JP5674255B2 - Cell culture substrate evaluation method - Google Patents
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Description
本発明は、生物学、医学等の分野において有用な細胞培養基材の評価方法に関するものである。 The present invention relates to a method for evaluating a cell culture substrate useful in fields such as biology and medicine.
今日、動物細胞培養技術が著しく進歩し、動物細胞を対象とした研究開発もさまざまな分野に広がって実施されるようになってきた。対象となる動物細胞の使われ方も、開発当初の細胞そのものを製品化したり、その産生物を製品化したりするだけでなく、今や細胞やその細胞表層蛋白質を分析することで有効な医薬品を設計したり、患者本人の細胞を生体外で再生したり、或いはその機能を高めたりしてから生体内へ戻し治療する等ということも実施されつつある。現在、動物細胞を培養する技術、並びに評価、解析、利用する技術は、研究者が注目している一分野である。 Today, animal cell culture technology has made significant progress, and research and development on animal cells has been extended to various fields. The target animal cells can be used not only to commercialize the original cells, but also to produce their products, and to design effective drugs by analyzing the cells and their cell surface proteins. It is also being practiced to regenerate the patient's own cells outside the living body, or to improve the function of the cell and then return it to the living body for treatment. Currently, techniques for culturing animal cells and techniques for evaluation, analysis, and utilization are one field that researchers are paying attention to.
ところで、ヒト細胞を含め動物細胞の多くは付着依存性のものである。すなわち、動物細胞を生体外で培養しようとするときは、それらを一度、どこかに付着させる必要性がある。そのような背景のもと、以前より多くの研究者らによって細胞にとってより好ましい基材表面の設計、考案がなされてきたが、これらの技術は何れも細胞培養時に関係するものばかりであった。付着依存性の培養細胞は何かに付着する際、自ら接着性蛋白質を産生する。従ってその細胞を剥離させるときには、従来技術ではその接着性蛋白質を破壊しなければならず、通常酵素処理が行われる。その際、細胞が培養中に産生した各種細胞固有の細胞表層蛋白も同時に破壊されてしまうという重大な課題であったにもかかわらず、現実には解決する手段が全くなく、特に検討されていなかった。この細胞回収時の課題の解決こそが、今後動物細胞を対象とした研究開発を飛躍的に発展させる上で強く求められるものと考えられる。 By the way, many animal cells including human cells are adhesion-dependent. That is, when cultivating animal cells in vitro, it is necessary to attach them once somewhere. Against this background, more and more researchers have designed and devised a substrate surface that is more favorable for cells than before. However, all of these techniques are related to cell culture. When an adhesion-dependent cultured cell attaches to something, it produces an adhesive protein by itself. Therefore, when the cells are detached, the adhesive protein must be destroyed in the prior art, and usually an enzyme treatment is performed. At that time, although there was a serious problem that the cell surface proteins inherent to various cells produced by the cells during the culture were destroyed at the same time, there was no means to solve the problem at all, and no particular investigation was made. It was. It is thought that the solution to the problem at the time of cell recovery will be strongly demanded for the dramatic development of research and development for animal cells.
このような背景のもと、特開平２−２１１８６５号公報には、水に対する上限若しくは下限臨界溶解温度が０〜８０℃である高分子で基材表面を被覆した細胞培養支持体上にて、細胞を上限臨界溶解温度以下または下限臨界溶解温度以上で培養し、その後上限臨界溶解温度以上または下限臨界溶解温度以下にすることにより酵素処理なくして培養細胞を剥離させる新規な細胞培養法が記載されている。また、特開平０５−１９２１３８号公報には、この温度応答性細胞培養基材を利用して皮膚細胞を上限臨界溶解温度以下或いは下限臨界溶解温度以上で培養し、その後上限臨界溶解温度以上或いは下限臨界溶解温度以下にすることにより培養皮膚細胞を低損傷で剥離させることが記載されている。さらに、特願２００７−１０５３１１号公報には、この温度応答性細胞培養基材を用いて培養細胞の表層蛋白質の修復方法が記載されている。温度応答性細胞培養基材を利用することにより、従来の培養技術に対しさまざまな新規な展開をはかれるようになってきた。 Under such background, JP-A-2-21865 discloses a cell culture support in which a substrate surface is coated with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. with respect to water. A novel cell culture method is described in which cells are cultured at or below the upper critical lysis temperature or above the lower critical lysis temperature and then detached from the cultured cells without enzyme treatment by bringing it to the upper or lower critical lysis temperature. ing. Japanese Patent Application Laid-Open No. 05-192138 discloses that this temperature-responsive cell culture substrate is used to cultivate skin cells at a temperature below the upper critical solution temperature or above the lower critical solution temperature, and then above or above the upper critical solution temperature. It is described that cultured skin cells are detached with low damage by setting the temperature to a critical dissolution temperature or lower. Furthermore, Japanese Patent Application No. 2007-105311 describes a method for repairing surface protein of cultured cells using this temperature-responsive cell culture substrate. By utilizing a temperature-responsive cell culture substrate, various new developments have been made with respect to conventional culture techniques.
温度応答性細胞培養基材を利用することにより、従来の培養技術に対しさまざまな新規な展開をはかれるようになった。そして、さらなる応用展開をはかる上で、今後は、この培養基材表面をより精密に設計する必要がある。そのためには、培養基材表面の状態を詳細に評価する方法の確立が望まれていた。 By using a temperature-responsive cell culture substrate, various new developments have been made with respect to conventional culture techniques. In order to further develop applications, it is necessary to design the culture substrate surface more precisely in the future. For this purpose, establishment of a method for evaluating in detail the state of the culture substrate surface has been desired.
この温度応答性細胞培養基材の評価方法に注目すると、上述した特開平２−２１１８６５号公報には、温度応答性細胞培養基材上にウシ大動脈血管内皮細胞を播種し、培養温度を変化させた時の細胞の剥離回収率が記載されている。しかしながら、ここで示される評価は温度応答性細胞培養基材の利用方法に関するものであり、本発明で示されるところの基材表面の評価に関するものではなく、また、評価に使用する好適な細胞の種類、播種密度、培養期間に関する記載もなく、さらに再現性の良い評価方法も記載されていなかった。 Focusing on this method for evaluating a temperature-responsive cell culture substrate, JP-A-2-21865 described above discloses that bovine aortic vascular endothelial cells are seeded on a temperature-responsive cell culture substrate and the culture temperature is changed. The cell detachment recovery rate is described. However, the evaluation shown here relates to the utilization method of the temperature-responsive cell culture substrate, and does not relate to the evaluation of the substrate surface shown in the present invention. There was no description on the type, seeding density, and culture period, and there was no description of a reproducible evaluation method.
特開２００７−０４９９１８号には、温度応答性培養表面上で培養した細胞の剥離率の求め方が記載されている。ここでは、温度を変化させた後、剥離せずに表面に付着している細胞数を顕微鏡下で計数することで剥離率を求めているが、温度応答性細胞培養基材の評価方法について言えば、基本的には上述した特開平２−２１１８６５号公報と同様に温度応答性細胞培養基材の利用方法に関するものであり、本発明で示されるところの基材表面の評価に関するものではなく、また、評価に使用する好適な細胞の種類、播種密度、培養期間に関する記載もなかった。さらに再現性の良い評価方法の記載もなく、今後、温度応答性細胞培養基材の表面を精密に設計する上で好適な培養基材表面の詳細な評価方法がなかったのが現状であった。 Japanese Patent Application Laid-Open No. 2007-049918 describes how to determine the detachment rate of cells cultured on a temperature-responsive culture surface. Here, after changing the temperature, the exfoliation rate is obtained by counting the number of cells adhering to the surface without exfoliation under a microscope, but the evaluation method for temperature-responsive cell culture substrate can be said. Basically, it relates to a method for using a temperature-responsive cell culture substrate as in JP-A-2-21865 described above, and is not related to the evaluation of the substrate surface as shown in the present invention. Moreover, there was no description regarding the suitable cell type, seeding density, and culture period used for evaluation. In addition, there was no description of an evaluation method with good reproducibility, and there was no detailed evaluation method for the surface of the culture substrate suitable for the precise design of the surface of the temperature-responsive cell culture substrate in the future. .
一方、米国特許５１８５４５０号公報では、培養中の細胞の生死（ｖｉａｂｉｌｉｔｙ）を判定するための物質としてのテトラゾリウム塩を提案し、当該物質を用いることで培養細胞中の生細胞を測れるようにした。この方法を利用することで大量の検体を簡便に、かつ再現性良く培養細胞中に生細胞数を計測できるようになるが、ここでの発明は培養細胞中の生細胞数の計測方法に関するものであり、本発明で示されるところの基材表面の評価に関するものではなかった。また、評価に使用する好適な細胞の種類、播種密度、培養期間に関する記載もなかった。 On the other hand, US Pat. No. 5,185,450 proposes a tetrazolium salt as a substance for determining viability of cells in culture, and by using the substance, it is possible to measure living cells in cultured cells. By using this method, it becomes possible to measure the number of living cells in cultured cells easily and reproducibly with a large amount of specimens. The invention here relates to a method for measuring the number of living cells in cultured cells. It was not related to the evaluation of the substrate surface as shown in the present invention. Moreover, there was no description regarding the suitable cell type, seeding density, and culture period used for evaluation.
本発明は、上述したような温度応答性細胞培養基材の表面評価方法に関する問題点を解決することを意図してなされたものである。すなわち、本発明は、従来技術と全く異なった発想からの新規な温度応答性細胞培養基材の表面評価方法を提供するものである。 The present invention has been made with the intention of solving the problems associated with the method for evaluating the surface of a temperature-responsive cell culture substrate as described above. That is, the present invention provides a novel method for evaluating the surface of a temperature-responsive cell culture substrate based on a completely different idea from the prior art.
本発明者らは、上記課題を解決するために、種々の角度から検討を加えて研究開発を行ってきた。その結果、０〜８０℃の温度範囲で水和力が変化する温度応答性ポリマーを被覆した細胞培養用基材表面の評価方法として、株化細胞を当該基材表面上に播種後、４８時間以内に蛋白質加水分解酵素を使わずに、温度処理だけで細胞を当該基材表面から剥離する方法が好適であることを見出した。そして、この方法は温度応答性細胞培養基材の生産管理を行う上でも有用であることが判明した。本発明はかかる知見に基づいて完成されたものである。 In order to solve the above-mentioned problems, the present inventors have conducted research and development by adding studies from various angles. As a result, as a method for evaluating the surface of a cell culture substrate coated with a temperature-responsive polymer whose hydration power changes in a temperature range of 0 to 80 ° C., 48 hours after seeding the cell line on the substrate surface. The present inventors have found that a method of peeling cells from the surface of the substrate only by temperature treatment without using a protein hydrolase is preferable. This method has been found to be useful in managing production of temperature-responsive cell culture substrates. The present invention has been completed based on such findings.
すなわち、本発明は、０〜８０℃の温度範囲で水和力が変化する温度応答性ポリマーを被覆した細胞培養用基材表面の評価方法として、株化細胞を当該基材表面上に播種後、４８時間以内に蛋白質加水分解酵素を使わずに、温度処理だけで細胞を当該基材表面から剥離する方法を提供する。
また、本発明は、温度応答性細胞培養基材の生産管理方法についても提供する。That is, the present invention is a method for evaluating the surface of a cell culture substrate coated with a temperature-responsive polymer whose hydration power changes in a temperature range of 0 to 80 ° C. After seeding a cell line on the surface of the substrate. The present invention provides a method for detaching cells from the surface of a substrate only by temperature treatment without using a protein hydrolase within 48 hours.
The present invention also provides a production management method for a temperature-responsive cell culture substrate.
本発明に記載される方法であれば、これまで計測することが困難であった温度応答性細胞培養基材の表面特性を詳細に評価できるようになる。また、この方法を利用することで温度応答性細胞培養基材の生産管理ができるようになる。 If it is the method described in this invention, it will come to be able to evaluate in detail the surface characteristic of the temperature-responsive cell culture base material which was difficult to measure until now. Moreover, production management of a temperature-responsive cell culture substrate can be performed by using this method.
本発明は、０〜８０℃の温度範囲で水和力が変化する温度応答性ポリマーを被覆した基材表面の評価方法に関するものである。本発明において、対象となる温度応答性細胞培養基材とは、０〜８０℃の温度範囲で水和力が変化するポリマーを表面に被覆されたものである。本発明に用いる温度応答性高分子はホモポリマー、コポリマーのいずれであってもよい。このようなポリマーとしては、例えば、特開平２−２１１８６５号公報に記載されているものが挙げられる。具体的には、例えば、以下のモノマーの単独重合または共重合によって得られる。使用し得るモノマーとしては、例えば、（メタ）アクリルアミド化合物、Ｎ−（若しくはＮ，Ｎ−ジ）アルキル置換（メタ）アクリルアミド誘導体、またはビニルエーテル誘導体が挙げられ、コポリマーの場合は、これらの中で任意の２種以上を使用することができる。更には、上記モノマー以外のモノマー類との共重合、ポリマー同士のグラフトまたは共重合、あるいはポリマー、コポリマーの混合物を用いてもよい。また、ポリマー本来の性質を損なわない範囲で架橋することも可能である。各種ポリマーの基材表面への被覆方法は、特に制限されないが、例えば、特開平２−２１１８６５号公報に記載されている方法に従ってよい。すなわち、かかる被覆は、基材と上記モノマーまたはポリマーを、電子線照射（ＥＢ）、γ線照射、紫外線照射、プラズマ処理、コロナ処理、有機重合反応のいずれかにより、または塗布、混練等の物理的吸着等により行うことができる。 The present invention relates to a method for evaluating a substrate surface coated with a temperature-responsive polymer whose hydration power changes in a temperature range of 0 to 80 ° C. In the present invention, the target temperature-responsive cell culture substrate is a material whose surface is coated with a polymer whose hydration power changes in a temperature range of 0 to 80 ° C. The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include those described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. The method for coating the surface of the base material with various polymers is not particularly limited. For example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption.
本発明における温度応答性ポリマーの固定化量は、細胞を培養させられ、かつ温度処理することだけで基材表面から剥離できるに十分な量が固定化されていれば良く特に限定されるものではないが、０．８〜１０．０μｇ／ｃｍ２、好ましくは１．３〜６．０μｇ／ｃｍ２、さらに好ましくは１．５〜３．０μｇ／ｃｍ２が良く、最も好ましくは２．０〜２．５μｇ／ｃｍ２が良い。ポリマー量が１０．０μｇ／ｃｍ２より多いと細胞の付着性が悪くなり、逆に０．８μｇ／ｃｍ２より少ないと温度を変えても剥離せず、本発明の技術を十分に達成できず好ましくない。ポリマーの固定化量の測定は常法に従えば良く、例えばＦＴ−ＩＲ−ＡＴＲを用いて直接測る方法、あらかじめラベル化したポリマーを同様な方法で固定化しラベル化ポリマー量より推測する方法などが挙げられるがいずれの方法を用いても良い。The immobilization amount of the temperature-responsive polymer in the present invention is not particularly limited as long as the cell is cultured and an amount sufficient to be detached from the substrate surface only by temperature treatment is immobilized. 0.8 to 10.0 μg / cm 2 , preferably 1.3 to 6.0 μg / cm 2 , more preferably 1.5 to 3.0 μg / cm 2 , most preferably 2.0 to 2.5 μg / cm 2 is good. If the amount of polymer is more than 10.0 μg / cm 2 , the adherence of cells is deteriorated. Conversely, if the amount is less than 0.8 μg / cm 2, peeling does not occur even if the temperature is changed, and the technique of the present invention cannot be sufficiently achieved. It is not preferable. The measurement of the amount of immobilized polymer may be carried out in accordance with a conventional method, for example, a method of directly measuring using FT-IR-ATR, a method of immobilizing a previously labeled polymer by the same method and estimating from the amount of labeled polymer, etc. Any method may be used.
本発明における培養基材の形状は特に制約されるものではないが、例えばディッシュ、マルチプレート、フラスコ、セルインサートマイクロビーズのような形態のもの、或いは平膜状のものなどが挙げられる。被覆を施される基材としては、通常細胞培養に用いられるガラス、改質ガラス、ポリスチレン、ポリメチルメタクリレート等の化合物を初めとして、一般に形態付与が可能である物質、例えば、上記以外の高分子化合物、セラミックス類など全て用いることができる。 The shape of the culture substrate in the present invention is not particularly limited, and examples thereof include dishes, multiplates, flasks, cell insert microbeads, and flat membranes. Examples of the base material to be coated include substances that can generally give form, such as glass, modified glass, polystyrene, polymethylmethacrylate, and the like, which are usually used for cell culture, such as polymers other than those described above. All compounds, ceramics, etc. can be used.
本発明の細胞培養支持体において、基材に被覆されている温度応答性ポリマーは温度を変えることで水和、脱水和を起こすものであり、その温度域は０℃〜８０℃、好ましくは１０℃〜５０℃、さらに好ましくは２０℃〜４５℃であることが判明した。８０℃を越えると細胞が死滅する可能性があるので好ましくない。また、０℃より低いと一般に細胞増殖速度が極度に低下するか、または細胞が死滅してしまうため、やはり好ましくない。 In the cell culture support of the present invention, the temperature-responsive polymer coated on the substrate is hydrated or dehydrated by changing the temperature, and the temperature range is 0 ° C. to 80 ° C., preferably 10 ° C. It has been found that the temperature is from 50 ° C to 50 ° C, more preferably from 20 ° C to 45 ° C. If the temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, if the temperature is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed, which is not preferable.
本発明とは、以上に示した温度応答性細胞培養基材に対して、特定の細胞を利用し、特定の条件下で培養することでその表面状態の詳細を計測しようとするものである。本発明とは、温度応答性細胞培養基材表面状態をその基材上に付着した細胞の付着の状態から判定する方法に関するものである。本発明では、温度応答性細胞培養基材表面上で細胞を培養することで、基材と細胞との相互作用だけの情報が得られるようにすべきであって、従って、培養細胞の状態は細胞が個々に分離されていた方が好ましく、例えば２個以上の細胞が結合した状態やコロニーを形成している状態等は好ましくない。培養細胞が個々に分離されていれば、培養細胞が温度応答性細胞培養基材から温度変化することで剥離した際は、基材と細胞との相互作用に反映された結果と考えられる。一方で、培養細胞が複数個で結合した状態であると、基材から温度変化することで剥離しても単純に基材と細胞との相互作用だけによる結果とは言い切れない。細胞と細胞が結合した状態で培養された細胞の場合、培養細胞が培養温度の変化で剥離しても、一部の細胞のみが剥離し、その細胞に結合した細胞が引きずられて剥離する場合も考えられ好ましくない。本発明では、温度応答性細胞培養基材上で培養される細胞は、個々の状態になっているものが８０％以上であることが望ましく、好ましくは８５％が良く、さらに好ましくは９０％以上が良い。個々の状態になっている細胞が８０％を満たないと、上述した理由により得られた結果は必ずしも基材と細胞との間の相互作用を反映したものと言い難く、本発明の技術として必ずしも好適なものではない。 The present invention is intended to measure the details of the surface state of the above-described temperature-responsive cell culture substrate by utilizing specific cells and culturing under specific conditions. The present invention relates to a method for determining the surface state of a temperature-responsive cell culture substrate from the state of attachment of cells attached on the substrate. In the present invention, by culturing cells on the surface of a temperature-responsive cell culture substrate, information on only the interaction between the substrate and the cells should be obtained. It is preferable that the cells are individually separated. For example, a state where two or more cells are combined or a state where a colony is formed is not preferable. If the cultured cells are individually separated, when the cultured cells are detached from the temperature-responsive cell culture substrate by temperature change, it is considered that the result is reflected in the interaction between the substrate and the cells. On the other hand, in the state where a plurality of cultured cells are combined, even if the cells are peeled off due to temperature change from the base material, it cannot be said that the result is simply due to the interaction between the base material and the cells. In the case of cells cultured in a state where the cells are bound to each other, even if the cultured cells are detached due to a change in the culture temperature, only some of the cells are detached, and the cells bound to the cells are dragged and separated Is also considered and is not preferable. In the present invention, the number of cells cultured on a temperature-responsive cell culture substrate is desirably 80% or more, preferably 85%, more preferably 90% or more. Is good. If the number of cells in an individual state does not reach 80%, the result obtained for the above-described reason cannot always be said to reflect the interaction between the base material and the cells. It is not preferred.
本発明において使用される細胞としては、例えば、動物、昆虫、植物等の細胞、細菌類が挙げられるが特に限定されるものではない。この中で、動物細胞は市販されているものが多く好都合である。動物細胞の由来として、ヒト、サル、イヌ、ネコ、ウサギ、ラット、ヌードマウス、マウス、モルモット、ブタ、ヒツジ、チャイニーズハムスター、ウシ、マーモセット、アフリカミドリザル等が挙げられるが特に限定されるものではない。また、特にその動物細胞の株化細胞であればそのもの自身の培養が安定に行え、本発明に使用する細胞として好ましい。このような細胞株としては、ＮＩＨ／３Ｔ３細胞株（マウス胎仔線維芽細胞）、３Ｔ３−Ｓｗｉｓｓ ａｌｂｉｎｏ細胞株（マウス胎仔線維芽細胞）、Ａ５４９細胞株（ヒト肺腺がん細胞）、ＨｅＬａ細胞株（ヒト子宮頸部類上皮腫細胞）、Ｖｅｒｏ細胞株（アフリカミドリザル正常腎細胞）、２９３（ヒト胎児腎細胞）、３Ｔ３−Ｌ１（マウス繊維芽細胞）、ＨｅｐＧ２（ヒト肝臓ガン由来細胞）、ＭＣＦ−７（ヒト乳癌由来細胞）、Ｖ７９（チャイニーズハムスター由来線維芽細胞）、ＣＯＳ−７（アフリカミトリザル腎臓由来細胞）、ＣＨＯ−Ｋ１（チャイニーズハムスター卵巣由来細胞）、ＷＩ−３８（ヒト肺線維芽細胞）、ＭＤＣＫ（イヌ腎由来細胞）、ＭＲＣ−５（正常肺線維芽細胞）等が挙げられるが特に限定されるものではない。この中で、線維芽細胞の細胞株が培養を行いやすく本発明に使用する細胞として好適である。 Examples of cells used in the present invention include, but are not limited to, cells of animals, insects, plants, and bacteria. Of these, many commercially available animal cells are advantageous. Examples of the origin of animal cells include, but are not limited to, humans, monkeys, dogs, cats, rabbits, rats, nude mice, mice, guinea pigs, pigs, sheep, Chinese hamsters, cattle, marmoset, and African green monkeys. . In particular, if it is a cell line of the animal cell, the cell itself can be stably cultured, and it is preferable as the cell used in the present invention. Such cell lines include NIH / 3T3 cell line (mouse fetal fibroblast), 3T3-Swiss albino cell line (mouse fetal fibroblast), A549 cell line (human lung adenocarcinoma cell), HeLa cell line. (Human cervical epithelioma cells), Vero cell line (African green monkey normal kidney cells), 293 (human embryonic kidney cells), 3T3-L1 (mouse fibroblasts), HepG2 (human liver cancer-derived cells), MCF -7 (human breast cancer-derived cells), V79 (Chinese hamster-derived fibroblasts), COS-7 (African mitrizal kidney-derived cells), CHO-K1 (Chinese hamster ovary-derived cells), WI-38 (human lung fibroblasts) Cells), MDCK (canine kidney-derived cells), MRC-5 (normal lung fibroblasts), etc. There. Of these, fibroblast cell lines are suitable for use in the present invention because they can be easily cultured.
本発明においては、培養細胞は個々の状態で温度応答性細胞培養基材表面上に付着していることが必要である。本発明では、このような状態になれば培養開始時の細胞播種数について特に限定されないが、一般的には、基材表面積あたり１００〜１００００個／ｃｍ２が良く、好ましくは５００〜８０００個／ｃｍ２が良く、さらに好ましくは１０００〜６０００個／ｃｍ２が良い。１００個／ｃｍ２を満たないとき、細胞培養する際に必要な細胞数を満たされず細胞の状態が極度に悪化することで好ましくない。また、１００００個／ｃｍ２以上となると細胞同士の接着頻度が高くなり、培養細胞が個々の状態になる確率が減少することで好ましくない。In the present invention, it is necessary that the cultured cells adhere to the surface of the temperature-responsive cell culture substrate in an individual state. In the present invention, the number of seeded cells at the start of culture is not particularly limited as long as such a state is reached, but in general, 100 to 10,000 cells / cm 2 per substrate surface area is good, preferably 500 to 8000 cells / cm 2. cm 2 is good, more preferably 1000 to 6000 / cm 2 . When less than 100 cells / cm 2 , it is not preferable because the number of cells necessary for cell culture is not satisfied and the state of the cells is extremely deteriorated. Moreover, when it becomes 10000 pieces / cm < 2 > or more, the adhesion frequency of cells will become high and it is unpreferable because the probability that a cultured cell will be in an individual state reduces.
本発明における細胞の培養時間は、温度応答性細胞培養基材上で細胞が個々の状態で付着していれば特に限定されないが、一般的には、４８時間以内が良く、好ましくは２４時間以内が良く、さらに好ましくは１２時間以内が良く、最も好ましくは６時間以内が良い。また、本発明を例えば温度応答性細胞培養基材の生産管理方法として利用するようなさらに短時間で評価しなくてはならない場合、培養時間が４時間以内となっても良い。４８時間以上であると、培養細胞が基材表面に付着し分裂し、２個以上の細胞が結合した状態になる確率が高くなり好ましくない。本発明で上述した株化動物細胞を用いれば、細胞は温度応答性細胞培養基材表面に速やかに付着する。付着後、細胞は基材表面上で扁平化し始めるが、本発明では、細胞が扁平化し始める前の段階で実施しても良く、その場合、培養時間が４時間以内となる。 The cell culture time in the present invention is not particularly limited as long as the cells adhere in an individual state on the temperature-responsive cell culture substrate, but generally it is within 48 hours, preferably within 24 hours. More preferably within 12 hours, and most preferably within 6 hours. In addition, when the present invention must be evaluated in a shorter time, for example, as a production management method for a temperature-responsive cell culture substrate, the culture time may be 4 hours or less. If it is 48 hours or longer, the probability that the cultured cells adhere to and divide on the surface of the base material and two or more cells are combined is increased. When the established animal cell described above in the present invention is used, the cell quickly adheres to the surface of the temperature-responsive cell culture substrate. After the attachment, the cells start to flatten on the surface of the substrate. In the present invention, the cell may start at a stage before the cells start to flatten, and in this case, the culture time is 4 hours or less.
本発明は、温度応答性細胞培養基材の表面状態を評価するものであり、基材上で培養された細胞は培養温度を変えるだけで剥離させることが良く、通常の培養操作で使われるトリプシン等の蛋白質加水分解酵素を使用することは好ましくない。培養細胞を剥離させる際のその他の条件は特に制約されるものではなく、温度変化時に静置されていても良く、適度に振とうさせてもが良いが、評価の際に操作を統一した方が好ましい。 The present invention evaluates the surface state of a temperature-responsive cell culture substrate, and the cells cultured on the substrate are preferably detached only by changing the culture temperature, and trypsin used in normal culture operations. It is not preferable to use a protein hydrolase such as Other conditions for exfoliating the cultured cells are not particularly limited and may be left standing at the time of temperature change or may be shaken appropriately. Is preferred.
本発明では、温度応答性細胞培養基材上で培養した細胞を温度変化させて剥離した細胞数を計測することで評価するものである。その際、剥離細胞数を計測する方法は特に限定されるものではないが、例えば、ＣｅｌｌＴｉｔｅｒ−９６ Ａｑｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製、Ｇ３５８０）のようにＮＡＤＨによるＭＴＳの還元により細胞数を測定する試薬、Ａｌａｍａｒ Ｂｌｕｅ（ＢｉｏＳｏｕｒｃｅ社製、ＤＡＬ１０２５）のようにＮＡＤＨによるテトラゾリウム塩の還元量により細胞数を計測する方法、ＭＴＴ（同仁化学研究所社製、３４５−０１８２１）のようにＮＡＤＨによるＭＴＴの還元量により細胞数を測定する試薬、ＣｅｌｌＴｉｔｅｒ−Ｇｌｏ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製、Ｇ７５７０）のように残存ＡＴＰ量から細胞数を測定する試薬、ＣｅｌｌＴｉｔｅｒ−Ｂｌｕｅ Ａｓｓａｙ（Ｐｒｏｍｅｇａ社製、Ｇ８０８０）のようにＮＡＤＨによるレサズリンの還元により細胞数を測定する試薬、ＣｙｔｏＴｏｘ−Ｏｎｅ Ａｓｓａｙのように漏出ＬＤＨ量から細胞数を測定する試薬、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ（同仁化学研究所社製、３４９−０６４６１）のようにＮＡＤＨによるＷＳＴ−１の還元量により細胞数を測定する試薬、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ−８（同仁化学研究所社製、３４１−０７７６１）のようにＮＡＤＨによるＷＳＴ−８の還元量により細胞数を測定する試薬、Ｃｅｌｌ Ｃｏｕｎｔｉｎｇ Ｋｉｔ−Ｆ（同仁化学研究所社製、３４３−０７７４３）のようにＣａｌｃｅｉｎ−ＡＭの加水分解量により細胞数を測定する試薬等の試薬を利用する方法、または、剥離した細胞を血球計算盤を用いて計測する方法、ＢｒＤＵ，ＣＦＳＥ，ＰＫＨ−２６を細胞に取り込ませ、その蛍光量からフローサイトメーターを用いて細胞数を測定する方法、３Ｈ−ｔｈｙｍｉｄｉｎｅを取り込ませＲＩにより検出し細胞数を計測する方法等が挙げられる。特に、簡便に再現良く評価する点から言えば、本発明では試薬を用いる方法が好適である。 In the present invention, evaluation is performed by measuring the number of detached cells by changing the temperature of cells cultured on a temperature-responsive cell culture substrate. At that time, the method for measuring the number of detached cells is not particularly limited. For example, the number of cells is measured by reducing MTS with NADH as in CellTiter-96 Aqueous One Solution Assay (Promega, G3580). Reagent, a method of measuring the number of cells based on the amount of tetrazolium salt reduced by NADH, such as Alamar Blue (manufactured by BioSource, DAL1025), MTT reduction by NADH, such as MTT (manufactured by Dojindo Laboratories, 345-01821) A reagent for measuring the number of cells by the amount, a reagent for measuring the number of cells from the amount of remaining ATP, such as CellTiter-Glo Assay (Promega, G7570), CellTiter-Blue Assay (Promega A reagent for measuring the number of cells by reducing resazurin with NADH as in G8080), a reagent for measuring the number of cells from the amount of leaked LDH as in CytoTox-One Assay, Cell Counting Kit (manufactured by Dojindo Laboratories, Inc., 349) A reagent for measuring the number of cells based on the amount of WST-1 reduced by NADH as in -06461), and the amount of WST-8 reduced by NADH as in Cell Counting Kit-8 (manufactured by Dojindo Laboratories, Inc., 341-07761) A method of using a reagent such as a reagent for measuring the number of cells by a reagent such as a reagent for measuring the number of cells by the amount of hydrolysis of Calcein-AM, such as Cell Counting Kit-F (manufactured by Dojindo Laboratories, Inc., 343-07743), Alternatively, measure detached cells using a hemocytometer. A method of incorporating BrDU, CFSE, PKH-26 into cells and measuring the number of cells using a flow cytometer from the amount of fluorescence; a method of incorporating 3H-thymidine and detecting by RI to measure the number of cells, etc. Can be mentioned. In particular, from the viewpoint of simple and good evaluation, a method using a reagent is preferred in the present invention.
細胞の剥離率の計算方法は特に限定されるものではなく、基材表面に付着した総細胞数に対する剥離した細胞数の比率に１００を乗じて％で表示しても良く、逆に基材表面に付着した総細胞数に対して剥離しなかった細胞数を減じ、得られた剥離細胞数の総細胞数に対する比率に１００を乗じて％で表示しても良い。こうして得られた剥離率は両現性の良いものであり、あらかじめその剥離率に対する、実際にその温度応答性細胞培養基材を利用して実施応用しようとする各組織の細胞の剥離挙動の相関性を調べておけば、その実施応用したい細胞に合わせて好適な温度応答性細胞培養基材を選択することができる。その際の剥離率は、基材を評価する細胞、実施応用したい細胞によって異なるが、例えば、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、角膜上皮組織移植用の培養口腔粘膜細胞シートや培養角膜上皮細胞シートを作製する上で好適なディッシュタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、５０％以上のものが良く、好ましくは６０％以上が良く、さらに好ましくは７０％以上のものが良い。剥離率が５０％を満たない場合、培養した培養口腔粘膜細胞シートや培養角膜上皮細胞シートを基材表面から損傷なく剥離させることが困難で、培養細胞の一部が基材に付着した状態となり、剥離したシートが欠損し好ましくない。同様に、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、角膜上皮組織移植用の培養口腔粘膜細胞シートや培養角膜上皮細胞シートを作製する上で好適なインサートタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、２０％以上のものが良く、好ましくは３０％以上が良く、さらに好ましくは４０％以上のものが良い。剥離率が２０％を満たない場合、培養した培養口腔粘膜細胞シートや培養角膜上皮細胞シートを基材表面から損傷なく剥離させることが困難で、培養細胞の一部が基材に付着した状態となり、剥離したシートが欠損し好ましくない。さらに、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、表皮組織移植用の培養表皮細胞シートを作製する上で好適なディッシュタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、５０％以上のものが良く、好ましくは６０％以上が良く、さらに好ましくは７０％以上のものが良い。剥離率が５０％を満たない場合、培養した培養表皮細胞シートを基材表面から損傷なく剥離させることが困難で、培養細胞の一部が基材に付着した状態となり、剥離したシートが欠損し好ましくない。同様に、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、表皮組織移植用の培養表皮細胞シートを作製する上で好適なインサートタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、２０％以上のものが良く、好ましくは３０％以上が良く、さらに好ましくは４０％以上のものが良い。剥離率が２０％を満たない場合、培養した培養表皮細胞シートを基材表面から損傷なく剥離させることが困難で、培養細胞の一部が基材に付着した状態となり、剥離したシートが欠損し好ましくない。さらに、心筋再生に好適な心筋細胞シート、骨格筋筋芽細胞シート、脂肪組織由来間葉系幹細胞シート及び骨髄由来間葉系幹細胞シート、歯根膜再生に有用な歯根膜細胞シート及び間葉系幹細胞シート、軟骨再生に有用な軟骨細胞シート及び滑膜細胞シートにおいても、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、上述した細胞シートを作製する上で好適なディッシュタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、５０％以上のものが良く、好ましくは６０％以上が良く、さらに好ましくは７０％以上のものが良い。剥離率が５０％を満たない場合、培養した上述の細胞シートを基材表面から損傷なく剥離させることが困難で、培養細胞の一部が基材に付着した状態となり、剥離したシートが欠損し好ましくない。一方、樹状細胞を培養し、細胞表層蛋白質を保持させたまま剥離させる目的の際には、評価用細胞として、ＮＩＨ／３Ｔ３細胞を用い、播種密度を６０００個／ｃｍ２とし、常法の培地を用い２４時間培養する方法を採用したとき、目的の樹状細胞を作製する上で好適なディッシュタイプの温度応答性細胞培養基材は本発明で示すところの剥離率は、２０％以上のものが良く、好ましくは３０％以上が良く、さらに好ましくは４０％以上のものが良い。剥離率が２０％を満たない場合、樹状細胞を基材表面から損傷なく剥離させることが困難となり好ましくない。The method for calculating the cell detachment rate is not particularly limited, and the ratio of the number of detached cells to the total number of cells attached to the substrate surface may be multiplied by 100 and displayed in%. The number of cells that have not detached can be reduced with respect to the total number of cells attached to the cell, and the ratio of the obtained number of detached cells to the total number of cells can be multiplied by 100 and displayed in%. The exfoliation rate obtained in this way has a good duality, and the exfoliation rate correlates with the exfoliation behavior of cells in each tissue to be actually applied using the temperature-responsive cell culture substrate. If the sex is examined, a suitable temperature-responsive cell culture substrate can be selected according to the cell to be applied. The exfoliation rate at that time varies depending on the cell for which the substrate is to be evaluated and the cell to be applied. For example, NIH / 3T3 cells are used as evaluation cells, the seeding density is 6000 cells / cm 2 , and a conventional medium is used. When adopting the method of culturing for 24 hours using a corneal epithelial tissue transplantation, a dish-type temperature-responsive cell culture substrate suitable for producing a cultured oral mucosal cell sheet or a cultured corneal epithelial cell sheet is used in the present invention. The peel rate shown is preferably 50% or more, preferably 60% or more, and more preferably 70% or more. When the peel rate is less than 50%, it is difficult to peel the cultured cultured oral mucosa cell sheet or cultured corneal epithelial cell sheet from the substrate surface without damage, and a part of the cultured cells is attached to the substrate. , The peeled sheet is not preferable. Similarly, when adopting a method in which NIH / 3T3 cells are used as evaluation cells, the seeding density is 6000 cells / cm 2, and culture is performed for 24 hours using a conventional medium, cultured oral mucosa for transplantation of corneal epithelial tissue An insert type temperature-responsive cell culture substrate suitable for producing a cell sheet or a cultured corneal epithelial cell sheet has a peeling rate of 20% or more, preferably 30% or more, as shown in the present invention. Good, more preferably 40% or more. When the peel rate is less than 20%, it is difficult to peel the cultured cultured oral mucosa cell sheet or cultured corneal epithelial cell sheet from the surface of the substrate without damage, and a part of the cultured cells is attached to the substrate. , The peeled sheet is not preferable. Furthermore, when using a method of culturing for 24 hours using a conventional medium using NIH / 3T3 cells as the cells for evaluation, with a seeding density of 6000 cells / cm 2 , a cultured epidermal cell sheet for epidermal tissue transplantation was used. The dish-type temperature-responsive cell culture substrate suitable for production has a peeling rate of 50% or more, preferably 60% or more, more preferably 70% or more, as shown in the present invention. Things are good. When the peel rate is less than 50%, it is difficult to peel the cultured cultured epidermal cell sheet from the substrate surface without damage, and a part of the cultured cells is attached to the substrate, and the peeled sheet is lost. It is not preferable. Similarly, when adopting a method in which NIH / 3T3 cells are used as evaluation cells, the seeding density is 6000 cells / cm 2, and culture is performed for 24 hours using a conventional medium, a cultured epidermal cell sheet for epidermal tissue transplantation is employed. The insert-type temperature-responsive cell culture substrate suitable for producing the present invention has a peeling rate of 20% or more, preferably 30% or more, more preferably 40% or more, as shown in the present invention. Things are good. When the peel rate is less than 20%, it is difficult to peel the cultured cultured epidermal cell sheet from the surface of the substrate without damage, and a part of the cultured cells is attached to the base material, and the peeled sheet is lost. It is not preferable. Furthermore, a myocardial cell sheet suitable for myocardial regeneration, a skeletal myoblast sheet, an adipose tissue-derived mesenchymal stem cell sheet and a bone marrow-derived mesenchymal stem cell sheet, a periodontal ligament cell sheet and a mesenchymal stem cell useful for periodontal regeneration The sheet, the chondrocyte sheet useful for cartilage regeneration, and the synovial cell sheet are also cultured for 24 hours using NIH / 3T3 cells as the cells for evaluation, with a seeding density of 6000 cells / cm 2, and using a conventional medium. When the method is employed, the dish-type temperature-responsive cell culture substrate suitable for producing the above-described cell sheet has a peeling rate of 50% or more as shown in the present invention, preferably 60%. The above is good, more preferably 70% or more. When the peel rate is less than 50%, it is difficult to peel the above-mentioned cultured cell sheet from the base material surface without damage, and a part of the cultured cells is attached to the base material, and the peeled sheet is lost. It is not preferable. On the other hand, for the purpose of culturing dendritic cells and detaching them while retaining the cell surface protein, NIH / 3T3 cells were used as evaluation cells, the seeding density was 6000 cells / cm 2 , and a conventional method was used. When adopting a method of culturing for 24 hours using a culture medium, the dish-type temperature-responsive cell culture substrate suitable for producing the desired dendritic cells has a peeling rate of 20% or more as shown in the present invention. Good, preferably 30% or more, more preferably 40% or more. When the peeling rate is less than 20%, it becomes difficult to peel the dendritic cells from the substrate surface without damage, which is not preferable.
以上のことを温度応答性ポリマーとしてポリ（Ｎ−イソプロピルアクリルアミド）を例にとり説明する。ポリ（Ｎ−イソプロピルアクリルアミド）は３１℃に下限臨界溶解温度を有するポリマーとして知られ、遊離状態であれば、水中で３１℃以上の温度で脱水和を起こしポリマー鎖が凝集し、白濁する。逆に３１℃以下の温度ではポリマー鎖は水和し、水に溶解した状態となる。本発明で対象となる基材は、このポリマーがシャーレなどの器材表面に被覆、固定されたものである。その場合、３１℃以上の温度であれば、基材表面のポリマーも同じように脱水和するが、ポリマー鎖が基材表面に被覆、固定されているため、基材表面が疎水性を示すようになる。逆に、３１℃以下の温度では、基材表面のポリマーは水和するが、ポリマー鎖が基材表面に被覆、固定されているため、基材表面が親水性を示すようになる。このときの疎水的な表面は細胞が付着、増殖できる適度な表面であり、また、親水的な表面は細胞が付着できないほどの表面となり、培養中の細胞も冷却するだけで剥離させられることになる。本発明では、このような機能を有する表面が基材表面に十分に構築されているのかどうかを評価するものであり、例えば、線維芽細胞であるＮＩＨ／３Ｔ３細胞を所定量播種し、２４時間以内に培養を中止し、培養温度を３１℃以下にすることで培養細胞を剥離させる。その後、剥離細胞をＣｅｌｌＴｉｔｅｒ ９６ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ（Ｐｒｏｍｅｇａ社製、Ｇ３５８０）を用いて生細胞数を計測すれば、温度応答性細胞培養基材表面の特性を評価できることとなる。 The above will be described by taking poly (N-isopropylacrylamide) as an example of a temperature-responsive polymer. Poly (N-isopropylacrylamide) is known as a polymer having a lower critical solution temperature at 31 ° C. In the free state, dehydration occurs in water at a temperature of 31 ° C. or more, and polymer chains aggregate and become cloudy. Conversely, at a temperature of 31 ° C. or lower, the polymer chain is hydrated and dissolved in water. The base material which is the object of the present invention is one in which this polymer is coated and fixed on the surface of a device such as a petri dish. In that case, if the temperature is 31 ° C. or higher, the polymer on the surface of the substrate is similarly dehydrated, but the polymer chain is coated and fixed on the surface of the substrate, so that the surface of the substrate is hydrophobic. become. Conversely, at a temperature of 31 ° C. or lower, the polymer on the substrate surface is hydrated, but the polymer chain is coated and fixed on the substrate surface, so that the substrate surface becomes hydrophilic. The hydrophobic surface at this time is an appropriate surface on which cells can attach and grow, and the hydrophilic surface becomes a surface on which cells cannot adhere, and the cells in culture can be detached only by cooling. Become. In the present invention, it is evaluated whether the surface having such a function is sufficiently constructed on the substrate surface. For example, a predetermined amount of NIH / 3T3 cells, which are fibroblasts, are seeded for 24 hours. The culture is stopped within 30 minutes, and the cultured cells are detached by setting the culture temperature to 31 ° C. or lower. Then, if the number of living cells is measured for the detached cells using CellTiter 96 AQueous One Solution (G3580, manufactured by Promega), the characteristics of the temperature-responsive cell culture substrate surface can be evaluated.
本発明により温度応答性細胞培養基材の表面を詳細に評価することができるようになり、しかもその操作は簡便で再現性の高いものである。従って、本発明で示す評価方法は、例えば温度応答性細胞培養基材の生産の際のスケールアップ時の表面機能を管理する規格化技術としても有用である。 According to the present invention, the surface of the temperature-responsive cell culture substrate can be evaluated in detail, and the operation is simple and highly reproducible. Therefore, the evaluation method shown in the present invention is also useful as a standardization technique for managing surface functions at the time of scale-up, for example, when producing a temperature-responsive cell culture substrate.
以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。 Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
３Ｔ３−Ｓｗｉｓｓ ａｌｂｉｎｏ細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、５．７×１０３個／ｃｍ２となるように、ポリ（Ｎ−イソプロピルアクリルアミド）が被覆されていない市販の細胞培養基材上へ播種した。これを３７℃、５％二酸化炭素中で２４時間培養した。培養後、細胞培養用ディッシュ内で培養した細胞に対して、リン酸緩衝生理食塩水（ＰＢＳ）で洗浄後、０．１％トリプシン溶液を加え、培養面に均一に行き渡らせ、過剰なトリプシンを吸引した。その後すぐに、３７℃、５％二酸化炭素中で、３分間、インキュベートし、接着している全ての細胞を剥離した。そのものに培地を加え再懸濁させた。この懸濁液の上清を１００μＬずつ回収し、測定用９６穴マイクロプレートに加えた。4. Subculture a 3T3-Swiss albino cell line (mouse fetal fibroblasts, working medium: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) to prepare a cell suspension; It seed | inoculated on the commercially available cell culture base material which is not coat | covered with poly (N-isopropylacrylamide) so that it might become 7 * 10 < 3 > piece / cm < 2 >. This was cultured at 37 ° C. in 5% carbon dioxide for 24 hours. After culturing, the cells cultured in the cell culture dish are washed with phosphate buffered saline (PBS), 0.1% trypsin solution is added, and evenly distributed over the culture surface to remove excess trypsin. Aspirated. Immediately thereafter, the cells were incubated at 37 ° C. in 5% carbon dioxide for 3 minutes to detach all the attached cells. The medium was added and resuspended. 100 μL of the supernatant of this suspension was collected and added to a 96-well microplate for measurement.
この測定用９６穴マイクロプレートを３７℃、５％二酸化炭素中で１時間プレインキュベートした。その後、ＣｅｌｌＴｉｔｅｒ ９６ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ（Ｐｒｏｍｅｇａ、Ｇ３５８０）を２０μＬ／ｗｅｌｌの割合で加え、３７℃、５％二酸化炭素中で２時間呈色反応を行った。２時間後、マイクロプレートリーダーで、４９０ｎｍの吸光度と参照波長７５０ｎｍを測定し、各ｗｅｌｌの吸光度を測定した。検量線用として細胞濃度既知の溶液も作製し、１００μＬずつ測定用９６穴マイクロプレートに加えた。得られた吸光度から、検量線を用いて浮遊細胞濃度を算出し、剥離度を下記の式に従って算出した。
剥離度（％）＝（剥離細胞数）／（培養基材上の細胞数（平均値））×１００
その結果、培養細胞の剥離度は１００±４．０％であった。得られた結果を図１に示す。The 96-well microplate for measurement was preincubated at 37 ° C. in 5% carbon dioxide for 1 hour. Thereafter, CellTiter 96 AQueous One Solution (Promega, G3580) was added at a rate of 20 μL / well, and a color reaction was carried out at 37 ° C. in 5% carbon dioxide for 2 hours. Two hours later, the absorbance at 490 nm and the reference wavelength of 750 nm were measured with a microplate reader, and the absorbance of each well was measured. A solution with a known cell concentration was prepared for the calibration curve, and 100 μL was added to a 96-well microplate for measurement. From the obtained absorbance, the floating cell concentration was calculated using a calibration curve, and the degree of detachment was calculated according to the following formula.
Degree of detachment (%) = (number of detached cells) / (number of cells on culture substrate (average value)) × 100
As a result, the degree of detachment of the cultured cells was 100 ± 4.0%. The obtained results are shown in FIG.
３Ｔ３−Ｓｗｉｓｓ ａｌｂｉｎｏ細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、５．７×１０３個／ｃｍ２となるように、ポリ（Ｎ−イソプロピルアクリルアミド）が被覆された温度応答性細胞培養基材上へ播種した。これを３７℃、５％二酸化炭素中で２４時間培養した。培養後、培養液の温度を２０℃とし、３０分静置させた。冷却後、剥離細胞に培地を加え再懸濁させた。この懸濁液の上清を１００μＬずつ回収し、測定用９６穴マイクロプレートに加えた。4. Subculture a 3T3-Swiss albino cell line (mouse fetal fibroblasts, working medium: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) to prepare a cell suspension; It seed | inoculated on the temperature-responsive cell culture base material coat | covered with the poly (N-isopropyl acrylamide) so that it might become 7 * 10 < 3 > piece / cm < 2 >. This was cultured at 37 ° C. in 5% carbon dioxide for 24 hours. After culturing, the temperature of the culture solution was set to 20 ° C. and allowed to stand for 30 minutes. After cooling, a medium was added to the exfoliated cells and resuspended. 100 μL of the supernatant of this suspension was collected and added to a 96-well microplate for measurement.
この測定用９６穴マイクロプレートを３７℃、５％二酸化炭素中で１時間プレインキュベートした。その後、ＣｅｌｌＴｉｔｅｒ ９６ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎ（Ｐｒｏｍｅｇａ、Ｇ３５８０）を２０μＬ／ｗｅｌｌの割合で加え、３７℃、５％二酸化炭素中で２時間呈色反応を行った。２時間後、マイクロプレートリーダーで、４９０ｎｍの吸光度と参照波長７５０ｎｍを測定し、各ｗｅｌｌの吸光度を測定した。その後、検量線を用いて、得られた吸光度から浮遊細胞濃度を算出し、剥離度を下記の式に従って算出した。
剥離度（％）＝（剥離細胞数）／（参考例１での培養基材上の細胞数（平均値））×１００
その結果、剥離度は、９５±１．０％であった。得られた結果を図１に示す。The 96-well microplate for measurement was preincubated at 37 ° C. in 5% carbon dioxide for 1 hour. Thereafter, CellTiter 96 AQueous One Solution (Promega, G3580) was added at a rate of 20 μL / well, and a color reaction was carried out at 37 ° C. in 5% carbon dioxide for 2 hours. Two hours later, the absorbance at 490 nm and the reference wavelength of 750 nm were measured with a microplate reader, and the absorbance of each well was measured. Thereafter, the floating cell concentration was calculated from the obtained absorbance using a calibration curve, and the degree of detachment was calculated according to the following formula.
Degree of peeling (%) = (number of peeled cells) / (number of cells on the culture substrate in Reference Example 1 (average value)) × 100
As a result, the degree of peeling was 95 ± 1.0%. The obtained results are shown in FIG.
３Ｔ３−Ｓｗｉｓｓ ａｌｂｉｎｏ細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、５．７×１０３個／ｃｍ２となるように、ポリ（Ｎ−イソプロピルアクリルアミド）が被覆されていない市販の細胞培養ディッシュ上へ播種した。これを３７℃、５％二酸化炭素中で２４時間培養した。培養後、培養液の温度を２０℃とし、３０分静置させた。その他の操作は実施例１と同様な方法に従った。その結果、剥離度は、３．５±０．６％であった。得られた結果を図１に示す。4. Subculture a 3T3-Swiss albino cell line (mouse fetal fibroblasts, working medium: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) to prepare a cell suspension; It seed | inoculated on the commercially available cell culture dish which is not coat | covered with poly (N-isopropylacrylamide) so that it might become 7 * 10 < 3 > piece / cm < 2 >. This was cultured at 37 ° C. in 5% carbon dioxide for 24 hours. After culturing, the temperature of the culture solution was set to 20 ° C. and allowed to stand for 30 minutes. Other operations were the same as in Example 1. As a result, the degree of peeling was 3.5 ± 0.6%. The obtained results are shown in FIG.
実施例１、参考例１、比較例１を比較することで、温度応答性細胞培養基材の場合、３Ｔ３−Ｓｗｉｓｓ ａｌｂｉｎｏ細胞（マウス胎仔線維芽細胞）を用い、播種濃度を５．７×１０３個／ｃｍ２とし、２４時間培養する方法を採用した場合、剥離度が９０％となり、温度応答性ポリマーであるポリ（Ｎ−イソプロピルアクリルアミド）が被覆されていない市販の培養基材では剥離度が３％程度であることが分かる。本発明の方法が、温度応答性細胞培養基材の評価方法として利用できることが分かる。By comparing Example 1, Reference Example 1, and Comparative Example 1, in the case of a temperature-responsive cell culture substrate, 3T3-Swiss albino cells (mouse fetal fibroblasts) were used, and the seeding concentration was 5.7 × 10. In the case of adopting a method of culturing for 24 hours at 3 / cm 2 , the degree of peeling is 90%, and the degree of peeling is obtained with a commercially available culture substrate that is not coated with the temperature-responsive polymer poly (N-isopropylacrylamide). Is about 3%. It can be seen that the method of the present invention can be used as a method for evaluating a temperature-responsive cell culture substrate.
ＮＩＨ／３Ｔ３細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、６．３×１０３個／ｃｍ２となるようにポリ（Ｎ−イソプロピルアクリルアミド）が被覆された温度応答性細胞培養基材へ播種し、３７℃、５％二酸化炭素中で２４時間培養した。２４時間培養後、位相差顕微鏡で接着状態を図２に示す。その後、２０℃、３０分間インキュベートし、培養細胞を剥離させた。得られた結果を図３に示す。図３より、培養細胞は２０℃で３０分間インキュベートすることで、シングルセルで丸くなっていることが分かる。この時の細胞数を、血球計算盤を用いて計数した結果、総細胞数は６．６×１０３個／ｃｍ２であった。本実施例のように、６．６×１０３個／ｃｍ２程度での細胞密度で回収する場合、低温処理後の細胞が個々の状態となるため、好ましいことが分かる。NIH / 3T3 cell line (mouse fetal fibroblasts, medium used: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) is subcultured to prepare a cell suspension, 6.3 The cells were seeded on a temperature-responsive cell culture substrate coated with poly (N-isopropylacrylamide) so as to have a density of 10 3 cells / cm 2 and cultured at 37 ° C. in 5% carbon dioxide for 24 hours. After 24 hours of incubation, the adhesion state is shown in FIG. 2 using a phase contrast microscope. Then, it incubated at 20 degreeC for 30 minutes, and the cultured cell was peeled. The obtained results are shown in FIG. From FIG. 3, it can be seen that the cultured cells are rounded as a single cell by incubating at 20 ° C. for 30 minutes. As a result of counting the number of cells at this time using a hemocytometer, the total number of cells was 6.6 × 10 3 cells / cm 2 . As shown in this example, it can be seen that when cells are collected at a cell density of about 6.6 × 10 3 cells / cm 2 , the cells after low-temperature treatment are in individual states.
ＮＩＨ／３Ｔ３細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、３．８×１０４個／ｃｍ２となるようにポリ（Ｎ−イソプロピルアクリルアミド）が被覆された温度応答性細胞培養基材へ播種し、３７℃、５％二酸化炭素中で７２時間培養した。７２時間培養後、位相差顕微鏡で接着状態を撮影した。得られた結果を図４に示す。２０℃、３０分間インキュベートした結果、図５に示すように、本比較例では培養細胞はシート状となって剥離したことが分かる。このシートの細胞数をトリプシン処理を行って血球計算盤を用いて計数したところ４．０×１０５個／ｃｍ２であった。本比較例のように、低温処理時に、細胞間が結合した状態で剥離する場合、浮遊細胞数を容易に定量できないため、本発明として好ましくない方法であることが分かった。NIH / 3T3 cell line (mouse fetal fibroblasts, medium used: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) is subcultured to prepare a cell suspension, and 3.8 The cells were inoculated on a temperature-responsive cell culture substrate coated with poly (N-isopropylacrylamide) so as to be × 10 4 cells / cm 2 and cultured in 37 ° C. and 5% carbon dioxide for 72 hours. After incubation for 72 hours, the adhesion state was photographed with a phase contrast microscope. The obtained results are shown in FIG. As a result of incubating at 20 ° C. for 30 minutes, as shown in FIG. 5, it can be seen that in this comparative example, the cultured cells were detached in a sheet form. The number of cells in this sheet was treated with trypsin and counted using a hemocytometer. The result was 4.0 × 10 5 cells / cm 2 . As in this comparative example, in the case of peeling in a state in which cells are bonded at the time of low-temperature treatment, the number of floating cells cannot be easily quantified.
ＮＩＨ／３Ｔ３細胞株（マウス胎仔線維芽細胞、使用培地：ウシ胎児血清（ＦＣＳ）を１０％含むダルベッコー改変イーグル培地（ＤＭＥＭ））を継代培養し、細胞懸濁液を調整し、５０個／ｃｍ２となるようにポリ（Ｎ−イソプロピルアクリルアミド）が被覆された温度応答性細胞培養基材へ播種し、３７℃、５％二酸化炭素中で２４時間培養した。２４時間培養後、２０℃、３０分間インキュベートし、細胞を剥離させ、この時の細胞数を、ＣｅｌｌＴｉｔｅｒ ９６ ＡＱｕｅｏｕｓ Ｏｎｅ Ｓｏｌｕｔｉｏｎを用いて計数した。その結果、０．９×１０２個／ｃｍ２で検出限界値以下であった。本比較例のように、回収時の細胞密度がより低い場合（例えば、１００個／ｃｍ２以下である場合）、回収した溶液中の細胞数が低くなり、ＣｅｌｌＴｉｔｅｒ ９６ ＡＱｕｅｏｕｓ Ｏｎｅ ｓｏｌｕｔｉｏｎの検出感度以下となるため、好ましくない実施例である。NIH / 3T3 cell line (mouse fetal fibroblasts, medium used: Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FCS)) is subcultured, and the cell suspension is adjusted to 50 cells / The cells were seeded on a temperature-responsive cell culture substrate coated with poly (N-isopropylacrylamide) so as to be cm 2 and cultured at 37 ° C. in 5% carbon dioxide for 24 hours. After culturing for 24 hours, the cells were incubated at 20 ° C. for 30 minutes to detach the cells, and the number of cells at this time was counted using CellTiter 96 AQueous One Solution. As a result, it was 0.9 × 10 2 pieces / cm 2 or less than the detection limit value. As in this comparative example, when the cell density at the time of recovery is lower (for example, 100 cells / cm 2 or less), the number of cells in the recovered solution becomes lower and is below the detection sensitivity of CellTiter 96 AQueous One solution. Therefore, this is an undesirable example.
本発明に記載される方法であれば、これまで計測することが困難であった温度応答性細胞培養基材の表面特性を詳細に評価できるようになる。また、この方法を利用することで温度応答性細胞培養基材の生産管理ができるようになる。 If it is the method described in this invention, it will come to be able to evaluate in detail the surface characteristic of the temperature-responsive cell culture base material which was difficult to measure until now. Moreover, production management of a temperature-responsive cell culture substrate can be performed by using this method.
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