JP2020110140A - Cell culture method - Google Patents
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Abstract
【課題】冷却処理による培養細胞の回収を高回収率で可能にする方法を提供すること。【解決手段】水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体とＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体とＨＬＢ値（グリフィン法）が０以上７未満の疎水性重合体を含む温度応答性ブロック共重合体が被覆された培養基材を用いた接着細胞の培養方法であって、培地中の血清濃度が２％以上であり、培養細胞をＬＣＳＴ以下の温度に冷却して回収することを特徴とする培養方法で前記課題を解決する。【選択図】 なしPROBLEM TO BE SOLVED: To provide a method capable of recovering cultured cells by cooling treatment at a high recovery rate. SOLUTION: A temperature-responsive polymer having a lower limit critical dissolution temperature (LCST) in the range of 0 ° C to 50 ° C and a hydrophilic polymer having an HLB value (Glyffin method) in the range of 7 or more and 20 or less. A method for culturing adherent cells using a culture substrate coated with a temperature-responsive block copolymer containing a hydrophobic polymer having a value (Glyffin method) of 0 or more and less than 7, and having a serum concentration of 2 in the medium. % Or more, and the above-mentioned problem is solved by a culture method characterized by cooling the cultured cells to a temperature of LCST or less and collecting them. [Selection diagram] None
Description
本発明は、高濃度血清培地中の培養細胞を、タンパク質分解酵素を用いずに高回収率で回収可能な方法に関する。 The present invention relates to a method capable of recovering cultured cells in a high-concentration serum medium at a high recovery rate without using a protease.
昨今、細胞を使用する医療や創薬の注目が高くなっており、細胞培養を工業規模で且つ高品質で行うことが重要となっている。細胞を増殖させるには培地が必要である。使用される培地は多種多様であり、ＤＭＥＭ培地やＦ１２培地などの基礎培地に、血清が加えられている。血清には成長因子や接着因子など必要不可欠な重要因子が含まれており１〜２０ｖｏｌ％の濃度で使用する。一方、注目を集めている細胞の多くは接着細胞であり、細胞を基材から剥離して回収する必要がある。トリプシンなどのタンパク質分解酵素を用い、接着因子を分解して細胞を基材から剥離しているが、タンパク質分解酵素が細胞表面のタンパク質も分解してしまうことで、細胞を高品質で回収することが困難であった。 BACKGROUND ART Recently, medical attention and drug discovery using cells have been attracting attention, and it is important to carry out cell culture on an industrial scale and with high quality. A medium is required to grow the cells. A variety of media are used, and serum is added to basal media such as DMEM medium and F12 medium. Serum contains essential and essential factors such as growth factors and adhesion factors, and is used at a concentration of 1 to 20% by volume. On the other hand, most of the cells attracting attention are adherent cells, and it is necessary to separate the cells from the base material and collect them. The cells are detached from the substrate by degrading the adhesion factor using a proteolytic enzyme such as trypsin, but the proteolytic enzyme also decomposes the protein on the cell surface, so that the cells can be collected with high quality. Was difficult.
以上の問題を温度応答性細胞培養基材の使用で解決できる。高温域では疎水性であるが、下限臨界溶液温度（Ｌｏｗｅｒ Ｃｒｉｔｉｃａｌ Ｓｏｌｕｔｉｏｎ Ｔｅｍｐｅｒａｔｕｒｅ：ＬＣＳＴ）よりも低温域において親水化する温度応答性細胞培養基材が特許文献１に開示されており、一例として製品名ＵｐＣｅｌｌとして製品化されている。このような基材によれば、タンパク質分解酵素を用いずに、冷却処理による親水化で細胞を剥離させ回収することができる。 The above problems can be solved by using a temperature-responsive cell culture substrate. Patent Document 1 discloses a temperature-responsive cell culture substrate that is hydrophobic in a high temperature range but becomes hydrophilic in a lower temperature range than a lower critical solution temperature (LCST), and as an example, a product name It is commercialized as UpCell. With such a base material, cells can be peeled and collected by hydrophilization by cooling treatment without using a proteolytic enzyme.
一方、細胞増殖を促進するために高血清濃度の培地を用いるが、先行技術の方法がしばしば直面する問題として、培養条件によっては冷却処理による細胞の剥離がうまくいかないことがあった。 On the other hand, although a medium with a high serum concentration is used to promote cell growth, a problem often encountered in the methods of the prior art is that the exfoliation of cells by cooling treatment is not successful depending on the culture conditions.
本発明の目的は、血清培地中の培養細胞を一定の比率以上に培養し、タンパク質分解酵素を用いずに高回収率で回収可能な方法を提供することにある。 An object of the present invention is to provide a method capable of culturing cultured cells in a serum medium at a certain ratio or more and recovering them at a high recovery rate without using a protease.
本発明者らは、以上の点を鑑み、鋭意研究を重ねた結果、培地中の血清濃度が２ｖｏｌ％以上であり、水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体とＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体とＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体を含む温度応答性ブロック共重合体が被覆された培養基材を用いた接着細胞の培養方法であって、培養細胞をＬＣＳＴ以下の温度に冷却して回収することを特徴とする培養方法が、培養細胞を高回収率で回収できることを見出し、本発明を完成した。
すなわち本発明は以下の態様を包含する。
＜１＞ 培地中の血清濃度が２ｖｏｌ％以上であり、下記成分（Ａ）〜（Ｃ）を含む温度応答性ブロック共重合体が被覆された培養基材を用いた接着細胞の培養方法であって、培養細胞をＬＣＳＴ以下の温度に冷却して回収することを特徴とする培養方法。
（Ａ）水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体。
（Ｂ）ＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体。
（Ｃ）ＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体。
＜２＞ ＬＣＳＴ以下の温度に冷却した培地での培地交換を伴うことを特徴とする＜１＞の培養方法。
＜３＞ 冷却時間が２０分以内であることを特徴とする＜１＞又は＜２＞に記載の培養方法。
＜４＞ 冷却後、ピペッティングを伴うことを特徴とする＜１＞から＜３＞いずれかに記載の培養方法。
＜５＞ 血清が牛胎児血清（ＦＢＳ）であることを特徴とする＜１＞から＜４＞いずれかに記載の培養方法。
＜６＞ 細胞が間葉系幹細胞であることを特徴とする＜１＞から＜５＞いずれかに記載の培養方法。
＜７＞ 間葉系幹細胞が骨髄由来であることを特徴とする＜６＞に記載の培養方法。
＜８＞ ＜１＞記載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とｎ−ブチルアクリレートとＮ−イソプロピルアクリルアミドからなることを特徴とする＜１＞から＜７＞いずれかに記載の培養方法。
The present inventors have conducted intensive studies in view of the above points, and as a result, the serum concentration in the medium was 2 vol% or more, and the lower critical solution temperature (LCST) in water was in the range of 0°C to 50°C. Temperature responsiveness including a temperature-responsive polymer and a hydrophilic polymer having an HLB value (Gryffin method) in the range of 7 to 20 and a hydrophobic polymer having an HLB value (Gryffin method) in the range of 0 to less than 7 A method for culturing adherent cells using a culture substrate coated with a block copolymer, which is characterized in that the cultured cells are cooled to a temperature of LCST or lower and recovered, and the cultured cells are highly recovered. The present invention has been completed based on the finding that it can be collected at a rate.
That is, the present invention includes the following aspects.
<1> A method for culturing adherent cells using a culture substrate having a serum concentration of 2 vol% or more in a medium and coated with a temperature-responsive block copolymer containing the following components (A) to (C). Then, the cultured cells are cooled to a temperature equal to or lower than LCST and collected.
(A) A temperature-responsive polymer having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
(B) A hydrophilic polymer having an HLB value (Griffin method) of 7 or more and 20 or less.
(C) A hydrophobic polymer having an HLB value (Griffin method) in the range of 0 or more and less than 7.
<2> The culture method according to <1>, which is accompanied by medium replacement with a medium cooled to a temperature of LCST or lower.
<3> The culture method according to <1> or <2>, wherein the cooling time is within 20 minutes.
<4> The culturing method according to any one of <1> to <3>, which is accompanied by pipetting after cooling.
<5> The culture method according to any one of <1> to <4>, wherein the serum is fetal bovine serum (FBS).
<6> The culture method according to any one of <1> to <5>, wherein the cells are mesenchymal stem cells.
<7> The culture method according to <6>, wherein the mesenchymal stem cells are derived from bone marrow.
<8> The temperature-responsive block copolymer according to <1> is composed of 2-methoxyethyl acrylate, n-butyl acrylate, and N-isopropylacrylamide. <1> to <7> Culture method.
培地中の血清濃度が２％以上であり、水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体とＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体とＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体を含む温度応答性ブロック共重合体が被覆された培養基材を用いた接着細胞の培養方法であって、培養細胞を一定の比率以上に培養し、ＬＣＳＴ以下の温度に冷却して回収することを特徴とする培養方法は、培養細胞を高回収率で回収できる。 The concentration of serum in the medium is 2% or more, the lower limit critical solution temperature (LCST) in water is in the range of 0°C to 50°C, and the HLB value (Griffin method) is in the range of 7 to 20. Method for culturing adherent cells using a culture substrate coated with a temperature-responsive block copolymer containing a hydrophilic polymer according to 1) and a HLB value (Gryffin method) in the range of 0 or more and less than 7 The culturing method characterized by culturing the cultured cells at a certain ratio or more, cooling to a temperature of LCST or lower, and recovering the cultured cells can be recovered at a high recovery rate.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明は、培地中の血清濃度が２ｖｏｌ％以上であり、水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体とＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体とＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体を含む温度応答性ブロック共重合体が被覆された培養基材を用いた接着細胞の培養方法であって、培養細胞を一定の比率以上に培養し、ＬＣＳＴ以下の温度に冷却して回収することを特徴とする培養方法であり、培養細胞を高回収率で回収を可能にする。 The present invention has a serum concentration of 2 vol% or more in a medium, a temperature-responsive polymer having a lower critical solution temperature (LCST) in the range of 0°C to 50°C in water, and an HLB value (Griffin method) of 7 or more. Adhesion using a culture substrate coated with a temperature-responsive block copolymer containing a hydrophilic polymer in the range of 20 or less and a hydrophobic polymer in which the HLB value (Griffin method) is 0 or more and less than 7 A method for culturing cells, which is characterized by culturing cells at a certain ratio or higher, cooling the cells to a temperature lower than LCST, and collecting the cells. To do.
本発明で用いる培地は、培地は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’ｓ Ｆ−１２、Ｈａｍ’ｓ Ｆ−１２、Ｈａｍ’ｓ Ｆ−１０、Ｍｅｄｉｕｍ １９９、ＲＰＭＩ １６４０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好ましくは２ｖｏｌ％以上であり、より好ましくは５ｖｏｌ％以上である。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％以上の濃度であっても良い。 The medium used in the present invention comprises a basal medium and serum, and may contain an antibiotic. 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 examples thereof include fetal bovine serum (FBS), calf serum, adult calf serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rabbit serum, human serum. The FBS is generally used because it is easily available. The serum concentration in the medium is preferably 2 vol% or more, more preferably 5 vol% or more. In general, it is often used at a concentration of 20 vol% or less for cost efficiency, but a concentration of 20 vol% or more may be used.
本発明で用いる細胞培養基材は、成分（Ａ）である水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体と、成分（Ｂ）であるＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体と、成分（Ｃ）であるＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体を含むブロック共重合体が被覆される。 The cell culture substrate used in the present invention comprises a temperature-responsive polymer having a lower critical solution temperature (LCST) in the range of 0° C. to 50° C. with respect to component (A), water, and an HLB value of component (B). A block copolymer containing a hydrophilic polymer having a (Griffin method) in the range of 7 or more and 20 or less and a hydrophobic polymer having a HLB value (Gryffin method) in the range of 0 or more and less than 7 as the component (C). Are coated.
本発明の培養方法は培養細胞を一定の比率以上に培養できれば特に限定は無い。一定の比率以上の培養とは培養細胞の増殖が概ね認められることであり、例えば、細胞の播種から７２時間培養後の細胞増殖率が１．４５以上を示すことである。細胞増殖率とは培養後の全細胞数を播種細胞数で割ったものである。 The culture method of the present invention is not particularly limited as long as the cultured cells can be cultured at a certain ratio or more. Culturing at a certain ratio or more means that the growth of cultured cells is generally recognized, and for example, the cell growth rate after culturing for 72 hours from seeding of cells is 1.45 or more. The cell proliferation rate is the total number of cells after culturing divided by the number of seeded cells.
本発明の成分（Ａ）である水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体の繰返し単位とその水に対するＬＣＳＴは、例えば、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−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は成分（Ａ）水溶液の濃度で発現する温度が前後するが、これらの中では、ＬＣＳＴ発現の濃度依存性が低いＮ−イソプロピルアクリルアミドの繰返し単位がより好ましい。本発明における成分（Ａ）は、前記繰り返し単位を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。また温度応答性を有するのであれば、前期温度応答性繰返し単位の他に、異なる繰返し単位を含んでも良い。
The repeating unit of the temperature-responsive polymer having a lower critical solution temperature (LCST) in water of the component (A) of the present invention in the range of 0°C to 50°C and its LCST in water are, for example, N-cyclopropylacrylamide. (LCST=46° C.), N-isopropyl acrylamide (LCST=32° C.), Nn-propyl methacrylamide (LCST=22° C.), N-tetrahydrofurfuryl acrylamide (LCST=28° C.), N-ethoxyethyl acrylamide. (LCST=35° C.), N,N-diethyl acrylamide (LCST=32° C.), N-isopropyl methacrylamide (LCST=44° C.), Nn-propyl methacrylamide (LCST=28° C.), N-tetrahydrofuran Furyl methacrylamide (LCST=35° C.), N-methyl-N-ethyl acrylamide (LCST=56° C.), N-methyl-N-isopropyl acrylamide (LCST=23° C.), N-methyl-Nn-propyl acrylamide (LCST=20° C.), N,N-dimethylaminoethyl methacrylate (LCST=47° C.) or the like can be exemplified. The temperature at which LCST is expressed varies depending on the concentration of the component (A) aqueous solution, but among these, a repeating unit of N-isopropylacrylamide, which has low concentration dependence of LCST expression, is more preferable. As the component (A) in the present invention, only one type of the repeating unit may be used, or two or more types may be used in combination. Further, as long as it has temperature responsiveness, different repeating units may be included in addition to the temperature responsive repeating unit.
本明細書において、ＨＬＢ値（ＨＬＢ；Ｈｙｄｒｏｐｈｉｌｅ−Ｌｉｐｏｐｈｉｌｅ Ｂａｌａｎｃｅ）とは、Ｗ．Ｃ．Ｇｒｉｆｆｉｎ， Ｊｏｕｒｎａｌ ｏｆ ｔｈｅ Ｓｏｃｉｅｔｙ ｏｆ Ｃｏｓｍｅｔｉｃ Ｃｈｅｍｉｓｔｓ， １， ３１１（１９４９）．に記載の、水と油への親和性の程度を表す値であり、０から２０までの値を取り、０に近いほど疎水性が高く、２０に近いほど親水性が高くなる。計算によって決定する方法として、アトラス法、グリフィン法、デイビス法、川上法があるが、本発明においてはグリフィン法で計算した値を使用し、繰り返し単位中の親水部の式量と繰り返し単位の総式量を元に、下記の計算式で求めた。
ＨＬＢ値＝２０×（親水部の式量）÷（総式量）
前述の、各ブロックの繰り返し単位中の親水部の定義として、スルホン部（−ＳＯ３−）、ホスホノ基部（−ＰＯ３−）、カルボキシル基部（−ＣＯＯＨ）、エステル部（−ＣＯＯ−）、アミド部（−ＣＯＮＨ−）、イミド部（−ＣＯＮ−）、アルデヒド基部（−ＣＨＯ）、カルボニル基部（−ＣＯ−）、ヒドロキシル基部（−ＯＨ）、アミノ基部（−ＮＨ２）、アセチル基部（−ＣＯＣＨ３）、エチレンアミン部（−ＣＨ２ＣＨ２Ｎ−）、エチレンオキシ部（−ＣＨ２ＣＨ２Ｏ−）、アルカリ金属イオン、アルカリ土類金属イオン、アンモニウムイオン、ハロゲン化物イオン、酢酸イオンを例示することができる。
In the present specification, the HLB value (HLB; Hydrophile-Lipophile Balance) refers to W.I. C. Griffin, Journal of the Society of Cosmetic Chemists, 1, 311 (1949). It is a value representing the degree of affinity to water and oil described in (3), and takes a value from 0 to 20. The closer to 0, the higher the hydrophobicity, and the closer to 20, the higher the hydrophilicity. Atlas method, Griffin method, Davis method, and Kawakami method are available as methods for determining by calculation.In the present invention, the value calculated by Griffin method is used, and the formula weight of the hydrophilic part in the repeating unit and the total number of repeating units are used. It was calculated by the following calculation formula based on the formula weight.
HLB value=20×(formula amount of hydrophilic part)÷(total formula amount)
Mentioned above, as the definition of the hydrophilic portion in the repeating units of each block, sulfone unit (-SO 3 -), phosphono base (-PO 3 -), a carboxyl base (-COOH), an ester unit (-COO-), an amide part (-CONH-), imido unit (-CON-), aldehyde base (-CHO), carbonyl base (-CO-), a hydroxyl base (-OH), amino base (-NH 2), acetyl base (-COCH 3), exemplified ethyleneamine unit (-CH 2 CH 2 N-), ethyleneoxy unit (-CH 2 CH 2 O-), an alkali metal ion, alkaline earth metal ions, ammonium ions, halide ions, acetate ions can do.
繰り返し単位中の親水部の算出では、親水部を構成する原子が、他の親水部を構成する原子として重複してはならない。繰り返し単位中のＨＬＢ値の算出例を以下に記載した。例えば、２−メトキシエチルアクリレート（分子量：１３０．１４）の場合、親水部は、エステル部が１部およびエチレンオキシ部が１部であり、親水部の分子量は８８．０３であるから、ＨＬＢ値は１３．５である。ｎ−ブチルアクリレート（分子量：１２８．１７）の場合、親水部は、エステル部が１部であり、親水部の分子量は４４．０１であるから、ＨＬＢ値は６．９である。
さらに、本発明のブロック共重合体を構成する各ブロックが、異なるモノマー（モノマー１、モノマー２・・・）からなる共重合体である場合は、それぞれのモノマーが重合して生成する繰り返し単位の共重合体中の比率（ｍｏｌ％）を分析し、下記の計算式で算出することができる。
ＨＬＢ値＝ＨＬＢ値１×比率１＋ＨＬＢ値２×比率２＋・・・・
ここで、ＨＬＢ値１はモノマー１が重合して生成する重合体のＨＬＢ値であり、組成１はモノマー１が重合して生成する繰り返し単位の共重合体中の比率（ｍｏｌ％）であり、ＨＬＢ値２はモノマー２が重合して生成する重合体のＨＬＢ値であり、組成２はモノマー２が重合して生成する繰り返し単位の共重合体中の比率（ｍｏｌ％）である。
ＨＬＢ値の取扱いは、小数点以下第２位を四捨五入して表示される小数点以下第１位までの数字から７以上か未満かを判断する。
In the calculation of the hydrophilic part in the repeating unit, atoms constituting the hydrophilic part must not overlap as atoms forming the other hydrophilic part. An example of calculating the HLB value in the repeating unit is described below. For example, in the case of 2-methoxyethyl acrylate (molecular weight: 130.14), the hydrophilic part has 1 part of the ester part and 1 part of the ethyleneoxy part, and the molecular weight of the hydrophilic part is 88.03. Is 13.5. In the case of n-butyl acrylate (molecular weight: 128.17), the hydrophilic portion has 1 part of the ester portion and the hydrophilic portion has a molecular weight of 44.01, and thus has an HLB value of 6.9.
Furthermore, when each block constituting the block copolymer of the present invention is a copolymer composed of different monomers (monomer 1, monomer 2...), the repeating units formed by polymerization of the respective monomers are The ratio (mol%) in the copolymer can be analyzed and calculated by the following formula.
HLB value=HLB value 1×ratio 1+HLB value 2×ratio 2+...
Here, the HLB value 1 is the HLB value of the polymer produced by the polymerization of the monomer 1, the composition 1 is the ratio (mol%) of the repeating units produced by the polymerization of the monomer 1 in the copolymer, The HLB value 2 is the HLB value of the polymer produced by the polymerization of the monomer 2, and the composition 2 is the ratio (mol%) of the repeating unit produced by the polymerization of the monomer 2 in the copolymer.
Regarding the handling of the HLB value, it is determined whether the number after the decimal point is rounded off to the first decimal place, and the value is 7 or more or less.
本発明の成分（Ｂ）であるＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体は、温度応答性ブロックの水和速度向上に寄与する。優れた水和速度を発現するために、好ましくは９以上２０以下の範囲に有り、さらに好ましくは１０以上２０以下の範囲にある。成分（Ｂ）に含まれる繰返し単位は、例えば、２−メタクリロイルオキシエチルホスホリルコリン（ＨＬＢ値＝１４．２）、ポリエチレングリコールアクリレート（ＨＬＢ値＝１５．３以上）、ポリエチレングリコールメタクリレート（ＨＬＢ値＝１３．７以上）、２−メトキシエチルアクリレート（ＨＬＢ値＝１３．５）、２−メトキシエ
チルメタクリレート（ＨＬＢ値＝１２．２）、テトラヒドロフルフリルアクリレート（ＨＬＢ値＝７．７）、テトラヒドロフルフリルメタクリレート（ＨＬＢ値＝７．１）等が例示できる。ＨＬＢ値が７未満である場合は、温度応答性ブロック（Ａ）の種類によっては優れた水和速度が発現できない。本発明における成分（Ｂ）は、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。
The hydrophilic polymer having an HLB value (Griffin method) of 7 or more and 20 or less, which is the component (B) of the present invention, contributes to the improvement of the hydration rate of the temperature-responsive block. In order to develop an excellent hydration rate, it is preferably in the range of 9 or more and 20 or less, more preferably in the range of 10 or more and 20 or less. The repeating unit contained in the component (B) is, for example, 2-methacryloyloxyethylphosphorylcholine (HLB value=14.2), polyethylene glycol acrylate (HLB value=15.3 or more), polyethylene glycol methacrylate (HLB value=13.3). 7 or more), 2-methoxyethyl acrylate (HLB value=13.5), 2-methoxyethyl methacrylate (HLB value=12.2), tetrahydrofurfuryl acrylate (HLB value=7.7), tetrahydrofurfuryl methacrylate ( HLB value=7.1) etc. can be illustrated. When the HLB value is less than 7, an excellent hydration rate cannot be expressed depending on the type of the temperature responsive block (A). As the component (B) in the present invention, only one type of repeating unit may be used, or two or more types may be used in combination.
本発明の成分（Ｃ）であるＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体は、膜の安定性に寄与する。基材に塗布して水中で剥離しない安定な膜を得るために、好ましくは０以上６以下の範囲にある。成分（Ｃ）に含まれる繰返し単位は、例えば、スチレン（ＨＬＢ値＝０．０）やその誘導体、ｎ−プロピルメタクリレート（ＨＬＢ値＝６．９）、ｎ−ブチルアクリレート（ＨＬＢ値＝６．９）、ｎ−ブチルメタクリレート（ＨＬＢ値＝６．２）等のアルキル（メタ）アクリレート等を例示できる。ＨＬＢ値が７以上である場合は、基材に塗布した場合に水中で剥離しやすく安定な膜を得ることができない。本発明における成分（Ｃ）は、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。 The hydrophobic polymer having an HLB value (Griffin method) in the range of 0 to less than 7 which is the component (C) of the present invention contributes to the stability of the film. In order to obtain a stable film which is applied to a base material and does not peel off in water, it is preferably in the range of 0 or more and 6 or less. The repeating unit contained in the component (C) is, for example, styrene (HLB value=0.0) or a derivative thereof, n-propyl methacrylate (HLB value=6.9), n-butyl acrylate (HLB value=6.9). ), n-butyl methacrylate (HLB value=6.2), and other alkyl (meth)acrylates. When the HLB value is 7 or more, it is difficult to peel off in water when applied to a substrate, and a stable film cannot be obtained. As the component (C) in the present invention, one type of repeating unit may be used, or two or more types may be used in combination.
ブロック共重合体を構成する全繰り返し単位の量に対する成分（Ａ）を構成する繰り返し単位の量の比率は１〜９９ｍｏｌ％であり、成分（Ｂ）を構成する繰返し単位の量の比率は１〜９９ｍｏｌ％であり、成分（Ｃ）を構成する繰返し単位の量の比率は１〜９９ｍｏｌ％である。温度応答性の発現と水和速度の向上を両立するために、成分（Ａ）を構成する繰り返し単位の量の比率は１０〜９０ｍｏｌ％であり、成分（Ｂ）を構成する繰返し単位の量の比率は１〜５０ｍｏｌ％であり、成分（Ｃ）を構成する繰返し単位の量の比率は１０〜７０ｍｏｌ％であることが好ましい。 The ratio of the amount of repeating units constituting the component (A) to the amount of all repeating units constituting the block copolymer is 1 to 99 mol %, and the ratio of the amount of repeating units constituting the component (B) is 1 to It is 99 mol%, and the ratio of the amount of repeating units constituting the component (C) is 1 to 99 mol %. In order to achieve both the expression of temperature responsiveness and the improvement of the hydration rate, the ratio of the amount of repeating units constituting the component (A) is 10 to 90 mol%, and the ratio of the repeating units constituting the component (B) is The ratio is preferably 1 to 50 mol %, and the ratio of the amount of repeating units constituting the component (C) is preferably 10 to 70 mol %.
本特許の温度応答性ブロック共重合体には成分（Ａ）、成分（Ｂ）、成分（Ｃ）の他に異なる成分を導入しても良い。例えば塗布膜による被覆において、膜の安定性を向上するためにｎ−ブチルアルコール繰返し単位からなる成分を導入しても良い。特許の温度応答性ブロック共重合体の数平均分子量（Ｍｎ）は３，０００以上１，０００，０００以下の範囲にあり、好ましくは１０，０００以上１，０００，０００以下である。３，０００未満の場合は膜の温度応答性が発現しにくい。 Different components may be introduced into the temperature responsive block copolymer of this patent in addition to the component (A), the component (B) and the component (C). For example, in coating with a coating film, a component composed of n-butyl alcohol repeating units may be introduced in order to improve the stability of the film. The number average molecular weight (Mn) of the patented temperature responsive block copolymer is in the range of 3,000 or more and 1,000,000 or less, preferably 10,000 or more and 1,000,000 or less. When it is less than 3,000, the temperature responsiveness of the film is difficult to develop.
ブロック共重合体の合成方法としては特に限定はなく、例えば、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、共重合する方法を用いることができる。 The method for synthesizing the block copolymer is not particularly limited, and examples thereof include "Radical Polymerization Handbook", p. The method of copolymerizing can be used using the living radical polymerization technique of 161-225 (2010).
ブロック共重合体の溶液は、膜作製のための表面処理剤として用いることができる。表面処理剤に用いる溶媒は、ブロック共重合体が溶解するものであれば特に限定はないが、コートする基板を侵食しない溶媒が好ましい。例えば、基材の材質がポリスチレンである場合はメタノール、エタノール、１−プロパノール、２−プロパノール、２−メトキシエタノールなどが挙げられ、さらに好ましくは、沸点が高いため高温に加温できるエタノール、１−プロパノール、２−プロパノール、２−メトキシエタノールが挙げられる。また表面処理剤の濃度は特に限定はないが、塗りムラの発生を低減できることから好ましくは０．０１ｗｔ％から１０ｗｔ％、より好ましくは０．０３ｗｔ％から１ｗｔ％である。 The solution of the block copolymer can be used as a surface treatment agent for forming a film. The solvent used for the surface treatment agent is not particularly limited as long as it can dissolve the block copolymer, but a solvent that does not corrode the substrate to be coated is preferable. For example, when the material of the base material is polystyrene, methanol, ethanol, 1-propanol, 2-propanol, 2-methoxyethanol and the like can be mentioned, and more preferably ethanol that can be heated to a high temperature because of its high boiling point, 1- Propanol, 2-propanol, 2-methoxyethanol are mentioned. The concentration of the surface treatment agent is not particularly limited, but is preferably 0.01 wt% to 10 wt%, and more preferably 0.03 wt% to 1 wt% because the occurrence of coating unevenness can be reduced.
上記表面処理剤を各種基材に塗布した後、乾燥することによって得られる本発明の膜の単位面積当たりの成分（Ａ）量は、温度応答性の発現を向上するために０．３μｇ／ｃｍ２以上であることが好ましく、０．４μｇ／ｃｍ２以上であることがより好ましい。上限は特に制限はないが、細胞が接着しやすい１０μｇ／ｃｍ２以下が好ましい。 The amount of the component (A) per unit area of the film of the present invention obtained by applying the surface treatment agent to various substrates and then drying is 0.3 μg/cm in order to improve the expression of temperature responsiveness. It is preferably 2 or more, and more preferably 0.4 μg/cm 2 or more. The upper limit is not particularly limited, but is preferably 10 μg/cm 2 or less where cells easily adhere.
また、本発明の膜厚に特に制限はないが、例えば１ｎｍ以上であってもよく、１０ｎｍ以上であってもよい。また、１μｍ以下であってもよく、１０μｍ以下であってもよい。 The film thickness of the present invention is not particularly limited, but may be, for example, 1 nm or more, or 10 nm or more. Further, it may be 1 μm or less, or 10 μm or less.
本発明における表面処理剤を基材にコートする方法としては特に限定はないが、例えば、ドロップキャスト、はけ塗り、ディップコーティング、スピンコーティング、バーコーティング、流し塗り、スプレー塗装、ロール塗装、エアーナイフコーティング、ブレードコーティングなど通常知られている各種の方法を用いることが可能であるが、均一にコートしやすいスピンコーティングが好ましい。 The method for coating the substrate with the surface treatment agent in the present invention is not particularly limited, and examples thereof include drop casting, brush coating, dip coating, spin coating, bar coating, flow coating, spray coating, roll coating, and air knife. Although various known methods such as coating and blade coating can be used, spin coating is preferable because it can be uniformly coated.
本発明で用いる細胞としては、好ましくは骨髄由来間葉系幹細胞であるが、温度降下による刺激付与前の表面に接着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、種々の組織に存在する幹細胞、さらにはそれらから分化誘導した細胞等を用いることができる。これら以外でも、血液、リンパ液、髄液、喀痰、尿又は便に含まれる細胞（生細胞）や、体内あるいは環境中に存在する微生物、ウイルス、原虫等を例示できる。 The cells used in the present invention are preferably bone marrow-derived mesenchymal stem cells, but are not particularly limited as long as they can adhere to the surface before stimulus application due to temperature decrease. For example, in addition to various cultured cell lines such as Chinese hamster ovary-derived CHO cells, mouse connective tissue L929 cells, human embryonic kidney-derived cells HEK293 cells and human cervical cancer-derived HeLa cells, for example, each tissue and organ in vivo is constituted. Epithelial cells and endothelial cells, contractile skeletal muscle cells, smooth muscle cells, cardiomyocytes, neuronal cells that make up the nervous system, glial cells, fibroblasts, liver parenchymal cells involved in the metabolism of the body, non-parenchymal liver cells As the adipocytes, cells having differentiating ability, stem cells existing in various tissues, and cells differentiated from them can be used. Other than these, cells (live cells) contained in blood, lymph, cerebrospinal fluid, sputum, urine or feces, microorganisms existing in the body or environment, viruses, protozoa and the like can be exemplified.
本発明の培養細胞をＬＣＳＴ以下の温度に冷却する方法としては特に限定はなく、冷却された培地で培地交換することや冷所保管が例示される。冷却された培地での培地交換は特に限定はなく、ピペットを用いて温かい培地を抜き取り、その後冷却した培地を注ぐ方法が例示される。 The method for cooling the cultured cells of the present invention to a temperature of LCST or lower is not particularly limited, and examples include replacement of the medium with a cooled medium and storage in a cold place. The medium exchange with the cooled medium is not particularly limited, and a method of extracting a warm medium with a pipette and then pouring the cooled medium is exemplified.
本発明の冷却時間は特に限定はないが、細胞剥離の発現を明瞭にするために２分以上が好ましく、冷却による細胞へのダメージを低減するために２０分以下が好ましい。 The cooling time of the present invention is not particularly limited, but it is preferably 2 minutes or more for clarifying the expression of cell exfoliation, and 20 minutes or less for reducing damage to cells due to cooling.
本発明の冷却後に行うピペッティングは、細胞−細胞間の接着を解くことができるため行うことが好ましい。ピペッティング方法は特に限定はないが、例えば１ｍＬピペットを用いて行う。細胞に物理的な刺激を与えるため、ピペッティング回数は少ない方が好ましく、６ｃｍΦディッシュであれば１０回程度であることが好ましい。 The pipetting performed after cooling according to the present invention is preferably performed because cell-cell adhesion can be released. The pipetting method is not particularly limited, but for example, a 1 mL pipette is used. Since the cells are physically stimulated, it is preferable that the number of pipettings is small, and it is preferably about 10 times in the case of 6 cmΦ dish.
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. In addition, unless otherwise stated, commercially available reagents were used.
＜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／ＬＩ）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。
<Composition of block copolymer>
It was determined by proton nuclear magnetic resonance spectroscopy ( 1 H-NMR) spectrum analysis using a nuclear magnetic resonance measuring device (trade name JNM-ECZ400S/LI manufactured by JEOL Ltd.).
＜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 ＴＳＫｇｅｌ Ｓｕｐｅｒ ＡＷＭ−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。
<Molecular weight and molecular weight distribution of block copolymer>
The weight average molecular weight (Mw), number average molecular weight (Mn), and molecular weight distribution (Mw/Mn) were measured by gel permeation chromatography (GPC). Tosoh Corporation HLC-8320GPC was used as the GPC device, two Tosoh TSKgel Super AWM-H columns were used, the column temperature was set to 40° C., and the eluent was 10 mM trifluoroacetate-containing 10 mM trifluoroacetate. It was measured using sodium acetate. The measurement sample was prepared and measured at 1.0 mg/mL. For the calibration curve of molecular weight, polymethylmethacrylate (manufactured by Polymer Laboratories) of known molecular weight was used.
＜細胞数の計測＞
細胞を播種し、培養３日後に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分間静置した。１０分後、１ｍＬピペッターを用いて１ｍＬの容量で、５回培養基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、１５ｍｌチューブに培養液ごと細胞を回収し、１６０ｒｃｆ、２５℃、５分の条件で遠心後、上清を除き、培養液を５００μＬ加え懸濁した。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 Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｃｈａｍｂｅｒ Ｓｌｉｄ）に添加し自動セルカウンター（Ｔｈｅｒｍｏ 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。これを冷却後回収細胞数とした。培養液を回収後、トリプシンを用いて培養基材に残った細胞を１５ｍｌチューブに回収し、１６０ｒｃｆ、２５℃、５分の条件で遠心後、上清を除き、培養液を１００μＬ加え懸濁し、同様の方法で細胞数を測定し、これを冷却後回収細胞数と足し合わせたものを全細胞数とした。細胞数は対数表記で測定され、小数点以下第二位までを結果として用いた。
<Measurement of cell number>
The cells were seeded, and after 3 days of culturing, the culture solution was removed, a new culture solution cooled to 4° C. was added, and the mixture was allowed to stand at room temperature for 10 minutes. After 10 minutes, using a 1 mL pipetter, pipetting was carried out 5 times in a volume of 1 mL so that the culture solution was applied to the entire culture surface of the culture substrate, and then the cells were collected together with the culture solution in a 15 ml tube. After centrifugation at 5° C. for 5 minutes, the supernatant was removed, and 500 μL of the culture solution was added to suspend. From the cell suspension, 10 μL was added to a slide for measuring cell number (Thermo Fisher Scientific Co., Ltd., trade name Count Cell Counting Chamber Slide), and an automatic cell counter (Thermo Fisher Scientific Co., Ltd., trade name Count). The cell number was measured using the Trademark) II). This was used as the number of recovered cells after cooling. After collecting the culture solution, cells remaining in the culture substrate were collected in a 15 ml tube using trypsin, centrifuged at 160 rcf, 25° C. for 5 minutes, the supernatant was removed, and 100 μL of the culture solution was added to suspend the cells. The number of cells was measured by the same method, and this was added to the number of cells recovered after cooling, which was taken as the total number of cells. The cell number was measured in logarithmic notation, and the second decimal place was used as the result.
＜細胞回収率の計算＞
細胞数の計測結果を用いて、細胞回収率（％）は冷却後回収細胞数を全細胞数で割って計算した。細胞回収率は小数点以下第一位を四捨五入して表示した。
<Calculation of cell recovery rate>
Using the measurement result of the number of cells, the cell recovery rate (%) was calculated by dividing the number of cells recovered after cooling by the total number of cells. The cell recovery rate was rounded off to the first decimal place.
＜ブロック共重合体の合成例＞
１００ｍＬ２口フラスコに成分（Ｂ）として２−メトキシエチルアクリレート（ＭＥＡ，ＨＬＢ値＝１１．５）０．６５０ｇ（５ｍｍｏｌ）を加え、さらに４−シアノ−４−［（ドデシルスルフォニルチオカルボニル）スルフォニル］ペンタノイックアシッドを３１．８ｍｇ（１００μｍｏｌ）とアゾビスイソブチロニトリル１．６ｍｇ（１０μｍｏｌ）とｔｅｒｔ−ブタノール１０ｍＬを加え、アルゴンガス置換後、６２℃で２４時間加熱撹拌した。
１回目の加熱撹拌後、上記に成分（Ｃ）として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ｔｅｒｔ−ブタノール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、上記に成分（Ａ）として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−ブタノール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１１．８×１０４、Ｍｗ／Ｍｎは１．４５であった。
<Synthesis example of block copolymer>
0.650 g (5 mmol) of 2-methoxyethyl acrylate (MEA, HLB value=11.5) was added as a component (B) to a 100 mL 2-necked flask, and further 4-cyano-4-[(dodecylsulfonylthiocarbonyl)sulfonyl]penta. 31.8 mg (100 μmol) of noic acid, 1.6 mg (10 μmol) of azobisisobutyronitrile and 10 mL of tert-butanol were added, and the mixture was heated and stirred at 62° C. for 24 hours after replacing with argon gas.
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 as the component (C), and further 1.6 mg (10 μmol) of azobisisobutyronitrile. And tert-butanol (5 mL) were added, and the atmosphere was replaced with argon gas, followed by heating with stirring at 62° C. for 48 hours.
After the second heating and stirring, 7.355 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST=32° C.) was added to the above as the component (A), and further 1.6 mg (10 μmol) of azobisisobutyronitrile and tert. -Butanol (35 mL) was added, the mixture was replaced with argon gas, and then heated and stirred at 62°C for 48 hours.
After heating and stirring for the third time, the reaction solution was reprecipitated and purified 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 by 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 11.8×10 4 , and Mw/Mn was 1.45.
＜表面処理剤の調製＞
ブロック共重合体８０ｍｇに２−メトキシエタノールを１９．９２０ｇ添加し、撹拌で全て溶解させ、ブロック共重合体の０．４０ｗｔ％表面処理剤を調製した。
<Preparation of surface treatment agent>
19.920 g of 2-methoxyethanol was added to 80 mg of the block copolymer, and all were dissolved by stirring to prepare a 0.40 wt% surface treatment agent of the block copolymer.
＜コート基材の調製＞
ＩＷＡＫＩ組織培養用ディッシュ（φ６ｃｍ）の中央に表面処理剤を１００μＬ加え、スピンコータ―（ミカサ製、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コートした基材を調製した。
<Preparation of coated substrate>
100 μL of a surface treatment agent was added to the center of an IWAKI tissue culture dish (φ6 cm), and spin coating was performed using a spin coater (Mikasa, trade name MS-B200) at a rotation speed of 2,000 rpm and a rotation time of 60 seconds. Thus, a base material coated with the block copolymer was prepared.
実施例１
合成例記載の温度応答性ブロック共重合体が被覆された培養基材（以降、培養基材１）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４．０×１０５ｃｅｌｌｓ／ｄｉｓｈ播種し、３７℃、ＣＯ２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２ｖｏｌ％含むダルベッコ・フォークト変法イーグル最小必須培地（２ｖｏｌ％ＦＢＳ／ＤＭＥＭ）を用いた。３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、１ｍＬピペッターを用いて１ｍＬの容量で、５回培養基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細胞数を測定した所、冷却によって回収できた細胞数（冷却後回収細胞数）は７．２３×１０５ｃｅｌｌｓであった。培養液を回収後、トリプシンを用いて培養基材に残った細胞を回収し全細胞数を測定した所７．２５×１０５ｃｅｌｌｓであった。以上より細胞増殖率は１．８１であり一定の比率以上の細胞増殖が認められた。冷却処理による細胞回収率は９９．７％であった。
Example 1
A culture substrate coated with the temperature-responsive block copolymer described in Synthesis Example (hereinafter, culture substrate 1) was added to bone marrow-derived human mesenchymal stem cells (Lonza Japan Co., Ltd., Product Code: PT-2501, Lot Number: 0000603525) was seeded at 4.0×10 5 cells/dish and cultured at 37° C. at a CO 2 concentration of 5%. The culture medium used was Dulbecco Voigt's modified Eagle's minimum essential medium (2 vol% FBS/DMEM) containing 2 vol% fetal bovine serum (produced in Colombia). After culturing for 3 days, the culture solution was removed, a culture solution newly cooled to 4° C. was added, and the mixture was cooled at room temperature for 10 minutes. After 10 minutes, using a 1 mL pipetter, pipetting was performed 5 times with a volume of 1 mL so that the culture solution was applied to the entire culture surface of the culture substrate, and then the culture solution was collected together with the cells and the number of cells was measured. The number of cells that could be recovered by cooling (the number of cells recovered after cooling) was 7.23×10 5 cells. After the culture solution was collected, the cells remaining on the culture substrate were collected using trypsin, and the total cell number was measured to be 7.25×10 5 cells. From the above, the cell growth rate was 1.81, and cell growth above a certain ratio was observed. The cell recovery rate by the cooling treatment was 99.7%.
実施例２
５ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は８．７４×１０５ｃｅｌｌｓであり、全細胞数は８．７６×１０５ｃｅｌｌｓであった。細胞増殖率は２．１９であり一定の比率以上の細胞増殖が認められた。細胞回収率は９９．８％であった。
Example 2
The cell recovery rate was evaluated in the same manner as in Example 1 except that 5 vol% FBS/DMEM was used. The number of recovered cells after cooling was 8.74×10 5 cells, and the total number of cells was 8.76×10 5 cells. The cell growth rate was 2.19, and cell growth above a certain ratio was observed. The cell recovery rate was 99.8%.
実施例３
１０ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は１．０４×１０６ｃｅｌｌｓであり、全細胞数は１．０５×１０６ｃｅｌｌｓであった。細胞増殖率は２．６３であり一定の比率以上の細胞増殖が認められた。細胞回収率は９９．０％であった。
Example 3
The cell recovery rate was evaluated in the same manner as in Example 1 except that 10 vol% FBS/DMEM was used. The number of cells recovered after cooling was 1.04×10 6 cells, and the total number of cells was 1.05×10 6 cells. The cell growth rate was 2.63, and cell growth above a certain ratio was observed. The cell recovery rate was 99.0%.
実施例４
１５ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は１．３５×１０６ｃｅｌｌｓであり、全細胞数は１．３５×１０６ｃｅｌｌｓであった。細胞増殖率は３．３８であり一定の比率以上の細胞増殖が認められた。細胞回収率は１００．０％であった。
Example 4
The cell recovery rate was evaluated by the same method as in Example 1 except that 15 vol% FBS/DMEM was used. The number of recovered cells after cooling was 1.35×10 6 cells, and the total number of cells was 1.35×10 6 cells. The cell growth rate was 3.38, and cell growth above a certain ratio was observed. The cell recovery rate was 100.0%.
実施例５
２０ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は１．４０×１０６ｃｅｌｌｓであり、全細胞数は１．４０×１０６ｃｅｌｌｓであった。細胞増殖率は３．５０であり一定の比率以上の細胞増殖が認められた。細胞回収率は１００．０％であった。
Example 5
The cell recovery rate was evaluated in the same manner as in Example 1 except that 20 vol% FBS/DMEM was used. The number of recovered cells after cooling was 1.40×10 6 cells, and the total number of cells was 1.40×10 6 cells. The cell growth rate was 3.50, and cell growth above a certain ratio was observed. The cell recovery rate was 100.0%.
比較例１
温度応答性培養基材としてＵｐＣｅｌｌ（（株）セルシード製）を用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は７．０３×１０５ｃｅｌｌｓであり、全細胞数は７．３５×１０５ｃｅｌｌｓであった。細胞増殖率は１．８４であり一定の比率以上の細胞増殖が認められた。細胞回収率は９５．６％であり、実施例１よりも回収率が低くなった。
Comparative Example 1
The cell recovery rate was evaluated by the same method as in Example 1 except that UpCell (manufactured by Cell Seed Co., Ltd.) was used as the temperature-responsive culture substrate. The number of cells recovered after cooling was 7.03×10 5 cells, and the total number of cells was 7.35×10 5 cells. The cell growth rate was 1.84, and cell growth above a certain ratio was observed. The cell recovery rate was 95.6%, which was lower than that in Example 1.
比較例２
５ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は比較例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は６．２５×１０５ｃｅｌｌｓであり、全細胞数は８．５５×１０５ｃｅｌｌｓであった。細胞増殖率は２．１４であり一定の比率以上の細胞増殖が認められた。細胞回収率は７３．１％であり、実施例２よりも回収率が低くなった。
Comparative example 2
The cell recovery rate was evaluated in the same manner as in Comparative Example 1 except that 5 vol% FBS/DMEM was used. The number of cells recovered after cooling was 6.25×10 5 cells, and the total number of cells was 8.55×10 5 cells. The cell growth rate was 2.14, and cell growth above a certain ratio was observed. The cell recovery rate was 73.1%, which was lower than that in Example 2.
比較例３
１０ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は比較例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は７．３２×１０５ｃｅｌｌｓであり、全細胞数は１．１６×１０６ｃｅｌｌｓであった。細胞増殖率は２．９０であり一定の比率以上の細胞増殖が認められた。細胞回収率は６３．１％であり、実施例３よりも回収率が低くなった。
Comparative Example 3
The cell recovery rate was evaluated in the same manner as in Comparative Example 1 except that 10 vol% FBS/DMEM was used. The number of recovered cells after cooling was 7.32×10 5 cells, and the total number of cells was 1.16×10 6 cells. The cell growth rate was 2.90, and cell growth above a certain ratio was observed. The cell recovery rate was 63.1%, which was lower than that in Example 3.
比較例４
１５ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は比較例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は５．７３×１０５ｃｅｌｌｓであり、全細胞数は１．１９×１０６ｃｅｌｌｓであった。細胞増殖率は２．９８であり一定の比率以上の細胞増殖が認められた。細胞回収率は４８．２％であり、実施例４よりも回収率が低くなった。
Comparative Example 4
The cell recovery rate was evaluated in the same manner as in Comparative Example 1 except that 15 vol% FBS/DMEM was used. The number of cells recovered after cooling was 5.73×10 5 cells, and the total number of cells was 1.19×10 6 cells. The cell growth rate was 2.98, and cell growth above a certain ratio was observed. The cell recovery rate was 48.2%, which was lower than that in Example 4.
比較例５
２０ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は比較例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は５．６７×１０５ｃｅｌｌｓであり、全細胞数は１．３９×１０６ｃｅｌｌｓであった。細胞増殖率は３．４８であり一定の比率以上の細胞増殖が認められた。細胞回収率は４０．８％であり、実施例５よりも回収率が低くなった。
Comparative Example 5
The cell recovery rate was evaluated in the same manner as in Comparative Example 1 except that 20 vol% FBS/DMEM was used. The number of cells recovered after cooling was 5.67×10 5 cells, and the total number of cells was 1.39×10 6 cells. The cell growth rate was 3.48, and cell growth above a certain ratio was observed. The cell recovery rate was 40.8%, which was lower than that in Example 5.
比較例６
１ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は実施例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は５．６１×１０５ｃｅｌｌｓであり、全細胞数は５．６５×１０５ｃｅｌｌｓであった。細胞増殖率は１．４１であり一定の比率以上の細胞増殖が認められなかった。細胞回収率は９９．３％であった。
Comparative Example 6
The cell recovery rate was evaluated in the same manner as in Example 1 except that 1 vol% FBS/DMEM was used. The number of recovered cells after cooling was 5.61×10 5 cells, and the total number of cells was 5.65×10 5 cells. The cell growth rate was 1.41, and cell growth above a certain ratio was not observed. The cell recovery rate was 99.3%.
比較例７
１ｖｏｌ％ＦＢＳ／ＤＭＥＭを用いたこと以外は比較例１と同様の方法で細胞回収率を評価した。冷却後回収細胞数は５．５９×１０５ｃｅｌｌｓであり、全細胞数は５．６３×１０５ｃｅｌｌｓであった。細胞増殖率は１．４１であり一定の比率以上の細胞増殖が認められなかった。細胞回収率は９９．３％であった。
Comparative Example 7
The cell recovery rate was evaluated in the same manner as in Comparative Example 1 except that 1 vol% FBS/DMEM was used. The number of cells recovered after cooling was 5.59×10 5 cells, and the total number of cells was 5.63×10 5 cells. The cell growth rate was 1.41, and cell growth above a certain ratio was not observed. The cell recovery rate was 99.3%.
Claims (8)
（Ａ）水に対する下限臨界溶解温度（ＬＣＳＴ）が０℃〜５０℃の範囲にある温度応答性重合体。
（Ｂ）ＨＬＢ値（グリフィン法）が７以上２０以下の範囲にある親水性重合体。
（Ｃ）ＨＬＢ値（グリフィン法）が０以上７未満の範囲にある疎水性重合体。 A method for culturing adherent cells using a culture substrate having a serum concentration in a medium of 2 vol% or more and coated with a temperature-responsive block copolymer containing the following components (A) to (C), A method for culturing, which comprises recovering cells by cooling them to a temperature of LCST or lower.
(A) A temperature-responsive polymer having a lower critical solution temperature (LCST) in water in the range of 0°C to 50°C.
(B) A hydrophilic polymer having an HLB value (Griffin method) of 7 or more and 20 or less.
(C) A hydrophobic polymer having an HLB value (Griffin method) in the range of 0 or more and less than 7.
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| WO2022202911A1 (en) | 2021-03-26 | 2022-09-29 | 東ソー株式会社 | Temperature-responsive polymer surface treatment agent |
| WO2024075722A1 (en) * | 2022-10-04 | 2024-04-11 | 東ソー株式会社 | Cell culture method using temperature-responsive microcarrier, and beads for temperature-responsive cell culture |
| WO2024253070A1 (en) * | 2023-06-06 | 2024-12-12 | 東ソー株式会社 | Temperature-responsive nonwoven fabric |
| EP4299198A4 (en) * | 2021-03-29 | 2025-03-12 | Tosoh Corporation | Surface treatment agent |
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| WO2018168983A1 (en) * | 2017-03-15 | 2018-09-20 | テルモ株式会社 | Method for producing sheet-shaped cell culture |
| JP2018174919A (en) * | 2017-04-12 | 2018-11-15 | 東ソー株式会社 | Block copolymer and surface treating agent using the same |
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| JP2018087316A (en) * | 2016-08-03 | 2018-06-07 | 東ソー株式会社 | Block copolymer and surface treatment agent using same |
| WO2018168983A1 (en) * | 2017-03-15 | 2018-09-20 | テルモ株式会社 | Method for producing sheet-shaped cell culture |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2022202911A1 (en) | 2021-03-26 | 2022-09-29 | 東ソー株式会社 | Temperature-responsive polymer surface treatment agent |
| EP4299716A4 (en) * | 2021-03-26 | 2025-07-09 | Tosoh Corp | TEMPERATURE-REACTIVE POLYMER SURFACE TREATMENT AGENT |
| EP4299198A4 (en) * | 2021-03-29 | 2025-03-12 | Tosoh Corporation | Surface treatment agent |
| WO2024075722A1 (en) * | 2022-10-04 | 2024-04-11 | 東ソー株式会社 | Cell culture method using temperature-responsive microcarrier, and beads for temperature-responsive cell culture |
| WO2024253070A1 (en) * | 2023-06-06 | 2024-12-12 | 東ソー株式会社 | Temperature-responsive nonwoven fabric |
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