JP7647047B2 - Low tissue factor activity cells and method for producing same - Google Patents

Description

Translated fromJapanese

本発明は、組織因子活性の低い細胞、およびその製法に関する。The present invention relates to cells with low tissue factor activity and a method for producing the same.

細胞治療は自身の細胞または他人の細胞を用いて疾患を治療する治療法であり、例えば、移植片対宿主疾患（ＧＶＨＤ）や心筋梗塞、脊髄損傷など、これまでに治療が困難と言われてきた疾患に対する新しい治療法として注目されている。細胞治療に用いる細胞は、シャーレなどの培養基材を使用してインビトロな環境で培養後、前記培養細胞を細胞懸濁液として回収し、生理食塩水などで濃度調整して製剤とし、患者に静脈注射する。一方、高濃度の細胞懸濁液を血管に投与するため、投与後の血栓生成が懸念されている。これに対し、特許文献１では、間葉系幹細胞を無血清培地で培養することで従来の培養条件下で得られる間葉系幹細胞に比べて間葉系幹細胞の組織因子（ＴＦ）の発現を６０％以下に低減できることが開示されている。しかしながら、間葉系幹細胞の組織因子（ＴＦ）の発現の抑制効果が十分とは言えず、さらなる間葉系幹細胞の組織因子（ＴＦ）の発現が低い細胞が求められる。Cell therapy is a method of treating a disease using one's own cells or cells from another person, and has attracted attention as a new treatment for diseases that have been considered difficult to treat, such as graft-versus-host disease (GVHD), myocardial infarction, and spinal cord injury. The cells used in cell therapy are cultured in an in vitro environment using a culture substrate such as a petri dish, and the cultured cells are collected as a cell suspension, and the concentration is adjusted with physiological saline or the like to prepare a formulation, which is then intravenously injected into the patient. On the other hand, since a highly concentrated cell suspension is administered into the blood vessel, there is a concern about the formation of thrombus after administration. In response to this, Patent Document 1 discloses that by culturing mesenchymal stem cells in a serum-free medium, the expression of tissue factor (TF) in mesenchymal stem cells can be reduced to 60% or less compared to mesenchymal stem cells obtained under conventional culture conditions. However, the effect of suppressing the expression of tissue factor (TF) in mesenchymal stem cells is not sufficient, and cells with a lower expression of tissue factor (TF) in mesenchymal stem cells are required.

ＷＯ２０１８／１５９４３２号WO2018/159432

本特許の目的は、ＴＦ活性が低い培養細胞、およびその製法を提供することにある。The purpose of this patent is to provide cultured cells with low TF activity and a method for producing the same.

本発明者らは、以上の点を鑑み、鋭意研究を重ねた結果、下限臨界溶解温度（ＬＣＳＴ）を示す培養器材上で培養し、前記培養器材をＬＣＳＴ以下に冷却して剥離・回収した細胞は組織因子（ＴＦ）活性が抑制されることを見出し、本発明を完成した。In view of the above, the inventors conducted extensive research and discovered that tissue factor (TF) activity was suppressed in cells cultured on a culture vessel exhibiting a lower critical solution temperature (LCST), and then the culture vessel was cooled to below the LCST, after which the cells were detached and collected, and thus completing the present invention.

すなわち本発明は以下の態様を含包する。
＜１＞インビトロで培養後、冷却処理によって培養器材から剥離された培養細胞であって、インビトロで培養後、タンパク質分解酵素を使用して剥離された培養細胞よりも組織因子（ＴＦ）活性の値が４０％以下であることを特徴とする細胞。
＜２＞前記細胞が、間葉系幹細胞であることを特徴とする＜１＞に記載の細胞。
＜３＞前記間葉系幹細胞が、ヒト骨髄由来であることを特徴とする＜２＞に記載の細胞。
＜４＞インビトロで培養後、冷却処理によって培養器材から剥離された培養細胞であって、インビトロで培養後、タンパク質分解酵素を使用して剥離された培養細胞よりもＴＦ活性の値が４０％以下であることを特徴とする細胞の生産方法。
＜５＞＜１＞～＜３＞記載のいずれか一項の細胞を、下限臨界溶解温度（ＬＣＳＴ）を示す培養器材上で培養後、冷却処理によって回収して得ることを特徴とする細胞回収方法。
＜６＞＜５＞記載の下限臨界溶解温度（ＬＣＳＴ）を示す培養器材が、温度応答性ブロック共重合体を支持材に被覆形成してなることを特徴とする細胞回収方法。 That is, the present invention includes the following aspects.
<1> A cultured cell that has been cultured in vitro and then detached from a culture vessel by cooling treatment, the cell being characterized in that the tissue factor (TF) activity is 40% or less than that of a cultured cell that has been cultured in vitro and then detached using a proteolytic enzyme.
<2> The cell described in <1>, characterized in that the cell is a mesenchymal stem cell.
<3> The cells described in <2>, characterized in that the mesenchymal stem cells are derived from human bone marrow.
<4> A method for producing cells, characterized in that the cultured cells are detached from a culture vessel by cooling treatment after in vitro culture, and have a TF activity value of 40% or less compared to cultured cells detached using a proteolytic enzyme after in vitro culture.
<5> A cell recovery method, comprising culturing the cells according to any one of <1> to <3> on a culture vessel exhibiting a lower critical solution temperature (LCST) and then recovering the cells by a cooling treatment.
<6> A cell recovery method, characterized in that the cultureware exhibiting the lower critical solution temperature (LCST) described in <5> is formed by coating a temperature-responsive block copolymer onto a support material.

本発明の構成を採用することにより、タンパク質分解酵素を使用した場合と同等の剥離時間にて、同酵素を使用して剥離された培養細胞よりも組織因子（ＴＦ）活性の値が低い培養細胞を得ることが可能となった。したがって、本発明の構成を採用して得られた培養細胞を患者に静脈注射する際、投与後の患者の血栓生成リスクを低下させることが可能となった。By adopting the configuration of the present invention, it is now possible to obtain cultured cells with lower tissue factor (TF) activity than cultured cells detached using a proteolytic enzyme, in the same detachment time as when the same enzyme is used. Therefore, when the cultured cells obtained by adopting the configuration of the present invention are intravenously injected into a patient, it is possible to reduce the risk of thrombus formation in the patient after administration.

以下、本発明を実施するための形態について詳細に説明するが、本発明を以下の内容に限定する趣旨ではない。本発明は、その趣旨の範囲内で適宜に変形して実施できる。The following describes in detail the form for carrying out the present invention, but it is not intended that the present invention be limited to the following content. The present invention can be carried out with appropriate modifications within the scope of its spirit.

本発明で用いる細胞としては、インビトロで培養器材に接着培養可能なものであれば特に限定されるものではない。例えばチャイニーズハムスター卵巣由来ＣＨＯ細胞やマウス結合組織Ｌ９２９細胞、ヒト胎児肺由来正常二倍体線維芽細胞（ＴＩＧ－３細胞）、ヒト胎児腎臓由来細胞（ＨＥＫ２９３細胞）やヒト子宮頸癌由来ＨｅＬａ細胞等の種々の培養細胞株に加え、例えば生体内の各組織、臓器を構成する上皮細胞や内皮細胞、収縮性を示す骨格筋細胞、平滑筋細胞、心筋細胞、神経系を構成するニューロン細胞、グリア細胞、繊維芽細胞、生体の代謝に関与する肝実質細胞、肝非実質細胞や脂肪細胞、分化能を有する細胞として、誘導多能性幹（ｉＰＳ）細胞、胚性幹（ＥＳ）細胞、胚性生殖（ＥＧ）細胞、胚性癌（ＥＣ）細胞、間葉系幹細胞、肝幹細胞、膵幹細胞、皮膚幹細胞、筋幹細胞、生殖幹細胞等の各種幹細胞、又は各組織の前駆細胞、さらにはそれらから分化誘導した細胞等が挙げられ、特に間葉系幹細胞は好適に用いられる。The cells used in the present invention are not particularly limited as long as they can be cultured in vitro by adhesion to culture equipment. For example, in addition to various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human fetal lung-derived normal diploid fibroblasts (TIG-3 cells), human fetal kidney-derived cells (HEK293 cells), and human cervical cancer-derived HeLa cells, for example, epithelial cells and endothelial cells that constitute various tissues and organs in the body, skeletal muscle cells that exhibit contractility, smooth muscle cells, cardiac muscle cells, neuron cells that constitute the nervous system, glial cells, fibroblasts, hepatic parenchymal cells involved in the metabolism of the body, non-parenchymal liver cells, and adipocytes, as well as cells with differentiation potential, such as induced pluripotent stem (iPS) cells, embryonic stem (ES) cells, embryonic germ (EG) cells, embryonic carcinoma (EC) cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells, skin stem cells, muscle stem cells, and germline stem cells, or precursor cells of various tissues, and further cells induced to differentiate from them, etc., are particularly preferably used.

間葉系幹細胞とは、軟骨細胞、骨芽細胞、脂肪細胞等の間葉系の細胞全て又はいくつかへの分化が可能な幹細胞並びにその前駆細胞の集団を意味する。間葉系幹細胞の由来として特に限定は無いが、骨髄、脂肪、歯髄、臍帯血、胎盤、滑膜などの組織由来やＥＳ細胞、ｉＰＳ細胞などの多能性幹細胞由来が挙げられ、特に骨髄由来が好ましい。また、間葉系幹細胞の由来種にも特に限定は無いが、ヒト、動物、植物が挙げられ、特にヒト由来が好ましい。Mesenchymal stem cells refer to a population of stem cells and their precursor cells that can differentiate into all or some of the mesenchymal cells, such as chondrocytes, osteoblasts, and adipocytes. There is no particular limit to the origin of mesenchymal stem cells, but examples include tissue origins such as bone marrow, fat, dental pulp, umbilical cord blood, placenta, and synovium, and pluripotent stem cells such as ES cells and iPS cells, with bone marrow origin being particularly preferred. There is also no particular limit to the species of origin of mesenchymal stem cells, but examples include humans, animals, and plants, with humans being particularly preferred.

組織因子（Ｔｉｓｓｕ ｆａｃｔｏｒ，ＴＦ）とは、ＶＩＩａ因子と複合体を形成する膜関連糖タンパク質である。またＶＩＩａ因子／ＴＦとの複合体は、血液凝固プロセスを開始させる働きがある。ＶＩＩａ因子／ＴＦ複合体は、一旦形成されると、凝血カスケードの外因系経路及び内因系経路に関与する一連の特定の酵素を活性化し、最終的にトロンビンの生成、フィブリンの生成、血小板の活性化、及び凝固の形成が起こり、血栓形成に繋がることが知られている。Tissue factor (TF) is a membrane-associated glycoprotein that forms a complex with factor VIIa. The factor VIIa/TF complex is known to initiate the blood clotting process. Once formed, the factor VIIa/TF complex activates a series of specific enzymes involved in the extrinsic and intrinsic pathways of the clotting cascade, ultimately resulting in the generation of thrombin, fibrin production, platelet activation, and clot formation, leading to thrombus formation.

なお、組織因子（ＴＦ）活性（「ＴＦ活性」ともいう）とは、血液凝固カスケードの開始因子であるＶＩＩａ因子活性化反応を指し、この活性化反応が低いほど血栓生成が抑えられることが知られている。Tissue factor (TF) activity (also called "TF activity") refers to the activation reaction of factor VIIa, which is the initiation factor of the blood coagulation cascade, and it is known that the lower this activation reaction is, the more suppressed thrombus formation is.

本発明の細胞は、ＴＦ活性の値が従来の培養細胞の４０％以下であればよく、好ましくは３５％以下、さらに好ましくは３０％以下から０％である。The cells of the present invention should have a TF activity level of 40% or less of that of conventional cultured cells, preferably 35% or less, and more preferably 30% or less to 0%.

ＴＦ活性の測定方法としては、当技術分野において一般に用いられる方法によって行われてよいが、操作の簡便さ、定量性、検出感度等の観点からＶＩＩａ因子／ＴＦ複合体によるＸ因子のＸａ因子への活性化作用をＸａ因子の発色基質を用いて直接的にＴＦ活性を測定するＴＦ活性比色定量法等が好ましい。また、ＴＦ活性を測定するための市販の測定キットを用いてもよい。市販の測定キットは、ＴＦ活性比色定量法の場合では、例えばＨｕｍａｎ Ｔｉｓｓｕｅ Ｆａｃｔｏｒ Ｃｈｒｏｍｏｇｅｎｉｃ Ａｃｔｉｖｉｔｙ Ａｓｓａｙ Ｋｉｔ（Ａｓｓａｙｐｒｏ，ＬＬＣ製）が挙げられる。ＴＦ活性比色定量法の場合、ＴＦ活性は、例えば、ＴＦ活性が既知のサンプルを測定し得られた検量線を用いることで算出できる。The TF activity may be measured by a method generally used in the art. From the viewpoints of ease of operation, quantitativeness, detection sensitivity, etc., a TF activity colorimetric assay in which the activation of factor X by factor Xa by the factor VIIa/TF complex is directly measured using a chromogenic substrate for factor Xa to measure TF activity is preferable. A commercially available assay kit for measuring TF activity may also be used. In the case of the TF activity colorimetric assay, for example, the Human Tissue Factor Chromogenic Activity Assay Kit (manufactured by Assaypro, LLC) can be used as a commercially available assay kit. In the case of the TF activity colorimetric assay, the TF activity can be calculated, for example, by measuring a sample with known TF activity and using a calibration curve obtained.

タンパク質分解酵素とは、トリプシン、アキュターゼ、コラゲナーゼ、天然プロテアーゼ、キモトリプシン、エラスターゼ、パパイン、プロナーゼ、もしくはそれらの組み換え体などの酵素を指す。また、タンパク質分解酵素を使用して剥離された培養細胞とは、これらのタンパク質分解酵素を用い、当技術分野において一般に用いられる方法によって剥離・回収された培養細胞である。Proteolytic enzymes refer to enzymes such as trypsin, accutase, collagenase, natural proteases, chymotrypsin, elastase, papain, pronase, or recombinant forms thereof. Cultured cells detached using proteolytic enzymes are cultured cells that have been detached and collected using methods commonly used in the art using these proteolytic enzymes.

培養器材の形状は特に限定は無く、ディッシュ、フラスコ、マイクロウエルプレート、培養バッグ、チューブ、トレイ、培養槽などが挙げられる。これらの器材の材質も、特に限定されず、ガラスやポリプロピレン、ポリスチレン、ステンレスなどやそれらの組み合わせが挙げられる。The shape of the culture equipment is not particularly limited, and examples include dishes, flasks, microwell plates, culture bags, tubes, trays, culture tanks, etc. The materials of these equipment are also not particularly limited, and examples include glass, polypropylene, polystyrene, stainless steel, etc., and combinations thereof.

培養器材表面にはさらに細胞外マトリックスを被覆してもよい。細胞外マトリックスの種類は特に限定は無く、例えば、コラーゲン、アテロコラーゲン、ヒアルロン酸、エラスチン、プロテオグリカン、グルコサミノグリカン、フィブロネクチン、ラミニン、ビトロネクチン、ゼラチン、又はラミニン、コラーゲンＩＶ、ヘパラン硫酸プロテオグリカン、エンタクチン／ニドジェン１，２等を主成分として含有するマトリゲルを用いてもよく、これらを１種類のみでもよく、２種類以上を組み合わせてあってもよい。また、これら細胞外マトリックスのセグメントであってもよい。The surface of the culture vessel may be further coated with an extracellular matrix. There are no particular limitations on the type of extracellular matrix, and for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glycosaminoglycan, fibronectin, laminin, vitronectin, gelatin, or matrigel containing laminin, collagen IV, heparan sulfate proteoglycan, entactin/nidogen 1, 2, etc. as the main component may be used, and one of these may be used alone, or two or more types may be combined. Also, it may be a segment of these extracellular matrices.

下限臨界溶解温度（ＬＣＳＴ；Ｌｏｗｅｒ Ｃｒｉｔｉｃａｌ Ｓｏｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ）とは、この温度よりも低い温度では高分子が水に溶解して透明の溶液になるが、この温度よりも高い温度では不溶化して白濁するか沈殿が生じ、相分離する温度であり、本願明細書ではＬＣＳＴを示すことを、温度応答性を有するということがある。ＬＣＳＴは２０℃～４０℃の範囲にあることが好ましく、２５℃～３５℃の範囲にあることがさらに好ましい。ＬＣＳＴを示す繰返し単位とその水に対するＬＣＳＴは、例えば、Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２２℃）、Ｎ－テトラヒドロフルフリルアクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－エトキシエチルアクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ，Ｎ－ジエチルアクリルアミド（ＬＣＳＴ＝３２℃）、Ｎ－ｎ－プロピルメタクリルアミド（ＬＣＳＴ＝２８℃）、Ｎ－テトラヒドロフルフリルメタクリルアミド（ＬＣＳＴ＝３５℃）、Ｎ－メチル－Ｎ－イソプロピルアクリルアミド（ＬＣＳＴ＝２３℃）、又はＮ－メチル－Ｎ－ｎ－プロピルアクリルアミド（ＬＣＳＴ＝２０℃）等が例示できる。ＬＣＳＴは水溶液の濃度で発現する温度が前後するが、Ｎ－イソプロピルアクリルアミドはＬＣＳＴ発現の濃度依存性が低いため好ましい。本発明におけるＬＣＳＴを示す培養器材に用いる繰返し単位は、１種類のみでもよく、２種類以上を組み合わせてあってもよい。また温度応答性を有するのであれば、ＬＣＳＴを示す繰返し単位の他に、異なる繰返し単位を含んでも良い。異なる繰返し単位を含む共重合体のＬＣＳＴは単独の繰返し単位からなる重合体のＬＣＳＴと異なる場合があり、共重合体のＬＣＳＴは実測で計測する。The lower critical solution temperature (LCST) is the temperature below which a polymer dissolves in water to form a transparent solution, but above which it becomes insoluble and cloudy, or precipitates and undergoes phase separation. In this specification, the presence of an LCST is sometimes referred to as having temperature responsiveness. The LCST is preferably in the range of 20°C to 40°C, and more preferably in the range of 25°C to 35°C. Examples of repeating units exhibiting LCST and their LCSTs with respect to water include N-isopropylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 22°C), N-tetrahydrofurfurylacrylamide (LCST = 28°C), N-ethoxyethylacrylamide (LCST = 35°C), N,N-diethylacrylamide (LCST = 32°C), N-n-propylmethacrylamide (LCST = 28°C), N-tetrahydrofurfurylmethacrylamide (LCST = 35°C), N-methyl-N-isopropylacrylamide (LCST = 23°C), and N-methyl-N-n-propylacrylamide (LCST = 20°C). The temperature at which the LCST appears varies depending on the concentration of the aqueous solution, but N-isopropylacrylamide is preferred because it has low concentration dependency of LCST appearance. The repeating units used in the cultureware exhibiting LCST in the present invention may be one type only, or two or more types may be combined. In addition, if the copolymer has temperature responsiveness, it may contain a different repeating unit in addition to the repeating unit that exhibits the LCST. The LCST of a copolymer containing different repeating units may differ from the LCST of a polymer consisting of a single repeating unit, and the LCST of the copolymer is measured by actual measurement.

ＬＣＳＴを示す培養器材は、その製造方法に特に限定は無く、例えばＬＣＳＴを示す繰返し単位を電子線照射でＬＣＳＴを示す繰返し単位を重合して器材表面に被覆する方法、ＬＣＳＴを示す繰返し単位を含有する重合体を溶媒に溶解させた表面処理剤を器材に塗布して、膜を被覆する方法が挙げられる。前記重合体は特に限定は無いがその合成方法としては、株式会社エヌ・ティー・エス発行、“ラジカル重合ハンドブック”、ｐ．１６１～２２５（２０１０）に記載のリビングラジカル重合技術を用いて、合成する方法を用いることができる。培養器材を用いて培養・増殖した細胞は、一般的にトリプシンのようなタンパク質分解酵素により処理することで容器表面から剥離・回収する必要があるが、ＬＣＳＴを示す培養器材を用いることで周囲環境の温度降下による温度応答性重合体のゾル転移で器材表面の接着力を弱めて、タンパク質分解酵素を使わずに細胞を剥離させ、回収することができる。There is no particular limitation on the method of producing a cultureware exhibiting LCST. Examples of the method include a method of polymerizing a repeating unit exhibiting LCST by irradiating it with an electron beam to coat the surface of the vessel, and a method of coating the vessel with a membrane by applying a surface treatment agent in which a polymer containing a repeating unit exhibiting LCST is dissolved in a solvent. The polymer is not particularly limited, but the synthesis method can be a method using the living radical polymerization technique described in "Radical Polymerization Handbook" pp. 161-225 (2010) published by NTS Co., Ltd. Cells cultured and grown using cultureware generally need to be treated with a protease such as trypsin to be peeled off and collected from the vessel surface. However, by using a cultureware exhibiting LCST, the adhesive force of the vessel surface can be weakened by the sol transition of the temperature-responsive polymer due to the temperature drop in the surrounding environment, and the cells can be peeled off and collected without using a protease.

ＬＣＳＴを示す繰返し単位を含有する重合体の膜を用いる場合は、膜を構成する重合体の成分として、ＬＣＳＴを示す繰返し単位の他に膜を器材に固定するための繰返し単位を含むことが好ましい。例えばスチレンやその誘導体、２－メトキシエチルアクリレート、ｎ－プロピルアクリレート、ｎ－プロピルメタクリレート、ｎ－ブチルアクリレート、ｎ－ブチルメタクリレート等を例示でき、２－メトキシエチルアクリレートの繰返し単位とｎ－ブチルアクリレートの繰返し単位とＮ－イソプロピルアクリルアミドの繰返し単位とを含んでなるブロック共重合体が特に好ましい。When using a polymer membrane containing a repeating unit exhibiting LCST, it is preferable that the polymer components constituting the membrane contain a repeating unit for fixing the membrane to the substrate in addition to the repeating unit exhibiting LCST. Examples include styrene and its derivatives, 2-methoxyethyl acrylate, n-propyl acrylate, n-propyl methacrylate, n-butyl acrylate, and n-butyl methacrylate, and block copolymers containing repeating units of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide are particularly preferred.

本発明の培養で使用される培地の組成については、細胞が接着・増殖すれば特に限定は無く、基礎培地と血清からなり、抗生物質が含まれていても良い。基礎培地の種類は特に限定はなく、例えば、ＭＥＭ、αＭＥＭ、ＤＭＥＭ、ＥＭＥＭ、ＧＭＥＭ、ＤＭＥＭ／Ｈａｍ’ｓＦ－１２、Ｈａｍ’ｓＦ－１２、Ｈａｍ’ｓＦ－１０、Ｍｅｄｉｕｍ１９９、ＲＰＭＩ１６４０などを用いることができる。血清の種類は特に限定はなく、例えば、牛胎児血清（Ｆｅｔａｌ Ｂｏｖｉｎｅ Ｓｅｒｕｍ：ＦＢＳ）、児牛血清、成牛血清、ウマ血清、ヒツジ血清、ヤギ血清、ブタ血清、ニワトリ血清、ウサギ血清、ヒト血清が使用されるが、入手の容易さから一般的にＦＢＳがよく用いられる。培地中の血清濃度は特に限定はない。費用体効果から一般的には２０ｖｏｌ％以下の濃度で用いられることが多いが、２０ｖｏｌ％超の濃度であっても良い。また、未処理又は未精製の血清をいずれも含まず、精製された血液由来成分又は動物組織由来成分（増殖因子など）を含有する無血清培地であってもよい。The composition of the medium used in the culture of the present invention is not particularly limited as long as the cells adhere and grow, and is composed of a basal medium and serum, and may contain antibiotics. The type of basal medium is not particularly limited, and for example, MEM, αMEM, DMEM, EMEM, GMEM, DMEM/Ham's F-12, Ham's F-12, Ham's F-10, Medium 199, RPMI 1640, etc. can be used. The type of serum is not particularly limited, and for example, fetal bovine serum (FBS), calf serum, adult bovine serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, and human serum are used, but FBS is generally used because of its ease of availability. The serum concentration in the medium is not particularly limited. From the perspective of cost-effectiveness, it is generally used at a concentration of 20 vol% or less, but it may be at a concentration of more than 20 vol%. Alternatively, the medium may be a serum-free medium that does not contain untreated or unpurified serum, but contains purified blood-derived components or animal tissue-derived components (such as growth factors).

細胞培養は、培養器材の表面に被覆された温度応答性重合体のＬＣＳＴよりも高い温度で行われるが、ヒト由来細胞を用いる場合は、高い培養効率を得ることを目的に体温付近で行うことが好ましく、３５～３９℃の温度範囲で行うことがより好ましく、３６～３８℃の温度範囲で行うことがさらに好ましい。Cell culture is performed at a temperature higher than the LCST of the temperature-responsive polymer that coats the surface of the cultureware. When using human-derived cells, it is preferable to perform the culture at temperatures close to body temperature in order to obtain high culture efficiency, more preferably in the temperature range of 35-39°C, and even more preferably in the temperature range of 36-38°C.

細胞の培養密度は、細胞が接着・増殖すれば特に限定は無いが、間葉系幹細胞の場合では、例えば１．０×１０^１～１．０×１０^５ｃｅｌｌｓ／ｃｍ^２が好ましく、１．０×１０^２～１．０×１０^４ｃｅｌｌｓ／ｃｍ^２がより好ましい。その他の培養条件は特に限定されず、当分野において通常行われる条件下で培養を行ってよい。 The cell culture density is not particularly limited as long as the cells adhere and grow, but in the case of mesenchymal stem cells, for example, 1.0×10¹ to 1.0×10⁵ cells/cm² is preferable, and 1.0×10² to 1.0×10⁴ cells/cm² is more preferable. Other culture conditions are not particularly limited, and culture may be performed under conditions normally used in this field.

ＬＣＳＴを示す培養器材から細胞を剥離・回収する方法は、周囲の温度を培養器材のＬＣＳＴよりも低い温度に変化させた後、細胞の自然剥離を待つ方法、タッピングや振盪による細胞に局所的に衝撃を与える方法、セルスクレーパーを用いる方法、ピペッティングを用いる方法が例示できるが、細胞へのダメージを抑えつつ、回収にかかる時間を短縮するために、ピペッティングを用いる方法が好ましい。Methods for detaching and recovering cells from cultureware that exhibits an LCST include changing the ambient temperature to a temperature lower than the LCST of the cultureware and then waiting for the cells to detach naturally, locally impacting the cells by tapping or shaking, using a cell scraper, and pipetting. However, the pipetting method is preferred in order to reduce the time required for recovery while minimizing damage to the cells.

培養器材の冷却時の温度はＬＣＳＴから１℃以上低温であることが好ましい。冷却によるダメージを低減するために、冷却する際の温度として好ましくは０℃～ＬＣＳＴから１℃低い温度、より好ましくは３℃～ＬＣＳＴから３℃低い温度、さらに好ましくは４℃～ＬＣＳＴから８℃低い温度である。培養器材の温度を降下させる方法（「冷却処理」ともいう）としては冷却した液体に交換することや冷所保管が例示されるが、冷却にかかる時間を短縮するために冷却した液体に交換することが好ましい。冷却した液体に特に限定はなく、培養液やその他の培地溶液、等張液など目的に応じて選択することができる。また、細胞へのダメージを低減するために、冷却時間は、６０分以下が好ましく、３０分以下がさらに好ましく、２０分以下が特に好ましく、１０分以下が最も好ましい。The temperature at which the cultureware is cooled is preferably 1°C or more lower than the LCST. In order to reduce damage caused by cooling, the temperature at which the cultureware is cooled is preferably 0°C to a temperature 1°C lower than the LCST, more preferably 3°C to a temperature 3°C lower than the LCST, and even more preferably 4°C to a temperature 8°C lower than the LCST. Methods for lowering the temperature of the cultureware (also called "cooling treatment") include replacing the cultureware with a cooled liquid or storing it in a cold place, but replacing the cultureware with a cooled liquid is preferable to shorten the time required for cooling. There are no particular limitations on the cooled liquid, and it can be selected according to the purpose, such as culture medium, other medium solution, isotonic solution, etc. In addition, in order to reduce damage to the cells, the cooling time is preferably 60 minutes or less, more preferably 30 minutes or less, particularly preferably 20 minutes or less, and most preferably 10 minutes or less.

以下に本発明の実施例を説明するが、本発明はこれら実施例により何ら制限されるものではない。なお、断りのない限り、試薬は市販品を用いた。
＜ブロック共重合体の組成＞
核磁気共鳴測定装置（日本電子（株）製、商品名：ＪＮＭ－ＥＣＺ４００Ｓ／Ｌ１）を用いたプロトン核磁気共鳴分光（^１Ｈ－ＮＭＲ）スペクトル分析より求めた。
＜ブロック共重合体の分子量、分子量分布＞
重量平均分子量（Ｍｗ）、数平均分子量（Ｍｎ）および分子量分布（Ｍｗ／Ｍｎ）は、ゲル・パーミエーション・クロマトグラフィー（ＧＰＣ）によって測定した。ＧＰＣ装置は東ソー（株）製ＨＬＣ－８３２０ＧＰＣを用い、カラムは東ソー製ＴＳＫｇｅｌＳｕｐｅｒＡＷＭ－Ｈを２本用い、カラム温度を４０℃に設定し、溶離液は１０ｍＭトリフルオロ酢酸ナトリウムを含む１０ｍＭトリフルオロ酢酸ナトリウムを用いて測定した。測定試料は１．０ｍｇ／ｍＬで調製して測定した。分子量の検量線は、分子量既知のポリメタクリル酸メチル（ポリマーラボラトリーズ製）を用いた。
＜ブロック共重合体の合成例＞
２００ｍＬ２口フラスコに２－メトキシエチルアクリレート（ＭＥＡ）０．６５０ｇ（５ｍｍｏｌ）を加え、さらにシアノメチルドデシルトリチオカルボナトを３１．８ｍｇ（１００μｍｏｌ）とアゾビスイソブチロニトリル１．６ｍｇ（１０μｍｏｌ）とｔｅｒｔ－ブチルアルコール１０ｍＬを加え、アルゴンガス置換後、６２℃で２４時間加熱撹拌した。 Examples of the present invention are described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used.
<Composition of block copolymer>
The values were determined by proton nuclear magnetic resonance spectroscopy (¹ H-NMR) spectrum analysis using a nuclear magnetic resonance measurement device (manufactured by JEOL Ltd., product name: JNM-ECZ400S/L1).
<Molecular weight and molecular weight distribution of block copolymer>
Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw/Mn) were measured by gel permeation chromatography (GPC). The GPC device used was a Tosoh Corporation HLC-8320GPC, and two Tosoh Corporation TSKgelSuperAWM-H columns were used. The column temperature was set to 40°C, and the eluent was 10 mM sodium trifluoroacetate containing 10 mM sodium trifluoroacetate. The measurement sample was prepared at 1.0 mg/mL and measured. For the molecular weight calibration curve, polymethylmethacrylate (Polymer Laboratories) with a known molecular weight was used.
<Synthesis example of block copolymer>
To a 200 mL two-neck flask, 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added, and then 31.8 mg (100 μmol) of cyanomethyl dodecyl trithiocarbonate, 1.6 mg (10 μmol) of azobisisobutyronitrile, and 10 mL of tert-butyl alcohol were added. After replacing with argon gas, the mixture was heated and stirred at 62° C. for 24 hours.

１回目の加熱撹拌後、ｎ－ブチルアクリレート（ＢＡ）３．８４５ｇ（３０ｍｍｏｌ）を加え、さらにアゾビスイソブチロニトリル１．６ｍｇ（１０μｍｏｌ）とｔｅｒｔ－ブチルアルコール５ｍＬを加え、アルゴンガス置換後、６２℃で２４時間加熱撹拌した。After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA) was added, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of tert-butyl alcohol. After replacing with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

２回目の加熱撹拌後、上記にＮ－イソプロピルアクリルアミド（ＩＰＡＡｍ ＬＣＳＴ＝３２℃）７．３５５ｇ（６５ｍｍｏｌ）を加え、さらにアゾビスイソブチロニトリル１．６ｍｇ（１０μｍｏｌ）とｔｅｒｔ－ブチルアルコール８５ｍＬを加え、アルゴンガス置換後、６２℃で２４時間加熱撹拌した。After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm LCST = 32°C) was added to the above, followed by 1.6 mg (10 μmol) of azobisisobutyronitrile and 85 mL of tert-butyl alcohol. After replacing with argon gas, the mixture was heated and stirred at 62°C for 24 hours.

３回目の加熱撹拌後、反応液を水で再沈精製し、減圧乾燥することで黄色固体を得た。得られた黄色固体をクロロホルムに溶解し、分液ロートを用いクロロホルム相を回収した。回収したクロロホルム相をエバポレーターで濃縮し、ヘプタンで再沈精製した。沈殿物をろ過で回収し、減圧乾燥することで、ブロック共重合体ｐｏｌｙ（ＭＥＡ－ＢＡ－ＩＰＡＡｍ）を８．２９５ｇ得た。得られたブロック共重合体の組成はＭＥＡ：ＢＡ：ＩＰＡＡｍ＝５：３０：６５（ｍｏｌ％）、Ｍｎは１１．８×１０^４、Ｍｗ／Ｍｎは１．４５、ＬＣＳＴは２８℃であった。
＜表面処理剤の調製＞
ブロック共重合体９０ｍｇに２－メトキシエタノールを１９．９１０ｇ添加し、撹拌で
全て溶解させ、ブロック共重合体の０．４５ｗｔ％表面処理剤を調製した。
＜温度応答性培養器材の調製＞
ＩＷＡＫＩ組織培養用ディッシュ（φ１０ｃｍ）の中央に表面処理剤を３００μＬ加え、スピンコータ―（ミカサ製、商品名ＭＳ－Ｂ２００）を用いて、回転数３，０００ｒｐｍ、回転時間６０秒の条件でスピンコートすることでブロック共重合体をコートした温度応答性培養器材を調製した。この器材表面のブロック共重合体の被覆量を全反射型フーリエ変換型赤外分光（ＡＴＲ／ＦＴ－ＩＲ）法により測定した所、温度応答性ブロック分として０．５５μｇ／ｃｍ^２であった。 After the third heating and stirring, the reaction solution was purified by reprecipitation with water and dried under reduced pressure to obtain a yellow solid. The obtained yellow solid was dissolved in chloroform, and the chloroform phase was collected using a separatory funnel. The collected chloroform phase was concentrated with an evaporator and purified by reprecipitation with heptane. The precipitate was collected by filtration and dried under reduced pressure to obtain 8.295 g of a block copolymer poly(MEA-BA-IPAAm). The composition of the obtained block copolymer was MEA:BA:IPAAm=5:30:65 (mol%), Mn was 11.8×10⁴ , Mw/Mn was 1.45, and LCST was 28° C.
<Preparation of Surface Treatment Agent>
19.910 g of 2-methoxyethanol was added to 90 mg of the block copolymer and completely dissolved by stirring to prepare a 0.45 wt % surface treatment agent for the block copolymer.
<Preparation of temperature-responsive cultureware>
A temperature-responsive culture vessel coated with a block copolymer was prepared by adding 300 μL of the surface treatment agent to the center of an IWAKI tissue culture dish (φ10 cm) and spin-coating the dish using a spin coater (Mikasa, product name MS-B200) at a rotation speed of 3,000 rpm for a rotation time of 60 seconds. The amount of the block copolymer coated on the vessel surface was measured by total reflection Fourier transform infrared spectroscopy (ATR/FT-IR) and found to be 0.55 μg/^cm2 in terms of the temperature-responsive block.

実施例１
上記温度応答性培養器材に、骨髄由来ヒト間葉系幹細胞（ロンザジャパン（株）製、Ｐｒｏｄｕｃｔ Ｃｏｄｅ：ＰＴ－２５０１、Ｌｏｔ Ｎｕｍｂｅｒ：００００６０３５２５）を１．０×１０^５ｃｅｌｌｓ／ｄｉｓｈ播種し、３７℃、ＣＯ_２濃度５％で培養した。培養液にはウシ胎児血清（コロンビア産）を１０ｖｏｌ％含むダルベッコ・フォークト変法イーグル最小必須培地（１０ｖｏｌ％ ＦＢＳ／ＤＭＥＭ）を用いた。 Example 1
Bone marrow-derived human mesenchymal stem cells (manufactured by Lonza Japan, Product Code: PT-2501, Lot Number: 0000603525) were seeded at 1.0 x¹⁰ cells/dish into the above-mentioned temperature-responsive culture equipment and cultured at 37°C and 5%_CO2 concentration. The culture medium used was Dulbecco-Voigt modified Eagle's minimum essential medium (10 vol% FBS/DMEM) containing 10 vol% fetal bovine serum (Colombia).

７日間培養後、培養液を抜き、新たに４℃に冷却した培養液を加え、室温で１０分間冷却した。１０分後、ピペッターを用いて培養器材の培養面の全面に培養液を当てるようにピペッティングした後、細胞ごと培養液を回収した。回収した培養液を、１６０ｒｃｆ、２５℃、５分の条件で遠心後、上清を除き、ＰＢＳ（－）を５００μＬ加え懸濁した。得られた細胞懸濁液中から１０μＬを細胞数測定用スライド（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ（株）製、商品名：Ｃｏｕｎｔｅｓｓ（登録商標） Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｃｈａｍｂｅｒ Ｓｌｉｄ）に添加し、自動セルカウンター（Ｔｈｅ
ｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ（株）製、商品名：Ｃｏｕｎｔｅｓｓ（登録商標） ＩＩ）を用いて、細胞数を測定した。細胞数測定後、再び１６０ｒｃｆ、２５℃、５分の条件で遠心、上清を除き、１％Ｔｒｉｔｏｎ Ｘ－１００／Ｔｒｉｓ－ＨＣｌ（ｐＨ８．０）を２００μＬ加え懸濁した。氷上で１５分間静置後、１４，０００ｒｃｆ、４℃、２０分の条件で遠心し、タンパク溶液を得た。得られたタンパク溶液の濃度をタンパク濃度測定キット（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ（株）製、商品名：Ｐｉｅｒｃｅ ＢＣＡ Ｐｒｏｔｅｉｎ Ａｓｓａｙ Ｋｉｔ）で測定した。濃度測定後、タンパク溶液のＴＦ活性をヒトＴＦ測定キット（Ａｓｓａｙｐｒｏ，ＬＬＣ製、商品名：Ｈｕｍａｎ Ｔｉｓｓｕｅ Ｆａｃｔｏｒ Ｃｈｒｏｍｏｇｅｎｉｃ Ａｃｔｉｖｉｔｙ Ａｓｓａｙ Ｋｉｔ）を用いて発色基質による波長４０５ｎｍの吸光度変化を測定し、活性が既知のＴＦサンプルから作成した検量線より算出した。その結果、全タンパク質量当たりのＴＦ活性の値は１.４ｐＭ／μｇであり、従来の培養方法で培養した比較例１の３０％の値であった。 After culturing for 7 days, the culture medium was removed, and fresh culture medium cooled to 4°C was added, followed by cooling at room temperature for 10 minutes. After 10 minutes, the culture medium was pipetted using a pipetter so as to apply the culture medium to the entire culture surface of the cultureware, and the culture medium was then recovered together with the cells. The recovered culture medium was centrifuged at 160 rcf, 25°C, and 5 minutes, after which the supernatant was removed and 500 μL of PBS (-) was added to suspend the cells. 10 μL of the obtained cell suspension was added to a slide for measuring cell counts (manufactured by Thermo Fisher Scientific, Inc., product name: Countess (registered trademark) Cell Counting Chamber Slide) and counted using an automatic cell counter (Thermo Fisher Scientific, Inc., product name: Countess (registered trademark) Cell Counting Chamber Slide).
The cell number was measured using Thermo Fisher Scientific Co., Ltd. (trade name: Countess (registered trademark) II). After measuring the cell number, the mixture was centrifuged again at 160 rcf, 25 ° C., and 5 minutes, the supernatant was removed, and 200 μL of 1% Triton X-100/Tris-HCl (pH 8.0) was added and suspended. After standing on ice for 15 minutes, the mixture was centrifuged at 14,000 rcf, 4 ° C., and 20 minutes to obtain a protein solution. The concentration of the obtained protein solution was measured using a protein concentration measurement kit (manufactured by Thermo Fisher Scientific Co., Ltd., trade name: Pierce BCA Protein Assay Kit). After measuring the concentration, the TF activity of the protein solution was measured by measuring the change in absorbance at a wavelength of 405 nm due to a chromogenic substrate using a human TF measurement kit (manufactured by Assaypro, LLC, product name: Human Tissue Factor Chromogenic Activity Assay Kit), and calculated from a calibration curve prepared from TF samples with known activity. As a result, the value of TF activity per total protein amount was 1.4 pM/μg, which was 30% of the value in Comparative Example 1, which was cultured by the conventional culture method.

実施例２
ヒト脂肪由来幹細胞（ロンザジャパン（株）製、Ｐｒｏｄｕｃｔ Ｃｏｄｅ：ＰＴ－５００６）を用いたこと以外は実施例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は２.９ｐＭ／μｇであり、従来の培養方法で培養した比較例２の３９％の値であった。 Example 2
TF activity was measured in the same manner as in Example 1, except that human adipose-derived stem cells (manufactured by Lonza Japan, Product Code: PT-5006) were used. As a result, the TF activity value per total protein amount was 2.9 pM/μg, which was 39% of the value in Comparative Example 2, where culture was performed by the conventional culture method.

実施例３
ヒト胎児肺由来正常二倍体線維芽細胞を用いて５日間培養したこと以外は実施例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は０.５ｐＭ／μｇであり、従来の培養方法で培養した比較例３の３６％の値であった。 Example 3
TF activity was measured in the same manner as in Example 1, except that normal diploid fibroblasts derived from human fetal lung were cultured for 5 days. As a result, the TF activity value per total protein amount was 0.5 pM/μg, which was 36% of the value in Comparative Example 3, which was cultured by the conventional culture method.

実施例４
７日間培養後、培養液を抜き、新たに２５℃の培養液を加え、室温で２０分間冷却したこと以外は実施例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は１.５ｐＭ／μｇであり、従来の培養方法で培養した比較例１の３３％の値であった。 Example 4
After culturing for 7 days, the culture medium was removed, new culture medium at 25° C. was added, and the TF activity was measured in the same manner as in Example 1, except that the culture medium was cooled to room temperature for 20 minutes. As a result, the TF activity value per total protein amount was 1.5 pM/μg, which was 33% of the value in Comparative Example 1, where culture was performed by the conventional culture method.

比較例１
ＩＷＡＫＩ組織培養用ディッシュ（φ１０ｃｍ）から間葉系幹細胞を剥離・回収する際に４℃に冷却した培養液を加える代わりに、ＰＢＳで洗浄後、タンパク質分解酵素（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ（株）製、商品名：Ｔｒｙｐｓｉｎ－ＥＤＴＡ （０．２５％））を１ｍＬ加え、３７℃で１０分間静置し、培地を添加後、細胞を回収した以外は実施例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は４.６ｐＭ／μｇであった。 Comparative Example 1
When detaching and recovering mesenchymal stem cells from an IWAKI tissue culture dish (φ10 cm), instead of adding culture medium cooled to 4°C, the cells were washed with PBS, 1 mL of protease (manufactured by Thermo Fisher Scientific, Inc., product name: Trypsin-EDTA (0.25%)) was added, left to stand at 37°C for 10 minutes, medium was added, and the cells were recovered. TF activity was measured in the same manner as in Example 1, except for this. As a result, the value of TF activity per total protein amount was 4.6 pM/μg.

比較例２
ヒト脂肪由来幹細胞（ロンザジャパン（株）製、Ｐｒｏｄｕｃｔ Ｃｏｄｅ：ＰＴ－５００６）を用いたこと以外は比較例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は７.４ｐＭ／μｇであった。 Comparative Example 2
TF activity was measured in the same manner as in Comparative Example 1, except that human adipose-derived stem cells (manufactured by Lonza Japan, Product Code: PT-5006) were used. As a result, the TF activity value per total protein amount was 7.4 pM/μg.

比較例３
ヒト胎児肺由来正常二倍体線維芽細胞を用いて５日間培養したこと以外は比較例１と同様の方法で、ＴＦ活性を測定した。その結果、全タンパク質量当たりのＴＦ活性の値は１.４ｐＭ／μｇであった。 Comparative Example 3
TF activity was measured in the same manner as in Comparative Example 1, except that normal diploid fibroblasts derived from human fetal lung were cultured for 5 days. As a result, the value of TF activity per total protein amount was 1.4 pM/μg.

Claims (2)

Translated fromJapanese

インビトロで培養後、冷却処理によって培養器材から剥離された培養細胞の回収方法であって、
回収された培養細胞が、タンパク質分解酵素を使用して剥離された培養細胞よりも組織因子（ＴＦ）活性の値が４０％以下である細胞の回収方法において、
前記培養細胞が、骨髄由来ヒト間葉系幹細胞、ヒト脂肪由来幹細胞及びヒト胎児肺由来正常二倍体線維芽細胞のいずれか一つの培養細胞であり、
温度応答性ブロック共重合体を支持材に被覆形成してなる下限臨界溶解温度（ＬＣＳＴ）を示す培養器材上で培養後に、
ＬＣＳＴよりも低い温度に変化させる処理である冷却処理によって回収して得ることを特徴とする細胞回収方法。A method for recovering cultured cells detached from a culture vessel by cooling treatment after in vitro culture, comprising:
A method forrecovering cells, the recovered cultured cellshaving a tissue factor (TF) activity value of 40% or less compared to cultured cells detached using a protease, comprising:
The cultured cells are any one of bone marrow-derived human mesenchymal stem cells, human adipose-derived stem cells, and human fetal lung-derived normal diploid fibroblast cells,
After culturing on a culture vessel exhibiting a lower critical solution temperature (LCST) in which a temperature-responsive block copolymer is coated on a support material,
A cell recovery method, comprising recovering cells by a cooling treatment, which is a treatment for changing the temperature to a temperature lower than the LCST .前記の温度応答性ブロック共重合体が、２－メトキシエチルアクリレートの繰返し単位とｎ－ブチルアクリレートの繰返し単位とＮ－イソプロピルアクリルアミドの繰返し単位とを含んでなる温度応答性ブロック共重合体である請求項１に記載の方法2. The method according to claim 1, wherein the temperature-responsive block copolymer comprises a repeating unit of 2-methoxyethyl acrylate, a repeating unit of n-butyl acrylate, and a repeating unit of N-isopropylacrylamide.