JP2007037426A - Serum-free culture medium for culturing animal stem cell - Google Patents
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本発明は、間葉系幹細胞のような動物幹細胞を血清非存在下において培養することが可能な動物幹細胞培養用無血清培地、及び、該無血清培地を用いた動物幹細胞の培養方法に関する。 The present invention relates to a serum-free medium for animal stem cell culture capable of culturing animal stem cells such as mesenchymal stem cells in the absence of serum, and a method for culturing animal stem cells using the serum-free medium.
医科歯科領域においては、組織再生医学に関する研究及び臨床応用が趨勢を極めており、バイオビジネスの分野においても、組織再生医療、すなわち事故や病気や生活習慣病で組織や臓器を欠損させてしまった場合、欠損組織等に分化する機能を保持した幹細胞を用いて欠損部分を元の組織に再生させる再生医療が注目を集めている。このような組織再生には、主として患者から採取した自己細胞（特に幹細胞）を体外で培養、増殖、分化させ、再生した組織を移植するという型がとられている。 In the field of medicine and dentistry, research and clinical applications related to tissue regenerative medicine are extremely popular, and even in the field of biobusiness, tissue regenerative medicine, that is, cases where tissues or organs are lost due to accidents, diseases or lifestyle-related diseases Regenerative medicine that uses stem cells that retain the function of differentiating into a defective tissue and the like to regenerate the defective portion into the original tissue has attracted attention. For such tissue regeneration, a type in which autologous cells (particularly stem cells) collected from a patient are cultured, expanded and differentiated outside the body, and the regenerated tissue is transplanted is used.
従来、哺乳動物細胞を培養するための培養用培地としては、生体に近い条件を作り出すために血清を加えたものを使用するのが主流であり、この血清としてはウシ胎児血清（ＦＢＳ）が最も一般的に用いられている。しかしながら、血清は狂牛病をはじめとする外来性の汚染やウイルスの感染源となり得るため、人体に用いるための細胞の増殖やワクチンの製造といった分野では問題が生じる虞があった。組織再生医療において、幹細胞の培養は、現在のところ、ヒトあるいは仔牛胎児血清を用いた培養が主流であるが、しかし、自己血清では十分な増殖を得られないケースもあり、また自己血清以外の血清を用いた培養は事業化・実用化の過程で、ＢＳＥやウィルス性疾患の水平感染などの様々な問題を含んでいる。 Conventionally, as a culture medium for culturing mammalian cells, it has been the mainstream to use a serum-added medium in order to create a condition close to that of a living body. As this serum, fetal bovine serum (FBS) is the most common. Commonly used. However, since serum can be a source of exogenous contamination such as mad cow disease and virus infection, there is a possibility that problems may arise in the fields of cell proliferation and vaccine production for use in the human body. In tissue regenerative medicine, stem cells are currently cultured mainly using human or calf fetal serum. However, there are cases where autologous serum does not provide sufficient growth. Culture using serum involves various problems such as BSE and horizontal infection of viral diseases in the process of commercialization and practical application.
従来より、上記のような問題を解決するために、動物細胞を血清の非存在下で培養するための無血清細胞培養培地の開発が行われている。例えば、特開平６−７８７５９号公報には、基礎培地に、ヒトトランスフェリン、ヒトインスリン、エタノールアミン及び亜セレン酸ナトリウムを添加した動物タンパク質を含まない無血清細胞培養培地が開示され、特開平７−８２７３号公報には、細胞接着活性を有するコラーゲン、ゼラチン、ケラチン、フィブロネクチン、ビトロネクチン及びラミニンからなる群より選択される天然高分子化合物を、細胞培養用担体の表面に存在させ、血清を含有せず、血清アルブミンを含有する無血清培地が開示されている。 Conventionally, in order to solve the above problems, a serum-free cell culture medium for culturing animal cells in the absence of serum has been developed. For example, JP-A-6-78759 discloses a serum-free cell culture medium containing no animal protein in which human transferrin, human insulin, ethanolamine and sodium selenite are added to a basal medium. No. 8273 discloses that a natural polymer compound selected from the group consisting of collagen, gelatin, keratin, fibronectin, vitronectin and laminin having cell adhesion activity is present on the surface of a cell culture carrier and does not contain serum. A serum-free medium containing serum albumin is disclosed.
また、特開平７−２３７８０号公報には、増殖因子であるタンパク質を全く含有せず、グリシルグリシンを含有する動物細胞の高密度培養用の完全無タンパク培地が、特開平９−２５２７６７号公報には、インスリン、ペプトン及びトランスフェリンを含有する動物細胞用の無血清培地が、特表平１１−５０６６１０号公報には、最小必須培地、血清アルブミン、鉄源、インスリン或いはインスリン状成長因子、及び、グルタミン、アルギニン及びシステインから選択される少なくとも１個のアミノ酸を含むヒト間葉幹細胞培養用無血清培地が、開示されている。 Japanese Patent Application Laid-Open No. 7-23780 discloses a complete protein-free medium for high-density culture of animal cells containing no glycylglycine and no protein as a growth factor. Is a serum-free medium for animal cells containing insulin, peptone and transferrin, and Japanese Patent Publication No. 11-506610 discloses a minimum essential medium, serum albumin, iron source, insulin or insulin-like growth factor, and A serum-free medium for culturing human mesenchymal stem cells comprising at least one amino acid selected from glutamine, arginine and cysteine is disclosed.
更に、特開２００４−１３５６７２号公報には、アルブミン代替物、トランスフェリン代替物及びインスリン代替物を含む組成物であって、アルブミン代替物が１重量％未満の量のポリエチレングリコールである、動物タンパク質を含まない動物細胞培養用の培地が、及び、特開２００４−２７５０４７号公報には、基礎培地に、アルギニン、アスパラギン酸、グルタミン酸、イソロイシン、ロイシン、リジン、セリン、スレオニン、バリンを添加した哺乳動物細胞培養用の無血清培地が開示されている。 Furthermore, JP 2004-135672 A discloses an animal protein comprising an albumin substitute, a transferrin substitute and an insulin substitute, wherein the albumin substitute is polyethylene glycol in an amount of less than 1% by weight. A mammalian cell culture medium that does not contain, and Japanese Patent Application Laid-Open No. 2004-275047 discloses a mammalian cell in which arginine, aspartic acid, glutamic acid, isoleucine, leucine, lysine, serine, threonine, and valine are added to the basic medium. A serum-free medium for culture is disclosed.
上記のとおり、動物細胞培養用の無血清培地は、種々のものが開示されているが、組織再生医療において、移植用の幹細胞を大量に、培養、増殖するためには、幹細胞の分化機能を保持したままの細胞を、血清非存在下において効率的に培養、増殖できる無血清培地が必要となり、そのための細胞増殖能の優れた培地の開発が更に望まれている。 As described above, various serum-free media for animal cell culture have been disclosed. In tissue regenerative medicine, in order to culture and proliferate stem cells for transplantation in large quantities, the differentiation function of stem cells is required. A serum-free medium capable of efficiently culturing and proliferating the retained cells in the absence of serum is required, and the development of a medium with excellent cell growth ability for this purpose is further desired.
本発明の課題は、組織再生医療等において用いられる動物幹細胞を培養、増殖するために、安全性を担保すること、すなわち、ＢＳＥやウィルス性疾患の水平感染などの血清を用いた場合の様々な問題の発生するのを防止し、しかも、血清を用いた場合に匹敵する十分な増殖を得られる、動物幹細胞培養用無血清培地を提供すること、及び、該無血清培地を用いた動物幹細胞の培養方法を提供することにある。 An object of the present invention is to ensure safety in order to culture and proliferate animal stem cells used in tissue regenerative medicine and the like, that is, various cases when using serum such as BSE or horizontal infection of viral diseases. Providing a serum-free medium for culturing animal stem cells that can prevent the occurrence of problems and can obtain sufficient growth comparable to that obtained when using serum, and of animal stem cells using the serum-free medium It is to provide a culture method.
本発明者は、上記課題を解決すべく、鋭意検討する中で、血清を含有しない動物細胞培養用基礎培地に、特定の抗菌ペプチドを添加することにより、動物幹細胞の培養において、血清の非存在下であっても、血清を用いた場合に匹敵する十分な増殖が得られ、しかも、分化能など動物幹細胞としての機能をそのまま保持した動物幹細胞の培養、増殖が可能であることを見い出し、該抗菌ペプチドを添加してなる動物幹細胞培養用無血清培地として調製して、本発明を完成するに至った。すなわち、本発明は、血清を含有しない動物細胞培養用基礎培地に、配列表の配列番号１に示される抗菌ペプチドを添加してなる動物幹細胞培養用無血清培地よりなる。本発明の抗菌ペプチドは、配列表の配列番号１に示される構造のペプチドであり、合成によって調製することができる。 In order to solve the above-mentioned problems, the present inventor has been diligently examining the absence of serum in animal stem cell culture by adding a specific antibacterial peptide to a basal medium for animal cell culture that does not contain serum. Even under such conditions, it was found that sufficient proliferation comparable to that obtained using serum was obtained, and that animal stem cells that retain the functions of animal stem cells such as differentiation ability can be cultured and expanded as they are. The present invention was completed by preparing a serum-free medium for animal stem cell culture to which an antimicrobial peptide was added. That is, the present invention comprises a serum-free medium for animal stem cell culture obtained by adding an antimicrobial peptide represented by SEQ ID NO: 1 in the sequence listing to a basal medium for animal cell culture that does not contain serum. The antibacterial peptide of the present invention is a peptide having a structure represented by SEQ ID NO: 1 in the sequence listing, and can be prepared by synthesis.
更に、本発明においては、該動物幹細胞培養用無血清培地に、ｂＦＧＦ（繊維芽細胞成長因子）及び／又はＰＤＧＦ（血小板由来成長因子）、及び、インスリン、トランスフェリン、セレン酸及びリノール酸からなるグループから選択される１又は２以上の成分を添加して、増殖能の高い動物幹細胞培養用無血清培地を提供することを可能とした。本発明において、血清を含有しない動物細胞培養用基礎培地に、配列表の配列番号１に示される抗菌ペプチドと、２種の発育因子すなわち、ｂＦＧＦ及びＰＤＦＧを応用して、間葉系幹細胞のような動物幹細胞を無血清で培養することを可能とし、従来技術による無血清培養に比べて約１００倍の増殖能を与える増殖能の高い動物幹細胞培養用無血清培地を提供することを可能とした。本発明は、本発明の動物幹細胞培養用無血清培地を用いて、間葉系幹細胞のような動物幹細胞の培養を行う方法を包含する。 Further, in the present invention, the serum-free medium for animal stem cell culture comprises a group consisting of bFGF (fibroblast growth factor) and / or PDGF (platelet-derived growth factor), and insulin, transferrin, selenate and linoleic acid. By adding one or more components selected from the above, it was possible to provide a serum-free medium for culturing animal stem cells having a high proliferation ability. In the present invention, an antibacterial peptide shown in SEQ ID NO: 1 in the sequence listing and two growth factors, namely bFGF and PDFG, are applied to a basal medium for animal cell culture that does not contain serum. It was possible to cultivate various animal stem cells without serum, and to provide a serum-free medium for culturing animal stem cells having a high proliferative ability that gave a proliferative ability approximately 100 times that of serum-free culture according to the prior art. . The present invention includes a method for culturing animal stem cells such as mesenchymal stem cells using the serum-free medium for animal stem cell culture of the present invention.
すなわち具体的には本発明は、（１）血清を含有しない動物細胞培養用基礎培地に、配列表の配列番号１に示される抗菌ペプチドを添加してなる動物幹細胞培養用無血清培地や、（２）上記（１）記載の動物幹細胞培養用無血清培地に、ｂＦＧＦ及び／又はＰＤＧＦを添加してなる動物幹細胞培養用無血清培地や、（３）上記(２)記載の動物幹細胞培養用無血清培地に、インスリン、トランスフェリン、セレン酸及びリノール酸からなるグループから選択される１又は２以上の成分を添加してなる動物幹細胞培養用無血清培地や、（４）上記（１）記載の動物幹細胞培養用無血清培地にｂＦＧＦ、ＰＤＧＦ、インスリン、トランスフェリン、セレン酸及びリノール酸を添加してなる上記（３）記載の動物幹細胞培養用無血清培地からなる。 Specifically, the present invention relates to (1) a serum-free medium for animal stem cell culture obtained by adding an antimicrobial peptide represented by SEQ ID NO: 1 in the sequence listing to a basal medium for animal cell culture that does not contain serum, 2) A serum-free medium for animal stem cell culture obtained by adding bFGF and / or PDGF to a serum-free medium for animal stem cell culture described in (1) above, or (3) no animal stem cell culture medium described in (2) above. A serum-free medium for animal stem cell culture obtained by adding one or more components selected from the group consisting of insulin, transferrin, selenate and linoleic acid to a serum medium; and (4) the animal according to (1) above It comprises the serum-free medium for animal stem cell culture described in (3) above, wherein bFGF, PDGF, insulin, transferrin, selenate and linoleic acid are added to the serum-free medium for stem cell culture.
また本発明は、（５）動物幹細胞が、間葉系幹細胞であることを特徴とする上記（１）〜（４）のいずれか記載の動物幹細胞培養用無血清培地や、（６）上記（１）〜（５）のいずれか記載の動物幹細胞培養用無血清培地を用いて、動物幹細胞を培養することを特徴とする動物幹細胞の培養方法や、（７）動物幹細胞を、血清を含む動物細胞用培地で培養し、培地を上記（１）〜（５）のいずれか記載の動物幹細胞培養用無血清培地に交換して、動物幹細胞培養することを特徴とする動物幹細胞の増殖、培養方法からなる。 The present invention also provides (5) a serum-free medium for animal stem cell culture according to any one of (1) to (4) above, wherein the animal stem cells are mesenchymal stem cells, and (6) the above ( An animal stem cell culture method characterized by culturing animal stem cells using the serum-free medium for animal stem cell culture according to any one of 1) to (5), and (7) an animal containing serum containing animal stem cells A method for growing and culturing animal stem cells, comprising culturing in a cell culture medium, replacing the medium with the serum-free medium for animal stem cell culture according to any one of (1) to (5) above, and culturing the animal stem cells. Consists of.
本発明の動物幹細胞培養用無血清培地は、血清の非存在下で、血清を添加した培地に匹敵する動物幹細胞の増殖効果を示し、しかも、動物幹細胞の幹細胞としての機能を保持しつつ、培養、増殖することが可能であり、組織再生医療等に用いる動物幹細胞用の培養用の培地として、実用上有用な無血清培地を提供する。本発明の動物幹細胞培養用無血清培地によって、血清を用いることによって発生する、ＢＳＥやウィルス性疾患の水平感染などの様々な問題を回避することができ、本発明の無血清培地は、特に、組織再生医療等に用いる間葉系幹細胞等、動物幹細胞の調製のための手段として大きく貢献することができる。 The serum-free medium for culturing animal stem cells of the present invention exhibits a proliferation effect of animal stem cells comparable to a medium supplemented with serum in the absence of serum, and further, while maintaining the function of animal stem cells as stem cells. It is possible to proliferate and provide a practically useful serum-free medium as a culture medium for animal stem cells used in tissue regeneration medicine and the like. The serum-free medium for animal stem cell culture of the present invention can avoid various problems such as horizontal infection of BSE and viral diseases caused by using serum. It can greatly contribute as a means for preparing animal stem cells such as mesenchymal stem cells used in tissue regenerative medicine.
更に、従来、動物の細胞培養には、ペニシリンや、ストレプトマイシン等の抗生物質或いはアンホテリシンなどの抗真菌物質の少量添加が必須とされており、これらの抗生物質、抗真菌物質には動物細胞増殖の抑制作用があるといわれていたが、これらの抗生物質、抗真菌物質は、細胞を再び生体にもどす再生医療を行う場合には、添加する物質として好ましい物質ではなかった。本発明の抗菌ペプチドはこのような動物細胞増殖の抑制作用はなく、抗菌性と抗真菌性があることが分かっており、本発明の無血清培地は、再生医療用の幹細胞の増殖培地として用いるに非常に好ましいものである。特に、本発明の抗菌ペプチド＋ＰＤＧＦ+ｂＦＧＦ＋ＩＴＳ（インスリン、トランスフェリン、セレン酸及びリノール酸）の組み合わせは１０％ＦＢＳに匹敵する細胞増殖能を示したことから、再生医療用の幹細胞の増殖培地として特に優れた動物幹細胞培養用無血清培地を提供する。 Furthermore, conventionally, in animal cell culture, it has been essential to add a small amount of antibiotics such as penicillin, streptomycin, or antifungal substances such as amphotericin, and these antibiotics and antifungal substances do not proliferate animal cells. Although said to have an inhibitory action, these antibiotics and antifungal substances were not preferable substances to be added when regenerative medicine for returning cells to the living body again. The antibacterial peptide of the present invention has no inhibitory effect on the growth of animal cells and is known to have antibacterial and antifungal properties. The serum-free medium of the present invention is used as a growth medium for stem cells for regenerative medicine. It is very preferable. In particular, the combination of antibacterial peptide + PDGF + bFGF + ITS (insulin, transferrin, selenate, and linoleic acid) of the present invention exhibited cell growth ability comparable to 10% FBS, and thus was particularly excellent as a growth medium for stem cells for regenerative medicine. A serum-free medium for culturing animal stem cells is provided.
本発明は、血清を含有しない動物細胞培養用基礎培地に、配列表の配列番号１に示される抗菌ペプチドを添加してなる動物幹細胞培養用無血清培地からなる。
本発明で使用する動物細胞培養用基礎培地としては、動物細胞培養用に使用されている適宜の基礎培地を使用することができるが、ＤＭＥＭ（Dulbeco改変Eagle培地）が有利に使用することができる。
The present invention comprises a serum-free medium for animal stem cell culture obtained by adding an antimicrobial peptide represented by SEQ ID NO: 1 in the sequence listing to a basal medium for animal cell culture that does not contain serum.
As the basal medium for animal cell culture used in the present invention, an appropriate basal medium used for animal cell culture can be used, but DMEM (Dulbeco modified Eagle medium) can be advantageously used. .
本発明の動物幹細胞培養用無血清培地の調製に使用される抗菌ペプチドは、Lys-Arg-Leu-Phe-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Tyrからなるペプチド（以下、「８１９４抗菌ペプチド」という）であり（（配列表の配列番号１）、抗菌ペプチドとして、公知のペプチドである（Oral Disease、10,4,221-228；特許第３４７２８２１号）。 The antimicrobial peptide used for the preparation of the serum-free medium for animal stem cell culture of the present invention is a peptide comprising Lys-Arg-Leu-Phe-Arg-Arg-Trp-Gln-Trp-Arg-Met-Lys-Lys-Tyr (Hereinafter referred to as “8194 antibacterial peptide”) ((SEQ ID NO: 1 in the sequence listing)), and is a known peptide as an antibacterial peptide (Oral Disease, 10, 4, 221-228; Patent No. 3472821).
本発明のペプチドは、ぺプチド合成法で取得することができる。ペプチド合成には、液相法及び固相法が存在するが、本発明のペプチドは、液相法及び固相法のいずれの方法も使用することができる。液相法は、反応を溶液状態で行い反応混合物から生成物を単離精製し、この生成物を中間体として次のペプチド伸長反応に用いる方法である。一方、固相法は、反応溶媒に不溶の固相担体にアミノ酸を結合させ、このアミノ酸に準じ縮合反応を行いペプチド鎖を伸長させていく方法である。 The peptide of the present invention can be obtained by a peptide synthesis method. There are a liquid phase method and a solid phase method for peptide synthesis, but the peptide of the present invention can use either a liquid phase method or a solid phase method. The liquid phase method is a method in which a reaction is performed in a solution state, a product is isolated and purified from a reaction mixture, and this product is used as an intermediate for the next peptide extension reaction. On the other hand, the solid phase method is a method in which an amino acid is bound to a solid phase carrier insoluble in a reaction solvent, and a peptide chain is elongated by performing a condensation reaction according to this amino acid.
ペプチドの化学合成は、カルボキシル基を保護したアミノ酸にアミノ基を保護したアミノ酸を脱水縮合させ、ペプチド結合を形成させ、次にアミノ保護基を除去後、遊離したアミノ基に次のアミノ基保護アミノ酸を順次、C末端からN末端に向かって一つずつ延長していく方法が基本である。脱水縮合反応では、カルボキシル基を活性化して、結合させようとするアミノ基と反応させる。この活性化には、ジシクロへキシカルボジイミド（ＤＣＣ）法、活性エステル法、酸無水物法、アジド法等があるがその反応性の高さとラセミ化その他の副反応を考慮して選ばれる。縮合反応時の副反応を防止するためにアミノ酸のアミノ基、カルボキシル基、側鎖(R)の官能基には保護基が導入される。これらの保護基は、縮合反応の条件で安定であり、必要なときには速やかに除去されるものが好ましい。また、アミノ基の保護基とカルボキシル基の保護基とは互いに選択的に除去されることが好ましい。 Chemical synthesis of peptides involves dehydration condensation of amino acid protected amino group to amino acid protected carboxyl group to form peptide bond, then remove amino protecting group, then free amino group to the next amino group protected amino acid The basic method is to sequentially extend from the C terminal to the N terminal one by one. In the dehydration condensation reaction, the carboxyl group is activated and reacted with an amino group to be bonded. This activation includes a dicyclohexylcarbodiimide (DCC) method, an active ester method, an acid anhydride method, an azide method and the like, and is selected in consideration of its high reactivity and racemization and other side reactions. In order to prevent side reactions during the condensation reaction, protective groups are introduced into the amino group, carboxyl group, and side chain (R) functional groups of the amino acid. These protecting groups are preferably stable under the conditions of the condensation reaction and can be removed quickly when necessary. Further, it is preferable that the amino protecting group and the carboxyl protecting group are selectively removed from each other.
縮合反応は、カルボジイミド等の縮合剤の存在下、あるいはN−保護アミノ酸活性エステル又はペプチド活性エステルを用いて実施する。縮合反応終了後、保護基は除去されるが、固相の場合は更に、ペプチドのC末端と樹脂との結合を切断する。製造されたペプチドは通常の方法に従い精製される。例えば、イオン交換クロマトグラフィー、逆相液体クロマトグラフィー、アフィニティークロマトグラフィー等が挙げられる。合成したペプチドは、エドマン分解法でC-末端からアミノ酸配列を読み取るプロティンシークエンサー、ＧＣ−ＭＳ、ＴＯＦ−ＭＳ等で分析される。 The condensation reaction is carried out in the presence of a condensing agent such as carbodiimide, or using an N-protected amino acid active ester or peptide active ester. After completion of the condensation reaction, the protecting group is removed, but in the case of a solid phase, the bond between the C-terminus of the peptide and the resin is further cleaved. The produced peptide is purified according to a usual method. For example, ion exchange chromatography, reverse phase liquid chromatography, affinity chromatography and the like can be mentioned. The synthesized peptide is analyzed by a protein sequencer that reads the amino acid sequence from the C-terminal by the Edman degradation method, GC-MS, TOF-MS, or the like.
本発明の動物幹細胞培養用無血清培地の調製において、動物細胞培養用基礎培地に添加される、ｂＦＧＦ（繊維芽細胞成長因子）、ＰＤＧＦ（血小板由来成長因子）、及び、ＩＴＳ（インスリン、トランスフェリン、セレン酸及びリノール酸）は、適宜市販のものを用いることができる。 In the preparation of a serum-free medium for animal stem cell culture of the present invention, bFGF (fibroblast growth factor), PDGF (platelet-derived growth factor), and ITS (insulin, transferrin, As selenic acid and linoleic acid, commercially available products can be used as appropriate.
本発明の無血清培地は、組織再生医療等の分野において、本発明の無血清培地を用いて、間葉系幹細胞のような動物幹細胞の培養、増殖を行うには、該無血清培地を用いて、採取した幹細胞を、動物細胞の通常の培養条件に従って培養することにより、行うことができる。また、採取した細胞を、予め、ウシ胎児血清（ＦＢＳ）を添加した培地で培養して、細胞を増殖し、その後、本発明の無血清培地に、培地交換することにより、効率的に動物幹細胞の無血清培養を行うことができる。 The serum-free medium of the present invention is used to culture and proliferate animal stem cells such as mesenchymal stem cells using the serum-free medium of the present invention in the field of tissue regeneration medicine and the like. Thus, the collected stem cells can be cultured by culturing them under normal culture conditions for animal cells. In addition, the collected cells are cultured in advance in a medium supplemented with fetal bovine serum (FBS) to proliferate the cells, and then the medium is replaced with the serum-free medium of the present invention. Serum-free culture can be performed.
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.
（材料と方法）
使用した細胞：ヒト腸骨由来骨髄間葉系幹細胞（CAMBREX社より購入）（以下「ＭＳＣ」という）。
培養条件：通法に従って培養したＭＳＣを２４穴培養皿に５０００細胞/穴になるような密度で１０％ウシ胎児血清（ＦＢＳ）と抗生物質を含むＤＭＥＭ（Sigma社）培地にて播種、培養した。翌日にＦＢＳを含まないＤＭＥＭ（抗生物質は含む）に培地交換し、被検因子を添加し、３日後に増殖した細胞を培養穴からトリプシンを用いて剥離し、コールターカウンター（ベックマン社）によって実細胞数を計測した。対照には、被検因子を希釈するのに用いた０．３％ＢＳＡ／ＰＢＳのみを添加した。Ｎ＝３で、平均と標準偏差をグラフ化した。
(Materials and methods)
Cells used: Human iliac bone marrow mesenchymal stem cells (purchased from CAMBREX) (hereinafter referred to as “MSC”).
Culture conditions: MSCs cultured according to a conventional method were seeded and cultured in a 24-well culture dish in a DMEM (Sigma) medium containing 10% fetal bovine serum (FBS) and antibiotics at a density of 5000 cells / well. . On the next day, change the medium to DMEM (containing antibiotics) without FBS, add the test factor, peel off the proliferated cells after 3 days using trypsin from the culture hole, and execute the test using a Coulter counter (Beckman). Cell number was counted. To the control, only 0.3% BSA / PBS used for diluting the test factor was added. The average and standard deviation were graphed with N = 3.
被検因子：濃度は培地添加後の終濃度をあらわす。（ａ）８１９４抗菌 ペプチド（合成依頼品）１０マイクロM；（ｂ）ｂＦＧＦ （Sigma社製）１０ｎｇ／ｍｌ；（ｃ）ＰＤＧＦ−ＢＢ（R&D社製）１０ｎｇ／ｍｌ（ここではＰＤＧＦと略す）；（ｄ）ＩＴＳ（ＢＤ Biosciences社製、Insulin, Transferrin, Selenious Acid,Linoleic Acidの混和物） メーカー指定で最終倍率１となるように希釈した。（ａ）〜（ｄ）の混和物をＤＭＥＭに添加した。 Test factor: The concentration represents the final concentration after addition of the medium. (A) 8194 antibacterial peptide (request for synthesis) 10 microM; (b) bFGF (manufactured by Sigma) 10 ng / ml; (c) PDGF-BB (manufactured by R & D) 10 ng / ml (abbreviated herein as PDGF); (D) ITS (mixture of BD Biosciences, Insulin, Transferrin, Selenious Acid, and Linoleic Acid) Diluted to a final magnification of 1 as specified by the manufacturer. The mixture of (a) to (d) was added to DMEM.
（結果）
結果を図に示す。縦軸は各条件での培養穴中の細胞数を、横軸には添加物を示す。横軸の添加物にさらに８１９４抗菌ペプチド、若しくは、ＩＴＳ＋８１９４抗菌ペプチドを添加したグラフを付している。１０％ＦＢＳとはウシ胎児血清を最終濃度１０％となるようにＤＭＥＭに混和した条件である。１０％ＦＢＳ添加群は対照（０．３％BSA in PBS）と比べて細胞増殖を引き起こした。８１９４抗菌ペプチド＋ＰＤＧＦ+ｂＦＧＦ＋ＩＴＳの４因子を混和したものは、１０％ＦＢＳに匹敵する増殖性を示した。各因子の欠けたものはそれぞれ増殖性が強く示されなかった。
(result)
The results are shown in the figure. The vertical axis represents the number of cells in the culture hole under each condition, and the horizontal axis represents the additive. A graph in which 8194 antibacterial peptide or ITS + 8194 antibacterial peptide is further added to the additive on the horizontal axis is attached. 10% FBS is a condition in which fetal bovine serum is mixed in DMEM to a final concentration of 10%. The group supplemented with 10% FBS caused cell proliferation compared to the control (0.3% BSA in PBS). A mixture containing 8194 antibacterial peptide + PDGF + bFGF + ITS, showed growth comparable to 10% FBS. Those lacking each factor did not show strong growth.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2008099561A1 (en) * | 2007-02-14 | 2008-08-21 | Japan Science And Technology Agency | Agent for promoting cell proliferation using antimicrobial peptide and method for promoting cell proliferation using serum-free medium containing the same |
| JP2013517292A (en) * | 2010-01-14 | 2013-05-16 | オーガノジェネシス・インコーポレイテッド | Bioengineered tissue constructs and methods for generating and using the same |
| CN111154717A (en) * | 2012-11-13 | 2020-05-15 | 基立福有限公司 | Culture medium for human mesenchymal stem cells |
| CN113412833A (en) * | 2021-08-23 | 2021-09-21 | 山东科金生物发展有限公司 | Cryopreservation protective agent for ultralow-temperature damage of mesenchymal stem cells |
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| JPH0678759A (en) * | 1992-06-12 | 1994-03-22 | Behringwerke Ag | Serum-free cell culture medium |
| JP2002179698A (en) * | 2000-12-08 | 2002-06-26 | Univ Hiroshima | New peptide and composition using the same peptide |
| JP2005154338A (en) * | 2003-11-26 | 2005-06-16 | Japan Science & Technology Agency | Cell proliferating agent containing basic antibacterial peptide as active ingredient |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| JPH0678759A (en) * | 1992-06-12 | 1994-03-22 | Behringwerke Ag | Serum-free cell culture medium |
| JP2002179698A (en) * | 2000-12-08 | 2002-06-26 | Univ Hiroshima | New peptide and composition using the same peptide |
| JP2005154338A (en) * | 2003-11-26 | 2005-06-16 | Japan Science & Technology Agency | Cell proliferating agent containing basic antibacterial peptide as active ingredient |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008099561A1 (en) * | 2007-02-14 | 2008-08-21 | Japan Science And Technology Agency | Agent for promoting cell proliferation using antimicrobial peptide and method for promoting cell proliferation using serum-free medium containing the same |
| JP2013517292A (en) * | 2010-01-14 | 2013-05-16 | オーガノジェネシス・インコーポレイテッド | Bioengineered tissue constructs and methods for generating and using the same |
| JP2018117643A (en) * | 2010-01-14 | 2018-08-02 | オーガノジェネシス・インコーポレイテッドＯｒｇａｎｏｇｅｎｅｓｉｓ Ｉｎｃ． | Bioengineered tissue constructs and methods for producing and using the same |
| CN111154717A (en) * | 2012-11-13 | 2020-05-15 | 基立福有限公司 | Culture medium for human mesenchymal stem cells |
| CN113412833A (en) * | 2021-08-23 | 2021-09-21 | 山东科金生物发展有限公司 | Cryopreservation protective agent for ultralow-temperature damage of mesenchymal stem cells |
| CN113412833B (en) * | 2021-08-23 | 2021-11-12 | 山东科金生物发展有限公司 | Cryopreservation protective agent for ultralow-temperature damage of mesenchymal stem cells |
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