JP2006000059A - Method for promoting proliferation and differentiation of animal cell by using extracellular substrate - Google Patents
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本発明は、多分化能を有する動物幹細胞等の新規な培養方法に関する。特に、組織の再生医療のための移植材料の調製等に有用な多種類の動物幹細胞等の培養のために、細胞外基質（Extracellular matrix,ECM）を用いて、動物幹細胞等の増殖や分化誘導を促進する動物細胞の培養方法に関する。 The present invention relates to a novel method for culturing animal stem cells having multipotency. In particular, for the culturing of various types of animal stem cells that are useful for the preparation of transplant materials for tissue regenerative medicine, etc., the proliferation and differentiation induction of animal stem cells, etc. using an extracellular matrix (ECM) The present invention relates to a method for culturing animal cells that promotes the above.
間葉系幹細胞のような動物細胞の幹細胞は、哺乳類の骨髄等に存在し、脂肪細胞、軟骨細胞、骨細胞に分化する多能性の幹細胞として知られている。該幹細胞は、その分化多能性の故に、骨、軟骨、腱、筋肉、脂肪、歯周組織など、多くの組織の再生医療のための移植材料として注目されている（遺伝子医学、Vol.４、No．２（2000）ｐ５８−６１）。最近、間葉系幹細胞研究の現状と展望についての総説が発行され、間葉系幹細胞の採取や培養に関する報告がなされている（実験医学、Vol．19、No．3（2月号）2001、ｐ350−356）。また、近年、間葉系幹細胞の培養、分化等に関しいくつかの特許出願が公開されている。例えば、特表平１１−５０６６１０公報には、無血清環境下でヒト間葉前駆細胞の生存を維持する組成物及び方法について、特表平１０−５１２７５６号公報には、間葉系幹細胞の分化を誘導するために、プロスタグランジン、アスコルビン酸、コラーゲン細胞外基質等からなる骨誘導因子、分化付随因子、軟骨誘導因子等の生物活性因子と接触させることよりなる方法について、特開２０００−２１７５７６号公報には、プロラクチン又はその同効物の共存下で多能性間葉系幹細胞を培養し、間葉系幹細胞を脂肪細胞へ分化させる方法について、それぞれ発明が開示されている。 Stem cells of animal cells such as mesenchymal stem cells exist in mammalian bone marrow and are known as pluripotent stem cells that differentiate into adipocytes, chondrocytes, and bone cells. Due to its pluripotency, the stem cells are attracting attention as transplant materials for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat, and periodontal tissue (Gene Medicine, Vol. 4). No. 2 (2000) p58-61). Recently, a review of the current status and prospects of mesenchymal stem cell research has been published, and reports on the collection and culture of mesenchymal stem cells have been made (Experimental Medicine, Vol. 19, No. 3 (February issue) 2001, p350-356). In recent years, several patent applications have been published regarding the culture and differentiation of mesenchymal stem cells. For example, JP-A-11-506610 discloses a composition and method for maintaining the survival of human mesenchymal progenitor cells in a serum-free environment, and JP-T-10-512756 discloses differentiation of mesenchymal stem cells. JP-A-2000-217576, which comprises contacting with a bioactive factor such as osteoinductive factor consisting of prostaglandin, ascorbic acid, collagen extracellular matrix, differentiation-associated factor, cartilage inducing factor, etc. The publication discloses a method for culturing pluripotent mesenchymal stem cells in the presence of prolactin or its equivalent and differentiating mesenchymal stem cells into adipocytes.
間葉系幹細胞のような幹細胞或いはその他の動物細胞を、組織の再生医療に利用するため、或いはその他のバイオ産業における動物細胞として利用するためには、まず、この動物細胞を生体組織から採取し、１）その細胞を大量の細胞数まで増殖させる、２）細胞増殖中に、将来分化できる能力を保持する、３）培養器（培養皿）上でその細胞を分化させる、ことが必要である。更にそれを用いて移植用の組織の調製を行うことが必要となる。
すなわち、間葉系幹細胞のような動物幹細胞は、骨髄や骨膜に存在するが、組織再生医療への実用化のためには、これらの組織から幹細胞を採取する安全で、手間のかからぬ方法を開発すること、且つ、培養等により幹細胞の十分な量を取得する方法を開発することが重要な課題となる。また、幹細胞の組織再生医療への実用化のためには、採取した幹細胞を分化能力を維持したまま培養増殖させることが重要である。更に、培養した幹細胞を目的の細胞へと試験管内で分化を誘導する技術があることが望ましい。
In order to use stem cells such as mesenchymal stem cells or other animal cells in tissue regenerative medicine or as animal cells in other bioindustries, first, these animal cells are collected from living tissue. It is necessary to 1) grow the cell to a large number of cells, 2) retain the ability to differentiate in the future during cell growth, and 3) differentiate the cell on a culture vessel (culture dish). . Furthermore, it is necessary to prepare a tissue for transplantation using it.
In other words, animal stem cells such as mesenchymal stem cells exist in the bone marrow and periosteum, but for practical application to tissue regeneration medicine, a safe and hassle-free method of collecting stem cells from these tissues is necessary. It is an important issue to develop and develop a method for obtaining a sufficient amount of stem cells by culturing or the like. In addition, for practical application of stem cells to tissue regeneration medicine, it is important to culture and proliferate the collected stem cells while maintaining the differentiation ability. Furthermore, it is desirable to have a technique for inducing differentiation of cultured stem cells into target cells in vitro.
したがって、動物幹細胞の組織再生医療への実用化のために、これらの要求を満足した動物幹細胞の効果的な培養方法を開発することが必要となる。更に、組織再生医療のための幹細胞の培養のためには、被治療対象に安全な自己血清を用いるのが望ましく、その場合、低濃度の血清で培養することは困難であるため、採取個体から大量の血清を採取する必要がある。しかしながら、採取個体から大量の血清を取得すること自体困難であることから、低濃度の血清下でも培養することが可能となる方法を開発することが必要となる。或いは、培養期間を短縮する必要がある。また、幹細胞を分化誘導培養する際においても、分化誘導能力に優れた培養方法を開発する必要がある。このように、間葉系幹細胞のような動物幹細胞を組織の再生医療等へ利用するに際して、その培養方法に関して、多くの課題の解決が要望されている。 Therefore, it is necessary to develop an effective method for culturing animal stem cells that satisfies these requirements in order to put animal stem cells into tissue regeneration medicine. Furthermore, for the culture of stem cells for tissue regeneration medicine, it is desirable to use autologous serum that is safe for the subject to be treated, and in that case, it is difficult to culture with low-concentration serum. A large amount of serum needs to be collected. However, since it is difficult to obtain a large amount of serum from a collected individual, it is necessary to develop a method capable of culturing even under a low concentration of serum. Alternatively, it is necessary to shorten the culture period. In addition, when culturing stem cells for differentiation induction, it is necessary to develop a culture method excellent in differentiation induction ability. As described above, when animal stem cells such as mesenchymal stem cells are used for tissue regenerative medicine and the like, there are demands for solutions to many problems relating to the culture method.
一方、細胞外基質は、上皮細胞、非上皮細胞を問わず体細胞の間に存在する物質で、単に組織の支持のみならず、すべての体細胞の生存に必要な内部環境の構成に関与しているものとして知られている。また、細胞外基質は、骨、歯、腱、皮膚などに多く含まれていることからもわかるように、組織の支持、結合、物理的境界、力の吸収、弾性要素といった物理的な役割が、その機能として古くから知られている。近年になって、これらの基質がその機能として、細胞増殖、細胞移動、細胞内代謝、細胞分化、細胞の形態などを細胞の外から調節する生理化学的役割などに関与し、その機能を担っていることが明らかになってきている。 On the other hand, extracellular matrix is a substance that exists between somatic cells, whether epithelial cells or non-epithelial cells, and is involved not only in supporting the tissue but also in the internal environment necessary for the survival of all somatic cells. Known as being. Extracellular matrix also has physical roles such as tissue support, bonding, physical boundaries, force absorption, and elastic elements, as can be seen from the fact that the extracellular matrix is abundant in bones, teeth, tendons, and skin. It has been known for a long time as its function. In recent years, these substrates have been involved in functions such as physiochemical roles that regulate cell proliferation, cell migration, intracellular metabolism, cell differentiation, cell morphology, etc. from outside the cell. It is becoming clear.
近年、細胞外基質の利用としては、例えば、生体細胞接着性材料や組織移植片組成物としての利用が知られており（特開平５−１５５８３号公報、特表２０００−５０８９２２号公報、特表２００１−５０５９１７号公報）、また、初代培養肝細胞の培養方法において、細胞外基質としてＩ型コラーゲンを用いる方法（特開平９−１０３２９１号公報）のような培養方法への利用が知られている。また、最近になって、生検採取細胞の培養に際して、支持体表面に細胞の接着が良好に行われるようにするために、タイプＩコラーゲンやタイプＩＶコラーゲンのようなコラーゲン等からなる細胞外基質を層状に塗布しておく方法が開示されている（特開２００３−９８５３号公報）。基底膜の利用に関しては、基底膜細胞外基質でコートした培養皿上で、ある特定の細胞の増殖が亢進することが報告されているが(Gospodarowicz D, Cohen, DC, Fujii DK, Regulation of cell growth by the basal lamina and plasma factors, in Cold Spring Harvor Conferences on Cell Proliferation vol 9. G. Sato, A. Pardee, and D. Sirbasku eds, Cold Spring Harbor, New York. 1982)、間葉系幹細胞等の動物細胞への報告はなされていない。 In recent years, the use of an extracellular matrix has been known, for example, as a biological cell adhesive material or a tissue graft composition (Japanese Patent Laid-Open No. 5-15583, Japanese Translation of PCT International Publication No. 2000-508922, Special Table) In addition, in the method for culturing primary cultured hepatocytes, use in culture methods such as a method using type I collagen as an extracellular matrix (Japanese Patent Laid-Open No. 9-103291) is known. . Recently, an extracellular matrix composed of collagen such as type I collagen or type IV collagen is used in order to ensure that cells adhere well to the support surface when culturing biopsy-collected cells. Has been disclosed (Japanese Unexamined Patent Publication No. 2003-9853). Regarding the use of basement membrane, it has been reported that proliferation of certain cells is enhanced on culture dishes coated with basement membrane extracellular matrix (Gospodarowicz D, Cohen, DC, Fujii DK, Regulation of cell growth by the basal lamina and plasma factors, in Cold Spring Harvor Conferences on Cell Proliferation vol 9.G. Sato, A. Pardee, and D. Sirbasku eds, Cold Spring Harbor, New York. 1982), mesenchymal stem cells, etc. There are no reports on animal cells.
最近、本発明者は、基底膜様の細胞外基質にてコートした培養皿が、ヒト間葉系幹細胞の増殖を促進して、更に細胞寿命を延長し、しかも幹細胞としての多分分化能を保持したまま大量に増殖させることを見出して、特許出願を行った（特開２００３−５２３６０号公報）。また、最近、本発明者は、基底膜細胞外基質培養皿は、間葉系幹細胞としての多分化能を保持したまま大量の細胞数まで増殖させることを報告した（Matsubara-T. et al. Biochem Biophys Res Commun 313, 503-508, 2004）。しかし、１）基底膜細胞外基質培養皿を利用して、動物細胞の分化を直接促進できるか、２）基底膜細胞外基質以外の細胞外基質が、細胞増殖と分化を促進できるかどうか、については不明であったので、使用できるのは基底膜細胞外基質培養皿に限られていた。
本発明の課題は、組織の再生医療のための間葉系幹細胞のような動物幹細胞の利用及びその他のバイオ産業における動物細胞の利用を目的として、各種動物幹細胞等の増殖培養及び分化誘導培養を行うに際して、その培養動物細胞の増殖や分化誘導を促進して、従来の培養方法と比較して顕著に多くの動物細胞を得ることができ、かつ、動物幹細胞の分化能力を維持したまま増殖させることができる動物細胞の培養方法を提供すること、更には、培養期間を短縮して血清使用量を少なくすること及び低濃度の血清でも増殖させることができ、ヒトの血清の使用も可能であり、そして、優れた分化誘導培養が可能な新規な動物幹細胞等の培養方法を提供することにある。 It is an object of the present invention to perform proliferation culture and differentiation induction culture of various animal stem cells for the purpose of using animal stem cells such as mesenchymal stem cells for tissue regenerative medicine and animal cells in other bioindustries. When performing, the proliferation and differentiation induction of the cultured animal cells are promoted, so that a significantly larger number of animal cells can be obtained as compared with the conventional culture method, and the cells are grown while maintaining the differentiation ability of the animal stem cells. It is possible to provide a method for culturing animal cells, to further reduce the amount of serum used by shortening the culture period and to grow even low-concentration serum, and to use human serum. Another object of the present invention is to provide a novel method for culturing animal stem cells and the like capable of excellent differentiation-inducing culture.
本発明者は、上記課題を解決すべく鋭意研究を進めた結果、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞由来の細胞外基質の存在下において、動物細胞を培養することにより、各種動物細胞に対して、その培養動物細胞の増殖を促進することができ、しかも、その増殖中は動物幹細胞等の分化能を効果的に維持することができること、更には、分化誘導を促進して、動物幹細胞の優れた分化誘導培養が可能であることを見出し、本発明を完成するに至った。更に、本発明の動物細胞の培養方法を用い、各種の添加物質を培地に加えることにより、培養期間を短縮できて、低濃度の血清でも効果的に増殖させることができ、ヒト血清を用いて培養することができ、しかも、培養によっても、増殖能力および分化能力が維持されることを見い出し、本発明をなした。 As a result of diligent research to solve the above-mentioned problems, the present inventor cultured animal cells in the presence of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage progenitor cells, or fibroblasts. Therefore, the growth of cultured animal cells can be promoted for various animal cells, and the differentiation ability of animal stem cells and the like can be effectively maintained during the growth. And the inventors have found that excellent differentiation-inducing culture of animal stem cells is possible, and have completed the present invention. Furthermore, by using the animal cell culture method of the present invention and adding various additives to the medium, the culture period can be shortened, and even low-concentration serum can be effectively proliferated. It was found that the cells can be cultured, and that the growth ability and differentiation ability are maintained by the culture, and the present invention has been made.
すなわち、従来、動物細胞の培養皿として一般には、プラスチック、ガラス製の培養皿が用いられ、特殊な場合、コラーゲン、フィブロネクチンなどの単一高分子でコートした培養皿が用いられていた。しかしこれらの培養皿を用いての動物幹細胞等の培養では、動物細胞の増殖、分化促進能は低く、しかも潜在的分化能力も培養中に著しく低下して、再生医療その他のバイオ産業における動物細胞の培養手段としては満足のいくものではなかった。
そこで、以前、本発明者は、基底膜様の細胞外基質にてコートした培養皿が、ヒト間葉系幹細胞の増殖を促進して、更に細胞寿命を延長し、しかも幹細胞としての多分化能を保持したまま大量に増殖させることができることを見出し、間葉系幹細胞の新規培養方法を開発した（特開２００３−５２３６０号公報）。しかし、基底膜以外の他の細胞外基質の影響や間葉系幹細胞以外で、再生医療やバイオ産業で役立つ各種の動物細胞の増殖に対する影響については不明であった。したがって、該増殖方法では、基底膜以外の他の細胞外基質の影響や間葉系幹細胞以外で、再生医療やバイオ産業で役立つ各種の動物細胞の増殖に対して適応ができず、これら各種の動物細胞の増殖に対応できる培養方法の開発が必要とされた。
That is, conventionally, plastic and glass culture dishes are generally used as culture dishes for animal cells, and in special cases, culture dishes coated with a single polymer such as collagen and fibronectin have been used. However, in the culture of animal stem cells and the like using these culture dishes, the proliferation and differentiation promoting ability of animal cells are low, and the potential differentiation ability is also significantly reduced during the cultivation. As a culture means, it was not satisfactory.
Therefore, in the past, the present inventor has shown that a culture dish coated with a basement membrane-like extracellular matrix promotes the proliferation of human mesenchymal stem cells, further prolongs the cell life, and multipotency as stem cells. And a new method for culturing mesenchymal stem cells was developed (Japanese Patent Laid-Open No. 2003-52360). However, the effects of extracellular matrix other than the basement membrane and the effects on the proliferation of various animal cells that are useful in the regenerative medicine and bioindustry other than mesenchymal stem cells were unknown. Therefore, this proliferation method cannot be applied to the effects of extracellular matrix other than the basement membrane or the proliferation of various animal cells useful in the regenerative medicine and bioindustry other than mesenchymal stem cells. Development of a culture method that can cope with the growth of animal cells was required.
そこで本発明においては、これらの点について追求して、基底膜細胞外基質以外にも、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞などの産生する細胞外基質が、各種幹細胞を含む動物細胞に対して、その増殖を促進することを見出した。しかも、これらの細胞外基質は、単に潜在的分化能を維持したまま増殖させるだけにとどまらず、骨分化、脂肪分化などの細胞分化に対して直接促進的に作用することを見出した。したがって、本発明の細胞外基質コートを用いた動物細胞の培養方法（培養皿、担体など）は、各種細胞外基質コートを利用して（各種細胞外基質コート培養皿を用いて）、各種幹細胞等に対する新規でかつ強力な動物細胞の増殖促進方法、分化誘導方法の提供を可能とし、再生医療とバイオ産業において、各種の動物細胞の増幅のみならず、動物組織（培養骨組織など）の製作と供給に有力な手段を提供することを可能とした。更に、本発明においては、本発明の動物細胞の培養方法において、各種の添加物質を培地に加えることにより低濃度の血清で培養しても、増殖能力および分化能力が維持されることを見い出し、本発明をなした。 Therefore, in the present invention, in pursuit of these points, in addition to the basement membrane extracellular matrix, various types of extracellular matrix produced by mesenchymal cells, epithelial cells, cartilage progenitor cells, fibroblasts, etc. It was found that the proliferation of animal cells including stem cells was promoted. Moreover, it has been found that these extracellular substrates not only proliferate while maintaining their potential differentiation ability, but also directly act on cell differentiation such as bone differentiation and adipose differentiation. Therefore, animal cell culture methods (culture dishes, carriers, etc.) using the extracellular matrix coat of the present invention utilize various extracellular matrix coats (using various extracellular matrix-coated culture dishes), and various stem cells. It is possible to provide new and powerful methods for promoting the proliferation and differentiation of animal cells, and in the regenerative medicine and biotechnology industries, not only the amplification of various animal cells but also the production of animal tissues (cultured bone tissues, etc.) And made it possible to provide powerful means for supply. Furthermore, in the present invention, in the method for culturing animal cells of the present invention, it is found that proliferation ability and differentiation ability are maintained even when cultured in a low concentration of serum by adding various additive substances to the medium. Made the present invention.
すなわち具体的には本発明は、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞由来の細胞外基質の存在下において、動物細胞を培養することを特徴とする哺乳動物細胞の培養方法（請求項１）や、動物細胞が、幹細胞であることを特徴する請求項１記載の哺乳動物細胞の培養方法（請求項２）や、間葉系細胞が骨髄間葉系幹細胞、上皮系細胞が胎児羊膜細胞、軟骨前駆細胞が胚性幹細胞由来軟骨前駆細胞、又は繊維芽細胞が胎児由来繊維芽細胞であることを特徴とする請求項１又は２記載の哺乳動物細胞の培養方法（請求項３）や、細胞外基質でコートした培養皿上で動物細胞を培養することを特徴とする請求項１〜３のいずれか記載の哺乳動物の培養方法（請求項４）や、細胞外基質が、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞を培養して形成したものであることを特徴とする請求項４記載の哺乳動物細胞の培養方法（請求項５）からなる。 Specifically, the present invention specifically relates to mammalian cells characterized by culturing animal cells in the presence of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage progenitor cells, or fibroblasts. A method of culturing (claim 1), a method of culturing mammalian cells according to claim 1 (claim 2), wherein the animal cells are stem cells, or a mesenchymal cell being a bone marrow mesenchymal stem cell, epithelium The method for culturing mammalian cells according to claim 1 or 2, wherein the systemic cell is a fetal amniotic cell, the cartilage progenitor cell is an embryonic stem cell-derived cartilage progenitor cell, or the fibroblast is a fetal fibroblast. A method for culturing a mammal according to any one of claims 1 to 3, characterized in that animal cells are cultured on a culture dish coated with an extracellular matrix (claim 3) or extracellular Matrix is mesenchymal cell, epithelial cell, cartilage progenitor cell Or it comprises a method for culturing mammalian cells of claim 4, wherein (claim 5) is obtained fibroblasts were formed by culturing.
また本発明は、細胞外基質が、ヒト骨髄間葉系細胞、ヒト胎児（羊膜）細胞、マウス３Ｔ３細胞、マウスＣ３Ｈ１０Ｔ１／２細胞、又はマウスＡＴＤＣ５細胞を培養して形成したものであることを特徴とする請求項４又は５記載の哺乳動物細胞の培養方法（請求項６）や、動物細胞の幹細胞の初代培養及び／又は継代培養を、ウシ胎児血清（ＦＢＳ）及び／又はヒト血清を含有する培地で行うことを特徴とする請求項１〜６のいずれか記載の哺乳動物細胞の培養方法（請求項７）や、動物細胞の幹細胞の初代培養及び／又は継代培養を、繊維芽細胞増殖因子（ＦＧＦ）を含有する培地で行うことを特徴とする請求項１〜６のいずれか記載の哺乳動物細胞の培養方法（請求項８）や、培地中に、トランスフェリン、インスリン、セレン酸、及びリノール酸からなるグループの１種又は２種以上の添加剤を添加することよりなる請求項１〜６のいずれか記載の哺乳動物細胞の培養方法（請求項９）や、動物細胞の幹細胞の組織細胞への分化誘導培養を、分化誘導培地を用いて行うことを特徴とする請求項１〜６のいずれか記載の哺乳動物細胞の培養方法（請求項１０）からなる。 In the present invention, the extracellular matrix is formed by culturing human bone marrow mesenchymal cells, human fetal (amniotic) cells, mouse 3T3 cells, mouse C3H10T1 / 2 cells, or mouse ATDC5 cells. The method for culturing mammalian cells according to claim 4 or 5 (Claim 6) or primary and / or subculture of stem cells of animal cells, comprising fetal bovine serum (FBS) and / or human serum The method for culturing mammalian cells according to any one of claims 1 to 6 and the primary culture and / or subculture of the stem cells of the animal cells according to claim 1, wherein fibroblasts are used. The method for culturing mammalian cells according to any one of claims 1 to 6 (claim 8) characterized in that it is carried out in a medium containing a growth factor (FGF), transferrin, insulin, selenate, as well as A method for culturing a mammalian cell according to any one of claims 1 to 6 or a tissue of stem cells of an animal cell, comprising adding one or more additives of a group consisting of nolic acid The method for culturing mammalian cells according to any one of claims 1 to 6, wherein the differentiation induction culture into cells is performed using a differentiation induction medium.
更に本発明は、動物細胞の幹細胞の骨芽細胞、軟骨細胞又は脂肪細胞への分化誘導培養を、骨分化誘導培地、軟骨細胞分化誘導培地又は脂肪細胞分化誘導培地を用いて行うことを特徴とする請求項１０記載の哺乳動物細胞の培養方法（請求項１１）や、動物細胞の幹細胞の分化誘導培養を、ウシ胎児血清（ＦＢＳ）を含有する培地で行うことを特徴とする請求項１０又は１１記載の哺乳動物細胞の培養方法（請求項１２）や、動物細胞の幹細胞の培養を、ヒト血清の存在下に行うことを特徴とする請求項１〜６のいずれか記載の哺乳動物細胞の培養方法（請求項１３）や、動物細胞の培養器の培養面に、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞由来の細胞外基質のコート層を設けたことを特徴とする哺乳動物細胞用の培養器（請求項１４）や、培養器が、細胞外基質でコートした培養皿であることを特徴とする請求項１４記載の哺乳動物細胞用の培養器（請求項１５）や、培養器が、細胞外基質でコートした細胞移植用担体であることを特徴とする請求項１４記載の哺乳動物細胞用の培養器（請求項１６）からなる。 Furthermore, the present invention is characterized in that differentiation induction culture of animal cells stem cells into osteoblasts, chondrocytes or adipocytes is performed using a bone differentiation induction medium, a chondrocyte differentiation induction medium or an adipocyte differentiation induction medium. The method for culturing mammalian cells according to claim 10 (claim 11) or the differentiation induction culture of stem cells of animal cells in a medium containing fetal bovine serum (FBS). The method of culturing mammalian cells according to claim 11 (claim 12) or culturing stem cells of animal cells in the presence of human serum. A coating method of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage progenitor cells, or fibroblasts is provided on a culture method (Claim 13) or on the culture surface of an incubator for animal cells. Incubator for mammalian cells 14. The incubator for mammalian cells (claim 15) or the incubator according to claim 14, wherein the incubator is a culture dish coated with an extracellular matrix. A cell transplant carrier coated with a substrate, comprising the incubator for mammalian cells according to claim 14 (claim 16).
本発明の細胞外基質を用いた動物細胞の培養方法は、組織の再生医療のための間葉系幹細胞のような各種動物幹細胞の利用及びその他のバイオ産業における動物細胞の利用のために、動物幹細胞等の増殖培養及び分化誘導培養を行うに際して、各種幹細胞を含む動物細胞の増殖や分化誘導を促進して、従来の培養方法と比較して顕著に多くの動物細胞を得ることができ、かつ、その培養においては、動物幹細胞の分化能力を維持したまま増殖させることができる。しかも、これらの細胞外基質は、各種幹細胞において、単に潜在的分化能を維持したまま増殖させるだけにとどまらず、骨分化、脂肪分化などの細胞分化に対して直接促進的に作用することを可能とする。更には、低濃度の血清でも増殖させることができ、ヒトの血清の使用も可能であり、そして、優れた分化誘導培養が可能である。したがって、本発明の動物細胞の培養方法は、各種幹細胞等に対する新規でかつ強力な動物細胞の増殖促進方法、分化誘導方法の提供を可能とし、再生医療とバイオ産業において、各種の動物細胞の増幅のみならず、動物組織（培養骨組織など）の製作と供給に有力な手段を提供する。 The method for culturing animal cells using the extracellular matrix of the present invention comprises the use of various animal stem cells such as mesenchymal stem cells for tissue regenerative medicine and the use of animal cells in other bioindustries. When performing proliferation culture and differentiation induction culture of stem cells, etc., the proliferation and differentiation induction of animal cells including various stem cells can be promoted, and significantly more animal cells can be obtained compared to conventional culture methods, and In the culture, it is possible to proliferate while maintaining the differentiation ability of animal stem cells. In addition, these extracellular matrices can not only proliferate while maintaining their potential differentiation potential in various stem cells, but can also directly act on cell differentiation such as bone differentiation and adipose differentiation. And Furthermore, even low-concentration serum can be grown, human serum can be used, and excellent differentiation-inducing culture is possible. Therefore, the animal cell culture method of the present invention can provide a novel and powerful method for promoting the proliferation of animal cells and a method for inducing differentiation for various stem cells and the like, and amplifying various animal cells in the regenerative medicine and biotechnology industries. In addition, it provides a powerful tool for the production and supply of animal tissues (such as cultured bone tissues).
本発明は、間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞由来の細胞外基質の存在下において、動物細胞を培養することよりなる。本発明において、
細胞外基質を形成するための間葉系細胞、上皮系細胞、軟骨前駆細胞、又は繊維芽細胞としては、間葉系細胞が骨髄間葉系幹細胞、上皮系細胞が胎児羊膜細胞、軟骨前駆細胞が胚性幹細胞由来軟骨前駆細胞、又は繊維芽細胞が胎児由来繊維芽細胞を挙げることができ、更に具体的には、ヒト骨髄間葉系細胞、ヒト胎児（羊膜）細胞、マウス３Ｔ３細胞、マウスＣ３Ｈ１０Ｔ１／２細胞、又はマウスＡＴＤＣ５細胞を挙げることができる。本発明の細胞外基質を用いた動物細胞の培養方法において、増殖培養及び分化誘導培養を行う動物細胞としては、組織の再生医療のための間葉系幹細胞のような各種動物幹細胞、或いはその他のバイオ産業における動物細胞を挙げることができる。
The present invention comprises culturing animal cells in the presence of an extracellular matrix derived from mesenchymal cells, epithelial cells, cartilage progenitor cells, or fibroblasts. In the present invention,
Mesenchymal cells, epithelial cells, cartilage progenitor cells or fibroblasts for forming extracellular matrix include mesenchymal cells as bone marrow mesenchymal stem cells, epithelial cells as fetal amniotic cells, cartilage progenitor cells Are embryonic stem cell-derived cartilage progenitor cells, or fibroblasts are fetal fibroblasts. More specifically, human bone marrow mesenchymal cells, human fetal (amniotic) cells, mouse 3T3 cells, mouse Mention may be made of C3H10T1 / 2 cells or mouse ATDC5 cells. In the method for culturing animal cells using the extracellular matrix of the present invention, the animal cells to be subjected to proliferation culture and differentiation induction culture are various animal stem cells such as mesenchymal stem cells for tissue regenerative medicine, or other Mention may be made of animal cells in the bio industry.
本発明において、間葉系幹細胞のような動物幹細胞は、骨芽細胞、軟骨細胞、脂肪細胞、筋肉細胞、腱細胞、歯根膜、セメント質などの細胞へと分化しうる又はそれらの修復を促進しうる多能性を有する未分化な細胞である。間葉系幹細胞のような動物幹細胞は、該細胞を有する任意の骨髄または骨膜から採取することができるが、採取しうる該細胞の量及び採取の容易性の理由から、大腿骨、脛骨および骨盤（腸骨）から採取するのが好ましい。ヒト以外の哺乳動物については、腸骨及び脛骨などから間葉系幹細胞のような動物幹細胞を採取することができる。骨髄等からの採取方法は、例えば医療において用いられている公知の任意の採取方法を用いることができる。本発明者らは、最近、口腔組織から間葉系幹細胞を分離して、再生医療に移植細胞として利用できる細胞数まで培養して増やす技術を開発した。したがって、このような口腔組織から分離した間葉系幹細胞も本発明において有利に利用することができる。また、脂肪組織からも間葉系幹細胞を分離、培養できる。 In the present invention, animal stem cells such as mesenchymal stem cells can differentiate into cells such as osteoblasts, chondrocytes, adipocytes, muscle cells, tendon cells, periodontal ligament, cementum, or promote their repair. It is an undifferentiated cell having pluripotency. Animal stem cells such as mesenchymal stem cells can be collected from any bone marrow or periosteum that contains the cells, but because of the amount of cells that can be collected and the ease of collection, the femur, tibia and pelvis It is preferable to collect from (iliac bone). For mammals other than humans, animal stem cells such as mesenchymal stem cells can be collected from the iliac and tibia. As a method for collecting from bone marrow or the like, for example, any known collecting method used in medicine can be used. The present inventors recently developed a technique for isolating mesenchymal stem cells from oral tissues and culturing them to increase the number of cells that can be used as transplanted cells in regenerative medicine. Therefore, mesenchymal stem cells isolated from such oral tissues can also be advantageously used in the present invention. In addition, mesenchymal stem cells can be isolated and cultured from adipose tissue.
本発明において、本発明の細胞外基質の存在下において、哺乳動物の幹細胞を培養する形態としては、培養器として公知の任意のものが利用できるが、細胞外基質でコートした培養皿上で細胞を培養する形態のものが特に有利に利用することが出来る（Proc. Natl. Acad. Sci. USA 77, 4094-4098, 1980）。また、細胞移植用担体（コラーゲン、リン酸カルシウム、生体吸収性高分子（ポリ乳酸など）、チタニウムブロック等）に細胞外基質をコートしたものに、動物細胞を播種して、試験管内で増殖或いは分化させて、再生医療用の移植用培養組織として用いることもできる。細胞外基質は、主として、プロテオグリカン、コラーゲン、エラスチン、フィブロネクチンなどの接着因子を構成成分として含有する。 In the present invention, any form known for use as an incubator can be used for culturing mammalian stem cells in the presence of the extracellular matrix of the present invention. However, cells can be cultured on a culture dish coated with the extracellular matrix. Can be used particularly advantageously (Proc. Natl. Acad. Sci. USA 77, 4094-4098, 1980). In addition, a cell transplant carrier (collagen, calcium phosphate, bioabsorbable polymer (polylactic acid, etc.), titanium block, etc.) coated with an extracellular matrix is seeded with animal cells and allowed to grow or differentiate in vitro. It can also be used as a transplanted tissue for regenerative medicine. The extracellular matrix mainly contains an adhesion factor such as proteoglycan, collagen, elastin, fibronectin as a constituent component.
動物幹細胞の初代培養及び／又は継代培養を行うには、採取分離した細胞を適当な培地（例えば、ＤＭＥＭ（Dulbecco's modified Eagle's medium）培地）を用い、組織培養用培養皿に細胞を播種して初代培養及び継代培養する。培養に用いる血清としては、ウシ胎児血清（ＦＢＳ）を用いることが出来る。本発明においては、培地への１０％以下の血清の添加量においても良好な増殖結果を得ることが出来る。また、ヒト血清を用いても、著明な増殖結果を得ることが出来る。更に、本発明においては、添加物質として、繊維芽細胞増殖因子（ＦＧＦ）を培地に添加することにより、本発明の培養方法の効果を高めることが出来る。 In order to perform primary culture and / or subculture of animal stem cells, the collected and separated cells are seeded on a tissue culture culture dish using an appropriate medium (for example, DMEM (Dulbecco's modified Eagle's medium) medium). Primary culture and subculture. As the serum used for the culture, fetal bovine serum (FBS) can be used. In the present invention, good growth results can be obtained even with the addition of 10% or less of serum to the medium. Moreover, even if human serum is used, a remarkable proliferation result can be obtained. Furthermore, in the present invention, the effect of the culture method of the present invention can be enhanced by adding fibroblast growth factor (FGF) as an additive substance to the medium.
本発明において、動物幹細胞の分化誘導培養を行うには、初代培養及び継代培養した動物幹細胞を、分化誘導培地を用いて培養し、細胞を分化誘導することにより行うことができる。分化誘導培地としては、分化誘導する組織細胞によって、適宜公知の分化誘導培地を用いることができる。培養動物幹細胞の骨芽細胞への分化誘導培養を行うには、骨分化誘導培地として、（αＭＥＭ、ＦＢＳ、デキサメサゾン、β−グリセロールリン酸及びアスコルビン酸−２−リン酸）からなる組成の誘導培地を用いることができる。間葉系幹細胞のような動物幹細胞の骨芽細胞への分化誘導培養は、ウシ胎児血清（ＦＢＳ）及び／又は繊維芽細胞増殖因子（ＦＧＦ）を含有する培地で増殖させた細胞を骨分化誘導培地へ切りかえることで、分化誘導効果を達成することができる。更に、本発明においては、増殖用の培地中に、トランスフェリン、インスリン、セレン酸、及びリノール酸からなるグループの１種又は２種以上の添加剤を添加することより、低濃度の血清を用いた場合でも、通常の血清の使用量と同等及びそれ以上の増殖効果を得る事ができ、更に骨分化誘導培地へ切りかえた時に優れた分化能力を発揮することができる。 In the present invention, differentiation induction culture of animal stem cells can be performed by culturing primary and subcultured animal stem cells using a differentiation induction medium and inducing differentiation of the cells. As the differentiation-inducing medium, a known differentiation-inducing medium can be appropriately used depending on the tissue cells to be differentiated. In order to conduct differentiation induction culture of cultured animal stem cells into osteoblasts, an induction medium having a composition comprising (αMEM, FBS, dexamethasone, β-glycerol phosphate and ascorbic acid-2-phosphate) is used as a bone differentiation induction medium. Can be used. Differentiation-inducing culture of animal stem cells such as mesenchymal stem cells into osteoblasts induces bone differentiation of cells grown in a medium containing fetal bovine serum (FBS) and / or fibroblast growth factor (FGF) By switching to a medium, a differentiation-inducing effect can be achieved. Furthermore, in the present invention, a low concentration of serum was used by adding one or more additives of the group consisting of transferrin, insulin, selenate, and linoleic acid to the growth medium. Even in such a case, it is possible to obtain a proliferation effect equivalent to or higher than the amount of normal serum used, and to exhibit excellent differentiation ability when switched to a bone differentiation-inducing medium.
以下、実施例により本発明をより具体的に説明するが、本発明の技術的範囲はこれらの例示に限定されるものではない。 EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
（間葉系細胞、軟骨前駆細胞、繊維芽細胞の産生した細胞外基質によりコートされた培養皿の製作とそれらの細胞増殖の促進効果）
マウスＣ３Ｈ１０Ｔ１／２細胞、マウスＡＴＤＣ５細胞、マウス３Ｔ３細胞は理化研の細胞バンクより得た。これらの培養細胞の産生する細胞外基質（Extracellular matrix,ＥＣＭ）でコートした培養皿については、基底膜細胞外基質コート培養皿の製作方法に準じて行った（特開２００３−５２３６０：文献（Gospodarowicz D, Cohen, DC, Fujii DK, Regulation of cell growth by the basal lamina and plasma factors, in Cold Spring Harvor Conferences on Cell Proliferation vol 9.G. Sato, A. Pardee and D. Sirbasku eds, 1982; Proc. Natl. Acad. Sci. USA 77, 4094-4098, 1980））。まず、上記の細胞を集密になるまで培養したのち、５％デキストラン（分子量２０万 Wako, Osaka, Japan）、５０μｇ／ｍｌアスコルビン酸−２−リン酸(Sigma)、１０％ＦＢＳ存在下でさらに１週間培養した。そして細胞層へ希アンモンニア溶液（２０ｍＭ〜５０ｍＭ）を添加して、細胞成分のみを除去した後、培養ディッシュ下層に接着して残存している細胞外基質をリン酸緩衝生理食塩液（ＰＢＳ）で数回洗浄したのち、４℃で保存した。細胞外基質がはがれやすい場合は、集密になる前にデキストラン処理をして、またアンモニア液も希釈した。
(Production of culture dishes coated with extracellular matrix produced by mesenchymal cells, cartilage progenitor cells, and fibroblasts, and the effect of promoting cell proliferation)
Mouse C3H10T1 / 2 cells, mouse ATDC5 cells, and mouse 3T3 cells were obtained from Rikaken's cell bank. The culture dish coated with the extracellular matrix (ECM) produced by these cultured cells was performed according to the production method of the basement membrane extracellular matrix-coated culture dish (Japanese Patent Laid-Open No. 2003-52360: literature (Gospodarowicz D, Cohen, DC, Fujii DK, Regulation of cell growth by the basal lamina and plasma factors, in Cold Spring Harvor Conferences on Cell Proliferation vol 9.G. Sato, A. Pardee and D. Sirbasku eds, 1982; Proc. Natl Acad. Sci. USA 77, 4094-4098, 1980)). First, after culturing the above cells until confluence, 5% dextran (molecular weight 200,000 Wako, Osaka, Japan), 50 μg / ml ascorbic acid-2-phosphate (Sigma), and 10% FBS were further added. Cultured for 1 week. Then, after adding a dilute ammonia solution (20 mM to 50 mM) to the cell layer to remove only the cellular components, the extracellular matrix remaining adhered to the lower layer of the culture dish is washed with phosphate buffered saline (PBS). After washing several times, it was stored at 4 ° C. When the extracellular matrix easily peeled off, dextran treatment was performed before confluence, and the ammonia solution was also diluted.
基底膜細胞外基質を産生するＰＹＳ−２細胞、ウシ角膜内皮細胞から細胞外基質でコートした培養皿が作成できることは判明していたが、間葉系細胞であるマウスＣ３Ｈ１０Ｔ１／２細胞、軟骨前駆細胞であるマウスＡＴＤＣ５細胞、繊維芽細胞であるマウス３Ｔ３細胞を用いても、細胞外基質コート培養皿を製作することができた。これらは、顕微鏡下で基底膜細胞外基質よりも網の目の細い構造を示した。これらの細胞外基質コート培養皿上にヒト間葉系幹細胞を播種して（１０００個／ｃｍ2）、ほぼ集密的になるまで培養した。そして細胞をトリプシン／ＥＤＴＡ溶液にて５分間処理して、単離して、別のそれぞれの培養皿に播種した。このような継代を繰り返して、６５日まで培養した。図１の各点は、継代時での、培養日数（横軸）と積算細胞数（縦軸）を示す。 Although it has been found that PYS-2 cells producing basement membrane extracellular matrix and bovine corneal endothelial cells can be used to prepare culture dishes coated with extracellular matrix, mouse C3H10T1 / 2 cells that are mesenchymal cells, cartilage precursors An extracellular matrix-coated culture dish could also be produced using mouse ATDC5 cells as cells and mouse 3T3 cells as fibroblasts. These showed a finer structure under the microscope than the basement membrane extracellular matrix. Human mesenchymal stem cells were seeded on these extracellular matrix-coated culture dishes (1000 cells / cm 2 ) and cultured until almost confluent. Cells were then treated with trypsin / EDTA solution for 5 minutes, isolated and seeded in separate respective culture dishes. Such passage was repeated and cultured until 65 days. Each point in FIG. 1 indicates the number of culture days (horizontal axis) and the number of accumulated cells (vertical axis) at the time of passage.
今回使用したヒト骨髄間葉系幹細胞株の成長因子と細胞外基質応答能は高くなかったものの、ｂＦＧＦ添加によりヒト骨髄間葉系幹細胞の増殖は促進されたことは、以前の報告（Tsutsumi S. et al., Biochem Biophys Res Commun 288, 413-419, 2001）と一致している。またＰＹＳ−２細胞、牛角膜内皮細胞の細胞外基質も細胞増殖を促進した。これも以前の報告と一致している。しかし、今回は、間葉系細胞であるＣ３Ｈ１０Ｔ１／２細胞、軟骨前駆細胞であるＡＴＤＣ５細胞、繊維芽細胞である３Ｔ３細胞の細胞外基質も、ヒト骨髄間葉系幹細胞の増殖を促進することが判明した（図１）。そしてこれらの新規細胞外基質は、ｂＦＧＦの作用よりも強く、またＣ３Ｈ１０Ｔ／２細胞の作用は、基底膜細胞外基質の作用に匹敵した（図１）。 Although the human bone marrow mesenchymal stem cell line used in this study did not have high growth factor and extracellular matrix responsiveness, the addition of bFGF promoted the proliferation of human bone marrow mesenchymal stem cells in a previous report (Tsutsumi S. et al., Biochem Biophys Res Commun 288, 413-419, 2001). The extracellular matrix of PYS-2 cells and bovine corneal endothelial cells also promoted cell proliferation. This is also consistent with previous reports. However, this time, the extracellular matrix of C3H10T1 / 2 cells, which are mesenchymal cells, ATDC5 cells, which are cartilage precursor cells, and 3T3 cells, which are fibroblasts, may also promote the proliferation of human bone marrow mesenchymal stem cells. It became clear (FIG. 1). These new extracellular matrixes were stronger than the action of bFGF, and the action of C3H10T / 2 cells was comparable to that of the basement membrane extracellular matrix (FIG. 1).
(細胞外基質による骨分化の直接促進効果−骨髄間葉系細胞に対する影響)
ヒト骨髄由来の間葉系幹細胞は、すでに記載した方法で３−６代目まで培養したものを用いた（Tsutsumi S. et al., Biochem Biophys Res Commun 288, 413-419, 2001; Matsubara-T. et al. Biochem Biophys Res Commun 313, 503-508, 2004）。これらの細胞を、ヒト骨髄間葉系幹細胞、ＰＹＳ−２細胞、或いはヒト胎児（羊膜）細胞の産生した細胞外基質にてコートした、或いは対照としてコートしていない通常のプラスチック培養皿上に播種して（４００００個／16 mmシャーレ）、骨分化誘導に適する下記の培地（文献Science 284, 143-147, 1999に記載の培地）を用いて１４−２８日間培養した。いずれの細胞外基質（ＥＣＭ）コート培養皿も対照のプラスチック培養皿に比較して、骨分化を促進した。なおこの実験では、骨分化は、アリザリン赤で染まる石灰化で評価した。
(Direct effect of bone differentiation by extracellular matrix-Effect on bone marrow mesenchymal cells)
Human bone marrow-derived mesenchymal stem cells were cultured in the manner described above until the 3rd to 6th generation (Tsutsumi S. et al., Biochem Biophys Res Commun 288, 413-419, 2001; Matsubara-T. et al. Biochem Biophys Res Commun 313, 503-508, 2004). These cells are seeded on normal plastic culture dishes coated with extracellular matrix produced by human bone marrow mesenchymal stem cells, PYS-2 cells, or human fetal (amniotic) cells, or uncoated as a control (40000 pieces / 16 mm petri dish) and cultured for 14 to 28 days using the following medium suitable for induction of bone differentiation (medium described in literature Science 284, 143-147, 1999). All extracellular matrix (ECM) coated culture dishes promoted bone differentiation compared to control plastic culture dishes. In this experiment, bone differentiation was evaluated by calcification stained with alizarin red.
骨分化誘導培地
αＭＥＭ
１０％ 牛胎児血清
１００ｎＭ デキサメサゾン
１０ｍＭ β−グリセロールリン酸
５０μｇ／ｍｌ アスコルビン酸−２−リン酸
Bone differentiation induction medium αMEM
10% fetal bovine serum 100 nM dexamethasone 10 mM β-glycerol phosphate 50 μg / ml ascorbic acid-2-phosphate
(細胞外基質による骨分化の直接促進効果−ヒト胎児細胞とヒト間葉系幹細胞に対する影響の詳しい解析)
本実施例では、ヒト骨髄由来間葉系幹細胞、対照としてヒト繊維芽細胞を用いた。更に、ヒト胎児細胞は、帝王切開した妊婦の羊膜間質より得た。これらの細胞をトリプシンとコラゲナーゼにて分散して、１０％血清＋ｂＦＧＦ含有ＤＭＥＭ培地にて培養したものを用いた。そして、これら三種類の細胞を、ヒト骨髄間葉系幹細胞、あるいはヒト胎児細胞の産生した細胞外基質（ＥＣＭ）にてコートした、あるいは対照としてコートしていない通常のプラスチック培養皿上に播種して（４００００個／16 mmシャーレ）、上記の骨分化誘導に適する培地を用いて３７℃、５％炭酸ガス存在下で７−２８日間培養した。培地は２日おきに交換した。
(Direct effect of bone differentiation by extracellular matrix-Detailed analysis of effects on human fetal cells and human mesenchymal stem cells)
In this example, human bone marrow-derived mesenchymal stem cells and human fibroblasts as controls were used. Furthermore, human fetal cells were obtained from the amnion stroma of pregnant women who had undergone cesarean section. These cells were dispersed in trypsin and collagenase and cultured in 10% serum + bFGF-containing DMEM medium. These three types of cells are then seeded on a normal plastic culture dish that is coated with human bone marrow mesenchymal stem cells or extracellular matrix (ECM) produced by human fetal cells, or as a control. (40000 pieces / 16 mm petri dish) and cultured at 37 ° C. in the presence of 5% carbon dioxide gas for 7 to 28 days using the medium suitable for inducing bone differentiation. The medium was changed every 2 days.
また、本実施例では、骨分化を評価するために、石灰化以外に、骨関連遺伝子のｍＲＮＡの発現レベルも測定した。培養した細胞のtotal ＲＮＡをTRIzol(Life Technologies Inc.)で抽出した。さらに、骨芽細胞に特異的な遺伝子マーカー：オステオカルシン、オステオポンチン及びアルカリホスファターゼの発現をＲＴ−ＰＣＲにて検討した。ＲＴ−ＰＣＲ kit: ReverTra Dash（Toyobo Co., Ltd, Osaka, Japan）。ＰＣＲの条件は９４℃３０秒、６８℃３分間の反複を２８サイクルで行った。ＰＣＲ産物の分析を電気泳動にて行った。
Human osteocalcin: forward 5'-CCACCGAGACACCATGAGAG-3'、
reverse 5'-CCATAGGGCTGGGAGGTCAG-3'。
Human osteopontin: forward 5'- CTAGGCATCACCTGTGCCATACC-3'、
reverse 5'-CAGTGACCAGTTCATCAGATTCATC-3'
Human alkaline phoaphatase : forward 5'- AGGCTGGAGATGGACAAGTT-3'、
reverse 5'- TGGTCAATTCTGCCTCCTTC -3'
Human GAPDH: forward 5'-GTCAAGGCCGAGAATGGGAA-3'、
reverse 5'-GCTTCACCACCTTCTTGATG-3'
（結果）
いずれの細胞外基質コート培養皿も対照と比較して、ヒト骨髄由来間葉系幹細胞とヒト胎児細胞の骨分化を促進した。一方、対照として用いたヒト繊維芽細胞にはほとんど影響しなかった。図２は、ヒト胎児細胞が、対照のプラスチック培養皿では、骨分化しないのに対して、ヒト胎児細胞（羊膜間質由来）の細胞外基質でコートした培養皿上では、培養初期から（７日）骨分化が開始していたことを示す（図２の上図）。またアリザリン赤で染まる石灰化で評価しても、この細胞外基質は骨分化を促進していた（図２の下図）。更に、図３は、同じ細胞外基質培養皿で、ヒト胎児細胞において、骨関連遺伝子であるosteocalcin（ＯＣ）、osteopontin（ＯＰ）、alkaline phosphatase（ＡＬＰ）のｍＲＮＡの発現が、対照のプラスチック培養皿に比較して、上昇していることを示している。図４は、ヒト骨髄由来間葉系幹細胞が、ヒト胎児（羊膜間質由来）細胞の細胞外基質でコートされた培養皿上で、対照のプラスチック培養皿に比較して、骨分化（アリザリン赤による石灰化の評価）が培養２８日で亢進していることを示している。
Moreover, in this example, in order to evaluate bone differentiation, the expression level of bone-related gene mRNA was also measured in addition to calcification. Total RNA of the cultured cells was extracted with TRIzol (Life Technologies Inc.). Furthermore, the expression of gene markers specific for osteoblasts: osteocalcin, osteopontin and alkaline phosphatase was examined by RT-PCR. RT-PCR kit: ReverTra Dash (Toyobo Co., Ltd, Osaka, Japan). The PCR conditions were 94 ° C for 30 seconds and 68 ° C for 3 minutes with 28 cycles. PCR products were analyzed by electrophoresis.
Human osteocalcin: forward 5'-CCACCGAGACACCATGAGAG-3 ',
reverse 5'-CCATAGGGCTGGGAGGTCAG-3 '.
Human osteopontin: forward 5'- CTAGGCATCACCTGTGCCATACC-3 ',
reverse 5'-CAGTGACCAGTTCATCAGATTCATC-3 '
Human alkaline phoaphatase: forward 5'- AGGCTGGAGATGGACAAGTT-3 ',
reverse 5'- TGGTCAATTCTGCCTCCTTC -3 '
Human GAPDH: forward 5'-GTCAAGGCCGAGAATGGGAA-3 ',
reverse 5'-GCTTCACCACCTTCTTGATG-3 '
(result)
All extracellular matrix-coated culture dishes promoted bone differentiation of human bone marrow-derived mesenchymal stem cells and human fetal cells as compared to the control. On the other hand, it hardly affected human fibroblasts used as a control. FIG. 2 shows that human embryonic cells do not differentiate into bone in the control plastic culture dish, whereas culture embryos coated with the extracellular matrix of human fetal cells (derived from the amniotic stroma) from the beginning of culture (7 Day) shows that bone differentiation has started (upper figure in FIG. 2). In addition, even when evaluated by calcification stained with alizarin red, this extracellular matrix promoted bone differentiation (lower figure in FIG. 2). Furthermore, FIG. 3 shows the same extracellular matrix culture dish in which the expression of mRNA for bone-related genes osteocalcin (OC), osteopontin (OP), and alkaline phosphatase (ALP) in human fetal cells is a control plastic culture dish. It shows that it is rising compared to FIG. 4 shows bone differentiation (alizarin red) on a culture dish in which human bone marrow-derived mesenchymal stem cells are coated with the extracellular matrix of human fetal (amniotic stroma) cells compared to a control plastic culture dish. (Evaluation of calcification by) is enhanced in 28 days of culture.
（ヒト胎児細胞由来細胞外基質コート培養皿での、間葉系幹細胞及び軟骨細胞の増殖の亢進）
使用するヒト胎児細胞としては、羊膜由来の細胞を用いた。インフォームドコンセントを得た妊婦の出産後（自然分娩、帝王切開を含む）の胎盤より羊膜を絨毛膜層からピンセットで剥離して分離した。羊膜細胞の分離はコラゲナーゼとトリプシン−ＥＤＴＡで２段階酵素分解法で行った。すなわち、得られた羊膜を１００ｍｌビーカーに入れて、リン酸緩衝液(ＰＢＳ)で３回洗った後、滅菌した手術用ハサミで細かく切って断片化し、それを１ｍｇ／ｍｌコラゲナーゼ (Sigma社)、０．０８％ＤＮＡ分解酵素(Sigma社)を含有するＤＭＥＭ−Ｆ１２培地(Sigma社)で３７℃、スターラーバーで回転（５００〜６００ｒｐｍ）しながら１時間振盪処理した。上皮細胞を除くために、回収した溶液を１００μｍ、４０μｍのフィルター網にそれぞれに通して、更に、２０００gで１０分間遠心して集め、間葉系細胞を分離した。
(Enhanced proliferation of mesenchymal stem cells and chondrocytes in human fetal cell-derived extracellular matrix-coated culture dishes)
As human fetal cells to be used, cells derived from amniotic membrane were used. The amnion was separated from the chorionic layer by tweezers and separated from the placenta after delivery of the pregnant woman who obtained informed consent (including spontaneous delivery and caesarean section). Amnion cells were separated by collagenase and trypsin-EDTA by a two-step enzymatic degradation method. That is, the obtained amniotic membrane was put in a 100 ml beaker and washed three times with a phosphate buffer (PBS), then finely cut with a sterile surgical scissors to fragment it, and 1 mg / ml collagenase (Sigma), The mixture was shaken with DMEM-F12 medium (Sigma) containing 0.08% DNA degrading enzyme (Sigma) at 37 ° C. for 1 hour while rotating with a stir bar (500 to 600 rpm). In order to remove epithelial cells, the collected solutions were passed through a filter network of 100 μm and 40 μm, respectively, and further collected by centrifugation at 2000 g for 10 minutes to separate mesenchymal cells.
得られた細胞の数を細胞計数分析装置（Ｚ１シングル、コールター社製）で計測した。間葉系細胞は、以上の方法で通常1グラム羊膜組織からおよそ１．０×１０6〜２×１０6 個の細胞が採取できた。初代培養は細胞を４×１０4個／ｃｍ2の密度で１０％牛胎児血清（ＦＢＳ）を含むＤＭＥＭ−Ｆ１２培地（ペニシリン:１００単位／ｍｌ、ストレプトマイシン: １００μｇ／ｍｌ）で１０ｃｍ直径組織培養ディッシュに播種し、３７℃、５％炭酸ガス存在下で行った。 The number of obtained cells was counted with a cell counting analyzer (Z1 single, manufactured by Coulter). As for mesenchymal cells, approximately 1.0 × 10 6 to 2 × 10 6 cells were usually collected from 1 gram of amniotic tissue by the above method. The primary culture is a 10 cm diameter tissue culture dish in DMEM-F12 medium (penicillin: 100 units / ml, streptomycin: 100 μg / ml) containing 10% fetal bovine serum (FBS) at a density of 4 × 10 4 cells / cm 2. And 37 ° C. in the presence of 5% carbon dioxide gas.
羊膜の上皮細胞は、間質細胞が完全に取り除かれた羊膜より分離した。フィルター網上に残された羊膜を０．０５％トリプシン＋０．２ｍＭ ＥＤＴＡで３７℃で５分間処理して、遠心して集め、上皮細胞を分離した。上皮細胞は、以上の方法で通常1グラム羊膜組織からおよそ８×１０6〜１×１０7の細胞が採取できた。得られた上皮細胞を５×１０4個／ｃｍ2の密度で１０ｃｍ直径組織培養ディッシュに播種し、１０％ＦＢＳを含むＤＭＥＭ−Ｆ１２培地（ペニシリン: １００単位／ｍｌ、ストレプトマイシン: １００μｇ／ｍｌ）で３７℃、５％炭酸ガス存在下で培養した。これらの細胞を用いて、上記の方法によって細胞外基質（ＥＣＭ）コート培養皿を製作した。 The amniotic epithelial cells were separated from the amniotic membrane from which the stromal cells had been completely removed. The amniotic membrane remaining on the filter net was treated with 0.05% trypsin + 0.2 mM EDTA at 37 ° C. for 5 minutes, collected by centrifugation, and epithelial cells were separated. With respect to the epithelial cells, approximately 8 × 10 6 to 1 × 10 7 cells were usually collected from 1 gram amnion tissue by the above method. The obtained epithelial cells were seeded in a 10 cm diameter tissue culture dish at a density of 5 × 10 4 cells / cm 2 , and DMEM-F12 medium containing 10% FBS (penicillin: 100 units / ml, streptomycin: 100 μg / ml). The cells were cultured at 37 ° C. in the presence of 5% carbon dioxide gas. Using these cells, an extracellular matrix (ECM) -coated culture dish was prepared by the method described above.
そしてヒト骨髄間葉系幹細胞（ｈＭＳＣ）を、ヒト胎児由来細胞（ＡｍＥ）ＥＣＭ或いは、ＰＹＳ−２基底膜ＥＣＭ培養皿あるいは対照として通常のプラスチック培養皿（ＰＬ）に播種して（５０００個／ｃｍ2）、ほぼ集密的になるまで培養した。そして細胞をトリプシン／ＥＤＴＡ溶液にて５分間処理して、単離して、別のそれぞれの培養皿に播種した。このような継代を繰り返して、６０日まで培養した。その結果、ヒト胎児細胞ECM上で培養したヒト腸骨骨髄由来間葉系幹細胞は、最初の細胞数と比較して、１０9倍以上に増殖した。この増殖能は従来法より優れており、更に、１２継代以上持続した。ヒト胎児細胞ＥＣＭは、基底膜ＥＣＭよりも強力にヒト間葉系幹細胞の増殖を促進した。図５では、ヒト胎児（羊膜上皮）ＥＣＭの強力な増殖促進作用を示した。なお図５の各点は、継代時での、培養日数（横軸）と積算細胞数（縦軸）を示す。 Then, human bone marrow mesenchymal stem cells (hMSC) are seeded on human embryonic cell (AmE) ECM, PYS-2 basement membrane ECM culture dish or normal plastic culture dish (PL) as a control (5000 cells / cm). 2 ) Incubated until almost confluent. Cells were then treated with trypsin / EDTA solution for 5 minutes, isolated and seeded in separate respective culture dishes. Such passage was repeated and cultured until 60 days. As a result, the human iliac bone marrow-derived mesenchymal stem cells cultured on human fetal cells ECM proliferated 10 9 times or more compared to the initial cell number. This proliferation ability was superior to that of the conventional method, and lasted more than 12 passages. Human fetal cell ECM promoted proliferation of human mesenchymal stem cells more strongly than basement membrane ECM. FIG. 5 shows the strong growth promoting effect of human fetal (amniotic epithelium) ECM. Each point in FIG. 5 indicates the number of culture days (horizontal axis) and the number of accumulated cells (vertical axis) at the time of passage.
次に、ウサギ肋軟骨から軟骨細胞を分離した（Kato-Y et al. J.Clin. Invest. 92:2323-2330, 1993）。そしてこれらの細胞を一旦凍結保存したものを、今回の実験に用いた。軟骨細胞は各種の培養皿に、（５０００個／ｃｍ2）で播種した。ヒト胎児細胞ＥＣＭで培養したウサギ軟骨細胞は、最初の細胞数と比較して、１０9倍以上に増殖した。この増殖能は従来法より優れており、更に、７継代以上持続した。図６は、凍結保存した軟骨細胞は、通常のプラスチック培養皿やＰＹＳ−２基底膜ＥＣＭ培養皿上では、増殖速度が遅いものの、ヒト胎児（羊膜上皮）ＥＣＭ培養皿では増殖が著しく亢進したことを示す。従って、ヒト胎児ＥＣＭを使用する細胞培養法は、様々な種類の細胞増殖に応用可能であることが示唆された。 Next, chondrocytes were isolated from rabbit costal cartilage (Kato-Y et al. J. Clin. Invest. 92: 2323-2330, 1993). Then, these cells once cryopreserved were used in this experiment. Chondrocytes were seeded in various culture dishes at (5000 cells / cm 2 ). Rabbit chondrocytes were cultured in human fetal cells ECM as compared to the initial number of cells were grown to more than 109 times. This proliferation ability was superior to the conventional method, and further persisted for more than 7 passages. FIG. 6 shows that cryopreserved chondrocytes showed a marked increase in proliferation in human fetal (amniotic epithelial) ECM culture dishes, although the growth rate was slow on normal plastic culture dishes and PYS-2 basement membrane ECM culture dishes. Indicates. Therefore, it was suggested that the cell culture method using human fetal ECM is applicable to various types of cell growth.
Claims (16)
15. The incubator for mammalian cells according to claim 14, wherein the incubator is a test tube containing a cell transplant carrier coated with an extracellular matrix.
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