JP2020174660A - Cell separation method - Google Patents
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Abstract
Description
本発明は、細胞分離方法に関する。 The present invention relates to a cell separation method.
再生医療やテーラーメード治療などの被検体由来の細胞が必要な分野において、目的細胞を効率よく分離・検出・培養する技術が必要とされる。これまでに目的細胞を分離する方法として、蛍光標識された抗体を目的細胞の表面タンパク質に修飾して分離するフローサイトメトリーや、磁気標識された抗体を目的細胞の表面タンパク質に修飾して外部磁場で分離する磁気細胞分離法など、イムノアフィニティに関する技術が主流である。しかし、使用する抗体などの試薬が高価であり、ランニングコストが高いという課題がある。また、イムノアフィニティ技術は細胞の表面タンパク質に抗体を修飾することで細胞にダメージを与えてしまう課題もある。将来的な再生医療分野等の発展のためには、安価に目的細胞を低侵襲で分離できる細胞分離技術が求められる。 In fields such as regenerative medicine and tailor-made treatment where cells derived from a subject are required, a technique for efficiently separating, detecting, and culturing target cells is required. So far, as a method for separating target cells, flow cytometry in which a fluorescently labeled antibody is modified to a surface protein of the target cell for separation, or a magnetically labeled antibody is modified to a surface protein in the target cell to form an external magnetic field. Technologies related to immunoaffinity, such as the magnetic cell separation method for separating with, are the mainstream. However, there is a problem that reagents such as antibodies used are expensive and running costs are high. In addition, immunoaffinity technology has a problem of damaging cells by modifying an antibody on the surface protein of cells. For the future development of the field of regenerative medicine and the like, a cell separation technology capable of separating target cells at low cost with minimal invasiveness is required.
本発明の目的は、目的細胞を低侵襲で分離できる細胞分離方法を提供することにある。 An object of the present invention is to provide a cell separation method capable of separating target cells with minimal invasiveness.
本発明者らは、以上の点を鑑み、鋭意研究を重ねた結果、本発明を完成した。 In view of the above points, the present inventors have completed the present invention as a result of repeated diligent research.
すなわち本発明の一態様は、下限臨界溶解温度（ＬＣＳＴ）を示すセグメントを含むブロック共重合体を被覆した細胞培養器材に２種類以上の細胞を播種する工程と、前記細胞培養器材から前記ブロック共重合体に非接着の細胞を除く工程と、前記細胞培養器材をＬＣＳＴ以下に冷却して前記ブロック共重合体に接着した細胞を回収する工程と、を含んでなることを特徴とする細胞分離方法である。 That is, one aspect of the present invention includes a step of seeding two or more types of cells in a cell culture equipment coated with a block copolymer containing a segment showing a lower limit critical dissolution temperature (LCST), and the block from the cell culture equipment. A cell separation method comprising a step of removing cells non-adherent to the polymer and a step of cooling the cell culture equipment to LCST or less and recovering the cells adhering to the block copolymer. Is.
本発明により、安価に目的細胞を低侵襲で分離できる。 According to the present invention, target cells can be isolated at low cost with minimal invasiveness.
以下、本発明の一態様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。 Hereinafter, one aspect of the present invention will be described in detail, but the present invention is not intended to be limited to the following contents. The present invention can be appropriately modified and implemented within the scope of the gist thereof.
ＬＣＳＴとは下限臨界溶解温度（Ｌｏｗｅｒ Ｃｒｉｔｉｃａｌ Ｓｏｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ：ＬＣＳＴ）であり、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る。 LCST is the lower critical dissolution temperature (LCST). At a temperature lower than this temperature, the polymer dissolves in water to become a transparent solution, but at a temperature higher than this temperature, it becomes insoluble. It is the temperature at which white turbidity or precipitation occurs and phase separation occurs.
ブロック共重合体は、２種類以上の繰り返し単位からなる重合体で、それぞれ同種の繰り返し単位からなる高分子鎖が、１本の鎖の中に結合している重合体をいう。 A block copolymer is a polymer composed of two or more types of repeating units, and refers to a polymer in which polymer chains composed of the same type of repeating units are bonded in one chain.
下限臨界溶解温度（ＬＣＳＴ）を示すセグメントを含むブロック共重合体は、特に限定はないが、重合が容易なことから、アクリロイル基あるいはメタクリロイル基を含む構造であることが好ましい。下限臨界溶解温度（ＬＣＳＴ）を示すセグメントの繰返し単位（以下、「ＬＣＳＴ繰返し単位」ということがある）とその水に対するＬＣＳＴは、例えば、Ｎ−エチルアクリルアミド（ＬＣＳＴ＝７２℃）、Ｎ−シクロプロピルアクリルアミド（ＬＣＳＴ＝４６℃）、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−シクロプロピルメタクリルアミド（ＬＣＳＴ＝５９℃）、Ｎ−イソプロピルメタクリルアミド（ＬＣＳＴ＝４４℃）、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−エチルアクリルアミド（ＬＣＳＴ＝５６℃）、Ｎ−メチル−Ｎ−イソプロピルアクリルアミド（ＬＣＳＴ＝２３℃）、Ｎ−メチル−Ｎ−ｎ−プロピルアクリルアミド（ＬＣＳＴ＝２０℃）、またはＮ，Ｎ−ジメチルアミノエチルメタクリレート（ＬＣＳＴ＝４７℃）等が例示できる。ＬＣＳＴの上限は、細胞培養温度は一般的に高温ではタンパク質変性に伴う細胞へのダメージが発生することから４２℃が好ましく、４０℃がより好ましい。また、ＬＣＳＴの下限は、低温での細胞活性の低下を避ける為に、１０℃が好ましく、２０℃がより好ましい。ＬＣＳＴ繰返し単位としては、その繰り返し単位を１種類のみ用いてもよく、２種類以上を組み合わせて用いてもよい。また、ブロック共重合体が温度変化によって親水性／疎水性の程度が変化する性質（以下、「温度応答性」ということがある）を有するのであれば、ＬＣＳＴ繰返し単位の他に、異なる繰返し単位を含んでも良い。 The block copolymer containing a segment showing the lower limit critical dissolution temperature (LCST) is not particularly limited, but is preferably a structure containing an acryloyl group or a methacryloyl group because polymerization is easy. The repetition unit of the segment indicating the lower limit critical dissolution temperature (LCST) (hereinafter, may be referred to as “LCST repetition unit”) and the LCST for water thereof are, for example, N-ethylacrylamide (LCST = 72 ° C.), N-cyclopropyl. Acrylamide (LCST = 46 ° C), N-isopropylacrylamide (LCST = 32 ° C), Nn-propylmethacrylamide (LCST = 22 ° C), N-tetrahydrofurfurylacrylamide (LCST = 28 ° C), N-ethoxyethyl Acrylamide (LCST = 35 ° C), N, N-diethylacrylamide (LCST = 32 ° C), N-cyclopropylmethacrylamide (LCST = 59 ° C), N-isopropylmethacrylamide (LCST = 44 ° C), Nn- Propylmethacrylamide (LCST = 28 ° C.), N-tetrahydrofurfurylmethacrylicamide (LCST = 35 ° C.), N-methyl-N-ethylacrylamide (LCST = 56 ° C.), N-methyl-N-isopropylacrylamide (LCST = 35 ° C.) 23 ° C.), N-methyl-Nn-propylacrylamide (LCST = 20 ° C.), N, N-dimethylaminoethyl methacrylate (LCST = 47 ° C.) and the like. The upper limit of LCST is preferably 42 ° C., more preferably 40 ° C., because the cell culture temperature is generally high and damage to cells due to protein denaturation occurs. The lower limit of LCST is preferably 10 ° C., more preferably 20 ° C., in order to avoid a decrease in cell activity at low temperatures. As the LCST repeating unit, only one type of the repeating unit may be used, or two or more types may be used in combination. Further, if the block copolymer has a property that the degree of hydrophilicity / hydrophobicity changes with a temperature change (hereinafter, may be referred to as "temperature responsiveness"), a different repeating unit in addition to the LCST repeating unit May include.
ブロック共重合体を構成するセグメントの数は２以上であればよく、３以上であっても構わない。ブロック共重合体中のＬＣＳＴ繰返し単位の構成割合は、５〜９５ｍｏｌ％であることが好ましく、３０〜９０ｍｏｌ％であることがより好ましく、４０〜８０ｍｏｌ％であることがさらに好ましい。ブロック共重合体を構成するセグメントには、温度応答性及び細胞培養器材への被覆性を向上させるために、ＬＣＳＴを示すセグメントのほかに、０℃〜５０℃の範囲にＬＣＳＴを持たない、ＨＬＢ値（グリフィン法）が９以上２０以下の範囲にある親水性重合体セグメントと、０℃〜５０℃の範囲にＬＣＳＴを持たない、ＨＬＢ値（グリフィン法）が０以上９未満の範囲にある疎水性重合体セグメントをさらに含んでいることが好ましい。これらのセグメントは、特開２０１８−０８７３１６号公報に記載されたセグメントを用いることができる。 The number of segments constituting the block copolymer may be 2 or more, and may be 3 or more. The composition ratio of the LCST repeating unit in the block copolymer is preferably 5 to 95 mol%, more preferably 30 to 90 mol%, still more preferably 40 to 80 mol%. In order to improve temperature responsiveness and coverage to cell culture equipment, the segments constituting the block copolymer do not have LCST in the range of 0 ° C to 50 ° C in addition to the segment showing LCST, HLB. Hydrophilic polymer segments with a value (Griffin method) in the range of 9 or more and 20 or less, and hydrophobic HLB values (Griffin method) in the range of 0 or more and less than 9 without LCST in the range of 0 ° C to 50 ° C. It preferably further contains a sex polymer segment. As these segments, the segments described in JP-A-2018-08731 can be used.
細胞培養器材へのブロック共重合体の被覆方法として特に限定はないが、例えば特開２０１８−０８７３１６号公報に記載された定法を用いることができる。ブロック共重合体のＬＣＳＴを示すセグメントの被覆量は、０．１μｇ／ｃｍ２以上２０．０μｇ／ｃｍ２以下の割合で被覆されることが好ましく、０．２μｇ／ｃｍ２以上１０．０μｇ／ｃｍ２以下の割合で被覆されることがより好ましい。被覆量の測定は定法に従えばよく、例えば、天秤を用いた秤量を利用すればよい。なお、細胞培養器材の形状としては、ディッシュ、プレート又はフラスコが例示できる。 The method for coating the block copolymer on the cell culture equipment is not particularly limited, and for example, the standard method described in JP-A-2018-08713 can be used. The coverage of the segment showing the LCST of the block copolymer is preferably coated at a rate of 0.1 [mu] g / cm 2 or more 20.0μg / cm 2 or less, 0.2 [mu] g / cm 2 or more 10.0 [mu] g / cm It is more preferable to cover with a ratio of 2 or less. The coating amount may be measured according to a conventional method, and for example, a weighing using a balance may be used. Examples of the shape of the cell culture equipment include dishes, plates, and flasks.
細胞培養用器材には、ブロック共重合体の表面に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、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／ナイドジェン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、これらを一種又は二種以上用いてもよい。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 cell culture equipment may be further coated with an extracellular matrix on the surface of the block copolymer. The type of extracellular matrix is not particularly limited, and for example, collagen, atelocollagen, hyaluronic acid, elastin, proteoglycan, glucosaminoglycan, fibronectin, laminin, vitronectin, gelatin, laminin, collagen IV, heparan sulfate proteoglycan, entactin / Nyidgen 1 , 2 and the like may be used as a main component of Matrigel, and one or more of these may be used. It may also be a segment of these extracellular matrices.
細胞の種類（以下、「細胞種」ということがある）は２種類以上であれば特に限定はなく、３種類以上であってもよい。用いる細胞種の組合せは特に限定はないが、線維芽細胞及び／又は上皮細胞は好適に本発明に用いられる。それ以外にも、血球系細胞、神経系や内臓系を構築する細胞といった異種細胞種が混合した状態や、間葉系幹細胞などの分化能を有した細胞の分化状態が異なる状態に対しても本発明を用いることは可能であるが、接着細胞であることが好ましい。また、ヒト以外の細胞であってもよく、例えば、マウスやハムスター、ウサギやサルといった哺乳類由来の細胞や、昆虫由来の細胞であってもよい。さらに継代数の異なる同種細胞であってもよい。 The type of cell (hereinafter, may be referred to as "cell type") is not particularly limited as long as it is two or more types, and may be three or more types. The combination of cell types used is not particularly limited, but fibroblasts and / or epithelial cells are preferably used in the present invention. In addition to that, even in a state where heterologous cell types such as blood cell lineage cells, cells that construct the nervous system and visceral system are mixed, and in a state where differentiated cells such as mesenchymal stem cells have different differentiation states. Although it is possible to use the present invention, it is preferably adherent cells. In addition, cells other than humans may be used, and for example, cells derived from mammals such as mice, hamsters, rabbits and monkeys, and cells derived from insects may be used. Further, allogeneic cells having different passage numbers may be used.
培地は特に限定はなく、イーグル最小必須培地（ＭＥＭ）などの基礎培地にウシ胎児血清等の血清や抗生物質などが添加された培地を用いてもよいし、細胞外マトリックスが添加された無血清培地を用いてもよい。培地中の血清濃度や細胞外マトリックス濃度は特に限定はなく、各々の細胞種で細胞分離に適した濃度を用いればよい。例えば、牛胎児由来血清の濃度であれば１〜３０ｖｏｌ％が好ましく、５〜２０ｖｏｌ％がより好ましい。 The medium is not particularly limited, and a medium in which serum such as fetal bovine serum or an antibiotic is added to a basal medium such as Eagle's minimum essential medium (MEM) may be used, or serum-free medium to which an extracellular matrix is added. A medium may be used. The serum concentration and extracellular matrix concentration in the medium are not particularly limited, and a concentration suitable for cell separation may be used for each cell type. For example, the concentration of fetal bovine serum is preferably 1 to 30 vol%, more preferably 5 to 20 vol%.
本発明の細胞分離方法は、上述したブロック共重合体を被覆した細胞培養器材に２種類以上の細胞を播種する工程（以下、「播種工程」ということがある）と、前記細胞培養器材から前記ブロック共重合体に非接着の細胞を除く工程（以下、「除去工程」ということがある）と、前記細胞培養器材をＬＣＳＴ以下に冷却して前記ブロック共重合体に接着した細胞を回収する工程（以下、「回収工程」ということがある）と、を含んでなる。 The cell separation method of the present invention includes a step of seeding two or more types of cells in the above-mentioned cell culture equipment coated with the block copolymer (hereinafter, may be referred to as a "seed step"), and the above-mentioned from the cell culture equipment. A step of removing cells non-adherent to the block copolymer (hereinafter, may be referred to as a "removal step") and a step of cooling the cell culture equipment to LCST or less and collecting cells adhering to the block copolymer. (Hereinafter, it may be referred to as a "recovery process") and.
播種工程では、一部の細胞種が接着しない又は接着率が低くなるよう、ブロック共重合体の被覆量、培地中の血清濃度、培養時間の調整などを一つ又は組み合わせて行う。ただし、培養時間に関しては、３０分間〜１２時間とすることが好ましく、１時間から３時間がより好ましい。 In the seeding step, one or a combination of the amount of the block copolymer coated, the serum concentration in the medium, the culture time, etc. are adjusted so that some cell types do not adhere or the adhesion rate is low. However, the culture time is preferably 30 minutes to 12 hours, more preferably 1 hour to 3 hours.
除去工程では、非接着の細胞を、例えばピペッターを用いて培地ごと回収する方法が例示できる。非接着の細胞除去後はＬＣＳＴ以上から４２℃以下のＰＢＳ又は培地（血清を含んでいても良い）を先述の細胞培養器材に加えて洗浄してもよい。 In the removal step, a method of collecting the non-adherent cells together with the medium using, for example, a pipettor can be exemplified. After removing the non-adherent cells, PBS or medium (which may contain serum) of LCST or higher and 42 ° C. or lower may be added to the above-mentioned cell culture equipment and washed.
回収工程では、ブロック共重合体に残存した細胞を回収する。冷却する方法は特に限定はなく、冷所で冷却しても良いし、冷却した培地で培地交換することで冷却しても良い。冷却する温度はＬＣＳＴ以下の温度であれば特に限定はないが、残存した細胞の回収を促すため、ＬＣＳＴより２℃以上低い温度が好ましく、ＬＣＳＴより４℃以上低い温度がより好ましい。冷却時間は、４０分未満が好ましく、２０分未満がより好ましく、１０分未満がさらに好ましい。冷却後は細胞培養器材から剥離した細胞を回収することができるが、細胞培養器材からの剥離が不完全な場合は、ピペッティングやタッピング、振とうなどの物理的衝撃を加えてもよい。 In the recovery step, the cells remaining in the block copolymer are recovered. The method of cooling is not particularly limited, and cooling may be performed in a cold place or by exchanging the medium with a cooled medium. The cooling temperature is not particularly limited as long as it is a temperature of LCST or less, but in order to promote the recovery of residual cells, a temperature lower than LCST by 2 ° C. or more is preferable, and a temperature lower than LCST by 4 ° C. or more is more preferable. The cooling time is preferably less than 40 minutes, more preferably less than 20 minutes, still more preferably less than 10 minutes. After cooling, the cells exfoliated from the cell culture equipment can be recovered, but if the exfoliation from the cell culture equipment is incomplete, physical impact such as pipetting, tapping, or shaking may be applied.
回収工程で回収した細胞は、さらに本発明の細胞分離方法を繰り返すことで、さらに細胞を分離・精製することができる。二回目の細胞分離を行う際は、一回目の細胞分離の際よりもブロック共重合体のＬＣＳＴを示すセグメントの被覆量を同量以上とした細胞培養器材を用いることが好ましい。三回目の細胞分離を行う際は、二回目の細胞分離の際よりもブロック共重合体のＬＣＳＴを示すセグメントの被覆量を同量以上とすることが好ましい。例えば３種類の細胞ａ、ｂ、ｃ（ブロック共重合体への接着性 ｃ＞ｂ＞ａ）を培養する場合において、一回目の操作で非接着細胞ａを分離し、冷却処理でｂ及びｃの混合物を回収できる。さらに二回目の操作でブロック共重合体のＬＣＳＴを示すセグメントの被覆量を上げた細胞培養器材を用いることで、非接着細胞ｂを分離し、冷却処理でｃの混合物を回収できる。 The cells recovered in the recovery step can be further separated and purified by repeating the cell separation method of the present invention. When performing the second cell separation, it is preferable to use a cell culture device having the same amount or more of the coating amount of the segment showing LCST of the block copolymer as compared with the case of the first cell separation. When the third cell separation is performed, it is preferable that the coating amount of the segment showing LCST of the block copolymer is the same or more than that in the second cell separation. For example, when culturing three types of cells a, b, and c (adhesion to block copolymer c> b> a), the non-adhesive cells a are separated by the first operation, and b and c are cooled. Mixtures can be recovered. Furthermore, by using a cell culture device in which the coating amount of the segment showing LCST of the block copolymer is increased in the second operation, the non-adherent cells b can be separated and the mixture of c can be recovered by the cooling treatment.
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。 Examples of the present invention will be described below, but the present invention is not limited to these examples. Unless otherwise specified, commercially available reagents were used.
＜ブロック共重合体の合成＞
１００ｍＬ２口フラスコに２−メトキシエチルアクリレート（ＭＥＡ，ＨＬＢ値＝１３．５）０．６５０ｇ（５ｍｍｏｌ）を加え、さらにシアノメチルドデシルカルボナトを３１．８ｍｇ（１００μｍｏｌ）とアゾビスイソブチロニトリル１．６ｍｇ（１０μｍｏｌ）と１，４−ジオキサン１０ｍＬを加え、アルゴンガス置換後、６２℃で２４時間加熱撹拌した。
１回目の加熱撹拌後、上記にｎ−ブチルアクリレート（ＢＡ，ＨＬＢ値＝６．９）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１，４−ジオキサン５ｍＬを加え、アルゴンガス置換後、６２℃で４８時間加熱撹拌した。
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℃、ＨＬＢ値＝７．６）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１，４−ジオキサン３５ｍＬを加え、アルゴンガス置換後、６２℃で４８時間加熱撹拌した。
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、ヘキサンで再沈精製した。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５．８０５ｇ得た。得られたブロック共重合体の組成比はＭＥＡ／ＢＡ／ＩＰＡＡｍ＝５／２６／６９（ｍｏｌ％）であり、数平均分子量Ｍｎは８．５万、分子量分布Ｍｗ／Ｍｎは１．７８であった。
<Synthesis of block copolymer>
To a 100 mL two-necked flask, 0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA, HLB value = 13.5) was added, and 31.8 mg (100 μmol) of cyanomethyldodecylcarbonate and azobisisobutyronitrile 1. 6 mg (10 μmol) and 10 mL of 1,4-dioxane were added, and after substitution with argon gas, the mixture was heated and stirred at 62 ° C. for 24 hours.
After the first heating and stirring, 3.845 g (30 mmol) of n-butyl acrylate (BA, HLB value = 6.9) was added to the above, and 1.6 mg (10 μmol) of azobisisobutyronitrile and 1,4- After adding 5 mL of dioxane and replacing with argon gas, the mixture was heated and stirred at 62 ° C. for 48 hours.
After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST = 32 ° C., HLB value = 7.6) was added, and 1.6 mg (10 μmol) of azobisisobutyronitrile was added. And 1,4-dioxane (35 mL) were added, and after substitution with argon gas, the mixture was heated and stirred at 62 ° C. for 48 hours.
After the third heating and stirring, the reaction solution was reprecipitated 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 recovered using a separating funnel. The recovered chloroform phase was concentrated on an evaporator and reprecipitated with hexane. The precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of block copolymer poly (MEA-BA-IPAAm). The composition ratio of the obtained block copolymer was MEA / BA / IPAAm = 5/26/69 (mol%), the number average molecular weight Mn was 85,000, and the molecular weight distribution Mw / Mn was 1.78. It was.
＜ブロック共重合体の組成＞
核磁気共鳴測定装置（日本電子製、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）及び分子量分布（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 ＴＳＫｇｅｌ ＳｕｐｅｒＡＷＭ−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２，２，２−トリフルオロエタノールを用いて測定した。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（Ｓｉｇｍａ−Ａｌｄｒｉｃｈ社製）を用いた。
＜細胞培養器材のＬＣＳＴを示すセグメントの被覆量＞
ブロック共重合体を被覆した細胞培養器材のＬＣＳＴを示すセグメント（ＩＰＡＡｍ）の被覆量はＩＰＡＡｍセグメントの被覆量を全反射型フーリエ変換型赤外分光（ＡＴＲ／ＦＴ−ＩＲ）法により解析した。解析には被覆量が既知のサンプルから作成した検量線を用い、単位面積当たりのＬＣＳＴを示すセグメントの被覆量（μｇ／ｃｍ２）で評価した。
<Composition of block copolymer>
It was obtained by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectral analysis using a nuclear magnetic resonance measuring device (manufactured by JEOL Ltd., trade name: JNM-ECZ400S / L1).
<Molecular weight and molecular weight distribution of block copolymers>
The 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 uses HLC-8320GPC manufactured by Tosoh Corporation, the column uses two TSKgel SuperAWM-H manufactured by Tosoh Co., Ltd., the column temperature is set to 40 ° C., and the eluent contains 2,2,2, which contains 10 mM sodium trifluoroacetate. Measured with 2-trifluoroethanol. The measurement sample was prepared at 1.0 mg / mL and measured. As the calibration curve of the molecular weight, polymethyl methacrylate (manufactured by Sigma-Aldrich) having a known molecular weight was used.
<Coverage of segment showing LCST of cell culture equipment>
The coating amount of the segment (IPAAm) showing LCST of the cell culture equipment coated with the block copolymer was analyzed by the total reflection Fourier transform infrared spectroscopy (ATR / FT-IR) method for the coating amount of the IPAAm segment. A calibration curve prepared from a sample having a known coverage was used for the analysis, and the coverage was evaluated by the coverage (μg / cm 2 ) of the segment showing LCST per unit area.
＜播種した細胞数の計測＞
播種する細胞の数は血球計算盤を用いて計測した。血球計算盤の四隅の１ｍｍ２区画内の全細胞を数え、全細胞数を（区画中の全生細胞の平均値）×希釈倍率×培地量（ｍＬ）×１００００で算出した。
＜細胞種の割合解析＞
予め、細胞の種類ごとに、色素の異なるＣｅｌｌＴｒａｃｋｅｒＴＭ（Ｔｈｅｍｏｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社製）を用い、細胞を染色した。混合済みの染色した細胞の懸濁液を細胞培養器材に播種し、３７℃、５％ＣＯ２条件で所定の時間培養後、非接着の細胞を培地ごと除去した後の培養器材上の残存細胞を、対物レンズ１０倍の共焦点定量イメージサイトメーターＣＱ１（横河電機社製）を用い画像撮影を行った。画像解析はＩｍａｇｅＪを用い、一例として、ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｂｌｕｅ ＣＭＡＣで染色した細胞は色相１２４〜２５５、ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色した細胞は色相６０〜１００、ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｏｒａｎｇｅ ＣＭＴＭＲで染色した細胞は色相０〜３０で各細胞の面積を割り出し、細胞当たりの染色面積で割ることで、細胞種の割合を解析した。
<Measurement of the number of seeded cells>
The number of cells to be seeded was measured using a hemocytometer. All cells in 1 mm 2 compartments at the four corners of the hemocytometer were counted, and the total number of cells was calculated by (mean value of all living cells in the compartment) x dilution ratio x medium volume (mL) x 10000.
<Percentage analysis of cell types>
In advance, cells were stained with CellTracker TM (manufactured by Themorphisher Scientific) having a different dye for each cell type. Residual cells on the culture equipment after seeding the mixed suspension of stained cells on the cell culture equipment, culturing at 37 ° C. for a predetermined time under 5% CO 2 conditions, and removing the non-adherent cells together with the medium. Was imaged using a cofocal quantitative image cytometer CQ1 (manufactured by Yokogawa Electric Co., Ltd.) with a 10x objective lens. Image J was used for image analysis. As an example, cells stained with CellTracker TM Blue CMAC had a hue of 124 to 255, cells stained with CellTracker TM Green CMFDA had a hue of 60 to 100, and cells stained with CellTracker TM Orange CMTMR had a hue of 0 to 0. The cell type ratio was analyzed by determining the area of each cell at 30 and dividing by the stained area per cell.
実施例１
２．０ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を調製した。ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央にブロック共重合体／２−メトキシエタノール溶液を１００μＬ加え、スピンコータ―（ミカサ社製、商品名ＭＳ−Ｂ２００）を用いて、回転数２，０００ｒｐｍ、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被覆した細胞培養器材（１）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は５．８μｇ／ｃｍ２であった。
調製した細胞培養器材（１）を用いてＴＩＧ３−２０細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ ｂｌｕｅ ＣＭＡＣで染色済み）とＡ５４９細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色済み）とＰＣ９細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ ＯＲＡＮＧＥ ＣＭＴＭＲ）を１×１０５個ずつ含む細胞懸濁液を播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。非接着細胞除去後の残存細胞の割合を表１に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ＰＣ９細胞の比率が７５．７％の細胞懸濁液を回収できた。
Example 1
A 2-methoxyethanol solution of 2.0 wt% block copolymer was prepared. Add 100 μL of block copolymer / 2-methoxyethanol solution to the center of the IWAKI tissue culture dish (φ6 cm), and use a spin coater (manufactured by Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time. The cell culture equipment (1) coated with the block copolymer was prepared by spin coating under the condition of 60 seconds. The coverage of the IPAAm segment on the culture surface of the equipment was 5.8 μg / cm 2 .
Using the prepared cell culture equipment (1), TIG3-20 cells (stained with CellTracker TM blue CMAC), A549 cells (stained with CellTracker TM Green CMFDA), and PC9 cells (CellTracker TM ORANGE CMTMR) 1 × 10 5 Cell suspensions containing individual cells were seeded. As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium. Table 1 shows the percentage of residual cells after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, and the cells were peeled off by pipetting after leaving for 10 minutes, and a cell suspension having a PC9 cell ratio of 75.7% could be recovered.
実施例２
４．０ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を用いたことは実施例１記載の方法でブロック共重合体を被覆した細胞培養器材（２）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は１３．３μｇ／ｃｍ２であった。
細胞培養器材（２）を用いたこと以外は実施例１と同様の方法で培養した。非接着細胞除去後の残存細胞の割合を表１に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ＰＣ９細胞の比率が８９．４％の細胞懸濁液を回収できた。
Example 2
Using a 2-methoxyethanol solution of 4.0 wt% block copolymer prepared a cell culture equipment (2) coated with the block copolymer by the method described in Example 1. The coverage of the IPAAm segment on the culture surface of the equipment was 13.3 μg / cm 2 .
The cells were cultured in the same manner as in Example 1 except that the cell culture equipment (2) was used. Table 1 shows the percentage of residual cells after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, and the cells were peeled off by pipetting after leaving for 10 minutes, and a cell suspension having a PC9 cell ratio of 89.4% could be recovered.
比較例１
細胞培養器材としてＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）を用いたこと以外は、実施例１と同様の方法で培養した。非接着細胞除去後の残存細胞率を表１に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行ったが、細胞が剥がれなかった。
Comparative Example 1
The cells were cultured in the same manner as in Example 1 except that an IWAKI tissue culture dish (φ6 cm) was used as the cell culture equipment. Table 1 shows the residual cell rate after removal of non-adherent cells. A medium cooled to 4 ° C. was added to the cell culture equipment containing the remaining cells, and the cells were left for 10 minutes and then pipetting was performed, but the cells did not peel off.
比較例２
細胞培養器材としてセルシード社製ＵｐＣｅｌｌR（φ６ｃｍ）を用いたこと以外は、実施例１と同様の方法で培養した。非接着細胞除去後の残存細胞率を表１に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれたが、細胞分離されていない細胞懸濁液の回収となった。
Comparative Example 2
The cells were cultured in the same manner as in Example 1 except that UpCell R (φ6 cm) manufactured by CellSeed was used as the cell culture equipment. Table 1 shows the residual cell rate after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, and the cells were peeled off by pipetting after leaving for 10 minutes, but the cell suspension in which the cells were not separated was recovered.
実施例３
細胞培養器材（１）を用いて、実施例１と同様の方法で培養した。非接着細胞除去後の残存細胞の割合を表２に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで、ＰＣ９細胞の比率が７６．３％の細胞懸濁液を回収した。
回収したＰＣ９細胞の比率が高い細胞懸濁液を細胞培養器材（２）に播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。
非接着細胞除去後の残存細胞率を表２に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで、ＰＣ９細胞の比率が９５．０％の細胞懸濁液を回収できた。
Example 3
Using the cell culture equipment (1), the cells were cultured in the same manner as in Example 1. Table 2 shows the percentage of residual cells after removal of non-adherent cells. A cell suspension having a PC9 cell ratio of 76.3% was recovered by adding a medium cooled to 4 ° C. to a cell culture device containing residual cells, leaving it for 10 minutes, and then pipetting.
A cell suspension having a high ratio of recovered PC9 cells was seeded in the cell culture equipment (2). As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium.
Table 2 shows the residual cell rate after removal of non-adherent cells. A cell suspension having a PC9 cell ratio of 95.0% could be recovered by adding a medium cooled to 4 ° C. to a cell culture device containing residual cells and leaving it for 10 minutes before pipetting.
実施例４
０．４ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を調製した。ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央にブロック共重合体／２−メトキシエタノール溶液を１００μＬ加え、スピンコータ―（ミカサ社製、商品名ＭＳ−Ｂ２００）を用いて、回転数２，０００ｒｐｍ、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被覆した細胞培養器材（３）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は０．５μｇ／ｃｍ２であった。
調製した細胞培養器材（３）を用いてヒト骨髄由来間葉系幹細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ ＯＲＡＮＧＥ ＣＭＴＭＲで染色済み）とＲＡＭＯＳ細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色済み）を１×１０５個ずつ含む細胞懸濁液を播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。非接着細胞除去後の残存細胞の割合を表３に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ヒト骨髄由来間葉系幹細胞の比率が９９．０％の細胞懸濁液を回収できた。
Example 4
A 2-methoxyethanol solution of 0.4 wt% block copolymer was prepared. Add 100 μL of block copolymer / 2-methoxyethanol solution to the center of the IWAKI tissue culture dish (φ6 cm), and use a spin coater (manufactured by Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time. The cell culture equipment (3) coated with the block copolymer was prepared by spin coating under the condition of 60 seconds. The coverage of the IPAAm segment on the culture surface of the equipment was 0.5 μg / cm 2 .
Using the prepared cell culture equipment (3), a cell suspension containing 1 × 10 5 human bone marrow-derived mesenchymal stem cells (stained with CellTracker TM ORANGE CMTMR) and RAMOS cells (stained with CellTracker TM Green CMFDA). The liquid was sown. As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium. Table 3 shows the percentage of residual cells after removal of non-adherent cells. A cell culture medium containing residual cells was added to a medium cooled to 4 ° C., left for 10 minutes, and then pipetting was performed to detach the cells. A cell suspension in which the ratio of human bone marrow-derived mesenchymal stem cells was 99.0%. Was able to be recovered.
実施例５
１．５ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を調製した。ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央にブロック共重合体／２−メトキシエタノール溶液を１００μＬ加え、スピンコータ―（ミカサ社製、商品名ＭＳ−Ｂ２００）を用いて、回転数２，０００ｒｐｍ、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被覆した細胞培養器材（４）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は４．０μｇ／ｃｍ２であった。
調製した細胞培養器材（４）を用いてヒト歯髄由来間葉系幹細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ ＯＲＡＮＧＥ ＣＭＴＭＲで染色済み）とヒト脂肪由来間葉系幹細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色済み）を１×１０５個ずつ含む細胞懸濁液を播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。非接着細胞除去後の残存細胞の割合を表４に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ヒト脂肪由来間葉系幹細胞の比率が８５．２％の細胞懸濁液を回収できた。
Example 5
A 2-methoxyethanol solution of 1.5 wt% block copolymer was prepared. Add 100 μL of block copolymer / 2-methoxyethanol solution to the center of the IWAKI tissue culture dish (φ6 cm), and use a spin coater (manufactured by Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time. The cell culture equipment (4) coated with the block copolymer was prepared by spin coating under the condition of 60 seconds. The coverage of the IPAAm segment on the culture surface of the equipment was 4.0 μg / cm 2 .
Using the prepared cell culture equipment (4), 1 × 10 5 human dental pulp-derived mesenchymal stem cells (stained with CellTracker TM ORANGE CMTMR) and human adipose-derived mesenchymal stem cells (stained with CellTracker TM Green CMFDA) Cell suspensions containing each were seeded. As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium. Table 4 shows the percentage of residual cells after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, left for 10 minutes, and then pipetting was performed to peel off the cells. A cell suspension containing 85.2% of human adipose-derived mesenchymal stem cells. Was able to be recovered.
実施例６
２．５ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を調製した。ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央にブロック共重合体／２−メトキシエタノール溶液を１００μＬ加え、スピンコータ―（ミカサ社製、商品名ＭＳ−Ｂ２００）を用いて、回転数２，０００ｒｐｍ、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被覆した細胞培養器材（５）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は６．９μｇ／ｃｍ２であった。
調製した細胞培養器材（５）を用いてヒト肝癌由来細胞ＨｅｐＧ２（ＣｅｌｌＴｒａｃｋｅｒＴＭ ＯＲＡＮＧＥ ＣＭＴＭＲで染色済み）とヒト結腸腺癌細胞ＨＣＴ１１６（ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色済み）を１×１０５個ずつ含む細胞懸濁液を播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。非接着細胞除去後の残存細胞の割合を表５に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ヒト結腸腺癌細胞ＨＣＴ１１６の比率が９８．１％の細胞懸濁液を回収できた。
Example 6
A 2-methoxyethanol solution of a 2.5 wt% block copolymer was prepared. Add 100 μL of block copolymer / 2-methoxyethanol solution to the center of the IWAKI tissue culture dish (φ6 cm), and use a spin coater (manufactured by Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time. The cell culture equipment (5) coated with the block copolymer was prepared by spin coating under the condition of 60 seconds. The coverage of the IPAAm segment on the culture surface of the equipment was 6.9 μg / cm 2 .
Using the prepared cell culture equipment (5), cells containing 1 × 10 5 human liver cancer-derived cells HepG2 (stained with CellTracker TM ORANGE CMTMR) and human colon adenocarcinoma cells HCT116 (stained with CellTracker TM Green CMFDA). The suspension was sown. As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium. Table 5 shows the percentage of residual cells after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, and the cells were peeled off by pipetting after leaving for 10 minutes. A cell suspension having a human colon adenocarcinoma cell HCT116 ratio of 98.1% was prepared. I was able to recover it.
実施例７
１．０ｗｔ％のブロック共重合体の２−メトキシエタノール溶液を調製した。ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央にブロック共重合体／２−メトキシエタノール溶液を１００μＬ加え、スピンコータ―（ミカサ社製、商品名ＭＳ−Ｂ２００）を用いて、回転数２，０００ｒｐｍ、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被覆した細胞培養器材（６）を調製した。器材培養面のＩＰＡＡｍセグメントの被覆量は２．６μｇ／ｃｍ２であった。
調製した細胞培養器材（６）を用いてＴＩＧ３−２０細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ ＯＲＡＮＧＥ ＣＭＴＭＲで染色済み）とＴＩＧ３−６０細胞（ＣｅｌｌＴｒａｃｋｅｒＴＭ Ｇｒｅｅｎ ＣＭＦＤＡで染色済み）を１×１０５個ずつ含む細胞懸濁液を播種した。培地はＤ−ＭＥＭ基礎培地にウシ胎児血清１０％と抗生物質を加えたものを用い、３７℃、５％ＣＯ２条件で１時間共培養した。ブロック共重合体に非接着の細胞を培地ごと除去した。非接着細胞除去後の残存細胞の割合を表６に示す。残存細胞を含む細胞培養器材へ４℃に冷却した培地を加え、１０分放置後にピペッティングを行うことで細胞が剥がれ、ＴＩＧ３−２０細胞の比率が８３．３％の細胞懸濁液を回収できた。
Example 7
A 2-methoxyethanol solution of 1.0 wt% block copolymer was prepared. Add 100 μL of block copolymer / 2-methoxyethanol solution to the center of the IWAKI tissue culture dish (φ6 cm), and use a spin coater (manufactured by Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time. The cell culture equipment (6) coated with the block copolymer was prepared by spin coating under the condition of 60 seconds. The coverage of the IPAAm segment on the culture surface of the equipment was 2.6 μg / cm 2 .
A cell suspension containing 1 × 10 5 TIG3-20 cells (stained with CellTracker TM ORANGE CMTMR) and 1 × 10 5 TIG3-60 cells (stained with CellTracker TM Green CMFDA) using the prepared cell culture equipment (6). Was sown. As the medium, a D-MEM basal medium supplemented with 10% fetal bovine serum and antibiotics was used, and co-cultured at 37 ° C. and 5% CO 2 conditions for 1 hour. Cells non-adherent to the block copolymer were removed together with the medium. Table 6 shows the percentage of residual cells after removal of non-adherent cells. A medium cooled to 4 ° C. was added to a cell culture device containing residual cells, and the cells were peeled off by pipetting after leaving for 10 minutes, and a cell suspension having a TIG3-20 cell ratio of 83.3% could be recovered. It was.
Claims (7)
前記細胞培養器材から前記ブロック共重合体に非接着の細胞を除く工程と、
前記細胞培養器材をＬＣＳＴ以下に冷却して前記ブロック共重合体に接着した細胞を回収する工程と、
を含んでなることを特徴とする細胞分離方法。 A step of seeding two or more types of cells in a cell culture device coated with a block copolymer containing a segment showing a lower critical lysis temperature (LCST).
A step of removing cells that are not adhered to the block copolymer from the cell culture equipment, and
A step of cooling the cell culture equipment below LCST to recover cells adhering to the block copolymer, and
A method for separating cells, which comprises.
（Ａ）水に対する下限臨界溶解温度（ＬＣＳＴ）が１０℃〜４２℃の範囲にある温度応答性重合体セグメント。
（Ｂ）０℃〜５０℃の範囲にＬＣＳＴを持たない、ＨＬＢ値（グリフィン法）が９以上２０以下の範囲にある親水性重合体セグメント。
（Ｃ）０℃〜５０℃の範囲にＬＣＳＴを持たない、ＨＬＢ値（グリフィン法）が０以上９未満の範囲にある疎水性重合体セグメント。 The cell separation method according to any one of claims 1 to 5, which comprises a segment of the following components (A), (B), and (C) as a constituent component of the block copolymer.
(A) A temperature-responsive polymer segment having a lower limit critical dissolution temperature (LCST) in water in the range of 10 ° C to 42 ° C.
(B) A hydrophilic polymer segment having no LCST in the range of 0 ° C. to 50 ° C. and having an HLB value (Griffin method) in the range of 9 or more and 20 or less.
(C) A hydrophobic polymer segment having no LCST in the range of 0 ° C. to 50 ° C. and having an HLB value (Griffin method) in the range of 0 or more and less than 9.
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| JP2016131561A (en) * | 2015-01-22 | 2016-07-25 | 国立大学法人山形大学 | Method of recovering cells, and polymer used for the same |
| JP2018087316A (en) * | 2016-08-03 | 2018-06-07 | 東ソー株式会社 | Block copolymer and surface treatment agent using same |
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| JP2006223195A (en) * | 2005-02-17 | 2006-08-31 | National Institute Of Advanced Industrial & Technology | Adsorbing material for cell separation, adsorbing material module for cell separation and method for separating cell |
| JP2016131561A (en) * | 2015-01-22 | 2016-07-25 | 国立大学法人山形大学 | Method of recovering cells, and polymer used for the same |
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