JP2018139497A - Cell culture vessel and cell culture method - Google Patents

Cell culture vessel and cell culture method Download PDF

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JP2018139497A
JP2018139497A JP2015117114A JP2015117114A JP2018139497A JP 2018139497 A JP2018139497 A JP 2018139497A JP 2015117114 A JP2015117114 A JP 2015117114A JP 2015117114 A JP2015117114 A JP 2015117114A JP 2018139497 A JP2018139497 A JP 2018139497A
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優史 丸山
Yuji Maruyama
優史 丸山
千鶴 平井
Chizuru Hirai
千鶴 平井
啓介 渋谷
Keisuke Shibuya
啓介 渋谷
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Abstract

PROBLEM TO BE SOLVED: To provide a cell culture vessel in which a cell is peeled with a change in temperature or pH.SOLUTION: A cell culture vessel cultures a cell. The cell culture vessel has, on its surface, a stimulus-responsive material containing temperature-responsive molecules, pH-responsive molecules and cell-adhesive molecules. In the stimulus-responsive material, there are a plurality of coupling structures of the temperature-responsive molecules and pH-responsive molecules.SELECTED DRAWING: Figure 1

Description

本発明は、細胞を培養する細胞培養装置及び細胞培養方法に関する。   The present invention relates to a cell culture apparatus and a cell culture method for culturing cells.

再生医療では患者から細胞を採取し、その細胞を体外で増殖させ、必要に応じて細胞組織として分化誘導を行い、患者の疾患に戻す治療法がある。細胞を体外で安定的に増殖培養するには、培養環境を一定に維持管理し、細胞を非侵襲的に回収できることが重要である。   In regenerative medicine, there is a treatment method in which cells are collected from a patient, the cells are proliferated outside the body, differentiation is induced as a cellular tissue as necessary, and the disease is returned to the patient. In order to stably proliferate and culture cells outside the body, it is important that the culture environment is maintained and managed so that the cells can be collected non-invasively.

細胞の品質に影響を与える培養環境因子としては、温度、ｐＨ、溶存酸素、溶存二酸化炭素などが挙げられる。多くの場合、専門知識を持った人による手作業での培養を行うが、より安定的に細胞を供給するためには自動細胞培養装置が必要となってくる。しかし、手作業や自動細胞培養装置で問題となるのは、ｐＨのオンラインモニタリングが困難であることである。手作業では、インキュベータ内の温度、ＣＯ２濃度を一定に維持するのみで、培養容器内のｐＨをオンラインでモニタリングすることはない。また自動培養装置にしても、無菌状態を保ったままｐＨ電極を取り付け、電極の校正まで行うことは困難である。そのため、万が一、培養中にｐＨ異常が発生してもそれを検知できず、下流工程に不良品細胞が混入してしまう。   Examples of culture environment factors that affect cell quality include temperature, pH, dissolved oxygen, and dissolved carbon dioxide. In many cases, manual culture is performed by a person with specialized knowledge, but an automatic cell culture apparatus is required to supply cells more stably. However, a problem with manual operations and automated cell culture devices is that online monitoring of pH is difficult. In manual operation, only the temperature and CO2 concentration in the incubator are maintained constant, and the pH in the culture vessel is not monitored online. Moreover, even in an automatic culture apparatus, it is difficult to attach a pH electrode while maintaining aseptic conditions and perform calibration of the electrode. Therefore, even if a pH abnormality occurs during the culture, it cannot be detected, and defective cells are mixed in the downstream process.

一方、細胞の回収にあたっては、通常はトリプシンなどの酵素を用い細胞と培養容器底面の接着を剥がすことが行われているが、細胞への損傷が問題となっている。そこで、近年、放射線照射や紫外線照射などの手法によって調製される温度応答性ポリマを用い、温度応答によって培養容器底面の親水性、疎水性を制御することで細胞を非侵襲的に剥離する材料が開発されている（特許文献１、２）。   On the other hand, in collecting cells, an enzyme such as trypsin is usually used to peel off the adhesion between the cell and the bottom of the culture container, but damage to the cell is a problem. Therefore, in recent years, a material that non-invasively detaches cells by controlling the hydrophilicity and hydrophobicity of the bottom surface of the culture vessel by temperature response using a temperature-responsive polymer prepared by a method such as radiation irradiation or ultraviolet irradiation has been developed. It has been developed (Patent Documents 1 and 2).

細胞を医療用材料などとして用いる場合、適切な環境下で培養されたかを管理することと細胞を非侵襲的に回収することを同時に満たす必要があるが、現在そのような培養容器は実現していない。例えば特許文献３では熱応答性ポリマとｐＨ応答性ポリマの複数の層を用いた培養容器の可能性について言及されているが、ｐＨ異常の際の細胞除去方法としては不十分である。   When cells are used as medical materials, it is necessary to satisfy both the management of whether the cells are cultured in an appropriate environment and the non-invasive recovery of the cells at the same time. Absent. For example, Patent Document 3 mentions the possibility of a culture vessel using a plurality of layers of a thermoresponsive polymer and a pH-responsive polymer, but it is insufficient as a method for removing cells when pH is abnormal.

特開２００８−２６３８６３号公報JP 2008-263863 A 特開２０１３−１６２７９６号公報JP 2013-162696 A 特表２０１４−５０１０９９号公報Special table 2014-501099 gazette

温度やｐＨの変化により細胞を剥離させる細胞培養容器を提供することを目的とする。   It aims at providing the cell culture container which peels a cell by the change of temperature or pH.

本発明は、細胞を培養する細胞培養容器であって、その細胞培養容器の表面に、温度応答性分子、ｐＨ応答性分子及び細胞接着性分子を含む刺激応答性材料を有する。また、刺激応答性材料において、温度応答性分子とｐＨ応答性分子の結合構造が複数存在する。   The present invention is a cell culture container for culturing cells, and has a stimulus-responsive material including a temperature-responsive molecule, a pH-responsive molecule, and a cell-adhesive molecule on the surface of the cell culture container. In addition, in the stimulus-responsive material, there are a plurality of binding structures of temperature-responsive molecules and pH-responsive molecules.

本発明により、温度やｐＨの変化により細胞を剥離させる細胞培養容器を提供することができる。従って、ｐＨ異常の有無を簡便に判別可能となる。また、温度変化による非侵襲的な細胞回収も可能となる。   According to the present invention, it is possible to provide a cell culture container in which cells are detached by a change in temperature or pH. Therefore, it is possible to easily determine whether there is a pH abnormality. In addition, non-invasive cell recovery by temperature change is also possible.

刺激応答性分子の部分構造例である。It is an example of a partial structure of a stimulus-responsive molecule. 細胞培養容器の製造工程の一部である。It is a part of manufacturing process of a cell culture container. 本発明で提供する細胞培養容器を用いる自動培養プロセスと自動培養装置の例である。It is an example of the automatic culture process and automatic culture apparatus which use the cell culture container provided by this invention. 細胞剥離の模式図である。It is a schematic diagram of cell detachment. 細胞剥離実験の培養容器写真である。It is a culture container photograph of a cell detachment experiment.

本発明は、細胞培養装置において、培養中の培養環境を維持管理し、正常に培養された細胞を非侵襲的に回収することを可能にする刺激応答性材料に関する。   The present invention relates to a stimulus-responsive material that maintains a culture environment during culture in a cell culture device and enables non-invasive recovery of normally cultured cells.

以下、図面等を用いて、本発明の実施形態について説明する。以下の説明は本発明の内容の具体例を示すものであり、本発明がこれらの説明に限定されるものではなく、本明細書に開示される技術的思想の範囲内において当業者による様々な変更および修正が可能である。また、本発明を説明するための全図において、同一の機能を有するものは、同一の符号を付け、その繰り返しの説明は省略する場合がある。   Hereinafter, embodiments of the present invention will be described with reference to the drawings. The following description shows specific examples of the contents of the present invention, and the present invention is not limited to these descriptions. Various modifications by those skilled in the art are within the scope of the technical idea disclosed in this specification. Changes and modifications are possible. In all the drawings for explaining the present invention, components having the same function are denoted by the same reference numerals, and repeated description thereof may be omitted.

本発明で提供する細胞培養容器は、表面に、刺激応答性分子（刺激応答性材料）を備える。刺激応答性分子は、温度応答性分子（温度応答性材料）とｐＨ応答性分子（ｐＨ応答性材料）と細胞接着性分子（細胞接着性材料）を備える。１種類の材料が温度応答性材料とｐＨ応答性材料と細胞接着性材料を兼ねていてもよいし、温度応答性材料とｐＨ応答性材料を兼ねる材料と細胞接着性材料の組み合わせでもよい。   The cell culture container provided in the present invention includes a stimulus-responsive molecule (stimulus-responsive material) on the surface. The stimulus-responsive molecule includes a temperature-responsive molecule (temperature-responsive material), a pH-responsive molecule (pH-responsive material), and a cell-adhesive molecule (cell-adhesive material). One kind of material may serve as a temperature responsive material, a pH responsive material, and a cell adhesive material, or a combination of a material that serves as a temperature responsive material and a pH responsive material and a cell adhesive material.

本発明に係る細胞培養容器によれば、（１）所定のｐＨ範囲を超えた培養環境の場合、培養細胞を剥離し、細胞を製品として回収しない、（２）適正な培養環境下で培養された細胞を回収する際、酵素を用いず、非侵襲的に細胞を回収することが可能となる。従って、培養後の検査工程への負荷を低減するとともに、安全性の高い細胞、特に医療用途の細胞を得ることができる。   According to the cell culture container of the present invention, (1) in the case of a culture environment exceeding a predetermined pH range, the cultured cells are detached and the cells are not collected as a product. (2) The cells are cultured in an appropriate culture environment. When the collected cells are collected, the cells can be collected non-invasively without using an enzyme. Therefore, it is possible to reduce the burden on the inspection process after culturing and obtain highly safe cells, particularly cells for medical use.

また、本発明に係る細胞培養方法によれば、放射線照射や紫外線照射を用いずに培養容器表面に機能性材料を導入できることから、安価で生産性の高い容器の生産プロセスを実現できる。   In addition, according to the cell culture method of the present invention, a functional material can be introduced onto the surface of the culture vessel without using radiation irradiation or ultraviolet irradiation, so that a low-cost and high-productivity container production process can be realized.

本発明で提供する細胞培養容器は、表面に導入されたいずれかの材料の水和状態が培養中のｐＨ異常の際もしくは温度低下の際に変化することで細胞が剥離する。ｐＨ異常の際の細胞剥離は、材料のイオン化の度合いがｐＨ正常範囲内におけるイオン化の度合いから外れることで水和状態がｐＨ正常範囲内から外れることを原理とするのが好ましいが、ｐＨに対して直接応答性を有さなくとも、ｐＨ異常に伴う電解質濃度の変化によって水和状態がｐＨ正常範囲内から外れるなどでもよい。なお、ｐＨ正常範囲およびｐＨ異常範囲は、適用する細胞および用途によって設定可能な範囲であるが、一例としてはｐＨ６．８−７．６がｐＨ正常範囲として挙げられる。温度低下による細胞剥離は、温度応答性材料がＬＣＳＴ以下になることで水和性が向上することを原理とすることが好ましいが、それ以外でもよい。   In the cell culture container provided in the present invention, the cells are detached by changing the hydration state of any material introduced to the surface when the pH is abnormal during the culture or when the temperature is lowered. Cell detachment in the event of abnormal pH is preferably based on the principle that the hydration state deviates from within the normal pH range by deviating from the degree of ionization within the normal pH range. Even if it does not have direct responsiveness, the hydration state may deviate from the normal pH range due to a change in the electrolyte concentration accompanying the abnormal pH. The normal pH range and the abnormal pH range are ranges that can be set depending on the cell to be applied and the intended use. As an example, a normal pH range of 6.8 to 7.6 can be mentioned. The cell detachment due to the temperature drop is preferably based on the principle that the temperature-responsive material is LCST or less to improve hydration, but other methods may be used.

本発明では、細胞培養容器の表面に、温度応答性分子、ｐＨ応答性分子及び細胞接着性分子を含む刺激応答性材料を有する。刺激応答性材料において、温度応答性分子とｐＨ応答性分子の結合構造が複数存在する。温度応答性分子とｐＨ応答性分子の結合構造が複数存在し、例えば温度応答性分子とｐＨ応答性分子が交互に存在することで、細胞培養容器の幅広い面に細胞剥離機能を持たせることができる。   In the present invention, the surface of the cell culture container has a stimulus-responsive material containing a temperature-responsive molecule, a pH-responsive molecule, and a cell adhesion molecule. In the stimulus-responsive material, there are a plurality of binding structures of temperature-responsive molecules and pH-responsive molecules. Multiple binding structures of temperature-responsive molecules and pH-responsive molecules exist. For example, the presence of alternating temperature-responsive molecules and pH-responsive molecules can give cell detachment function to a wide range of cell culture vessels. it can.

図１は刺激応答性分子の部分構造例を示す。図１の刺激応答性分子１０は、温度応答性分子３０とｐＨ応答性分子４０で構成される。   FIG. 1 shows an example of a partial structure of a stimulus-responsive molecule. The stimulus-responsive molecule 10 in FIG. 1 is composed of a temperature-responsive molecule 30 and a pH-responsive molecule 40.

多官能性分子として細胞接着性分子を利用してもよく、ｐＨ応答性分子と細胞接着性分子の両方を用いることも可能であり、細胞接着性ｐＨ温度応答性材料が得られる。図１の刺激応答性分子２０は、温度応答性分子３０とｐＨ応答性分子４０と細胞接着性分子５０で構成される。   A cell adhesive molecule may be used as the multifunctional molecule, and both a pH responsive molecule and a cell adhesive molecule can be used, and a cell adhesive pH temperature responsive material is obtained. The stimulus-responsive molecule 20 in FIG. 1 is composed of a temperature-responsive molecule 30, a pH-responsive molecule 40, and a cell adhesion molecule 50.

いずれにせよ、温度応答材料は温度変化もしくはｐＨ変化によって水和状態が変化するが、特に、ｐＨ応答性分子と細胞接着分子の両方を用いた場合、１種類の材料のみで目的の機能を達成可能なため、好ましいと言える。なお、温度応答材料は、直鎖ポリマでもよいし、ブランチポリマ、架橋ポリマであってもよい。   In any case, the temperature-responsive material changes its hydration state due to temperature change or pH change. In particular, when both pH-responsive molecules and cell adhesion molecules are used, the desired function can be achieved with only one type of material. This is preferable because it is possible. The temperature responsive material may be a linear polymer, a branch polymer, or a crosslinked polymer.

温度応答材料とｐＨ応答材料を１種類の材料が兼ねない場合でも、別々に温度応答材料とｐＨ応答材料を導入することで細胞培養容器表面に温度応答性とｐＨ応答性を備えさせることは可能だが、均一性の観点から望ましくない。すなわち、細胞接着部位の周辺に温度応答材料しか存在しない場合や、逆にｐＨ応答材料しか存在しない場合が生じ得るため、細胞の確実な剥離は必ずしも実現できない。   Even if one kind of temperature-responsive material and pH-responsive material does not work, it is possible to provide temperature-responsiveness and pH-responsiveness on the cell culture vessel surface by introducing temperature-responsive material and pH-responsive material separately. However, it is not desirable from the viewpoint of uniformity. That is, since there may be a case where only a temperature responsive material is present around the cell adhesion site, or a case where only a pH responsive material is present, reliable cell detachment cannot always be realized.

温度応答材料は下限臨界溶液温度（Lower Critical Solution temperature、 以下、ＬＣＳＴという）を示し得ることが望ましく、そのＬＣＳＴは０℃以上４０℃以下の範囲内であり得ることが好ましい。本発明は細胞を培養する培養容器の発明であり、細胞が生存できる環境を作る必要がある。ＬＣＳＴが０℃より小さい、または４０℃より大きい場合には、細胞の生存が困難になるので、ＬＣＳＴは０℃以上４０℃以下の範囲内にすることが望ましい。   The temperature-responsive material may desirably exhibit a lower critical solution temperature (hereinafter referred to as LCST), and the LCST may preferably be within a range of 0 ° C. or higher and 40 ° C. or lower. The present invention is an invention of a culture vessel for culturing cells, and it is necessary to create an environment in which the cells can survive. When LCST is smaller than 0 ° C. or larger than 40 ° C., cell survival becomes difficult. Therefore, it is desirable that LCST be in the range of 0 ° C. or more and 40 ° C. or less.

温度応答性分子と多官能性分子の結合は、共有結合であることが好ましいが、水素結合やイオン結合等の可逆な結合の可能性を否定するものではない。   The bond between the temperature-responsive molecule and the polyfunctional molecule is preferably a covalent bond, but the possibility of a reversible bond such as a hydrogen bond or an ionic bond is not denied.

温度応答性分子と多官能性分子の間の共有結合生成には、求核部位と被求核部位の反応、求電子部位と被求電子部位の反応、付加反応、転位反応、ラジカル反応、シグマトロピー反応、メタセシス反応、カップリング反応、酸化的結合生成反応、脱水縮合やそれらの組み合わせなどを利用することが出来るが、生産性や簡便性の観点から、求核部位と被求核部位の反応が好ましい。   For covalent bond formation between temperature-responsive molecule and polyfunctional molecule, reaction of nucleophilic site and nucleophilic site, reaction of electrophilic site and electrophilic site, addition reaction, rearrangement reaction, radical reaction, sigmatropy Reactions, metathesis reactions, coupling reactions, oxidative bond formation reactions, dehydration condensation and combinations thereof can be used, but from the viewpoint of productivity and simplicity, the reaction between the nucleophilic site and the nucleophilic site preferable.

求核部位の例としては、アミン、チオール、チオラート、アルコール、アルコキシド、カルボキシレート、有機金属化合物部位、エノール、アセタール、アミナール、などが挙げられるが、これらに限定されるものではない。また、被求核部位の反応の例としては、エポキシド、酸塩化物、酸無水物、エステル、カルボニル、イミン、共役カルボニル、アリールハライド、などが挙げられるが、これらに限定されるものではない。   Examples of nucleophilic moieties include, but are not limited to, amines, thiols, thiolates, alcohols, alkoxides, carboxylates, organometallic compound moieties, enols, acetals, aminals, and the like. Examples of the reaction at the nucleophilic site include, but are not limited to, epoxide, acid chloride, acid anhydride, ester, carbonyl, imine, conjugated carbonyl, aryl halide, and the like.

温度応答材料の原料となる温度応答性分子は、合成上の観点から、両末端に求核部位または被求核部位を有することが好ましいが、これに限定されるものではない。   The temperature-responsive molecule that is a raw material of the temperature-responsive material preferably has a nucleophilic site or a nucleophilic site at both ends from the viewpoint of synthesis, but is not limited thereto.

温度応答材料の原料となる温度応答性分子は、ＬＣＳＴを示し得ることが好ましいが、温度応答性分子が多官能性分子と交互に結合した際にＬＣＳＴを示し得るならば、原料がＬＣＳＴを示す必要はない。   The temperature-responsive molecule that is the raw material of the temperature-responsive material is preferably capable of exhibiting LCST. However, if the temperature-responsive molecule can exhibit LCST when alternately bonded with a polyfunctional molecule, the raw material exhibits LCST. There is no need.

温度応答性分子としては、ε−ポリリジン吉草酸アミド、ε−ポリリジン吉草酸アミド共重合体、ε−ポリリジン酪酸アミド共重合体、ε−ポリリジンプロピオン酸アミド共重合体、Ｎ−ヒドロキシペンチルε−ポリリジン、Ｎ−ヒドロキシペンチルε−ポリリジン共重合体、Ｎ−ヒドロキシブチルε−ポリリジン、Ｎ−ヒドロキシブチルε−ポリリジン共重合体、γ−ポリグルタミン酸ヒドロキシヘキシルアミド、γ−ポリグルタミン酸ヒドロキシヘキシルアミド共重合体、γ−ポリグルタミン酸ヒドロキシペンチルアミド、γ−ポリグルタミン酸ヒドロキシペンチルアミド共重合体、γ−ポリグルタミン酸ヒドロキシブチルアミド共重合体、δ−ポリアスパラギン酸ヒドロキシヘキシルアミド、δ−ポリアスパラギン酸ヒドロキシヘキシルアミド共重合体、δ−ポリアスパラギン酸ヒドロキシペンチルアミド、δ−ポリアスパラギン酸ヒドロキシペンチルアミド共重合体、ポリ乳酸、ポリエチレングリコール共重合体、ポリプロピレングリコール、ポリプロピレングリコール共重合体、ヒドロキシプロピルセルロース誘導体、ポリ−Ｎ−イソプロピルアクリルアミド、ポリ−Ｎ，Ｎ−ジエチルアクリルアミド、ポリメタクリル酸ブチルもしくは、それらの複合体やそれらを部分構造として備えるデンドリマーやゲルや架橋ゲルを含む分子が挙げられるが、それらに限定されるものではない。なお、温度応答性は、温度応答分子の種類や分子量、多官能性分子の種類や量を変えることで制御できる。   As temperature-responsive molecules, ε-polylysine valeric acid amide, ε-polylysine valeric acid amide copolymer, ε-polylysine butyric acid amide copolymer, ε-polylysine propionic acid amide copolymer, N-hydroxypentyl ε-polylysine N-hydroxypentyl ε-polylysine copolymer, N-hydroxybutyl ε-polylysine, N-hydroxybutyl ε-polylysine copolymer, γ-polyglutamic acid hydroxyhexylamide, γ-polyglutamic acid hydroxyhexylamide copolymer, γ-polyglutamic acid hydroxypentylamide, γ-polyglutamic acid hydroxypentylamide copolymer, γ-polyglutamic acid hydroxybutylamide copolymer, δ-polyaspartic acid hydroxyhexylamide, δ-polyaspartic acid hydroxyhexylamide copolymer Polymer, δ-polyaspartic acid hydroxypentylamide, δ-polyaspartic acid hydroxypentylamide copolymer, polylactic acid, polyethylene glycol copolymer, polypropylene glycol, polypropylene glycol copolymer, hydroxypropyl cellulose derivative, poly-N- Examples include, but are not limited to, isopropylacrylamide, poly-N, N-diethylacrylamide, polybutyl methacrylate, or complexes thereof, and dendrimers, gels and cross-linked gels comprising them as a partial structure. Absent. The temperature responsiveness can be controlled by changing the type and molecular weight of the temperature-responsive molecule and the type and amount of the polyfunctional molecule.

多官能性分子が有する官能基の数は、２つ以上であり、直鎖ポリマを調製する場合には２つであることが好ましい。   The number of functional groups possessed by the polyfunctional molecule is 2 or more, and preferably 2 when preparing a linear polymer.

温度応答材料は水中かつ細胞が生存可能なｐＨ領域内でｐＨ応答性を有するべきであることから、多官能性分子としてｐＨ応答分子を利用する場合、ｐＨ応答分子は、ｐＫａが２以上１３以下の酸もしくはｐＫａが２以上１３以下の共役酸を生じ得る塩基を含むことが好ましい。ｐＫａが２以上１３以下の酸としては、カルボン酸、フェノール性ヒドロキシル基、ホウ酸、スルホン酸、ホスホン酸、ホスフィン酸、などが挙げられ、ｐＫａが２以上１３以下の共役酸を生じ得る塩基としては、アミン、イミン、ホスファゼン、グアニジン、ヘテロ芳香族化合物、ヘテロ環状化合物、などが挙げられるが、これらに限定されるものではない。ｐＨ応答性分子の一例としてはエチレンジアミンが挙げられる。なお、ｐＨ応答性は、導入するｐＨ応答分子の種類や量を変えることで制御することができる。   Since the temperature responsive material should have pH responsiveness in water and in a pH range where cells can survive, when using a pH responsive molecule as a multifunctional molecule, the pH responsive molecule has a pKa of 2 or more and 13 or less. It is preferable that an acid or a base having a pKa of 2 or more and 13 or less can be produced. Examples of the acid having a pKa of 2 or more and 13 or less include carboxylic acid, phenolic hydroxyl group, boric acid, sulfonic acid, phosphonic acid, phosphinic acid, and the like, and a base capable of producing a conjugate acid having a pKa of 2 or more and 13 or less. Examples include, but are not limited to, amines, imines, phosphazenes, guanidines, heteroaromatic compounds, and heterocyclic compounds. An example of a pH-responsive molecule is ethylenediamine. The pH responsiveness can be controlled by changing the type and amount of the pH responsive molecule to be introduced.

温度応答材料は、培養容器器材表面への導入の観点から、末端もしくは側鎖に反応性の部位を有する必要がある。反応性の部位としては求核性官能基が好ましく、１級アミンがより好ましいが、これに限定されるものではない。   The temperature-responsive material needs to have a reactive site at the terminal or side chain from the viewpoint of introduction to the surface of the culture vessel equipment. The reactive site is preferably a nucleophilic functional group, more preferably a primary amine, but is not limited thereto.

温度応答材料の培養容器表面への導入は、共有結合を介することが好ましいが、静電吸着や物理吸着などの可能性を否定するものではない。なお、共有結合を介する場合は、求核性官能基と被求核性官能基の反応を用いることが好ましく、その場合、培養容器表面側の官能基は被求核性官能基であることが好ましい。このとき被求核性官能基としては、カルボジイミドやＤＭＴ−ＭＭのような縮合剤によって活性化されたカルボン酸、エポキシド、ＮＨＳエステル、ＨＯＢｔエステル、ＨＯＡtエステル、アルデヒド、酸塩化物、酸無水物、などが挙げられるがそれらに限定されるものではない。   The introduction of the temperature-responsive material to the culture vessel surface is preferably via a covalent bond, but it does not deny the possibility of electrostatic adsorption or physical adsorption. When a covalent bond is used, it is preferable to use a reaction between a nucleophilic functional group and a nucleophilic functional group. In that case, the functional group on the surface of the culture vessel is a nucleophilic functional group. preferable. At this time, as the nucleophilic functional group, carboxylic acid activated by a condensing agent such as carbodiimide or DMT-MM, epoxide, NHS ester, HOBt ester, HOAt ester, aldehyde, acid chloride, acid anhydride, However, it is not limited to them.

細胞接着材料の原料となる細胞接着性分子は、温度応答材料と共有結合していてもよいが、結合していなくてもよいし、共有結合している材料と共有結合していない材料の混合物でもよい。細胞接着性分子を温度応答材料と共有結合させる場合には、温度応答材料と多官能性分子との結合様式と同様の結合様式を利用することが生産性の観点から好ましい。細胞接着性分子を温度応答材料と共有結合させない場合には、細胞接着性分子の溶液塗布による導入が生産性の観点から好ましい。   The cell adhesion molecule as a raw material of the cell adhesion material may be covalently bonded to the temperature-responsive material, but may not be bonded, or a mixture of the covalently bonded material and the non-covalently bonded material. But you can. When the cell adhesion molecule is covalently bonded to the temperature-responsive material, it is preferable from the viewpoint of productivity to use a bonding mode similar to the bonding mode between the temperature-responsive material and the multifunctional molecule. In the case where the cell adhesion molecule is not covalently bonded to the temperature-responsive material, introduction of the cell adhesion molecule by solution application is preferable from the viewpoint of productivity.

細胞接着性分子としては、ゼラチン、コラーゲン、ＲＤＧペプチド、ラミニン、フィブロネクチン、ビトロネクチン、コンドロイチン硫酸塩、ヒアルロン酸、フィブリン、インテグリン、カドヘリン、クローディン、ポリリジン、免疫グロブリン、トロンボスポンジもしくはそれらの部分構造を含む分子が挙げられるが、それらに限定されるものではない。   Cell adhesion molecules include gelatin, collagen, RDG peptide, laminin, fibronectin, vitronectin, chondroitin sulfate, hyaluronic acid, fibrin, integrin, cadherin, claudin, polylysine, immunoglobulin, thrombosponge or partial structures thereof Include, but are not limited to, molecules.

温度応答性材料を導入する細胞培養容器の器材は、細胞毒性がない限り特に制限はなく、例えばポリスチレン、ガラス、ポリペプチド、ポリサッカライド、エポキシ樹脂、アクリル樹脂、メタクリレート樹脂、ビニル樹脂、フェノール樹脂、シリコーン樹脂、フルオロ系樹脂、ポリエステル、無機酸化物、などを用いることが出来る。   The equipment of the cell culture container into which the temperature responsive material is introduced is not particularly limited as long as there is no cytotoxicity. For example, polystyrene, glass, polypeptide, polysaccharide, epoxy resin, acrylic resin, methacrylate resin, vinyl resin, phenol resin, Silicone resin, fluororesin, polyester, inorganic oxide, and the like can be used.

細胞培養容器の表面は、温度応答材料、ｐＨ応答材料、細胞接着材料以外の物質によって修飾されていてもよい。   The surface of the cell culture vessel may be modified with a substance other than the temperature responsive material, the pH responsive material, and the cell adhesion material.

図２は細胞培養容器の製造する工程の一部である。細胞培養容器６０の表面に被求核性官能基７０が存在する。固定化処理により、刺激応答性分子２０と被求核性官能基７０が結合する。   FIG. 2 shows a part of a process for manufacturing a cell culture container. A nucleophilic functional group 70 exists on the surface of the cell culture vessel 60. By the immobilization treatment, the stimulus-responsive molecule 20 and the nucleophilic functional group 70 are bonded.

本発明で提供する細胞培養容器の製造方法は、細胞培養容器の表面活性化、表面活性化された細胞培養容器への導入する材料の溶液の接触、材料導入後の細胞培養容器の洗浄、を工程として含む。一方で、放射線照射や紫外線照射を材料の導入に工程として含まない。   The method for producing a cell culture container provided by the present invention comprises surface activation of a cell culture container, contact of a solution of a material to be introduced into the surface-activated cell culture container, and washing of the cell culture container after introduction of the material. Included as a process. On the other hand, irradiation and ultraviolet irradiation are not included as a process in the introduction of materials.

本発明で提供する細胞培養容器の滅菌は、紫外線照射、γ線照射、エタノール処理、などで実施することが出来る。   Sterilization of the cell culture container provided in the present invention can be carried out by ultraviolet irradiation, γ-ray irradiation, ethanol treatment, or the like.

本発明で提供する細胞培養容器を用いた細胞培養には、一般的な条件を用いることが出来る。   General conditions can be used for cell culture using the cell culture vessel provided in the present invention.

本発明で提供する細胞培養容器を用いた非浸襲的細胞回収は、培養温度にある細胞培養容器の温度を室温もしくは室温以下０℃以上の温度に低下させる工程を含むことが好ましい。また、このときに培養時の培地のまま温度を変化させてもよいし、培地を、あらかじめ一定の温度にされた細胞毒性のない緩衝液に入れ替えて温度を変化させてもよい。細胞毒性のない緩衝液としては、リン酸緩衝液、リン酸緩衝生理食塩水、トリス緩衝液、トリス緩衝生理食塩水、グッド緩衝液（ＭＥＳ、Ｂｉｓ−Ｔｒｉｓ、ＡＤＡ、ＰｌＰＥＳ、ＡＣＥＳ、ＭＯＰＳＯ、ＢＥＳ、ＭＯＰＳ、ＴＥＳ、ＨＥＰＥＳ、ＤｌＰＳＯ、ＴＡＰＳＯ、ＰＯＰＳＯ、ＨＥＰＰＳＯ、ＥＰＰＳ、Ｔｒｉｃｉｎｅ、Ｂｉｃｉｎｅ、ＴＡＰＳ、ＣＨＥＳ、ＣＡＰＳＯ、ＣＡＰＳ）、生理食塩水、培地などが挙げられるが、これらに限定されるものではない。   The noninvasive cell recovery using the cell culture container provided in the present invention preferably includes a step of lowering the temperature of the cell culture container at the culture temperature to room temperature or below 0 ° C. At this time, the temperature may be changed as it is in the culture medium, or the temperature may be changed by replacing the culture medium with a non-cytotoxic buffer solution that has been previously set to a constant temperature. Non-cytotoxic buffers include phosphate buffer, phosphate buffered saline, Tris buffer, Tris buffered saline, Good buffer (MES, Bis-Tris, ADA, PlPES, ACES, MOPSO, BES , MOPS, TES, HEPES, DLPSO, TAPSO, POPSO, HEPPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPSO, CAPS), physiological saline, medium, and the like, but are not limited thereto.

図３は自動培養プロセスと自動培養装置を示す。自動培養装置３２０は自動培養プロセス３１０のように、細胞播種、培地交換、継代、培地交換、継代、細胞剥離の順で処理する。この過程で、温度管理、ＣＯ２濃度管理、光学観察を行う。 FIG. 3 shows an automatic culture process and an automatic culture apparatus. As in the automatic culture process 310, the automatic culture apparatus 320 performs processing in the order of cell seeding, medium exchange, passage, medium exchange, passage, and cell detachment. In this process, temperature management, CO 2 concentration management, and optical observation are performed.

本発明で提供する細胞培養容器は、培地交換、温度管理、ＣＯ２濃度管理、光学観察の機能を有する自動培養装置に適用できる。自動培養装置は、播種機能や継代機能を備えていてもよい。 The cell culture vessel provided in the present invention can be applied to an automatic culture apparatus having functions of medium exchange, temperature management, CO 2 concentration management, and optical observation. The automatic culture apparatus may have a seeding function and a passage function.

自動培養装置３２０は、細胞培養容器６０を置くステージ３３０と、ポンプ３４０と、ポンプ３４０を用いて細胞培養容器６０から培地を吸い出すノズル３５０と、緩衝液を保存する保存部３６０と、細胞培養容器６０に緩衝液を導入するノズル３７０と、保存中の緩衝液の温度を制御する温度制御部３８０と、培養環境の温度を計測する温度計測部３９０と、培養環境のＣＯ２濃度を計測する濃度計測部４００と、培養容器の状態を光学的に観察する観察部４１０を有する。 The automatic culture apparatus 320 includes a stage 330 on which the cell culture container 60 is placed, a pump 340, a nozzle 350 that sucks out the medium from the cell culture container 60 using the pump 340, a storage unit 360 that stores a buffer solution, and a cell culture container. 60, a nozzle 370 for introducing a buffer solution, a temperature control unit 380 for controlling the temperature of the buffer solution during storage, a temperature measurement unit 390 for measuring the temperature of the culture environment, and a concentration for measuring the CO 2 concentration of the culture environment It has the measurement part 400 and the observation part 410 which observes the state of a culture container optically.

これらの機器により、温度やｐＨを制御できる。例えば培養終了時には、細胞剥離のために培地を２５℃の緩衝液に交換した後に２５℃でインキュベートすることができる。また、例えばｐＨ異常によって細胞が剥離した際にも光学系によって自動または手動にて検知できる。   These devices can control temperature and pH. For example, at the end of the culture, the medium can be replaced with a 25 ° C. buffer for cell detachment, and then incubated at 25 ° C. For example, even when cells are detached due to an abnormal pH, it can be detected automatically or manually by an optical system.

実施例１〜２および比較例１〜２で細胞培養容器を作成した。また、実施例３〜４および比較例３〜５では温度およびｐＨ応答による細胞剥離の実証実験を実施した（図４、図５）。   Cell culture containers were prepared in Examples 1-2 and Comparative Examples 1-2. In Examples 3 to 4 and Comparative Examples 3 to 5, demonstration experiments of cell detachment by temperature and pH response were performed (FIGS. 4 and 5).

図４は細胞剥離工程を示す。細胞培養容器６０の表面に被求核性官能基７０及び刺激応答性分子２０の結合物が存在する。さらに、刺激応答分子２０に細胞５２０が接着している。温度およびｐＨ応答による細胞剥離処理により、細胞５５０は剥離する。   FIG. 4 shows a cell detachment process. A binding product of the nucleophilic functional group 70 and the stimulus-responsive molecule 20 is present on the surface of the cell culture vessel 60. Further, the cell 520 is adhered to the stimulus response molecule 20. The cells 550 are detached by the cell detachment treatment based on the temperature and pH response.

図５は細胞剥離実験の培養容器写真を示す。   FIG. 5 shows a culture container photograph of the cell detachment experiment.

＜実施例１＞
実施例１では、温度応答性分子と二官能性ｐＨ応答分子と細胞接着分子を含む材料Ａを合成した。 <Example 1>
In Example 1, a material A containing a temperature-responsive molecule, a bifunctional pH-responsive molecule, and a cell adhesion molecule was synthesized.

ポリプロピレングリコールジグリシジルエーテル（１当量）、エチレンジアミン（１．１当量）、ゼラチン（５ｗｔ％）を含む水/エタノール溶液を６０℃で６時間加熱した。得られた反応溶液に５倍量の水を加え、再度６０℃で加熱することで温度応答性を示す成分のみ沈殿させ、上澄みを捨てることで精製した。この精製操作を３回繰り返した。この材料ＡのＬＣＳＴは約３０℃であった。   A water / ethanol solution containing polypropylene glycol diglycidyl ether (1 equivalent), ethylenediamine (1.1 equivalent), and gelatin (5 wt%) was heated at 60 ° C. for 6 hours. Five times the amount of water was added to the obtained reaction solution, and the mixture was heated again at 60 ° C. to precipitate only the component exhibiting temperature responsiveness, and the supernatant was discarded for purification. This purification operation was repeated three times. The LCST of this material A was about 30 ° C.

＜比較例１＞
比較例１では、温度応答性分子と二官能性ｐＨ応答分子を含み、細胞接着性分子を含まない材料Ｂを合成した。 <Comparative Example 1>
In Comparative Example 1, a material B including a temperature-responsive molecule and a bifunctional pH-responsive molecule and not including a cell adhesion molecule was synthesized.

ポリプロピレングリコールジグリシジルエーテル（１当量）、エチレンジアミン（１．１当量）を含む水/エタノール溶液を６０℃で６時間加熱した。得られた反応溶液に５倍量の水を加え、再度６０℃で加熱することで温度応答性を示す成分のみ沈殿させ、上澄みを捨てることで精製した。この精製操作を３回繰り返した。この材料ＢのＬＣＳＴは約３０℃であった。   A water / ethanol solution containing polypropylene glycol diglycidyl ether (1 equivalent) and ethylenediamine (1.1 equivalent) was heated at 60 ° C. for 6 hours. Five times the amount of water was added to the obtained reaction solution, and the mixture was heated again at 60 ° C. to precipitate only the component exhibiting temperature responsiveness, and the supernatant was discarded for purification. This purification operation was repeated three times. The LCST of this material B was about 30 ° C.

＜実施例２＞
実施例２では、材料Ａを用いて培養容器Ａを作成した。 <Example 2>
In Example 2, the culture container A was prepared using the material A.

表面にカルボン酸を有する親水化ポリスチレン容器（以下、無修飾培養容器）に、ＥＤＣ・ＨＣｌのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で２時間加熱した。その後、容器表面をＰＢＳ緩衝液（ｐＨ５．８）にて洗浄し、材料ＡのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で１２時間加熱した。得られた培養容器Ａは、ＰＢＳ緩衝液（ｐＨ５．８）、純水の順で洗浄した後、紫外線照射によって滅菌した。   A PBS solution (pH 5.8) of EDC · HCl was added to a hydrophilic polystyrene container (hereinafter referred to as “unmodified culture container”) having carboxylic acid on the surface, and heated at 40 ° C. for 2 hours. Thereafter, the surface of the container was washed with a PBS buffer (pH 5.8), a PBS solution (pH 5.8) of material A was added, and the mixture was heated at 40 ° C. for 12 hours. The obtained culture vessel A was washed with PBS buffer (pH 5.8) and pure water in this order, and then sterilized by ultraviolet irradiation.

＜比較例２＞
比較例２では、材料Ｂを用いて培養容器Ｂを作成した。 <Comparative Example 2>
In Comparative Example 2, a culture vessel B was prepared using the material B.

無修飾培養容器に、ＥＤＣ・ＨＣｌのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で２時間加熱した。その後、容器表面をＰＢＳ緩衝液（ｐＨ５．８）にて洗浄し、材料ＢのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で１２時間加熱した。得られた培養容器Ｂは、ＰＢＳ緩衝液（ｐＨ５．８）、純水の順で洗浄した後、紫外線照射によって滅菌した。   A PBS solution (pH 5.8) of EDC · HCl was added to an unmodified culture vessel and heated at 40 ° C. for 2 hours. Thereafter, the container surface was washed with a PBS buffer (pH 5.8), a PBS solution of material B (pH 5.8) was added, and the mixture was heated at 40 ° C. for 12 hours. The obtained culture vessel B was washed with PBS buffer (pH 5.8) and pure water in this order, and then sterilized by ultraviolet irradiation.

＜実施例３＞（温度応答による剥離実験）
実施例３では、培養容器Ａを用いてＭＥＦ細胞（マウス胚性線維芽細胞）の温度応答による剥離実験を実施した。 <Example 3> (Peeling experiment by temperature response)
In Example 3, an exfoliation experiment was performed using the culture vessel A by temperature response of MEF cells (mouse embryonic fibroblasts).

標準的なプロトコルにて、培養容器Ａに培養液を入れ、培養容器Ａ上でＭＥＦ細胞を培養した。細胞が培養容器Ａに十分に接着したことを確認し、培養容器Ａをインキュベータから取り出し、３７℃に温めた培地と交換した後に、４℃に温度を低下させたところ、培養容器Ａから細胞は剥離し、回収率９５％で細胞が回収された。   By using a standard protocol, the culture solution was put into the culture vessel A, and the MEF cells were cultured on the culture vessel A. After confirming that the cells were sufficiently adhered to the culture vessel A, removing the culture vessel A from the incubator and replacing the medium with a medium warmed to 37 ° C, the temperature was lowered to 4 ° C. The cells were detached and recovered at a recovery rate of 95%.

本発明の主な目的は細胞を剥離させて回収することである。細胞の回収率が７０％以上あれば、回収率が高いと言える。なお、本発明で用いる回収率は生死細胞オートアナライザーＶｉ−ＣＥＬＬによって測定した結果である。   The main object of the present invention is to detach and collect cells. If the cell recovery rate is 70% or more, it can be said that the recovery rate is high. In addition, the recovery rate used in the present invention is a result of measurement by viable cell autoanalyzer Vi-CELL.

＜実施例４＞（ｐＨ応答による剥離実験）
実施例４では、培養容器Ａを用いてＭＥＦ細胞のｐＨ応答による剥離実験を実施した。 <Example 4> (Peeling experiment by pH response)
In Example 4, a peeling experiment based on the pH response of MEF cells was performed using the culture vessel A.

標準的なプロトコルにて、培養容器Ａに培養液を入れ、培養容器Ａ上でＭＥＦ細胞を培養した。細胞が培養容器Ａに十分に細胞が接着したことを確認し、培養容器Ａをインキュベータから取り出し、３７℃に温めたｐＨ６．５リン酸緩衝生理食塩水と交換した後に、再度インキュベータ中で静置したところ、培養容器Ａから細胞は剥離し、回収率９５％で細胞が回収された。   By using a standard protocol, the culture solution was put into the culture vessel A, and the MEF cells were cultured on the culture vessel A. After confirming that the cells were sufficiently adhered to the culture vessel A, the culture vessel A was removed from the incubator, replaced with pH 6.5 phosphate buffered saline heated to 37 ° C., and then left in the incubator again. As a result, the cells were detached from the culture vessel A, and the cells were recovered at a recovery rate of 95%.

＜比較例３＞（温度応答による剥離実験）
比較例３では、培養容器Ｂを用いてＭＥＦ細胞の温度応答による剥離実験を実施した。 <Comparative example 3> (Peeling experiment by temperature response)
In Comparative Example 3, an exfoliation experiment based on the temperature response of MEF cells was performed using the culture vessel B.

標準的なプロトコルにて、培養容器Ｂに培養液を入れ、培養容器Ｂ上でＭＥＦ細胞を培養した。細胞が培養容器Ｂに十分に細胞が接着したことを確認し、培養容器Ｂをインキュベータ中で一晩静置したものの、接着した細胞の数は播種した細胞の数の５％以下であり、細胞接着性は低かった。   By using a standard protocol, the culture solution was put into the culture vessel B, and the MEF cells were cultured on the culture vessel B. Although it was confirmed that the cells were sufficiently adhered to the culture vessel B and the culture vessel B was allowed to stand overnight in an incubator, the number of adhered cells was 5% or less of the number of seeded cells. The adhesion was low.

比較例３では、実施例３、４と比べて回収率が非常に低かった。これは、培養容器Ｂが温度応答性分子と二官能性ｐＨ応答分子を含み、細胞接着性分子を含まない材料を用いるからである。培養容器Ｂが細胞接着性分子を有することで、多くの細胞を培養容器Ｂに十分に収集することができ、十分に収集できたからこそ細胞の回収率が向上するものである。   In Comparative Example 3, the recovery rate was very low as compared with Examples 3 and 4. This is because the culture vessel B uses a material that contains a temperature-responsive molecule and a bifunctional pH-responsive molecule, but does not contain a cell adhesion molecule. Since the culture vessel B has cell adhesion molecules, a large number of cells can be sufficiently collected in the culture vessel B, and the collection rate of the cells is improved because the cells are sufficiently collected.

＜比較例４＞（温度応答による剥離実験）
比較例４では、無修飾培養容器を用いてＭＥＦ細胞の温度応答による剥離実験を実施した。 <Comparative example 4> (Peeling experiment by temperature response)
In Comparative Example 4, an exfoliation experiment based on the temperature response of MEF cells was performed using an unmodified culture vessel.

標準的なプロトコルにて、無修飾培養容器に培養液を入れ、無修飾培養容器上でＭＥＦ細胞を培養した。細胞が無修飾培養容器に十分に細胞が接着したことを確認し、無修飾培養容器をインキュベータから取り出し、３７℃に温めた培地と交換した後に、４℃に温度を低下させたが、細胞は回収されなかった。   By using a standard protocol, the culture solution was put into an unmodified culture vessel, and MEF cells were cultured on the unmodified culture vessel. After confirming that the cells were sufficiently adhered to the unmodified culture vessel, the unmodified culture vessel was removed from the incubator and replaced with a medium warmed to 37 ° C., and the temperature was lowered to 4 ° C. It was not recovered.

＜比較例５＞（ｐＨ応答による剥離実験）
比較例５では、無修飾培養容器を用いてＭＥＦ細胞のｐＨ応答による剥離実験を実施した。 <Comparative Example 5> (Peeling experiment by pH response)
In Comparative Example 5, a peeling experiment based on the pH response of MEF cells was performed using an unmodified culture vessel.

標準的なプロトコルにて、無修飾培養容器に培養液を入れ、無修飾培養容器上でＭＥＦ細胞を培養した。細胞が無修飾培養容器に十分に細胞が接着したことを確認し、無修飾培養容器をインキュベータから取り出し、３７℃に温めたｐＨ６．５緩衝液と交換した後に、再度インキュベータ中で静置したところ、回収率４０％で細胞が回収された。   By using a standard protocol, the culture solution was put into an unmodified culture vessel, and MEF cells were cultured on the unmodified culture vessel. After confirming that the cells were sufficiently adhered to the unmodified culture vessel, the unmodified culture vessel was removed from the incubator, replaced with a pH 6.5 buffer warmed to 37 ° C., and then allowed to stand again in the incubator. Cells were recovered at a recovery rate of 40%.

＜実施例５＞
実施例５では、培養容器Ａを自動培養装置に適用し、温度制御による細胞剥離を実証した。 <Example 5>
In Example 5, the culture vessel A was applied to an automatic culture apparatus to demonstrate cell detachment by temperature control.

自動培養装置中にて培養容器Ａを用いてｉＰＳ細胞を培養した後に、培地交換機能を用いて培地の大部分を取り除き、あらかじめ２５℃にされたｐＨ７．４リン酸緩衝生理食塩水と交換した。その後、培養容器Ａを２５℃にてインキュベートしたところ、顕微鏡観察にて、ｉＰＳ細胞の剥離が確認された。   After culturing iPS cells using the culture vessel A in an automatic culture apparatus, most of the medium was removed using the medium exchange function, and replaced with pH 7.4 phosphate buffered saline that had been brought to 25 ° C. in advance. . Thereafter, when the culture vessel A was incubated at 25 ° C., iPS cell detachment was confirmed by microscopic observation.

＜実施例６＞
実施例６では、培養容器Ａを自動培養装置に適用し、ｐＨ異常時の細胞剥離を再現した実証を行った。 <Example 6>
In Example 6, the culture container A was applied to an automatic culture apparatus, and a demonstration that reproduced cell detachment when pH was abnormal was performed.

自動培養装置中にて培養容器Ａを用いてｉＰＳ細胞を培養した後に、培地交換機能を用いて培地の大部分を取り除き、あらかじめ３７℃にされたｐＨ６．５リン酸緩衝生理食塩水と交換することでｐＨ低下を再現した。その後、培養容器Ａを３７℃にてインキュベートしたところ、顕微鏡観察にて、ｉＰＳ細胞の剥離が確認された。   After culturing iPS cells using the culture vessel A in an automatic culture apparatus, most of the medium is removed using the medium exchange function, and replaced with pH 6.5 phosphate buffered saline that has been previously brought to 37 ° C. The pH drop was reproduced. Thereafter, when the culture vessel A was incubated at 37 ° C., iPS cell detachment was confirmed by microscopic observation.

実施例３、４ではＭＥＦ細胞を用いて細胞が培養容器から剥離することを確認した。また、実施例５、６ではｉＰＳ細胞を用いて細胞が培養容器から剥離するかを確認した。以上により、実施例２で作成した培養容器を用いることで、細胞の種類によらず高い回収率で細胞を回収できることが確認できた。   In Examples 3 and 4, it was confirmed that cells were detached from the culture vessel using MEF cells. In Examples 5 and 6, iPS cells were used to confirm whether the cells were detached from the culture vessel. As described above, it was confirmed that by using the culture container prepared in Example 2, it was possible to recover cells with a high recovery rate regardless of the type of cells.

＜比較例６＞
比較例６では、温度応答性分子と二官能性ｐＨ応答分子と細胞接着分子を含むが、下限溶解臨界温度が０℃より小さい材料Ｃを合成した。 <Comparative Example 6>
In Comparative Example 6, a material C containing a temperature-responsive molecule, a bifunctional pH-responsive molecule, and a cell adhesion molecule, but having a lower limit dissolution critical temperature lower than 0 ° C. was synthesized.

ポリプロピレングリコールジグリシジルエーテル（１当量）、エチレンジアミン（５当量）、ゼラチン（５ｗｔ％）を含む水/エタノール溶液を６０℃で６時間加熱した。得られた反応溶液は透析により精製した。この材料ＣはＬＣＳＴを示さなかった。   A water / ethanol solution containing polypropylene glycol diglycidyl ether (1 equivalent), ethylenediamine (5 equivalents), and gelatin (5 wt%) was heated at 60 ° C. for 6 hours. The obtained reaction solution was purified by dialysis. This material C showed no LCST.

＜比較例７＞
比較例７では、材料Ｃを用いて培養容器Ｃを作成した。 <Comparative Example 7>
In Comparative Example 7, a culture vessel C was prepared using the material C.

無修飾培養容器に、ＥＤＣ・ＨＣｌのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で２時間加熱した。その後、容器表面をＰＢＳ緩衝液（ｐＨ５．８）にて洗浄し、材料ＣのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で１２時間加熱した。得られた培養容器Ｃは、ＰＢＳ緩衝液（ｐＨ５．８）、純水の順で洗浄した後、紫外線照射によって滅菌した。   A PBS solution (pH 5.8) of EDC · HCl was added to an unmodified culture vessel and heated at 40 ° C. for 2 hours. Thereafter, the surface of the container was washed with a PBS buffer (pH 5.8), a PBS solution of material C (pH 5.8) was added, and the mixture was heated at 40 ° C. for 12 hours. The obtained culture container C was washed with PBS buffer (pH 5.8) and pure water in this order, and then sterilized by ultraviolet irradiation.

＜比較例８＞
比較例８では、培養容器Ｃを用いてＭＥＦ細胞の温度応答による剥離実験を実施した。標準的なプロトコルにて、培養容器Ｃに培養液を入れ、培養容器Ｃ上でＭＥＦ細胞を培養した。細胞が培養容器Ｃに十分に接着したことを確認し、培養容器Ｃをインキュベータから取り出し、３７℃に温めたｐＨ６．５リン酸緩衝生理食塩水と交換した後に、再度インキュベータ中で静置したところ、培養容器Ｃから細胞は剥離し、回収率１０％で細胞が回収された。 <Comparative Example 8>
In Comparative Example 8, an exfoliation experiment based on the temperature response of MEF cells was performed using the culture vessel C. Using a standard protocol, the culture solution was put into the culture vessel C, and the MEF cells were cultured on the culture vessel C. After confirming that the cells adhered sufficiently to the culture vessel C, the culture vessel C was removed from the incubator, replaced with pH 6.5 phosphate buffered saline heated to 37 ° C., and then left to stand in the incubator again. The cells were detached from the culture vessel C, and the cells were recovered at a recovery rate of 10%.

＜実施例７＞
実施例７では、温度応答性分子と二官能性ｐＨ応答分子と細胞接着分子を含む材料Ｄを合成した。 <Example 7>
In Example 7, a material D containing a temperature-responsive molecule, a bifunctional pH-responsive molecule, and a cell adhesion molecule was synthesized.

Ｎ−アシル化ポリリジン（１当量）、ポリエチレングリコールジグリシジルエーテル（１．１当量）、ゼラチン（５ｗｔ％）を含む水/エタノール溶液を６０℃で６時間加熱した。得られた反応溶液は透析により精製した。この材料ＤのＬＣＳＴは約３０℃であった。   A water / ethanol solution containing N-acylated polylysine (1 equivalent), polyethylene glycol diglycidyl ether (1.1 equivalent) and gelatin (5 wt%) was heated at 60 ° C. for 6 hours. The obtained reaction solution was purified by dialysis. The LCST of material D was about 30 ° C.

＜実施例８＞
実施例８では、材料Ｄを用いて培養容器Ｄを作成した。 <Example 8>
In Example 8, a culture vessel D was prepared using the material D.

無修飾培養容器に、ＥＤＣ・ＨＣｌのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で２時間加熱した。その後、容器表面をＰＢＳ緩衝液（ｐＨ５．８）にて洗浄し、材料ＤのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で１２時間加熱した。得られた培養容器Ｄは、ＰＢＳ緩衝液（ｐＨ５．８）、純水の順で洗浄した後、紫外線照射によって滅菌した。   A PBS solution (pH 5.8) of EDC · HCl was added to an unmodified culture vessel and heated at 40 ° C. for 2 hours. Thereafter, the surface of the container was washed with a PBS buffer (pH 5.8), a PBS solution (pH 5.8) of material D was added, and the mixture was heated at 40 ° C. for 12 hours. The obtained culture vessel D was washed with PBS buffer (pH 5.8) and pure water in this order, and then sterilized by ultraviolet irradiation.

＜実施例９＞（温度応答による剥離実験）
実施例９では、培養容器Ｄを用いてＣＨＯ細胞の温度応答による剥離実験を実施した。 <Example 9> (Peeling experiment by temperature response)
In Example 9, an exfoliation experiment based on the temperature response of CHO cells was performed using the culture vessel D.

標準的なプロトコルにて、培養容器Ｄに培養液を入れ、培養容器Ｄ上でＣＨＯ細胞を培養した。細胞が培養容器Ｄに十分に接着したことを確認し、培養容器Ｄをインキュベータから取り出し、３７℃に温めたｐＨ６．５リン酸緩衝生理食塩水と交換した後に、再度インキュベータ中で静置したところ、培養容器Ａから細胞は剥離し、回収率７０％で細胞が回収された。   Using a standard protocol, the culture solution was put into the culture vessel D, and CHO cells were cultured on the culture vessel D. After confirming that the cells were sufficiently adhered to the culture vessel D, the culture vessel D was removed from the incubator, replaced with pH 6.5 phosphate buffered saline heated to 37 ° C., and then allowed to stand again in the incubator. The cells were detached from the culture vessel A, and the cells were recovered at a recovery rate of 70%.

＜実施例１０＞（ｐＨ応答による剥離実験）
実施例１０では、培養容器Ｄを用いてＣＨＯ細胞のｐＨ応答による剥離実験を実施した。 <Example 10> (Peeling experiment by pH response)
In Example 10, a peeling experiment based on the pH response of CHO cells was performed using the culture vessel D.

標準的なプロトコルにて、培養容器Ｄに培養液を入れ、培養容器Ｄ上でＣＨＯ細胞を培養した。細胞が培養容器Ｄに十分に接着したことを確認し、培養容器Ｄをインキュベータから取り出し、３７℃に温めたｐＨ６．５緩衝液と交換した後に、再度インキュベータ中で静置したところ、培養容器Ｄから細胞は剥離し、回収率８０％で細胞が回収された。   Using a standard protocol, the culture solution was put into the culture vessel D, and CHO cells were cultured on the culture vessel D. After confirming that the cells were sufficiently adhered to the culture vessel D, the culture vessel D was removed from the incubator, replaced with a pH 6.5 buffer solution warmed to 37 ° C., and then allowed to stand again in the incubator. The cells were detached, and the cells were collected at a recovery rate of 80%.

＜実施例１１＞
実施例１１では、温度応答性分子と二官能性ｐＨ応答分子と細胞接着分子を含む材料Ｅを合成した。 <Example 11>
In Example 11, a material E containing a temperature-responsive molecule, a bifunctional pH-responsive molecule, and a cell adhesion molecule was synthesized.

Ｎ−アルキル化ポリリジン（１当量）、ポリエチレングリコールジグリシジルエーテル（１．１当量）、ゼラチン（５ｗｔ％）を含む水/エタノール溶液を６０℃で６時間加熱した。得られた反応溶液は透析により精製した。この材料ＥのＬＣＳＴは約３０℃であった。   A water / ethanol solution containing N-alkylated polylysine (1 equivalent), polyethylene glycol diglycidyl ether (1.1 equivalent) and gelatin (5 wt%) was heated at 60 ° C. for 6 hours. The obtained reaction solution was purified by dialysis. The LCST of this material E was about 30 ° C.

＜実施例１２＞
実施例１２では、材料Ｅを用いて培養容器Ｅを作成した。 <Example 12>
In Example 12, a culture vessel E was prepared using the material E.

無修飾培養容器に、ＥＤＣ・ＨＣｌのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で２時間加熱した。その後、容器表面をＰＢＳ緩衝液（ｐＨ５．８）にて洗浄し、材料ＥのＰＢＳ溶液（ｐＨ５．８）を加え、４０℃で１２時間加熱した。得られた培養容器Ｅは、ＰＢＳ緩衝液（ｐＨ５．８）、純水の順で洗浄した後、紫外線照射によって滅菌した。   A PBS solution (pH 5.8) of EDC · HCl was added to an unmodified culture vessel and heated at 40 ° C. for 2 hours. Thereafter, the container surface was washed with a PBS buffer (pH 5.8), a PBS solution (pH 5.8) of material E was added, and the mixture was heated at 40 ° C. for 12 hours. The obtained culture vessel E was washed with PBS buffer solution (pH 5.8) and pure water in this order, and then sterilized by ultraviolet irradiation.

＜実施例１３＞（Ph応答による薄利実験）
実施例１３では、培養容器Ｅを用いてＭＥＦ細胞のｐＨ応答による剥離実験を実施した。 <Example 13> (Slight experiment by Ph response)
In Example 13, a peeling experiment based on the pH response of MEF cells was performed using the culture vessel E.

標準的なプロトコルにて、培養容器Ｅに培養液を入れ、培養容器Ｅ上でＭＥＦ細胞を培養した。細胞が培養容器Ｅに十分に接着したことを確認し、培養容器Ｅをインキュベータから取り出し、３７℃に温めたｐＨ６．５緩衝液と交換した後に、再度インキュベータ中で静置したところ、培養容器Ｅから細胞は剥離し、回収率８０％で細胞が回収された。   Using a standard protocol, the culture solution was put into the culture vessel E, and the MEF cells were cultured on the culture vessel E. After confirming that the cells were sufficiently adhered to the culture vessel E, the culture vessel E was removed from the incubator, replaced with a pH 6.5 buffer solution warmed to 37 ° C., and then allowed to stand again in the incubator. The cells were detached, and the cells were collected at a recovery rate of 80%.

＜実施例１４＞（ｐＨ応答による剥離実験）
実施例１４では、培養容器Ｅを用いてＭＥＦ細胞のｐＨ応答による剥離実験を実施した。 <Example 14> (Peeling experiment by pH response)
In Example 14, a peeling experiment based on the pH response of MEF cells was performed using the culture vessel E.

標準的なプロトコルにて、培養容器Ｅに培養液を入れ、培養容器Ｅ上でＭＥＦ細胞を培養した。細胞が培養容器Ｅに十分に接着したことを確認し、培養容器Ｅをインキュベータから取り出し、３７℃に温めたｐＨ６．５緩衝液と交換した後に、再度インキュベータ中で静置したところ、培養容器Ｅから細胞は剥離し、回収率７０％で細胞が回収された。   Using a standard protocol, the culture solution was put into the culture vessel E, and the MEF cells were cultured on the culture vessel E. After confirming that the cells were sufficiently adhered to the culture vessel E, the culture vessel E was removed from the incubator, replaced with a pH 6.5 buffer solution warmed to 37 ° C., and then allowed to stand again in the incubator. The cells were detached from the cells, and the cells were recovered at a recovery rate of 70%.

１０ 刺激応答性分子
２０ 刺激応答性分子
３０ 温度応答性分子
４０ ｐＨ応答性分子
５０ 細胞接着性分子
６０ 細胞培養容器
７０ 被求核性官能基
３１０ 自動培養プロセス
３２０ 自動培養装置
３３０ ステージ
３４０ ポンプ
３５０ ノズル
３６０ 保存部
３７０ ノズル
３８０ 温度制御部
３９０ 温度計測部
４００ 濃度計測部
４１０ 観察部
５１０ 細胞が接着した細胞培養容器表面
５２０ 細胞
５５０ 細胞 DESCRIPTION OF SYMBOLS 10 Stimulus responsive molecule 20 Stimulus responsive molecule 30 Temperature responsive molecule 40 pH responsive molecule 50 Cell adhesion molecule 60 Cell culture vessel 70 Nucleophilic functional group 310 Automatic culture process 320 Automatic culture device 330 Stage 340 Pump 350 Nozzle 360 Storage Unit 370 Nozzle 380 Temperature Control Unit 390 Temperature Measurement Unit 400 Concentration Measurement Unit 410 Observation Unit 510 Cell Culture Container Surface 520 Cells 550 Cells Adhered to Cells

Claims (11)

細胞を培養する細胞培養容器であって、
前記細胞培養容器の表面に、温度応答性分子、ｐＨ応答性分子及び細胞接着性分子を含む刺激応答性材料を有し、
前記刺激応答性材料において、前記温度応答性分子と前記ｐＨ応答性分子の結合構造が複数存在することを特徴とする細胞培養容器。 A cell culture vessel for culturing cells,
On the surface of the cell culture container, there is provided a stimulus-responsive material containing a temperature-responsive molecule, a pH-responsive molecule and a cell adhesion molecule,
The cell culture container, wherein the stimulus-responsive material includes a plurality of binding structures of the temperature-responsive molecule and the pH-responsive molecule. 請求項１に記載の細胞培養容器であって、
前記温度応答性分子は下限臨界溶解温度を示す分子であることを特徴とする細胞培養容器。 The cell culture container according to claim 1,
The cell culture vessel, wherein the temperature-responsive molecule is a molecule exhibiting a lower critical solution temperature. 請求項２に記載の細胞培養容器であって、
前記下限臨界溶解温度を示す分子は、ポリアルキレングリコール、Ｎ−アシル化ポリリジン、Ｎ−アルキル化ポリリジン、γ−ポリグルタミン酸ヒドロキシヘキシルアミド、δ−ポリアスパラギン酸ヒドロキシヘキシルアミド、ヒドロキシプロピルセルロース、ポリ−Ｎ−イソプロピルアクリルアミド、ポリ−Ｎ，Ｎ−ジエチルアクリルアミド、のいずれかを含むことを特徴とする細胞培養容器。 A cell culture container according to claim 2,
The molecules exhibiting the lower critical solution temperature are polyalkylene glycol, N-acylated polylysine, N-alkylated polylysine, γ-polyglutamic acid hydroxyhexylamide, δ-polyaspartic acid hydroxyhexylamide, hydroxypropylcellulose, poly-N. A cell culture vessel comprising any of isopropylacrylamide and poly-N, N-diethylacrylamide. 請求項１に記載の細胞培養容器であって、
前記ｐＨ応答性分子は、ｐＫａが２以上１３以下の酸、またはｐＫａが２以上１３以下の共役酸を生じ得る塩基を含むことを特徴とする細胞培養容器。 The cell culture container according to claim 1,
The cell-culture container, wherein the pH-responsive molecule contains an acid having a pKa of 2 or more and 13 or less, or a base capable of generating a conjugate acid having a pKa of 2 or more and 13 or less. 請求項４に記載の細胞培養容器であって、
前記ｐＫａが２以上１３以下の酸は、カルボン酸、フェノール性ヒドロキシル基、ホウ酸、スルホン酸、ホスホン酸、ホスフィン酸のいずれかを含み、
前記ｐＫａが２以上１３以下の共役酸を生じ得る塩基は、アミン、イミン、ホスファゼン、グアニジン、ヘテロ芳香族化合物、ヘテロ環状化合物、のいずれかを含むことを特徴とする細胞培養容器。 The cell culture container according to claim 4, wherein
The acid having a pKa of 2 or more and 13 or less includes any of carboxylic acid, phenolic hydroxyl group, boric acid, sulfonic acid, phosphonic acid, and phosphinic acid,
The cell culture vessel, wherein the base capable of producing a conjugate acid having a pKa of 2 or more and 13 or less includes any of amine, imine, phosphazene, guanidine, heteroaromatic compound, and heterocyclic compound. 請求項１に記載の細胞培養容器であって、
前記細胞接着性分子は、細胞膜上の分子と相互作用し得る分子であることを特徴とする細胞培養容器。 The cell culture container according to claim 1,
The cell culture container, wherein the cell adhesion molecule is a molecule capable of interacting with a molecule on a cell membrane. 請求項６に記載の細胞培養容器であって、
前記細胞膜上の分子と相互作用し得る分子は、ゼラチン、コラーゲン、ＲＤＧペプチド、ラミニン、フィブロネクチン、ビトロネクチン、コンドロイチン硫酸塩、ヒアルロン酸、フィブリン、インテグリン、カドヘリン、クローディン、ポリリジン、免疫グロブリン、トロンボスポンジ、のいずれかを含むことを特徴とする細胞培養容器。 The cell culture container according to claim 6,
Molecules that can interact with molecules on the cell membrane are gelatin, collagen, RDG peptide, laminin, fibronectin, vitronectin, chondroitin sulfate, hyaluronic acid, fibrin, integrin, cadherin, claudin, polylysine, immunoglobulin, thrombosponge, A cell culture vessel comprising any of the above. 細胞培養容器を用いて細胞を培養する細胞培養方法であって、
前記細胞培養容器の表面に、温度応答性分子、ｐＨ応答性分子及び細胞接着性分子を含む刺激応答性材料を有し、
前記刺激応答性材料において、前記温度応答性分子と前記ｐＨ応答性分子の結合構造が複数存在し、
前記多官能性分子はｐＨ応答性分子及び細胞接着性分子を含み、
前記細胞培養容器に培養液を入れて細胞を培養する培養工程と、
前記細胞培養容器に所定の処理を加えて、前記細胞培養容器から細胞を剥離させる剥離工程を有することを特徴とする細胞培養方法。 A cell culture method for culturing cells using a cell culture container,
On the surface of the cell culture container, there is provided a stimulus-responsive material containing a temperature-responsive molecule, a pH-responsive molecule and a cell adhesion molecule,
In the stimulus-responsive material, there are a plurality of binding structures of the temperature-responsive molecule and the pH-responsive molecule,
The multifunctional molecule includes a pH responsive molecule and a cell adhesion molecule;
A culture step of culturing cells by placing a culture solution in the cell culture vessel;
A cell culture method comprising a peeling step of applying a predetermined treatment to the cell culture container to peel the cells from the cell culture container. 請求項８に記載の細胞培養方法であって、
前記所定の処理は、前記培養工程の時から温度を低下させる処理であることを特徴とする細胞培養方法。 The cell culture method according to claim 8, wherein
The cell culturing method, wherein the predetermined treatment is a treatment for lowering the temperature from the time of the culturing step. 請求項８に記載の細胞培養方法であって、
前記所定の処理は、前記培養液を細胞毒性のない緩衝液に交換する処理であることを特徴とする細胞培養方法。 The cell culture method according to claim 8, wherein
The cell culture method, wherein the predetermined treatment is a treatment for exchanging the culture solution with a buffer solution having no cytotoxicity. 請求項１０に記載の細胞培養方法であって、
前記緩衝液は、リン酸緩衝液、リン酸緩衝生理食塩水、トリス緩衝液、トリス緩衝生理食塩水、グッド緩衝液、生理食塩水、培地、のいずれかであることを特徴とする細胞培養方法。 The cell culture method according to claim 10, wherein
The cell culture method, wherein the buffer solution is any one of a phosphate buffer solution, a phosphate buffered saline solution, a Tris buffer solution, a Tris buffered saline solution, a Good buffer solution, a physiological saline solution, and a medium. .
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