are known.
また、・毛母細胞を単離培養する方法も従来より試みら
れている。かかる方法としては、例えば、適当な方法に
よって単離した毛母細胞を、適当な塩類ｉｉ（培養液）
甲で培養する方法が知られている。また、この方法では
成育が悪いため、培養液中に牛胎児血清又は脳下垂体抽
出物などを添加して培養を行う方法が提案されている（
特開昭６３−３０１７８５号公報）。Furthermore, methods for isolating and culturing hair matrix cells have also been attempted. As such a method, for example, hair matrix cells isolated by an appropriate method are mixed with an appropriate salt ii (culture solution).
A method of culturing in the instep is known. In addition, since growth is poor with this method, a method has been proposed in which culture is performed by adding fetal bovine serum or pituitary gland extract to the culture solution (
(Japanese Patent Application Laid-Open No. 63-301785).
しかしながら、上記器官培養法は、得られる細胞の量が
少ない、個体間の差が大きい、長期的に培養できないな
どの欠点があった。However, the organ culture method described above has drawbacks such as a small amount of cells obtained, large differences between individuals, and inability to culture for a long period of time.
また、上記公報に記載された毛母細胞の単離培養方法は
、一定条件下で定常的に毛母細胞を培養し得るが、その
増殖率は低く、毛の成長を生理的に正しく反映したもの
とは言い難い、即ち、この方法は、毛包組織中の微小環
境の影響を複雑に受けつつ増殖する毛母細胞を、その毛
包組織から切り離しているため、生理的な状態の毛母細
胞の増殖を行えない欠点があった。In addition, although the method for isolating and culturing hair matrix cells described in the above-mentioned publication can steadily culture hair matrix cells under certain conditions, the proliferation rate is low and does not physiologically accurately reflect hair growth. In other words, this method separates the hair matrix cells, which proliferate under the influence of the microenvironment in the hair follicle tissue, from the hair follicle tissue, so the hair matrix in a physiological state is separated from the hair follicle tissue. The drawback was that cells could not grow.
従って、本発明の目的は、生理的な状態の増殖分化能力
を有した毛母細胞を定常的に培養し得る毛母細胞培養法
を提供することにある。Therefore, an object of the present invention is to provide a method for culturing hair matrix cells capable of constantly culturing hair matrix cells having the ability to proliferate and differentiate in a physiological state.
本発明は、上記目的を、毛母細胞を、毛乳頭細胞、毛乳
頭細胞を含有する細胞画分、又は毛乳頭細胞膜画分の存
在下に培養することを特徴とする毛母細胞培養法を提供
することにより達成したものである。The present invention achieves the above object by providing a method for culturing hair matrix cells, which comprises culturing hair matrix cells in the presence of dermal papilla cells, a cell fraction containing dermal papilla cells, or a dermal papilla cell membrane fraction. This was achieved by providing
以下、本発明の毛母細胞培養法について詳述する。Hereinafter, the hair matrix cell culture method of the present invention will be described in detail.
本発明の培養対象である毛母細胞としては、その由来に
制限されるものではなく、ヒトに限らず毛髪を有する種
の毛母細胞であれば良く、またその部位、雌雄、齢、毛
髪の発育状態を問わず用いることかできるが、好ましく
は鰯歯類動物の新生児体毛から採取した毛母細胞を用い
る。材料からの毛母細胞調製法としては、外科的手法（
Ｊｏｎｅｓ、Ｌ、＋Ｎ、＋ｅｔ ａｌ、Ｊ Ｉｎｖｅｓ
ｔ Ｄｅｒｗａｔｏｌ、ｗ、５８．１９８８）、酵素処
理を用いた手法（Ｒｏｇｅｒｓ、Ｇ、、ｅｔ ａｌ、Ｊ
Ｉｎｖｅｓｔ Ｄｅｒｍａｔｏｌ、８９，３６９，
１９８７； Ｇｒｅｅｎ、門、、Ｒ，、ｅｔ ａｌ
、ＪＩｎｖｅｓｔ Ｄｅｒｓａｔｏｌ、 ８７，７６８
．１９８６ ）等があるが、本発明においてはいずれの
手法を用いても良い。The hair matrix cells to be cultured in the present invention are not limited to their origin, and may be hair matrix cells of any species that has hair, not just humans, and may be based on their site, sex, age, or type of hair. Although it can be used regardless of the developmental state, hair matrix cells collected from newborn baby hair of a rodent are preferably used. As a method for preparing hair matrix cells from materials, surgical methods (
Jones, L, +N, +et al, J Inves
t Derwatol, W, 58.1988), a method using enzyme treatment (Rogers, G., et al., J.
Invest Dermatol, 89,369,
1987; Green, R., et al.
, JInvest Dersatol, 87,768
．． 1986), but any method may be used in the present invention.
本発明の培養対象である毛母細胞は、例えば、次のよう
にして調製することができる。Hair matrix cells to be cultured in the present invention can be prepared, for example, as follows.
皮下組織等を取り除き、約３ｍ幅の短冊状に裁断した皮
膚をリン酸緩衝生理食塩水（ＰＢＳ）で洗浄した後、ト
リプシンなど蛋白質分解酵素溶液による化学的処理方法
やビンセットなどによる物理的方法を組合せて表皮を剥
離除去し、真皮組織を得る。得られた真皮組織をコラゲ
ナーゼ溶液や核酸分解酵素で処理し、さらに物理的な方
法により細組縁断片とし、濾過法により残存＆［Ｉ微片
を除去した組織懸濁液を培地で洗浄する。After removing subcutaneous tissue, etc., and washing the skin with phosphate buffered saline (PBS), which is cut into strips about 3 m wide, chemical treatment with a proteolytic enzyme solution such as trypsin, or physical treatment with a bottle set, etc. The epidermis is exfoliated and removed to obtain the dermal tissue. The obtained dermal tissue is treated with a collagenase solution or a nucleolytic enzyme, and then cut into fine margin fragments by a physical method, and the tissue suspension from which residual &[I particles are removed by a filtration method is washed with a culture medium.
この組織懸濁液をフィコール（Ｆｉｃｏｌｌ）やパーコ
ル（Ｐｅｒｃｏｌｌ）等による密度勾配による遠心分離
を行う、この方法により、より下層に得られた細胞を毛
母細胞として用いる。得られた細胞をさらに上記密度勾
配遠心分前を繰り返し、より純度を高めることもできる
。This tissue suspension is centrifuged using a density gradient using Ficoll, Percoll, etc. By this method, the cells obtained in the lower layer are used as hair matrix cells. The purity of the obtained cells can be further increased by repeating the above-mentioned density gradient centrifugation.
また、本発明で用いられる毛乳頭細胞としては、毛母細
胞の場合と同様にその由来に制限されるものではなく、
種々の種属の毛乳頭細胞を用いることができ、また毛母
細胞と組織の由来を同一にする必要はなく、異なる種間
、部位であっても良い。Furthermore, the dermal papilla cells used in the present invention are not limited to their origin, as in the case of hair matrix cells;
Dermal papilla cells of various species and genera can be used, and the origins of the hair matrix cells and tissues do not have to be the same, and they may be of different species or sites.
上記毛乳頭細胞の調製法としては、専ら外科的手法（Ｊ
ａｈｏｄａ、Ｃ，、ｅｔ ａｌ、Ｂｒ Ｊ Ｄｅｒｍａ
ｔｏｌ、 １０５．６２３＋１９８１： Ｋａｔｓｕｏ
ｋａ、Ｍ、、Ａｒｃｈ ＤｅｒｗａＬｏｌ Ｒｅｓ、２
７９，２０．１９８６）が用いられるが、真皮＆［Ｉ織
のコラゲナーゼ処理等を用いた上記毛母細胞の調製と同
様な手法によっても代用できる。The method for preparing the dermal papilla cells described above is exclusively performed by a surgical method (J
ahoda, C, et al, Br J Derma
tol, 105.623+1981: Katsuo
ka, M,, Arch DerwaLol Res, 2
79, 20, 1986), but it can also be substituted by a method similar to the above-mentioned preparation of hair matrix cells using collagenase treatment of dermis & [I weave.
即ち、上記毛母細胞の調製における場合と同様にして調
製した組織懸濁液をフィコール（Ｆｉｃｏｌｌ）やパー
コール（Ｐｅｒｃｏｌｌ）等による密度勾配による遠心
分離を行う゛ことにより、よ゛り上層に得られた細胞を
毛乳頭細胞又は該細胞を含有する細胞画分として用いる
ことができる。得られた細胞をさらに上記密度勾配遠心
分離を繰り返し、より純度を高めることもできる。さら
に、このようにして得られた細胞から一般的に用いられ
ている細胞膜画分調製法により得られた試料、即ち毛乳
頭細胞膜両分も、上記の毛乳頭細胞又は該細胞を含有す
る細胞画分と同しように用いることができる。That is, by centrifuging a tissue suspension prepared in the same manner as in the preparation of hair matrix cells above using a density gradient using Ficoll, Percoll, etc., more of the tissue obtained in the upper layer is obtained. The cells can be used as dermal papilla cells or a cell fraction containing the cells. The obtained cells can be further subjected to the density gradient centrifugation described above to further increase the purity. Furthermore, a sample obtained from the cells thus obtained by a commonly used cell membrane fraction preparation method, that is, both dermal papilla cell membrane fractions, may also be used as the dermal papilla cell or cell containing cell fraction as described above. It can be used in the same way as minutes.
而して、本発明を実施するには、上記毛母細胞と、上記
毛乳頭細胞、上記毛乳頭細胞を含有する細胞画分、又は
上記毛乳頭細胞膜両分とを、細胞数、蛋白質量又はＤＮ
Ａ量の比が好ましくは１：９９〜９９：１、より好まし
くは１：９〜９：ｊの範囲で混合し培養する。培養は、
いずれが一方の細胞をあらかしめ培養し、他方を後から
加えても良く、またあらかしめ両者を混合しておき、同
時に培養しても良い。Accordingly, in carrying out the present invention, the hair matrix cells, the dermal papilla cells, the cell fraction containing the dermal papilla cells, or both the dermal papilla cell membrane fractions are determined according to the cell number, protein amount, or D.N.
They are mixed and cultured at a ratio of A amounts preferably in the range of 1:99 to 99:1, more preferably in the range of 1:9 to 9:j. The culture is
One of the cells may be pre-cultivated and the other may be added later, or both may be pre-mixed and cultured at the same time.
培養方法としては、浮遊培養法、付着培養法、及び包埋
培養法等の通常の培養法を用いることができるが、コラ
ーゲン、寒天などの支持体を用いた包埋培養法が好まし
い、また、その培養条件としては、２０〜３８°Ｃ１０
〜１０％ＣＯｚ及びＯ〜９５％Ｏ１含有気相下が好まし
い。As a culture method, normal culture methods such as floating culture method, attached culture method, and embedding culture method can be used, but embedding culture method using a support such as collagen or agar is preferable. The culture conditions are 20-38°C10
A gas phase containing ~10% COz and O~95% O1 is preferred.
本発明の毛母細胞培養法によれば、生理的な状態の増殖
・分化能力を有した毛母細胞が定常的に培養される。According to the hair matrix cell culture method of the present invention, hair matrix cells having the ability to proliferate and differentiate in a physiological state are constantly cultured.
［実施例］ 以下に実施例を挙げ、本発明をさらに詳しく説明する。[Example] The present invention will be explained in more detail with reference to Examples below.
実施例１
［毛母細胞、毛乳頭細胞を得る方法〕
新生マウスを水冷麻酔し、７０％エタノールで皮膚を消
毒した。皮膚を採取し、皮下組織等を取り除いた後、約
３ｗ幅の短冊状に裁断した。この皮膚をＰＢＳで洗浄し
た後、０１２５％のトリプシン溶液を用い、４°Ｃ下で
一昼夜消化した。トリブンン処理後、ビンセントで丁寧
に表皮を剥離除去して真皮組織を得た。得られた真皮Ｕ
織をＰＢＳで洗浄した後、３７°Ｃ下で０．２５％コラ
ゲナーゼ溶液で撹拌しながら処理した。さらに、［１Ｎ
ａｓｅ１（５０００単位）加え、ナイロン網（１５０Ｍ
／Ｓ）に濾過することで残存組織片を除去した組織懸濁
液を液体培地（Ｍｅｄｉａａ１９９ ）で洗浄した。Example 1 [Method for obtaining hair matrix cells and dermal papilla cells] A newborn mouse was anesthetized with water cooling, and its skin was disinfected with 70% ethanol. The skin was collected, subcutaneous tissues etc. were removed, and then cut into strips approximately 3W wide. After washing the skin with PBS, it was digested with 0.125% trypsin solution at 4°C overnight. After the tribun treatment, the epidermis was carefully peeled off using Vincent to obtain dermal tissue. Obtained dermis U
After washing the tissue with PBS, it was treated with a 0.25% collagenase solution at 37°C with stirring. Furthermore, [1N
In addition to ase1 (5000 units), nylon net (150M
The tissue suspension, from which residual tissue debris was removed by filtration with /S), was washed with a liquid medium (Mediaa199).
この組織懸濁液を１０％（ｗ／ｖ）Ｆｉｃｏｌｌ ４
００での密度勾配による遠心分離を行った。これにより
、１０％Ｆｉｃｏｌｌ上層には毛乳頭細胞が、下層（沈
澱物）には毛母細胞が、それぞれ分離された。得られた
沈澱物をさらに上記密度勾配遠心分離を繰り返し、遠心
処理（５０ｇ、４分）で二回洗浄し、毛母細胞懸濁液を
得た。一方、上層より回収された細胞は、洗浄した後、
ステンレス網（２００Ｍ／Ｓ）でｍ過し、毛乳頭細胞懸
濁液を得た。This tissue suspension was mixed with 10% (w/v) Ficoll 4
Centrifugation was performed through a density gradient at 00. As a result, dermal papilla cells were separated from the 10% Ficoll upper layer, and hair matrix cells were separated from the lower layer (precipitate). The resulting precipitate was further subjected to the density gradient centrifugation described above and washed twice by centrifugation (50 g, 4 minutes) to obtain a hair matrix cell suspension. On the other hand, after washing the cells collected from the upper layer,
The mixture was passed through a stainless steel net (200M/S) to obtain a suspension of dermal papilla cells.
コラーゲン・ゲル溶液（Ｍｅｄｉａｍ１９９で平衡化し
たもの）２容に対し、上記毛母細胞懸濁液２容及び上記
毛乳頭細胞懸濁液】容を加えてよく撹拌し、マイクロ・
ウェル・プレートに植え込み、ゲルの硬化が観察された
ところでゲルと同量のＭｅｄｉａｍ１９９を添加した。To 2 volumes of collagen gel solution (equilibrated with Medium 199), add 2 volumes of the above hair matrix cell suspension and 1 volume of the above dermal papilla cell suspension, stir well, and mix well.
It was implanted into a well plate, and when hardening of the gel was observed, the same amount of Medium 199 as the gel was added.
これを５％Ｃ０２インキユペター内（３７“Ｃ）で培養
し、３日−チミジン取り込み等による細胞増殖能の検討
を行ったところ、各々組織単独培養時に比べて、混合培
養時の方が著しく高陽していた。This was cultured in a 5% C02 incubator (37"C), and the cell proliferation ability was examined by thymidine incorporation for 3 days. It was found that the growth rate was significantly higher in the mixed culture than in the culture of each tissue alone. was.
実施例２ ［混合比の検討］
実施例１の混合培養法における毛母細胞と毛乳頭細胞と
の混合比を変えてコラーゲン・ゲル包埋培養を行った。Example 2 [Study of mixing ratio] Collagen gel embedding culture was performed by changing the mixing ratio of hair matrix cells and dermal papilla cells in the mixed culture method of Example 1.
各細胞混合比での相対的細胞増殖能を下記第１表に示す
。The relative cell proliferation ability at each cell mixing ratio is shown in Table 1 below.
第１表
上記第１表に示す結果から判るように、本発明の培養法
は、毛母細胞と毛乳頭細胞との混合比を変化させること
により、培養下における毛母細胞の増殖促進率を調節し
得る。Table 1 As can be seen from the results shown in Table 1 above, the culture method of the present invention improves the growth promotion rate of hair matrix cells under culture by changing the mixing ratio of hair matrix cells and dermal papilla cells. Can be adjusted.
実施例３ 〔異種間混合培養〕
ラット真皮より毛乳頭細胞を、マウス真皮より毛母細胞
を調製し、これらの細胞を実施例１と同様にして混合培
養した。この場合のマウス由来の毛母細胞の増殖能を、
毛母細胞（マウス）を単独培養した場合の細胞増殖能を
基準とした増殖比により下記第２表に示す。また、下記
第２表には、毛母細胞及び毛乳頭細胞のいずれもマウス
由来のものを用いた場合の増殖比も併せて示す。Example 3 [Heterogeneous mixed culture] Dermal papilla cells were prepared from rat dermis and hair matrix cells were prepared from mouse dermis, and these cells were mixed and cultured in the same manner as in Example 1. In this case, the proliferation ability of mouse-derived hair matrix cells is
Table 2 below shows the proliferation ratio based on the cell proliferation ability when hair matrix cells (mouse) were cultured alone. Table 2 below also shows the proliferation ratio when both hair matrix cells and dermal papilla cells were derived from mice.
第２表
異種間混合での相対的細胞増殖能
上記第２表に示す結果から判るように、本発明の培養法
は、種を隔てて活用できる。Table 2: Relative Cell Proliferation Ability in Mixed Mixtures of Different Species As can be seen from the results shown in Table 2 above, the culture method of the present invention can be used regardless of species.
実施例４ 〔毛乳頭細胞膜百分の利用〕毛乳頭細胞懸濁
液の代わりに３ Ｘ １０’ ｃｅｌｌｓの毛乳頭細胞
から得られた毛乳頭細胞膜画分を用いた以外は、実施例
１と同様にして混合培養した。Example 4 [Using dermal papilla cell membrane percentage] Same as Example 1 except that the dermal papilla cell membrane fraction obtained from 3 x 10' cells of dermal papilla cells was used instead of the dermal papilla cell suspension. A mixed culture was performed.
この場合の細胞増殖能（増殖比）を、毛乳頭細胞（３Ｘ
１０ ’ ｃｅｌｌｓ）を用いた場合の増殖能と共に
下記第３表に示す。In this case, the cell proliferation ability (proliferation ratio) was calculated using dermal papilla cells (3X
Table 3 below shows the proliferation ability when using 10' cells).
第３表
〔発明の効果〕
本発明の毛母細胞培養法は、生理的な状態の増殖・分化
能力を有した毛母細胞を定常的に培養し得るため、育毛
剤開発研究や、毛包組繊細胞間相互作用など多方面にわ
たる毛の基礎研究に利用され、毛の発生に関与し得る因
子をとらえ、永年未解決であった脱毛の機構の解明の平
文てとなる。Table 3 [Effects of the Invention] The hair matrix cell culture method of the present invention can steadily culture hair matrix cells that have the ability to proliferate and differentiate in a physiological state. It will be used in basic research on hair in a wide range of fields, including the interactions between tissue cells, and will help us understand factors that may be involved in hair development, and will provide a clue to the long-unsolved mechanism of hair loss.
Claims (1)
分、又は毛乳頭細胞膜画分の存在下に培養することを特
徴とする毛母細胞培養法。A method for culturing hair matrix cells, which comprises culturing hair matrix cells in the presence of dermal papilla cells, a cell fraction containing dermal papilla cells, or a dermal papilla cell membrane fraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2214181A JPH0732706B2 (en) | 1990-08-13 | 1990-08-13 | Hair mother cell culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2214181A JPH0732706B2 (en) | 1990-08-13 | 1990-08-13 | Hair mother cell culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0499482A true JPH0499482A (en) | 1992-03-31 |
| JPH0732706B2 JPH0732706B2 (en) | 1995-04-12 |
Family
ID=16651583
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2214181A Expired - Fee Related JPH0732706B2 (en) | 1990-08-13 | 1990-08-13 | Hair mother cell culture method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0732706B2 (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH08248027A (en) * | 1995-02-17 | 1996-09-27 | L'oreal Sa | Testing method of active material for extracted hair |
| USH1610H (en) * | 1993-12-03 | 1996-11-05 | The Procter & Gamble Company | Methods for culturing hair follicle epithelial matrix cells |
| JP2008306938A (en) * | 2007-06-12 | 2008-12-25 | Shiseido Co Ltd | Method for culturing hair papilla cell |
| CN114891724A (en) * | 2022-07-01 | 2022-08-12 | 南方医科大学南方医院 | Method for extracting hair follicle and hair papilla cells of mouse hair |
-
1990
- 1990-08-13 JP JP2214181A patent/JPH0732706B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| USH1610H (en) * | 1993-12-03 | 1996-11-05 | The Procter & Gamble Company | Methods for culturing hair follicle epithelial matrix cells |
| JPH08248027A (en) * | 1995-02-17 | 1996-09-27 | L'oreal Sa | Testing method of active material for extracted hair |
| JP2008306938A (en) * | 2007-06-12 | 2008-12-25 | Shiseido Co Ltd | Method for culturing hair papilla cell |
| CN114891724A (en) * | 2022-07-01 | 2022-08-12 | 南方医科大学南方医院 | Method for extracting hair follicle and hair papilla cells of mouse hair |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0732706B2 (en) | 1995-04-12 |
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