JP2008220205A - Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate - Google Patents

Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate Download PDF

Info

Publication number
JP2008220205A
JP2008220205A JP2007059745A JP2007059745A JP2008220205A JP 2008220205 A JP2008220205 A JP 2008220205A JP 2007059745 A JP2007059745 A JP 2007059745A JP 2007059745 A JP2007059745 A JP 2007059745A JP 2008220205 A JP2008220205 A JP 2008220205A
Authority
JP
Japan
Prior art keywords
neural stem
water
stem cell
container
cell aggregate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2007059745A
Other languages
Japanese (ja)
Inventor
Hayao Tanaka
速雄 田中
Yoshiaki Watanabe
芳明 渡辺
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sumitomo Bakelite Co Ltd
Original Assignee
Sumitomo Bakelite Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Bakelite Co Ltd filed Critical Sumitomo Bakelite Co Ltd
Priority to JP2007059745A priority Critical patent/JP2008220205A/en
Publication of JP2008220205A publication Critical patent/JP2008220205A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/20—Material Coatings

Landscapes

  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Microbiology (AREA)
  • Clinical Laboratory Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Sustainable Development (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Immunology (AREA)

Abstract

<P>PROBLEM TO BE SOLVED: To provide a container for forming a neural stem cell aggregate, capable of efficiently and readily affording the neural stem cell aggregate in a step of forming the neural stem cell aggregate for obtaining neural stem cells from a biological tissue containing the neural stem cells, to provide a method for producing the container, and to provide a method for preparing the neural stem cell aggregate. <P>SOLUTION: The method for producing the container for forming the neural stem cell aggregate is characterized by comprising a water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin and forming a water-soluble resin coating layer and a water-insoluble cured film-modifying step of curing the water-soluble resin coating layer and modifying the coating layer into a water-insoluble cured film layer after the step. The container for forming the neural stem cell aggregate is produced by the method for production. The method for preparing the neural stem cell aggregate is characterized by suspending a tissue containing multifunctional neural stem cells in a culture medium containing at least one kind of stem cell growth factor, carrying out seeding and culturing thereof in the container for forming the neural stem cell aggregate and thereby forming the neural stem cell aggregate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、神経幹細胞凝集塊形成用容器、その製造方法、及び神経幹細胞凝集塊の作成方法に関する。   The present invention relates to a container for forming neural stem cell aggregates, a method for producing the same, and a method for producing neural stem cell aggregates.

哺乳動物生体組織内には、自己複製能とさまざまなタイプのニューロン、グリア細胞を生産する多能性を有する神経系の幹細胞が存在する。
神経系の幹細胞は個体の発生初期から生体に至るまで骨髄のような活発に分裂する組織内に存在し、その構築及び機能維持に関与している。
ところが、殆どの哺乳類においてその生体内においては神経幹細胞の多能性は制限され、新規ニューロン細胞の発生能力が低い為、例えば障害や病気によりニューロン細胞が失われた場合、新たな神経組織を再生し、補う、いわゆる自然治癒は困難である。 In the mammalian biological tissue, there are neural stem cells having self-replicating ability and pluripotency to produce various types of neurons and glial cells.
Nervous stem cells are present in actively dividing tissues such as bone marrow from the early stage of development to the living body, and are involved in the construction and maintenance of functions.
However, in most mammals, the pluripotency of neural stem cells is limited in vivo, and the ability to generate new neuronal cells is low, so if neuronal cells are lost due to a disorder or disease, for example, new neural tissue is regenerated. However, so-called natural healing is difficult.

近年の研究において、神経幹細胞がｉｎ ｖｉｔｒｏにおける特定の条件下で分化させることによってニューロン、グリア細胞に分化する即ち多能性を発揮する事が明らかになり、これまで困難であった特定の神経変性疾患および外傷の治療に新しい可能性を与えるものとして期待されている。   Recent research has revealed that neural stem cells differentiate into neurons and glial cells by differentiating them under specific conditions in vitro, that is, they exhibit pluripotency. It is expected to provide new possibilities for the treatment of disease and trauma.

胎児や生体から中枢神経系の細胞の中に僅かに含まれる神経幹細胞を未分化の状態で単離・生成し、大量に培養する事が出来れば、分化誘導因子の研究や、それを応用した薬剤のスクリーニング、細胞移植による治療、遺伝子治療のプローブ等、幅広い用途への応用が期待できる。
生体組織から未分化の神経幹細胞を単離する事が、前述の研究のファーストステップであり、ニューロスフェア法という選択的培養方法が現在最も一般的な神経幹細胞の単離方法として知られている。 If neural stem cells that are slightly contained in cells of the central nervous system from fetuses and living bodies can be isolated and generated in an undifferentiated state and cultured in large quantities, research on differentiation-inducing factors and its application Applications to a wide range of applications such as drug screening, cell transplantation, and gene therapy probes can be expected.
Isolating undifferentiated neural stem cells from biological tissue is the first step in the above-mentioned research, and a selective culture method called neurosphere method is currently known as the most common method for isolating neural stem cells.

ニューロスフェア法とはＥＧＦあるいはＦＧＦ−２という増殖因子存在下で神経幹細胞を含む中枢神経系の細胞を浮遊系で培養する事で、神経幹細胞が分裂を繰り替えして自分と同じ細胞集団を作っていき、神経幹細胞凝集塊を形成する性質を利用したもので、そのようにして得られた神経幹細胞凝集塊は組織から単離された未分化の神経幹細胞の集合体であり、そうして得られた集合体は次の分化誘導ステップに供する事が出来る。
前述の神経幹細胞凝集塊形成において、神経幹細胞が培養容器表面に接着すると非特異な分化が誘引されてしまうため、完全な浮遊状態で培養することが重要な事となる。 The neurosphere method is a culture of central nervous system cells, including neural stem cells, in the presence of EGF or FGF-2 in the presence of growth factors. The neural stem cell aggregate obtained in this way is an aggregate of undifferentiated neural stem cells isolated from the tissue and thus obtained using the property of forming neural stem cell aggregates. The aggregate can be used for the next differentiation induction step.
In the above-described formation of neural stem cell aggregates, non-specific differentiation is induced when neural stem cells adhere to the surface of the culture vessel. Therefore, it is important to culture in a completely floating state.

現在培養用容器として、浮遊培養系の細胞を培養する際に使用される浮遊培養用容器と呼ばれるものが使用されており、ポリスチレン成形品またはポリスチレン成形品表面に酸素プラズマ処理やコロナ処理によって濡れ性を変化させて細胞接着を少なくしたものであるが、それらの容器では完全に神経幹細胞の接着を防止する事が困難で、培養日数を経るに従い、形成した幹細胞凝集塊の一部が培養容器表面に接着してしまうと共に一回の培養で得られる幹細胞の数、即ち形成される幹細胞凝集塊の数とサイズが充分ではなく、必要量の神経幹細胞を得るために、幹細胞の自己複製能力を利用して形成した幹細胞凝集塊をバラバラにして再度同様の培養工程を繰り返す作業が必要となる。
一方、分化誘導性増殖因子を用いて、神経幹細胞から神経前駆細胞を生成／分化させて分化神経細胞を生成する方法が開示されている。（例えば、特許文献１参照） Currently, the culture vessel is a suspension culture vessel that is used when culturing cells in suspension culture. The polystyrene molded product or the surface of the polystyrene molded product is wettable by oxygen plasma treatment or corona treatment. However, in these containers, it is difficult to completely prevent neural stem cell adhesion, and as the number of days of culture passes, a part of the aggregated stem cell aggregates form on the surface of the culture container. The number of stem cells obtained in one culture, that is, the number and size of stem cell aggregates formed is insufficient, and the self-replication ability of stem cells is used to obtain the required amount of neural stem cells. Thus, it is necessary to separate the stem cell aggregates formed in this manner and repeat the same culturing process again.
On the other hand, a method for generating differentiated neurons by generating / differentiating neural progenitor cells from neural stem cells using differentiation-inducing growth factors is disclosed. (For example, see Patent Document 1)

特開２００７−１４３５２号公報JP 2007-14352 A

本発明は、上記事情に鑑みてなされたものであり、神経幹細胞を含む生体組織から神経幹細胞を得るための神経幹細胞凝集塊形成工程において、神経幹細胞凝集塊を効率よく容易に得ることが出来る神経幹細胞凝集塊形成用容器、その製造方法、及び神経幹細胞凝集塊の作成方法を提供することにある。   The present invention has been made in view of the above circumstances, and a nerve stem cell aggregate can be efficiently and easily obtained in a neural stem cell aggregate formation step for obtaining neural stem cells from a biological tissue containing neural stem cells. An object of the present invention is to provide a container for forming a stem cell aggregate, a method for producing the same, and a method for producing a neural stem cell aggregate.

このような目的は、下記（１）から（９）に記載の本発明により達成される。
（１）神経幹細胞凝集塊形成用容器の製造方法であって、
水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、
前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、を含むことを特徴とする神経幹細胞凝集塊形成用容器の製造方法。
（２）前記水溶性樹脂は、側鎖に放射線反応性、感光性、熱反応性の中から選ばれる官能基を有するものである（１）に記載の神経幹細胞凝集塊形成用容器の製造方法。
（３）前記官能基は、アジド基を有するものを含む（１）又は（２）に記載の神経幹細胞凝集塊形成用容器の製造方法。
（４）前記水溶性樹脂被覆層を硬化させる方法は、光照射による硬化方法を含む（１）に記載の神経幹細胞凝集塊形成用容器の製造方法。
（５）神経幹細胞凝集塊形成用容器であって、
水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、
前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、によって内面に非水溶性硬化皮膜を有することを特徴とする神経幹細胞凝集塊形成用容器。
（６）前記水溶性樹脂は、側鎖に感光性の官能基を有するものを含む（５）に記載の神経幹細胞凝集塊形成用容器。
（７）前記官能基は、アジド基を有するものを含む（６）に記載の神経幹細胞凝集塊形成用容器。
（８）前記水溶性樹脂被覆層を硬化させる方法は、光照射による硬化方法を含む（５）に記載の神経幹細胞凝集塊形成用容器。
（９）多能性神経幹細胞を含む組織を少なくとも一種の幹細胞増殖因子を含む培地に懸濁し、（５）ないし（８）のいずれかに記載の神経幹細胞凝集塊形成用容器に播種、培養する事により神経幹細胞凝集塊を形成させることを特徴とする神経幹細胞凝集塊の作成方法。 Such an object is achieved by the present invention described in the following (1) to (9).
(1) A method for producing a neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
A method for producing a neural stem cell aggregate formation container, comprising: a water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the step to modify the water-insoluble cured film layer.
(2) The method for producing a neural stem cell aggregate formation container according to (1), wherein the water-soluble resin has a functional group selected from radiation reactivity, photosensitivity, and heat reactivity in a side chain. .
(3) The method for producing a neural stem cell aggregate formation container according to (1) or (2), wherein the functional group includes one having an azide group.
(4) The method for curing the water-soluble resin coating layer includes the method for forming a neural stem cell aggregate formation according to (1), including a curing method by light irradiation.
(5) A neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
A neural stem cell aggregation characterized by having a water-insoluble cured film on the inner surface by the water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the step to modify the water-insoluble cured film layer Container for lump formation.
(6) The container for forming neural stem cell aggregates according to (5), wherein the water-soluble resin includes one having a photosensitive functional group in a side chain.
(7) The container for neural stem cell aggregate formation according to (6), wherein the functional group includes one having an azide group.
(8) The neural stem cell aggregate formation container according to (5), wherein the method of curing the water-soluble resin coating layer includes a curing method by light irradiation.
(9) A tissue containing pluripotent neural stem cells is suspended in a medium containing at least one kind of stem cell growth factor, and seeded and cultured in the neural stem cell aggregate formation container according to any one of (5) to (8). A method for producing a neural stem cell aggregate, which comprises forming a neural stem cell aggregate.

本発明によれば、神経幹細胞を含む生体組織から神経幹細胞を得るための神経幹細胞凝集塊形成工程において、神経幹細胞凝集塊を効率よく容易に得ることが出来る神経幹細胞凝集塊形成用容器、その製造方法、及び神経幹細胞凝集塊の作成方法を得ることができる。   According to the present invention, a neural stem cell aggregate formation container capable of efficiently and easily obtaining neural stem cell aggregates in a neural stem cell aggregate formation step for obtaining neural stem cells from a biological tissue containing neural stem cells, and its production A method and a method for producing neural stem cell aggregates can be obtained.

本発明は、水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、を含むことを特徴とする神経幹細胞凝集塊形成用容器の製造方法であり、上記製造方法によって得られた神経幹細胞凝集塊形成用容器であり、また、この容器に多能性神経幹細胞を含む組織を、少なくとも一種の幹細胞増殖因子を含む培地に懸濁し、神経幹細胞凝集塊形成用容器に播種、培養する事により神経幹細胞凝集塊を形成させることを特徴とする神経幹細胞凝集塊の作成方法である。   The present invention includes a water-soluble resin coating step in which a water-soluble resin is coated on the inner surface of the container to form a water-soluble resin coating layer, and after the step, the water-soluble resin coating layer is cured to form a water-insoluble cured coating layer. A method for producing a neural stem cell agglomerate forming vessel characterized by comprising: In this container, a tissue containing pluripotent neural stem cells is suspended in a medium containing at least one kind of stem cell growth factor, and seeded and cultured in a neural stem cell aggregate forming container to form neural stem cell aggregates. This is a method for producing a characteristic neural stem cell aggregate.

まず、本発明による神経幹細胞凝集塊形成用容器の製造方法（以下、単に「製造方法」ということがある）について説明する。
本発明の製造方法においては、水溶性樹脂を容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程を有することを特徴とする。
本発明に用いられる水溶性樹脂とは、水分子とのイオンもしくは水素結合により水和し、その結果として水に溶解するものであり、言い換えれば、水溶性樹脂とは水に溶解するために分子内の主鎖に対して必要充分な量のイオン性もしくは極性の側鎖を持つ樹脂である。なお、ここで水溶性樹脂とは、２５℃の水１００ｇに対して１．０ｇ以上溶解可能なものをいう。 First, a method for producing a neural stem cell aggregate formation container according to the present invention (hereinafter sometimes simply referred to as “manufacturing method”) will be described.
The production method of the present invention includes a water-soluble resin coating step in which a water-soluble resin is coated on the inner surface of the container to form a water-soluble resin coating layer.
The water-soluble resin used in the present invention is hydrated by an ion or hydrogen bond with a water molecule, and as a result, dissolves in water. In other words, the water-soluble resin is a molecule that dissolves in water. It is a resin having a necessary and sufficient amount of ionic or polar side chains with respect to the main chain. In addition, water-soluble resin means what can melt | dissolve 1.0g or more with respect to 100g of 25 degreeC water here.

上記水溶性樹脂の平均重合度は、特に限定されないが、１００以上、１０，０００以下が好ましく、特に２００以上、５，０００以下が好ましい。平均重合度が１００以上であると、均一な皮膜を成形することができ、また、平均重合度が１０，０００以下であれば作業性に適した水溶性の粘度とすることができる。   The average degree of polymerization of the water-soluble resin is not particularly limited, but is preferably 100 or more and 10,000 or less, particularly preferably 200 or more and 5,000 or less. When the average degree of polymerization is 100 or more, a uniform film can be formed, and when the average degree of polymerization is 10,000 or less, a water-soluble viscosity suitable for workability can be obtained.

水溶性樹脂としては、例えば、ポリ酢酸ビニルのケン化物、ポリビニルピロリドン、ポリエチレングリコール、ポリアクリルアミド、ポリメタアクリルアミド、ポリヒドロキシエチルメタアクリレート、ポリペンタエリスリトールトリアクリレート、ポリペンタエリスリトールテトラアクリレート、ポリジエチレングリコールジアクリレート、およびそれらを構成するモノマー同士の共重合体、また２−メタクリロイルオキシエチルホスホリルコリンと他のモノマー（例えばブチルメタクリレート等）との共重合体等が挙げられる。これらの中でもポリ酢酸ビニルのケン化物、ポリビニルピロリドン、ポリエチレングリコールの中から選ばれる１種以上と上記反応基からなる構造が好ましい。これにより、神経幹細胞凝集塊の形成率、および形成速度を向上することができる。   Examples of water-soluble resins include saponified polyvinyl acetate, polyvinyl pyrrolidone, polyethylene glycol, polyacrylamide, polymethacrylamide, polyhydroxyethyl methacrylate, polypentaerythritol triacrylate, polypentaerythritol tetraacrylate, polydiethylene glycol diacrylate. And copolymers of monomers constituting them, and copolymers of 2-methacryloyloxyethyl phosphorylcholine with other monomers (for example, butyl methacrylate). Among these, a structure comprising at least one selected from saponified products of polyvinyl acetate, polyvinyl pyrrolidone, and polyethylene glycol and the reactive group is preferable. Thereby, the formation rate and formation rate of neural stem cell aggregates can be improved.

ここで、ポリ酢酸ビニルのケン化物とは、例えば、ポリビニルアルコールまたはビニルアルコールと他の化合物との共重合体をいう。さらには、例えば、ビニルアルコールと、親水基変性、疎水基変性、アニオン変性、カチオン変性、アミド基変性またはアセトアセチル基のような反応基変性させた変性酢酸ビニルのケン化物等も含まれる。   Here, the saponified product of polyvinyl acetate refers to, for example, polyvinyl alcohol or a copolymer of vinyl alcohol and another compound. Furthermore, for example, saponified products of vinyl alcohol and modified vinyl acetate modified with a reactive group such as hydrophilic group modification, hydrophobic group modification, anion modification, cation modification, amide group modification or acetoacetyl group are included.

また、上記ポリ酢酸ビニルのケン化物を用いる場合、ポリ酢酸ビニルのケン化物のケン化度は特に限定されないが、該ポリ酢酸ビニル全体の２０ｍｏｌ％以上、１００ｍｏｌ％以下が好ましく、特に５０ｍｏｌ％以上、９５ｍｏｌ％以下が好ましい。ポリ酢酸ビニルのケン化度が上記範囲内であると、神経幹細胞凝集塊の形成速度、形成率を特に向上することができる。   Further, when the saponified product of polyvinyl acetate is used, the saponification degree of the saponified product of polyvinyl acetate is not particularly limited, but is preferably 20 mol% or more and 100 mol% or less of the entire polyvinyl acetate, particularly 50 mol% or more, 95 mol% or less is preferable. When the saponification degree of polyvinyl acetate is within the above range, the formation rate and formation rate of neural stem cell aggregates can be particularly improved.

上記水溶性樹脂は、特に限定されないが、２０℃における粘度が１ｍＰａ・ｓ以上、１０ｍＰａ・ｓ以下が好ましく、更に２ｍＰａ・ｓ以上、７ｍＰａ・ｓ以下となるよう溶媒を用いて調製されたものを使用する事が好ましい。その際に使用する溶媒は水もしくは溶解度を高めるために、水と有機溶媒との混合物を使用することができる。水溶性樹脂の粘度が上記範囲内であると、細胞の接着量が少なく、神経細胞凝集塊形成効果が特に優れる。充分な細胞の接着低減効果により、良好な神経細胞凝集塊形成性が得られ、接着性の共雑細胞の増殖が抑えられるため選択培養効率に優れる。被覆層の厚みとしては、特に限定されないが、１００ｎｍ以上５，０００ｎｍ以下が好ましく、１５０以上１，０００ｎｍ以下がより好ましい。
被覆層の厚みを上記下限値以上にする事により細胞が基材から受ける物理的な刺激をより抑える事ができ、厚みを上記上限値以下とする事により被覆層に取り込まれる蛋白質の量を少なくし、蛋白質を介した細胞の接着を抑えることが出来るため細胞凝集塊形成率がより向上する。 The water-soluble resin is not particularly limited, but is preferably prepared using a solvent so that the viscosity at 20 ° C. is 1 mPa · s or more and 10 mPa · s or less, and further 2 mPa · s or more and 7 mPa · s or less. It is preferable to use it. The solvent used in that case may be water or a mixture of water and an organic solvent in order to increase solubility. When the viscosity of the water-soluble resin is within the above range, the cell adhesion amount is small, and the effect of forming nerve cell aggregates is particularly excellent. A sufficient neuron agglomerate formation property is obtained due to a sufficient cell adhesion reduction effect, and the growth of adherent confluent cells is suppressed, so that selective culture efficiency is excellent. Although it does not specifically limit as thickness of a coating layer, 100 nm or more and 5,000 nm or less are preferable, and 150 or more and 1,000 nm or less are more preferable.
By making the thickness of the coating layer more than the above lower limit value, the physical stimulation that the cells receive from the base material can be further suppressed, and by making the thickness less than the above upper limit value, the amount of protein taken into the coating layer is reduced. In addition, since cell adhesion through proteins can be suppressed, the cell aggregate formation rate is further improved.

水溶性樹脂を容器内面に被覆させる方法としては、例えば、スピンコート、ディッピング、または上記水溶性樹脂溶液を容器内面に分注した後、容器を傾けて溶液を排出する方法を用いることができる。その様な方法で容器内面に水溶性樹脂を接触させた後、容器内面に残留した水溶性樹脂溶液を乾燥させることで水溶性樹脂被覆層を形成することができる。   As a method for coating the inner surface of the container with the water-soluble resin, for example, spin coating, dipping, or a method of dispensing the water-soluble resin solution onto the inner surface of the container and then discharging the solution by tilting the container can be used. After bringing the water-soluble resin into contact with the inner surface of the container by such a method, the water-soluble resin coating layer can be formed by drying the water-soluble resin solution remaining on the inner surface of the container.

本発明の製造方法においては、上記工程後に、上記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程を有することを特徴とする。上記水溶性樹脂被覆層を非水溶性硬化皮膜層とする事で、密度の高いイオン性もしくは極性の側鎖を持つ表面を構築することができる。この表面に構築されたイオン性もしくは極性の側鎖は、培養液と接触した際に、静電相互作用もしくは水素結合により水分子と水和し、容器表面は実質的に水分子の密な水和層となり、この水和層は細胞に対する基材表面からの刺激を抑制し、神経幹細胞凝集塊が迅速に形成されることとなる。こうすることで、培養液を接触させた際に、水溶性樹脂の被覆層が溶解、遊離することを防ぎ、容器として必要な耐水性を獲得することができる。   The production method of the present invention is characterized by having a water-insoluble cured film modifying step of curing the water-soluble resin coating layer after the above step to modify the water-insoluble cured film layer. By using the water-soluble resin coating layer as a water-insoluble cured film layer, a surface having a high density ionic or polar side chain can be constructed. The ionic or polar side chain constructed on this surface hydrates with water molecules by electrostatic interaction or hydrogen bonding when in contact with the culture solution, and the container surface is substantially water-dense water molecules. A hydration layer suppresses the stimulation from the surface of the base material to cells, and a neural stem cell aggregate is rapidly formed. By doing so, it is possible to prevent the water-soluble resin coating layer from being dissolved and released when the culture solution is brought into contact, and to obtain water resistance necessary for the container.

上記水溶性皮膜層を硬化させる方法としては特に限定するものではなく、側鎖に、硬化させるための官能基、例えば放射線反応性、感光性、熱反応性の官能基を有する水溶性樹脂を用いることができる。例えば、感光性の官能基であれば、ジアゾ基、アジド基、シンモナイル基等が挙げられ、また、熱反応性および放射線反応性の官能基であれば、ビニル基、エポキシ基等を挙げることができる。これらの中でも硬化処理を迅速におこなうことができ、簡易な設備で硬化させることができる感光性の官能基を有する水溶性樹脂が特に好ましい。   The method for curing the water-soluble film layer is not particularly limited, and a water-soluble resin having a functional group for curing, for example, a radiation-reactive, photosensitive, or heat-reactive functional group, in the side chain is used. be able to. For example, if it is a photosensitive functional group, a diazo group, an azide group, a simmonyl group, etc. may be mentioned, and if it is a thermally reactive and radiation reactive functional group, a vinyl group, an epoxy group, etc. may be mentioned. it can. Among these, a water-soluble resin having a photosensitive functional group that can be cured rapidly and can be cured with simple equipment is particularly preferable.

光照射により硬化させる場合の光源は、特に限定するものではなく、照度が５．０ｍＷ／ｃｍ2程度の超高圧水銀灯または０．１ｍＷ／ｃｍ2程度のＵＶランプを使用することができる。光照射による硬化は照度と照射時間で制御することができるため、照度の低い光源を用いる場合は照射時間を長くすればよく、反応性の高い感光基を選択した場合は蛍光灯下で硬化させることも可能である。例えば、５．０ｍＷ／ｃｍ2の超高圧水銀灯を使用した場合は１ないし１０秒間の照射で、０．１ｍＷ／ｃｍ2のＵＶランプを使用した場合は３ないし１０分間の照射で充分に硬化させることができる。 A light source in the case of curing by light irradiation is not particularly limited, can illuminance using 5.0 mW / cm 2 of about ultra-high pressure mercury lamp or 0.1 mW / cm 2 about UV lamps. Curing by light irradiation can be controlled by illuminance and irradiation time, so when using a light source with low illuminance, the irradiation time can be lengthened, and when a highly reactive photosensitive group is selected, it is cured under a fluorescent lamp. It is also possible. For example, when using an extra-high pressure mercury lamp with 5.0 mW / cm 2 irradiation 1 to 10 seconds, when using a UV lamp 0.1 mW / cm 2 is sufficiently cured by irradiation of 3 to 10 minutes be able to.

上記感光性の官能基としては、アジド基を含む官能基が特に好ましい。これにより、実用的な２３０〜５００ｎｍの波長で反応させる事ができ、更に優れた解像性により皮膜の形成性を向上することができる。このように、表面に予め水溶性樹脂被覆層を形成し、上記被覆層を硬化させて非水溶性硬化皮膜層に変性する工程によって上記の厚みの被覆層を得ることができる。
水溶性樹脂を用いるもう一つの利点としては、硬化後に表面を水で洗浄する事で、未反応の樹脂を容易に洗い流す事ができるという点である。もし、硬化反応性が悪い等の原因で溶出物が確認された場合は、硬化後に洗浄工程を入れることにより、溶出物を低減し、更に良好なＥＢ体形成率を得る事ができる。 As the photosensitive functional group, a functional group containing an azide group is particularly preferable. Thereby, it can be made to react with a practical wavelength of 230-500 nm, and the formability of a film | membrane can be improved with the further outstanding resolution. Thus, a coating layer having the above thickness can be obtained by forming a water-soluble resin coating layer on the surface in advance and curing the coating layer to modify it into a water-insoluble cured coating layer.
Another advantage of using a water-soluble resin is that the unreacted resin can be easily washed away by washing the surface with water after curing. If the eluate is confirmed due to poor curing reactivity or the like, the eluate can be reduced by adding a washing step after curing, and a better EB body formation rate can be obtained.

次に、本発明の神経幹細胞凝集塊形成用容器（以下、単に「容器」ということがある）について説明する。
本発明の容器は、上記本発明の製造方法によって製造されることを特徴とする。即ち、水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、によって内面に非水溶性硬化皮膜を有することを特徴とする。 Next, the neural stem cell aggregate formation container of the present invention (hereinafter sometimes simply referred to as “container”) will be described.
The container of the present invention is manufactured by the manufacturing method of the present invention. That is, a water-soluble resin coating step of forming a water-soluble resin coating layer by coating the inner surface of the container with a water-soluble resin, and after the step, the water-soluble resin coating layer is cured to be modified into a water-insoluble cured coating layer. And having a water-insoluble cured film on the inner surface by the water-insoluble cured film modifying step.

本発明の容器は、樹脂製の材料で成形することができる。この樹脂材料は、上記容器をディスポーザルタイプにすることができるのに加え、種々の形状を容易に成形できるものである。上記樹脂材料としては、例えば、ポリプロピレン樹脂、ポリエチレン樹脂、エチレン-プロピレン共重合体等のポリオレフィン系樹脂または環状ポリオレフィン系樹脂、ポリスチレン、アクリロニトリル−ブタジエン−スチレン系樹脂等のポリスチレン系樹脂、ポリカーボネート樹脂、ポリエチレンテレフタレート樹脂、ポリメチルメタクリレート樹脂等のメタクリル系樹脂、塩化ビニル樹脂、ポリブチレンテレフタレート樹脂、ポリアリレート樹脂、ポリサルホン樹脂、ポリエーテルサルホン樹脂、ポリエーテルエーテルケトン樹脂、ポリエーテルイミド樹脂、ポリテトラフルオロエチレン等のフッ素系樹脂、ポリメチルペンテン樹脂、ポリアクリロニトリル等のアクリル系樹脂、プロピオネート樹脂等の繊維素系樹脂等が挙げられる。これらの中でも容器に求められる成形性、透明性、放射線耐性の点においてポリスチレン樹脂が特に好ましい。   The container of the present invention can be formed of a resin material. In addition to making the container into a disposable type, this resin material can easily form various shapes. Examples of the resin material include polypropylene resins, polyethylene resins, polyolefin resins such as ethylene-propylene copolymers or cyclic polyolefin resins, polystyrene resins such as acrylonitrile-butadiene-styrene resins, polycarbonate resins, polyethylene Methacrylic resins such as terephthalate resin, polymethyl methacrylate resin, vinyl chloride resin, polybutylene terephthalate resin, polyarylate resin, polysulfone resin, polyethersulfone resin, polyetheretherketone resin, polyetherimide resin, polytetrafluoroethylene And fluorine resin such as polymethylpentene resin and polyacrylonitrile, and acrylic resin such as polyacrylonitrile, and fiber resin such as propionate resin. Among these, a polystyrene resin is particularly preferable in terms of moldability, transparency, and radiation resistance required for the container.

上記樹脂材料の重量平均分子量は、特に限定されないが、１０，０００以上５００，０００以下が好ましく、特に２０，０００以上１００，０００以下が好ましい。重量平均分子量が前記範囲内であると、容器の成形性に優れる。   The weight average molecular weight of the resin material is not particularly limited, but is preferably 10,000 or more and 500,000 or less, particularly preferably 20,000 or more and 100,000 or less. When the weight average molecular weight is within the above range, the moldability of the container is excellent.

上記重量平均分子量は、例えばサイズ排除クロマトグラフィー法（Ｇｅｌ Ｐｅｒｍｅａｔｉｏｎ Ｃｈｒｏｍａｔｏｇｒａｐｈｙ システム、Ｓｈｏｄｅｘ ＫＦ−８００ カラム、何れも昭和電工社製、溶出溶媒：テトラヒドロフラン）を用いて測定することができる。
上記樹脂材料には成形性向上、耐候性向上を目的として、本発明の目的を損なわない範囲で、例えば、炭化水素系、脂肪酸アミド系の滑剤やフェノール系、アミン系の酸化防止剤等の添加剤を添加することができる。
上記樹脂材料から本発明の容器を製造する場合、例えば射出成形、ブロー成形、インジェクションブロー成形により、製造することができる。 The weight average molecular weight can be measured using, for example, a size exclusion chromatography method (Gel Permeation Chromatography system, Shodex KF-800 column, both manufactured by Showa Denko KK, elution solvent: tetrahydrofuran).
For the purpose of improving moldability and weather resistance, the resin material is added within the range not impairing the object of the present invention, for example, hydrocarbon-based, fatty acid amide-based lubricants, phenol-based, amine-based antioxidants, etc. An agent can be added.
When manufacturing the container of this invention from the said resin material, it can manufacture by injection molding, blow molding, injection blow molding, for example.

本発明の容器の形態としては、例えば、マルチウェルプレートおよびシャーレ（ディッシュ）、フラスコ等の容器類が挙げられ、更にシート状の成形品であっても、容器底面等の細胞が培養できる環境下に設置して使用する事ができる。これらの中でも、バイオリアクターの生成または薬効や毒物の評価、人工臓器の開発研究等で用いられる６〜３８４穴のマルチウェルプレートやシャーレが好ましい。これにより、神経幹細胞凝集塊を用いた評価、研究の精度を向上させることができる。   Examples of the form of the container of the present invention include containers such as a multiwell plate, a petri dish (dish), and a flask. Furthermore, even in the case of a sheet-shaped molded article, the environment such that cells such as the bottom of the container can be cultured. It can be installed and used. Among these, a multi-well plate or petri dish having 6 to 384 holes, which is used for bioreactor generation or medicinal effect and toxicological evaluation, artificial organ development research, and the like is preferable. Thereby, the accuracy of evaluation and research using neural stem cell aggregates can be improved.

本発明の容器の必須条件である滅菌に関しては、例えば、エチレンオキサイドガス滅菌、感熱滅菌、蒸気滅菌、放射線滅菌等が挙げられるが、この中でもγ線あるいは電子線を用いた放射線滅菌が好ましく、大量生産を行う場合は放射線透過性の点でγ線滅菌が特に好ましい。
放射線の吸収線量については特に限定するものではないが、吸収線量が低すぎると滅菌性は確保されず、高すぎると細胞容器および被覆層が劣化してしまう場合がある。
本発明の容器における放射線の吸収線量としては、１ｋＧｙ以上、５０ｋＧｙ以下が好ましく、５ｋＧｙ以上、３０ｋＧｙ以下が特に好ましい。これによって本発明の容器の特性を充分に保持したまま滅菌性を付与する事ができる。 Examples of the sterilization that is an essential condition of the container of the present invention include ethylene oxide gas sterilization, heat-sensitive sterilization, steam sterilization, and radiation sterilization. Among them, radiation sterilization using γ rays or electron beams is preferable, For production, γ-ray sterilization is particularly preferred from the viewpoint of radiolucency.
The absorbed dose of radiation is not particularly limited, but if the absorbed dose is too low, sterility is not ensured, and if it is too high, the cell container and the coating layer may be deteriorated.
The absorbed dose of radiation in the container of the present invention is preferably 1 kGy or more and 50 kGy or less, particularly preferably 5 kGy or more and 30 kGy or less. As a result, sterility can be imparted while sufficiently maintaining the characteristics of the container of the present invention.

次に、本発明の容器を用いた本発明の神経幹細胞凝集塊の作成方法について説明する。
本発明の神経幹細胞凝集塊の作成方法は、多能性神経幹細胞を含む組織を少なくとも一種の幹細胞増殖因子を含む培地に懸濁し、上記本発明の神経幹細胞凝集塊形成用容器に播種、培養する事により神経幹細胞凝集塊を形成させることを特徴とする。 Next, a method for producing the neural stem cell aggregate of the present invention using the container of the present invention will be described.
In the method for producing a neural stem cell aggregate of the present invention, a tissue containing pluripotent neural stem cells is suspended in a medium containing at least one stem cell growth factor, and seeded and cultured in the above-mentioned container for forming a neural stem cell aggregate of the present invention. It is characterized by forming neural stem cell aggregates.

本発明の神経幹細胞凝集塊の作成方法の具体的な一例を示すと、まず、線条体等から採取した神経幹細胞を含む中枢神経系の細胞をＥＧＦあるいはＦＧＦ−２という増殖因子を添加した既知の培養液に任意の濃度で分散させた細胞懸濁液を本発明の容器に播種し、炭酸ガスインキュベーター等の環境下で培養することで、通常１日〜３日間で神経幹細胞凝集塊の形成が確認することができる。   A specific example of the method for producing the neural stem cell aggregate of the present invention will be described. First, cells of the central nervous system including neural stem cells collected from the striatum and the like are added with a growth factor such as EGF or FGF-2. The cell suspension dispersed at an arbitrary concentration in the culture medium is seeded in the container of the present invention and cultured in an environment such as a carbon dioxide incubator, so that a neural stem cell aggregate is usually formed in 1 to 3 days. Can be confirmed.

以下、本発明を実施例および比較例に基づいて詳細に説明するが、本発明はこれに限定されるものではない。   EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example and a comparative example, this invention is not limited to this.

（実施例１）
樹脂材料としてポリスチレン樹脂（ＰＳジャパン社製、ＨＦ７７）を用いて、射出成形により２４穴のマルチウェルプレート（２４穴プレート）を成形した。得られた２４穴プレートにプラズマ処理装置（ＢＲＡＮＳＯＮ／ＩＰＣ社製 ＳＥＲＩＥＳ７０００）を用いてプラズマ処理（酸素プラズマ５分間）を行い、２４穴プレート表面に濡れ性を付与した。
得られた２４穴プレートのウェル形状は、高さ２０ｍｍ、内径１６ｍｍであった。
次に、水溶性樹脂として側鎖にアジド基を有するポリビニルアルコール（東洋合成工業社製 ＡＷＰ、水溶性樹脂の平均重合度１６００、感光基の導入率０．６５ｍｏｌ％）をアルミ箔で遮光をしたガラス容器中で、２０容量％エタノール水溶液に溶解し、１．０重量％の溶液を調整した。
上述の２４穴プレートを前記アルミ箔で遮光したガラス容器に１分間、浸漬した後、取り出し、２４穴プレートを裏返して溶液を充分廃棄し、４０℃で６０分間一次乾燥した後、ＵＶランプで２５０ｎｍのＵＶ光を０．１ｍＷ／ｃｍ2×３分間照射して水溶性樹脂を硬化した後、純水で３回繰り返し洗浄し、乾燥後、γ線を吸収線量１０ｋＧｙで照射（ラジエ工業株式会社）して、本発明の培養容器（２４穴プレート）を得た。
得られた２４穴プレートの表面には、上記水溶性樹脂で形成される層が厚さ５５０ｎｍで形成されていた。なお、層の厚さは液体窒素中で破断した２４穴プレートの破断面を電子顕微鏡（ＦＥＩ社製 Ｑｕａｎｔａ４００Ｆ）を用いて測定した。 (Example 1)
A 24-well multiwell plate (24-well plate) was formed by injection molding using polystyrene resin (manufactured by PS Japan, HF77) as the resin material. The obtained 24-hole plate was subjected to plasma treatment (oxygen plasma for 5 minutes) using a plasma processing apparatus (SERIES7000 manufactured by BRANSON / IPC) to impart wettability to the 24-hole plate surface.
The well shape of the obtained 24-well plate had a height of 20 mm and an inner diameter of 16 mm.
Next, polyvinyl alcohol having an azide group in the side chain as a water-soluble resin (AWP manufactured by Toyo Gosei Kogyo Co., Ltd., average polymerization degree of water-soluble resin 1600, introduction rate of photosensitive group 0.65 mol%) was shielded with aluminum foil. In a glass container, it was dissolved in a 20 vol% ethanol aqueous solution to prepare a 1.0 wt% solution.
The above-mentioned 24-hole plate was immersed in the glass container shielded with aluminum foil for 1 minute, then taken out, and the 24-hole plate was turned upside down, and the solution was thoroughly discarded. UV light of 0.1 mW / cm 2 × 3 minutes to cure the water-soluble resin, then repeatedly washed with pure water three times, dried, and then irradiated with γ rays at an absorbed dose of 10 kGy (Radie Industries, Ltd.) Thus, the culture container (24-well plate) of the present invention was obtained.
On the surface of the obtained 24-hole plate, a layer formed of the water-soluble resin was formed with a thickness of 550 nm. In addition, the thickness of the layer was measured using an electron microscope (Quanta 400F manufactured by FEI Co., Ltd.) on the fracture surface of a 24-hole plate broken in liquid nitrogen.

（実施例２）
樹脂材料として環状オレフィン共重合系樹脂（ポリプラスチックス社製、ＴＯＰＡＳR ６０１３）を用いた以外は実施例１と同様にして培養容器（２４穴プレート）を得た。
得られた２４穴プレートの表面には、上記側鎖に水溶性樹脂で形成される層が厚さ５５０ｎｍで形成されていた。 (Example 2)
A culture container (24-well plate) was obtained in the same manner as in Example 1 except that a cyclic olefin copolymer resin (manufactured by Polyplastics, TOPAS R 6013) was used as the resin material.
On the surface of the obtained 24-hole plate, a layer formed of a water-soluble resin on the side chain was formed with a thickness of 550 nm.

（比較例）
実施例１の工程におけるプラズマ処理により親水性を付与する工程から水溶性樹脂への浸漬、及びＵＶランプによる硬化、洗浄、乾燥までの工程を除いた以外は実施例１と同様にして培養容器（２４穴プレート）を得た。 (Comparative example)
A culture vessel (similar to Example 1) except for the steps from the step of imparting hydrophilicity by plasma treatment in the step of Example 1 to the steps of immersion in a water-soluble resin, and curing, washing, and drying with a UV lamp. 24-well plate) was obtained.

得られた容器について、以下の方法により凝集塊の形成状態の評価を行った。結果を表１に示す。
定法に従いラット胎児線条体組織から分離した神経細胞をＥＧＦ２０ｎｇ／ｍＬ、ＦＧＦ２ ２０ｎｇ／ｍＬ（ＳＩＧＭＡ製）およびＮ−２添加物（ＧＩＢＣＯ社製）を含むＤＭＥＭ／Ｆ−１２培地に１×１０４ｃｅｌｌｓ／ｍＬの濃度で懸濁し、実施例１、２の容器および比較例の容器に２ｍＬづつ播種し、５％の炭酸ガス雰囲気下で培養し、接着細胞の存在の有無及び形態を播種５日後に倒立型顕微鏡（オリンパス社製ＢＸ５１）下１００倍及び４０倍の倍率で観察した。 About the obtained container, the formation method of the aggregate was evaluated with the following method. The results are shown in Table 1.
Nerve cells isolated from rat fetal striatum according to a standard method were added to DMEM / F-12 medium containing 20 ng / mL EGF, 20 ng / mL FGF2 (manufactured by SIGMA) and N-2 additive (manufactured by GIBCO) at 1 × 10 4 cells / Suspended at a concentration of mL, seeded 2 mL each in the containers of Examples 1 and 2 and the comparative example, cultured in a 5% carbon dioxide atmosphere, and inverted the presence or absence and form of adherent cells 5 days after seeding. The images were observed at a magnification of 100 times and 40 times under a scanning microscope (Olympus BX51).

表１から明らかなように、本発明の製造方法によって得られた本発明の容器を用いた実施例１、２は、これを用いなかった比較例に比べて多数個の凝集塊が形成されており、更にそのサイズも大きい事から、本発明の容器を用いることで神経幹細胞の増殖が非常に早く効率的に凝集塊が形成される事が確認された。   As is clear from Table 1, Examples 1 and 2 using the container of the present invention obtained by the production method of the present invention formed a larger number of agglomerates than the comparative examples not using this. In addition, since the size thereof is also large, it was confirmed that the use of the container of the present invention resulted in very rapid proliferation of neural stem cells and efficient formation of aggregates.

実施例１の７日後の顕微鏡写真（×１００）、サイズの大きな多数個の神経幹細胞凝集塊が確認される。A micrograph (× 100) after 7 days of Example 1 confirms a large number of large neural stem cell aggregates. 実施例１の７日後の顕微鏡写真（×２０）A micrograph after 7 days of Example 1 (× 20) 比較例の７日後の顕微鏡写真（×１００）、神経幹細胞凝集塊のサイズは小さく、その数も少ない。A micrograph (× 100) after 7 days of the comparative example, the size of neural stem cell aggregates is small, and the number is also small. 比較例の７日後の顕微鏡写真（×２０）Photomicrograph after 7 days of comparative example (× 20)

Claims (9)

神経幹細胞凝集塊形成用容器の製造方法であって、
水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、
前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、
を含むことを特徴とする神経幹細胞凝集塊形成用容器の製造方法。 A method for producing a neural stem cell aggregate formation container,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
After the step, the water-soluble resin coating layer is cured to modify the water-insoluble cured film layer to a water-insoluble cured film layer; and
The manufacturing method of the container for neural stem cell aggregate formation characterized by including this. 前記水溶性樹脂は、側鎖に放射線反応性、感光性、熱反応性の中から選ばれる官能基を有するものである請求項１に記載の神経幹細胞凝集塊形成用容器の製造方法。   The method for producing a container for forming a neural stem cell aggregate according to claim 1, wherein the water-soluble resin has a functional group selected from radiation reactivity, photosensitivity, and heat reactivity in a side chain. 前記官能基は、アジド基を有するものを含む請求項１又は２に記載の神経幹細胞凝集塊形成用容器の製造方法。   The method for producing a neural stem cell aggregate formation container according to claim 1 or 2, wherein the functional group includes an azide group. 前記水溶性樹脂被覆層を硬化させる方法は、光照射による硬化方法を含む請求項１に記載の神経幹細胞凝集塊形成用容器の製造方法。   The method for producing a neural stem cell aggregate formation container according to claim 1, wherein the method for curing the water-soluble resin coating layer includes a curing method by light irradiation. 神経幹細胞凝集塊形成用容器であって、
水溶性樹脂を前記容器内面に被覆させて水溶性樹脂被覆層を形成する水溶性樹脂被覆工程と、
前記工程後に、前記水溶性樹脂被覆層を硬化させて非水溶性硬化皮膜層に変性する非水溶性硬化皮膜変性工程と、
によって内面に非水溶性硬化皮膜を有することを特徴とする神経幹細胞凝集塊形成用容器。 A container for forming neural stem cell aggregates,
A water-soluble resin coating step of coating the inner surface of the container with a water-soluble resin to form a water-soluble resin coating layer;
After the step, the water-soluble resin coating layer is cured to modify the water-insoluble cured film layer to a water-insoluble cured film layer; and
A neural stem cell aggregate formation container characterized by having a water-insoluble cured film on the inner surface. 前記水溶性樹脂は、側鎖に感光性の官能基を有するものを含む請求項５に記載の神経幹細胞凝集塊形成用容器。   The said water-soluble resin is a container for neural stem cell aggregate formation of Claim 5 containing what has a photosensitive functional group in a side chain. 前記官能基は、アジド基を有するものを含む請求項６に記載の神経幹細胞凝集塊形成用容器。   The neural stem cell aggregate formation container according to claim 6, wherein the functional group includes one having an azide group. 前記水溶性樹脂被覆層を硬化させる方法は、光照射による硬化方法を含む請求項５に記載の神経幹細胞凝集塊形成用容器。   The neural stem cell aggregate formation container according to claim 5, wherein the method of curing the water-soluble resin coating layer includes a curing method by light irradiation. 多能性神経幹細胞を含む組織を少なくとも一種の幹細胞増殖因子を含む培地に懸濁し、請求項５ないし８のいずれかに記載の神経幹細胞凝集塊形成用容器に播種、培養する事により神経幹細胞凝集塊を形成させることを特徴とする神経幹細胞凝集塊の作成方法。   Aggregating neural stem cells by suspending a tissue containing pluripotent neural stem cells in a medium containing at least one kind of stem cell growth factor, and seeding and culturing them in a container for forming neural stem cell aggregates according to any one of claims 5 to 8. A method for producing a neural stem cell aggregate comprising forming a mass.
JP2007059745A 2007-03-09 2007-03-09 Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate Pending JP2008220205A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2007059745A JP2008220205A (en) 2007-03-09 2007-03-09 Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2007059745A JP2008220205A (en) 2007-03-09 2007-03-09 Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate

Publications (1)

Publication Number Publication Date
JP2008220205A true JP2008220205A (en) 2008-09-25

Family

ID=39839539

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2007059745A Pending JP2008220205A (en) 2007-03-09 2007-03-09 Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate

Country Status (1)

Country Link
JP (1) JP2008220205A (en)

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010147122A1 (en) 2009-06-15 2010-12-23 株式会社資生堂 Container for formation of aggregated cell mass, and method for formation of aggregated cell mass
WO2012081470A1 (en) 2010-12-13 2012-06-21 株式会社資生堂 Method for forming cell aggregate
JP2013138629A (en) * 2011-12-28 2013-07-18 Dainippon Printing Co Ltd Method for producing surface hydrophilic substrate
JP2013138622A (en) * 2011-12-28 2013-07-18 Dainippon Printing Co Ltd Method for producing surface hydrophilic substrate
EP2759592A4 (en) * 2011-09-20 2015-04-29 Kuraray Co Adherent cell culture method
WO2018135289A1 (en) * 2017-01-23 2018-07-26 東洋製罐グループホールディングス株式会社 Culture container and cell culture method

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2003210156A (en) * 2002-01-24 2003-07-29 Sumitomo Bakelite Co Ltd Cell culture substrate, method for cell culture of the same and bioassay thereof
WO2005001019A1 (en) * 2003-06-25 2005-01-06 Nof Corporation Container for germ layer formation and method of forming germ layer
JP2006055069A (en) * 2004-08-20 2006-03-02 National Institute Of Advanced Industrial & Technology Cell culture vessel and culture method
JP2006204232A (en) * 2005-01-31 2006-08-10 Sumitomo Bakelite Co Ltd Method for producing cell culture vessel and cell culture vessel
JP2007014352A (en) * 1992-10-28 2007-01-25 Neurospheres Holdings Ltd Biological factors and neural stem cells

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007014352A (en) * 1992-10-28 2007-01-25 Neurospheres Holdings Ltd Biological factors and neural stem cells
JP2003210156A (en) * 2002-01-24 2003-07-29 Sumitomo Bakelite Co Ltd Cell culture substrate, method for cell culture of the same and bioassay thereof
WO2005001019A1 (en) * 2003-06-25 2005-01-06 Nof Corporation Container for germ layer formation and method of forming germ layer
JP2006055069A (en) * 2004-08-20 2006-03-02 National Institute Of Advanced Industrial & Technology Cell culture vessel and culture method
JP2006204232A (en) * 2005-01-31 2006-08-10 Sumitomo Bakelite Co Ltd Method for producing cell culture vessel and cell culture vessel

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010147122A1 (en) 2009-06-15 2010-12-23 株式会社資生堂 Container for formation of aggregated cell mass, and method for formation of aggregated cell mass
US9347031B2 (en) 2009-06-15 2016-05-24 Shiseido Company, Ltd. Container for forming a cell aggregate and a method for forming a cell aggregate
WO2012081470A1 (en) 2010-12-13 2012-06-21 株式会社資生堂 Method for forming cell aggregate
KR101355899B1 (en) 2010-12-13 2014-02-06 가부시키가이샤 시세이도 Method for forming cell aggregate
EP2759592A4 (en) * 2011-09-20 2015-04-29 Kuraray Co Adherent cell culture method
US10655107B2 (en) 2011-09-20 2020-05-19 Corning Incorporated Adherent cell culture method
JP2013138629A (en) * 2011-12-28 2013-07-18 Dainippon Printing Co Ltd Method for producing surface hydrophilic substrate
JP2013138622A (en) * 2011-12-28 2013-07-18 Dainippon Printing Co Ltd Method for producing surface hydrophilic substrate
WO2018135289A1 (en) * 2017-01-23 2018-07-26 東洋製罐グループホールディングス株式会社 Culture container and cell culture method

Similar Documents

Publication Publication Date Title
JP2008061609A (en) Method for producing cell-culturing container and cell culturing container
Aisenbrey et al. Synthetic alternatives to Matrigel
JP2009050194A (en) Vessel for cell aggregate-forming culture
Dattola et al. Development of 3D PVA scaffolds for cardiac tissue engineering and cell screening applications
JP4967238B2 (en) Cell culture container manufacturing method and cell culture container
Wang et al. Modulation of human multipotent and pluripotent stem cells using surface nanotopographies and surface-immobilised bioactive signals: A review
Zhu et al. Effect of argon‐plasma treatment on proliferation of human‐skin–derived fibroblast on chitosan membrane in vitro
JP6826244B1 (en) Culture materials and their uses
US20090191634A1 (en) (meth)acrylate surfaces for cell culture, methods of making and using the surfaces
JP6678934B2 (en) Culture container for single cell aggregate formation
JP2008220205A (en) Container for forming neural stem cell aggregate, method for producing the same and method for preparing neural stem cell aggregate
WO2012133514A1 (en) Culture vessel for forming embryoid body
Turunen et al. Chemical and topographical patterning of hydrogels for neural cell guidance in vitro
Heath et al. Regenerating the cell resistance of micromolded PEG hydrogels
Li et al. Chemically crosslinked alginate porous microcarriers modified with bioactive molecule for expansion of human hepatocellular carcinoma cells
JP5151892B2 (en) Multiwell plate
JP2009017809A (en) Substrate for cell culture, method for producing the same, and method for culturing cell
US20150247126A1 (en) Method of culturing induced pluripotent stem cell and material for culturing the same
CA3170695A1 (en) Multilayered membrane for spheroid culture
Du et al. Mediating the migration of mesenchymal stem cells by dynamically changing the density of cell-selective peptides immobilized on β-cyclodextrin-modified cell-resisting polymer brushes
Sharma et al. Technologies for the fabrication of crosslinked polysaccharide-based hydrogels and its role in microbial three-dimensional bioprinting–A review
JP2008178367A (en) Culturing container for developing embryoid, method for producing the same and method for developing the embryoid
WO2022196444A1 (en) Sterile container for cell culture
JP2022099831A (en) Culture member and applications thereof
Fernandez et al. Thin film coatings for stem cell technologies

Legal Events

Date Code Title Description
A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20091210

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20120228

A02 Decision of refusal

Free format text: JAPANESE INTERMEDIATE CODE: A02

Effective date: 20120626