JP6523373B2 - Method for producing medical cell sheet - Google Patents
Method for producing medical cell sheet Download PDFInfo
- Publication number
- JP6523373B2 JP6523373B2 JP2017114371A JP2017114371A JP6523373B2 JP 6523373 B2 JP6523373 B2 JP 6523373B2 JP 2017114371 A JP2017114371 A JP 2017114371A JP 2017114371 A JP2017114371 A JP 2017114371A JP 6523373 B2 JP6523373 B2 JP 6523373B2
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- Prior art keywords
- cell
- cells
- cell sheet
- serum
- growth factor
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Description
本発明は、有効量の成長因子を用いることなく細胞シートを製造する方法、およびかかる方法により製造された細胞シート、特に、製造工程由来不純物を実質的に含まない細胞シートに関する。 The present invention relates to a method of producing a cell sheet without using an effective amount of growth factor, and a cell sheet produced by such a method, in particular, a cell sheet substantially free of production process-derived impurities.
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 ischemic heart diseases such as angina pectoris and myocardial infarction, sufficient oxygen can not be distributed to myocardial tissue, and if this condition continues for a long time, myocardial tissue damage occurs. Originally, adult cardiomyocytes have poor self-replication ability, so once damaged, heart muscle can not be repaired, or even if it can be repaired, only limited recovery can be expected and heart failure eventually occurs.
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 treatment method for heart failure, in many cases, a left ventricular assist artificial heart is attached 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 coronary arteriosclerosis at a distant stage, and absolute donor shortage, etc. Also, the current assisted artificial heart is thromboembolic The patient's QOL is extremely limited due to complications such as illness and infection, and the durability of the device, making long-term assistance 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 these circumstances, skeletal myoblast transplantation into myocardial tissue is being studied as a new therapeutic method.
Myoblasts contained in skeletal muscle divide and repair when the muscle is damaged. Myocardium and skeletal muscle have many similarities in structure, function and so on, so it is thought that skeletal muscle-derived myoblasts can also repair damaged myocardium. Overseas, transplantation of autologous skeletal myoblasts 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 suppression of cardiac function depression due to transplantation of skeletal myoblasts is not clear, transplantation of myoblasts into beating cardiac muscle results in orientation of the cell arrangement at the site where mechanical stretch is added. It is believed that proper differentiation is taking place, and also 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、いわゆる「細胞シート」としての提供が渇望された。 However, in the transplantation of myoblast suspension as a single cell to an infarcted heart, damage to the transplanted cells, tissue damage at the time of injection into the recipient heart, tissue supply efficiency to the recipient heart, occurrence of arrhythmia, infarct Problems such as treatment difficulties for the entire site have been pointed out, and in response to these, there has been a demand for providing myoblasts as a sheet, so-called "cell sheet".
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, by growing cells under specific culture conditions, an organization develops more than expected and an artificial tissue having a property of being easily detached from the culture dish is found, and it is found in parts other than adult myocardium. There has been provided a cell sheet as a three-dimensional tissue structure applicable to the heart including cells derived from it, and a method for producing the same (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 solution used in the known production method is considered to be any medium as long as the target cells are proliferated, and even a medium to which a serum such as a known fetal bovine serum is added may be used. It is considered to be 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, equine serum, vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) 2 are used as a cell culture solution for proliferation of cardiac muscle precursor cells etc. (patented) Reference 2).
このように、細胞シートの形成は、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通常ステロイド剤が添加される。ステロイド剤を添加しないと、後述の実験が示すとおり、骨格筋芽細胞は１６時間程度の培養で分化を始めてしまう。骨格筋芽細胞は分化すると管状の筋管を形成してしまい、細胞シート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。
Thus, formation of a cell sheet is usually performed by growing cells, and addition of growth factors is generally performed in addition to heterologous serum components to promote cell growth, while, for example, confluence In the case of skeletal myoblasts and the like which tend to differentiate as they become, a steroid agent is usually added to the culture solution of skeletal myoblasts in order to suppress the transition of proliferated cells to differentiation. Without the addition of steroids, skeletal myoblasts begin to differentiate in about 16 hours of culture, as the experiments below show. When differentiated, skeletal myoblasts form tubular myotubes, making it impossible to obtain a cell sheet.
Furthermore, addition of selenium may be performed to the culture solution for the purpose of antioxidative action.
しかし臨床への適用を考えると、異種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 the clinical application, heterologous serum components may contain pathogens such as viruses that can infect the recipient, and growth factors are usually produced recombinantly using microorganisms. In view of safety, it can not be used for transplantation as it is because it may become an impurity derived from the manufacturing process if it remains.
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。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 antioxidative enzymes for the living body, and is incorporated into proteins as selenocysteine, mainly acting as selenoproteins, and cooperates with vitamins E and C to protect the living body from active oxygen and radicals It is believed that. However, selenium has a very narrow range of appropriate and toxic levels, and symptoms such as nausea, nausea, diarrhea, anorexia, headache, immunosuppression, high density lipoprotein (HDL) reduction, etc. are known as excess disease. There is. On the other hand, as steroids, adrenal cortical dysfunction, Cushing's syndrome and the like are known as side effects thereof, and it is a component which is preferably removed at the time of transplantation as an impurity derived from the production process.
これらの製造工程由来不純物は、通常、細胞シート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、細胞シート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、細胞シート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。 Although these process-related impurities are usually removed by washing the cell sheet with a medium free of these substances, the cell sheet is extremely fragile mechanically and is easily destroyed by the water flow at the time of washing etc. Therefore, it has been extremely difficult to wash the manufacturing process-derived impurities in the cell sheet to a level that does not cause clinical hindrance.
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細胞シート、およびその製造方法の提供を課題とする。 An object of the present invention is to provide a cell sheet which does not contain a production process-derived impurity component which may be an obstacle to clinical application, and a method for producing the same.
培養する細胞の増殖能促進を目的として、これまで述べたとおり細胞培養液には異種血清成分に加え、成長因子が成分として含有される。また、細胞種によってはステロイド剤が添加され、適用する基礎培地によってはセレンを含有するケースもある。
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。
For the purpose of promoting the growth ability of cells to be cultured, as described above, the cell culture solution contains, as a component, a growth factor in addition to the heterologous serum component. Moreover, depending on the cell type, a steroid agent is added, and depending on the basal medium to be applied, it may contain selenium.
However, these components are impurities in the manufacturing process that can not be denied to cause side effects such as anaphylactic shock to recipients in the clinic, and should be excluded in the clinical application.
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、細胞シート製造プロセスにおいて、播種細胞数をコントロールすることで細胞増殖の考慮は不要と考え、一般に細胞増殖に必要な因子として添加する成長因子を実質的に含有しない非細胞増殖系の培養液中で細胞を培養したところ、意外にも細胞シート作製が可能であることを見出した。
さらに研究を続けたところ、培養液を非細胞増殖系とすることにより、細胞シートの作製において、骨格筋芽細胞の分化抑制のため添加するステロイド剤が不要となること、セレンが不要となること、また、異種血清の代わりにレシピエント由来の血清が利用可能であることなどを見出し、本発明を完成させた。
In the cell sheet production process, the present inventors consider that the consideration of cell proliferation is unnecessary by controlling the number of seeded cells in the process of earnestly research in order to solve the above problems, and as a factor necessary for cell proliferation in general. When the cells were cultured in a culture medium of a non-cell growth system substantially free of the growth factor to be added, it was unexpectedly found that cell sheet preparation is possible.
Furthermore, when the research was continued, by making the culture solution into a non-cell growth system, in preparation of a cell sheet, the steroid agent to be added for suppression of differentiation of skeletal myoblasts becomes unnecessary, and selenium becomes unnecessary. Also, the inventors have found that recipient-derived serum can be used instead of heterologous serum, etc. to complete the present invention.
より詳細には、本発明者らは、アミノ酸、ビタミン剤、電解質を主成分とするＭＣＤＢ１３１培地（Invitrogen製）を基礎培地とし、これに異種血清成分ではなくレシピエント由来の血清を想定して、５〜４０％のヒト血清（Cambrex製、または研究採血由来）を含有した細胞培養液を調製し、ヒト骨格筋芽細胞による細胞シート形成の検討を行った。その結果、適度な強度を有する三次元組織構造体としての細胞シートを形成することができ、且つこれが安定した機能性を有することが判明した。
ここで含有するヒト血清は、高い安全性の確保という観点からレシピエント由来の血清を適用し、その濃度は１０〜２０％であることが望ましい。
More specifically, the present inventors use MCDB 131 medium (manufactured by Invitrogen) containing amino acids, vitamins and electrolyte as the main components as a basal medium, and assuming serum from the recipient instead of different serum components. A cell culture solution containing 5 to 40% of human serum (Cambrex or from research blood samples) was prepared, and cell sheet formation by human skeletal myoblasts was examined. As a result, it was found that a cell sheet as a three-dimensional tissue structure having appropriate strength can be formed, and it has stable functionality.
The human serum contained here applies the serum derived from the recipient from the viewpoint of securing high safety, and its concentration is preferably 10 to 20%.
次に本発明者らは、１０〜２０％ヒト血清を含むＭＣＤＢ１３１培地に、上皮成長因子、リン酸デキサメタゾンナトリウムを含有した細胞培養液と、これらの成分を含有しない細胞培養液を用い、一定量以上の細胞を播種し細胞シートを作製したところ、上皮成長因子、リン酸デキサメタゾンナトリウムを含有しない細胞培養液で作製した細胞シートの純度が優位であることを確認した。
これは、細胞培養液を非増殖系とすることで、同じ接着系細胞でありながら骨格筋芽細胞より増殖能が高い目的外細胞である線維芽細胞の増殖を抑える効果も示唆される結果となった。
Next, we use a cell culture solution containing epidermal growth factor, dexamethasone sodium phosphate and a cell culture solution not containing these components in MCDB 131 medium containing 10 to 20% human serum, and a fixed amount When the above cells were seeded to prepare a cell sheet, it was confirmed that the purity of the cell sheet prepared with a cell culture solution containing no epidermal growth factor and dexamethasone sodium phosphate was superior.
It is also suggested that the non-proliferation type of the cell culture solution has the effect of suppressing the proliferation of fibroblasts which are non-target cells which are the same adhesion type cells but have higher proliferation ability than skeletal myoblasts. became.
また、上皮成長因子、リン酸デキサメタゾンナトリウムを含有する細胞培養液において、培養２５時間後に骨格筋芽細胞の分化を示す多核化が認められたのに対し、上皮成長因子、リン酸デキサメタゾンナトリウムを含有しない細胞培養液では多核化は認められなかった。
これらの結果は、先に述べた細胞シートの作製において細胞増殖の考慮は不要とする仮定の裏付けとなる結果であり、そのため細胞分化抑制のためのステロイド剤も不要であることが明らかとなった。
In addition, in the cell culture solution containing epidermal growth factor, dexamethasone sodium phosphate, multinucleation indicating differentiation of skeletal myoblasts was observed after 25 hours of culture, whereas epidermal growth factor, sodium phosphate dexamethasone was contained. No nucleating was observed in the cell culture solution without
These results support the hypothesis that consideration of cell proliferation is not necessary in the preparation of the cell sheet described above, and thus it has become clear that steroid agents for suppressing cell differentiation are also unnecessary. .
ところで、本発明者らが基礎培地として使用したＭＣＤＢ１３１培地には微量であるが亜セレン酸が含まれる。本成分は「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。そのため、ＭＣＤＢ１３１培地と同様にアミノ酸、ビタミン剤、電解質を主成分とし、且つセレン成分を含まないＤＭＥＭ培地（Invitrogen製）を基礎培地とし、前述と同様の検討を実施したところ、得られた細胞シートに差異は認められなかったため、細胞シートの作製にセレンの影響はないものと考えられた。
したがって、成長因子やステロイド剤に加え、細胞シートの作製においてはセレン成分の添加も不要であることが示唆された。
表１にＭＣＤＢ１３１培地とＤＭＥＭ培地の組成を示す。
By the way, in the MCDB 131 medium used by the present inventors as a basal medium, although selenium is contained in a small amount, it is a slight amount. This ingredient is designated as a poison designated item in the "Toxic and Dramatic Substances Designation Order" (January 4, 1981 Cabinet Order No. 2). Therefore, similar to MCDB 131, amino acids, vitamins and electrolytes as the main components, and a DMEM medium (manufactured by Invitrogen) containing no selenium component is used as a basal medium, and the same examination as described above is carried out. There was no difference between them, so it was considered that there was no influence of selenium on the preparation of the cell sheet.
Therefore, in addition to growth factors and steroids, it was suggested that addition of a selenium component is also unnecessary in preparation of a cell sheet.
Table 1 shows the compositions of MCDB 131 medium and DMEM medium.
したがって、本発明は以下の（１）〜（１８）に示されるものである。
（１）実質的に増殖することなく細胞シートを形成し得る密度の細胞を、有効量の成長因子を含まない細胞培養液中で培養することを含む、細胞シートの製造方法。
（２）細胞が、単層の細胞シートを形成する、（１）の製造方法。
（３）培養期間中、細胞が未分化の状態に維持される、（１）または（２）の製造方法。
（４）細胞培養液が、ステロイド剤成分を実質的に含まない、（１）〜（３）の製造方法。
（５）細胞培養液が、異種血清成分を実質的に含まない、（１）〜（４）の製造方法。
（６）細胞培養液が、同種血清成分を含む、（１）〜（５）の製造方法。
（７）同種血清成分が、レシピエント由来である、（６）の製造方法。
（８）細胞培養液が、セレン成分を実質的に含まない、（１）〜（７）の製造方法。
（９）細胞培養液が、アミノ酸およびビタミン剤を成分とする基礎培地を含む、（１）〜（８）の製造方法。
（１０）アミノ酸が、少なくとも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−バリンを含む、（９）の製造方法。
（１１）アミノ酸の濃度が、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、（１０）の製造方法。
（１２）ビタミン剤が、少なくとも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、ピリドキシンを含む、（９）の製造方法。
（１３）ビタミン剤の濃度が、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、（１２）の製造方法。
（１４）製造工程由来不純物を除去する工程を含まない、（１）〜（１３）の製造方法。
（１５）（１）〜（１４）の製造方法で製造された細胞シート。
（１６）疾病、傷病の治療に用いる細胞シートであって、成長因子、ステロイド剤、セレン成分を実質的に含まない細胞シート。
（１７）異種血清成分を含有しない、（１５）または（１６）の細胞シート。
（１８）アミノ酸およびビタミン剤を成分とする基礎培地、および同種血清は含むが、成長因子、ステロイド剤、セレン成分を実質的に含まない、細胞シート作製用培養液。
Accordingly, the present invention is as shown in the following (1) to (18).
(1) A method for producing a cell sheet, comprising culturing cells in a density capable of forming a cell sheet without substantially proliferating in a cell culture medium free of an effective amount of growth factor.
(2) The method according to (1), wherein the cells form a monolayer cell sheet.
(3) The method of (1) or (2), wherein the cells are maintained in an undifferentiated state during the culture period.
(4) The method for producing (1) to (3), wherein the cell culture solution is substantially free of a steroid agent component.
(5) The method for producing (1) to (4), wherein the cell culture solution is substantially free of heterologous serum components.
(6) The method for producing (1) to (5), wherein the cell culture solution contains the homologous serum component.
(7) The method for producing (6), wherein the allogeneic serum component is derived from a recipient.
(8) The method for producing (1) to (7), wherein the cell culture solution is substantially free of the selenium component.
(9) The method for producing (1) to (8), wherein the cell culture medium comprises a basal medium containing an amino acid and a vitamin agent as a component.
(10) The amino acids are at least L-arginine, L-cystine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L -The manufacturing method of (9) which contains threonine, L-tryptophan, L-tyrosine, L-valine.
(11) 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 to 30 mg / L, L-histidine: 42 mg / L, L-isoleucine: 66 to 105 mg / L, L-leucine: 105 to 131 mg / L, L-lysine: 146 to 182 mg / L, L-methionine: 15 to 30 mg / L, L-phenylalanine: 33 to 66 mg / L, L-serine: 32 to 42 mg / L, L-threonine: 12 to 95 mg / L, L-tryptophan: 4.1 to 16 mg / L, L-tyrosine: 18.1 to 1 The manufacturing method of (10) which is 104 mg / L and L-valine: 94-117 mg / L.
(12) The method for producing (9), wherein the vitamin preparation comprises at least calcium D-pantothenate, choline chloride, folic acid, i-inositol, niacinamide, riboflavin, thiamine, pyridoxine.
(13) Vitamin D concentration: 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, Preparation of (12) which is niacinamide: 4 to 6.1 mg / L, riboflavin: 0.0038 to 0.4 mg / L, thiamine: 3.4 to 4 mg / L, pyridoxine: 2.1 to 4 mg / L Method.
(14) The manufacturing method of (1)-(13) which does not include the process of removing the manufacturing process origin impurity.
(15) A cell sheet produced by the method of (1) to (14).
(16) A cell sheet for use in the treatment of a disease or disease, wherein the cell sheet is substantially free of a growth factor, a steroid and a selenium component.
(17) The cell sheet of (15) or (16) which does not contain heterologous serum components.
(18) A culture medium for producing a cell sheet, which comprises a basal medium containing an amino acid and a vitamin agent, and allogeneic serum but substantially free of a growth factor, a steroid agent and a selenium component.
本発明により、三次元組織構造体としての細胞シートの製造にあたって、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、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、骨格筋芽細胞の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り、また、異種血清の代わりにレシピエント由来の血清を用いることが可能となる。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シートを提供することが可能となる。 According to the present invention, since production of cell sheets as three-dimensional tissue structures can generally be carried out with a culture solution of non-cell growth system which does not contain a growth factor to be added as a factor necessary for cell growth, Contamination with endotoxin and the like that may be contained in the growth factor which is a recombinant product can be avoided, and a steroid agent added as a differentiation inhibitor for skeletal myoblasts becomes unnecessary. Furthermore, the present invention eliminates the need for selenium or derivatives thereof for oxidation prevention, and also allows recipient-derived serum to be used instead of heterologous serum. As a result, it is possible to provide a clinically safe cell sheet free of manufacturing process derived impurities.
また、作製した細胞シート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、細胞シートのより確実で安定した製造が可能となる。
さらに、本発明の方法により、所望の大きさ・形状の細胞シートが短期間で製造できるため、細胞シート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, operations such as washing for the purpose of removing impurities from the production process which may cause damage to the produced cell sheet become unnecessary, and more reliable and stable production of the cell sheet becomes possible.
Furthermore, according to the method of the present invention, a cell sheet of desired size and shape can be produced in a short period of time, so that treatment of a living body using the cell sheet can be performed more flexibly and easily.
図１Ａに従来の細胞シート製造フロー、図１Ｂに本発明による細胞シート製造フローの非限定例を示す。
本発明は、実質的に増殖することなく細胞シートを形成し得る密度の細胞を、有効量の成長因子を含まない細胞培養液中で培養することを含む、細胞シートの製造方法に関する。
本発明における細胞には、細胞シート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、単層の細胞シートを形成するもの、例えば、筋芽細胞が好ましい。細胞は、細胞シート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、細胞シートを形成する細胞が２種類以上ある場合、最も多い細胞の比率（純度）は、細胞シート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。
FIG. 1A shows a conventional cell sheet production flow, and FIG. 1B shows a non-limiting example of the cell sheet production flow according to the present invention.
The present invention relates to a method for producing a cell sheet, comprising culturing cells in a density capable of forming a cell sheet substantially without proliferation, in a cell culture solution free of an effective amount of growth factor.
The cells in the present invention include any cells capable of forming a cell sheet. Examples of such cells include, but are not limited to, myoblasts (eg, skeletal myoblasts), cardiomyocytes, fibroblasts, synoviocytes, epithelial cells, endothelial cells and the like. Among these, in the present invention, those which form a monolayer cell sheet, for example, myoblasts are preferred. The cells can be derived from any organism that can be treated by a cell sheet. Such organisms include, but are not limited to, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like. Further, although only one type of cells may be used in the method of the present invention, 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 the cell sheet, the ratio (purity) of the largest number of cells is 65% or more, preferably 70% or more, more preferably It is 75% or more.
本発明において、「実質的に増殖することなく細胞シートを形成し得る密度」とは、成長因子を含まない培養液で培養した場合に、細胞シート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、細胞シートを形成するために、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細胞シートを形成することはできない。したがって、本発明の方法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、細胞シート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、細胞シートは形成するが、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、細胞シート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、細胞シート形成後の細胞数は、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 the present invention, the "density capable of forming a cell sheet without substantially proliferating" means a cell density capable of forming a cell sheet when cultured in a growth factor-free medium. For example, in the case of skeletal myoblasts, in the conventional method using a medium containing a growth factor, cells with a density of about 6,500 cells / cm 2 were seeded on the plate to form a cell sheet ( For example, it is not possible to form a cell sheet by culturing cells of such a density in a culture medium containing no growth factor (see Patent Document 1). Therefore, the cell density in the method of the present invention is higher than in the conventional method using a medium containing a growth factor. Specifically, for example, for skeletal myoblasts, such a density is typically 300,000 cells / cm 2 or more. The upper limit of cell density is, for example, 1,000,000 cells / cm 2 for skeletal myoblasts, although the formation of the cell sheet is not impaired and the cells are not particularly limited unless differentiation takes place. Those 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 do, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start to differentiate when confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a cell sheet but does not go into differentiation. In a preferred embodiment of the invention, the cells do not grow beyond the range of measurement error. 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 sheet. In this embodiment, the number of cells after cell sheet formation is typically 300% or less, preferably 200% or less, more preferably 150% or less, still more preferably 125% or less, particularly preferably the number of cells at the time of seeding. It is 100% or less.
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, the culture of the cells is performed within a predetermined period of time, preferably within a period of time in which the cells do not go into differentiation. Thus, in this embodiment, the cells are maintained in an undifferentiated state during the culture period. The transition to cell differentiation can be assessed by any method known to one skilled in the art. For example, in the case of skeletal myoblasts, expression of MHC or multinucleation of cells can be used as an indicator of differentiation. In a preferred embodiment of the present invention, the culture period is within 48 hours, more preferably within 40 hours, even more preferably within 24 hours.
本発明において、「細胞シート」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、細胞シート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。
本発明の細胞シートは、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、細胞シート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、本発明の細胞シートは、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、本発明の細胞シートは、好ましくは、細胞シート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。
In the present invention, "cell sheet" refers to a sheet in which cells are linked to each other, and typically consists of one cell layer, but is composed of two or more cell layers. Also includes. The cells may be linked to each other directly and / or via an mediator. The mediator is not particularly limited as long as it is a substance capable of at least mechanically connecting the cells, and examples thereof include extracellular matrix. The mediator is preferably of cell origin, in particular of the cells constituting the cell sheet. The cells are at least mechanically linked, but may be further functionally, eg chemically, electrically linked.
The cell sheet 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 their surface and maintain the physical integrity of the cell sheet, eg, made of polyvinylidene difluoride (PVDF) However, the cell sheet of the present invention can maintain its physical integrity without such a scaffold. In addition, the cell sheet of the present invention preferably comprises only the substance derived from cells constituting the cell sheet, and does not contain any other substance.
本発明において、「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。
本発明において、「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 the present invention, "growth factor" means any substance that promotes the proliferation of cells as compared to in the absence thereof, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibers Including blast growth factor (FGF) and the like.
In the present invention, the term "effective amount of growth factor" refers to the amount of growth factor that significantly promotes cell growth as compared to in the absence of growth factor or, for convenience, cell growth in the art. Mean the amount usually added for the purpose. The significance of cell growth promotion can be appropriately evaluated, for example, by any statistical method known in the art, such as t-test, etc., and the usual addition amount can be varied according to various art. It can be known from known literature. Specifically, the effective amount of EGF in the culture of skeletal myoblasts 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、細胞シート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。 Thus, "containing no effective amount of growth factor" means that the concentration of growth factor in the culture medium in the present invention is less than the effective amount. For example, the concentration of EGF in the culture medium in the culture of skeletal myoblasts is preferably less than 0.005 μg / mL, more preferably less than 0.001 μg / mL. In a preferred embodiment of the invention, the concentration of growth factor in the culture medium is less than the normal concentration in the organism. In such an embodiment, for example, the concentration of EGF in the culture medium in the culture of skeletal myoblasts is preferably less than 5.5 ng / mL, more preferably less than 1.3 ng / mL, still more preferably 0.5 ng / mL. It is less than mL. In a further preferred embodiment, the culture medium in the present invention is substantially free of growth factors. Here, the term "substantially free" means that the growth factor content in the culture solution is such that the cell sheet is not adversely affected when applied to a living body, and preferably, the growth factor in the culture solution is positively Means not added to Thus, in this aspect, the culture medium does not contain other components therein, such as growth factors at concentrations above that contained in serum or the like.
本発明に用いる細胞培養液（単に「培養液」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、前記表１にＭＣＤＢ１３１およびＤＭＥＭの組成を示した。基礎培地は、標準的な組成のまま（例えば、市販されたままの状態で）用いてもよいし、細胞種や細胞条件に応じてその組成を適宜変更してもよい。したがって、本発明に用いる基礎培地は、公知の組成のものに限定されず、１または２以上の成分が追加、除去、増量もしくは減量されたものを含む。 The cell culture solution used in the present invention (sometimes referred to simply as "culture solution") is not particularly limited as long as it can maintain the survival of cells, but typically, it mainly contains amino acids, vitamins and electrolytes. The thing which was taken is available. In one aspect of the invention, the culture medium is based on a basal medium for cell culture. Such a basal medium includes, but is not limited to, for example, DMEM, MEM, F12, DME, RPMI 1640, MCDB (MCDBs 102, 104, 107, 131, 153, 199 etc.), L15, SkBM, RITC80-7, etc. . Many of these basal media are commercially available, and their compositions are also known. As an example, the composition of MCDB 131 and DMEM is shown in Table 1 above. The basal medium may be used as a standard composition (for example, as it is marketed), or its composition may be changed as appropriate depending on the cell type and cell conditions. Accordingly, the basal medium used in the present invention is not limited to those of known composition, but includes those in which one or more components are added, removed, increased or decreased.
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。 The amino acids contained in the basal medium include, but are not limited to, for example, 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 as vitamins, and examples thereof include calcium D-pantothenate, choline chloride, folic acid, i -Inositol, niacinamide, riboflavin, thiamine, pyridoxine, biotin, lipoic acid, vitamin B 12 , adenine, thymidine etc. and as electrolytes, 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 ) 6Mo 7 O 24 , NaVO 3 , NiCl 2 , ZnSO 4 and the like are included, respectively. The basal medium may contain, in addition to these components, saccharides such as D-glucose, sodium pyruvate, pH indicators such as phenol red, putrescine, and the like.
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、細胞シート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 aspect of the invention, the cell culture fluid is substantially free of a steroid agent component. Here, the "steroid agent component" refers to a compound having a steroid nucleus that can exert adverse effects such as adrenocortical dysfunction, Cushing's syndrome and the like on a living body. Such compounds include, but are not limited to, eg, 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 not adversely affected when the cell sheet is applied to a living body, and preferably to the culture solution. It means that these compounds are not actively added, that is, the culture solution does not contain the steroid component at a concentration higher than that of other components contained therein, such as serum.
本発明の一態様において、細胞培養液は、異種血清成分を実質的に含まない。ここで「異種血清成分」は、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。したがって、「異種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、細胞シート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、細胞シート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。 In one aspect of the invention, the cell culture fluid is substantially free of heterologous serum components. Here, "heterologous serum component" means a serum component derived from an organism of a species different from that of the recipient. For example, when the recipient is a human, serum derived from bovine or horse, such as fetal bovine serum (FBS, FCS), calf serum (CS), equine serum (HS), etc. corresponds to different serum components. Therefore, "substantially free of heterologous serum components" means that the content of these sera in the culture solution is not adversely affected when the cell sheet is applied to a living body (for example, the serum albumin content in the cell sheet) Is preferably less than 50 ng, which preferably means that these substances are not actively added to the culture solution.
本発明の一態様において、細胞培養液は同種血清成分を含む。ここで「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。同種血清成分が、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であることが好ましい。同種血清成分の含量は、細胞シートの形成を可能とする量であれば特に限定されないが、好ましくは５〜４０％、より好ましくは１０〜２０％である。なお、本明細書中では、特に断らない限り、「％」は、「ｖ／ｖ（容量／容量）％」を意味する。 In one aspect of the invention, the cell culture medium comprises allogeneic serum components. Here, "homologous serum component" means a serum component derived from an organism of the same species as the recipient. For example, when the recipient is a human, human serum corresponds to the homologous serum component. It is preferred that the allogeneic serum component is an autologous serum component, ie a serum component derived from the recipient. The content of the homologous serum component is not particularly limited as long as it enables formation of a cell sheet, but it is preferably 5 to 40%, more preferably 10 to 20%. In the present specification, unless otherwise specified, "%" means "v / v (volume / volume)%".
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、細胞シート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 aspect of the invention, the cell culture medium is substantially free of selenium components. 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 selenium acid and the like. Therefore, the phrase "substantially free of selenium component" means that the content of these substances in the culture solution is not adversely affected when the cell sheet is applied to a living body, and preferably in the culture solution. In other words, the culture solution does not contain the selenium component at a concentration higher than that contained in other components such as serum etc. Specifically, for example, in the case of human, the selenium concentration in the culture solution is the normal value (for example, 10.6 to 17.4 μg / dL) in human serum, and the proportion of human serum contained in the medium is It is lower than the multiplied value (ie, if the content of human serum is 10%, the selenium concentration is, for example, less than 1.0 to 1.7 μg / dL).
本発明の方法は、典型的には、（１）細胞を培養液に播種する工程、（２）細胞を培養して細胞シートを形成させる工程、および（３）細胞シートを剥離する工程を含む。成長因子を含む培養液を用いる従来の方法においては、生体に適用する細胞シートを作製する場合、工程（３）の後に、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必須であった。しかしながら、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）。
The method of the present invention typically comprises the steps of (1) seeding the cells in a culture medium, (2) culturing the cells to form a cell sheet, and (3) detaching the cell sheet . In the conventional method using a culture solution containing a growth factor, when producing a cell sheet to be applied to a living body, after the step (3), manufacturing process-derived impurities such as growth factor, steroid agent component, heterologous serum component, etc. The process of removing by washing etc. was essential. However, one embodiment of the method of the present invention does not include the step of removing this manufacturing process-derived impurity.
Here, the term "manufacturing process derived impurities" typically includes those listed below which are derived from each manufacturing process. That is, those derived from the cell substrate (for example, host cell-derived protein, host cell-derived DNA), those derived from the cell culture solution (for example, inducer, antibiotics, medium components), or steps after cell culture These include those derived from extraction, separation, processing, and purification steps of certain target substances (see, for example, Japanese Patent Laid-Open 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、細胞シートの十分な形成、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シート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シートを短期間で得ることが可能となる。細胞シートの大きさや形状は、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。
細胞シートの剥離法は、細胞シート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剥離できれば特に限定されないが、典型的には、細胞を、温度によって表面の親水性が変化する温度反応性培養皿上で培養し、温度変化により、非酵素的に剥離する。
Culturing of the cells can be performed under conditions that are commonly used in the art. For example, typical culture conditions include culture at 37 ° C., 5% CO 2 . The culture period is preferably within 48 hours, more preferably within 40 hours, still more preferably within 24 hours, from the viewpoint of sufficient formation of the cell sheet and prevention of cell differentiation. Culturing can be performed in vessels of any size and shape. In the method of the present invention, since the cells do not substantially proliferate, the cell sheet of the desired size and shape can be obtained in a short period of time without waiting for the cell sheet to grow to the desired size as in the conventional method. It becomes possible to obtain. The size and shape of the cell sheet can be adjusted by adjusting the size and shape of the cell attachment surface of the culture vessel, or by placing a mold of the desired size and shape on the cell attachment surface of the culture vessel, It can be optionally regulated by culturing cells or the like.
The cell sheet peeling method is not particularly limited as long as the cell sheet can be peeled at least partially from the scaffolding substrate while maintaining the sheet structure, but typically, the cells are subjected to surface hydrophilicity depending on temperature. They are cultured on temperature-reactive culture dishes of varying sex, and non-enzymatically exfoliated by temperature change.
本発明の方法は、細胞の採取から、細胞の増殖および細胞シートの作製を経て、細胞シートの適用に至る、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、
（１）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、
（２）単離した細胞を増殖させる工程、
（３）増殖した細胞を、実質的に増殖することなく細胞シートを形成し得る密度で、有効量の成長因子を含まない細胞培養液中で培養して、細胞シートを形成する工程、
（４）対象への適用のために、細胞シートを剥離する工程、
を含む、再生治療用細胞シートの製造方法にも関する。
The method of the present invention can also be regarded as one step of regenerative therapy, from the collection of cells, through the proliferation of cells and the preparation of cell sheets, to the application of cell sheets. Therefore, the present invention
(1) isolating desired cells from a tissue or biological fluid collected from a subject;
(2) proliferating the isolated cells,
(3) culturing the grown cells in a cell culture medium free of an effective amount of growth factor to form a cell sheet at a density capable of forming a cell sheet substantially without proliferation.
(4) peeling the cell sheet for application to a subject;
The present invention also relates to a method for producing a cell sheet for regenerative therapy, comprising
本発明はまた、上記製造方法によって作製された細胞シート、さらには、成長因子、ステロイド剤、セレン成分を実質的に含まない細胞シートに関する。好ましい態様において、本発明の細胞シートは、異種血清成分を実質的に含まない。この細胞シートは、細胞を、成長因子、ステロイド剤、セレン成分、好ましくはさらに異種血清成分を実質的に含まない培養液で培養して、細胞シートを形成させることにより作製することができる。ここで、細胞シートが成長因子、ステロイド剤、セレン成分、または異種血清成分を実質的に含まないとは、細胞シートが、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、細胞シートの形成を、成長因子、ステロイド剤、セレン成分、好ましくはさらに異種血清成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。
本発明の細胞シートは、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シートは、心疾患、例えば、心筋梗塞、拡張型心筋症などに用いることができる。
The present invention also relates to a cell sheet produced by the above-mentioned production method, and further to a cell sheet substantially free of a growth factor, a steroid agent and a selenium component. In a preferred embodiment, the cell sheet of the invention is substantially free of heterologous serum components. This cell sheet can be prepared by culturing the cells in a culture medium substantially free of growth factors, steroids, selenium components, and preferably, further, different serum components to form cell sheets. Here, the cell sheet is substantially free of growth factors, steroids, selenium components, or different serum components, at least that the cell sheet does not contain these components at a concentration that adversely affects the recipient. Although meaning, such conditions can be satisfied by carrying out the formation of the cell sheet in a culture solution substantially free of growth factors, steroids, selenium components, preferably, further, heterologous serum formation.
The cell sheet of the present invention can be used to treat a subject's disease or injury. For example, the cell sheet by skeletal myoblasts can be used for heart disease such as myocardial infarction, dilated cardiomyopathy and the like.
本発明はまた、アミノ酸およびビタミン剤を成分とする基礎培地、および同種血清は含むが、成長因子、ステロイド剤、セレン成分を実質的に含まない、細胞シート作製用培養液に関する。本発明の培養液に関する各用語の定義は、上記定義のとおりである。 The present invention also relates to a culture medium for producing a cell sheet, which contains an amino acid and a vitamin agent as a component, and allogeneic serum but is substantially free of a growth factor, a steroid agent and a selenium component. The definition of each term regarding the culture solution of the present invention is as defined above.
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。
１．同種血清成分の検討
従来の方法では、ヒト用細胞シート作製にあたり、培養液中に公知のウシ胎仔由来血清などを加えていたが、こうした異種血清成分にはヒト感染性のウイルスなどが含まれる恐れがあるため、安全性に対する危険性が低いヒト血清成分の可能性を検討した。
Hereinafter, the present invention will be further described based on specific examples, but such specific examples are examples of the present invention and are not intended to limit the present invention.
1. Examination of allogeneic serum components In the conventional method, a known fetal bovine serum or the like was added to the culture solution in preparation of the cell sheet for human, but it is feared that such foreign serum components may contain human infectious virus and the like. Therefore, we examined the possibility of human serum components with a low risk for safety.
ヒト血清（Cambrex製または研究採血由来）５％、１０％、２０％、４０％をそれぞれ含有するＭＣＤＢ１３１培地とＤＭＥＭ培地を用意し、各々２ｍＬあたりヒト筋芽細胞を３．０×１０６〜３．１×１０６個ずつ懸濁し、φ３．５ｃｍ温度応答性培養皿（株式会社セルシード製）にそれぞれ播種した。
播種後、３７℃、５％ＣＯ２の条件で培養を行い４０時間後に状態観察を実施した結果、全ての培養細胞において、三次元組織構造体としての細胞シート形成が可能であった。また、シート作製後の細胞回収率は８３〜９８％と、播種細胞数と比べてほぼ同一であり、細胞の増殖は実質的に認められなかった。
Prepare MCDB 131 medium and DMEM medium containing 5%, 10%, 20%, 40% of human serum (Cambrex or research blood source derived) respectively, and 3.0 × 10 6 to 3 human myoblasts per 2 mL. 1 × 10 6 pieces were suspended, and each was seeded on a φ3.5 cm temperature-responsive culture dish (made by Cellseed Co., Ltd.).
After seeding, the cells were cultured under conditions of 37 ° C., 5% CO 2 and subjected to state observation after 40 hours. As a result, in all cultured cells, cell sheet formation as a three-dimensional tissue structure was possible. In addition, the cell recovery rate after sheet preparation was 83 to 98%, which was almost the same as the number of seeded cells, and cell proliferation was substantially not observed.
図２に、ヒト血清を含有する細胞培養液で作製した細胞の外観図を示す。同図より、細胞が敷石状に並んでおり、細胞シートを形成していることが分かる。 FIG. 2 shows an external view of cells produced with a cell culture solution containing human serum. From the figure, it can be seen that the cells are arranged in a cobble-like shape, forming a cell sheet.
次に、ヒト血清を含有する細胞培養液で作製した細胞シートと、公知のウシ胎仔由来血清（Invitrogen製）を含有する細胞培養液で作製した細胞シートとの比較を行い、細胞機能への影響確認を検証した。
比較検証には、細胞生存率と純度を指標とした。
Next, the cell sheet prepared with a cell culture solution containing human serum is compared with the cell sheet prepared with a cell culture solution containing known fetal bovine serum (manufactured by Invitrogen), and the influence on the cell function I verified the confirmation.
Cell viability and purity were used as indicators for comparative verification.
細胞生存率の測定は以下の手順に従った。
形成した細胞シートをトリプシン様蛋白分解酵素（TrypLE Select、Invitrogen製）で解離させた後、同量のTrypan Blue Stain０．４％液（Invitrogen製）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（エルマ製）に注入した。注入後、直ちに倒立型光学顕微鏡（オリンパス製）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。
The measurement of cell viability followed the following procedure.
The formed cell sheet was dissociated with trypsin-like protease (TrypLE Select, Invitrogen), and mixed with the same amount of Trypan Blue Stain 0.4% solution (Invitrogen).
After mixing, 10 μL of the cell suspension was collected before cells were allowed to settle and was injected into a hemocytometer (manufactured by Elma). Immediately after injection, the number of cells observed in the entire 9 mm 2 frame of the two chambers of the hemacytometer was measured with an inverted light microscope (manufactured by Olympus).
After measurement, the average number of viable and dead cells in the two chambers was determined, 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。
形成した細胞シート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。
Measurement of cell purity was performed according to the following procedure.
The formed cell sheet was dissociated with a trypsin-like protease and centrifuged to discard the supernatant.
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, one to which a negative control antibody (manufactured by Becton Dickinson) diluted 10-fold in a PBS solution containing 0.5% BSA was added and mixed was prepared.
After mixing the respective antibodies, they were reacted immediately in a cool dark place for about 1 hour and 0.5% BSA-containing PBS was added to rinse the cells, and 0.5% BSA-containing PBS was added for analysis.
The analysis used the flow cytometer (made by Becton Dickinson), and measured the ratio of the antibody positive cell contained in the cell which mixed each antibody. In the measurement, the positive rate of the negative control was corrected, and the number of cells was analyzed to 5,000 to 10,000.
After analysis, the purity was determined from the difference in the proportion of positive cells in cells mixed with each antibody.
比較検討の結果、５％〜４０％のヒト血清を用いて培養した全ての細胞と、対照とした公知のウシ胎仔由来血清を用いて培養した細胞との間に、細胞生存率と純度の差は認められなかった。表２に一覧表を示す。
２．成長因子、ステロイド剤の要否に関する検討
細胞の増殖に対する成長因子の効果を確認するため、ヒト筋芽細胞３．５×１０４個（２００個／ｃｍ２）を、０〜０．０１μｇ／ｍＬ濃度の上皮成長因子（Invitrogen製）を添加した２０％ウシ胎仔由来血清、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培地に播種し、培養１０日後の細胞数を計測しその増殖能をみた。
2. Examination about the necessity of growth factor and steroid agent In order to confirm the effect of growth factor on cell growth, human myoblasts 3.5 × 10 4 (200 cells / cm 2 ), 0 to 0.01 μg / mL Seed on MCDB131 medium containing 20% fetal bovine serum derived from epidermal growth factor (manufactured by Invitrogen) at a concentration of 4 μg / mL dexamethasone sodium phosphate injection (manufactured by Daiichi Sanko Pharmaceutical Co., Ltd.) The number was counted and the proliferation ability was observed.
その結果、上皮成長因子を含まない培地では明らかに細胞増殖が低かったことから、上皮成長因子が細胞増殖を目的とした培養液には必要な成分であることが判った。表３に結果を示す。
これに対し、２０％ヒト血清を含有するＭＣＤＢ１３１培地と、対照として２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液を含有するＭＣＤＢ１３１培地を用意し、各々２ｍＬあたりヒト筋芽細胞３．０×１０６〜３．１×１０６個ずつ懸濁し、φ３．５ｃｍ温度応答性培養皿にそれぞれ播種した。
播種後、３７℃、５％ＣＯ２の条件で培養を行い４０時間後に状態観察を実施した結果、いずれの細胞培養液においても、三次元組織構造体としての細胞シート形成が可能であった。
この結果から、φ３．５ｃｍ温度応答性培養皿に対し播種細胞数を３．０×１０６個、すなわち約３．０×１０５個／ｃｍ２以上にコントロールすることで、細胞増殖の考慮は不要となることが明らかとなった。
なお、使用する培養皿の有効面積に対する播種細胞数は１つの目安であり、ここに例示した培養皿有効面積と細胞数に限定されない。
On the other hand, prepare MCDB 131 medium containing 20% human serum, and MCDB 131 medium containing 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL dexamethasone sodium phosphate injection as a control. Each 2 mL of human myoblasts 3.0 × 10 6 to 3.1 × 10 6 were suspended, and each was seeded on a φ3.5 cm temperature-responsive culture dish.
After seeding, the cells were cultured under conditions of 37 ° C., 5% CO 2 and subjected to state observation for 40 hours. As a result, in any cell culture solution, it was possible to form a cell sheet as a three-dimensional tissue structure.
From this result, by controlling the number of seeded cells to 3.0 × 10 6 cells, ie, about 3.0 × 10 5 cells / cm 2 or more, in the φ3.5 cm temperature-responsive culture dish, consideration of cell proliferation is It became clear that it became unnecessary.
In addition, the number of seeded cells with respect to the effective area of the culture dish to be used is one standard, and it is not limited to the culture dish effective area and the number of cells illustrated here.
図３Ａに２０％ヒト血清を含有するＭＣＤＢ１３１培地からなる細胞培養液で作製した細胞の外観図を、図３Ｂに２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液を含有するＭＣＤＢ１３１培地からなる細胞培養液で作製した細胞の外観図を示す。
外観上、細胞の形態に差が認められなかったことから、成長因子、ステロイド剤の排除に対する細胞への影響を検証した。検証には、細胞生存率と純度を指標とした。
FIG. 3A shows the appearance of cells prepared with a cell culture solution consisting of MCDB 131 medium containing 20% human serum, and FIG. 3B shows 20% fetal calf serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL phosphoric acid. The external view of the cell produced with the cell culture solution which consists of MCDB131 culture medium containing a dexamethasone sodium injection solution is shown.
From the appearance that no difference was found in the cell morphology, we examined the influence of cells on the exclusion of growth factors and steroids. Cell viability and purity were used as indicators for verification.
細胞生存率の測定は以下の手順に従った。
形成した細胞シート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。
The measurement of cell viability followed the following procedure.
The formed cell sheet was dissociated with a trypsin-like protease, and the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of cell suspension was collected before cells were allowed to sink and was injected into a hemocytometer. Immediately after 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 light microscope.
After measurement, the average number of viable and dead cells in the two chambers was determined, 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。
形成した細胞シート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。
Measurement of cell purity was performed according to the following procedure.
The formed cell sheet was dissociated with a trypsin-like protease and centrifuged to discard the supernatant.
To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, one to which a negative control antibody diluted 10-fold with a 0.5% BSA-containing PBS solution was added and mixed was prepared.
After mixing the respective antibodies, they were reacted immediately in a cool dark place for about 1 hour and 0.5% BSA-containing PBS was added to rinse the cells, and 0.5% BSA-containing PBS was added for analysis.
The analysis used the flow cytometer and measured the ratio of the antibody positive cell contained in the cell which mixed each antibody. In the measurement, the positive rate of the negative control was corrected, and the number of cells was analyzed to 5,000 to 10,000.
After analysis, the purity was determined from the difference in the proportion of positive cells in cells mixed with each antibody.
比較検討の結果、成長因子、ステロイド剤を含有する細胞培養液で作製した細胞シートの純度は６３％であった。これに対し、成長因子、ステロイド剤を含有しない細胞培養液で作製した細胞シートの純度は７５％と優位に高い値を得た。表４に一覧表を示す。
これは、細胞培養液を非増殖系とすることで、同じ接着系細胞でありながら骨格筋芽細胞より増殖能が高い目的外細胞である線維芽細胞の増殖を抑える効果も示唆される結果となった。 It is also suggested that the non-proliferation type of the cell culture solution has the effect of suppressing the proliferation of fibroblasts which are non-target cells which are the same adhesion type cells but have higher proliferation ability than skeletal myoblasts. became.
さらに、細胞シート作製の過程で培養中の細胞性状を観察したところ、上皮成長因子、リン酸デキサメタゾンナトリウムを含有する細胞培養液において、培養２５時間後に骨格筋芽細胞の分化を示す多核化が認められた。そのため、ＭＨＣを標識とした細胞分化状態の確認を行ったところ、培養１６時間後には分化を示す発現が認められた。
これに対し、上皮成長因子、リン酸デキサメタゾンナトリウムを含有しない細胞培養液で作製した細胞シートでは、培養２５時間後においても細胞の多核化は認められなかった。
Furthermore, when the cell properties during culture were observed in the process of preparing the cell sheet, multinucleation indicating differentiation of skeletal myoblasts was observed after 25 hours of culture in a cell culture solution containing epidermal growth factor and dexamethasone sodium phosphate It was done. Therefore, when the cell differentiation state labeled with MHC was confirmed, expression showing differentiation was observed after 16 hours of culture.
On the other hand, in the cell sheet prepared from the cell culture medium not containing epidermal growth factor and dexamethasone sodium phosphate, no multinucleation of cells was observed even after 25 hours of culture.
図４Ａに上皮成長因子、リン酸デキサメタゾンナトリウムを含有する細胞培養液にて作製した培養２５時間後の細胞シート外観性状図を、図４Ｂに前記細胞シートの培養１６時間後のＭＨＣ発現像に関する図を、図４Ｃに上皮成長因子、リン酸デキサメタゾンナトリウムを含有しない細胞培養液にて作製した培養２５時間後の細胞シート外観性状図を示す。 Fig. 4A shows the appearance of the cell sheet after 25 hours of culture prepared with a cell culture solution containing epidermal growth factor and sodium dexamethasone phosphate, and Fig. 4B shows the MHC expression image of the cell sheet after 16 hours of culture. FIG. 4C shows the appearance of a cell sheet after 25 hours of culture prepared using a cell culture solution containing neither epidermal growth factor nor dexamethasone sodium phosphate.
３．セレンの要否に関する検討
２０％ヒト血清を含有するＤＭＥＭ培地（セレン成分を含まない培地）と、対照として２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液を含有するＭＣＤＢ１３１培地を用意し、各々２ｍＬあたりヒト筋芽細胞３．０×１０６個ずつ懸濁し、φ３．５ｃｍ温度応答性培養皿にそれぞれ播種した。
播種後、３７℃、５％ＣＯ２の条件で培養を行い４０時間後に状態観察を実施した結果、いずれの細胞培養液においても、三次元組織構造体としての細胞シート形成が可能であった。
3. Examination about necessity of selenium DMEM medium containing 20% human serum (medium without selenium component) and 20% fetal calf serum as a control, 0.01 μg / mL epidermal growth factor, 4 μg / mL sodium dexamethasone phosphate An MCDB 131 medium containing an injection solution was prepared, suspended to 3.0 × 10 6 human myoblasts per 2 mL, and each was seeded in a φ3.5 cm temperature-responsive culture dish.
After seeding, the cells were cultured under conditions of 37 ° C., 5% CO 2 and subjected to state observation for 40 hours. As a result, in any cell culture solution, it was possible to form a cell sheet as a three-dimensional tissue structure.
図５Ａに２０％ヒト血清を含有するＤＭＥＭ培地からなる細胞培養液で作製した細胞の外観図を、図５Ｂに２０％ウシ胎仔由来血清、０．０１μｇ／ｍＬ上皮成長因子、４μｇ／ｍＬリン酸デキサメタゾンナトリウム注射液を含有するＭＣＤＢ１３１培地からなる細胞培養液で作製した細胞の外観図を示す。 FIG. 5A shows the appearance of cells prepared with a cell culture solution consisting of DMEM medium containing 20% human serum, and FIG. 5B shows 20% fetal bovine serum, 0.01 μg / mL epidermal growth factor, 4 μg / mL phosphoric acid. The external view of the cell produced with the cell culture solution which consists of MCDB131 culture medium containing a dexamethasone sodium injection solution is shown.
外観上、細胞の形成状態に差が認められなかったことから、成長因子、ステロイド剤に加え、亜セレン酸の排除に対する細胞への影響を検証した。検証には、細胞生存率と純度を指標とした。 Since there was no difference in appearance of cells in appearance, in addition to the growth factors and steroids, the influence on cells for the elimination of selenite was examined. Cell viability and purity were used as indicators for verification.
細胞生存率の測定は以下の手順に従った。
形成した細胞シート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。
The measurement of cell viability followed the following procedure.
The formed cell sheet was dissociated with a trypsin-like protease, and the same amount of Trypan Blue Stain 0.4% solution was added and mixed.
After mixing, 10 μL of cell suspension was collected before cells were allowed to sink and was injected into a hemocytometer. Immediately after 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 light microscope.
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。 After measurement, the average number of viable and dead cells in the two chambers was determined, 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。
形成した細胞シート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。
Measurement of cell purity was performed according to the following procedure.
The formed cell sheet was dissociated with a trypsin-like protease and centrifuged to discard the supernatant.
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を添加混和したものを用意した。 To this was added 0.5% BSA-containing PBS solution to rinse the cells, and then anti-human CD56 antibody diluted 10-fold with 0.5% BSA-containing PBS solution was added and mixed. As a control, one to which a negative control antibody diluted 10-fold with a 0.5% BSA-containing PBS solution was added and mixed was prepared.
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。 After mixing the respective antibodies, they were reacted immediately in a cool dark place for about 1 hour and 0.5% BSA-containing PBS was added to rinse the cells, and 0.5% BSA-containing PBS was added for analysis.
解析はフローサイトメーターを用い、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。
The analysis used the flow cytometer and measured the ratio of the antibody positive cell contained in the cell which mixed each antibody. In the measurement, the positive rate of the negative control was corrected, and the number of cells was analyzed to 5,000 to 10,000.
After analysis, the purity was determined from the difference in the proportion of positive cells in cells mixed with each antibody.
比較検討の結果、亜セレン酸の排除に対する細胞への影響は認められなかった。表５に結果を示す。
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