JP2020074704A - Eluate testing method - Google Patents
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Abstract
Description
本発明は、インビトロで培養した細胞を含む培地に溶出した高分子化合物を定量する方法に関する。 The present invention relates to a method for quantifying a polymer compound eluted in a medium containing cells cultured in vitro.
インビトロでの細胞培養では、従来から用いられている培養基材に機能性高分子をコーティングした培養基材が用いられている。これら素材の溶出は培養細胞に悪影響を与える可能性が懸念されることから、悪影響を与えないことを担保するための手段として溶出物試験がある。 In in vitro cell culture, a culture substrate obtained by coating a conventionally used culture substrate with a functional polymer is used. Since it is feared that the elution of these materials may adversely affect the cultured cells, an eluate test is available as a means for ensuring that they do not have an adverse effect.
溶出物試験法は、第十七改正日本薬局方一般試験法＿プラスチック医薬品容器試験法＿溶出物試験に記載されている。本記載の方法によれば、例えば、幅０．５ｃｍに細断した約１，２００ｃｍ２の試料（φ１０ｃｍディッシュ１０枚相当）を、水２００ｍＬと共にガラス製瓶に加え、７０℃２４時間加熱した液を試験液として用いる。試験液は空試験液と共に、泡立ち試験、ｐＨ試験、過マンガン酸カリウム還元性試験、紫外吸収スペクトル試験、蒸発残留物試験が行われる。蒸発残留物試験では、試験液２０ｍＬを水浴上で蒸発乾固し、残留物を１０５℃で１時間乾燥し、その質量を量ることで、不揮発性溶出物を定量評価できる。しかしながら、不揮発性蒸発物が１ｍｇを下回る場合は定量が困難であった。また本溶出物試験法で溶出しなくとも、特許文献１記載の技術では、細胞を基材から回収する際に高分子膜が細胞ごと剥離するため、従来の溶出物試験法では細胞に対し必ずしも安全であることは言えなかった。 The leachate test method is described in 17th Amended Japanese Pharmacopoeia General Test Method_Plastic Drug Container Test Method_Eluate Test. According to the method of the present description, for example, a liquid of about 1,200 cm 2 sample (equivalent to 10 φ10 cm dishes) shredded to a width of 0.5 cm was added to a glass bottle together with 200 mL of water, and heated at 70 ° C. for 24 hours. Is used as a test solution. The test solution is subjected to a foaming test, a pH test, a potassium permanganate reducing property test, an ultraviolet absorption spectrum test, and an evaporation residue test together with the blank test solution. In the evaporation residue test, 20 mL of the test solution is evaporated to dryness on a water bath, the residue is dried at 105 ° C. for 1 hour, and the mass thereof is weighed, whereby the nonvolatile eluate can be quantitatively evaluated. However, it was difficult to quantify when the nonvolatile evaporate was less than 1 mg. Further, even if the eluate test method does not elute, in the technique described in Patent Document 1, the polymer membrane is peeled off together with the cells when the cells are collected from the base material. I couldn't say it was safe.
本発明の目的は、インビトロで培養した細胞を含む培地に含まれる培養基材由来の高分子化合物を定量評価する方法を提供することにある。 An object of the present invention is to provide a method for quantitatively evaluating a polymeric compound derived from a culture substrate contained in a medium containing cells cultured in vitro.
本発明者らは、以上の点を鑑み、鋭意研究を重ねた結果、インビトロで培養した細胞を含む培地に溶出した１μｇ／ｍＬ以上１０ｍｇ／ｍＬ以下の培養基材由来の高分子化合物を、有機溶媒を用いて高分子化合物を培地から抽出する工程、サイズ排除クロマトグラフィーを用いて高分子化合物を分離する工程、高分子化合物由来の検出強度から高分子化合物を定量する工程、それぞれを含む方法で定量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＞ インビトロで培養した細胞を含む培地に溶出した１μｇ／ｍＬ以上１０ｍｇ／ｍＬ以下の培養基材由来の高分子化合物を、下記（Ａ）〜（Ｃ）の工程を含む方法で定量する方法。
（Ａ）有機溶媒を用いて高分子化合物を培地から抽出する工程。
（Ｂ）サイズ排除クロマトグラフィーを用いて高分子化合物を分離する工程。
（Ｃ）高分子化合物由来の検出強度から高分子化合物を定量する工程。
＜２＞ ＜１＞記載の（Ａ）工程で、冷却で水を凍結させ、高分子化合物が抽出された有機溶液のみを回収する工程を含む＜１＞記載の方法。
＜３＞ ＜１＞記載の有機溶媒が、水と非相溶性であることを特徴とする＜１＞〜＜２＞いずれかに記載の方法。
＜４＞ ＜１＞記載の（Ｂ）工程で、試料の注入量が１０μＬ以上５００μＬ以下であることを特徴とする＜１＞〜＜３＞のいずれかに記載の方法。
＜５＞ ＜１＞記載の（Ｂ）工程で、検出器が示差屈折率検出器であること特徴とする＜１＞〜＜４＞いずれかに記載の方法。
＜６＞ ＜１＞記載の（Ｃ）工程で、濃度が既知の高分子化合物溶液の試料から作成した検量線を用いて定量する＜１＞〜＜５＞いずれかに記載の方法。
In view of the above points, the inventors of the present invention have conducted extensive studies, and as a result, the polymer compound derived from a culture substrate of 1 μg / mL or more and 10 mg / mL or less eluted in a medium containing cells cultured in vitro was A method comprising a step of extracting the polymer compound from the medium using a solvent, a step of separating the polymer compound using size exclusion chromatography, a step of quantifying the polymer compound from the detection intensity derived from the polymer compound, The inventors have found that it can be quantified and have completed the present invention. That is, the present invention includes the following aspects.
<1> A method for quantifying a polymeric substrate-derived polymer compound of 1 μg / mL or more and 10 mg / mL or less eluted in a medium containing cells cultured in vitro by a method including the following steps (A) to (C) ..
(A) A step of extracting a polymer compound from a medium using an organic solvent.
(B) A step of separating a polymer compound using size exclusion chromatography.
(C) A step of quantifying the polymer compound from the detection intensity derived from the polymer compound.
<2> The method according to <1>, which comprises the step (A) described in <1> including freezing water by cooling and collecting only the organic solution from which the polymer compound has been extracted.
<3> The method according to any one of <1> and <2>, wherein the organic solvent described in <1> is incompatible with water.
<4> The method according to any one of <1> to <3>, wherein in the step (B) described in <1>, the injection amount of the sample is 10 μL or more and 500 μL or less.
<5> The method according to any one of <1> to <4>, wherein in the step (B) described in <1>, the detector is a differential refractive index detector.
<6> The method according to any one of <1> to <5>, wherein in the step (C) described in <1>, quantification is performed using a calibration curve prepared from a sample of a polymer compound solution having a known concentration.
本発明によれば、インビトロで培養した細胞を含む培地に溶出した１μｇ／ｍＬ以上１０ｍｇ／ｍＬ以下の培養基材由来の高分子化合物を定量できる。 According to the present invention, it is possible to quantify 1 μg / mL or more and 10 mg / mL or less of a polymer compound derived from a culture substrate, which is eluted in a medium containing cells cultured in vitro.
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、インビトロで培養した細胞を含む培地に溶出した１μｇ／ｍＬ以上１０ｍｇ／ｍＬ以下の培養基材由来の高分子化合物を、有機溶媒を用いて高分子化合物を培地から抽出する工程、サイズ排除クロマトグラフィーを用いて高分子化合物を分離する工程、高分子化合物由来の検出強度から高分子化合物を定量する工程、それぞれを含む方法で定量できる。 The present invention comprises a step of extracting a polymer compound derived from a culture substrate of 1 μg / mL or more and 10 mg / mL or less, which is eluted in a medium containing cells cultured in vitro, from the medium using an organic solvent, and a size. It can be quantified by a method including a step of separating a high molecular compound using exclusion chromatography and a step of quantifying the high molecular compound from the detection intensity derived from the high molecular compound.
本発明の培養基材由来の高分子化合物は特に限定はないが、一般的な培養基材として用いられるポリスチレンやポリエチレンに加え、これらの表面にコーティングした高分子化合物を例示できる。表面にコーティングする高分子化合物としては、一例として、温度応答性を有するＮ−イソプロピルアクリルアミドからなる、およびまたは、を含む合成高分子に加え、糖鎖やタンパク、アミノ酸や核酸などの生体由来高分子を例示できる。 The polymer compound derived from the culture substrate of the present invention is not particularly limited, and in addition to polystyrene and polyethylene used as a general culture substrate, polymer compounds coated on the surface thereof can be exemplified. Examples of the polymer compound to be coated on the surface include, as an example, a synthetic polymer including and / or containing temperature-responsive N-isopropylacrylamide, as well as a biopolymer such as a sugar chain, protein, amino acid or nucleic acid Can be illustrated.
本発明の高分子化合物は特に限定はないが、サイズ排除クロマトグラフィーで分離できるのであれば、合成高分子でも生体高分子で有っても良い。高分子化合物の分子量はサイズ排除クロマトグラフィーで分離するため、５００以上１０，０００，０００以下であり、より好ましくは１，０００以上１，０００，０００である。また含まれる高分子化合物の種類は１種類であっても良く、２種類以上であっても良い。 The polymer compound of the present invention is not particularly limited, but may be a synthetic polymer or a biopolymer as long as it can be separated by size exclusion chromatography. The molecular weight of the polymer compound is 500 or more and 10,000,000 or less, and more preferably 1,000 or more and 1,000,000 because it is separated by size exclusion chromatography. The type of the polymer compound contained may be one type or two or more types.
本発明のインビトロで培養した細胞を含む培地には、細胞を含んでいれば特に制限はない。培地には細胞の他に塩や有機化合物、生体材料を含んでも良い。細胞の種類は特に限定はないが、例えば、ヒト骨髄由来間葉系幹細胞、ヒト脂肪組織由来間葉系幹細胞、ヒト肺由来繊維芽細胞、ヒト皮膚繊維芽細胞、チャイニーズハムスター卵巣由来ＣＨＯ細胞、マウス結合組織Ｌ９２９細胞、ヒト胎児腎臓由来細胞ＨＥＫ２９３細胞、ヒト子宮頸癌由来ＨｅＬａ細胞等の種々の培養細胞株に加え、生体内の各組織や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など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 medium containing cells cultured in vitro of the present invention is not particularly limited as long as it contains cells. In addition to cells, the medium may contain salts, organic compounds, and biomaterials. The type of cell is not particularly limited, and examples thereof include human bone marrow-derived mesenchymal stem cells, human adipose tissue-derived mesenchymal stem cells, human lung-derived fibroblasts, human skin fibroblasts, Chinese hamster ovary-derived CHO cells, and mice. In addition to various cultured cell lines such as connective tissue L929 cells, human embryonic kidney-derived cells HEK293 cells, and human cervical cancer-derived HeLa cells, epithelial cells and endothelial cells constituting various tissues and organs in vivo, and exhibit contractility Skeletal muscle cells, smooth muscle cells, cardiomyocytes, neuronal cells constituting the nervous system, glial cells, fibroblasts, hepatic parenchymal cells, hepatic nonparenchymal cells and adipocytes involved in the metabolism of the living body can be used. Other than these, cells contained in blood, lymph, spinal fluid, sputum, urine or feces, microorganisms existing in the body or environment, viruses, protozoa and the like can be exemplified.
本発明の培養基材の形状は特に限定はないが、ディッシュ形状、フラスコ形状、フィルム状、球状などを例示できる。 The shape of the culture substrate of the present invention is not particularly limited, and examples thereof include a dish shape, a flask shape, a film shape, and a spherical shape.
本発明の高分子化合物を抽出する有機溶媒は、溶液あるいは懸濁に含まれる高分子を抽出できるものであれば特に限定はないが、高分子が抽出された有機溶液の回収を容易にすることから、水と非相溶の溶媒を用いることが好ましい。一例として、クロロホルム、トルエン、酢酸エチルが挙げられる。また、水と相溶な溶媒であっても、水のみを凍結させることで、高分子が抽出された液体の有機溶液のみを回収できるため好ましい。本高分子が抽出された液体の有機溶液を試料として取り扱う。抽出を行う際に用いる容器の材質は、用いる有機溶媒に溶出しないものであれば良く、有機溶媒としてクロロホルムを用いる場合は、一例として、ガラス製の容器が挙げられる。 The organic solvent for extracting the polymer compound of the present invention is not particularly limited as long as it can extract the polymer contained in the solution or suspension, but it should facilitate the recovery of the organic solution from which the polymer has been extracted. Therefore, it is preferable to use a solvent incompatible with water. Examples include chloroform, toluene and ethyl acetate. Even if the solvent is compatible with water, it is preferable to freeze only water so that only the liquid organic solution in which the polymer is extracted can be recovered. The liquid organic solution from which the polymer is extracted is treated as a sample. The material of the container used for the extraction may be any as long as it does not elute in the organic solvent used, and when chloroform is used as the organic solvent, a glass container can be given as an example.
本発明のサイズ排除クロマトグラフィーとは、高分子の分子サイズの違いによってカラムへの担持時間が変わってくる性質を利用し、分離・精製をする方法である。適用される評価試料は先述の高分子化合物を抽出した有機溶媒を用いる。フィルター濾過で不溶物を除去することが好ましい。適用される溶離液は、検出する高分子化合物ごとに最適なものが異なり、検出する高分子化合物が溶解する溶媒を使用する。本特許の実施例では、一例として、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使用している。適用されるカラムも検出する高分子化合物ごとに最適なものが異なり、検出する高分子化合物の極性や溶解性を考慮して最適なカラムを使用する。本特許の実施例では、一例として、東ソー製 ＴＳＫｇｅｌ ＳｕｐｅｒＡＷＭ−Ｈを使用している。適用される分離された高分子は、示差屈折、紫外／可視／赤外検出、核磁気共鳴などの検出器を用い検出できる。検出強度は試料中の高分子濃度に相関するため、高分子濃度と検出強度の検量線をあらかじめ作成しておくことで、試料中の高分子濃度を定量できる。試料の注入量は、１０μＬ以上５００μＬ以下が好ましく、試料中の高分子化合物濃度が高い場合は１０μＬ以上１００μＬ以下、高分子化合物濃度が低い場合は３００μＬ以上５００μＬ以下であることがそれぞれ好ましい。 The size exclusion chromatography of the present invention is a method for separating and purifying by utilizing the property that the loading time on the column changes depending on the molecular size of the polymer. As the evaluation sample to be applied, an organic solvent obtained by extracting the above-mentioned polymer compound is used. Insoluble matter is preferably removed by filtration with a filter. The optimum eluent varies depending on the polymer compound to be detected, and a solvent in which the polymer compound to be detected is dissolved is used. In the examples of this patent, 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate is used as an example. The optimum column also differs depending on the polymer compound to be detected, and the optimum column is used in consideration of the polarity and solubility of the polymer compound to be detected. In the examples of this patent, TSKgel SuperAWM-H manufactured by Tosoh Corporation is used as an example. The separated macromolecules applied can be detected using a detector such as differential refraction, ultraviolet / visible / infrared detection, nuclear magnetic resonance. Since the detection intensity correlates with the polymer concentration in the sample, the polymer concentration in the sample can be quantified by preparing a calibration curve for the polymer concentration and the detection intensity in advance. The injection amount of the sample is preferably 10 μL or more and 500 μL or less, preferably 10 μL or more and 100 μL or less when the polymer compound concentration is high, and 300 μL or more and 500 μL or less when the polymer compound concentration is low.
本発明の検量線作成方法は特に限定はないが、試料をサイズ排除クロマトグラフィーで測定する際のカラム温度や溶離液流量と同一な条件が好ましい。カラム温度は特に限定なく、室温以上、適用する溶離液の沸点未満を例示できる。溶離液流速は特に限定なく、測定時間の短縮とカラムの分離能のバランスをとって、０．１ｍＬ／ｍｉｎ〜２ｍＬ／ｍｉｎを例示できる。検量線作成に用いるピークは、ピークトップ高さやピーク面積を用いることができる。 The method for preparing the calibration curve of the present invention is not particularly limited, but the same conditions as the column temperature and the eluent flow rate when measuring the sample by size exclusion chromatography are preferable. The column temperature is not particularly limited and can be, for example, room temperature or higher and lower than the boiling point of the applied eluent. The flow rate of the eluent is not particularly limited, and 0.1 mL / min to 2 mL / min can be exemplified by balancing the shortening of the measurement time and the resolution of the column. A peak top height or a peak area can be used for the peak used for preparing the calibration curve.
本発明は、先述の検量線を用いることで、試料中の高分子化合物の濃度を１μｇ／ｍＬ以上１０ｍｇ／ｍＬ以下で解析できるが、用いる高分子化合物やカラムの種類、試料注入量、溶離液の流速によっては１μｇ／ｍＬ未満の濃度や１０ｍｇ／ｍＬを超える濃度であっても測定できる。 INDUSTRIAL APPLICABILITY The present invention can analyze the concentration of the polymer compound in the sample at 1 μg / mL or more and 10 mg / mL or less by using the calibration curve described above, but the polymer compound used, the type of column, the sample injection amount, and the eluent Depending on the flow rate, the concentration of less than 1 μg / mL or the concentration of more than 10 mg / mL can be measured.
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.
＜細胞培養基材コーティング用高分子化合物の組成＞
核磁気共鳴測定装置（日本電子製、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 polymer compound for coating cell culture substrate>
It was determined by proton nuclear magnetic resonance spectroscopy (1H-NMR) spectrum analysis using a nuclear magnetic resonance measurement device (manufactured by JEOL Ltd., trade name JNM-ECZ400S / LI).
＜細胞培養基材コーティング用高分子化合物の分子量、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、サイズ排除クロマトグラフィー（ＳＥＣ）によって測定した。ＳＥＣ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。
<Molecular weight and molecular weight distribution of polymer compound for coating cell culture substrate>
Weight average molecular weight (Mw), number average molecular weight (Mn) and molecular weight distribution (Mw / Mn) were measured by size exclusion chromatography (SEC). HLC-8320GPC manufactured by Tosoh Corporation was used as the SEC device, two TSKgel SuperAWM-H manufactured by Tosoh were used as the column, the column temperature was set to 40 ° C., and the eluent contained 10 mM sodium trifluoroacetate 2,2,2. It was measured using 2-trifluoroethanol. The measurement sample was prepared and measured at 1.0 mg / mL. For the calibration curve of the molecular weight, polymethylmethacrylate (manufactured by Sigma-Aldrich) having a known molecular weight was used.
＜細胞培養基材コーティング用高分子化合物の被覆量＞
濃度が既知の表面処理剤を細胞培養基材（ＩＷＡＫＩ製Φ６ｃｍ組織培養用ディッシュ）に塗布し、室温で５分間放置した後、加えた表面処理剤をパスツールピペットで回収し、細胞培養基材上に残った表面処理剤の量を電子天秤で秤量した。表面処理剤の濃度と表面処理剤の量、および細胞培養基材への被覆面積から、高分子化合物の被覆量を単位μｇ／ｃｍ２で算出した。
<Coating amount of polymer compound for coating cell culture substrate>
A surface treatment agent having a known concentration is applied to a cell culture substrate (Φ6 cm tissue culture dish made by IWAKI), left at room temperature for 5 minutes, and then the added surface treatment agent is collected with a Pasteur pipette to obtain a cell culture substrate. The amount of the surface treatment agent remaining on the top was measured by an electronic balance. From the concentration of the surface treatment agent and the amount of the surface treatment agent, and the coating area on the cell culture substrate, the coating amount of the polymer compound was calculated in the unit of μg / cm 2 .
＜細胞培養基材コーティング用高分子化合物（１）の合成＞
１００ｍＬ２口フラスコにＮ，Ｎ−ジメチルアミノエチルメタクリレート（ＤＭＡＥＭＡ）０．９４ｇ（６ｍｍｏｌ）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４１ｍｇ（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。
<Synthesis of polymer compound (1) for coating cell culture substrate>
0.94 g (6 mmol) of N, N-dimethylaminoethylmethacrylate (DMAEMA) was added to a 100 mL two-necked flask, and 41 mg (100 μmol) of 4-cyano-4-[(dodecylsulfonylthiocarbonyl) sulfonyl] pentanoic acid was further added. 1.6 mg (10 μmol) of azobisisobutyronitrile and 10 mL of 1,4-dioxane were added, and the mixture was heated and stirred at 62 ° C. for 24 hours after replacing with argon gas.
１回目の加熱撹拌後、上記にｎ−ブチルメタクリレート（ＢＭＡ）４．２６ｇ（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１０ｍＬ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, 4.26 g (30 mmol) of n-butyl methacrylate (BMA) was added to the above, further 1.6 mg (10 μmol) of azobisisobutyronitrile and 10 mL of 1,4-dioxane were added, and argon gas was added. After the replacement, 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）６．７８ｇ（６０ｍｍｏｌ）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。 After the second heating and stirring, 6.78 g (60 mmol) of N-isopropylacrylamide (IPAAm) was added to the above, 1.6 mg (10 μmol) of azobisisobutyronitrile and 35 mL of 1,4-dioxane were further added, and argon gas was added. After the replacement, the mixture was heated and stirred at 62 ° C. for 48 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（１）ｐｏｌｙ（ＤＭＡＥＭＡ−ＢＭＡ−ＩＰＡＡｍ）を４．７０５ｇ得た。得られたブロック共重合体の組成はＤＭＡＥＭＡ／ＢＭＡ／ＩＰＡＡｍ＝６／３２／６２（ｍｏｌ％）、Ｍｎ＝１１．０×１０４、およびＭｗ／Ｍｎ＝１．５であった。 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 hexane. The precipitate was collected by filtration, and dried under reduced pressure to obtain 4.705 g of block copolymer (1) poly (DMAEMA-BMA-IPAAm). The composition of the obtained block copolymer was DMAEMA / BMA / IPAAm = 6/32/62 (mol%), Mn = 11.0 × 10 4 , and Mw / Mn = 1.5.
＜細胞培養基材コーティング用高分子化合物（２）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）０．６５ｇ（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。
<Synthesis of polymer compound (2) for coating cell culture substrate>
0.65 g (5 mmol) of 2-methoxyethyl acrylate (MEA) was added to a 100 mL two-necked flask, and 31.8 mg (100 μmol) of cyanomethyldodecylcarbonato and 1.6 mg (10 μmol) of azobisisobutyronitrile and 1, 4-dioxane (10 mL) was added, and the mixture was replaced with argon gas, and then heated and stirred at 62 ° C. for 24 hours.
１回目の加熱撹拌後、上記にｎ−ブチルアクリレート（ＢＡ）３．８５ｇ（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。 After the first heating and stirring, 3.85 g (30 mmol) of n-butyl acrylate (BA) was added to the above, 1.6 mg (10 μmol) of azobisisobutyronitrile and 5 mL of 1,4-dioxane were further added, and argon gas was added. After the replacement, the mixture was heated and stirred at 62 ° C. for 48 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℃）７．３６ｇ（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。 After the second heating and stirring, 7.36 g (65 mmol) of N-isopropylacrylamide (IPAAm, LCST = 32 ° C.) was added to the above, and further 1.6 mg (10 μmol) of azobisisobutyronitrile and 35 mL of 1,4-dioxane. Was added, and the atmosphere was replaced with argon gas, followed by heating with stirring at 62 ° C. for 48 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（２）ｐｏｌｙ（ＭＥＡ−ＢＡ−ＩＰＡＡｍ）を５．８０５ｇ得た。得られたブロック共重合体の組成はＭＥＡ／ＢＡ／ＩＰＡＡｍ＝５／２６／６９（ｍｏｌ％）、Ｍｎ＝１１．７×１０４、およびＭｗ／Ｍｎ＝１．５であった。 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 with an evaporator and purified by reprecipitation with hexane. The precipitate was collected by filtration and dried under reduced pressure to obtain 5.805 g of block copolymer (2) poly (MEA-BA-IPAAm). The composition of the obtained block copolymer was MEA / BA / IPAAm = 5/26/69 (mol%), Mn = 11.7 × 10 4 , and Mw / Mn = 1.5.
＜高分子化合物の定量評価装置＞
定量評価装置は、東ソー（株）製 ＨＬＣ−８３２０ＧＰＣを用いた。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た。溶離液の流量は０．６ｍＬ／ｍｉｎとし、検出器として示差屈折計を用いた。
<Quantitative evaluation device for polymer compounds>
As a quantitative evaluation device, HLC-8320GPC manufactured by Tosoh Corporation was used. Two TSKgel SuperAWM-H manufactured by Tosoh were used as a column, the column temperature was set to 40 ° C., and 2,2,2-trifluoroethanol containing 10 mM sodium trifluoroacetate was used as an eluent. The flow rate of the eluent was 0.6 mL / min, and a differential refractometer was used as a detector.
実施例１
［定量評価用検量線の作成］
高分子化合物（１）をクロロホルムに溶解し、濃度０．００５ｍｇ／ｍＬ〜３．５８４ｍｇ／ｍＬの検量線用試料を得た。本試料を定量評価装置に１００μＬ注入した。各ピーク面積から検量線作成を試みたところ、濃度０．００８ｍｇ／ｍＬ〜３．５８４ｍｇ／ｍＬの範囲において直線性の良い検量線を得た。結果を表１および図１に示す。
Example 1
[Creation of calibration curve for quantitative evaluation]
The polymer compound (1) was dissolved in chloroform to obtain a calibration curve sample having a concentration of 0.005 mg / mL to 3.584 mg / mL. 100 μL of this sample was injected into the quantitative evaluation device. When an attempt was made to create a calibration curve from each peak area, a calibration curve with good linearity was obtained in the concentration range of 0.008 mg / mL to 3.584 mg / mL. The results are shown in Table 1 and FIG.
［本評価］
高分子化合物（１）被覆量１．０μｇ／ｃｍ２、６．０μｇ／ｃｍ２、２０．０μｇ／ｃｍ２のコートディッシュを得た。それぞれにヒト骨髄由来間葉系幹細胞２．０×１０５ｃｅｌｌｓを播種し、４ｍＬの培地（ＤＭＥＭ＋１０％牛胎児血清）を加え、３７℃５ＣＯ２雰囲気下で４日間培養した。その後、４℃の冷蔵庫で１時間放置し、細胞を剥離させ、細胞を含む培地４ｍＬをスクリュー瓶に回収した。さらに４ｍＬのクロロホルムを加え、栓をし、超音波振動装置で２分間処理した。１時間静置後、−１０℃の冷凍庫に２時間放置した。冷凍庫から取り出し、液体のクロロホルム層をパスツールピペットで回収し、定量評価装置に１００μＬ注入した。高分子化合物（１）被覆量２０．０μｇ／ｃｍ２のコートディッシュから回収した試料において、０．０１０ｍｇ／ｍＬの高分子化合物（１）の溶出が確認できた。結果を表２に示す。
[Main evaluation]
The polymer compound (1) coverage of 1.0μg / cm 2, 6.0μg / cm 2, to obtain a coated dish 20.0μg / cm 2. Human bone marrow-derived mesenchymal stem cells (2.0 × 10 5 cells) were seeded on each, 4 mL of a medium (DMEM + 10% fetal bovine serum) was added, and the cells were cultured at 37 ° C. under 5 CO 2 atmosphere for 4 days. After that, the cells were left to stand in a refrigerator at 4 ° C. for 1 hour, and 4 mL of a medium containing cells was collected in a screw bottle. Further, 4 mL of chloroform was added, the stopper was added, and the mixture was treated with an ultrasonic vibration device for 2 minutes. After standing for 1 hour, it was left in a freezer at -10 ° C for 2 hours. The sample was taken out from the freezer, the liquid chloroform layer was collected with a Pasteur pipette, and 100 μL was injected into the quantitative evaluation device. In the sample collected from the coated dish having the coating amount of the polymer compound (1) of 20.0 μg / cm 2 , it was confirmed that 0.010 mg / mL of the polymer compound (1) was eluted. The results are shown in Table 2.
比較例１
［本評価］
高分子化合物（１）被覆量１．０μｇ／ｃｍ２、６．０μｇ／ｃｍ２、２０．０μｇ／ｃｍ２のコートディッシュを得た。それぞれにヒト骨髄由来間葉系幹細胞２．０×１０５ｃｅｌｌｓを播種し、４ｍＬの培地（ＤＭＥＭ＋１０％牛胎児血清）を加え、３７℃５ＣＯ２雰囲気下で４日間培養した。その後、４℃の冷蔵庫で１時間放置し、細胞を剥離させ、細胞を含む培地４ｍＬをスクリュー瓶に回収した。さらに４ｍＬのクロロホルムを加え、栓をし、超音波振動装置で２分間処理した。１時間静置後、−１０℃の冷凍庫に２時間放置した。冷凍庫から取り出し、液体のクロロホルム層をパスツールピペットで回収した。それぞれを１０５℃で蒸発乾固させ、残存物の質量を最小標示０．１ｍｇの電子天秤で測定した。いずれのサンプルも残留物を検出できず、試料が同じ実施例１よりも低感度であった。結果を表２に示す。
Comparative Example 1
[Main evaluation]
The polymer compound (1) coverage of 1.0μg / cm 2, 6.0μg / cm 2, to obtain a coated dish 20.0μg / cm 2. Human bone marrow-derived mesenchymal stem cells (2.0 × 10 5 cells) were seeded on each, 4 mL of a medium (DMEM + 10% fetal bovine serum) was added, and the cells were cultured at 37 ° C. under 5 CO 2 atmosphere for 4 days. After that, the cells were left to stand in a refrigerator at 4 ° C. for 1 hour, and 4 mL of a medium containing cells was collected in a screw bottle. Further, 4 mL of chloroform was added, the stopper was added, and the mixture was treated with an ultrasonic vibration device for 2 minutes. After standing for 1 hour, it was left in a freezer at -10 ° C for 2 hours. It was taken out of the freezer and the liquid chloroform layer was collected with a Pasteur pipette. Each was evaporated to dryness at 105 ° C. and the mass of the residue was measured by an electronic balance with a minimum label of 0.1 mg. No residue could be detected in any of the samples, and the samples were less sensitive than the same Example 1. The results are shown in Table 2.
実施例２
［定量評価用検量線の作成］
高分子化合物（２）をクロロホルムに溶解し、濃度０．００１ｍｇ／ｍＬ〜１．０００ｍｇ／ｍＬの検量線用試料を得た。本試料を定量評価装置に３００μＬ注入した。各ピークトップ高さから検量線作成を試みたところ、濃度０．００５ｍｇ／ｍＬ〜１．０００ｍｇ／ｍＬの範囲において直線性の良い検量線を得た。結果を表３および図２に示す。
Example 2
[Creation of calibration curve for quantitative evaluation]
The polymer compound (2) was dissolved in chloroform to obtain a calibration curve sample having a concentration of 0.001 mg / mL to 1.000 mg / mL. 300 μL of this sample was injected into the quantitative evaluation device. When an attempt was made to create a calibration curve from each peak top height, a calibration curve with good linearity was obtained in the concentration range of 0.005 mg / mL to 1.000 mg / mL. The results are shown in Table 3 and FIG.
［本評価］
高分子化合物（２）被覆量５．０μｇ／ｃｍ２、１００．０μｇ／ｃｍ２のコートディッシュを準備した。それぞれにヒト骨髄由来間葉系幹細胞２．０×１０５ｃｅｌｌｓを播種し、４ｍＬの培地（ＤＭＥＭ＋１０％牛胎児血清）を加え、３７℃５ＣＯ２雰囲気下で４日間培養した。その後、４℃の冷蔵庫で１時間放置し、細胞を剥離させ、細胞を含む培地４ｍＬをスクリュー瓶に回収した。さらに４ｍＬのクロロホルムを加え、栓をし、超音波振動装置で２分間処理した。１時間静置後、−１０℃の冷凍庫に２時間放置した。冷凍庫から取り出し、液体のクロロホルム層をパスツールピペットで回収し、定量評価装置に３００μＬ注入した。高分子化合物（２）被覆量１００．０μｇ／ｃｍ２のコートディッシュから回収した試料において、０．０２２ｍｇ／ｍＬの高分子化合物（２）の溶出が確認できた。結果を表４に示す。
[Main evaluation]
Polymer compound (2) coverage of 5.0 [mu] g / cm 2, were prepared coated dish 100.0 / cm 2. Human bone marrow-derived mesenchymal stem cells (2.0 × 10 5 cells) were seeded on each, 4 mL of a medium (DMEM + 10% fetal bovine serum) was added, and the cells were cultured at 37 ° C. under 5 CO 2 atmosphere for 4 days. After that, the cells were left to stand in a refrigerator at 4 ° C. for 1 hour, and 4 mL of a medium containing cells was collected in a screw bottle. Further, 4 mL of chloroform was added, the stopper was added, and the mixture was treated with an ultrasonic vibration device for 2 minutes. After standing for 1 hour, it was left in a freezer at -10 ° C for 2 hours. The sample was taken out from the freezer, the liquid chloroform layer was collected with a Pasteur pipette, and 300 μL was injected into the quantitative evaluation device. Elution of 0.022 mg / mL of the polymer compound (2) was confirmed in the sample collected from the coated dish having the polymer compound (2) coating amount of 100.0 μg / cm 2 . The results are shown in Table 4.
比較例２
［本評価］
高分子化合物（２）被覆量５．０μｇ／ｃｍ２、１００．０μｇ／ｃｍ２のコートディッシュを準備した。それぞれにヒト骨髄由来間葉系幹細胞２．０×１０５ｃｅｌｌｓを播種し、４ｍＬの培地（ＤＭＥＭ＋１０％牛胎児血清）を加え、３７℃５ＣＯ２雰囲気下で４日間培養した。その後、４℃の冷蔵庫で１時間放置し、細胞を剥離させ、細胞を含む培地４ｍＬをスクリュー瓶に回収した。さらに４ｍＬのクロロホルムを加え、栓をし、超音波振動装置で２分間処理した。１時間静置後、−１０℃の冷凍庫に２時間放置した。冷凍庫から取り出し、液体のクロロホルム層をパスツールピペットで回収した。それぞれを１０５℃で蒸発乾固させ、残存物の質量を最小標示０．１ｍｇの電子天秤で測定した。いずれのサンプルも残留物を検出できず、試料が同じ実施例２よりも低感度であった。結果を表４に示す。
Comparative example 2
[Main evaluation]
Polymer compound (2) coverage of 5.0 [mu] g / cm 2, were prepared coated dish 100.0 / cm 2. Human bone marrow-derived mesenchymal stem cells (2.0 × 10 5 cells) were seeded on each, 4 mL of a medium (DMEM + 10% fetal bovine serum) was added, and the cells were cultured at 37 ° C. under 5 CO 2 atmosphere for 4 days. After that, the cells were left to stand in a refrigerator at 4 ° C. for 1 hour, and 4 mL of a medium containing cells was collected in a screw bottle. Further, 4 mL of chloroform was added, the stopper was added, and the mixture was treated with an ultrasonic vibration device for 2 minutes. After standing for 1 hour, it was left in a freezer at -10 ° C for 2 hours. It was taken out of the freezer and the liquid chloroform layer was collected with a Pasteur pipette. Each was evaporated to dryness at 105 ° C. and the mass of the residue was measured by an electronic balance with a minimum label of 0.1 mg. No residue could be detected in any of the samples and the samples were less sensitive than the same Example 2. The results are shown in Table 4.
Claims (6)
（Ａ）有機溶媒を用いて高分子化合物を培地から抽出する工程。
（Ｂ）サイズ排除クロマトグラフィーを用いて高分子化合物を分離する工程。
（Ｃ）高分子化合物由来の検出強度から高分子化合物を定量する工程。 A method for quantifying a polymeric compound derived from a culture substrate, which is 1 μg / mL or more and 10 mg / mL or less, eluted in a medium containing cells cultured in vitro by a method including the following steps (A) to (C).
(A) A step of extracting a polymer compound from a medium using an organic solvent.
(B) A step of separating a polymer compound using size exclusion chromatography.
(C) A step of quantifying the polymer compound from the detection intensity derived from the polymer compound.
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07113742A (en) * | 1993-10-19 | 1995-05-02 | Tosoh Corp | Concentration detecting method for polyarylene-sulfide solution |
| JP2004189800A (en) * | 2002-12-09 | 2004-07-08 | Mitsubishi Rayon Co Ltd | Thermoplastic resin composition |
| JP2005060593A (en) * | 2003-08-18 | 2005-03-10 | Kaneka Corp | Thickener for amorphous polyester resin, amorphous polyester resin composition containing the same, and molding molded therefrom |
| WO2007060891A2 (en) * | 2005-11-24 | 2007-05-31 | Asahi Kasei Chemicals Corp | Methacrylic resin and method for producing same |
| JP2016512459A (en) * | 2013-03-11 | 2016-04-28 | テレフレックス メディカル インコーポレイテッドＴｅｌｅｆｌｅｘ Ｍｅｄｉｃａｌ Ｉｎｃｏｒｐｏｒａｔｅｄ | Devices with antithrombotic and antibacterial treatment |
| JP2016214644A (en) * | 2015-05-22 | 2016-12-22 | 株式会社Kri | Chitin/chitosan porous body and production method therefor |
| WO2018003569A1 (en) * | 2016-06-30 | 2018-01-04 | 備前化成株式会社 | Method for producing ω3 and ω6 unsaturated fatty acid oxides |
| JP2018087316A (en) * | 2016-08-03 | 2018-06-07 | 東ソー株式会社 | Block copolymer and surface treatment agent using same |
| WO2018116904A1 (en) * | 2016-12-22 | 2018-06-28 | Dic株式会社 | Cell culture substrate |
| JP2018154752A (en) * | 2017-03-17 | 2018-10-04 | 東ソー株式会社 | Copolymer and manufacturing method therefor |
| WO2018198495A1 (en) * | 2017-04-25 | 2018-11-01 | ダイキン工業株式会社 | Temperature-responsive cell culture substrate, and method for producing same |
-
2018
- 2018-11-07 JP JP2018209426A patent/JP7262206B2/en active Active
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07113742A (en) * | 1993-10-19 | 1995-05-02 | Tosoh Corp | Concentration detecting method for polyarylene-sulfide solution |
| JP2004189800A (en) * | 2002-12-09 | 2004-07-08 | Mitsubishi Rayon Co Ltd | Thermoplastic resin composition |
| JP2005060593A (en) * | 2003-08-18 | 2005-03-10 | Kaneka Corp | Thickener for amorphous polyester resin, amorphous polyester resin composition containing the same, and molding molded therefrom |
| WO2007060891A2 (en) * | 2005-11-24 | 2007-05-31 | Asahi Kasei Chemicals Corp | Methacrylic resin and method for producing same |
| JP2016512459A (en) * | 2013-03-11 | 2016-04-28 | テレフレックス メディカル インコーポレイテッドＴｅｌｅｆｌｅｘ Ｍｅｄｉｃａｌ Ｉｎｃｏｒｐｏｒａｔｅｄ | Devices with antithrombotic and antibacterial treatment |
| JP2016214644A (en) * | 2015-05-22 | 2016-12-22 | 株式会社Kri | Chitin/chitosan porous body and production method therefor |
| WO2018003569A1 (en) * | 2016-06-30 | 2018-01-04 | 備前化成株式会社 | Method for producing ω3 and ω6 unsaturated fatty acid oxides |
| JP2018087316A (en) * | 2016-08-03 | 2018-06-07 | 東ソー株式会社 | Block copolymer and surface treatment agent using same |
| WO2018116904A1 (en) * | 2016-12-22 | 2018-06-28 | Dic株式会社 | Cell culture substrate |
| JP2018154752A (en) * | 2017-03-17 | 2018-10-04 | 東ソー株式会社 | Copolymer and manufacturing method therefor |
| WO2018198495A1 (en) * | 2017-04-25 | 2018-11-01 | ダイキン工業株式会社 | Temperature-responsive cell culture substrate, and method for producing same |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US12031128B2 (en) | 2022-04-07 | 2024-07-09 | Battelle Memorial Institute | Rapid design, build, test, and learn technologies for identifying and using non-viral carriers |
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