JP5436905B2 - Method for producing sheet cell culture - Google Patents
Method for producing sheet cell culture Download PDFInfo
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- JP5436905B2 JP5436905B2 JP2009077220A JP2009077220A JP5436905B2 JP 5436905 B2 JP5436905 B2 JP 5436905B2 JP 2009077220 A JP2009077220 A JP 2009077220A JP 2009077220 A JP2009077220 A JP 2009077220A JP 5436905 B2 JP5436905 B2 JP 5436905B2
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Description
本発明は、血清で被覆された培養基材上に細胞を播種する工程を含む、シート状細胞培養物を製造する方法、同方法により製造されたシート状細胞培養物、特に、製造工程由来不純物を実質的に含まないシート状細胞培養物、上記方法に用いる血清がコートされた培養基材、前記培養基材の製造方法および製造キット、ならびに、細胞培養物の炎症性サイトカイン産生を抑制する方法に関する。 The present invention relates to a method for producing a sheet-shaped cell culture comprising a step of seeding cells on a culture substrate coated with serum, and a sheet-shaped cell culture produced by the method, particularly impurities derived from the production process. Cell culture material substantially free of serum, culture substrate coated with serum used in the above method, method and kit for producing the culture substrate, and method for suppressing inflammatory cytokine production in cell culture About.
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、この状態が長時間続くと心筋組織に傷害が生じる。元来成体の心筋細胞は自己複製能に乏しいため、一旦傷害を受けると心筋の修復はできないか、例え修復できたとしてもごく限られた回復しか期待できず最終的に心不全に陥ってしまう。 In an ischemic heart disease such as angina pectoris and myocardial infarction, sufficient oxygen does not reach the myocardial tissue, and if this state continues for a long time, the myocardial tissue is damaged. Originally, adult cardiomyocytes are poor in self-replicating ability, so once damaged, the myocardium cannot be repaired, or even if it can be repaired, only limited recovery can be expected and eventually heart failure will occur.
心不全の有効な治療方法として心臓移植があるが、移植までの待機中にブリッジとして左室補助人工心臓を装着するケースが多い。
しかし心臓移植は、免疫抑制治療に伴う感染症の危険性、遠隔期の冠動脈硬化病変の出現、絶対的なドナー不足などが深刻な問題となっており、また現在の補助人工心臓は、血栓塞栓症・感染などの合併症、装置の耐久性の問題から患者ＱＯＬが非常に制限され、長期補助が困難である。
Although heart transplantation is an effective method for treating heart failure, a left ventricular assist device is often used as a bridge while waiting for transplantation.
However, heart transplantation has serious problems such as the risk of infection associated with immunosuppressive treatment, the appearance of distant coronary sclerosis, and the lack of absolute donors. Patient QOL is very limited due to complications such as illness and infection, and device durability, and long-term assistance is difficult.
このような中で新たな治療法として研究が進められているのが、心筋組織への骨格筋芽細胞移植である。
骨格筋に含まれる筋芽細胞は、筋肉が損傷を受けたとき分裂し修復を行う。心筋と骨格筋は構造、機能などに類似する部分が多く、そのため骨格筋由来の筋芽細胞は傷害心筋も修復し得ると考えられている。海外では自己骨格筋芽細胞の心筋への移植が臨床的に応用されつつある。
Under such circumstances, skeletal myoblast transplantation into myocardial tissue is being studied as a new treatment method.
Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. The myocardium and skeletal muscle have many parts that are similar in structure and function, and it is therefore considered that myoblasts derived from skeletal muscle can repair damaged myocardium. Overseas, autologous skeletal myoblast transplantation into the myocardium is being clinically applied.
骨格筋芽細胞の移植による心機能の低下抑制に関するメカニズムは明らかでないが、筋芽細胞を拍動している心筋に移植することで、メカニカル・ストレッチが加わる場で細胞の配列に配向性が生じ適切な分化が行われていることが考えられ、またＶＥＧＦ（血管内皮増殖因子）などのサイトカイン・デリバリー・システムとして機能している可能性も考えられている。 Although the mechanism for suppressing the decline in cardiac function due to transplantation of skeletal myoblasts is not clear, transplantation of myoblasts into beating myocardium causes orientation of the cell arrangement when mechanical stretch is applied Appropriate differentiation is considered to be performed, and it is also considered that it may function as a cytokine delivery system such as VEGF (vascular endothelial growth factor).
しかしながら、梗塞心臓に対するシングル・セルとしての筋芽細胞懸濁液の移植では、移植細胞の障害損失、レシピエント心の注入時の組織障害、レシピエント心への組織供給効率、不整脈の発生、梗塞部位全体への治療困難などの欠点が指摘されており、これらに対応すべく筋芽細胞のシート化が渇望された。 However, transplantation of myoblast suspension as a single cell to the infarcted heart causes loss of transplanted cells, tissue damage during injection of the recipient heart, tissue supply efficiency to the recipient heart, occurrence of arrhythmia, infarct Disadvantages, such as difficulty in treatment of the entire region, have been pointed out, and there has been a craving for making myoblasts into a sheet to deal with these.
これに対し、特定の培養条件によって細胞を増殖させることにより予想以上に組織化が進展し、且つ培養皿から剥離し易いという性質をもった人工組織が見出され、成体の心筋以外の部分に由来する細胞を含む心臓に適用可能な三次元組織構造体としての細胞シートと、その製造方法が提供された（特許文献１参照）。 On the other hand, an artificial tissue with the property that the organization is progressed more than expected by proliferating the cells under specific culture conditions and is easy to peel off from the culture dish, and is found in parts other than the adult myocardium. There has been provided a cell sheet as a three-dimensional tissue structure applicable to the heart including cells derived therefrom, and a production method thereof (see Patent Document 1).
ところで、公知の製造方法で使用される細胞培養液は、目的とする細胞が増殖する限りどのような培地でも良いとされており、公知のウシ胎仔由来血清等の血清が添加されている培地でも良いとされている（例えば、特許文献１参照）。 By the way, the cell culture medium used in the known production method may be any medium as long as the target cells proliferate, and may be a medium supplemented with a serum such as a known bovine fetal serum. It is considered good (see, for example, Patent Document 1).
また、心筋前駆細胞等の増殖に用いる細胞培養液には、ウシ胎仔血清、ウマ血清、血管内皮成長因子（ＶＥＧＦ）、線維芽細胞成長因子（ＦＧＦ）２を用いることが公知されている（特許文献２参照）。 In addition, it is known that fetal bovine serum, horse serum, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) 2 are used as cell culture media used for the proliferation of myocardial progenitor cells (patent) Reference 2).
このように、シート状の細胞培養物の形成は、通常、細胞を増殖させて行っており、細胞の増殖促進のため、異種血清成分以外に成長因子の添加が一般に行われているが、一方で、例えば、コンフルエントになると分化する傾向がある骨格筋芽細胞などでは、増殖した細胞の分化への移行を抑制するため、骨格筋芽細胞の培養液にはステロイド剤を添加することが一般的である。ステロイド剤を添加しないと、骨格筋芽細胞は１６時間程度の培養で分化を始めて、管状の筋管を形成してしまい、シート状の細胞培養物を得ることが不可能となる。
さらに、培養液には、抗酸化作用を目的としたセレンの添加が行われる場合がある。
As described above, the formation of a sheet-shaped cell culture is usually carried out by proliferating cells, and in order to promote cell proliferation, growth factors are generally added in addition to heterogeneous serum components. For example, in skeletal myoblasts that tend to differentiate when confluent, it is common to add a steroid to the culture of skeletal myoblasts in order to suppress the transition of the proliferated cells to differentiation. It is. If a steroid is not added, skeletal myoblasts will begin to differentiate in culture for about 16 hours to form tubular myotubes, making it impossible to obtain a sheet-like cell culture.
Further, selenium may be added to the culture solution for the purpose of antioxidant action.
しかし臨床への適用を考えると、異種血清成分などの他家血清成分にはレシピエントに感染し得るウイルスなどの病原体が含まれる恐れがあり、成長因子も、通常は微生物を利用して組換え的に製造されることを考えると、残存した場合に製造工程由来不純物となり得るため、安全性の観点からそのまま移植に利用することはできない。 However, considering clinical application, other family serum components such as heterologous serum components may contain pathogens such as viruses that can infect recipients, and growth factors are also usually recombined using microorganisms. In view of the fact that it can be produced, it can become an impurity derived from the production process if it remains, so it cannot be used for transplantation as it is from the viewpoint of safety.
また、セレンは生体にとって抗酸化酵素の合成に必要な必須元素であり、セレノシステインとして蛋白質に組み込まれ、主にセレノプロテインとして働き、ビタミンＥやＣと協調して活性酸素やラジカルから生体を防御すると考えられている。しかし、セレンは、適正量と中毒量との幅が非常に狭く、過剰症として悪心、吐き気、下痢、食欲不振、頭痛、免疫抑制、高比重リポ蛋白（ＨＤＬ）減少などの症状が知られている。さらに、亜セレン酸は「毒物及び劇物指定令」（昭和４０年１月４日 政令第２号）で毒物指定品目に指定されている。一方ステロイド剤は、その副作用として副腎皮質機能不全、クッシング症候群などが知られており、いずれも製造工程由来不純物として移植に際して除去することが好ましい成分である。 In addition, selenium is an essential element necessary for the synthesis of antioxidant enzymes for the living body. It is incorporated into proteins as selenocysteine and works mainly as selenoprotein, and protects the living body from active oxygen and radicals in cooperation with vitamins E and C. It is considered to be. However, selenium has a very narrow range of appropriate and toxic doses, and symptoms such as nausea, nausea, diarrhea, loss of appetite, headache, immunosuppression, and high-density lipoprotein (HDL) decrease are known as excess symptoms. Yes. Furthermore, selenite is designated as a poisonous designated item in the “Poisonous and Deleterious Substances Designation Order” (Decree No. 2 of January 4, 1965). On the other hand, steroids are known to have side effects such as adrenal cortex dysfunction and Cushing's syndrome, and all of them are preferable components to be removed at the time of transplantation as impurities derived from the production process.
これらの製造工程由来不純物は、通常、細胞培養物をこれらの物質を含まない媒体で洗浄することによって除去するが、細胞培養物は機械的に極めて脆弱であり、洗浄時の水流などにより容易に破壊されるため、細胞培養物における製造工程由来不純物を臨床上障害とならないレベルまで洗浄することはこれまで極めて困難であった。
さらに、他家血清の代わりにレシピエントへの悪影響の少ない自己血清を用いることもできるが、自己血清は、事前にレシピエントから採血して調製する必要があるなど、レシピエントや医療従事者に身体的・時間的負担を強いるものであり、レシピエントの状態によっては、入手が困難なケースもある。
したがって、臨床的に安全性が高い、良質なシート状の細胞培養物を簡便に得るための方法的改善が求められていた。
These process-derived impurities are usually removed by washing the cell culture with a medium that does not contain these substances, but the cell culture is mechanically extremely fragile and can be easily removed by water flow during washing. Due to the destruction, it has been extremely difficult to wash the impurities from the manufacturing process in the cell culture to a level that does not cause clinical problems.
In addition, autologous serum that has little adverse effect on recipients can be used in place of allogeneic serum, but autologous serum needs to be collected from the recipient in advance and prepared for recipients and healthcare professionals. It imposes a physical and time burden and may be difficult to obtain depending on the recipient's condition.
Therefore, there has been a demand for a method improvement for simply obtaining a high-quality sheet-like cell culture that is clinically safe.
本発明は、臨床への適用に障害となり得る製造工程由来不純物成分を含まない良質な細胞培養物、およびその製造方法の提供を課題とする。 It is an object of the present invention to provide a high-quality cell culture that does not contain impurity components derived from a manufacturing process that can hinder clinical application, and a method for manufacturing the same.
本発明者らは、上記課題解決のために鋭意研究を続けたところ、培養基材を血清で被覆した後、細胞をこの培養基材上で培養すると、血清要求性の細胞種においても無血清培地でシート状の細胞培養物を得ることができることを見出した。さらに、細胞をかかる培養基材上、無血清培地で培養することにより、血清含有培地で培養した細胞培養物と比べて炎症性サイトカインの産生を顕著に低減できることを見出し、本発明を完成させた。 The inventors of the present invention have continued intensive research to solve the above-mentioned problems. After coating the culture substrate with serum and then culturing the cells on the culture substrate, the serum-free cell type is also serum-free. It has been found that a sheet-like cell culture can be obtained with a medium. Furthermore, the inventors have found that by culturing cells in a serum-free medium on such a culture substrate, the production of inflammatory cytokines can be significantly reduced compared to cell cultures cultured in a serum-containing medium, and the present invention has been completed. .
すなわち、本発明は以下に関する。
（１）（ｉ）血清で被覆された培養基材上に細胞を播種する工程、および
（ｉｉ）細胞を培養してシート状の細胞培養物を形成する工程、
を含む、シート状細胞培養物の製造方法。
（２）細胞が、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される、上記（１）の方法。
（３）細胞が、筋芽細胞を含む、上記（１）または（２）の方法。
（４）培養基材が、成長因子によりさらに被覆されている、上記（１）〜（３）のいずれかの方法。
（５）培養基材が、ステロイド剤によりさらに被覆されている、上記（１）〜（４）のいずれかの方法。
（６）血清で被覆された培養基材が、培養基材を血清と共にインキュベートし、その後血清を廃棄することにより得られる、上記（１）〜（５）のいずれかの方法。
（７）血清で被覆された培養基材が、培養基材を血清と共にインキュベートし、その後血清を廃棄し、次いで培養基材を無血清洗浄液で洗浄することにより得られる、上記（１）〜（６）のいずれかの方法。
That is, the present invention relates to the following.
(1) (i) a step of seeding cells on a culture substrate coated with serum, and (ii) a step of culturing the cells to form a sheet-like cell culture,
A method for producing a sheet-shaped cell culture.
(2) The method according to (1) above, wherein the cells are seeded at a density capable of forming a sheet-like cell culture without substantially growing.
(3) The method according to (1) or (2) above, wherein the cell comprises a myoblast.
(4) The method according to any one of (1) to (3) above, wherein the culture substrate is further coated with a growth factor.
(5) The method according to any one of (1) to (4) above, wherein the culture substrate is further coated with a steroid agent.
(6) The method according to any one of (1) to (5) above, wherein the culture substrate coated with serum is obtained by incubating the culture substrate with serum and then discarding the serum.
(7) The above-mentioned (1) to (1), wherein the culture substrate coated with serum is obtained by incubating the culture substrate with serum, then discarding the serum, and then washing the culture substrate with a serum-free washing solution. 6) Any one of the methods.
（８）培養が、無血清培地中で行われる、上記（１）〜（７）のいずれかの方法。
（９）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）〜（８）のいずれかの方法。
（１０）上記（１）〜（９）のいずれかの方法により製造された細胞培養物。
（１１）疾病、傷病の治療に用いる、上記（１０）の細胞培養物。
（１２）製造工程由来不純物を実質的に含まない、上記（１０）または（１１）の細胞培養物。
（１３）上記（１０）〜（１２）のいずれかの細胞培養物を含む移植片。
（１４）血清がコートされた、上記（１）〜（９）のいずれかの方法に用いる培養基材。
（１５）成長因子がさらにコートされた、上記（１４）の培養基材。
（１６）ステロイド剤がさらにコートされた、上記（１４）または（１５）の培養基材。
(8) The method according to any one of (1) to (7) above, wherein the culture is performed in a serum-free medium.
(9) The method according to any one of (1) to (8), further comprising the step of isolating the cell culture from the culture substrate.
(10) A cell culture produced by the method according to any one of (1) to (9) above.
(11) The cell culture according to (10), which is used for treatment of diseases and injuries.
(12) The cell culture according to the above (10) or (11), which is substantially free from impurities from the production process.
(13) A graft containing the cell culture of any one of (10) to (12) above.
(14) A culture substrate used in any one of the above (1) to (9), which is coated with serum.
(15) The culture substrate according to (14), further coated with a growth factor.
(16) The culture substrate according to (14) or (15), further coated with a steroid agent.
（１７）（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（１４）〜（１６）のいずれかの培養基材の製造方法。
（１８）（ｉｉｉ）培養基材を洗浄する工程
をさらに含む、上記（１７）の方法。
（１９）培養基材と、血清とを含む、上記（１４）〜（１６）のいずれかの培養基材の製造キット。
（２０）（ｉ）血清で被覆された培養基材上に細胞を播種する工程、および
（ｉｉ）細胞を無血清培地で培養して細胞培養物を形成する工程、
を含む、細胞培養物の炎症性サイトカイン産生を抑制する方法。
(17) (i) incubating the culture substrate with serum, and (ii) discarding the serum,
The manufacturing method of the culture base material in any one of said (14)-(16) containing.
(18) The method according to (17), further comprising the step of (iii) washing the culture substrate.
(19) The kit for producing a culture substrate according to any one of (14) to (16), comprising a culture substrate and serum.
(20) (i) seeding cells on a culture substrate coated with serum, and (ii) culturing the cells in a serum-free medium to form a cell culture,
A method for suppressing inflammatory cytokine production in a cell culture, comprising:
本発明により、シート状細胞培養物の製造にあたって、一般に細胞増殖に必要な因子として添加する血清や成長因子を含有しない非細胞増殖系の培養液での培養が可能となるため、血清中の有害成分や、通常は組換え品である成長因子に含まれ得るエンドトキシンなどの混入を避けることができるうえ、分化抑制剤として添加するステロイド剤が不要となる。さらに本発明により、酸化防止のためのセレンまたはその誘導体が不要となる。この結果、製造工程由来不純物を含まない、臨床において安全性の高い細胞培養物を提供することが可能となるとともに、異種血清の代替物としての自己血清を調製する必要もなくなる。 According to the present invention, in the production of a sheet-shaped cell culture, it is possible to culture in a serum that is generally added as a factor necessary for cell proliferation or in a non-cell proliferation culture medium that does not contain a growth factor. In addition to avoiding contamination of components and endotoxin that can be contained in growth factors that are usually recombinant products, a steroid agent added as a differentiation inhibitor is not necessary. Further, the present invention eliminates the need for selenium or its derivatives for preventing oxidation. As a result, it is possible to provide a clinically safe cell culture that does not contain impurities from the manufacturing process, and it is not necessary to prepare autologous serum as a substitute for heterologous serum.
また、無血清培地での培養を可能とすることで、血清含有培地で培養した細胞培養物と比べて炎症性サイトカインの産生を顕著に低減でき、細胞培養物の臨床における安全性をさらに高めることが可能となる。
さらに、血清や成長因子などを含まない培地で培養した場合、作製した細胞培養物の損傷が懸念される製造工程由来不純物の除去を目的とした洗浄などの操作が不要となり、細胞培養物のより確実で安定した製造が可能となる。
さらにまた、本発明の方法により、所望の大きさ・形状の細胞培養物が短期間で製造できるため、細胞培養物を利用した生体の処置をより柔軟かつ容易に行うことが可能となる。
In addition, by allowing culture in a serum-free medium, the production of inflammatory cytokines can be significantly reduced compared to cell cultures cultured in serum-containing medium, further enhancing the clinical safety of cell cultures. Is possible.
Furthermore, when cultured in a medium that does not contain serum, growth factors, etc., operations such as washing for the purpose of removing impurities from the manufacturing process that may cause damage to the produced cell culture are no longer necessary. Secure and stable production is possible.
Furthermore, since a cell culture having a desired size and shape can be produced in a short period of time by the method of the present invention, it becomes possible to perform treatment of a living body using the cell culture more flexibly and easily.
本発明は、（ｉ）血清で被覆された培養基材上に細胞を播種する工程、および（ｉｉ）細胞を培養してシート状の細胞培養物を形成する工程、を含む、シート状細胞培養物の製造方法に関する。
本発明における細胞には、細胞培養物、特にシート状の細胞培養物を形成し得る任意の細胞が含まれる。かかる細胞の例としては、限定されずに、筋芽細胞（例えば、骨格筋芽細胞）、心筋細胞、線維芽細胞、滑膜細胞、上皮細胞、内皮細胞などが含まれる。これらのうち、本発明においては、単層の細胞培養物を形成するもの、例えば、筋芽細胞が好ましい。細胞は、細胞培養物による治療が可能な任意の生物に由来し得る。かかる生物には、限定されずに、例えば、ヒト、非ヒト霊長類、イヌ、ネコ、ブタ、ウマ、ヤギ、ヒツジなどが含まれる。また、本発明の方法に用いる細胞は１種類のみであってもよいが、２種類以上の細胞を用いることもできる。本発明の好ましい態様において、細胞培養物を形成する細胞が２種類以上ある場合、最も多い細胞の比率（純度）は、細胞培養物製造終了時において、６５％以上、好ましくは７０％以上、より好ましくは７５％以上である。
The present invention includes (i) a step of seeding cells on a culture substrate coated with serum, and (ii) a step of culturing the cells to form a sheet-like cell culture. The present invention relates to a method for manufacturing a product.
The cell in the present invention includes any cell that can form a cell culture, particularly a sheet-shaped cell culture. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, synovial cells, epithelial cells, endothelial cells, and the like. Among these, in the present invention, those that form a monolayer cell culture, such as myoblasts, are preferred. The cells can be derived from any organism capable of being treated with cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like. Further, only one type of cell may be used in the method of the present invention, but two or more types of cells can also be used. In a preferred embodiment of the present invention, when there are two or more types of cells forming a cell culture, the most cell ratio (purity) is 65% or more, preferably 70% or more at the end of cell culture production. Preferably it is 75% or more.
本発明において、「培養基材」は、細胞がその上で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。 In the present invention, the “culture substrate” is not particularly limited as long as cells can form a cell culture thereon, and examples thereof include containers of various materials, solid or semi-solid surfaces in containers, and the like. including. The container preferably has a structure / material that does not allow permeation of 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し、その後血清を廃棄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 and then discarding the 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.
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。本発明の好ましい態様においては、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）攪拌後、廃棄する手法などを用いることができる。 As a method for discarding serum, a conventional liquid disposal method such as suction with a pipette or decantation can be used. In a preferred 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 a 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 discarding 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、血清と成長因子とステロイド剤、または、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 may be coated with any one of serum, growth factor and steroid agent, any combination of these: serum and growth factor, serum and steroid agent, serum and growth factor and steroid agent, Alternatively, it may be coated with a combination of a growth factor and a steroid. 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.
本発明の好ましい態様において、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、本態様における細胞密度は、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。具体的には、例えば、骨格筋芽細胞については、かかる密度は典型的には３００，０００個／ｃｍ２以上である。細胞密度の上限は、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、例えば、１，０００，０００個／ｃｍ２である。当業者であれば、本発明に適した細胞密度を、実験により適宜決定することができる。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。 In a preferred embodiment of the invention, the cells are seeded at a density that can form a sheet cell culture without substantial growth. “The density at which a sheet-like cell culture can be formed without substantially growing” means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain growth factors. Refers to cell density. 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 cell density in this embodiment is higher than that in the conventional method using a culture solution containing a growth factor. Specifically, for example, for skeletal myoblasts, such density is typically 300,000 cells / cm 2 or more. The upper limit of the cell 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, for example, 1,000,000 cells / cm 2 . A person skilled in the art can appropriately determine the cell density suitable for the present invention by experiments. 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、「シート状の細胞培養物」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, a “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. Includes what is composed. 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の発現や、細胞の多核化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 and cell multinucleation 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.
本発明の好ましい一態様において、細胞培養液は血清成分を実質的に含まない。血清成分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。血清成分としては、異種血清成分および同種血清成分が挙げられる、ここで「異種血清成分」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成分を意味する。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）などが異種血清成分に該当する。また、「同種血清成分」は、レシピエントと同一の種の生物に由来する血清成分を意味する。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成分に該当する。同種血清成分は、自己血清成分、すなわち、レシピエントに由来する血清成分を含む。したがって、「血清成分を実質的に含まない」とは、培養液におけるこれらの血清の含量が、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。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他家血清または非自己血清と総称することもある。 In a preferred embodiment of the present invention, the cell culture medium is substantially free of serum components. A cell culture medium substantially free from serum components may be referred to herein as “serum-free medium”. Serum components include heterogeneous serum components and allogeneic serum components, where “heterologous serum components” refers to serum components derived from an organism of a species different from the recipient when the cell culture is used for transplantation. To do. 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 components. In addition, “same serum component” means a serum component derived from the same species of organism as the recipient. For example, when the recipient is a human, human serum corresponds to the homologous serum component. Allogeneic serum components include autologous serum components, ie, serum components derived from the recipient. Therefore, “substantially free of serum components” means that the content of these sera in the culture solution does not have an adverse effect when the cell culture is applied to a living body (for example, serum albumin in the cell culture). It means that the content is less than 50 ng), preferably that these substances are not actively added to the culture solution. In the present specification, sera other than autoserum, that is, heterologous serum and allogeneic autologous serum may be collectively referred to as autologous serum or non-autologous 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、宿主細胞由来蛋白質、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 process 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).
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できれば特に限定されず、例えば、蛋白分解酵素、例えばトリプシン等による酵素処理および／または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、形成された細胞培養物を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 substrate is not particularly limited as long as the cell culture can be released from the substrate serving as a scaffold while at least partially maintaining the sheet structure, for example, a proteolytic enzyme such as It can be carried out by enzyme treatment with trypsin or the like and / or mechanical treatment such as 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. The formed cell culture 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剥離する工程、
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 on a culture substrate coated with serum;
(Iv) culturing cells to form a sheet-shaped cell culture, and (v) detaching the formed culture from the substrate,
And a method for producing a sheet-shaped cell culture for regenerative treatment.
本発明はまた、上記製造方法によって作製された細胞培養物、さらには、他家血清、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細胞培養物、特にシート状の細胞培養物に関する。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、他家血清、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、細胞培養物が他家血清、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、他家血清、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。 The present invention also provides a cell culture produced by the above production method, and further a cell culture substantially free of allogeneic serum, growth factor, steroid agent and / or selenium component, particularly a sheet-shaped cell culture. About. In a 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 produced by culturing cells in a culture solution substantially free of allogeneic serum, growth factors, steroids and / or selenium components to form a cell culture. Here, the cell culture is substantially free of allogeneic 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 the cell culture can be formed in a culture solution that is substantially free of allogeneic 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−６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、血清含有培地で形成した細胞培養物の場合に比べて、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、遺伝子の転写から蛋白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、蛋白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、血清含有培地で形成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, and examples thereof include, but are not limited to, TNF-α, IL-1, and IL-6. Therefore, “inflammatory cytokines are reduced” means that the abundance or production (secretory amount) of these cytokines is reduced compared to the case of cell cultures formed in a serum-containing medium. Means. Therefore, the cytokine may be present in any form in the process from gene transcription to protein secretion. For example, the cytokine may be present 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, compared to a cell culture formed with a serum-containing medium. More preferably, it is 60% or more, and particularly preferably 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、蛋白質レベルでは、免疫沈降法、ウェスタンブロッティング法、ＥＩＡ、ＥＬＩＳＡ、ＲＩＡ、免疫組織化学法、免疫細胞化学法等の任意の公知の蛋白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、（ｉ）血清で被覆された培養基材上に細胞を播種する工程、および（ｉｉ）細胞を無血清培地で培養して細胞培養物を形成する工程、を含む、細胞培養物の炎症性サイトカイン産生を抑制する方法に関する。
本方法における各工程は、基本的に、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。本方法において、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−６などが挙げられる。したがって、「炎症性サイトカイン産生を抑制する」とは、これらのサイトカインの産生を、血清含有培地で細胞培養物を形成した場合に比べて、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、血清含有培地で細胞培養物を形成した場合に比べて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％以上である。
The present invention also includes (i) seeding cells on a culture substrate coated with serum, and (ii) culturing the cells in a serum-free medium to form a cell culture. The present invention relates to a method for suppressing the production of inflammatory cytokines.
Each step in this method is basically as described above for the method for producing a sheet-shaped cell culture of the present invention. In the present method, the inflammatory cytokine is a general term for cytokines produced with inflammation, and examples thereof include, but are not limited to, TNF-α, IL-1, and IL-6. Therefore, “suppressing the production of inflammatory cytokines” means that the production of these cytokines is reduced as compared to the case where a cell culture is formed in a serum-containing medium. 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%, compared to the case where a cell culture is formed with a serum-containing medium. Above, more preferably 60% or more, particularly preferably 70% or more.
サイトカインの産生量は、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、蛋白質レベルでは、免疫沈降法、ウェスタンブロッティング法、ＥＩＡ、ＥＬＩＳＡ、ＲＩＡ、免疫組織化学法、免疫細胞化学法等の任意の公知の蛋白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。 Cytokine production can be determined at the gene level by any known method such as Northern blotting, Southern blotting, RNase protection assay, PCR methods such as RT-PCR, real-time PCR, in situ hybridization, in vitro transcription, etc. And at the protein level, it can be detected by any known protein detection method such as immunoprecipitation, Western blotting, EIA, ELISA, RIA, immunohistochemistry, immunocytochemistry, etc. it can. 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、本発明を具体例に基づいてさらに説明するが、かかる具体例は、本発明の例示であり、本発明を限定するものではない。 Hereinafter, the present invention will be further described based on specific examples. However, the specific examples are examples of the present invention and do not limit the present invention.
比較例１ 無血清培地の検討
血清成分を含まないＭＣＤＢ１３１培地、ＩＭＤＭ培地（GIBCO製）、ＣＤハイブリドーマ培地（GIBCO製）、乳腺上皮細胞用培地（GIBCO製）または神経幹細胞用培地（GIBCO製）に０．０１μｇ／ｍＬ上皮成長因子（Invitrogen製）、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液（第一三共製薬製）を添加した後、各々２ｍＬにヒト筋芽細胞３．０×１０６〜３．１×１０６個ずつを懸濁し、無処理のφ３．５ｃｍ温度応答性培養皿（セルシード製）にそれぞれ播種した。
播種後、３７℃、５％ＣＯ２の条件で培養を行い１８時間後に状態観察を実施した結果、いずれの培養細胞においても、シート状の細胞培養物の形成は認められなかった。
図１に、無血清培地を用いた細胞培養１８時間後の外観図を示す。この結果から、従来の手法では、シート状細胞培養物の作製に血清成分が不可欠であることが明らかとなった。
Comparative Example 1 Examination of serum-free medium MCDB131 medium without serum components, IMDM medium (GIBCO), CD hybridoma medium (GIBCO), mammary epithelial cell medium (GIBCO) or neural stem cell medium (GIBCO) After adding 0.01 μg / mL epidermal growth factor (manufactured by Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sankyo Pharmaceutical Co., Ltd.), human myoblasts 3.0 × 10 6 -3 in 2 mL each. .1 × 10 6 pieces were suspended and seeded on untreated φ3.5 cm temperature-responsive culture dishes (manufactured by Cellseed).
After seeding, the cells were cultured under conditions of 37 ° C. and 5% CO 2 , and the state was observed 18 hours later. As a result, no sheet-like cell culture was formed in any of the cultured cells.
FIG. 1 shows an external view after 18 hours of cell culture using a serum-free medium. From these results, it has been clarified that serum components are indispensable for the production of sheet-like cell cultures by conventional techniques.
比較例２ 従来の手法によるシート状細胞培養物の作製
２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子（Invitrogen製）、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液（第一三共製薬製）を含有するＭＣＤＢ１３１培地２ｍＬに、ヒト筋芽細胞３．０×１０６〜３．１×１０６個ずつを懸濁し、無処理のφ３．５ｃｍ温度応答性培養皿（セルシード製）にそれぞれ播種した。播種後、３７℃、５％ＣＯ２濃度の条件で培養を行い２４時間後に状態観察を実施した結果、シート状細胞培養物が形成されていた（図２）。
Comparative Example 2 Production of Sheet Cell Culture by Conventional Technique 20% Fetal Calf Serum, 0.01 μg / mL Epidermal Growth Factor (Invitrogen), 4 μg / mL Dexamethasone Sodium Phosphate Injection (Daiichi Sankyo Pharmaceutical) ) Suspension of 3.0 × 10 6 to 3.1 × 10 6 human myoblasts in 2 mL of MCDB131 medium containing 2) and seeded in untreated φ3.5 cm temperature-responsive culture dishes (manufactured by Cellseed). did. After seeding, the cells were cultured under conditions of 37 ° C. and 5% CO 2 concentration, and the state was observed after 24 hours. As a result, a sheet-like cell culture was formed (FIG. 2).
実施例１ 無血清培地を用いたシート状細胞培養物の作製
２０％ウシ胎仔由来血清（Invitrogen製）を含有するＭＣＤＢ１３１培地（Invitrogen製）を、２４ウェル温度応答性培養皿（セルシード製）に４００μＬ添加し、３７℃、５％ＣＯ２濃度の条件でインキュベーションを行い、７時間後にピペッティングにより除去した。
血清除去後の培養皿にHanks’ Balanced Salt Solution（Invitrogen製）を１ｍＬ添加し、２０秒間ゆっくりと撹拌洗浄を行った後、これをピペッティングにより廃棄した。これを２回繰り返し行った。
洗浄後、血清、成長因子、ステロイド剤、セレンなどの因子を一切添加していないＦ１２/ＤＭＥＭ培地（Invitrogen製）４００μＬを培養皿に加え、３．０×１０６個の骨格筋芽細胞（ヒト由来）を播種した。
播種後、３７℃、５％ＣＯ２濃度の条件で培養を行い２４時間後に状態観察を実施した結果、シート状細胞培養物が形成されていた（図３）。形成されたシート状細胞培養物は、血清成分を含有した培地を用いた既知の方法によって作製したシート状細胞培養物（比較例２、図２）と比較し、目視観察において何ら遜色は認められないものだった。
Example 1 Production of Sheet Cell Culture Using Serum-free Medium MCDB131 medium (Invitrogen) containing 20% fetal bovine serum (Invitrogen) was transferred to a 24-well temperature-responsive culture dish (Cellseed) at 400 μL. The mixture was added, incubated at 37 ° C. and 5% CO 2 , and removed by pipetting after 7 hours.
1 mL of Hanks' Balanced Salt Solution (manufactured by Invitrogen) was added to the culture dish after removing the serum, and after gently washing with stirring for 20 seconds, this was discarded by pipetting. This was repeated twice.
After washing, 400 μL of F12 / DMEM medium (manufactured by Invitrogen) to which no factors such as serum, growth factors, steroids, and selenium have been added is added to the culture dish, and 3.0 × 10 6 skeletal myoblasts (human Origin).
After seeding, the cells were cultured under conditions of 37 ° C. and 5% CO 2 concentration, and the state was observed after 24 hours. As a result, a sheet-like cell culture was formed (FIG. 3). The formed sheet-shaped cell culture is visually inferior to the sheet-shaped cell culture (Comparative Example 2, FIG. 2) prepared by a known method using a medium containing a serum component. It was not.
次に、作製したシート状細胞培養物の細胞生存率および骨格筋芽細胞純度を比較した。
細胞生存率の測定は以下の手順に従った。
形成したシート状細胞培養物をトリプシン様蛋白分解酵素で解離させた後、同量のTrypan Blue Stain０．４％液を加え混和した。
混和後、細胞浮遊液を細胞が沈まないうちに１０μＬずつ採取し、血球計算盤に注入した。注入後、直ちに倒立型光学顕微鏡にて、血球計算盤の２つのチャンバーの９ｍｍ２枠全体に観察される細胞数の計測を行った。
計測後、２つのチャンバーの生死細胞数の平均を求め、染色された細胞を含む全細胞数に対する無染色細胞の割合を算出した。
Next, the cell viability and skeletal myoblast purity of the prepared sheet-shaped cell cultures were compared.
The cell viability was measured according to the following procedure.
The formed sheet-like cell culture was dissociated with trypsin-like proteolytic enzyme, and the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of the cell suspension was collected before the cells settled and injected into a hemocytometer. Immediately after the injection, the number of cells observed in the entire 9 mm 2 frame of the two chambers of the hemocytometer was measured with an inverted optical microscope.
After the measurement, the average number of viable and dead cells in the two chambers was obtained, and the ratio of unstained cells to the total number of cells including stained cells was calculated.
細胞純度の測定は以下の手順に従った。
まず、形成したシート状細胞培養物をトリプシン様蛋白分解酵素で解離させた後、遠心処理を行い上清を廃棄した。
これに０．５％ＢＳＡ含ＰＢＳ液を加え細胞をリンスした後、０．５％ＢＳＡ含ＰＢＳ液で１０倍希釈した抗ヒトＣＤ５６抗体（ベクトン・ディッキンソン製）を添加し混和した。対照として０．５％ＢＳＡ含ＰＢＳ液で１０倍希釈した陰性コントロール用抗体（ベクトン・ディッキンソン製）を添加混和したものを用意した。
各抗体を混和した後、直ちに冷暗所で約１時間反応させ０．５％ＢＳＡ含ＰＢＳ液を加え細胞をリンスした後、０．５％ＢＳＡ含ＰＢＳ液を加え解析に供した。
解析はフローサイトメーター（ベクトン・ディッキンソン製）を用い、各抗体を混和した細胞に含まれる抗体陽性細胞の割合を計測した。計測にあたっては、陰性コントロールの陽性率の補正を行い、細胞数５，０００〜１０，０００個を解析した。
解析後、各抗体を混和した細胞の陽性細胞率の割合の差から純度を求めた。
表２の結果が示すとおり、実施例１と比較例２のシート状細胞培養物の細胞生存率および骨格筋芽細胞純度に違いは見られなかった。
Cell purity was measured according to the following procedure.
First, the formed sheet-shaped cell culture was dissociated with trypsin-like proteolytic enzyme, centrifuged, and the supernatant was discarded.
To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody (Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed.
After each antibody was mixed, it was immediately reacted in a cool dark place for about 1 hour, and a 0.5% BSA-containing PBS solution was added to rinse the cells. Then, a 0.5% BSA-containing PBS solution was added for analysis.
For analysis, a flow cytometer (manufactured by Becton Dickinson) was used to measure the ratio of antibody-positive cells contained in the cells mixed with each antibody. In the measurement, the positive rate of the negative control was corrected, and 5,000 to 10,000 cells were analyzed.
After the analysis, the purity was determined from the difference in the ratio of the positive cell ratio of the cells mixed with each antibody.
As the results in Table 2 show, there was no difference in cell viability and skeletal myoblast purity between the sheet-like cell cultures of Example 1 and Comparative Example 2.
実施例２ 無血清培地で作製したシート状細胞培養物の安全性に関する検討
無血清培地を用いた効果を検証するため、以下の比較試験を実施した。
まず、２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子（Invitrogen製）、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液（第一三共製薬製）を含有するＭＣＤＢ１３１培地を、２４ウェル温度応答性培養皿に４００μＬ添加し、３７℃、５％ＣＯ２濃度の条件でインキュベーションを行い、７時間後にピペッティングにより除去した。これを６枚用意した。
このうち３枚は、既知の方法として、前述と同様の組成からなる培地を４００μＬ加え、６．７×１０５個の骨格筋芽細胞を播種した。播種後、３７℃、５％ＣＯ２濃度の条件で培養を行い２４時間後に形成されたシート状細胞培養物が含浸された前記の培地を２００μＬ採取し、これを検体１とした。
Example 2 Examination on safety of sheet-like cell culture prepared in serum-free medium In order to verify the effect of using serum-free medium, the following comparative test was performed.
First, MCDB131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor (manufactured by Invitrogen), 4 μg / mL dexamethasone sodium phosphate injection (Daiichi Sankyo Pharmaceutical Co., Ltd.) was subjected to a 24-well temperature response. 400 μL was added to the sex culture dish, incubated at 37 ° C. and 5% CO 2 concentration, and removed by pipetting after 7 hours. Six of these were prepared.
Three of them were, as a known method, added 400 μL of a medium having the same composition as described above, and seeded with 6.7 × 10 5 skeletal myoblasts. After seeding, the cells were cultured under the conditions of 37 ° C. and 5% CO 2 concentration, and 200 μL of the medium impregnated with the sheet-like cell culture formed 24 hours later was collected.
残りの３枚には、本発明の方法として、前述のインキュベーションを経た温度応答性培養皿に、Hanks' Balanced Salt Solution液を１ｍＬ添加し、２０秒間ゆっくりと撹拌洗浄した後、ピペッティングによりこれを廃棄した。この撹拌洗浄を２回繰り返し行った。洗浄後、ウシ胎仔由来血清、上皮成長因子、リン酸デキサメタゾンナトリウムを一切含有しないＤＭＥＭ／Ｆ１２培地４００μＬを加え、６．７×１０５個の骨格筋芽細胞を播種した。播種後、３７℃、５％ＣＯ２濃度の条件で培養を行い２４時間後に形成されたシート状細胞培養物が含浸された前記の培地を２００μＬ採取し、これを検体２とした。 For the remaining 3 sheets, as a method of the present invention, 1 mL of Hanks' Balanced Salt Solution solution was added to the temperature-responsive culture dish that had been incubated as described above, and after gently stirring and washing for 20 seconds, this was pipetted. Discarded. This stirring and washing was repeated twice. After washing, 400 μL of DMEM / F12 medium containing no fetal bovine serum, epidermal growth factor, or dexamethasone sodium phosphate was added, and 6.7 × 10 5 skeletal myoblasts were seeded. After seeding, the cells were cultured under the conditions of 37 ° C. and 5% CO 2 concentration, and 200 μL of the medium impregnated with the sheet-like cell culture formed 24 hours later was collected.
各検体に対し、炎症性サイトカインとして知られるインターロイキン−６（ＩＬ−６）を指標として、細胞培養過程で産生される同サイトカインの濃度を測定した。インターロイキン−６濃度の測定は、ＥＬＩＳＡ（Enzyme-Linked Immunosorbent Assay）法とし、測定キットにはHuman IL-6 Immunoassay（R&D Systems製）を用いた。このキットでの測定方法は以下の通りである。
あらかじめＲＤ１Ｗ液を１００μＬずつ添加したＥＬＩＳＡ用プレートの各ウェルに、各検体をそれぞれ１００μＬずつアプライし、２時間反応させた。ウェルを４回洗浄した後、２００μＬのConjugateを入れ、２時間反応させた。ウェルを４回洗浄した後、Substrate Solution２００μＬを加え発色させ、３０分後に発色停止液５０μＬを加えた後、４５０ｎｍの吸光度を測定し（対照波長は５４０ｎｍ）、検量線より各検体のインターロイキン−６濃度を算出した。
表３の結果が示すとおり、検体２について算出されたインターロイキン−６濃度は検体１に比べ明らかに低かった。この結果は、本発明による無血清培地を用いたシート状細胞培養物の作製方法が、細胞へ与える刺激が低いことを示すものである。したがって、本発明の方法により、炎症発症の危険が極めて低い、臨床において高い安全性を有するシート状細胞培養物を提供できることが明らかとなった。
For each specimen, the concentration of the cytokine produced in the cell culture process was measured using interleukin-6 (IL-6), which is known as an inflammatory cytokine, as an index. Interleukin-6 concentration was measured by ELISA (Enzyme-Linked Immunosorbent Assay) method, and Human IL-6 Immunoassay (manufactured by R & D Systems) was used as a measurement kit. The measurement method using this kit is as follows.
100 μL of each sample was applied to each well of an ELISA plate to which 100 μL of RD1W solution had been added in advance, and reacted for 2 hours. After the well was washed 4 times, 200 μL of Conjugate was added and allowed to react for 2 hours. After the wells were washed 4 times, 200 μL of Substrate Solution was added to cause color development. After 30 minutes, 50 μL of color stop solution was added, and the absorbance at 450 nm was measured (control wavelength was 540 nm). Concentration was calculated.
As the results in Table 3 show, the interleukin-6 concentration calculated for Sample 2 was clearly lower than Sample 1. This result shows that the method for producing a sheet-shaped cell culture using a serum-free medium according to the present invention has a low stimulation to cells. Therefore, it has been clarified that the method of the present invention can provide a sheet-like cell culture having a very low risk of inflammation and having high safety in clinical practice.
Claims (19)
（ｉｉ）細胞を増殖させずに培養してシート状の細胞培養物を形成する工程、
を含む、シート状細胞培養物の製造方法。 (I) a step of seeding cells on a culture substrate coated with serum at a density capable of forming a sheet-like cell culture without substantially growing ; and (ii) culturing the cells without growing. Forming a sheet-like cell culture,
A method for producing a sheet-shaped cell culture.
（ｉｉ）血清を廃棄する工程、
を含む、請求項１４〜１６のいずれかに記載の培養基材の製造方法。 (I) incubating the culture substrate with serum; and (ii) discarding the serum;
The manufacturing method of the culture base material in any one of Claims 14-16 containing this.
をさらに含む、請求項１７に記載の方法。 The method according to claim 17, further comprising the step of (iii) washing the culture substrate.
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