JP5727665B2 - Method for producing sheet cell culture - Google Patents
Method for producing sheet cell culture Download PDFInfo
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- JP5727665B2 JP5727665B2 JP2014250745A JP2014250745A JP5727665B2 JP 5727665 B2 JP5727665 B2 JP 5727665B2 JP 2014250745 A JP2014250745 A JP 2014250745A JP 2014250745 A JP2014250745 A JP 2014250745A JP 5727665 B2 JP5727665 B2 JP 5727665B2
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- cell culture
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- sheet
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Description
本発明は、シート状細胞培養物の製造方法、および該製造方法により製造されたシート状細胞培養物に関する。 The present invention relates to a method for producing a sheet-like cell culture, and a sheet-like cell culture produced by the production method.
心臓はそのポンプ機能により全身に酸素を運搬しており、生命の維持に必須の役割を担っている。そのため、心疾患は死亡原因の上位をしめており、狭心症、心筋梗塞などの虚血性心疾患では、心筋組織に十分な酸素が行き渡らなくなり、この状態が長時間つづくと壊死をおこしてしまう。成人の心筋細胞は自己複製能に乏しいため、一度壊死すると再生することはなく心不全に陥ってしまう。このような状態に至ると、左心補助人工心臓を装着するか、最終的には心臓移植を受けるしか治療法がないのが現状である。
このような中で心不全の新たな治療法として研究が進められているのが、心臓への細胞移植である。これまでに、胎児心筋細胞、骨格筋芽細胞、ＥＳ細胞、骨髄由来の細胞等の利用が、動物を用いた実験で報告されている。
The heart transports oxygen throughout the body by its pump function and plays an essential role in maintaining life. For this reason, heart disease is the leading cause of death. In an ischemic heart disease such as angina pectoris and myocardial infarction, sufficient oxygen is not distributed to the myocardial tissue, and necrosis occurs if this state continues for a long time. Adult cardiomyocytes are poorly self-replicating, so once necrotic they do not regenerate and fall into heart failure. In this situation, the only treatment available is to wear a left ventricular assist device or eventually receive a heart transplant.
Under such circumstances, cell transplantation into the heart is being studied as a new treatment method for heart failure. So far, the use of fetal cardiomyocytes, skeletal myoblasts, ES cells, bone marrow-derived cells and the like has been reported in experiments using animals.
このうち骨格筋芽細胞は、骨格筋組織中に存在し、筋組織が障害を受けた際に活性化され、増殖、融合することにより骨格筋組織を再生する。骨格筋芽細胞は、心筋と構造、機能などに類似する部分が多く、増殖して梗塞巣を修復できない心筋細胞の代わりに当該病巣を物理的に補填し、心臓のポンプ機能を改善できるのではないか、という考え方から心疾患への適応が期待されてきた。一方、近年ではその有効性にも関わらず移植細胞の生着率が低いことから、移植細胞の産生する液性因子が心筋組織に作用し、細胞死の抑制、血管新生などの作用によりその有効性を示すのではないかとも考えられている。また、骨格筋芽細胞を利用した細胞移植の場合、患者自身の細胞を用いることで免疫適合性の問題を回避することが可能であるという利点があり、海外では臨床試験も行われている。
しかし、移植細胞を細胞懸濁液の状態で組織へと投与した場合には、移植細胞の注入効率が悪いこと、レシピエント組織への穿刺による障害、広範囲な組織修復が困難である等の問題点が指摘されており、これを克服すべくスキャフォールドを利用した細胞構築物や細胞をシート状に形成した細胞シートが開発されてきた。
Among these, skeletal myoblasts are present in skeletal muscle tissue and are activated when the muscle tissue is damaged, and regenerate skeletal muscle tissue by proliferating and fusing. Skeletal myoblasts have many parts similar to the myocardium, structure, function, etc., so that they can physically compensate for the lesion instead of myocardial cells that cannot proliferate and repair the infarct, and can improve the pump function of the heart From the idea of whether or not there is an expectation for adaptation to heart disease. On the other hand, in recent years, the engraftment rate of transplanted cells is low despite its effectiveness, so humoral factors produced by transplanted cells act on myocardial tissue and are effective by suppressing cell death and angiogenesis. It is thought that it may show sex. In the case of cell transplantation using skeletal myoblasts, there is an advantage that it is possible to avoid the problem of immunocompatibility by using the patient's own cells, and clinical trials are being conducted overseas.
However, when transplanted cells are administered to a tissue in the form of a cell suspension, problems such as poor injection efficiency of transplanted cells, obstacles caused by puncture of the recipient tissue, and difficulty in extensive tissue repair In order to overcome this problem, cell constructs using scaffolds and cell sheets in which cells are formed into sheets have been developed.
骨格筋芽細胞についても、適切な培養液中で一定期間増殖させることにより細胞シートの形成が可能であることが知られている（特許文献1）。同文献において、骨格筋芽細胞シートは、細胞を約９×１０４細胞／ｃｍ２の密度で播種し、成長因子等を含む培養液中で十分な期間増殖させることにより形成している。しかしながら、このような製造方法においては、培養液中に含まれる成長因子、ステロイド剤、異種血清等の添加因子が製造物に残存する可能性があるという安全性の問題があった。また、かかる従来の製造方法においては、形成される細胞シートが物理的強度に乏しく、洗浄操作や移植手術、輸送の際の扱いが困難であるという問題点があった。さらに、近年では治療法としての細胞移植の作用メカニズムとして、移植細胞から産生される液性因子の働きが注目されているが、移植細胞からは治療の有効性に関わる因子の他、炎症性サイトカイン等、レシピエントに対して不利益な効果をもたらす因子も産生される可能性があるため、細胞シートの臨床応用にむけては、血管新生、細胞死の抑制等、有効性に関わる因子を多く産生し、炎症性サイトカイン産生の抑制された細胞シートを得ることが望ましい。したがって、上記のような従来技術の問題点を有しない、強度、安全性および有効性の高い細胞シートの製造が求められていた。 It is known that skeletal myoblasts can also form cell sheets by growing for a certain period in an appropriate culture solution (Patent Document 1). In the same document, the skeletal myoblast sheet is formed by seeding cells at a density of about 9 × 10 4 cells / cm 2 and allowing them to grow for a sufficient period in a culture solution containing growth factors and the like. However, in such a production method, there is a safety problem that additional factors such as growth factors, steroids, and heterogeneous serum contained in the culture medium may remain in the product. In addition, such a conventional production method has a problem that the formed cell sheet has poor physical strength and is difficult to handle during washing operation, transplantation surgery, and transportation. Furthermore, in recent years, the action of humoral factors produced from transplanted cells has attracted attention as an action mechanism of cell transplantation as a therapeutic method. For example, factors that have adverse effects on recipients may be produced, so there are many factors related to efficacy such as angiogenesis and suppression of cell death for clinical application of cell sheets. It is desirable to obtain a cell sheet that is produced and suppressed in production of inflammatory cytokines. Therefore, there has been a demand for the production of a cell sheet having high strength, safety and effectiveness, which does not have the problems of the prior art as described above.
本発明は、従来技術の問題点を有しない、強度、安全性および有効性に優れたシート状細胞培養物の製造を目的とする。 The object of the present invention is to produce a sheet-shaped cell culture excellent in strength, safety and effectiveness, which does not have the problems of the prior art.
本発明者らは、上記課題を解決すべく鋭意研究を行う中で、骨格筋芽細胞を１．０×１０６個／ｃｍ２を超える密度で播種した場合、播種された細胞が完全にシートを形成する以前に剥離が生じ、十分な強度をもつシート状細胞培養物を形成することができなかったが、前処理として培養基材に血清を被覆させた後に高密度の細胞を播種したところ、自然に剥離することなくシート状細胞培養物を形成することが可能であることを見出した。また、より低密度の播種密度３．５×１０５個／ｃｍ２の場合において、血清被覆処理をした培養皿を使用することにより、かかる処理を行っていない培養皿を使用した場合に比べてシートを構成する細胞数が増加、すなわち、細胞回収率が上昇し、シートの強度も高まることを見出した。そして、さらに研究を続けたところ、多核化していない骨格筋芽細胞のシート状細胞培養物からは、分化が進み多核化した骨格筋芽細胞のシート状細胞培養物と比較して血管新生等の有効性に関わる作用を有する因子の産生が多い一方で、炎症性サイトカインの産生が低いこと、および、上記製造方法で作製されたシート状細胞培養物について分化の程度を評価したところ、分化の進行がみられないことを見出し、本発明を完成させた。 When the present inventors have conducted intensive research to solve the above-mentioned problems, when seeding skeletal myoblasts at a density exceeding 1.0 × 10 6 cells / cm 2 , the seeded cells are completely sheeted. When a cell culture with sufficient strength was not able to be formed before detachment, it was impossible to form a sheet-shaped cell culture with sufficient strength. The present inventors have found that it is possible to form a sheet-shaped cell culture without spontaneous peeling. In addition, in the case of a lower density seeding density of 3.5 × 10 5 cells / cm 2 , the use of a culture dish that has been subjected to a serum coating treatment, compared to the case of using a culture dish that has not been subjected to such treatment. It has been found that the number of cells constituting the sheet increases, that is, the cell recovery rate increases and the strength of the sheet also increases. And when further research was carried out, from the skeletal myoblast sheet-like cell culture that was not multinucleated, compared with the sheet-like cell culture of skeletal myoblast that has been differentiated and multinucleated, angiogenesis, etc. While the production of factors having an effect on effectiveness is large, the production of inflammatory cytokines is low, and the degree of differentiation of the sheet-like cell culture prepared by the above production method was evaluated. As a result, the present invention was completed.
したがって、本発明は以下の（１）〜（１０）に示されるものである。
（１）血清で被覆された培養基材上に、実質的に増殖することなくシート状細胞培養物を形成し得る密度の細胞を播種する工程を含む、シート状細胞培養物の製造方法。
（２）細胞の密度が３．５×１０５以上、３．４×１０６個／ｃｍ２未満である、上記（１）に記載の製造方法。
（３）シート状細胞培養物を培養基材から単離する工程をさらに含む、上記（１）または（２）に記載の製造方法。
（４）単離したシート状細胞培養物を洗浄する工程をさらに含む、上記（１）〜（３）のいずれかに記載の製造方法。
（５）シート状細胞培養物が、筋管への分化が抑制された骨格筋芽細胞を含む、上記（１）〜（４）のいずれかに記載の製造方法。
（６）上記（１）〜（５）のいずれかに記載の方法により製造された、シート状細胞培養物。
（７）洗浄操作に対して十分な物理的強度を有する、上記（６）に記載のシート状細胞培養物。
（８）移植操作に耐え得る物理的強度を有する、上記（６）または（７）に記載のシート状細胞培養物。
（９）疾病または傷病の治療に用いる、上記（６）〜（８）のいずれかに記載のシート状細胞培養物。
（１０）製造由来不純物を実質的に含まない、上記（６）〜（９）のいずれかに記載のシート状細胞培養物。
Therefore, this invention is shown by the following (1)-(10).
(1) A method for producing a sheet-shaped cell culture, comprising a step of seeding cells having a density capable of forming a sheet-shaped cell culture without substantially growing on a culture substrate coated with serum.
(2) The production method according to (1) above, wherein the cell density is 3.5 × 10 5 or more and less than 3.4 × 10 6 cells / cm 2 .
(3) The production method according to (1) or (2), further including a step of isolating the sheet-shaped cell culture from the culture substrate.
(4) The production method according to any one of (1) to (3), further comprising a step of washing the isolated sheet cell culture.
(5) The manufacturing method in any one of said (1)-(4) in which a sheet-like cell culture contains the skeletal myoblast in which the differentiation to a myotube was suppressed.
(6) A sheet-shaped cell culture produced by the method according to any one of (1) to (5) above.
(7) The sheet-shaped cell culture according to (6), which has sufficient physical strength for the washing operation.
(8) The sheet-shaped cell culture according to (6) or (7), which has physical strength capable of withstanding a transplantation operation.
(9) The sheet-shaped cell culture according to any one of (6) to (8), which is used for treatment of a disease or injury.
(10) The sheet-like cell culture according to any one of (6) to (9), which is substantially free from production-derived impurities.
本発明の製造方法により、浸漬撹拌などの洗浄操作や、取出し、保持、移送などの移植操作に耐え得る十分な物理的強度を有するシート状細胞培養物が得られるため、従来の製法で得られた細胞シートよりもより容易かつ確実に製造・移植操作を行うことが可能となる。また、本発明の製造方法により、高い細胞回収率でシート状細胞培養物を得ることができるため、使用する細胞の有効利用が可能となり、コストの削減などに寄与する。さらに、本発明の製造方法により得られたシート状細胞培養物は、血管新生等の有効性に関わる作用を有する因子の産生が多い一方で、炎症性サイトカインの産生が低いため、治療上の有効性も、レシピエントに対する安全性も極めて高く、臨床的に極めて有用である。このため、ヒト医療および獣医療において多大な貢献が期待できる。 According to the production method of the present invention, a sheet-like cell culture having sufficient physical strength that can withstand a washing operation such as immersion stirring and a transplanting operation such as removal, holding, and transfer can be obtained. Manufacturing and transplantation can be performed more easily and reliably than cell sheets. In addition, since the sheet-like cell culture can be obtained with a high cell recovery rate by the production method of the present invention, the cells to be used can be effectively used, which contributes to cost reduction and the like. In addition, the sheet-like cell culture obtained by the production method of the present invention has a high production of factors having an effect on the effectiveness such as angiogenesis, while the production of inflammatory cytokines is low, so that it is therapeutically effective. Sexuality and safety to recipients are extremely high and are clinically very useful. For this reason, a great contribution can be expected in human medicine and veterinary medicine.
本発明は、血清で被覆された培養基材上に、実質的に増殖することなくシート状細胞培養物を形成し得る密度の細胞を播種する工程を含む、シート状細胞培養物の製造方法に関する。
本発明において、「培養基材」は、細胞がその上でシート状細胞培養物を形成し得るものであれば特に限定されず、例えば、種々の材質の容器、容器中の固形もしくは半固形の表面などを含む。容器は、培養液などの液体を透過させない構造・材料が好ましい。かかる材料としては、限定することなく、例えば、ポリエチレン、ポリプロピレン、テフロン（登録商標）、ポリエチレンテレフタレート、ポリメチルメタクリレート、ナイロン６，６、ポリビニルアルコール、セルロース、シリコン、ポリスチレン、ガラス、ポリアクリルアミド、ポリジメチルアクリルアミド、金属（例えば、鉄、ステンレス、アルミニウム、銅、真鍮）等が挙げられる。また、容器は、少なくとも１つの平坦な面を有することが好ましい。かかる容器の例としては、限定することなく、例えば、細胞培養皿、細胞培養ボトルなどが挙げられる。また、容器は、その内部に固形もしくは半固形の表面を有してもよい。固形の表面としては、上記のごとき種々の材料のプレートや容器などが、半固形の表面としては、ゲル、軟質のポリマーマトリクスなどが挙げられる。
The present invention relates to a method for producing a sheet-shaped cell culture, comprising the step of seeding cells having a density capable of forming a sheet-shaped cell culture without substantially growing on a culture substrate coated with serum. .
In the present invention, the “culture substrate” is not particularly limited as long as cells can form a sheet-like cell culture thereon, and examples thereof include containers of various materials, solid or semi-solid in containers. Including the surface. The container preferably has a structure / material that does not allow permeation of a liquid such as a culture solution. Examples of such materials include, but are not limited to, polyethylene, polypropylene, Teflon (registered trademark), polyethylene terephthalate, polymethyl methacrylate, nylon 6,6, polyvinyl alcohol, cellulose, silicon, polystyrene, glass, polyacrylamide, polydimethyl. Examples include acrylamide and metals (for example, iron, stainless steel, aluminum, copper, brass). The container preferably has at least one flat surface. Examples of such containers include, but are not limited to, cell culture dishes and cell culture bottles. Further, the container may have a solid or semi-solid surface therein. Examples of solid surfaces include plates and containers of various materials as described above, and examples of semi-solid surfaces include gels and soft polymer matrices.
培養基材は、刺激、例えば、温度や光に応答して物性が変化する材料で表面が被覆されていてもよい。かかる材料としては、限定されずに、例えば、（メタ）アクリルアミド化合物、Ｎ−アルキル置換（メタ）アクリルアミド誘導体（例えば、Ｎ−エチルアクリルアミド、Ｎ−ｎ−プロピルアクリルアミド、Ｎ−ｎ−プロピルメタクリルアミド、Ｎ−イソプロピルアクリルアミド、Ｎ−イソプロピルメタクリルアミド、Ｎ−シクロプロピルアクリルアミド、Ｎ−シクロプロピルメタクリルアミド、Ｎ−エトキシエチルアクリルアミド、Ｎ−エトキシエチルメタクリルアミド、Ｎ−テトラヒドロフルフリルアクリルアミド、Ｎ−テトラヒドロフルフリルメタクリルアミド等）、Ｎ，Ｎ−ジアルキル置換（メタ）アクリルアミド誘導体（例えば、Ｎ，Ｎ−ジメチル（メタ）アクリルアミド、Ｎ，Ｎ−エチルメチルアクリルアミド、Ｎ，Ｎ−ジエチルアクリルアミド等）、環状基を有する（メタ）アクリルアミド誘導体（例えば、１−（１−オキソ−２−プロペニル）−ピロリジン、１−（１−オキソ−２−プロペニル）−ピペリジン、４−（１−オキソ−２−プロペニル）−モルホリン、１−（１−オキソ−２−メチル−２−プロペニル）−ピロリジン、１−（１−オキソ−２−メチル−２−プロペニル）−ピペリジン、４−（１−オキソ−２−メチル−２−プロペニル）−モルホリン等）、またはビニルエーテル誘導体（例えば、メチルビニルエーテル）のホモポリマーまたはコポリマーからなる温度応答性材料、アゾベンゼン基を有する光吸収性高分子、トリフェニルメタンロイコハイドロオキシドのビニル誘導体とアクリルアミド系単量体との共重合体、および、スピロベンゾピランを含むＮ−イソプロピルアクリルアミドゲル等の光応答性材料などの公知のものを用いることができる（例えば、特開平2-211865、特開2003-33177参照）。これらの材料に所定の刺激を与えることによりその物性、例えば、親水性や疎水性を変化させ、同材料上に付着した細胞培養物の剥離を促進することができる。
上記培養基材は、種々の形状であってもよいが、平坦であることが好ましい。また、その面積は特に限定されないが、典型的には、１〜２００ｃｍ２、好ましくは２〜１００ｃｍ２、より好ましくは３〜５０ｃｍ２である。
The surface of the culture substrate may be coated with a material whose physical properties change in response to stimulation, for example, temperature or light. Examples of such materials include, but are not limited to, (meth) acrylamide compounds, N-alkyl substituted (meth) acrylamide derivatives (for example, N-ethylacrylamide, Nn-propylacrylamide, Nn-propylmethacrylamide, N-isopropylacrylamide, N-isopropylmethacrylamide, N-cyclopropylacrylamide, N-cyclopropylmethacrylamide, N-ethoxyethylacrylamide, N-ethoxyethylmethacrylamide, N-tetrahydrofurfurylacrylamide, N-tetrahydrofurfurylmethacrylate Amides), N, N-dialkyl-substituted (meth) acrylamide derivatives (eg, N, N-dimethyl (meth) acrylamide, N, N-ethylmethylacrylamide, N, N-diethyl) Chloramide and the like), (meth) acrylamide derivatives having a cyclic group (for example, 1- (1-oxo-2-propenyl) -pyrrolidine, 1- (1-oxo-2-propenyl) -piperidine, 4- (1-oxo -2-propenyl) -morpholine, 1- (1-oxo-2-methyl-2-propenyl) -pyrrolidine, 1- (1-oxo-2-methyl-2-propenyl) -piperidine, 4- (1-oxo -2-methyl-2-propenyl) -morpholine), or a vinyl ether derivative (for example, methyl vinyl ether) homopolymer or copolymer, a temperature-responsive material, a light-absorbing polymer having an azobenzene group, triphenylmethane leucohydro Copolymer of vinyl derivative of oxide and acrylamide monomer, and spirobenzopyra It can be used to include N- and isopropyl acrylamide gels known, such as photoresponsive materials (e.g., JP-A-2-211865, see JP-2003-33177). By giving a predetermined stimulus to these materials, the physical properties, for example, hydrophilicity and hydrophobicity can be changed, and peeling of the cell culture adhered on the materials can be promoted.
The culture substrate may have various shapes, but is preferably flat. Although the area is not particularly limited, typically, 1～200Cm 2, preferably 2～100Cm 2, more preferably 3~50cm 2.
本発明において、培養基材は血清でコート（被覆またはコーティング）されている。「血清でコートされている」とは、培養基材の表面に血清成分が付着している状態を意味する。かかる状態は、限定されずに、例えば、培養基材を血清と共にインキュベートすることにより得ることができる。インキュベートとは、血清を培養基材に接触させることを含む。血清としては、異種血清および同種血清を用いることができる。異種血清は、細胞培養物を移植する場合、レシピエントとは異なる種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ウシやウマに由来する血清、例えば、ウシ胎仔血清（ＦＢＳ、ＦＣＳ）、仔ウシ血清（ＣＳ）、ウマ血清（ＨＳ）などが異種血清に該当する。また、「同種血清」は、レシピエントと同一の種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ヒト血清が同種血清に該当する。同種血清は、自己血清、すなわち、レシピエントに由来する血清を含む。
培養基材をコートするための血清は、市販されているか、または、所望の生物から採取した血液から定法により調製することができる。具体的には、例えば、採取した血液を室温で２０〜６０分程度放置して凝固させ、これを１０００〜１２００×ｇ程度で遠心分離し、上清を採取する方法などが挙げられる。
In the present invention, the culture substrate is coated (coated or coated) with serum. “Coated with serum” means a state in which serum components are attached to the surface of a culture substrate. Such a state is not limited, and can be obtained, for example, by incubating a culture substrate with serum. Incubating includes contacting the serum with a culture substrate. As the serum, heterologous serum and allogeneic serum can be used. Xenogeneic serum refers to serum derived from a different species of organism than the recipient when the cell culture is transplanted. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum. “Allogeneic serum” means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum, ie serum derived from the recipient.
Serum for coating the culture substrate is commercially available or can be prepared from blood collected from a desired organism by a conventional method. Specifically, for example, the collected blood is allowed to stand at room temperature for about 20 to 60 minutes to be coagulated, centrifuged at about 1000 to 1200 × g, and the supernatant is collected.
培養基材上でインキュベートする場合、血清は原液で用いても、希釈して用いてもよい。希釈は、任意の媒体、例えば、限定することなく、水、生理食塩水、種々の緩衝液（例えば、ＰＢＳ、ＨＢＳなど）、種々の液体培地（例えば、ＤＭＥＭ、ＭＥＭ、Ｆ１２、ＤＭＥ、ＲＰＭＩ１６４０、ＭＣＤＢ（ＭＣＤＢ１０２、１０４、１０７、１３１、１５３、１９９など）、Ｌ１５、ＳｋＢＭ、ＲＩＴＣ８０−７、ＤＭＥＭ／Ｆ１２など）等で行うことができる。希釈濃度は、血清成分が培養基材上に付着することができれば特に限定されず、例えば、０．５〜９０％（ｖ／ｖ）、好ましくは１〜６０％（ｖ／ｖ）、より好ましくは５〜４０％（ｖ／ｖ）である。
インキュベート時間も、血清成分が培養基材上に付着することができれば特に限定されず、例えば、１〜７２時間、好ましくは４〜４８時間、より好ましくは５〜２４時間、さらに好ましくは６〜１２時間である。インキュベート温度も、血清成分が培養基材上に付着することができれば特に限定されず、例えば、０〜６０℃、好ましくは４〜４５℃、より好ましくは室温〜４０℃である。
When incubating on a culture substrate, serum may be used as a stock solution or diluted. Dilution can be any medium such as, without limitation, water, saline, various buffers (eg, PBS, HBS, etc.), various liquid media (eg, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.) can be used. The dilution concentration is not particularly limited as long as the serum component can adhere to the culture substrate. For example, the dilution concentration is 0.5 to 90% (v / v), preferably 1 to 60% (v / v), and more preferably. Is 5-40% (v / v).
The incubation time is not particularly limited as long as the serum component can adhere to the culture substrate. For example, the incubation time is 1 to 72 hours, preferably 4 to 48 hours, more preferably 5 to 24 hours, and further preferably 6 to 12 hours. It's time. The incubation temperature is not particularly limited as long as the serum component can adhere to the culture substrate, and is, for example, 0 to 60 ° C, preferably 4 to 45 ° C, more preferably room temperature to 40 ° C.
インキュベート後に血清を廃棄してもよい。血清の廃棄手法としては、ピペットなどによる吸引や、デカンテーションなどの慣用の液体廃棄手法を用いることができる。本発明の一態様においては、血清廃棄後に、培養基材を無血清洗浄液で洗浄してもよい。無血清洗浄液としては、血清を含まず、培養基材に付着した血清成分に悪影響を与えない液体媒体であれば特に限定されず、例えば、限定することなく、水、生理食塩水、種々の緩衝液（例えば、ＰＢＳ、ＨＢＳなど）、種々の液体培地（例えば、ＤＭＥＭ、ＭＥＭ、Ｆ１２、ＤＭＥ、ＲＰＭＩ１６４０、ＭＣＤＢ（ＭＣＤＢ１０２、１０４、１０７、１３１、１５３、１９９など）、Ｌ１５、ＳｋＢＭ、ＲＩＴＣ８０−７、ＤＭＥＭ／Ｆ１２など）等で行うことができる。洗浄手法としては、慣用の培養基材洗浄手法、例えば、限定することなく、培養基材上に無血清洗浄液を加えて所定時間（例えば、５〜６０秒間）撹拌後、廃棄する手法などを用いることができる。 Serum may be discarded after incubation. As a method for discarding serum, a conventional liquid disposal method such as suction with a pipette or decantation can be used. In one embodiment of the present invention, the culture substrate may be washed with a serum-free washing solution after serum is discarded. The serum-free washing solution is not particularly limited as long as it is a liquid medium that does not contain serum and does not adversely affect the serum components attached to the culture substrate. For example, without limitation, water, physiological saline, various buffers Liquid (for example, PBS, HBS, etc.), various liquid media (for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7 , DMEM / F12, etc.). As the washing method, a conventional culture substrate washing method, for example, without limitation, a method of adding a serum-free washing solution on the culture substrate, stirring for a predetermined time (for example, 5 to 60 seconds), and then discarding it is used. be able to.
本発明において、培養基材を、成長因子でコートしてもよい。ここで、「成長因子」は、細胞の増殖を、それがない場合に比べて促進する任意の物質を意味し、例えば、上皮細胞成長因子（ＥＧＦ）、血管内皮成長因子（ＶＥＧＦ）、線維芽細胞成長因子（ＦＧＦ）などを含む。成長因子による培養基材のコート手法、廃棄手法および洗浄手法は、インキュベーション時の希釈濃度が、例えば、０．０００１μｇ／ｍＬ〜１μｇ／ｍＬ、好ましくは０．０００５μｇ／ｍＬ〜０．０５μｇ／ｍＬ、より好ましくは０．００１μｇ／ｍＬ〜０．０１μｇ／ｍＬである以外は、基本的に血清と同じである。 In the present invention, the culture substrate may be coated with a growth factor. As used herein, “growth factor” means any substance that promotes cell proliferation as compared to the case without it, such as epithelial cell growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast, and the like. Cell growth factor (FGF) and the like. The culture substrate coating method, the discarding method and the washing method using a growth factor have a dilution concentration at the time of incubation of, for example, 0.0001 μg / mL to 1 μg / mL, preferably 0.0005 μg / mL to 0.05 μg / mL, More preferably, it is basically the same as serum except that it is 0.001 μg / mL to 0.01 μg / mL.
本発明において、培養基材を、ステロイド剤でコートしてもよい。ここで「ステロイド剤成分」は、ステロイド核を有する化合物のうち、生体に、副腎皮質機能不全、クッシング症候群などの悪影響を及ぼし得るものをいう。かかる化合物としては、限定されずに、例えば、コルチゾール、プレドニゾロン、トリアムシノロン、デキサメタゾン、ベタメタゾン等が含まれる。ステロイド剤による培養基材のコート手法、廃棄手法および洗浄手法は、インキュベーション時の希釈濃度が、デキサメタゾンとして、例えば、０．１μｇ／ｍＬ〜１００μｇ／ｍＬ、好ましくは０．４μｇ／ｍＬ〜４０μｇ／ｍＬ、より好ましくは１μｇ／ｍＬ〜１０μｇ／ｍＬである以外は、基本的に血清と同じである。 In the present invention, the culture substrate may be coated with a steroid agent. Here, the “steroid component” refers to a compound having a steroid nucleus that can adversely affect a living body such as adrenal cortex dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. The culture substrate coating method, discarding method, and washing method using a steroid agent have a dilution concentration at the time of incubation of, for example, 0.1 μg / mL to 100 μg / mL, preferably 0.4 μg / mL to 40 μg / mL. It is basically the same as serum except that the dose is 1 μg / mL to 10 μg / mL.
培養基材は、血清、成長因子およびステロイド剤のいずれか１つでコートしても、これらの任意の組合わせ、すなわち、血清と成長因子、血清とステロイド剤、または血清と成長因子とステロイド剤の組合わせでコートしてもよい。複数の成分でコートする場合、これらの成分を混合して同時にコートしてもよいし、別々の工程でコートしてもよい。 The culture substrate can be coated with any one of serum, growth factor and steroid agent, any combination thereof, ie serum and growth factor, serum and steroid agent, or serum and growth factor and steroid agent You may coat by the combination of. When coating with a plurality of components, these components may be mixed and coated simultaneously, or may be coated in separate steps.
培養基材は、血清等でコートした後直ちに細胞を播種してもよいし、コートした後に保存しておき、その後細胞を播種することもできる。コートした基材は、例えば４℃以下、好ましくは−２０℃以下、より好ましくは−８０℃以下に保つことにより長期間保存することができる。 The culture substrate may be seeded with cells immediately after coating with serum or the like, or may be stored after coating and then seeded with cells. The coated substrate can be stored for a long period of time by keeping it at, for example, 4 ° C. or lower, preferably −20 ° C. or lower, more preferably −80 ° C. or lower.
本発明の製造方法において、細胞は、実質的に増殖することなくシート状細胞培養物を形成し得る密度で播種する。「実質的に増殖することなくシート状細胞培養物を形成し得る密度」とは、成長因子を実質的に含まない非増殖系の培養液で培養した場合に、シート状細胞培養物を形成することができる細胞密度を意味する。例えば、骨格筋芽細胞の場合、成長因子を含む培養液を用いる従来法では、シート状細胞培養物を形成するために、約６，５００個／ｃｍ２の密度の細胞をプレートに播種していたが（例えば、特許文献１参照）、かかる密度の細胞を、成長因子を含まない培養液で培養してもシート状の細胞培養物を形成することはできない。したがって、本発明の製造方法における播種密度は、成長因子を含む培養液を用いる従来法におけるものよりも高いものである。このため、本明細書中では、かかる播種密度を、単に「高密度」と記すこともある。かかる密度は、具体的には、例えば、骨格筋芽細胞について典型的には３００，０００個／ｃｍ２以上である。播種密度の上限は、細胞培養物の形成が損なわれず、細胞が分化に移行しなければ特に制限されないが、骨格筋芽細胞については、３．４×１０６個／ｃｍ２未満である。 In the production method of the present invention, the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing. “The density at which a sheet-like cell culture can be formed without substantially growing” means that a sheet-like cell culture is formed when cultured in a non-proliferating culture medium substantially free of growth factors. It means the cell density that can be. For example, in the case of skeletal myoblasts, in the conventional method using a culture solution containing a growth factor, cells having a density of about 6,500 cells / cm 2 are seeded on a plate in order to form a sheet-like cell culture. However (for example, refer to Patent Document 1), even if cells having such a density are cultured in a culture solution containing no growth factor, a sheet-like cell culture cannot be formed. Therefore, the seeding density in the production method of the present invention is higher than that in the conventional method using a culture solution containing a growth factor. For this reason, in this specification, such seeding density may be simply referred to as “high density”. Specifically, such density is typically 300,000 cells / cm 2 or more for skeletal myoblasts, for example. The upper limit of the seeding density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not shift to differentiation, but for skeletal myoblasts, it is less than 3.4 × 10 6 cells / cm 2 .
したがって、本発明の製造方法における骨格筋芽細胞の播種密度は、ある態様では３．０×１０５〜３．４×１０６個／ｃｍ２、別の態様では３．５×１０５〜３．４×１０６個／ｃｍ２、さらに別の態様では１．０×１０６〜３．４×１０６個／ｃｍ２、さらに別の態様では３．０×１０５〜１．７×１０６個／ｃｍ２、別の態様では３．５×１０５〜１．７×１０６個／ｃｍ２、さらに別の態様では１．０×１０６〜１．７×１０６個／ｃｍ２である。上記範囲は、上限が３．４×１０６個／ｃｍ２未満である限り、上限および下限の両方、または、そのいずれか一方を含んでもよい。したがって、本発明の製造方法における骨格筋芽細胞の播種密度は、例えば、３．０×１０５個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、３．５×１０５個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、１．０×１０６個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、１．０×１０６個／ｃｍ２超３．４×１０６個／ｃｍ２未満（下限も上限も含まない）、１．０×１０６個／ｃｍ２超１．７×１０６個／ｃｍ２以下（下限は含まないが、上限は含む）であってもよい。当業者であれば、骨格筋芽細胞以外の細胞について、本発明に適した細胞密度を、本明細書の教示に従い、実験により適宜決定することができる。 Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is 3.0 × 10 5 to 3.4 × 10 6 cells / cm 2 in one embodiment, and 3.5 × 10 5 to 3 in another embodiment. 4 × 10 6 pieces / cm 2 , in yet another embodiment 1.0 × 10 6 to 3.4 × 10 6 pieces / cm 2 , and in yet another embodiment 3.0 × 10 5 to 1.7 × 10 6 pieces / cm 2 , in another embodiment, 3.5 × 10 5 to 1.7 × 10 6 pieces / cm 2 , and in another embodiment, 1.0 × 10 6 to 1.7 × 10 6 pieces / cm 2 It is. As long as an upper limit is less than 3.4 * 10 < 6 > piece / cm < 2 >, the said range may include both an upper limit and a lower limit, or any one thereof. Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0 × 10 5 cells / cm 2 or more and less than 3.4 × 10 6 cells / cm 2 (including the lower limit and not including the upper limit). ), 3.5 × 10 5 pieces / cm 2 or more and less than 3.4 × 10 6 pieces / cm 2 (including the lower limit, not including the upper limit), 1.0 × 10 6 pieces / cm 2 or more and 3.4 × 10 less than 6 / cm 2 (including the lower and the upper limit is not included), 1.0 × 10 6 cells / cm 2 ultra 3.4 × 10 than 6 / cm 2 (lower limit also contains no upper limit), 1 It may be more than 0.0 × 10 6 pieces / cm 2 and 1.7 × 10 6 pieces / cm 2 or less (not including the lower limit but including the upper limit). A person skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teaching of the present specification.
培養期間中、細胞は増殖してもしなくてもよいが、増殖するとしても、細胞の性状が変化する程には増殖しない。例えば、骨格筋芽細胞はコンフルエントになると分化を開始するが、本発明においては、骨格筋芽細胞は、細胞培養物は形成するが、分化に移行しない密度で播種される。本発明の好ましい態様において、細胞は計測誤差の範囲を超えて増殖しない。細胞が増殖したか否かは、例えば、播種時の細胞数と、細胞培養物形成後の細胞数とを比較することにより評価することができる。本態様において、細胞培養物形成後の細胞数は、典型的には播種時の細胞数の３００％以下、好ましくは２００％以下、より好ましくは１５０％以下、さらに好ましくは１２５％以下、特に好ましくは１００％以下である。 During the culture period, the cells may or may not proliferate, but even if they proliferate, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start to differentiate when they become confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a cell culture but does not transition to differentiation. In a preferred embodiment of the invention, the cells do not grow beyond the range of measurement errors. Whether or not the cells have proliferated can be evaluated, for example, by comparing the number of cells at the time of seeding with the number of cells after formation of the cell culture. In this embodiment, the number of cells after the formation of the cell culture is typically 300% or less, preferably 200% or less, more preferably 150% or less, even more preferably 125% or less, particularly preferably the number of cells at the time of seeding. Is 100% or less.
本発明において、細胞の培養は、対象となる細胞がシート状の細胞培養物を形成するのに適した条件で行われる。
本発明において、「シート状細胞培養物」は、細胞が互いに連結してシート状になったものをいい、典型的には１つの細胞層からなるものであるが、２以上の細胞層から構成されるものも含む。細胞同士は、直接および／または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも機械的に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、細胞培養物を構成する細胞に由来するものである。細胞は少なくとも機械的に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。
In the present invention, the cells are cultured under conditions suitable for the target cells to form a sheet-like cell culture.
In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it is a substance capable of mechanically connecting cells to each other, and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, in particular, derived from the cells constituting the cell culture. The cells are at least mechanically linked, but may be further functionally, eg, chemically or electrically linked.
本発明のシート状細胞培養物は、好ましくはスキャフォールド（支持体）を含まない。スキャフォールドは、その表面上および／またはその内部に細胞を付着させ、細胞培養物の物理的一体性を維持するために当該技術分野において用いられることがあり、例えば、ポリビニリデンジフルオリド（ＰＶＤＦ）製の膜等が知られているが、本発明の細胞培養物は、かかるスキャフォールドがなくともその物理的一体性を維持することができる。また、本発明の細胞培養物は、好ましくは、細胞培養物を構成する細胞由来の物質のみからなり、それら以外の物質を含まない。 The sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Although manufactured membranes are known, the cell culture of the present invention can maintain its physical integrity even without such a scaffold. In addition, the cell culture of the present invention preferably consists only of substances derived from the cells constituting the cell culture and does not contain any other substances.
本発明の一態様において、細胞の培養は、所定の期間内、好ましくは、細胞が分化に移行しない期間内に行われる。したがって、この態様において、細胞は、培養期間中、未分化の状態に維持される。細胞の分化への移行は、当業者に知られた任意の方法で評価することができる。例えば、骨格筋芽細胞の場合は、ＭＨＣの発現、クレアチンキナーゼ（ＣＫ）活性、細胞の多核化、筋管の形成などを分化の指標とすることができる。本発明の好ましい態様において、培養期間は４８時間以内、より好ましくは４０時間以内、さらに好ましくは２４時間以内である。 In one embodiment of the present invention, cell culture is performed within a predetermined period, preferably within a period in which cells do not shift to differentiation. Thus, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition to cell differentiation can be evaluated by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, cell multinucleation, myotube formation, etc. can be used as indicators of differentiation. In a preferred embodiment of the present invention, the culture period is within 48 hours, more preferably within 40 hours, and even more preferably within 24 hours.
培養に用いる細胞培養液（単に「培養液」もしくは「培地」と呼ぶ場合もある）は、細胞の生存を維持できるものであれば特に限定されないが、典型的には、アミノ酸、ビタミン類、電解質を主成分としたものが利用できる。本発明の一態様において、培養液は、細胞培養用の基礎培地をベースにしたものである。かかる基礎培地には、限定されずに、例えば、ＤＭＥＭ、ＭＥＭ、Ｆ１２、ＤＭＥ、ＲＰＭＩ１６４０、ＭＣＤＢ（ＭＣＤＢ１０２、１０４、１０７、１３１、１５３、１９９など）、Ｌ１５、ＳｋＢＭ、ＲＩＴＣ８０−７などが含まれる。これらの基礎培地の多くは市販されており、その組成も公知となっている。一例として、下記表１にＭＣＤＢ１３１およびＤＭＥＭの組成を示す。 The cell culture medium used for the culture (sometimes simply referred to as “culture medium” or “medium”) is not particularly limited as long as it can maintain cell survival, but typically, amino acids, vitamins, electrolytes are used. Can be used. In one embodiment of the present invention, the culture solution is based on a basal medium for cell culture. Such basal media include, but are not limited to, for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, and the like. . Many of these basal media are commercially available, and their compositions are also known. As an example, the composition of MCDB131 and DMEM is shown in Table 1 below.
基礎培地に含まれるアミノ酸としては、限定されずに、例えば、Ｌ−アルギニン、Ｌ−シスチン、Ｌ−グルタミン、グリシン、Ｌ−ヒスチジン、Ｌ−イソロイシン、Ｌ−ロイシン、Ｌ−リジン、Ｌ−メチオニン、Ｌ−フェニルアラニン、Ｌ−セリン、Ｌ−トレオニン、Ｌ−トリプトファン、Ｌ−チロシン、Ｌ−バリンなどが、ビタミン類としては、限定されずに、例えば、Ｄ−パントテン酸カルシウム、塩化コリン、葉酸、ｉ−イノシトール、ナイアシンアミド、リボフラビン、チアミン、ピリドキシン、ビオチン、リポ酸、ビタミンＢ１２、アデニン、チミジンなどが、そして、電解質としては、限定されずに、例えば、ＣａＣｌ２、ＫＣｌ、ＭｇＳＯ４、ＮａＣｌ、ＮａＨ２ＰＯ４、ＮａＨＣＯ３、Ｆｅ（ＮＯ３）３、ＦｅＳＯ４、ＣｕＳＯ４、ＭｎＳＯ４、Ｎａ２ＳｉＯ３、（ＮＨ４）６Ｍｏ７Ｏ２４、ＮａＶＯ３、ＮｉＣｌ２、ＺｎＳＯ４などがそれぞれ含まれる。基礎培地には、これらの成分のほか、Ｄ−グルコースなどの糖類、ピルビン酸ナトリウム、フェノールレッドなどのｐＨ指示薬、プトレシンなどを含んでもよい。 Examples of amino acids contained in the basal medium include, but are not limited to, L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine and the like are not limited to vitamins, for example, calcium D-pantothenate, choline chloride, folic acid, i - inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B 12, adenine, thymidine and then, as the electrolyte, without limitation, for example, CaCl 2, KCl, MgSO 4 , NaCl, NaH 2 PO 4 , NaHCO 3 , Fe (NO 3 ) 3 , FeS O 4 , CuSO 4 , MnSO 4 , Na 2 SiO 3 , (NH 4 ) 6 Mo 7 O 24 , NaVO 3 , NiCl 2 , ZnSO 4 and the like are included. In addition to these components, the basal medium may contain sugars such as D-glucose, pH indicators such as sodium pyruvate and phenol red, putrescine and the like.
本発明の一態様において、基礎培地に含まれるアミノ酸の濃度は、Ｌ−アルギニン：６３．２〜８４ｍｇ／Ｌ、Ｌ−シスチン：３５〜６３ｍｇ／Ｌ、Ｌ−グルタミン：４．４〜５８４ｍｇ／Ｌ、グリシン：２．３〜３０ｍｇ／Ｌ、Ｌ−ヒスチジン：４２ｍｇ／Ｌ、Ｌ−イソロイシン：６６〜１０５ｍｇ／Ｌ、Ｌ−ロイシン：１０５〜１３１ｍｇ／Ｌ、Ｌ−リジン：１４６〜１８２ｍｇ／Ｌ、Ｌ−メチオニン：１５〜３０ｍｇ／Ｌ、Ｌ−フェニルアラニン：３３〜６６ｍｇ／Ｌ、Ｌ−セリン：３２〜４２ｍｇ／Ｌ、Ｌ−トレオニン：１２〜９５ｍｇ／Ｌ、Ｌ−トリプトファン：４．１〜１６ｍｇ／Ｌ、Ｌ−チロシン：１８．１〜１０４ｍｇ／Ｌ、Ｌ−バリン：９４〜１１７ｍｇ／Ｌである。
また、本発明の一態様において、基礎培地に含まれるビタミン剤の濃度は、Ｄ−パントテン酸カルシウム：４〜１２ｍｇ／Ｌ、塩化コリン：４〜１４ｍｇ／Ｌ、葉酸：０．６〜４ｍｇ／Ｌ、ｉ−イノシトール：７．２ｍｇ／Ｌ、ナイアシンアミド：４〜６．１ｍｇ／Ｌ、リボフラビン：０．００３８〜０．４ｍｇ／Ｌ、チアミン：３．４〜４ｍｇ／Ｌ、ピリドキシン：２．１〜４ｍｇ／Ｌである。
In one embodiment of the present invention, the concentration of amino acids contained in the basal medium is as follows: L-arginine: 63.2 to 84 mg / L, L-cystine: 35 to 63 mg / L, L-glutamine: 4.4 to 584 mg / L Glycine: 2.3-30 mg / L, L-histidine: 42 mg / L, L-isoleucine: 66-105 mg / L, L-leucine: 105-131 mg / L, L-lysine: 146-182 mg / L, L -Methionine: 15-30 mg / L, L-phenylalanine: 33-66 mg / L, L-serine: 32-42 mg / L, L-threonine: 12-95 mg / L, L-tryptophan: 4.1-16 mg / L L-tyrosine: 18.1 to 104 mg / L, L-valine: 94 to 117 mg / L.
In one embodiment of the present invention, the concentration of the vitamin preparation contained in the basal medium is as follows: calcium D-pantothenate: 4 to 12 mg / L, choline chloride: 4 to 14 mg / L, folic acid: 0.6 to 4 mg / L , I-inositol: 7.2 mg / L, niacinamide: 4-6.1 mg / L, riboflavin: 0.0038-0.4 mg / L, thiamine: 3.4-4 mg / L, pyridoxine: 2.1- 4 mg / L.
細胞培養液は、上記のほか、血清、成長因子、ステロイド剤成分、セレン成分などの１種または２種以上の添加物を含んでもよい。しかし、これらの成分は臨床においてはレシピエントに対するアナフィラキシーショック等の副作用要因となり得ることが否定できない製造工程由来不純物であり、臨床への適用にあたっては排除すべき成分である。したがって、本発明の好ましい態様において、細胞培養液は、これらの添加物の少なくとも１種の有効量を含まない。また、本発明のより好ましい態様において、細胞培養液は、これらの添加物の少なくとも１種を実質的に含まない。さらに、本発明の特に好ましい態様において、細胞培養液は、添加物を実質的に含まない。したがって、細胞培養液は、基礎培地のみを含んでもよい。 In addition to the above, the cell culture medium may contain one or more additives such as serum, growth factor, steroid component, and selenium component. However, these components are impurities derived from the manufacturing process that cannot be denied that they can cause side effects such as anaphylactic shock to the recipient in the clinic, and should be excluded in clinical application. Accordingly, in a preferred embodiment of the invention, the cell culture medium does not contain an effective amount of at least one of these additives. In a more preferred embodiment of the present invention, the cell culture medium is substantially free of at least one of these additives. Furthermore, in a particularly preferred embodiment of the present invention, the cell culture medium is substantially free of additives. Therefore, the cell culture medium may contain only the basal medium.
細胞培養液に含まれる血清としては、異種血清および同種血清が挙げられる、ここで「異種血清」は、細胞培養物を移植に用いる場合、そのレシピエントとは異なる種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ウシやウマに由来する血清、例えば、ウシ胎仔血清（ＦＢＳ、ＦＣＳ）、仔ウシ血清（ＣＳ）、ウマ血清（ＨＳ）などが異種血清に該当する。また、「同種血清」は、レシピエントと同一の種の生物に由来する血清を意味する。例えば、レシピエントがヒトである場合、ヒト血清が同種血清に該当する。同種血清は、自己血清、すなわち、レシピエントに由来する血清、およびレシピエント以外の同種個体に由来する同種他家血清を含む。なお、本明細書中で、自己血清以外の血清、すなわち、異種血清と同種他家血清を非自己血清と総称することもある。本発明においては、移植時の副作用を回避するために、同種血清が好ましく、自己血清がさらに好ましい。 Serum contained in the cell culture medium includes heterologous serum and allogeneic serum, where “heterologous serum” refers to serum derived from an organism of a species different from the recipient when the cell culture is used for transplantation. means. For example, when the recipient is a human, serum derived from bovine or horse, for example, fetal calf serum (FBS, FCS), calf serum (CS), horse serum (HS), etc. corresponds to the heterologous serum. “Allogeneic serum” means serum derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to allogeneic serum. Allogeneic serum includes autologous serum, ie serum derived from the recipient, and allogeneic serum derived from allogeneic individuals other than the recipient. In the present specification, sera other than autoserum, that is, heterologous serum and allogeneic sera are sometimes collectively referred to as non-self serum. In the present invention, in order to avoid side effects during transplantation, allogeneic serum is preferable, and autologous serum is more preferable.
本発明の一態様において、細胞培養液は血清を実質的に含まない。血清を実質的に含まない細胞培養液のことを、本明細書中で「無血清培地」と呼ぶこともある。ここで、「血清を実質的に含まない」とは、培養液における血清の含量が、細胞培養物を生体に適用した場合に悪影響を及ぼさない程度（例えば、細胞培養物中の血清アルブミン含量が５０ｎｇ未満となる量）であること、好ましくは、培養液にこれらの物質を積極的に添加しないことを意味する。本発明においては、移植時の副作用を回避するために、細胞培養液は異種血清を実質的に含まないことが好ましく、非自己血清を実質的に含まないことがさらに好ましい。 In one embodiment of the present invention, the cell culture medium is substantially free of serum. A cell culture medium substantially free of serum may be referred to herein as “serum-free medium”. Here, “substantially free of serum” means that the serum content in the culture solution does not have an adverse effect when the cell culture is applied to a living body (for example, the serum albumin content in the cell culture is It means that these substances are not positively added to the culture medium. In the present invention, in order to avoid side effects at the time of transplantation, the cell culture medium preferably contains substantially no heterogeneous serum, and more preferably contains substantially no non-self serum.
本発明の一態様において、細胞培養液は有効量の成長因子を含まない。ここで、「成長因子」は、細胞の増殖を、それがない場合に比べて促進する任意の物質を意味し、例えば、上皮細胞成長因子（ＥＧＦ）、血管内皮成長因子（ＶＥＧＦ）、線維芽細胞成長因子（ＦＧＦ）などを含む。また、「有効量の成長因子」とは、細胞の増殖を、成長因子がない場合に比べて、有意に促進する成長因子の量、または、便宜的に、当該技術分野において細胞の増殖を目的として通常添加する量を意味する。細胞増殖促進の有意性は、例えば、当該技術分野で知られた任意の統計学的手法、例えば、ｔ検定などにより適宜評価することができ、また、通常の添加量は当該技術分野の種々の公知文献から知ることができる。具体的には、骨格筋芽細胞の培養におけるＥＧＦの有効量は、例えば０．００５μｇ／ｍＬ以上である。 In one embodiment of the invention, the cell culture fluid does not contain an effective amount of growth factor. As used herein, “growth factor” means any substance that promotes cell proliferation as compared to the case without it, such as epithelial cell growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast, and the like. Cell growth factor (FGF) and the like. In addition, an “effective amount of growth factor” refers to the amount of growth factor that significantly promotes cell proliferation compared to the absence of growth factor, or for the purpose of cell proliferation in the art for convenience. Means the amount usually added. The significance of cell growth promotion can be appropriately evaluated, for example, by any statistical method known in the art, for example, t-test, and the usual addition amount is various in the art. It can be known from known literature. Specifically, the effective amount of EGF in skeletal myoblast culture is, for example, 0.005 μg / mL or more.
したがって、「有効量の成長因子を含まない」とは、本発明における培養液における成長因子の濃度がかかる有効量未満であることを意味する。例えば、骨格筋芽細胞の培養におけるＥＧＦの培養液中の濃度は、好ましくは０．００５μｇ／ｍＬ未満、より好ましくは０．００１μｇ／ｍＬ未満である。本発明の好ましい態様においては、培養液における成長因子の濃度は、生体における通常の濃度未満である。かかる態様においては、例えば、骨格筋芽細胞の培養におけるＥＧＦの培養液中の濃度は、好ましくは５．５ｎｇ／ｍＬ未満、より好ましくは１．３ｎｇ／ｍＬ未満、さらに好ましくは、０．５ｎｇ／ｍＬ未満である。さらに好ましい態様において、本発明における培養液は、成長因子を実質的に含まない。ここで、実質的に含まないとは、培養液中の成長因子の含量が、細胞培養物を生体に適用した場合に悪影響を及ぼさない程度であること、好ましくは、培養液に成長因子を積極的に添加しないことを意味する。したがって、この態様においては、培養液は、その中の他の成分、例えば血清などに含まれる以上の濃度の成長因子を含まない。 Therefore, “not containing an effective amount of growth factor” means that the concentration of the growth factor in the culture medium of the present invention is less than the effective amount. For example, the concentration of EGF in the culture medium in skeletal myoblast culture is preferably less than 0.005 μg / mL, more preferably less than 0.001 μg / mL. In a preferred embodiment of the present invention, the concentration of the growth factor in the culture solution is less than the normal concentration in the living body. In such an embodiment, for example, the concentration of EGF in the culture medium in skeletal myoblast culture is preferably less than 5.5 ng / mL, more preferably less than 1.3 ng / mL, and even more preferably 0.5 ng / mL. Less than mL. In a further preferred embodiment, the culture medium in the present invention is substantially free from growth factors. Here, “substantially free” means that the content of the growth factor in the culture solution is such that the cell culture is not adversely affected when applied to a living body, and preferably the growth factor is positively added to the culture solution. Means that it is not added. Therefore, in this embodiment, the culture solution does not contain a growth factor at a concentration higher than that contained in other components such as serum.
本発明の一態様において、細胞培養液は、ステロイド剤成分を実質的に含まない。ここで「ステロイド剤成分」は、ステロイド核を有する化合物のうち、生体に、副腎皮質機能不全、クッシング症候群などの悪影響を及ぼし得るものをいう。かかる化合物としては、限定されずに、例えば、コルチゾール、プレドニゾロン、トリアムシノロン、デキサメタゾン、ベタメタゾン等が含まれる。したがって、「ステロイド剤成分を実質的に含まない」とは、培養液におけるこれらの化合物の含量が、細胞培養物を生体に適用した場合に悪影響を及ぼさない程度であること、好ましくは、培養液にこれらの化合物を積極的に添加しないこと、すなわち、培養液が、その中の他の成分、例えば血清などに含まれる以上の濃度のステロイド剤成分を含まないことを意味する。 In one embodiment of the present invention, the cell culture medium is substantially free of steroid component. Here, the “steroid component” refers to a compound having a steroid nucleus that can adversely affect a living body such as adrenal cortex dysfunction and Cushing's syndrome. Such compounds include, but are not limited to, for example, cortisol, prednisolone, triamcinolone, dexamethasone, betamethasone and the like. Therefore, “substantially free of steroid component” means that the content of these compounds in the culture solution is such that the cell culture is not adversely affected when applied to a living body. This means that these compounds are not actively added, that is, the culture solution does not contain a steroid component at a concentration higher than that contained in other components such as serum.
本発明の一態様において、細胞培養液は、セレン成分を実質的に含まない。ここで「セレン成分」は、セレン分子、およびセレン含有化合物、特に、生体内でセレン分子を遊離し得るセレン含有化合物、例えば、亜セレン酸などを含む。したがって、「セレン成分を実質的に含まない」とは、培養液におけるこれらの物質の含量が、細胞培養物を生体に適用した場合に悪影響を及ぼさない程度であること、好ましくは、培養液にこれらの物質を積極的に添加しないこと、すなわち、培養液が、その中の他の成分、例えば血清などに含まれる以上の濃度のセレン成分を含まないことを意味する。具体的には、例えば、ヒトの場合、培養液中のセレン濃度は、ヒト血清中の正常値（例えば、１０．６〜１７．４μｇ／ｄＬ）に、培地中に含まれるヒト血清の割合を乗じた値よりも低い（すなわち、ヒト血清の含量が１０％であれば、セレン濃度は、例えば、１．０〜１．７μｇ／ｄＬ未満である）。 In one embodiment of the present invention, the cell culture solution is substantially free of a selenium component. Here, the “selenium component” includes a selenium molecule and a selenium-containing compound, in particular, a selenium-containing compound capable of releasing a selenium molecule in vivo, such as selenite. Therefore, “substantially free of selenium component” means that the content of these substances in the culture solution is such that the cell culture is not adversely affected when applied to a living body. This means that these substances are not actively added, that is, the culture solution does not contain a selenium component at a concentration higher than that contained in other components such as serum. Specifically, for example, in the case of humans, the selenium concentration in the culture solution is the normal value in human serum (for example, 10.6 to 17.4 μg / dL), and the ratio of human serum contained in the medium is It is lower than the multiplied value (that is, if the content of human serum is 10%, the selenium concentration is, for example, 1.0 to less than 1.7 μg / dL).
本発明の上記好ましい態様においては、生体に適用する細胞培養物を作製する場合に従来必要であった、成長因子、ステロイド剤成分、異種血清成分などの製造工程由来不純物を、洗浄などにより除去する工程が不要となる。したがって、本発明の方法の一態様は、この製造工程由来不純物を除去する工程を含まない。
ここで、「製造工程由来不純物」とは、典型的には、製造各工程に由来する以下に列挙するものが含まれる。すなわち、細胞基材に由来するもの（例えば、宿主細胞由来タンパク質、宿主細胞由来ＤＮＡ）、細胞培養液に由来するもの（例えば、インデューサー、抗生物質、培地成分）、あるいは細胞培養以降の工程である目的物質の抽出、分離、加工、精製工程に由来するものなどである（例えば、医薬審発第５７１号参照）。
In the above preferred embodiment of the present invention, impurities derived from production processes such as growth factors, steroid components, and heterogeneous serum components, which have been conventionally required when preparing a cell culture to be applied to a living body, are removed by washing or the like. A process becomes unnecessary. Therefore, one embodiment of the method of the present invention does not include the step of removing impurities derived from the production process.
Here, “manufacturing process-derived impurities” typically include those listed below, which are derived from each manufacturing process. That is, a substance derived from a cell substrate (for example, host cell-derived protein, host cell-derived DNA), a substance derived from a cell culture medium (for example, inducer, antibiotic, medium component), or a step after cell culture It is derived from the extraction, separation, processing, and purification steps of a certain target substance (see, for example, Pharmaceutical Examination No. 571).
また、本発明の方法により製造されたシート状細胞培養物は、洗浄操作に耐え得る物理的強度を有しているため、本発明の方法は、上記製造工程由来不純物等を洗浄などにより除去する工程を含んでもよい。したがって、本発明の一態様は、単離したシート状細胞培養物を洗浄する工程をさらに含む。かかる工程を加えることにより、上記製造工程由来不純物を実質的に含む細胞培養液を用いても、かかる不純物を実質的に含まないシート状細胞培養物を得ることが可能となる。 In addition, since the sheet-shaped cell culture produced by the method of the present invention has physical strength that can withstand a washing operation, the method of the present invention removes impurities and the like derived from the production process by washing or the like. A process may be included. Accordingly, one embodiment of the present invention further includes a step of washing the isolated sheet-shaped cell culture. By adding such a step, it is possible to obtain a sheet-shaped cell culture that does not substantially contain such impurities, even if a cell culture solution that substantially contains the impurities derived from the above production steps is used.
細胞の培養は、当該技術分野で通常なされている条件で行うことができる。例えば、典型的な培養条件としては、３７℃、５％ＣＯ２での培養が挙げられる。培養期間は、細胞培養物の十分な形成、および、細胞分化防止の観点から、好ましくは４８時間以内、より好ましくは４０時間以内、さらに好ましくは２４時間以内である。培養は任意の大きさおよび形状の容器で行うことができる。本発明の方法において、細胞は実質的に増殖しないため、従来の方法のように細胞培養物が所望の大きさに成長するのを待つことなく、所望の大きさおよび形状の細胞培養物を短期間で得ることが可能となる。細胞培養物の大きさや形状は、培養容器の細胞付着面の大きさ・形状を調整すること、または、培養容器の細胞付着面に、所望の大きさ・形状の型枠を設置し、その内部で細胞を培養することなどにより任意に調節することができる。 Cell culture can be performed under conditions usually used in the art. For example, typical culture conditions include culture at 37 ° C. and 5% CO 2 . The culture period is preferably within 48 hours, more preferably within 40 hours, and even more preferably within 24 hours, from the viewpoint of sufficient formation of a cell culture and prevention of cell differentiation. Culturing can be performed in containers of any size and shape. In the method of the present invention, since the cells do not substantially proliferate, the cell culture of the desired size and shape can be rapidly transferred without waiting for the cell culture to grow to the desired size as in the conventional method. It becomes possible to get between. The size and shape of the cell culture can be adjusted by adjusting the size and shape of the cell adhesion surface of the culture vessel, or by installing a mold of the desired size and shape on the cell adhesion surface of the culture vessel. It can be arbitrarily adjusted by culturing the cells with, for example.
本方法は、細胞培養物を培養基材から単離する工程をさらに含んでもよい。細胞培養物の基材からの単離は、細胞培養物が少なくとも部分的に、シート構造を保ったまま、足場となっている基材から遊離（剥離）できれば特に限定されず、例えば、タンパク分解酵素、例えばトリプシンによる酵素処理および／または浸漬撹拌、ピペッティングなどの機械的処理によって行うことができる。また、細胞を、刺激、例えば、温度や光に応答して物性が変化する材料で表面を被覆した培養基材上で培養して細胞培養物を形成させることにより、所定の刺激を加えることで、非酵素的に遊離することもできる。 The method may further comprise the step of isolating the cell culture from the culture substrate. The isolation of the cell culture from the base material is not particularly limited as long as the cell culture can be released (peeled) from the base material serving as a scaffold while at least partially maintaining the sheet structure. It can be carried out by enzyme treatment with an enzyme such as trypsin and / or mechanical treatment such as immersion stirring and pipetting. In addition, by applying a predetermined stimulus by culturing cells on a culture substrate whose surface is coated with a material that changes physical properties in response to stimulation, for example, temperature or light, to form a cell culture. It can also be released non-enzymatically.
本発明の方法は、細胞の採取から、細胞の増殖および細胞培養物の作製を経て、細胞培養物の適用に至る、再生治療の一工程として位置づけることもできる。したがって、本発明は、
（ｉ）対象から採取した組織または生体液から所望の細胞を単離する工程、
（ｉｉ）単離した細胞を増殖させる工程、
（ｉｉｉ）血清で被覆された培養基材上に、実質的に増殖することなくシート状細胞培養物を形成し得る密度の細胞を播種する工程、
（ｉｖ）細胞を培養してシート状の細胞培養物を形成する工程、および
（ｖ）形成された培養物を基材から単離する工程、および任意に
（ｖｉ）単離したシート状細胞培養物を洗浄する工程
を含む、再生治療用シート状細胞培養物の製造方法にも関する。
The method of the present invention can also be positioned as one step of regenerative treatment from cell collection to cell growth and cell culture preparation to cell culture application. Therefore, the present invention
(I) isolating desired cells from tissue or biological fluid collected from the subject,
(Ii) growing the isolated cells;
(Iii) seeding cells at a density capable of forming a sheet-like cell culture without substantially growing on a culture substrate coated with serum;
(Iv) culturing the cells to form a sheet-shaped cell culture, and (v) isolating the formed culture from the substrate, and optionally (vi) the isolated sheet-shaped cell culture. The present invention also relates to a method for producing a sheet-shaped cell culture for regenerative treatment, comprising a step of washing the product.
本発明はまた、上記製造方法によって作製されたシート状細胞培養物に関する。
好ましい態様において、本発明のシート状細胞培養物は、浸漬撹拌などの洗浄操作や、取出し、保持、移送などの移植操作に対して十分な物理的強度を有する。十分な物理的強度を有するとは、上記操作を施しても細胞培養物のシート状構造が損なわれないことを意味し、これは、例えば、得られたシート状細胞培養物に実際に上記操作を施し、シート状構造が保たれていることを肉眼的または顕微鏡的に調査することにより確認することができる。ここで、浸漬撹拌による洗浄操作とは、典型的には、シート状細胞培養物の入った培養容器に同培養物が十分浸漬する量の緩衝液を加え、培養容器ごと振とう撹拌することをいう。したがって、十分な物理的強度を有することは、かかる浸漬撹拌による洗浄操作と同様の物理的ストレスをシート状細胞培養物に加えることによっても確認することができる。また、上記製造方法によって作製されたシート状細胞培養物であれば、通常かかる十分な物理的強度を有する。
The present invention also relates to a sheet-like cell culture produced by the above production method.
In a preferred embodiment, the sheet-like cell culture of the present invention has sufficient physical strength for washing operations such as immersion stirring and transplantation operations such as removal, holding and transfer. Having sufficient physical strength means that even if the above operation is performed, the sheet-like structure of the cell culture is not impaired. For example, the obtained sheet-like cell culture is actually subjected to the above operation. It can be confirmed by conducting a macroscopic or microscopic investigation that the sheet-like structure is maintained. Here, the washing operation by immersing and stirring typically means adding a sufficient amount of a buffer solution in which the culture is immersed in a culture vessel containing a sheet-like cell culture, and stirring and agitating the whole culture vessel. Say. Therefore, having sufficient physical strength can also be confirmed by applying physical stress similar to the washing operation by soaking and stirring to the sheet-like cell culture. In addition, a sheet-like cell culture produced by the above production method usually has sufficient physical strength.
本発明の細胞培養物の好ましい態様において、細胞培養物は非自己血清、成長因子、ステロイド剤および／またはセレン成分を実質的に含まない。より好ましい態様において、本発明の細胞培養物は、上記成分を含む製造工程由来不純物を実質的に含まない。この細胞培養物は、細胞を、非自己血清、成長因子、ステロイド剤および／またはセレン成分を実質的に含まない培養液で培養して、細胞培養物を形成させることにより作製することができる。ここで、細胞培養物が非自己血清、成長因子、ステロイド剤および／またはセレン成分を実質的に含まないとは、細胞培養物が、これらの成分を、レシピエントに悪影響を与える濃度で含まないことを少なくとも意味するが、細胞培養物の形成を、非自己血清、成長因子、ステロイド剤および／またはセレン成分を実質的に含まない培養液で行うことにより、かかる条件を充足することができる。さらに好ましい態様において、本発明の細胞培養物は、製造工程由来不純物のほか、自己血清も実質的に含まない。ここで、「実質的に含まない」の意味は、上記と同様である。 In a preferred embodiment of the cell culture of the present invention, the cell culture is substantially free of non-autologous serum, growth factors, steroidal agents and / or selenium components. In a more preferred embodiment, the cell culture of the present invention is substantially free of manufacturing process-derived impurities containing the above components. This cell culture can be prepared by culturing cells in a culture medium substantially free of non-autologous serum, growth factors, steroids and / or selenium components to form a cell culture. Here, the cell culture is substantially free of non-autologous serum, growth factors, steroids and / or selenium components, so that the cell culture does not contain these components at concentrations that adversely affect the recipient. At least that means that cell culture can be formed in a culture medium that is substantially free of non-autologous serum, growth factors, steroids and / or selenium components. In a further preferred embodiment, the cell culture of the present invention is substantially free from autologous serum in addition to impurities originating from the manufacturing process. Here, the meaning of “substantially free” is the same as described above.
本発明の細胞培養物の好ましい態様において、細胞培養物は炎症性サイトカインが低減されている。炎症性サイトカインとは、炎症に伴い産生されるサイトカインの総称であり、例えば、限定することなく、ＴＮＦ−α、ＩＬ−１（ＩＬ−１α、ＩＬ−１βなど）、ＩＬ−４、ＩＬ−５、ＩＬ−６、ＩＬ−１３などが挙げられる。したがって、「炎症性サイトカインが低減されている」とは、これらのサイトカインの存在量または産生量（分泌量）が、分化した細胞培養物に比べて低減していることを意味する。したがって、サイトカインは、遺伝子の転写からタンパク質の分泌に至る過程のいずれにおける形態で存在してもよく、例えば、ｍＲＮＡなどの転写物の形態や、タンパク質の形態で存在していてもよい。低減の程度は、誤差の範囲を超えるものであれば特に限定されないが、例えば、分化した細胞培養物に比べて１５％以上、好ましくは２５％以上、より好ましくは５０％以上、さらに好ましくは６０％以上、特に好ましくは７０％以上の低減である。 In a preferred embodiment of the cell culture of the present invention, the cell culture has reduced inflammatory cytokines. Inflammatory cytokine is a general term for cytokines produced along with inflammation. For example, without limitation, TNF-α, IL-1 (IL-1α, IL-1β, etc.), IL-4, IL-5 , IL-6, IL-13 and the like. Therefore, “the inflammatory cytokine is reduced” means that the abundance or production (secretory amount) of these cytokines is reduced as compared to a differentiated cell culture. Therefore, the cytokine may exist in any form in the process from gene transcription to protein secretion. For example, the cytokine may exist in the form of a transcript such as mRNA or in the form of a protein. The degree of reduction is not particularly limited as long as it exceeds the error range. For example, it is 15% or more, preferably 25% or more, more preferably 50% or more, and still more preferably 60% compared to differentiated cell culture. % Or more, particularly preferably a reduction of 70% or more.
サイトカインの量は、遺伝子レベルでは、例えば、ノーザンブロッティング法、サザンブロッティング法、ＲＮａｓｅプロテクションアッセイ、ＲＴ−ＰＣＲ、リアルタイムＰＣＲ等のＰＣＲ法、in situハイブリダイゼーション法、in vitro転写法等の任意の公知の遺伝子発現解析法により、また、タンパク質レベルでは、免疫沈降法、ウェスタンブロッティング法、ＥＩＡ、ＥＬＩＳＡ、ＲＩＡ、免役組織化学法、免疫細胞化学法等の任意の公知のタンパク質検出法により検出することができる。検体としては、細胞培養物が含浸された培地や、細胞培養物の一部などを用いることができる。 At the gene level, the amount of cytokine can be any known, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, real-time PCR, in situ hybridization method, in vitro transcription method, etc. It can be detected by gene expression analysis, and at the protein level by any known protein detection method such as immunoprecipitation, Western blotting, EIA, ELISA, RIA, immunohistochemistry, immunocytochemistry, etc. . As the specimen, a medium impregnated with a cell culture, a part of the cell culture, or the like can be used.
本発明の好ましい態様において、細胞培養物は治療上有用な生理活性物質を含む。かかる物質は、治療上何らかの有用性を有するものであれば特に限定されないが、例えば、血管新生、細胞死の抑制、組織リモデリング、心臓発生、幹細胞の誘導等に関与する物質を含み、より具体的には、限定されずに、例えば、ＶＥＧＦ、ＰＩＧＦ、アンギオゲニン、アンギオポエチン、ＨＧＦ、ＰＤＧＦ（ＰＤＧＦ−ＡＢ、ＰＤＧＦ−ＢＢなど）、ＢＡＧ−１、ＢＣＬ−２、ＭＭＰ（ＭＭＰ−２、ＭＭＰ−３、ＭＭＰ−９、ＭＭＰ−１０など）、ＴＩＭＰ、ＴＮＮＴ２、ＴＮＮＣ１、ＩＧＦ−１などが挙げられる。 In a preferred embodiment of the invention, the cell culture contains a therapeutically useful physiologically active substance. Such a substance is not particularly limited as long as it has some therapeutic utility, but includes, for example, substances involved in angiogenesis, suppression of cell death, tissue remodeling, cardiac development, stem cell induction, and the like. Specifically, without limitation, for example, VEGF, PIGF, angiogenin, angiopoietin, HGF, PDGF (PDGF-AB, PDGF-BB, etc.), BAG-1, BCL-2, MMP (MMP-2, MMP-3, MMP-9, MMP-10, etc.), TIMP, TNNT2, TNNC1, IGF-1 and the like.
これらの物質を「含む」とは、これらの存在量または産生量（分泌量）が細胞培養物において検出できることを意味する。本発明の好ましい態様において、細胞培養物は、これらの物質を分化した細胞培養物よりも多く含む。増大の程度は、誤差の範囲を超えるものであれば特に限定されないが、例えば、分化した細胞培養物に比べて１．２倍以上、好ましくは１．５倍以上、より好ましくは２倍以上、さらに好ましくは３倍以上である。これらの物質は、遺伝子の転写からタンパク質の分泌に至る過程のいずれにおける形態で存在してもよく、例えば、ｍＲＮＡなどの転写物の形態や、タンパク質の形態で存在していてもよい。これらの物質の存在は、上記のサイトカインと同様に検出および／または定量できる。 “Contains” these substances means that their abundance or production (secreted amount) can be detected in the cell culture. In a preferred embodiment of the invention, the cell culture contains more of these substances than the differentiated cell culture. The degree of increase is not particularly limited as long as it exceeds the range of error, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, compared to differentiated cell culture, More preferably, it is 3 times or more. These substances may exist in a form in any of the processes from gene transcription to protein secretion. For example, the substance may exist in the form of a transcript such as mRNA or in the form of a protein. The presence of these substances can be detected and / or quantified in the same manner as the above cytokines.
本発明の好ましい態様において、細胞培養物は未分化の状態である。細胞の分化への移行は、当業者に知られた任意の方法で評価することができる。例えば、骨格筋芽細胞の場合は、ＭＨＣの発現、クレアチンキナーゼ（ＣＫ）活性、細胞の多核化などを分化の指標とすることができる。また、未分化細胞では、分化細胞に比べて炎症性サイトカインが少なく、治療上有用な生理活性物質が多いことから、これを指標に分化の有無を判断することもできる。未分化の状態の細胞培養物を得る手法としては、例えば、細胞を本発明の方法に従って、４８時間以内、より好ましくは４０時間以内、さらに好ましくは２４時間以内の期間培養することが挙げられる。 In a preferred embodiment of the invention, the cell culture is in an undifferentiated state. The transition to cell differentiation can be evaluated by any method known to those skilled in the art. For example, in the case of skeletal myoblasts, MHC expression, creatine kinase (CK) activity, cell multinucleation, etc. can be used as indicators of differentiation. In addition, since undifferentiated cells have fewer inflammatory cytokines than differentiated cells and there are many therapeutically useful physiologically active substances, the presence or absence of differentiation can also be determined using this as an index. Examples of a technique for obtaining an undifferentiated cell culture include culturing cells in a period of 48 hours, more preferably 40 hours, and even more preferably 24 hours according to the method of the present invention.
本発明の細胞培養物は、対象の疾病、傷病の治療に用いることができる。例えば、骨格筋芽細胞による細胞培養物は、心疾患、例えば、心筋梗塞、拡張型心筋症などに、移植片などの形態で用いることができる。したがって、本発明の別の態様は、上記細胞培養物を含む移植片に関する。 The cell culture of the present invention can be used for treatment of a target disease or injury. For example, a cell culture using skeletal myoblasts can be used in the form of a graft for heart diseases such as myocardial infarction and dilated cardiomyopathy. Accordingly, another aspect of the present invention relates to a graft comprising the above cell culture.
本発明はまた、血清および／または成長因子および／またはステロイド剤がコートされた培養基材に関する。本発明の培養基材は、このましくは、上記の本発明によるシート状細胞培養物の製造に用いられる。コートの手法などは、本発明のシート状細胞培養物の製造方法に関して上記したとおりである。
本発明はさらに、（ｉ）培養基材を血清と共にインキュベートする工程、および（ｉｉ）血清を廃棄する工程を含む、上記培養基材の製造方法に関する。具体的な製造手法は、本発明のシート状細胞培養物の製造方法に関して上記したとおりである。本発明の一態様において、工程（ｉｉ）の後に、（ｉｉｉ）培養基材を無血清洗浄液で洗浄する工程が追加されてもよい。
The invention also relates to a culture substrate coated with serum and / or growth factors and / or steroids. The culture substrate of the present invention is preferably used for producing the sheet-shaped cell culture according to the present invention. The coating method and the like are as described above for the method for producing a sheet-shaped cell culture of the present invention.
The present invention further relates to a method for producing the culture substrate, comprising (i) a step of incubating the culture substrate with serum, and (ii) a step of discarding the serum. The specific production method is as described above with respect to the production method of the sheet-shaped cell culture of the present invention. In one embodiment of the present invention, a step of (iii) washing the culture substrate with a serum-free washing solution may be added after step (ii).
本発明はさらにまた、培養基材と、血清、成長因子、ステロイド剤からなる群から選択されるコーティング剤とを含む、上記培養基材の製造キットに関する。同キットにおいて、コーティング剤は、種々の保存可能な形態、例えば、冷蔵、冷凍または凍結乾燥された状態で、適切な容器、例えば、種々の材質（ガラス、プラスチックなど）のバイアル、アンプル、ボトル、チューブ等に収納されていてもよい。本発明のキットは、上記のほか、コーティング剤を希釈および／または再構成するための媒体、例えば、水、生理食塩水、種々の緩衝液（例えば、ＰＢＳ、ＨＢＳなど）、種々の液体培地（例えば、ＤＭＥＭ、ＭＥＭ、Ｆ１２、ＤＭＥ、ＲＰＭＩ１６４０、ＭＣＤＢ（ＭＣＤＢ１０２、１０４、１０７、１３１、１５３、１９９など）、Ｌ１５、ＳｋＢＭ、ＲＩＴＣ８０−７、ＤＭＥＭ／Ｆ１２など）、ならびに、培養基材のコーティング手法に関する情報を含む説明書や、ＣＤ、ＤＶＤ等の電子記録媒体などを含んでいてもよい。本キットに含まれる培養基材やコーティング剤等は、好ましくは滅菌された状態で提供することができる。滅菌手法としては、限定することなく、エチレンオキサイドガスなどによるガス滅菌、紫外線や放射線（例えばγ線）による滅菌などが挙げられる。 The present invention further relates to a kit for producing the above culture substrate, comprising a culture substrate and a coating agent selected from the group consisting of serum, growth factor, and steroid. In the kit, the coating agent can be stored in various storable forms, such as refrigerated, frozen, or lyophilized, in a suitable container, such as vials, ampoules, bottles of various materials (glass, plastic, etc.) It may be stored in a tube or the like. In addition to the above, the kit of the present invention includes a medium for diluting and / or reconstituting the coating agent, such as water, physiological saline, various buffers (for example, PBS, HBS, etc.), various liquid media ( For example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDB102, 104, 107, 131, 153, 199, etc.), L15, SkBM, RITC80-7, DMEM / F12, etc.) and culture substrate coating techniques It may include an instruction including information on the electronic recording medium or an electronic recording medium such as a CD or a DVD. The culture substrate, coating agent, and the like included in the kit can be preferably provided in a sterilized state. Examples of the sterilization method include, but are not limited to, gas sterilization using ethylene oxide gas and the like, and sterilization using ultraviolet rays and radiation (for example, γ rays).
本発明はまた、細胞培養物を未分化状態に維持することによる、細胞培養物における炎症性サイトカインを低減する方法に関する。本発明はさらに、細胞培養物を未分化状態に維持することによる、細胞培養物に含まれる治療上有用な生理活性物質の低減を抑制する方法に関する。これらの方法において、細胞培養物を未分化状態に維持する手法としては、例えば、細胞の培養を、所定の期間内、好ましくは、細胞が分化に移行しない期間内に行うことが挙げられる。これらの方法の好ましい態様において、培養期間は４８時間以内、より好ましくは４０時間以内、さらに好ましくは２４時間以内である。これらの方法における各工程、炎症性サイトカインおよび治療上有用な生理活性物質は、基本的に、本発明のシート状細胞培養物の製造方法およびシート状細胞培養物に関して上記したとおりである。これらの方法において、「炎症性サイトカインを低減する」とは、これらのサイトカインの存在量または産生量（分泌量）を、分化した細胞培養物に比べて低減させることを意味する。低減の程度は、誤差の範囲を超えるものであれば特に限定されないが、例えば、分化した細胞培養物に比べて１５％以上、好ましくは２５％以上、より好ましくは５０％以上、さらに好ましくは６０％以上、特に好ましくは７０％以上の低減である。また、「治療上有用な生理活性物質の低減を抑制する」とは、これらの物質の存在量または産生量（分泌量）を、分化した細胞培養物に比べて増大した状態に維持することを意味する。増大の程度は、誤差の範囲を超えるものであれば特に限定されないが、例えば、分化した細胞培養物に比べて１．２倍以上、好ましくは１．５倍以上、より好ましくは２倍以上、さらに好ましくは３倍以上である。 The present invention also relates to a method of reducing inflammatory cytokines in a cell culture by maintaining the cell culture in an undifferentiated state. The present invention further relates to a method for suppressing reduction of a therapeutically useful physiologically active substance contained in a cell culture by maintaining the cell culture in an undifferentiated state. In these methods, as a technique for maintaining the cell culture in an undifferentiated state, for example, cell culture is performed within a predetermined period, preferably within a period in which the cells do not shift to differentiation. In a preferred embodiment of these methods, the culture period is within 48 hours, more preferably within 40 hours, and even more preferably within 24 hours. Each step, inflammatory cytokine, and therapeutically useful physiologically active substance in these methods are basically as described above for the method for producing a sheet-shaped cell culture and the sheet-shaped cell culture of the present invention. In these methods, “reducing inflammatory cytokines” means reducing the abundance or production (secretory amount) of these cytokines compared to differentiated cell cultures. The degree of reduction is not particularly limited as long as it exceeds the error range. For example, it is 15% or more, preferably 25% or more, more preferably 50% or more, and still more preferably 60% compared to differentiated cell culture. % Or more, particularly preferably a reduction of 70% or more. In addition, “suppressing the reduction of therapeutically useful physiologically active substances” means that the abundance or production (secretory quantity) of these substances is maintained in an increased state compared to differentiated cell cultures. means. The degree of increase is not particularly limited as long as it exceeds the range of error, for example, 1.2 times or more, preferably 1.5 times or more, more preferably 2 times or more, compared to differentiated cell culture, More preferably, it is 3 times or more.
炎症性サイトカインまたは治療上有用な生理活性物質の量は、遺伝子レベルでは、例えば、ノーザンブロッティング法、サザンブロッティング法、ＲＮａｓｅプロテクションアッセイ、ＲＴ−ＰＣＲ、リアルタイムＰＣＲ等のＰＣＲ法、in situハイブリダイゼーション法、in vitro転写法等の任意の公知の遺伝子発現解析法により、また、タンパク質レベルでは、免疫沈降法、ウェスタンブロッティング法、ＥＩＡ、ＥＬＩＳＡ、ＲＩＡ、免役組織化学法、免疫細胞化学法等の任意の公知のタンパク質検出法により検出することができる。検体としては、細胞培養物が含浸された培地や、細胞培養物の一部などを用いることができる。 The amount of inflammatory cytokine or therapeutically useful physiologically active substance is determined at the gene level, for example, Northern blotting method, Southern blotting method, RNase protection assay, PCR method such as RT-PCR, real-time PCR, in situ hybridization method, By any known gene expression analysis method such as in vitro transcription method, and at the protein level, any known method such as immunoprecipitation method, Western blotting method, EIA, ELISA, RIA, immunohistochemistry method, immunocytochemistry method, etc. It can be detected by the protein detection method. As the specimen, a medium impregnated with a cell culture, a part of the cell culture, or the like can be used.
以下に、本発明を実施例に基づいてさらに説明するが、かかる実施例は、本発明の例示であり、本発明を限定するものではない。また、下記実施例においては、特に別記しない限り、骨格筋芽細胞はヒト由来のものであり、細胞の培養は３７℃、５％ＣＯ２の条件で行うものとする。 Hereinafter, the present invention will be further described based on examples, but these examples are illustrative of the present invention and do not limit the present invention. In the following examples, unless otherwise specified, skeletal myoblasts are derived from humans, and the cells are cultured under conditions of 37 ° C. and 5% CO 2 .
実施例１ 播種密度／血清被覆処理によるシート状細胞培養物の強度への影響
温度応答性培養皿（２４ウェルプレート、セルシード製）を用意し、一部のウェルには血清を含有する培地（ＤＭＥＭ／Ｆ１２に２０％のヒト血清（Cambrex製または研究採血由来）を含むもの、以下、血清含有培地と記す）を添加し、３７℃、５％ＣＯ２の条件でインキュベートすることにより、血清被覆処理を施した。次に骨格筋芽細胞（Lonza製）を血清含有培地に懸濁し、血清を被覆した培養皿、もしくは無処理の培養皿に３．５×１０５、１．０×１０６、１．４×１０６、１．７×１０６、３．４×１０６個／ｃｍ２の各密度で播種した。２４時間培養後、温度応答性培養皿より細胞培養物を剥離し、シート形成の可否およびその強度について評価した。その結果、無処理の培養皿を用いた場合には、１．０×１０６個／ｃｍ２を超える密度で播種した場合、播種された細胞が完全にシートを形成する以前に剥離が生じ、十分な強度を有するシート状細胞培養物を形成することができなかった。一方、血清を被覆処理した培養皿を使用した場合には自然に剥離することなくシート状細胞培養物を形成することが可能であった。なお、細胞播種密度３．４×１０６個／ｃｍ２ではシート状細胞培養物を得ることができなかった。
Example 1 Effect of Seeding Density / Serum Coating Treatment on Strength of Sheet-like Cell Culture A temperature-responsive culture dish (24-well plate, manufactured by Cellseed) was prepared, and a medium containing serum (DMEM) was prepared in some wells. / F12 containing 20% human serum (manufactured by Cambrex or derived from research blood collection, hereinafter referred to as serum-containing medium), and incubated at 37 ° C. under 5% CO 2 for serum coating treatment Was given. Next, skeletal myoblasts (Lonza) are suspended in a serum-containing medium and placed in a culture dish coated with serum, or an untreated culture dish, 3.5 × 10 5 , 1.0 × 10 6 , 1.4 ×. 10 6, 1.7 × 10 6, were seeded at the density of 3.4 × 10 6 cells / cm 2. After culturing for 24 hours, the cell culture was peeled from the temperature-responsive culture dish and evaluated for the possibility of sheet formation and its strength. As a result, when an untreated culture dish was used, when seeded at a density exceeding 1.0 × 10 6 cells / cm 2 , detachment occurred before the seeded cells completely formed a sheet, A sheet-like cell culture having sufficient strength could not be formed. On the other hand, when a culture dish coated with serum was used, it was possible to form a sheet-shaped cell culture without spontaneous separation. In addition, a sheet-like cell culture could not be obtained at a cell seeding density of 3.4 × 10 6 cells / cm 2 .
さらに、上記と同様に血清被覆処理を施した、または施さない温度応答性培養皿（φ３．５ｃｍ、セルシード製）を用意し、骨格筋芽細胞を血清含有培地に懸濁して３．５×１０５個／ｃｍ２の密度で播種した。２４時間培養後、温度応答性培養皿よりシート状細胞培養物を剥離し、その強度を浸漬撹拌による損傷の程度に基づいて評価したところ、血清を被覆した培養皿を用いた場合に、より強度に優れたシート状細胞培養物を得ることができた。また、剥離したシート状細胞培養物を構成する細胞数について検討したところ、血清を被覆した培養皿を用いた場合には、無処理の培養皿を用いた場合に比べより多くの細胞からシート状細胞培養物が形成されており、細胞の回収率が、無処理培養皿を用いた場合の６９．８％に比べ、９６．３％と顕著に高いことが明らかとなった（表３）。
さらに、形成されたシート状細胞培養物について、播種密度による強度を評価したところ、播種密度を増加させることにより、より強度に優れたシート状細胞培養物を得ることができた。
実施例２ 分化の評価
温度応答性培養皿（２４ウェルプレート、セルシード製）を用意し、一部のウェルには、実施例１と同様に血清の被覆処理を施した。次に骨格筋芽細胞を血清含有培地に懸濁し、血清を被覆したウェル、もしくは無処理のウェルに１．０×１０６、１．４×１０６、１．７×１０６個／ｃｍ２の各密度で播種した。２４時間培養後、形成されたシート状細胞培養物からタンパク質を抽出し、クレアチンキナーゼ（ＣＫ）の活性を測定した。ＣＫは骨格筋の分化指標の１つであり、分化が進むとＣＫの活性が高値を示す。具体的には、まず形成されたシート状細胞培養物に細胞可溶化試薬（CelLyticM、sigma製）と、プロテアーゼ阻害剤（Protease Inhibitor Cocktail、sigma社）とを加え、シェーカーで振とうしながら室温で１５分間インキュベートした。細胞可溶化液を回収し、４℃、１５０００ｇの条件で１５分間遠心し、上清を回収してタンパク質抽出液とし、測定に供した。ＣＫ活性の測定は、EnzyChromTM Creatine Kinase Assay Kit（BioAssay Systems製）を使用し、添付のプロトコールに従って行った。また、抽出液のタンパク質濃度をＢＣＡ法により測定し、タンパク濃度あたりのＣＫ活性値を算出した。測定の結果、血清を被覆した培養皿を用いたシート状細胞培養物では、播種細胞密度を増加させても活性の上昇は認められず、分化の進行はみられなかった（図１）。
Example 2 Evaluation of Differentiation A temperature-responsive culture dish (24-well plate, cell seed) was prepared, and some of the wells were coated with serum in the same manner as in Example 1. Next, skeletal myoblasts are suspended in a serum-containing medium, and 1.0 × 10 6 , 1.4 × 10 6 , 1.7 × 10 6 cells / cm 2 are added to wells coated with serum or untreated wells. Of each density. After culturing for 24 hours, protein was extracted from the formed sheet-shaped cell culture, and the activity of creatine kinase (CK) was measured. CK is one of skeletal muscle differentiation indices, and the activity of CK increases as differentiation progresses. Specifically, a cell solubilizing reagent (CelLyticM, manufactured by sigma) and a protease inhibitor (Protease Inhibitor Cocktail, sigma) are first added to the formed sheet-shaped cell culture, and the mixture is shaken on a shaker at room temperature. Incubated for 15 minutes. The cell lysate was recovered and centrifuged at 4 ° C. and 15000 g for 15 minutes, and the supernatant was recovered and used as a protein extract for measurement. The CK activity was measured using EnzyChrom ™ Creatine Kinase Assay Kit (manufactured by BioAssay Systems) according to the attached protocol. Further, the protein concentration of the extract was measured by the BCA method, and the CK activity value per protein concentration was calculated. As a result of the measurement, in the sheet-like cell culture using a culture dish coated with serum, no increase in activity was observed even when the seeded cell density was increased, and no progression of differentiation was observed (FIG. 1).
実施例３ 液性因子の測定
温度応答性培養皿（φ３．５ｃｍ、セルシード製）に骨格筋芽細胞を３．５×１０５個／ｃｍ２の密度で播種し、４日間培養した後に培養上清を回収した（未分化シート状細胞培養物上清）。また、温度応答性培養皿に播種した骨格筋芽細胞を分化誘導培地（ＤＭＥＭ２００ｍｌに１００μｇ／ｍｌのＩＧＦ−１を１００μｌ、７．５％のＢＳＡ溶液を２．６ｍｌ混合したもの）にて４日間培養することにより多核化細胞（分化した細胞）とした後、さらに４日間培養して多核化細胞の培養上清を回収した（多核化シート状細胞培養物上清）。抗体アレイ（RayBio(R) Human Cytokine Antibody Array G Series 2000、RayBiotech製）を用いて、未分化シート状細胞培養物および多核化シート状細胞培養物の培養上清中に含まれる液性因子の測定を行った。その結果、多核化シート状細胞培養物では骨格筋芽シート状細胞培養物に比べてＩＬ−１α、ＩＬ−４、ＩＬ−５、ＩＬ−１３などの炎症性サイトカインの産生が多いことが明らかとなった（図２）。ＩＬ−１αは炎症反応の誘導、Ｔ細胞やＢ細胞の活性化に関与し、ＩＬ−４、ＩＬ−１３はＢ細胞の活性化や抗体産生を誘導し、ＩＬ−５は主として好酸球の活性化に働くが、いずれもアレルギー反応の誘導に関わりの深いサイトカインである。
Example 3 Measurement of humoral factor Skeletal myoblasts were seeded at a density of 3.5 × 10 5 cells / cm 2 in a temperature-responsive culture dish (φ3.5 cm, manufactured by Cellseed), cultured for 4 days, and then cultured. The supernatant was collected (undifferentiated sheet cell culture supernatant). In addition, skeletal myoblasts seeded in a temperature-responsive culture dish were cultured in a differentiation-inducing medium (100 ml of 100 μg / ml IGF-1 in 2.6 ml of DMEM and 2.6 ml of 7.5% BSA solution) for 4 days. After culturing into multinucleated cells (differentiated cells), the culture supernatant of the multinucleated cells was collected by further culturing for 4 days (multinucleated sheet-like cell culture supernatant). Measurement of humoral factors in the culture supernatant of undifferentiated and multinucleated sheet cell cultures using an antibody array (RayBio (R) Human Cytokine Antibody Array G Series 2000, manufactured by RayBiotech) Went. As a result, it is clear that multinucleated sheet-like cell cultures produce more inflammatory cytokines such as IL-1α, IL-4, IL-5, and IL-13 than skeletal myoblast sheet-like cell cultures. (Fig. 2). IL-1α is involved in the induction of inflammatory responses and activation of T cells and B cells, IL-4 and IL-13 induce B cell activation and antibody production, and IL-5 is mainly an eosinophil. Although they are active, they are cytokines that are closely related to the induction of allergic reactions.
また、幹細胞移植における有効性メカニズムの１つとして考えられている各種液性因子については、未分化シート状細胞培養物において多核化シート状細胞培養物と比較してＰＤＧＦ−ＡＢ、ＰＤＧＦ−ＢＢといった有効性に関わる作用を有する因子の産生が顕著に多いことが明らかとなった（図３）。
したがって、培養皿表面を血清で被覆した場合、細胞の播種数を多くしてもシート状細胞培養物の形成が可能であり、さらに治療上有効に作用する因子の産生が期待される。
Various humoral factors considered as one of the effective mechanisms in stem cell transplantation are PDGF-AB and PDGF-BB in undifferentiated sheet cell cultures compared to multinucleated sheet cell cultures. It was revealed that the production of factors having an effect on the effectiveness was remarkably large (FIG. 3).
Therefore, when the surface of the culture dish is coated with serum, a sheet-like cell culture can be formed even if the number of seeded cells is increased, and production of factors that act therapeutically effectively is expected.
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