JP6510163B2 - Method for producing sheet-like cell culture - Google Patents
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Description
本発明は、簡便にシート状の細胞培養物を製造する方法に関する。 The present invention relates to a method for conveniently producing sheet-like cell cultures.
近年の心臓病に対する治療の革新的進歩にかかわらず、重症心不全に対する治療体系は未だ確立されていない。心不全の治療法としては、βブロッカーやＡＣＥ阻害剤による内科治療が行われるが、これらの治療が奏功しないほど重症化した心不全には、補助人工心臓や心臓移植などの置換型治療、つまり外科治療が行われる。 Despite recent advances in the treatment of heart disease, a treatment system for severe heart failure has not yet been established. Treatment of heart failure includes medical treatment with β-blockers and ACE inhibitors, but for heart failure that has become so severe that these treatments are not effective, replacement-type treatments such as assisted artificial heart and heart transplantation, that is, surgical treatment Is done.
このような外科治療の対象となる重症心不全には、進行した弁膜症や高度の心筋虚血に起因するもの、急性心筋梗塞やその合併症、急性心筋炎、虚血性心筋症（ＩＣＭ）、拡張型心筋症（ＤＣＭ）などによる慢性心不全やその急性憎悪など、多種多様の原因がある。
これらの原因と重症度に応じて弁形成術や置換術、冠動脈バイパス術、左室形成術、機械的補助循環などが適用される。
The severe heart failure targeted for such surgical treatment includes those caused by advanced valvular disease and severe myocardial ischemia, acute myocardial infarction and its complications, acute myocarditis, ischemic cardiomyopathy (ICM), and dilation. There are a wide variety of causes such as chronic heart failure due to cardiomyopathy (DCM) etc. and its acute exacerbation.
Valvuloplasty, replacement, coronary artery bypass grafting, left ventricular angioplasty, mechanical assisted circulation, etc. are applied according to the cause and severity of these.
この中で、ＩＣＭやＤＣＭによる高度の左室機能低下から心不全を来たしたものについては、心臓移植や人工心臓による置換型治療のみが有効な治療法とされてきた。しかしながら、これら重症心不全患者に対する置換型治療は、慢性的なドナー不足、継続的な免疫抑制の必要性、合併症の発症など解決すべき問題が多く、すべての重症心不全に対する普遍的な治療法とは言い難い。 Among them, for those with heart failure due to severe left ventricular dysfunction due to ICM and DCM, only heart transplantation and replacement treatment with an artificial heart have been considered as effective treatments. However, replacement therapy for these patients with severe heart failure has many problems to be solved such as chronic donor shortage, the need for continuous immunosuppression, onset of complications, and universal treatment for all severe heart failure Is hard to say.
その一方、最近、重症心不全治療の解決策として新しい再生医療の展開が不可欠と考えられている。
重症心筋梗塞等においては、心筋細胞が機能不全に陥り、さらに線維芽細胞の増殖、間質の線維化が進行し心不全を呈するようになる。心不全の進行に伴い、心筋細胞は傷害されてアポトーシスに陥るが、心筋細胞は殆ど細胞分裂をおこさないため、心筋細胞数は減少し心機能の低下もさらに進む。
このような重症心不全患者に対する心機能回復には細胞移植法が有用とされ、既に自己骨格筋芽細胞による臨床応用が開始されている。
On the other hand, recently, development of new regenerative medicine is considered indispensable as a solution for treatment of severe heart failure.
In severe myocardial infarction and the like, cardiomyocytes become dysfunctional, and further, proliferation of fibroblasts and fibrosis of stroma progress to exhibit heart failure. With the progress of heart failure, cardiomyocytes are damaged and fall into apoptosis, but since cardiomyocytes cause almost no cell division, the number of cardiomyocytes decreases and cardiac function declines further.
Cell transplantation is considered useful for recovery of cardiac function in patients with such severe heart failure, and clinical application using autologous skeletal myoblasts has already been started.
近年、その一例として、組織工学を応用した温度応答性培養皿を用いることによって、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に構成された細胞培養物と、その製造方法が提供された（特許文献１）。 In recent years, a three-dimensional cell culture applicable to the heart including cells derived from parts other than adult myocardium by using a temperature-responsive culture dish to which tissue engineering is applied as an example thereof, and its cell culture A manufacturing method has been provided (Patent Document 1).
特許文献２には、細胞培養面に細胞が潜入できない寸法の凹部を有する凹凸構造を形成することにより、損傷が少なく、シートの剥離が容易であり、シート形成速度も遅くならない細胞培養支持体が記載されている。特許文献３には、細胞接着性領域と細胞接着阻害性領域を有する細胞培養基板が開示され、該培養基板を用いて培養すると、細胞接着性領域のみに細胞が接着することにより、細胞のパターニングを行うことができることが記載されている。 In Patent Document 2, a cell culture support is formed by forming a concavo-convex structure having a recess with a size that can not infiltrate cells in a cell culture surface, thereby reducing damage, facilitating sheet peeling, and not slowing the sheet formation rate. Have been described. Patent Document 3 discloses a cell culture substrate having a cell adhesive region and a cell adhesion inhibitory region, and when cultured using the culture substrate, the cells adhere to only the cell adhesive region, thereby patterning the cells. It is stated that it can do.
良質で安全性の高いシート状細胞培養物を製造する方法においては、シートの物理的強度やシート形成の速度は重要な要素であるが、剥離の容易さなどもまた重要な要素である。特に脆弱なシート状細胞培養物の製造においては、剥離の工程においてシート状細胞培養物の損傷が生じる場合が少なからず存在する。したがって培養したシート状細胞培養物を容易に剥離できる培養基材または容易に剥離可能とする方法が求められている。本発明の目的は、良質で安全性の高いシート状細胞培養物を、迅速かつ容易に製造する方法を提供することにある。 In the method of producing a high quality and highly safe sheet-like cell culture, the physical strength of the sheet and the speed of sheet formation are important factors, but the ease of peeling is also an important factor. In the production of particularly fragile sheet-like cell cultures, damage to the sheet-like cell cultures often occurs in the step of exfoliation. Therefore, there is a need for a culture substrate capable of easily exfoliating a cultured sheet-like cell culture or a method of facilitating exfoliation. An object of the present invention is to provide a method for rapidly and easily producing a high-quality, high-safety sheet-like cell culture.
本発明者らは、非接着性の表面にある程度の間隔で血清をプロットして接着性領域を形成したディッシュにおいてシート化培養を行うと、細胞非接着性の領域が存在しても、細胞がパターニングされずに均一な一枚のシートが形成され、さらに該シートは容易に剥離可能であることを見出した。かかる知見に基づき、さらに鋭意研究を重ねた結果、本発明を完成させるに至った。 The present inventors plot the serum at a certain distance on the non-adhesive surface and perform the sheeting culture in the dish in which the adhesive area is formed, and even if the cell non-adhesive area exists, the cells It has been found that a uniform single sheet is formed without being patterned, and the sheet can be easily peeled off. As a result of further intensive research based on such findings, the present invention has been completed.
すなわち本発明は、以下に関する。
［１］シート状細胞培養物の製造方法であって、細胞非接着性領域と細胞接着性領域とがパターン形状を形成する、略平面状の表面を有する培養基材上に、細胞を播種し、インキュベートすることを含む、前記方法。
［２］さらに、培養基材上で形成されたシート状細胞培養物を該基材から剥離することを含む、［１］の方法。
［３］１つの細胞接着領域と、該細胞接着領域に隣接する他の細胞接着領域との間の少なくとも一部における間隔が、播種される細胞の長径よりも大きい、［１］または［２］の方法。
［４］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、［１］〜［３］の方法。
［５］細胞が、骨格筋芽細胞である、［１］〜［４］の方法。
［６］パターン形状が、細胞非接着領域または細胞接着領域のいずれか一方を島とし、他方を海とする海島構造である、［１］〜［５］の方法。
［７］細胞接着性領域が島であり、細胞非接着性領域が海である、［６］の方法。
［８］細胞接着性領域の割合が、表面全体の４％〜６５％である、［１］〜［７］の方法。
That is, the present invention relates to the following.
[1] A method for producing a sheet-like cell culture, which comprises seeding cells on a culture substrate having a substantially planar surface, in which a cell non-adhesive region and a cell adhesive region form a pattern shape. , Said method comprising incubating.
[2] The method of [1], further comprising peeling the sheet-like cell culture formed on the culture substrate from the substrate.
[3] [1] or [2], wherein the spacing in at least a part between one cell adhesion area and another cell adhesion area adjacent to the cell adhesion area is larger than the major axis of the seeded cells, [1] or [2] the method of.
[4] The method of [1]-[3], wherein the cells are seeded at a density capable of forming a sheet-like cell culture without substantially proliferating.
[5] The method of [1] to [4], wherein the cell is a skeletal myoblast.
[6] The method according to [1] to [5], wherein the pattern shape is a sea-island structure in which either one of a cell non-adhesion area or a cell adhesion area is an island and the other is an ocean.
[7] The method of [6], wherein the cell adhesive area is an island and the cell non-adhesive area is a sea.
[8] The method of [1] to [7], wherein the proportion of the cell adhesive area is 4% to 65% of the entire surface.
本発明により、浸漬撹拌などの洗浄操作や、取出し、保持、移送などの移植操作に耐え得る十分な物理的強度を有するシート状細胞培養物が得られるため、従来の製法で得られた細胞シートよりもより容易かつ確実に製造・移植操作を行うことが可能となる。また、本発明の製造方法により、高い細胞回収率でシート状細胞培養物を得ることができるため、使用する細胞の有効利用が可能となり、コストの削減などに寄与する。さらに、本発明の製造方法により得られたシート状細胞培養物は、血管新生等の有効性に関わる作用を有する因子の産生が多い一方で、炎症性サイトカインの産生が低いため、治療上の有効性も、レシピエントに対する安全性も極めて高く、臨床的に極めて有用である。このため、ヒト医療および獣医療において多大な貢献が期待できる。さらにまた、本発明の製造方法によれば、シート形成までに必要な時間が短く、剥離も容易に行えるため、製造工程におけるシートの破損のリスクを低減し、良質なシート状細胞培養物を大量に製造するのに好適である。 According to the present invention, a sheet-like cell culture having sufficient physical strength to withstand washing operations such as immersion and stirring, and transplantation operations such as removal, retention, and transfer can be obtained. Thus, the cell sheet obtained by the conventional production method It becomes possible to carry out the manufacturing / transplanting operation more easily and reliably. In addition, since the sheet-like cell culture can be obtained at a high cell recovery rate by the production method of the present invention, effective use of cells to be used becomes possible, which contributes to cost reduction and the like. Furthermore, the sheet-like cell culture obtained by the production method of the present invention has a high production of factors having an action related to the efficacy such as angiogenesis, and a low production of inflammatory cytokines, so that it is therapeutically effective. Both sex and recipient safety are extremely high and clinically very useful. Therefore, a great contribution can be expected in human and veterinary medicine. Furthermore, according to the production method of the present invention, the time required for sheet formation is short, and peeling can be easily performed, so that the risk of sheet breakage in the production process is reduced, and a large amount of high quality sheet-like cell culture can be obtained. It is suitable for manufacturing in
本発明は、細胞非接着性領域と細胞接着性領域がパターン形状を形成する、略平面状の表面を有する培養基材上に細胞を播種し、インキュベートすることを含む、シート状細胞培養物の製造方法に関する。
本発明において、「シート状細胞培養物」は、細胞が互いに連結してシート状になったものをいい、典型的には１つの細胞層からなるものであるが、２以上の細胞層から構成されるものも含む。細胞同士は、直接および／または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも機械的に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、細胞培養物を構成する細胞に由来するものである。細胞は少なくとも機械的に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。
The present invention relates to a sheet-like cell culture comprising seeding and incubating cells on a culture substrate having a substantially planar surface, in which the cell non-adhesive area and the cell adhesive area form a pattern shape. It relates to the manufacturing method.
In the present invention, "sheet-like cell culture" refers to cells in which cells are linked to each other to form a sheet, and typically consist of one cell layer, but are composed of two or more cell layers. Including those to be 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 culture. The cells are at least mechanically linked, but may be further functionally, eg chemically, 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-like 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 their surfaces and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Membranes and the like are known, but the cell culture of the present invention can maintain its physical integrity without such scaffolds. In addition, the cell culture of the present invention preferably comprises only the substance derived from cells constituting the cell culture, and does not contain any other substance.
本発明において、「細胞接着性」とは、接着細胞が接着可能であるかまたは接着しやすいことを意味する。したがって逆に「細胞非接着性」は、接着細胞が接着可能でないかまたは接着しにくいことを意味する。本発明において「細胞接着性領域」とは、培養基材の培養表面において、細胞接着性である部分を意味し、「細胞非接着性領域」とは、培養基材の培養表面において、非細胞接着性である部分を意味する。したがって、本発明において「細胞接着性表面」という語は「細胞接着性領域」と同義に用いられ、同様に「細胞非接着性表面」という語は「細胞非接着性領域」と同義に用いられる。
接着細胞は、接する表面の疎水性または親水性がある程度以上高いと接着しにくくなることが知られている。したがって、本発明の一態様において、細胞非接着性表面は、ある程度以上の疎水性または親水性を有している。表面の疎水性または親水性の程度は、例えば、水接触角で表すことができるが、本発明において、細胞非接着性表面の水接触角は、好ましくは７０°以上または５０°以下、特に７５°以上または４０°以下である。
In the present invention, "cell adhesion" means that adherent cells can or can easily adhere. Thus, conversely, "cell non-adherent" means that adherent cells can not or do not easily adhere. In the present invention, "cell-adhesive area" means a portion which is cell-adhesive on the culture surface of the culture substrate, and "cell non-adhesive area" means a non-cell on the culture surface of the culture substrate It means a part that is adhesive. Therefore, in the present invention, the term "cell adhesive surface" is used synonymously with "cell adhesive area", and similarly, the term "cell nonadhesive surface" is used synonymously with "cell nonadhesive area". .
It is known that adherent cells become difficult to adhere when the hydrophobicity or hydrophilicity of the contact surface is higher than a certain level. Thus, in one aspect of the invention, the non-cell-adhesive surface is more or less hydrophobic or hydrophilic. The degree of hydrophobicity or hydrophilicity of the surface can be expressed, for example, by the water contact angle, but in the present invention, the water contact angle of the non-cell-adherent surface is preferably 70 ° or more or 50 ° or less, in particular 75 It is more than ° or less than 40 °.
本発明の方法において用いられる培養基材は、その表面に細胞非接着性領域と細胞接着性領域とを具備している。そして該細胞非接着性領域と該細胞接着性領域とは、パターン形状を形成している。本発明において「パターン形状」とは、ある程度のばらつきをもって両領域が培養表面全体に分散して存在している状態を意味し、繰り返しパターンなどがあげられるが、必ずしも繰り返しパターンを形成している必要はない。パターン形状の例としては、これに限定するものではないが、例えば海島状、格子状、ストライプ状、同心円状、放射状、市松状、または１もしくは２以上のこれらの組み合わせなどが挙げられる。 The culture substrate used in the method of the present invention has a cell non-adhesive area and a cell adhesive area on its surface. The cell non-adhesive region and the cell adhesive region form a pattern shape. In the present invention, “pattern shape” means a state in which both regions are dispersed and present over the entire culture surface with a certain degree of variation, and a repetitive pattern etc. can be mentioned, but it is necessary to form a repetitive pattern. There is no. Examples of the pattern shape include, but are not limited to, sea-island, lattice, stripe, concentric, radial, checkered, or a combination of one or more, and the like.
細胞非接着性領域と細胞接着性領域とのパターン形状は、当該技術分野において知られた任意の方法を用いて形成してよい。パターン形状を形成する方法の例としては、これに限定するものではないが、細胞非接着性の培養表面に細胞接着性成分を塗布する方法、細胞接着性の培養表面に細胞非接着性成分を塗布する方法、細胞非接着性の培養表面に細胞接着性処理を施す方法などが挙げられる。細胞接着性処理とは、これに限定するものではないが、例えばプラズマ処理（電荷処理）などがあげられる。
「細胞接着性成分」とは、細胞が接着可能なあらゆる成分を意味する。逆に「細胞非接着性成分」とは、細胞が接着できないか、またはしづらいあらゆる成分を意味する。細胞接着性成分としては、これに限定するものではないが、例えばフィブロネクチン、ビトロネクチン、ラメニン、コラーゲン、プロテオグリカンなどの血清・細胞外マトリクス成分、ＲＧＤ、ＣＳ−1などの細胞接着ペプチド、ポリリジン、ポリエチレンイミン、ポリアリルアミンなどのポリマー化合物などが挙げられる。細胞非接着性成分としては、ポリメタクリル酸ヒドロキシエチル（ポリＨＥＭＡ）、２−メタクリロイルオキシエチルホスホリスコリン（ＭＰＣ）などのハイドロゲルなどが挙げられる。
The pattern of cell non-adherent regions and cell adhesive regions may be formed using any method known in the art. Examples of methods for forming a pattern shape include, but are not limited to, a method of applying a cell adhesive component to a cell non-adhesive culture surface, a cell non-adhesive component on a cell adhesive culture surface A method of applying, a method of applying a cell adhesion treatment to a cell non-adhesive culture surface, and the like can be mentioned. The cell adhesion treatment is not limited to this, and examples thereof include plasma treatment (charge treatment) and the like.
By "cell adhesive component" is meant any component to which cells can adhere. Conversely, "cell non-adherent component" means any component to which cells can not adhere or are difficult to attach. Cell adhesion components include, but are not limited to, for example, serum / extracellular matrix components such as fibronectin, vitronectin, lamenin, collagen, proteoglycans, cell adhesion peptides such as RGD and CS-1, polylysine, polyethyleneimine And polymer compounds such as polyallylamine. The cell non-adhesive component may, for example, be a hydrogel such as polyhydroxyethyl methacrylate (polyHEMA) or 2-methacryloyloxyethylphosphorischoline (MPC).
本発明の一態様において、培養基材の培養表面は凹凸形状を有していてもよいが、好ましくは培養表面が略平面状である。本発明において「略平面状」とは、細胞間接着および細胞の均等分布に支障をきたす程度の高低差がない状態をいい、これに限定するものではないが、例えば１０μｍ以上の凹凸を有さないものである。 In one aspect of the present invention, the culture surface of the culture substrate may have an uneven shape, but preferably, the culture surface is substantially planar. In the present invention, “substantially planar” refers to a state where there is no difference in height that interferes with cell adhesion and uniform distribution of cells, and is not limited to this, for example, it has unevenness of 10 μm or more. It is nothing.
本発明の方法におけるインキュベートは、シート状細胞培養物を形成するためのインキュベートである。したがって細胞間接着を形成することができる条件であればいかなる条件であってもよいが、通常は一般的な細胞培養条件と同様の条件であればよい。当該技術分野において通常の知識を有するものであれば、播種する細胞の種類に応じて最適な条件を選択することが出来る。 The incubation in the method of the present invention is an incubation to form a sheet-like cell culture. Therefore, any conditions may be used as long as they can form cell-to-cell adhesion, but the conditions may generally be the same as general cell culture conditions. Optimal conditions can be selected according to the type of cells to be seeded, as long as the person has ordinary knowledge in the art.
本発明の方法では、細胞は非接着性領域上でもパターニングされずに細胞間接着を形成し、一枚のシート状細胞培養物を製造できる。この事実は、通常細胞非接着性領域には細胞が接着できないため、十分に細胞間接着を形成できないと考えられていたこと、実際その事実を利用して、細胞のパターニングのために細胞非接着性領域および細胞接着性領域を培養表面上に形成する技術が公知であったこと（特許文献３参照）に鑑みると、驚くべきものである。 In the method of the present invention, cells are not patterned even on nonadherent regions to form intercellular adhesion, and a sheet-like cell culture can be produced. This fact was considered to be incapable of forming sufficient cell-cell adhesion because cells can not usually adhere to cell non-adherent regions, and the fact is used to make cell non-adhesion for cell patterning. It is surprising in view of the fact that techniques for forming sexual and cell-adherent regions on culture surfaces are known (see Patent Document 3).
本発明の方法においては、形成されたシート状細胞培養物と培養基材とは、培養表面上の細胞接着性領域のみで結合しているため、従来の方法で形成されるシート状細胞培養物と比較して、培養表面との接着力が弱くなっている。そのため長時間培養するとシート状細胞培養物の細胞間結合力がシート状細胞培養物と培養表面との結合力を上回り、自然と剥離が起こる。したがって、本発明の一態様において、自然に剥離が起こるまでインキュベートを行ってもよい。しかしながら、剥離されたシート状細胞培養物は非常にもろく、また、そのままインキュベートを続けると細胞間結合力によりシート状細胞培養物の収縮によりよれやしわが出来たり、めくれた状態で張り付いてしまうため、自然に剥離が起こる前に能動的に剥離を行うのが好ましい。 In the method of the present invention, since the formed sheet-like cell culture and the culture substrate are bound only by the cell adhesive region on the culture surface, the sheet-like cell culture formed by the conventional method In comparison with, the adhesion to the culture surface is weak. Therefore, when cultured for a long time, the intercellular cohesion of the sheet-like cell culture exceeds the cohesion of the sheet-like cell culture to the culture surface, and peeling occurs spontaneously. Thus, in one aspect of the invention, the incubation may be carried out until detachment naturally occurs. However, the exfoliated sheet-like cell culture is very fragile, and if the incubation is continued as it is, the cell-cell cohesion causes the contraction of the sheet-like cell culture to cause wrinkles or wrinkles or stick in a curled state. Therefore, it is preferable to actively perform the exfoliation before the exfoliation naturally occurs.
上述の通り、本発明のインキュベートはシートを形成するためのインキュベートであるため、細胞増殖が起こる必要はない。したがって細胞間接着が形成されるのに十分な時間インキュベートを行えばよい。インキュベートの時間としては、好ましくは１〜２４時間、より好ましくは２〜１２時間である。１時間より短いと十分に細胞間接着が形成されないため、シート化が不十分となり、２４時間より多いと剥離したシート状細胞培養物によれやしわが出来たり、シートの形状が崩れてきたりしてしまう。 As mentioned above, because the incubation of the present invention is an incubation to form a sheet, cell proliferation need not occur. Therefore, incubation may be performed for a sufficient time to form cell-cell adhesion. The incubation time is preferably 1 to 24 hours, more preferably 2 to 12 hours. If it is shorter than 1 hour, the adhesion between cells is not sufficiently formed, so that the sheeting is insufficient, and if it is more than 24 hours, the peeled sheet-like cell culture may cause wrinkles or wrinkles, or the sheet may be deformed. It will
本発明の好ましい一態様において、１つの細胞接着領域と、該細胞接着領域に隣接する他の細胞接着領域との間隔が、少なくとも１箇所において播種される細胞の長径よりも大きい。すなわち、培養基材表面全体で破損無くシートを形成するためには、細胞非接着領域を隙間無く覆う必要があり、そのためには、少なくとも１箇所の細胞非接着領域について２つ以上の細胞が必要となる。より好ましくは、少なくとも１つの細胞非接着領域内に、少なくとも１つの細胞が存在する。細胞非接着領域内に細胞が少なくとも１つ存在するとは、細胞非接着領域に接触している細胞のうち、細胞接着領域に接触していない細胞が少なくとも１つあることを意味する。本発明の方法によれば、細胞接着領域に接触していない細胞が存在していても、培養基材全面で破損のない一枚のシートを形成可能となる。 In a preferred embodiment of the present invention, the distance between one cell adhesion area and the other cell adhesion area adjacent to the cell adhesion area is larger than the major axis of cells seeded at at least one location. That is, in order to form a sheet without breakage over the entire surface of the culture substrate, it is necessary to cover the cell non-adherent area without gaps, and for this purpose, at least one cell non-adherent area requires at least two cells. It becomes. More preferably, at least one cell is present in the at least one cell non-adherent region. The presence of at least one cell in the non-cell-adhesive area means that there are at least one cell in contact with the non-cell-adhesive area but not in contact with the cell-adhesive area. According to the method of the present invention, even if there are cells not in contact with the cell adhesion region, it is possible to form a single sheet without breakage on the entire surface of the culture substrate.
本発明の好ましい一態様において、播種した細胞がインキュベートの間実質的に増殖しない。「実質的に増殖しない」とは、計測誤差の範囲を超えて増殖しないことを意味し、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。本発明において、シート状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 seeded cells do not substantially proliferate during the incubation. The phrase "does not substantially proliferate" means that proliferation does not occur beyond the range of measurement error, and whether or not cells proliferated is, for example, the number of cells at the time of seeding and the number of cells after formation of the cell culture. It can be evaluated by comparing In the present invention, the number of cells after formation of the sheet-like cell culture is typically 300% or less, preferably 200% or less, more preferably 150% or less, still more preferably 125% or less of the number of cells at the time of seeding. Particularly preferably, it is 100% or less.
細胞が実質的に増殖するか否かは、様々な条件、例えば播種細胞数（播種細胞密度）、培地、インキュベート条件などにより決定される。本発明においては、播種した細胞がシート状細胞培養物を形成することが必要であり、細胞の生物学的な活性を低下させることなく増殖をコントロールする必要があるため、播種細胞密度により増殖をコントロールするのが好ましい。したがって本発明の好ましい一態様においては、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される。 Whether or not cells substantially proliferate is determined by various conditions such as the number of seeded cells (seeded cell density), culture medium, incubation conditions, and the like. In the present invention, it is necessary for the seeded cells to form a sheet-like cell culture, and it is necessary to control the growth without reducing the biological activity of the cells. It is preferable to control. Thus, in a preferred embodiment of the present invention, the cells are seeded at a density capable of forming a sheet-like cell culture without substantial growth.
「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方法では、シート状細胞培養物を形成するために、約６，５００個／ｃｍ２の密度の細胞をプレートに播種していたが（例えば、特許文献１参照）、かかる密度の細胞を、成長因子を含まない培養液で培養してもシート状の細胞培養物を形成することはできない。したがって、本発明におけるシート形成細胞の播種密度は、成長因子を含む培養液を用いる方法、すなわち細胞の増殖を少なくとも目的の一部とする培養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、３．４×１０６個／ｃｍ２未満である。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、シート状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。 The "density capable of forming a sheet-like cell culture substantially without proliferation" means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain a growth factor. Mean cell density. For example, in the case of skeletal myoblasts, in the method using a medium containing growth factor, cells were seeded with a density of about 6,500 cells / cm 2 in order to form a sheet-like cell culture. However, even if cells of such a density are cultured in a culture medium containing no growth factor, no sheet-like cell culture can be formed. Therefore, the seeding density of the sheet-forming cells in the present invention is higher than that in the method using a culture medium containing a growth factor, that is, culture in which cell proliferation is at least a part of the object. 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 not particularly limited unless the formation of cell culture is impaired and the cells go into differentiation, but for skeletal myoblasts, for example, less than 3.4 × 10 6 cells / cm 2 . 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 sheet-like cell culture but does not shift to differentiation.
本発明の製造方法における細胞の播種密度は、ある態様では３．０×１０５〜３．４×１０６個／ｃｍ２、別の態様では３．５×１０５〜３．４×１０６個／ｃｍ２、さらに別の態様では１．０×１０６〜３．４×１０６個／ｃｍ２、さらに別の態様では３．０×１０５〜１．７×１０６個／ｃｍ２、別の態様では３．５×１０５〜１．７×１０６個／ｃｍ２、さらに別の態様では１．０×１０６〜１．７×１０６個／ｃｍ２であってよい。上記範囲は、上限が３．４×１０６個／ｃｍ２未満である限り、上限および下限の両方、または、そのいずれか一方を含んでもよい。したがって、本発明の製造方法における骨格筋芽細胞の播種密度は、例えば、３．０×１０５個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、３．５×１０５個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、１．０×１０６個／ｃｍ２以上３．４×１０６個／ｃｍ２未満（下限を含み、上限は含まない）、１．０×１０６個／ｃｍ２超３．４×１０６個／ｃｍ２未満（下限も上限も含まない）、１．０×１０６個／ｃｍ２超１．７×１０６個／ｃｍ２以下（下限は含まないが、上限は含む）であってもよい。当業者であれば、骨格筋芽細胞以外の細胞について、本発明に適した細胞密度を、本明細書の教示に従い、実験により適宜決定することができる。 The seeding density of cells in the production method of the present invention is, in one aspect, 3.0 × 10 5 to 3.4 × 10 6 cells / cm 2 , and in another aspect, 3.5 × 10 5 to 3.4 × 10 6 Pieces / cm 2 , in still another embodiment 1.0 × 10 6 to 3.4 × 10 6 pieces / cm 2 , and in still another embodiment 3.0 × 10 5 to 1.7 × 10 6 pieces / cm 2 In another embodiment, it may be 3.5 × 10 5 to 1.7 × 10 6 / cm 2 , and in still another embodiment 1.0 × 10 6 to 1.7 × 10 6 / cm 2 . The above range may include both the upper limit and the lower limit, or any one of them, as long as the upper limit is less than 3.4 × 10 6 pieces / cm 2 . Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0 × 10 5 / cm 2 or more and 3.4 × 10 6 / cm 2 or less (including the lower limit and the upper limit) ), 3.5 × 10 5 pieces / cm 2 or more and 3.4 × 10 6 pieces / cm 2 (including the lower limit and not including the upper limit), 1.0 × 10 6 pieces / cm 2 or more and 3.4 × 10 less than 6 / cm 2 (including the lower and the upper limit is not included), 1.0 × 10 6 cells / cm 2 ultra 3.4 × 10 than 6 / cm 2 (lower limit also contains no upper limit), 1 More than 0 × 10 6 pieces / cm 2 and not more than 1.7 × 10 6 pieces / cm 2 (not including the lower limit but including the upper limit). Those skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teaching of the present specification.
上述の通り、本発明のインキュベートはシート状細胞培養物を形成するためのインキュベートであり、したがって細胞はシート状細胞培養物を形成することが出来る細胞である。また、シート状細胞培養物を形成するためのインキュベートを、単に「シート化培養」という場合もある。細胞は具体的には、これに限定するものではないが、例えば骨格筋芽細胞、皮膚細胞、角膜上皮細胞、歯根膜細胞、心筋細胞、肝細胞、膵細胞、口腔粘膜上皮細胞などが挙げられ、好ましくは骨格筋芽細胞が挙げられる。 As mentioned above, the incubation of the present invention is an incubation to form a sheet-like cell culture, thus the cells are cells capable of forming a sheet-like cell culture. Moreover, the incubation for forming a sheet-like cell culture may be simply referred to as "sheeting culture". Specific examples of the cells include, but are not limited to, skeletal myoblasts, skin cells, corneal epithelial cells, periodontal ligament cells, cardiomyocytes, hepatocytes, pancreatic cells, oral mucosal epithelial cells and the like. And preferably skeletal myoblasts.
本発明の好ましい一態様において、パターン形状が海島構造である。本明細書において「海島構造」とは、比較的連続的な一方の領域（「海」という）の中に、不連続的に他方の領域（「島」という）が混在している構造をいう。また本明細書においては、「海島構造」を有する形状を特に「海島状」と表現する場合がある。海側の領域は、比較的連続的であれば完全に連続的である必要は必ずしも無いが、形成されたシート状細胞培養物の剥離容易性の観点から、島側の領域が培養表面の一部の領域に偏在するのは好ましくない。 In a preferred embodiment of the present invention, the pattern shape is a sea-island structure. As used herein, “sea-island structure” refers to a structure in which the other region (referred to as “island”) is discontinuously mixed in one region (referred to as “sea”) that is relatively continuous. . Further, in the present specification, a shape having the “sea-island structure” may be particularly expressed as “the sea-island”. The seaward region does not necessarily have to be completely continuous if it is relatively continuous, but from the viewpoint of the peelability of the formed sheet-like cell culture, the island-side region is a part of the culture surface. It is not preferable to be unevenly distributed in the area of the department.
本発明の海島構造は、細胞非接着性領域が海であっても細胞接着性領域が海であってもよい。海島構造を形成する方法は、当該技術分野において知られたあらゆる方法を用いることが出来、これに限定するものではないが、例えば細胞非接着性の基材にスポット状に血清をプロットして部分的に被覆する方法、細胞接着性の基材にスポット状に細胞非接着性ポリマーをプロットして部分的に被覆する方法、などが挙げられる。しかしながら、形成の容易さ、シート状細胞培養物の形成後の剥離容易性などに鑑みると、好ましくは細胞接着性領域が島である。 In the sea-island structure of the present invention, the cell non-adhesive region may be the sea or the cell adhesive region may be the sea. Any method known in the art can be used to form the islands-in-the-sea structure, including but not limited to, for example, plotting serum as spots on a non-cell-adhesive substrate A method of coating, a method of plotting and partially covering a cell non-adhesive polymer in a spot shape on a cell adhesive substrate, and the like. However, in view of easiness of formation, ease of exfoliation after formation of a sheet-like cell culture, etc., preferably, the cell adhesive area is an island.
本発明の培養表面に存在する細胞接着領域は、広すぎるとシート状細胞培養物との接着性が上がるため、シートの形成には都合がよいが、シート状細胞培養物の剥離性は低下する。逆に狭すぎると、シート状細胞培養物との接着性が低下するためシート状細胞培養物の形成が困難となるが、形成されれば剥離が容易となる。したがって本発明の細胞接着性領域は、好ましくは表面全体の４％〜６５％であり、より好ましくは８％〜５２％である。 If the cell adhesion area present on the culture surface of the present invention is too wide, the adhesion to the sheet-like cell culture will be increased, which is convenient for forming a sheet, but the peelability of the sheet-like cell culture will decrease . On the other hand, if the width is too narrow, the adhesion to the sheet-like cell culture decreases, and formation of the sheet-like cell culture becomes difficult, but if it is formed, peeling becomes easy. Therefore, the cell adhesive area of the present invention is preferably 4% to 65% of the entire surface, more preferably 8% to 52%.
本発明はまた、製造方法によって作製されたシート状の細胞培養物にも関する。本発明のシート状細胞培養物は、浸漬撹拌などの洗浄操作や、取出し、保持、移送などの移植操作に対して十分な物理的強度を有する。十分な物理的強度を有するとは、上記操作を施しても細胞培養物のシート状構造が損なわれないことを意味し、これは、例えば、得られたシート状細胞培養物に実際に上記操作を施し、シート状構造が保たれていることを肉眼的または顕微鏡的に調査することにより確認することができる。ここで、浸漬撹拌による洗浄操作とは、典型的には、シート状細胞培養物の入った培養容器に同培養物が十分浸漬する量の緩衝液を加え、培養容器ごと振とう撹拌することをいう。したがって、十分な物理的強度を有することは、かかる浸漬撹拌による洗浄操作と同様の物理的ストレスをシート状細胞培養物に加えることによっても確認することができる。また、上記製造方法によって作製されたシート状細胞培養物であれば、通常かかる十分な物理的強度を有する。 The present invention also relates to a sheet-like cell culture produced by the production method. The sheet-like cell culture of the present invention has sufficient physical strength for washing operations such as immersion agitation, and transplantation operations such as removal, retention, and transfer. Having sufficient physical strength means that the sheet-like structure of the cell culture is not impaired even if the above-mentioned operation is carried out, which means that, for example, the above-mentioned operation is actually carried out on the obtained sheet-like cell culture. And the sheet-like structure is maintained by visual or microscopic examination. Here, a washing operation by immersion stirring typically involves adding a buffer solution in an amount sufficient to immerse the same culture in a culture vessel containing a sheet-like cell culture, and shaking and stirring the entire culture vessel. Say. Therefore, having sufficient physical strength can also be confirmed by applying the same physical stress to the sheet-like cell culture as the immersion stirring washing operation. Moreover, if it is the sheet-like cell culture produced by the said manufacturing method, it has such sufficient physical strength normally.
以下の実施例に基づいて、本発明を具体的な態様を挙げて本発明をさらに詳細に説明するが、本発明はこれらの具体例に限定されるものではない。 The present invention will be described in more detail by way of specific embodiments based on the following examples, but the present invention is not limited to these specific examples.
実施例１
細胞非接着性表面を有する浮遊系細胞用のペトリディッシュ（BD製、code:35-1008）の非接着性表面に、４ｍｍの間隔で２０％ＦＢＳ含有ＭＣＤＢ１３１培地を約１ｍｍの径となるようにプロットし、４０時間インキュベートして接着性表面を形成した。全培養表面に対する接着性表面の割合は約１０％であった。２０％（ｖ／ｖ）ＦＢＳ含有のＭＣＤＢ１３１培地で懸濁した細胞を９．３×１０６個／枚となるようにディッシュに播種した。細胞を３７℃、５％ＣＯ２で６時間培養後、ピペッティングによりシート状になった細胞培養物をプレートから剥離させた。
図１に示す通り、６時間培養したものは、ピペッティングにより容易に剥離可能であり、そのためよれ、しわの少ない上質のシート状細胞培養物が得られた。また同様の条件で１５時間培養した場合は、シート状細胞培養物を形成後ピペッティングすることなく自然に剥離したが、よれやしわができてしまっていた。
Example 1
On a non-adhesive surface of a petri dish for floating cells with cell non-adhesive surface (BD, code: 35-1008), MCDB 131 medium containing 20% FBS at a distance of 4 mm to a diameter of about 1 mm Plotted and incubated for 40 hours to form an adhesive surface. The ratio of adhesive surface to total culture surface was about 10%. The cells suspended in MCDB 131 medium containing 20% (v / v) FBS were seeded in a dish at 9.3 × 10 6 cells / sheet. After culturing the cells at 37 ° C., 5% CO 2 for 6 hours, the sheeted cell culture by pipetting was detached from the plate.
As shown in FIG. 1, those cultured for 6 hours can be easily peeled off by pipetting, so that a high quality sheet-like cell culture with less wrinkles is obtained. When cultured under the same conditions for 15 hours, the sheet-like cell culture was peeled off without pipetting after formation, but wrinkles and wrinkles were formed.
実施例２
細胞培養用マルチウェルプレート（BD製、code: 353046）の接着性表面に、４ｍｍの間隔で非接着性ポリマー（ポリＨＥＭＡ）を約３ｍｍの径となるようにプロットし、室温で１時間ほど乾燥させた。全培養表面に対する接着性表面の割合は６０％以下であった。２０％（ｖ／ｖ）ＦＢＳ含有のＭＣＤＢ１３１培地で懸濁した細胞を、９．３×１０６個／枚となるようにディッシュに播種した。細胞を３７℃、５％ＣＯ２で６時間培養後、ピペッティングによりシート状になった細胞培養物をプレートから剥離させた。
図２に示す通り、６時間培養したものは、ピペッティングにより容易に剥離可能であり、そのためよれ、しわの少ない上質のシート状細胞培養物が得られた。
Example 2
Non-adhesive polymer (poly-HEMA) is plotted at an interval of 4 mm on the adhesive surface of a multi-well plate for cell culture (BD, code: 353046) to a diameter of about 3 mm, and dried at room temperature for about 1 hour I did. The proportion of adhesive surface to total culture surface was less than 60%. The cells suspended in MCDB131 medium containing 20% (v / v) FBS were seeded at 9.3 × 10 6 cells / plate. After culturing the cells at 37 ° C., 5% CO 2 for 6 hours, the sheeted cell culture by pipetting was detached from the plate.
As shown in FIG. 2, those cultured for 6 hours can be easily peeled off by pipetting, so that a high quality sheet-like cell culture with less wrinkles is obtained.
本発明によれば、浸漬撹拌などの洗浄操作や、取出し、保持、移送などの移植操作に耐え得る十分な物理的強度を有する良質のシート状細胞培養物を簡便かつ迅速に製造することが可能となる。また、本発明の方法を用いると、通常製造工程において最もシート状細胞培養物に破損の生じやすい剥離工程が非常に容易に行えるか、または行う必要がないため、高い再現性で良質のシート状細胞培養物を製造可能となる。 According to the present invention, it is possible to simply and rapidly produce a sheet-like cell culture of good quality having sufficient physical strength to withstand washing operations such as immersion and agitation, and transplantation operations such as removal, retention and transfer. It becomes. In addition, when the method of the present invention is used, it is very easy to carry out the peeling step which is most likely to cause breakage in the sheet-like cell culture in the ordinary manufacturing step, or it is not necessary to carry out. Cell cultures can be produced.
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