JP2014183825A - Method of stable and large-scale culture and storage of cardiomyocyte or human cardiomyocyte derived from human ips cell (induced pluripotent stem cell) contributing to regenerative medicine - Google Patents
Method of stable and large-scale culture and storage of cardiomyocyte or human cardiomyocyte derived from human ips cell (induced pluripotent stem cell) contributing to regenerative medicine Download PDFInfo
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本発明は、再生医療に資するヒトｉＰＳ細胞由来の心筋細胞あるいはヒト心筋細胞を安定的に大量培養して保存する方法に関する。 The present invention relates to a method for stably culturing and storing human iPS cell-derived cardiomyocytes or human cardiomyocytes that contribute to regenerative medicine.
重症心不全患者などに対して心筋の再生医療を行う場合、遺伝子レベルで細胞破壊の無いヒト心筋細胞を安定的に大量に培養し保存できる技術の確立が必須である。２０１０年に山本らは、Ａｎｎ Ｔｈｏｒａｃ Ｃａｒｄｉｏｖａｓｃ Ｓｕｒｇ誌にて従来の定説を覆し、ヒト心筋細胞は凍結保存することで大量培養できることを示した。しかし、凍結−融解後のヒト心筋細胞の生存率は５０％弱と低く、本当に再生医療に資するレベルで大量培養および保存が可能か否かは不明である。さらに、凍結−融解後に生存したヒト心筋細胞が凍結前に比べて遺伝子レベルで細胞破壊が無く、それらがヒト生体内に移植された後に正常な機能を有し、再生医療に資することができるのか否かも全く不明であった。 When performing myocardial regenerative medicine for patients with severe heart failure, it is essential to establish a technique capable of stably culturing and storing a large amount of human cardiomyocytes without cell destruction at the gene level. In 2010, Yamamoto et al. Reversed the conventional theory in Ann Thorac Cardiovas Surg and showed that human cardiomyocytes can be cultured in large quantities by cryopreserving them. However, the survival rate of human cardiomyocytes after freeze-thaw is as low as 50%, and it is unclear whether mass culture and storage are possible at a level that really contributes to regenerative medicine. Furthermore, human cardiomyocytes surviving after freezing and thawing have no cell disruption at the gene level compared to those before freezing. It was not clear at all.
本発明は、再生医療に好ましいヒトｉＰＳ細胞由来の心筋細胞またはヒト心筋細胞を含む試料を安定的に大量に得て保存するための新たな方法に関する。 The present invention relates to a new method for stably obtaining and storing human iPS cell-derived cardiomyocytes or samples containing human cardiomyocytes, which are preferable for regenerative medicine.
本発明の一実施形態は、凍結する試料を、−５℃以下の過冷却状態において、一定時間保持することを特徴とする大量培養方法である。前記試料を、４℃から−５℃に冷却するのに、１分あたり０．５℃以上、好ましくは、１℃以上の速度で低下させてもよい。前記試料がヒトｉＰＳ細胞由来の心筋細胞またはヒト心筋細胞であることが好ましい。また、この方法において前記試料を過冷却状態において、２０分以上保持することが好ましい。 One embodiment of the present invention is a mass culture method characterized by holding a sample to be frozen in a supercooled state of −5 ° C. or lower for a certain period of time. To cool the sample from 4 ° C. to −5 ° C., it may be reduced at a rate of 0.5 ° C. or more, preferably 1 ° C. or more per minute. The sample is preferably a human iPS cell-derived cardiomyocyte or a human cardiomyocyte. In this method, it is preferable to hold the sample for 20 minutes or more in a supercooled state.
本発明によって、再生医療に好ましいヒトｉＰＳ細胞由来の心筋細胞またはヒト心筋細胞を安定的に大量に得て保存するための新たな方法を提供できるようになった。 According to the present invention, a new method for stably obtaining and storing human iPS cell-derived cardiomyocytes or human cardiomyocytes, which are preferable for regenerative medicine, can be provided.
以下、上記知見に基づき完成した本発明の実施の形態を、実施例を挙げながら詳細に説明する。
実施の形態及び実施例に特に説明がない場合には、Ｊ．Ｓａｍｂｒｏｏｋ，Ｅ．Ｆ．Ｆｒｉｔｓｃｈ＆Ｔ．Ｍａｎｉａｔｉｓ（Ｅｄ．），Ｍｏｌｅｃｕｌａｒ ｃｌｏｎｉｎｇ，ａ ｌａｂｏｒａｔｏｒｙ ｍａｎｕａｌ（３ｒｄ ｅｄｉｔｉｏｎ），Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ Ｐｒｅｓｓ，Ｃｏｌｄ Ｓｐｒｉｎｇ Ｈａｒｂｏｒ，Ｎｅｗ Ｙｏｒｋ（２００１）；Ｆ．Ｍ．Ａｕｓｕｂｅｌ，Ｒ．Ｂｒｅｎｔ，Ｒ．Ｅ．Ｋｉｎｇｓｔｏｎ，Ｄ．Ｄ．Ｍｏｏｒｅ，Ｊ．Ｇ．Ｓｅｉｄｍａｎ，Ｊ．Ａ．Ｓｍｉｔｈ，Ｋ．Ｓｔｒｕｈｌ（Ｅｄ．），Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｍｏｌｅｃｕｌａｒ Ｂｉｏｌｏｇｙ，Ｊｏｈｎ Ｗｉｌｅｙ＆Ｓｏｎｓ Ｌｔｄ．；Ｊｕａｎ Ｓ．Ｂｏｎｉｆａｃｉｎｏ（Ｂｅｔｈｅｓｄａ，Ｍａｒｙｌａｎｄ）；Ｍａｒｙ Ｄａｓｓｏ（Ｂｅｔｈｅｓｄａ，Ｍａｒｙｌａｎｄ）；Ｊ．Ｂ．Ｈａｒｆｏｒｄ，Ｊ．Ｌ−Ｓｃｈｗａｒｔｚ，Ｋ．Ｍ．Ｙａｍａｄａ（Ｅｄ．），Ｃｕｒｒｅｎｔ Ｐｒｏｔｏｃｏｌｓ ｉｎ Ｃｅｌｌ Ｂｉｏｌｏｇｙ，Ｊｏｈｎ Ｗｉｌｅｙ＆Ｓｏｎｓ Ｌｔｄ．等の標準的なプロトコール集に記載の方法、あるいはそれを修飾したり、改変した方法を用いる。また、市販の試薬キットや測定装置を用いる場合には、特に説明が無い場合、それらに添付のプロトコールを用いる。
なお、本発明の目的、特徴、利点、及びそのアイデアは、本明細書の記載により、当業者には明らかであり、本明細書の記載から、当業者であれば、容易に本発明を再現できる。以下に記載された発明の実施の形態及び具体的に実施例などは、本発明の好ましい実施態様を示すものであり、例示又は説明のために示されているのであって、本発明をそれらに限定するものではない。本明細書で開示されている本発明の意図並びに範囲内で、本明細書の記載に基づき、様々な改変並びに修飾ができることは、当業者にとって明らかである。
＝＝本発明の方法によって凍結保存されたヒトｉＰＳ細胞由来の心筋細胞＝＝
本発明の方法（Ｓ．Ｃ．法）によって凍結されたヒトｉＰＳ細胞由来の心筋細胞は、融解後、その特異的マーカーが遺伝子レベルで、先行技術のＣ．Ｓ．Ｃ．法によって凍結−融解されたヒトｉＰＳ細胞由来の心筋細胞よりも高レベルで、そして凍結前のヒトｉＰＳ細胞由来の心筋細胞と同様のレベルで安定的に発現していることがはじめて確認された。また、凍結前の細胞や、Ｃ．Ｓ．Ｃ．法によって凍結−融解されたヒトｉＰＳ細胞由来の心筋細胞よりも、培養条件での細胞増殖能が著しく増していることもはじめて確認された。なお、凍結−融解後のヒトｉＰＳ細胞由来の心筋細胞の形状も、凍結前と変化は無い。さらに、生体内に細胞移植後も心筋細胞として正常に機能していることも、はじめて確認された。従って本発明の方法によって、ヒトｉＰＳ細胞由来の心筋細胞が再生医療に資するレベルで安定的に大量培養および保存できることが、はじめて示された。
ここで、ヒト心筋細胞の由来は特に限定されず、生体から単離した心筋組織や心筋細胞でも良く、人工誘導多能性幹細胞（ｉＰＳ細胞）、胚幹細胞、間充織幹細胞、心筋前駆細胞などの未分化な細胞をｉｎ ｖｉｔｒｏで分化させて得られた細胞であっても良い。Hereinafter, embodiments of the present invention completed based on the above knowledge will be described in detail with reference to examples.
If there is no particular description in the embodiments and examples, J.I. Sambrook, E .; F. Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); M.M. Ausubel, R.A. Brent, R.M. E. Kingston, D.M. D. Moore, J. et al. G. Seidman, J .; A. Smith, K.M. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Juan S .; Bonifacino (Bethesda, Maryland); Mary Dasso (Bethesda, Maryland); B. Harford, J .; L-Schwartz, K.M. M.M. Yamada (Ed.), Current Protocols in Cell Biology, John Wiley & Sons Ltd. A method described in a standard protocol collection such as the above, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.
The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.
== Human iPS cell-derived cardiomyocytes cryopreserved by the method of the present invention ==
The human iPS cell-derived cardiomyocytes frozen by the method of the present invention (SC method) have a specific marker at the gene level after thawing, and the prior art C.I. S. C. It was confirmed for the first time that it was stably expressed at a higher level than human iPS cell-derived cardiomyocytes frozen and thawed by the method, and at a level similar to that of human iPS cell-derived cardiomyocytes before freezing. Further, cells before freezing, C.I. S. C. It was also confirmed for the first time that the cell proliferation ability under culture conditions was significantly increased compared to cardiomyocytes derived from human iPS cells frozen and thawed by the method. In addition, the shape of the cardiomyocytes derived from human iPS cells after freezing-thawing is not changed from that before freezing. Furthermore, it was confirmed for the first time that the cells function normally as cardiomyocytes even after cell transplantation in vivo. Therefore, it has been shown for the first time that cardiomyocytes derived from human iPS cells can be stably cultured and stored in large quantities at a level that contributes to regenerative medicine by the method of the present invention.
Here, the origin of human cardiomyocytes is not particularly limited, and may be myocardial tissue or cardiomyocytes isolated from a living body, such as artificially induced pluripotent stem cells (iPS cells), embryonic stem cells, mesenchymal stem cells, myocardial progenitor cells, etc. A cell obtained by differentiating an undifferentiated cell in vitro may be used.
以下の実施例では、このように過冷却装置を用いた新しいＳ．Ｃ．法によって、再生医療に好ましいヒト心筋細胞を得るための例を示す。
（１）ヒトｉＰＳ細胞由来の心筋細胞の調製
ヒトｉＰＳ細胞は、患者由来の肝臓前駆細胞から樹立された細胞株（ＣｈｉＰｓ ｃｅｌｌ ｌｉｎｅｓ）（ＵＳ２０１２／０００９６０１）を用い、４ｎｇ／ｍＬｂＦＧＦ含有ＭＥＦ−ＣＭを用いて、マトリジェルでコートした培養皿で培養した。
そのヒトｉＰＳ細胞をヒト心筋細胞に分化させる場合（Ｎａｔ Ｂｉｏｔｅｃｈｎｏｌ ｖｏｌ．２５，Ｐ．１０１５−１０２４，２００７）、ヒトｉＰＳ細胞を、培地をＢ２７含有ＲＰＭＩ１６４０（１００ｎｇ／ｍＬヒト組換えアクチビンＡ含有）に置換して２４時間培養し、その後、培地をＢ２７含有ＲＰＭＩ１６４０（１０ｎｇ／ｍＬヒトＢＭＰ４含有）に置換して４日間培養した。その後は、サイトカインの含まないＢ２７含有ＲＰＭＩ１６４０で、１日おきに培地交換しながら細胞を維持した。
継代２回目のヒトｉＰＳ細胞由来の心筋細胞を２ｍＬの凍結用培地Ｍｅ２ＳＯ（７０％ＩＭＤＭ−２０％ＦＢＳ−１０％ＤＭＳＯ）に懸濁し、２０ｍＬのバイアルに移して、バイアルを４℃にし、その後、１．０℃／分の速度で−５℃まで市販の過冷却装置を用いて冷却して、２０分間にわたり過冷却状態で維持する。そして、その後、上記の細胞をすぐに市販のプログラムフリーザーに移し、１．０℃／分の速度で−４０．０℃まで冷却し、さらに１０．０℃／分の速度で−８０．０℃まで冷却する。さらに、その後すぐに、上記の細胞を液体窒素中で−１９６．０℃まで冷却して維持した。
なお、上記の細胞を融解させるときは、バイアルを２分間室温に放置し、２５度の温水中に２分間浸した。その後、１０％Ｍｅ２ＳＯ含有培地、５％Ｍｅ２ＳＯ含有培地、２．５％Ｍｅ２ＳＯ含有培地、Ｍｅ２ＳＯの含有しない培地の順で、遠心−回収を繰りかえしながら、各５分ずつ処理した。
（２）ＲＴ−ＰＣＲによるヒト心筋細胞マーカー遺伝子の発現レベルの測定
試料となる細胞には、（１）で得られたヒトｉＰＳ細胞由来の心筋細胞を用い、本発明のＳ．Ｃ．法による凍結−融解を行う前のヒトＰＳ細胞由来の心筋細胞（コントロール群またはＰｒｅ−Ｓ．Ｃ．）、Ｓ．Ｃ．法による凍結−融解を行った後のヒトＰＳ細胞由来の心筋細胞（Ｐｏｓｔ−Ｓ．Ｃ．）、Ｃ．Ｓ．Ｃ．法による凍結−融解を行った後のヒトｉＰＳ細胞由来の心筋細胞（Ｐｏｓｔ−Ｃ．Ｓ．Ｃ．）、を調製した。
これらの細胞から調製したＲＮＡに対し、ＲＴ−ＰＣＴを行った。以下に用いたプライマー配列を示す。
図１に示すように凍結前のヒト心筋細胞マーカー遺伝子の発現は、本発明のＳ．Ｃ．法による凍結−融解後でも安定的に維持された。一方、Ｃ．Ｓ．Ｃ．法によって凍結−融解されたヒトｉＰＳ細胞由来の心筋細胞では、それらのマーカー遺伝子の発現は、ほとんど観察されなかった。これは、Ｃ．Ｓ．Ｃ．法によるヒトｉＰＳ細胞由来の心筋細胞の凍結−融解では、ヒトｉＰＳ細胞由来の心筋細胞の遺伝子レベルでの破壊（ＤＮＡの破壊あるいは、遺伝子間制御の破壊など）が生じている証拠である。しかし、Ｓ．Ｃ．法によるヒトｉＰＳ細胞由来の心筋細胞の凍結−融解では、凍結前と比べても、ヒト心筋細胞マーカー遺伝子の発現が同等の高レベルで安定的であることが示された。すなわち山本らによる先行技術（Ａｎｎ Ｔｈｏｒａｃ Ｃａｒｄｉｏｖａｓｃ Ｓｕｒｇ誌（２０１０）；１６：１０５−１１２．）によるＣ．Ｓ．Ｃ法では、ヒトｉＰＳ細胞由来の心筋細胞の遺伝子レベルでの破壊を回避することはできなかった。しかし、本発明による過冷却装置を用いたＳ．Ｃ法によってはじめて、ヒトｉＰＳ細胞由来の心筋細胞が凍結−融解による遺伝子破壊が無く安定的に保存できることが証明された。
（３）ヒトｉＰＳ細胞由来の心筋細胞の増殖速度の測定
上記で調製したヒトｉＰＳ細胞由来の心筋細胞を、Ｂ２７含有ＲＰＭＩ１６４０で培養し、０、３、５、７、９、１１、１３、１５日目に電子細胞カウンター（Ｚ１ Ｃｅｌｌ ａｎｄ Ｐａｒｔｉｃｌｅ Ｃｏｕｎｔｅｒ；Ｂｅｃｋｍａｎ Ｃｏｕｌｔｅｒ Ｉｎｃ．）で細胞数を計測し、増殖速度を調べた。コントロールの細胞としては通常培養方法で維持されているヒトｉＰＳ細胞由来の心筋細胞が用いられた。図２に示すように本発明のＳ．Ｃ．法によって得られた細胞のほうが、Ｃ．Ｓ．Ｃ．法によって得られた細胞よりも、はるかに増殖速度が高かった。なお、１５日目の細胞生存率は、コントロールを１００％とした時、Ｃ．Ｓ．Ｃ．法によって得られた細胞は３０％、Ｓ．Ｃ．法によって得られた細胞は９０％であった。このことから、凍結・融解の影響は、山本らによる先行技術（Ａｎｎ Ｔｈｏｒａｃ Ｃａｒｄｉｏｖａｓｃ Ｓｕｒｇ誌（２０１０）；１６：１０５−１１２．）によるＣ．Ｓ．Ｃ．法よりもＳ．Ｃ．法のほうがはるかに小さいことがわかる。
すなわち、本発明による過冷却装置を用いたＳ．Ｃ法によってはじめて、ヒトｉＰＳ細胞由来の心筋細胞が遺伝子破壊が無く安定的に大量培養および保存できることが証明された。
（４）ヒト心筋細胞マーカータンパクの発現測定
本発明の過冷却装置を用いたＳ．Ｃ．法による凍結−解凍後のヒトｉＰＳ細胞由来のヒト心筋細胞を重症心不全患者に冠動脈バイパス手術と併用して移植し、６ヶ月後、治療前に心筋壊死していた部分から患者の心筋細胞を検査目的で採取した。その際の患者由来の心筋細胞におけるヒト心筋細胞マーカータンパクの発現程度を免疫染色法によって調べた。方法は下記に示すとおりである。
Ｂｅａｔｉｎｇ ｈｕｍａｎ ｃａｒｄｉｏｍｙｏｃｙｔｅｓ ｄｅｒｉｖｅｄ ｂｙ ｍｙｏｃａｒｄｉａｃ ｂｉｏｐｓｙ ｗｅｒｅ ｆｉｘｅｄ ｗｉｔｈ ４％−ｐａｒａｆｏｒｍａｌｄｅｈｙｄｅ，ｐｅｒｍｅａｂｉｌｉｚｅｄ ｗｉｔｈ １％−Ｔｒｉｔｏｎ−Ｘ−１００（Ｓｉｇｍａ），ｂｌｏｃｋｅｄ ｗｉｔｈ ５％−ｈｏｒｓｅ ｓｅｒｕｍ，ａｎｄ ｉｎｃｕｂａｔｅｄ ｏｖｅｒｎｉｇｈｔ ａｔ ４８℃ ｗｉｔｈ ｐｒｉｍａｒｙ ａｎｔｉｂｏｄｉｅｓ ｔａｒｇｅｔｉｎｇ：ｃａｒｄｉａｃ−ｔｒｏｐｏｎｉｎ−Ｉ（ｃＴｎＩ，１：２００，Ｃｈｅｍｉｃｏｎ），ｃＴｎＴ（１：１５０ Ｒ＆Ｄ），ａｎｄ ｓａｒｃｏｍｅｒｉｃ−ａ−ａｃｔｉｎｉｎ（１：２００，Ｓｉｇｍａ）．Ｐｒｅｐａｒａｔｉｏｎｓ ｗｅｒｅ ｉｎｃｕｂａｔｅｄ ｗｉｔｈ ｓｅｃｏｎｄａｒｙ ａｎｔｉｂｏｄｉｅｓ（Ｊａｃｋｓｏｎ）ａｔ １：２００ ｄｉｌｕｔｉｏｎ ｆｏｒ １ｈ．Ｎｕｃｌｅｉ ｗｅｒｅ ｃｏｕｎｔｅｒｓｔａｉｎｅｄ ｗｉｔｈ ＤＡＰＩ（１：５００，Ｓｉｇｍａ）．Ｐｒｅｐａｒａｔｉｏｎｓ ｗｅｒｅ ｅｘａｍｉｎｅｄ ｕｓｉｎｇ ａ ｌａｓｅｒ ｓｃａｎｎｉｎｇ ｃｏｎｆｏｃａｌ ｍｉｃｒｏｓｃｏｐｅ（Ｚｅｉｓｓ ＬＳＭ−５１０−ＰＡＳＣＡＬ）．
Ａｓ ｆｏｒ ｔｈｅ ｒｅｓｕｌｔｓ ｏｆ ｔｈｅ ｉｍｍｕｎｏｓｔａｉｎｉｎｇ ａｎａｌｙｓｅｓ ｏｆ ｈｕｍａｎ ｃａｒｄｉｏｍｙｏｃｙｔｅｓ，ｔｈｅ ｈｕｍａｎ ｃａｒｄｉｏｍｙｏｃｙｔｅｓ ｉｎ ｔｈｅ ａｂｏｖｅ−ｍｅｎｔｉｏｎｅｄ ｐｏｓｉｔｉｏｎ ｗｅｒｅ ｓｔａｉｎｅｄ ｐｏｓｉｔｉｖｅｌｙ ｆｏｒ ｓａｒｃｏｍｅｒｉｃ−ａ−ａｃｔｉｎｉｎ（Ａ，Ｄ），ｃＴｎＩ（Ｂ，Ｅ）ａｎｄ ｃＴｎＴ（Ｃ，Ｆ）．Ｎｕｃｌｅｉ ｗｅｒｅ ｃｏｕｎｔｅｒｓｔａｉｎｅｄ ｗｉｔｈ ＤＡＰＩ（ｂｌｕｅ）．Ｔｈｅｓｅ ｄａｔａ ｗｅｒｅ ｓｈｏｗｎ ａｓ Ｆｉｇｕｒｅ ３．
図３に示すように凍結前のヒト心筋細胞マーカータンパクの発現は、本発明のＳ．Ｃ．法による凍結−融解後でも安定的に維持され、生体内に細胞移植後も心筋細胞として正常に機能していることが確認された。
すなわち、本発明による過冷却装置を用いたＳ．Ｃ法によってはじめて、凍結−融解後に細胞移植されたヒトｉＰＳ細胞由来の心筋細胞にはｃＴｎＴ（心筋トロポニンＴ）といった機能性を持つ心筋であることを示すマーカータンパクが存在することを確認できた。In the following examples, a new S.P. C. An example for obtaining human cardiomyocytes preferable for regenerative medicine by the method will be described.
(1) Preparation of cardiomyocytes derived from human iPS cells Human iPS cells were prepared from 4Pg / mLbFGF-containing MEF-CM using a cell line established from patient-derived liver progenitor cells (ChiPs cell lines) (US2012 / 0009601). Incubated on a culture dish coated with matrigel.
When the human iPS cells are differentiated into human cardiomyocytes (Nat Biotechnol vol. 25, P. 1015-1024, 2007), the human iPS cells are transformed into RPMI 1640 containing B27 (containing 100 ng / mL human recombinant activin A). The medium was replaced and cultured for 24 hours, and then the medium was replaced with B27-containing RPMI1640 (containing 10 ng / mL human BMP4) and cultured for 4 days. Thereafter, the cells were maintained with B27-containing RPMI1640 containing no cytokine while the medium was changed every other day.
Passage 2 human iPS cell-derived cardiomyocytes were suspended in 2 mL of freezing medium Me2SO (70% IMDM-20% FBS-10% DMSO), transferred to a 20 mL vial, the vial was brought to 4 ° C., and then Cool at a rate of 1.0 ° C./min to −5 ° C. using a commercially available supercooling device and maintain in a supercooled state for 20 minutes. Thereafter, the cells are immediately transferred to a commercial program freezer, cooled to −40.0 ° C. at a rate of 1.0 ° C./min, and further −80.0 ° C. at a rate of 10.0 ° C./min. Allow to cool. Furthermore, immediately after that, the cells were cooled and maintained in liquid nitrogen to -196.0 ° C.
When thawing the cells, the vial was left at room temperature for 2 minutes and immersed in warm water at 25 degrees for 2 minutes. Thereafter, the medium was treated for 5 minutes each with repeated centrifugation and recovery in the order of a medium containing 10% Me2SO, a medium containing 5% Me2SO, a medium containing 2.5% Me2SO, and a medium not containing Me2SO.
(2) Measurement of expression level of human cardiomyocyte marker gene by RT-PCR As a sample cell, the human iPS cell-derived cardiomyocyte obtained in (1) is used. C. Human PS cell-derived cardiomyocytes (control group or Pre-SC) before freeze-thawing by the method, S. C. Human PS cell-derived cardiomyocytes (Post-SC) after freeze-thawing by the method, C.I. S. C. Human iPS cell-derived cardiomyocytes (Post-CSC) after freeze-thawing by the method were prepared.
RT-PCT was performed on RNA prepared from these cells. The primer sequences used are shown below.
As shown in FIG. 1, the expression of the human cardiomyocyte marker gene before freezing is shown in S. of the present invention. C. It remained stable even after freeze-thaw by the method. On the other hand, C.I. S. C. In cardiomyocytes derived from human iPS cells frozen and thawed by the method, expression of these marker genes was hardly observed. This is because C.I. S. C. This is evidence that human iPS cell-derived cardiomyocytes are disrupted at the gene level (DNA disruption or inter-gene regulation disruption) in human iPS cell-derived cardiomyocytes by the method. However, S. C. In the freeze-thaw of cardiomyocytes derived from human iPS cells by the method, it was shown that the expression of the human cardiomyocyte marker gene is stable at the same high level as compared to before freezing. That is, C. by the prior art by Yamamoto et al. (Ann Thorac Cardiovas Surg (2010); 16: 105-112.). S. In the method C, it was impossible to avoid the destruction of cardiomyocytes derived from human iPS cells at the gene level. However, S. using the supercooling device according to the present invention. It was proved only by the C method that cardiomyocytes derived from human iPS cells can be stably stored without gene disruption due to freezing and thawing.
(3) Measurement of proliferation rate of human iPS cell-derived cardiomyocytes The human iPS cell-derived cardiomyocytes prepared above were cultured in B27-containing RPMI1640, and 0, 3, 5, 7, 9, 11, 13, 15 On the day, the number of cells was counted with an electronic cell counter (Z1 Cell and Particle Counter; Beckman Coulter Inc.) to examine the growth rate. As control cells, cardiomyocytes derived from human iPS cells that were normally maintained by a culture method were used. As shown in FIG. C. The cells obtained by the method are C.I. S. C. The growth rate was much higher than the cells obtained by the method. The cell viability on the 15th day was calculated as C.I. S. C. The cells obtained by the method were 30% S. C. The cells obtained by the method were 90%. From this, the effect of freezing and thawing is described in C. by Yamamoto et al. (Ann Thorac Cardiovas Surg (2010); 16: 105-112.). S. C. S. C. You can see that the law is much smaller.
That is, S. using the supercooling device according to the present invention. Only by the C method, it was proved that cardiomyocytes derived from human iPS cells can be stably cultured and stored in large quantities without gene disruption.
(4) Expression measurement of human cardiomyocyte marker protein S. cerevisiae using the supercooling device of the present invention. C. Transplanted human iPS cell-derived cardiomyocytes after freezing and thawing by the method together with coronary artery bypass surgery in patients with severe heart failure, and after 6 months, examine myocardial cells from the area where myocardial necrosis occurred before treatment Collected for purpose. The degree of expression of human cardiomyocyte marker protein in patient-derived cardiomyocytes at that time was examined by immunostaining. The method is as shown below.
Beating human cardiomyocytes derived by myocardiac biopsy were fixed with 4% -paraformaldehyde, permeabilized with 1% -Triton-X-100 (Sigma), blocked with 5% -horse serum, and incubated overnight at 48 ℃ with primary antibodies targeting: cardiac- troponin-I (cTnI, 1: 200, Chemicon), cTnT (1: 150 R & D), and sarcomeric-a-actinin (1: 200, Sigma). Preparations in incubation with secondary activities (Jackson) at 1: 200 dilution for 1h. Nuclei wear counterstained with DAPI (1: 500, Sigma). Preparations were used using a laser scanning confocal microscope (Zeiss LSM-510-PASCAL).
As for the results of the immunostaining analyses of human cardiomyocytes, the human cardiomyocytes in the above-mentioned position were stained positively for sarcomeric-a-actinin (A, D), cTnI (B, E) and cTnT (C, F). Nuclei wear counterstained with DAPI (blue). These data we have as Figure 2.
As shown in FIG. 3, the expression of the human cardiomyocyte marker protein before freezing is shown in S. of the present invention. C. It was confirmed that the cells were stably maintained even after freezing and thawing by the method, and functioned normally as cardiomyocytes even after cell transplantation in vivo.
That is, S. using the supercooling device according to the present invention. For the first time, it was confirmed by the method C that a marker protein indicating a functional myocardium such as cTnT (myocardial troponin T) was present in cardiomyocytes derived from human iPS cells transplanted after freeze-thawing.
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| WO2017010544A1 (en) * | 2015-07-15 | 2017-01-19 | テルモ株式会社 | Cryopreservation method for myocardial cells derived from pluripotent stem cells or from mesenchymal stem cells derived from adipose tissue or bone marrow |
| WO2017159862A1 (en) * | 2016-03-18 | 2017-09-21 | 国立大学法人京都大学 | Freezing method for aggregates of pluripotent stem cell-derived myocardial cells |
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| WO2017010544A1 (en) * | 2015-07-15 | 2017-01-19 | テルモ株式会社 | Cryopreservation method for myocardial cells derived from pluripotent stem cells or from mesenchymal stem cells derived from adipose tissue or bone marrow |
| JPWO2017010544A1 (en) * | 2015-07-15 | 2018-05-24 | テルモ株式会社 | Method for cryopreserving cardiomyocytes derived from pluripotent stem cells or mesenchymal stem cells derived from adipose tissue or bone marrow |
| WO2017159862A1 (en) * | 2016-03-18 | 2017-09-21 | 国立大学法人京都大学 | Freezing method for aggregates of pluripotent stem cell-derived myocardial cells |
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| KR20190059850A (en) * | 2017-11-23 | 2019-05-31 | 한국식품연구원 | Method for improving viability and storage stability by supercooling cold adaptation of Weissella cibaria WiKim28 |
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