WO2015146560A1 - Cell culture instrument for producing sheet-shaped cell culture and method for producing sheet-shaped cell culture using same - Google Patents
Cell culture instrument for producing sheet-shaped cell culture and method for producing sheet-shaped cell culture using same Download PDFInfo
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- the present invention relates to a cell culture instrument for easily producing a sheet-shaped cell culture and a method for producing a sheet-shaped cell culture using the instrument.
- the incubation of the present invention is an incubation for forming a sheet, it is not necessary for cell proliferation to occur. Therefore, the incubation may be performed for a time sufficient for the formation of 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, cell-cell adhesion is not sufficiently formed, so that the sheet formation becomes insufficient. If it is longer than 24 hours, the peeled sheet-shaped cell culture may wrinkle or wrinkle, or the shape of the sheet may collapse. End up.
- “Present with a certain regularity” means that a specific arrangement pattern is repeated, and examples include arrangement at regular intervals.
- the cells are not arranged at equal intervals, there is a portion where the ratio of the region not locally treated with cell adhesion is increased, and the formation of the sheet-shaped cell culture may be inhibited in that portion.
- the cell-adhesive treated regions are arranged at equal intervals.
- the pattern arranged at equal intervals is not limited to this, and examples thereof include lattice points and concentric circles, and it is preferable that the patterns be arranged in lattice points for ease of arrangement.
- “lattice-like” refers to a state in which the grids are arranged vertically and horizontally.
- ultraviolet irradiation is preferable in that cell adhesion can be controlled by changing the exposure time.
- the treatment time in the case of performing the ultraviolet irradiation treatment is sufficient as long as it does not unnecessarily deteriorate the substrate, and varies depending on the material of the culture substrate used, the wavelength of the mercury lamp used for irradiation, and the irradiation height.
- the substrate When the substrate is irradiated with a wavelength of 184.9 nm at an irradiation height of 20 mm, the substrate may be exposed for at least 15 seconds, preferably about 30 seconds to 100 seconds.
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Abstract
The purpose of the present invention is to provide a cell culture container for easily and quickly producing a sheet-shaped cell culture with excellent qualities and a method for producing a sheet-shaped cell culture using the aforesaid cell culture container.
To achieve this purpose, provided are: a cell culture container having a nearly planar surface wherein a cell non-adhesion region and a cell adhesion region form a pattern shape; and a method for producing a sheet-shaped cell culture, said method comprising seeding cells on a culture substrate of the aforesaid cell culture container and then incubating the cells.
Description
本発明は、簡便にシート状の細胞培養物を製造するための細胞培養器具および該器具を用いたシート状細胞培養物の製造方法に関する。
The present invention relates to a cell culture instrument for easily producing a sheet-shaped cell culture and a method for producing a sheet-shaped cell culture using the instrument.
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. For the treatment of heart failure, medical treatment with β-blockers or ACE inhibitors is performed. For heart failure that has become so severe that these treatments are not successful, replacement therapy such as an artificial heart or a heart transplant, 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。 Severe heart failure that is the subject of 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), dilation There are a wide variety of causes such as chronic heart failure due to dilated cardiomyopathy (DCM) and acute aversion.
Depending on these causes and severity, valvuloplasty and replacement, coronary artery bypass surgery, left ventricular plastic surgery, mechanical assisted circulation, etc. are applied.
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。 Severe heart failure that is the subject of 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), dilation There are a wide variety of causes such as chronic heart failure due to dilated cardiomyopathy (DCM) and acute aversion.
Depending on these causes and severity, valvuloplasty and replacement, coronary artery bypass surgery, left ventricular plastic surgery, mechanical assisted circulation, etc. are applied.
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。
Of these, only heart transplantation or replacement treatment with an artificial heart has been regarded as an effective treatment for those who have suffered heart failure due to severe deterioration of left ventricular function caused by ICM or DCM. However, replacement therapy for these patients with severe heart failure has many problems to be solved, such as chronic donor shortages, the need for continuous immunosuppression, and the development of complications. 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 the treatment of severe heart failure.
In severe myocardial infarction and the like, cardiomyocytes become dysfunctional, and fibroblast proliferation and interstitial fibrosis progress, resulting in heart failure. As the heart failure progresses, the cardiomyocytes are damaged and fall into apoptosis, but the cardiomyocytes hardly undergo cell division, so the number of cardiomyocytes decreases and the cardiac function further decreases.
Cell transplantation is considered useful for the recovery of cardiac function in such patients with severe heart failure, and clinical application with autologous skeletal myoblasts has already been started.
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 the treatment of severe heart failure.
In severe myocardial infarction and the like, cardiomyocytes become dysfunctional, and fibroblast proliferation and interstitial fibrosis progress, resulting in heart failure. As the heart failure progresses, the cardiomyocytes are damaged and fall into apoptosis, but the cardiomyocytes hardly undergo cell division, so the number of cardiomyocytes decreases and the cardiac function further decreases.
Cell transplantation is considered useful for the recovery of cardiac function in such patients with severe heart failure, and clinical application with 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, as an example, a three-dimensional cell culture that can be applied to the heart including cells derived from parts other than the adult myocardium by using a temperature-responsive culture dish that applies tissue engineering, and its A manufacturing method was 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。
Patent Document 2 discloses a cell culture support that is less damaged, easily peels off, and does not slow down the sheet formation speed by forming a concave-convex structure having a concave portion with a size that prevents cells from entering the cell culture surface. Are listed. Patent Document 3 discloses a cell culture substrate having a cell adhesion region and a cell adhesion inhibitory region. When cells are cultured using the culture substrate, the cells adhere to only the cell adhesion region, thereby patterning the cells. It is described that can be performed.
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およびそのために有用な細胞培養器具を提供することにある。
In the method for producing a high-quality and highly safe sheet-shaped 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 particular, in the production of a fragile sheet-shaped cell culture, there are many cases in which the sheet-shaped cell culture is damaged in the peeling process. Accordingly, there is a need for a culture substrate that can easily peel a cultured sheet-like cell culture or a method that allows easy peeling. An object of the present invention is to provide a method for quickly and easily producing a high-quality and highly safe sheet-shaped cell culture and a cell culture instrument useful for the method.
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。
When the present inventors performed sheet culture in a dish in which serum is plotted at a certain interval on a non-adhesive surface to form an adhesive region, the cells are not affected even if the cell non-adhesive region exists. It was found that a uniform single sheet was formed without patterning, and that the sheet was easily peelable. As a result of further earnest research based on this knowledge, 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。
［９］シート状細胞培養物を製造するための細胞培養器具であって、シート状細胞培養物を形成するための略平面状の細胞非接着性の培養基材表面を有し、該培養基材表面は細胞接着性処理された領域を複数有し、該領域が前記培養基材表面に均一に分散して存在している、前記細胞培養器具。
［１０］細胞接着性処理が、紫外線照射、プラズマ放電またはコロナ放電により行われる、［９］の細胞培養器具。
［１１］細胞接着性処理が、紫外線照射により行われる、［１０］の細胞培養器具。
［１２］細胞接着性処理された領域が、２～４ｍｍ間隔で存在する直径１～４ｍｍの点状の領域である、［９］～［１１］の細胞培養器具。
［１３］細胞接着性処理された領域が、培養基材表面全体の４％～６５％である、［９］～［１２］の細胞培養器具。
［１４］シート状細胞培養物の製造方法であって、［９］～［１３］の細胞培養器具の培養基材表面に細胞を播種し、インキュベートすることを含む、前記方法。
［１５］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、［１４］の方法。
［１６］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、［１４］または［１５］の方法。
［１７］細胞が、骨格筋芽細胞である、［１４］～［１６］の方法。 That is, the present invention relates to the following.
[1] A method for producing a sheet-shaped cell culture, in which cells are seeded 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. Incubating said method.
[2] The method according to [1], further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
[3] The interval in at least a part between one cell adhesion region and another cell adhesion region adjacent to the cell adhesion region is larger than the major axis of the seeded cells [1] or [2] the method of.
[4] The method of [1] to [3], wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
[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 the cell non-adhesion region or the cell adhesion region is an island and the other is the sea.
[7] The method of [6], wherein the cell adhesion region is an island and the cell non-adhesion region is the sea.
[8] The method according to [1] to [7], wherein the ratio of the cell adhesion region is 4% to 65% of the entire surface.
[9] A cell culture device for producing a sheet-shaped cell culture, comprising a substantially planar cell non-adhesive culture substrate surface for forming a sheet-shaped cell culture, The cell culture instrument, wherein the material surface has a plurality of cell-adhesive treated regions, and the regions are uniformly dispersed on the culture substrate surface.
[10] The cell culture instrument according to [9], wherein the cell adhesion treatment is performed by ultraviolet irradiation, plasma discharge or corona discharge.
[11] The cell culture instrument according to [10], wherein the cell adhesion treatment is performed by ultraviolet irradiation.
[12] The cell culture instrument according to [9] to [11], wherein the cell-adhesive treated region is a dot-like region having a diameter of 1 to 4 mm present at intervals of 2 to 4 mm.
[13] The cell culture instrument according to [9] to [12], wherein the area subjected to cell adhesion treatment is 4% to 65% of the entire culture substrate surface.
[14] A method for producing a sheet-shaped cell culture, which comprises seeding and incubating cells on the surface of a culture substrate of the cell culture device according to [9] to [13].
[15] The method according to [14], further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
[16] The method according to [14] or [15], wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
[17] The method of [14] to [16], wherein the cell is a skeletal myoblast.
［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。
［９］シート状細胞培養物を製造するための細胞培養器具であって、シート状細胞培養物を形成するための略平面状の細胞非接着性の培養基材表面を有し、該培養基材表面は細胞接着性処理された領域を複数有し、該領域が前記培養基材表面に均一に分散して存在している、前記細胞培養器具。
［１０］細胞接着性処理が、紫外線照射、プラズマ放電またはコロナ放電により行われる、［９］の細胞培養器具。
［１１］細胞接着性処理が、紫外線照射により行われる、［１０］の細胞培養器具。
［１２］細胞接着性処理された領域が、２～４ｍｍ間隔で存在する直径１～４ｍｍの点状の領域である、［９］～［１１］の細胞培養器具。
［１３］細胞接着性処理された領域が、培養基材表面全体の４％～６５％である、［９］～［１２］の細胞培養器具。
［１４］シート状細胞培養物の製造方法であって、［９］～［１３］の細胞培養器具の培養基材表面に細胞を播種し、インキュベートすることを含む、前記方法。
［１５］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、［１４］の方法。
［１６］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、［１４］または［１５］の方法。
［１７］細胞が、骨格筋芽細胞である、［１４］～［１６］の方法。 That is, the present invention relates to the following.
[1] A method for producing a sheet-shaped cell culture, in which cells are seeded 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. Incubating said method.
[2] The method according to [1], further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
[3] The interval in at least a part between one cell adhesion region and another cell adhesion region adjacent to the cell adhesion region is larger than the major axis of the seeded cells [1] or [2] the method of.
[4] The method of [1] to [3], wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
[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 the cell non-adhesion region or the cell adhesion region is an island and the other is the sea.
[7] The method of [6], wherein the cell adhesion region is an island and the cell non-adhesion region is the sea.
[8] The method according to [1] to [7], wherein the ratio of the cell adhesion region is 4% to 65% of the entire surface.
[9] A cell culture device for producing a sheet-shaped cell culture, comprising a substantially planar cell non-adhesive culture substrate surface for forming a sheet-shaped cell culture, The cell culture instrument, wherein the material surface has a plurality of cell-adhesive treated regions, and the regions are uniformly dispersed on the culture substrate surface.
[10] The cell culture instrument according to [9], wherein the cell adhesion treatment is performed by ultraviolet irradiation, plasma discharge or corona discharge.
[11] The cell culture instrument according to [10], wherein the cell adhesion treatment is performed by ultraviolet irradiation.
[12] The cell culture instrument according to [9] to [11], wherein the cell-adhesive treated region is a dot-like region having a diameter of 1 to 4 mm present at intervals of 2 to 4 mm.
[13] The cell culture instrument according to [9] to [12], wherein the area subjected to cell adhesion treatment is 4% to 65% of the entire culture substrate surface.
[14] A method for producing a sheet-shaped cell culture, which comprises seeding and incubating cells on the surface of a culture substrate of the cell culture device according to [9] to [13].
[15] The method according to [14], further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
[16] The method according to [14] or [15], wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
[17] The method of [14] to [16], wherein the cell is a skeletal myoblast.
本発明の細胞培養器具は、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を培養基材表面上に形成することができ、かつかかるシート状細胞培養物を容易に剥離することができるものである。また、本発明の製造方法によれば、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。
The cell culture instrument of the present invention forms on the surface of a culture substrate a sheet-like cell culture having sufficient physical strength that can withstand a washing operation such as immersion stirring and a transplanting operation such as removal, holding, and transfer. And the sheet-like cell culture can be easily detached. In addition, according to the production method of the present invention, since a sheet-like cell culture can be obtained with a high cell recovery rate, the cells to be used can be effectively used, which contributes to cost reduction and the like. In addition, the sheet-like cell culture obtained by the production method of the present invention has a high production of factors having an effect on the effectiveness such as angiogenesis, while the production of inflammatory cytokines is low, so that it is therapeutically effective. Sexuality and safety to recipients are extremely high and are clinically very useful. For this reason, a great contribution can be expected in human medicine and veterinary medicine. Furthermore, according to the production method of the present invention, the time required to form a sheet is short and can be easily peeled off, thereby reducing the risk of sheet breakage in the production process and producing a large amount of high-quality sheet-like cell cultures. It is suitable for manufacturing.
本発明は一側面において、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。 In one aspect, the present invention provides a sheet-like cell comprising seeding and incubating a cell 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 The present invention relates to a method for producing a culture.
In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it is a substance capable of mechanically connecting cells to each other, and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, in particular, derived from the cells constituting the cell culture. The cells are at least mechanically linked, but may be further functionally, eg, chemically or electrically linked.
本発明において、「シート状細胞培養物」は、細胞が互いに連結してシート状になったものをいい、典型的には１つの細胞層からなるものであるが、２以上の細胞層から構成されるものも含む。細胞同士は、直接および／または介在物質を介して、互いに連結していてもよい。介在物質としては、細胞同士を少なくとも機械的に連結し得る物質であれば特に限定されないが、例えば、細胞外マトリックスなどが挙げられる。介在物質は、好ましくは細胞由来のもの、特に、細胞培養物を構成する細胞に由来するものである。細胞は少なくとも機械的に連結されるが、さらに機能的、例えば、化学的、電気的に連結されてもよい。 In one aspect, the present invention provides a sheet-like cell comprising seeding and incubating a cell 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 The present invention relates to a method for producing a culture.
In the present invention, the “sheet-shaped cell culture” refers to a sheet in which cells are connected to each other and is typically composed of one cell layer, but is composed of two or more cell layers. Including those that are made. The cells may be linked to each other directly and / or via an intervening substance. The intervening substance is not particularly limited as long as it is a substance capable of mechanically connecting cells to each other, and examples thereof include an extracellular matrix. The intervening substance is preferably derived from cells, in particular, derived from the cells constituting the cell culture. The cells are at least mechanically linked, but may be further functionally, eg, chemically or electrically linked.
本発明のシート状細胞培養物は、好ましくはスキャフォールド（支持体）を含まない。スキャフォールドは、その表面上および／またはその内部に細胞を付着させ、細胞培養物の物理的一体性を維持するために当該技術分野において用いられることがあり、例えば、ポリビニリデンジフルオリド（ＰＶＤＦ）製の膜等が知られているが、本発明の細胞培養物は、かかるスキャフォールドがなくともその物理的一体性を維持することができる。また、本発明の細胞培養物は、好ましくは、細胞培養物を構成する細胞由来の物質のみからなり、それら以外の物質を含まない。
The sheet-shaped cell culture of the present invention preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within its surface and maintain the physical integrity of the cell culture, eg, polyvinylidene difluoride (PVDF) Although manufactured membranes are known, the cell culture of the present invention can maintain its physical integrity even without such a scaffold. In addition, the cell culture of the present invention preferably consists only of substances derived from the cells constituting the cell culture and does not contain any other substances.
本発明において、「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８０°以上または５０°以下、特に７０°以上または４０°以下である。 In the present invention, “cell adhesion” means that adherent cells can adhere or are easily adhered. Thus, conversely, “cell non-adhesive” means that adherent cells are not adherent or difficult to adhere. In the present invention, the “cell adhesive region” means a portion that is cell adhesive on the culture surface of the culture substrate, and the “cell non-adhesive region” means a non-cell on the culture surface of the culture substrate. It means the part that is adhesive. Accordingly, in the present invention, the term “cell adhesive surface” is used synonymously with “cell adhesive region”, and similarly the term “cell nonadhesive surface” is used synonymously with “cell nonadhesive region”. .
It is known that adherent cells are difficult to adhere if the surface or surface to be contacted has a higher hydrophobicity or hydrophilicity than a certain level. Therefore, in one embodiment of the present invention, the non-cell-adhesive surface has a certain degree of hydrophobicity or hydrophilicity. The degree of hydrophobicity or hydrophilicity of the surface can be expressed by, for example, a water contact angle. In the present invention, the water contact angle of the non-cell-adhesive surface is preferably 80 ° or more or 50 ° or less, particularly 70. It is more than or less than 40 degrees.
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８０°以上または５０°以下、特に７０°以上または４０°以下である。 In the present invention, “cell adhesion” means that adherent cells can adhere or are easily adhered. Thus, conversely, “cell non-adhesive” means that adherent cells are not adherent or difficult to adhere. In the present invention, the “cell adhesive region” means a portion that is cell adhesive on the culture surface of the culture substrate, and the “cell non-adhesive region” means a non-cell on the culture surface of the culture substrate. It means the part that is adhesive. Accordingly, in the present invention, the term “cell adhesive surface” is used synonymously with “cell adhesive region”, and similarly the term “cell nonadhesive surface” is used synonymously with “cell nonadhesive region”. .
It is known that adherent cells are difficult to adhere if the surface or surface to be contacted has a higher hydrophobicity or hydrophilicity than a certain level. Therefore, in one embodiment of the present invention, the non-cell-adhesive surface has a certain degree of hydrophobicity or hydrophilicity. The degree of hydrophobicity or hydrophilicity of the surface can be expressed by, for example, a water contact angle. In the present invention, the water contact angle of the non-cell-adhesive surface is preferably 80 ° or more or 50 ° or less, particularly 70. It is more than or less than 40 degrees.
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 region and a cell adhesive region on its surface. The cell non-adhesive region and the cell adhesive region form a pattern shape. In the present invention, the “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 examples include a repeated pattern, but it is necessary to form a repeated pattern. There is no. Examples of the pattern shape include, but are not limited to, a sea island shape, a lattice shape, a stripe shape, a concentric circle shape, a radial shape, a checkered shape, or a combination of one or two or more thereof.
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、例えば紫外線照射処理、プラズマ処理（電荷処理）、コロナ放電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。 You may form the pattern shape of a cell non-adhesion area | region and a cell adhesion area | region using the arbitrary methods known in the said technical field. 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, and a cell non-adhesive component to a cell adhesive culture surface. Examples thereof include a method of applying, and a method of performing cell adhesion treatment on a non-cell-adhesive culture surface. Examples of the cell adhesion treatment include, but are not limited to, ultraviolet irradiation treatment, plasma treatment (charge treatment), corona discharge treatment, and the like.
“Cell-adhesive component” means any component to which cells can adhere. Conversely, the “cell non-adhesive component” means any component to which cells cannot adhere or are difficult to adhere. Examples of cell adhesion components include, but are not limited to, serum / extracellular matrix components such as fibronectin, vitronectin, ramenin, collagen, proteoglycan, cell adhesion peptides such as RGD and CS-1, polylysine, polyethyleneimine And polymer compounds such as polyallylamine. Examples of the cell non-adhesive component include hydrogels such as polyhydroxyethyl methacrylate (polyHEMA) and 2-methacryloyloxyethylphosphorischoline (MPC).
「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。 You may form the pattern shape of a cell non-adhesion area | region and a cell adhesion area | region using the arbitrary methods known in the said technical field. 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, and a cell non-adhesive component to a cell adhesive culture surface. Examples thereof include a method of applying, and a method of performing cell adhesion treatment on a non-cell-adhesive culture surface. Examples of the cell adhesion treatment include, but are not limited to, ultraviolet irradiation treatment, plasma treatment (charge treatment), corona discharge treatment, and the like.
“Cell-adhesive component” means any component to which cells can adhere. Conversely, the “cell non-adhesive component” means any component to which cells cannot adhere or are difficult to adhere. Examples of cell adhesion components include, but are not limited to, serum / extracellular matrix components such as fibronectin, vitronectin, ramenin, collagen, proteoglycan, cell adhesion peptides such as RGD and CS-1, polylysine, polyethyleneimine And polymer compounds such as polyallylamine. Examples of the cell non-adhesive component include hydrogels such as polyhydroxyethyl methacrylate (polyHEMA) and 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 embodiment of the present invention, the culture surface of the culture substrate may have an uneven shape, but the culture surface is preferably substantially planar. In the present invention, “substantially planar” means a state where there is no difference in level that hinders cell-cell adhesion and uniform distribution of cells, and is not limited thereto, but has, for example, irregularities of 10 μm or more. There 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 for forming a sheet-like cell culture. Therefore, any conditions can be used as long as they can form cell-cell adhesion, but usually the same conditions as general cell culture conditions may be used. Any person who has ordinary knowledge in the art can select optimal conditions according to the type of cells to be seeded.
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 form an intercellular adhesion without being patterned even on a non-adhesive region, and a single sheet-like cell culture can be produced. This fact was thought to be due to the fact that cells cannot normally adhere to non-cell-adherent areas, so that it was thought that cell-cell adhesion could not be formed sufficiently. In view of the fact that the technology for forming the sex region and the cell adhesion region on the culture surface has been known (see Patent Document 3), it is surprising.
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-shaped cell culture and the culture substrate are bound only by the cell adhesive region on the culture surface, the sheet-shaped cell culture formed by the conventional method Compared with, the adhesive force with the culture surface is weak. Therefore, when the cells are cultured for a long time, the cell-cell binding force of the sheet-shaped cell culture exceeds the binding force between the sheet-shaped cell culture and the culture surface, and peeling occurs naturally. Therefore, in one embodiment of the present invention, incubation may be performed until separation occurs spontaneously. However, the peeled sheet-shaped cell culture is very fragile, and if the incubation is continued as it is, the cell-like cell culture may cause wrinkles or wrinkles due to the contraction of the sheet-shaped cell culture, or it will stick in a folded state. Therefore, it is preferable to actively perform the peeling before the peeling 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 described above, since the incubation of the present invention is an incubation for forming a sheet, it is not necessary for cell proliferation to occur. Therefore, the incubation may be performed for a time sufficient for the formation of 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, cell-cell adhesion is not sufficiently formed, so that the sheet formation becomes insufficient. If it is longer than 24 hours, the peeled sheet-shaped cell culture may wrinkle or wrinkle, or the shape of the sheet may collapse. End up.
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 region and another cell adhesion region adjacent to the cell adhesion region is larger than the major axis of the cells to be seeded in at least one place. That is, in order to form a sheet on the entire culture substrate surface without breakage, it is necessary to cover the cell non-adherent region without any gap, and for that purpose, at least one cell non-adherent region requires two or more cells. It becomes. More preferably, at least one cell is present in at least one cell non-adherent region. The presence of at least one cell in the cell non-adherent region means that there is at least one cell that is not in contact with the cell adhesive region among the cells in contact with the cell non-adherent region. According to the method of the present invention, even if cells that are not in contact with the cell adhesion region exist, it is possible to form a single sheet that is not damaged 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 grow during the incubation. “Substantially does not proliferate” means that the cell does not proliferate beyond the range of measurement error, and whether or not the cell has proliferated is, for example, the number of cells at the time of seeding and the number of cells after formation of the cell culture. And can be evaluated by comparing. In the present invention, the number of cells after forming the sheet-shaped cell culture is typically 300% or less of the number of cells at the time of seeding, preferably 200% or less, more preferably 150% or less, more preferably 125% or less, 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 the cells substantially proliferate is determined by various conditions such as the number of seeded cells (seed cell density), the culture medium, and the incubation conditions. In the present invention, it is necessary for the seeded cells to form a sheet-like cell culture, and it is necessary to control proliferation 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 that can form a sheet 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 at which a sheet-like cell culture can be formed without substantially growing” means that a sheet-like cell culture can be formed when cultured in a non-proliferating culture medium that does not contain growth factors. Refers to cell density. For example, in the case of skeletal myoblasts, in a method using a culture solution containing a growth factor, cells having a density of about 6,500 cells / cm 2 were seeded on a plate in order to form a sheet-like cell culture. However, even if cells having such a density are cultured in a culture solution not containing a growth factor, a sheet-like cell culture cannot be formed. Accordingly, the seeding density of the sheet-forming cells in the present invention is higher than that in a method using a culture solution containing a growth factor, that is, in a culture in which cell proliferation is at least a part of the purpose. Specifically, for example, for skeletal myoblasts, such density is typically 300,000 cells / cm 2 or more. The upper limit of the cell density is not particularly limited as long as the formation of the cell culture is not impaired and the cells do not shift to differentiation, but for skeletal myoblasts, for example, it is less than 3.4 × 10 6 cells / cm 2. . A person skilled in the art can appropriately determine the cell density suitable for the present invention by experiments. During the culture period, the cells may or may not proliferate, but even if they proliferate, they do not proliferate to the extent that the properties of the cells change. For example, skeletal myoblasts start to differentiate when they become confluent, but in the present invention, skeletal myoblasts are seeded at a density that forms a 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 cell seeding density in the production method of the present invention is 3.0 × 10 5 to 3.4 × 10 6 cells / cm 2 in one embodiment, and 3.5 × 10 5 to 3.4 × 10 6 in another embodiment. Pieces / cm 2 , 1.0 × 10 6 to 3.4 × 10 6 pieces / cm 2 in still another embodiment, and 3.0 × 10 5 to 1.7 × 10 6 pieces / cm 2 in still another embodiment. In another embodiment, it may be 3.5 × 10 5 to 1.7 × 10 6 pieces / cm 2 , and in another embodiment, 1.0 × 10 6 to 1.7 × 10 6 pieces / cm 2 . As long as an upper limit is less than 3.4 * 10 < 6 > piece / cm < 2 >, the said range may include both an upper limit and a lower limit, or any one thereof. Therefore, the seeding density of skeletal myoblasts in the production method of the present invention is, for example, 3.0 × 10 5 cells / cm 2 or more and less than 3.4 × 10 6 cells / cm 2 (including the lower limit and not including the upper limit). ), 3.5 × 10 5 pieces / cm 2 or more and less than 3.4 × 10 6 pieces / cm 2 (including the lower limit, not including the upper limit), 1.0 × 10 6 pieces / cm 2 or more and 3.4 × 10 less than 6 / cm 2 (including the lower and the upper limit is not included), 1.0 × 10 6 cells / cm 2 ultra 3.4 × 10 than 6 / cm 2 (lower limit also contains no upper limit), 1 It may be more than 0.0 × 10 6 pieces / cm 2 and 1.7 × 10 6 pieces / cm 2 or less (not including the lower limit but including the upper limit). A person skilled in the art can appropriately determine the cell density suitable for the present invention for cells other than skeletal myoblasts by experiments according to the teaching of the present specification.
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 described above, the incubation of the present invention is an incubation for forming a sheet-shaped cell culture, and thus the cells are cells capable of forming a sheet-shaped cell culture. In addition, the incubation for forming the sheet-shaped cell culture may be simply referred to as “sheet 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. Preferably, skeletal myoblasts are used.
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. In the present specification, the “sea-island structure” refers to a structure in which one region that is relatively continuous (referred to as “the sea”) is discontinuously mixed with the other region (referred to as “island”). . In this specification, a shape having a “sea-island structure” may be expressed as a “sea-island shape”. The sea side region does not necessarily need to be completely continuous as long as it is relatively continuous. However, from the viewpoint of ease of separation of the formed sheet-shaped cell culture, the island side region is a part of the culture surface. It is not preferable to be unevenly distributed in the region of the part.
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 as a method for forming the sea-island structure, and the method is not limited to this. For example, a portion of the serum is plotted by spotting on a non-cell-adhesive substrate. And a method of partially coating a non-cell-adhesive polymer in a spot shape on a cell-adhesive substrate. However, in view of ease of formation, ease of peeling after formation of the sheet-shaped cell culture, and the like, the cell adhesion region is preferably 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 region existing on the culture surface of the present invention is too wide, the adhesion with the sheet-shaped cell culture is increased, which is convenient for the formation of the sheet, but the peelability of the sheet-shaped cell culture is reduced. . On the other hand, if it is too narrow, the adhesiveness with the sheet-shaped cell culture is lowered, so that it becomes difficult to form the sheet-shaped cell culture, but if formed, peeling becomes easy. Accordingly, the cell adhesion region of the present invention is preferably 4% to 65% of the entire surface, more preferably 8% to 52%.
本発明はまた、上記製造方法によって作製されたシート状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 above production method. The sheet-shaped cell culture of the present invention has sufficient physical strength for washing operations such as immersion stirring and transplantation operations such as removal, holding and transfer. Having sufficient physical strength means that even if the above operation is performed, the sheet-like structure of the cell culture is not impaired. For example, the obtained sheet-like cell culture is actually subjected to the above operation. It can be confirmed by conducting a macroscopic or microscopic investigation that the sheet-like structure is maintained. Here, the washing operation by immersing and stirring typically means adding a sufficient amount of a buffer solution in which the culture is immersed in a culture vessel containing a sheet-like cell culture, and stirring and agitating the whole culture vessel. Say. Therefore, having sufficient physical strength can also be confirmed by applying physical stress similar to the washing operation by soaking and stirring to the sheet-like cell culture. In addition, a sheet-like cell culture produced by the above production method usually has sufficient physical strength.
本発明は別の一側面において、シート状細胞培養物を製造するための細胞培養器具であって、シート状細胞培養物を形成するための略平面状の細胞非接着性の培養基材表面を有し、該培養基材表面は細胞接着性処理された領域を複数有し、該領域が前記培養基材表面に均一に分散して存在している、前記細胞培養器具に関する。
In another aspect of the present invention, there is provided a cell culture device for producing a sheet-shaped cell culture, comprising a substantially planar cell non-adhesive culture substrate surface for forming the sheet-shaped cell culture. And the culture substrate surface has a plurality of cell-adhesive treated regions, and the regions are uniformly dispersed on the culture substrate surface.
本側面における細胞培養器具は、細胞非接着性の細胞培養基材表面に、部分的に、細胞接着性処理された領域を複数有している。したがって細胞接着性処理された領域が細胞接着性領域であり、処理されていない領域が細胞接着性領域となる。この細胞接着性領域が複数存在し、細胞培養基材表面に均一に分散して存在しているものである。
The cell culture instrument in this aspect has a plurality of cell adhesion treated regions partially on the surface of the cell culture substrate that is not cell adhesive. Therefore, the cell-adhesive treated region is the cell-adhesive region, and the untreated region is the cell-adhesive region. A plurality of the cell adhesion regions exist and are uniformly dispersed on the surface of the cell culture substrate.
本側面において、「均一に分散して存在」するとは、細胞接着性領域が、細胞培養基材表面全体に偏りなく存在している状態をいう。偏りなく存在しているか否かは当業者であれば容易に判断できるものであり、これに限定するものではないが例えば、細胞培養基材表面を所定の数に等分に分割した際に、全ての分割された表面において細胞接着性領域の占める割合がほぼ同一であれば、偏りなく存在しているといえる。
In the present aspect, “uniformly distributed” means a state in which the cell adhesion region exists evenly on the entire cell culture substrate surface. Whether or not there is no bias can be easily determined by those skilled in the art, and is not limited thereto, for example, when dividing the cell culture substrate surface into a predetermined number of equal parts, If the proportion of the cell adhesion region is almost the same in all the divided surfaces, it can be said that the cell adhesion region is present evenly.
本発明の細胞培養器具に用いられる細胞接着性処理としては、細胞培養基材を不必要に劣化させない処理であれば特に限定されない。かかる細胞接着性処理の例としては、これに限定するものではないが、紫外線照射処理、プラズマ放電処理、コロナ放電処理などが挙げられ、簡便さ、細胞接着性の制御のしやすさなどの観点から、好ましくは紫外線照射処理である。
The cell adhesion treatment used in the cell culture instrument of the present invention is not particularly limited as long as the treatment does not unnecessarily deteriorate the cell culture substrate. Examples of such cell adhesion treatment include, but are not limited to, ultraviolet irradiation treatment, plasma discharge treatment, corona discharge treatment, and the like, from the viewpoints of simplicity and ease of control of cell adhesion. Therefore, an ultraviolet irradiation treatment is preferable.
上述のとおり、本発明の好ましい一態様において、細胞接着性処理は紫外線照射によって行われる。かかる態様において、細胞接着性処理が十分に行われる紫外線照射条件には、照射する紫外線の波長、照度、光源との距離（照射高）、照射時間などが含まれ、培養基材の材質や求める細胞接着性の度合いに応じて変化し得るが、当業者であれば最適な条件を適宜選択して組み合わせることが可能である。例えばポリスチレン製の培養基材に対して細胞接着性処理を行う場合、照射する紫外線波長としては、１００ｎｍ～４００ｎｍ、好ましくは１５０ｎｍ～２００ｎｍ、より好ましくは約１８５ｎｍなどが挙げられる。また紫外線の照度としては、１～１０ｍＷ／ｃｍ２、好ましくは３～７ｍＷ／ｃｍ２、より好ましくは約５ｍＷ／ｃｍ２などが挙げられる。また光源の照射高としては、１～１０００ｍｍ、好ましくは５～２００ｍｍ、より好ましくは１０～８０ｍｍが挙げられる。また紫外線の照射時間としては、１～３００秒、好ましくは５～１００秒、より好ましくは１５～６０秒が挙げられる。
As described above, in a preferred embodiment of the present invention, the cell adhesion treatment is performed by ultraviolet irradiation. In such an embodiment, the ultraviolet irradiation conditions under which cell adhesion treatment is sufficiently performed include the wavelength of the ultraviolet rays to be irradiated, the illuminance, the distance from the light source (irradiation height), the irradiation time, etc. Although it may vary depending on the degree of cell adhesion, those skilled in the art can appropriately select and combine optimum conditions. For example, when cell adhesion treatment is performed on a culture substrate made of polystyrene, the ultraviolet wavelength to be irradiated is 100 nm to 400 nm, preferably 150 nm to 200 nm, more preferably about 185 nm. The illuminance of ultraviolet rays is 1 to 10 mW / cm 2 , preferably 3 to 7 mW / cm 2 , more preferably about 5 mW / cm 2 . The irradiation height of the light source is 1 to 1000 mm, preferably 5 to 200 mm, more preferably 10 to 80 mm. The irradiation time of ultraviolet rays is 1 to 300 seconds, preferably 5 to 100 seconds, more preferably 15 to 60 seconds.
本発明の細胞培養器具の培養基材の材質としては、接着性処理が可能なものであれば特に限定されず、当該技術分野において一般的に培養基材に用いられている材質を使用することができる。かかる材質としては、これに限定するものではないが、例えばポリスチレン、ポリエチレン、ポリエチレンテレフタラート（ＰＥＴ）などが挙げられる。
The material of the culture substrate of the cell culture instrument of the present invention is not particularly limited as long as it can be subjected to adhesive treatment, and a material generally used for a culture substrate in the technical field should be used. Can do. Examples of such materials include, but are not limited to, polystyrene, polyethylene, polyethylene terephthalate (PET), and the like.
上述のとおり本発明においては、パターン形状が海島状であることが好ましく、とくに偏りなく存在しているためには、細胞接着性処理された領域が一定の形状であり、かつ一定の規則性で存在している島であることが好ましい。「一定の形状」は、統一された形状であれば特に限定されないが、対称性を有した形状が好ましく、細胞接着性処理された領域の形成の簡便さの観点から、点状または円状であることがより好ましい。
As described above, in the present invention, it is preferable that the pattern shape is a sea-island shape. In particular, since the pattern shape is present without unevenness, the cell-adhesive treated region has a constant shape and a constant regularity. It is preferable that the island exists. The “certain shape” is not particularly limited as long as it is a unified shape, but a symmetrical shape is preferable, and in terms of ease of formation of the cell-adhesive treated region, it is a dot shape or a circular shape. More preferably.
「一定の規則性で存在している」とは、特定の配置パターンを繰り返していることを意味し、典型的には等間隔での配置などが挙げられる。とくに、等間隔で配置されていない場合には、局所的に細胞接着性処理されていない領域の割合が大きくなる部分が生じ、その部分ではシート状細胞培養物の形成が阻害される可能性が高まるため、細胞接着性処理された領域は等間隔で配置されることが好ましい。等間隔で配置されるパターンとしては、これに限定するものではないが、例えば格子点状、同心円状などが挙げられ、配置の簡便さから格子点状に配置されるのが好ましい。なお、本発明において「格子点状」とは、縦横に整列するように配置された状態をいう。
“Present with a certain regularity” means that a specific arrangement pattern is repeated, and examples include arrangement at regular intervals. In particular, in the case where the cells are not arranged at equal intervals, there is a portion where the ratio of the region not locally treated with cell adhesion is increased, and the formation of the sheet-shaped cell culture may be inhibited in that portion. In order to increase, it is preferable that the cell-adhesive treated regions are arranged at equal intervals. The pattern arranged at equal intervals is not limited to this, and examples thereof include lattice points and concentric circles, and it is preferable that the patterns be arranged in lattice points for ease of arrangement. In the present invention, “lattice-like” refers to a state in which the grids are arranged vertically and horizontally.
したがって、より好ましい一態様において、細胞接着性処理された領域は、等間隔で配置された点状の領域である。かかる態様においては、細胞接着性処理された領域は、１～１０ｍｍ程度の間隔で存在することが好ましく、２～４ｍｍ程度の間隔で存在することがより好ましく、例えば１ｍｍ、２ｍｍ、３ｍｍ、４ｍｍ、５ｍｍ、６ｍｍ、７ｍｍ、８ｍｍ、９ｍｍまたは１０ｍｍなどの間隔で存在する。
Therefore, in a more preferred embodiment, the cell-adhesive treated regions are dot-like regions arranged at equal intervals. In such an embodiment, the cell adhesion treated regions are preferably present at an interval of about 1 to 10 mm, more preferably at an interval of about 2 to 4 mm, such as 1 mm, 2 mm, 3 mm, 4 mm, It exists at intervals such as 5 mm, 6 mm, 7 mm, 8 mm, 9 mm or 10 mm.
細胞接着性処理された領域の大きさは特に限定されないが、あまり大きいと培養基材表面全体に対して偏りが生じやすくなり、あまり小さいと領域の形成が困難で煩雑となる。したがって細胞接着性処理された領域は直径が１ｍｍ～６ｍｍ程度の点状の領域であることが好ましく、直径１～４ｍｍ程度の点状領域であることがより好ましく、例えば直径１ｍｍ、２ｍｍ、３ｍｍ、４ｍｍ、５ｍｍまたは６ｍｍなどである。
したがって、特に好ましい一態様において、細胞接着性処理された領域は、２～４ｍｍ程度の間隔で存在する直径１～４ｍｍ程度の点状の領域であり、さらに好ましくは格子点状に配置されている。 The size of the cell adhesion treated region is not particularly limited, but if it is too large, it tends to be biased with respect to the entire culture substrate surface, and if it is too small, formation of the region becomes difficult and complicated. Accordingly, the cell-adhesive treated region is preferably a punctiform region having a diameter of about 1 mm to 6 mm, more preferably a punctiform region having a diameter of about 1 to 4 mm, for example, a diameter of 1 mm, 2 mm, 3 mm, For example, 4 mm, 5 mm or 6 mm.
Therefore, in a particularly preferred embodiment, the cell-adhesive treated region is a dot-like region having a diameter of about 1 to 4 mm, which is present at intervals of about 2 to 4 mm, and more preferably arranged in a grid-point shape. .
したがって、特に好ましい一態様において、細胞接着性処理された領域は、２～４ｍｍ程度の間隔で存在する直径１～４ｍｍ程度の点状の領域であり、さらに好ましくは格子点状に配置されている。 The size of the cell adhesion treated region is not particularly limited, but if it is too large, it tends to be biased with respect to the entire culture substrate surface, and if it is too small, formation of the region becomes difficult and complicated. Accordingly, the cell-adhesive treated region is preferably a punctiform region having a diameter of about 1 mm to 6 mm, more preferably a punctiform region having a diameter of about 1 to 4 mm, for example, a diameter of 1 mm, 2 mm, 3 mm, For example, 4 mm, 5 mm or 6 mm.
Therefore, in a particularly preferred embodiment, the cell-adhesive treated region is a dot-like region having a diameter of about 1 to 4 mm, which is present at intervals of about 2 to 4 mm, and more preferably arranged in a grid-point shape. .
上述のとおり、細胞接着領域は、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。したがって、細胞非接着領域は、好ましくは３５～９６％であり、より好ましくは４８％～９２％である。
As described above, the cell adhesion region is preferably 4% to 65% of the entire surface, and more preferably 8% to 52%. Therefore, the cell non-adhesion region is preferably 35 to 96%, more preferably 48% to 92%.
本発明の細胞培養器具は、例えば以下のようにして製造することができる。
浮遊細胞培養用ディッシュなどの、接着細胞用の表面処理を施されていない培養基材表面に、細胞接着性処理を施したい領域のみを露出させるようにマスキングを行う。マスキング方法については、当該技術分野において知られた通常の方法を用いることができ、例えばマスキングシールなどを貼付する、フォトマスクを用いるなどの方法で行うことができる。次にマスキングした細胞培養表面に紫外線照射などを行い、露出領域に細胞接着性処理を施す。プラズマ処理やコロナ放電処理で細胞接着性処理を行う場合は、１、２秒で処理が完了するが、紫外線照射で細胞接着性処理を行う場合はある程度の時間露光する必要がある。したがって紫外線照射は、露光時間を変更することで細胞接着性の制御を行うことができる点で好ましい。紫外線照射処理を行う場合の処理時間は不必要に基材を劣化しない程度であればよく、用いる培養基材の材質および照射に用いる水銀ランプの波長、さらに照射高によっても異なるが、例えばポリスチレン製の基材に対して１８４．９ｎｍの波長を照射高２０ｍｍで照射する場合は、少なくとも１５秒以上、好ましくは３０秒～１００秒程度露光すればよい。一般的には、処理表面に照射される紫外線量(積算光量)を目安とし、照度を上げれば時間が短くなり、照度を下げれば時間が長くなることとなる。さらに、波長によりエネルギー量が変化するため、最適な時間は異なることとなる。一般的に、波長が短いほうがエネルギー量を大きいため、照射時間は短くなる。 The cell culture instrument of the present invention can be produced, for example, as follows.
Masking is performed on the surface of a culture substrate that has not been subjected to surface treatment for adherent cells, such as a dish for suspension cell culture, so that only the region to be subjected to cell adhesion treatment is exposed. As a masking method, a normal method known in the technical field can be used, and for example, a masking seal or the like is attached, or a photomask is used. Next, the masked cell culture surface is irradiated with ultraviolet rays, and the exposed region is subjected to cell adhesion treatment. When the cell adhesion treatment is performed by plasma treatment or corona discharge treatment, the treatment is completed in 1 or 2 seconds. However, when the cell adhesion treatment is performed by ultraviolet irradiation, it is necessary to expose to some extent. Therefore, ultraviolet irradiation is preferable in that cell adhesion can be controlled by changing the exposure time. The treatment time in the case of performing the ultraviolet irradiation treatment is sufficient as long as it does not unnecessarily deteriorate the substrate, and varies depending on the material of the culture substrate used, the wavelength of the mercury lamp used for irradiation, and the irradiation height. When the substrate is irradiated with a wavelength of 184.9 nm at an irradiation height of 20 mm, the substrate may be exposed for at least 15 seconds, preferably about 30 seconds to 100 seconds. Generally, the amount of ultraviolet rays (integrated light amount) irradiated on the treatment surface is used as a guide, and the time is shortened when the illuminance is increased, and the time is lengthened when the illuminance is decreased. Furthermore, since the amount of energy changes depending on the wavelength, the optimum time differs. In general, the shorter the wavelength, the greater the amount of energy, and the shorter the irradiation time.
浮遊細胞培養用ディッシュなどの、接着細胞用の表面処理を施されていない培養基材表面に、細胞接着性処理を施したい領域のみを露出させるようにマスキングを行う。マスキング方法については、当該技術分野において知られた通常の方法を用いることができ、例えばマスキングシールなどを貼付する、フォトマスクを用いるなどの方法で行うことができる。次にマスキングした細胞培養表面に紫外線照射などを行い、露出領域に細胞接着性処理を施す。プラズマ処理やコロナ放電処理で細胞接着性処理を行う場合は、１、２秒で処理が完了するが、紫外線照射で細胞接着性処理を行う場合はある程度の時間露光する必要がある。したがって紫外線照射は、露光時間を変更することで細胞接着性の制御を行うことができる点で好ましい。紫外線照射処理を行う場合の処理時間は不必要に基材を劣化しない程度であればよく、用いる培養基材の材質および照射に用いる水銀ランプの波長、さらに照射高によっても異なるが、例えばポリスチレン製の基材に対して１８４．９ｎｍの波長を照射高２０ｍｍで照射する場合は、少なくとも１５秒以上、好ましくは３０秒～１００秒程度露光すればよい。一般的には、処理表面に照射される紫外線量(積算光量)を目安とし、照度を上げれば時間が短くなり、照度を下げれば時間が長くなることとなる。さらに、波長によりエネルギー量が変化するため、最適な時間は異なることとなる。一般的に、波長が短いほうがエネルギー量を大きいため、照射時間は短くなる。 The cell culture instrument of the present invention can be produced, for example, as follows.
Masking is performed on the surface of a culture substrate that has not been subjected to surface treatment for adherent cells, such as a dish for suspension cell culture, so that only the region to be subjected to cell adhesion treatment is exposed. As a masking method, a normal method known in the technical field can be used, and for example, a masking seal or the like is attached, or a photomask is used. Next, the masked cell culture surface is irradiated with ultraviolet rays, and the exposed region is subjected to cell adhesion treatment. When the cell adhesion treatment is performed by plasma treatment or corona discharge treatment, the treatment is completed in 1 or 2 seconds. However, when the cell adhesion treatment is performed by ultraviolet irradiation, it is necessary to expose to some extent. Therefore, ultraviolet irradiation is preferable in that cell adhesion can be controlled by changing the exposure time. The treatment time in the case of performing the ultraviolet irradiation treatment is sufficient as long as it does not unnecessarily deteriorate the substrate, and varies depending on the material of the culture substrate used, the wavelength of the mercury lamp used for irradiation, and the irradiation height. When the substrate is irradiated with a wavelength of 184.9 nm at an irradiation height of 20 mm, the substrate may be exposed for at least 15 seconds, preferably about 30 seconds to 100 seconds. Generally, the amount of ultraviolet rays (integrated light amount) irradiated on the treatment surface is used as a guide, and the time is shortened when the illuminance is increased, and the time is lengthened when the illuminance is decreased. Furthermore, since the amount of energy changes depending on the wavelength, the optimum time differs. In general, the shorter the wavelength, the greater the amount of energy, and the shorter the irradiation time.
本側面の別の一態様において、培養基材として本側面の細胞培養容器を用いた、上記シート状細胞培養物の製造方法もまた、本発明に包含される。また、該製造方法により製造されたシート状細胞培養物もまた、本発明に包含される。これらの各態様におけるその他の各構成については、上記で詳述した内容を同様に利用可能である。
In another embodiment of the present aspect, a method for producing the sheet-shaped cell culture using the cell culture container of the present aspect as a culture substrate is also encompassed in the present invention. Moreover, the sheet-like cell culture manufactured by this manufacturing method is also included by this invention. About each other structure in each of these aspects, the content explained in full detail above can be utilized similarly.
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。
Based on the following examples, the present invention will be described in more detail with reference to specific embodiments, 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
A non-adherent surface of a Petri dish (BD, code: 35-1008) for floating cells having a cell non-adhesive surface, with 20% FBS-containing MCDB131 medium having a diameter of about 1 mm at intervals of 4 mm. Plotted and incubated for 40 hours to form an adhesive surface. The ratio of adhesive surface to total culture surface was about 10%. Cells suspended in MCDB131 medium containing 20% (v / v) FBS were seeded in a dish at 9.3 × 10 6 cells / plate. After culturing the cells at 37 ° C. and 5% CO 2 for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 1, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained. In addition, when cultured for 15 hours under the same conditions, the sheet-like cell culture was formed and spontaneously detached without pipetting, but it was wrinkled or wrinkled.
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
A non-adherent surface of a Petri dish (BD, code: 35-1008) for floating cells having a cell non-adhesive surface, with 20% FBS-containing MCDB131 medium having a diameter of about 1 mm at intervals of 4 mm. Plotted and incubated for 40 hours to form an adhesive surface. The ratio of adhesive surface to total culture surface was about 10%. Cells suspended in MCDB131 medium containing 20% (v / v) FBS were seeded in a dish at 9.3 × 10 6 cells / plate. After culturing the cells at 37 ° C. and 5% CO 2 for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 1, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained. In addition, when cultured for 15 hours under the same conditions, the sheet-like cell culture was formed and spontaneously detached without pipetting, but it was wrinkled or wrinkled.
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の径となるように格子点状にプロットし、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
On the adhesive surface of a multiwell plate for cell culture (BD, code: 353046), a non-adhesive polymer (poly-HEMA) is plotted in a lattice point shape at an interval of 4 mm so as to have a diameter of about 3 mm. It was dried for about 1 hour. The ratio of the adhesive surface to the entire culture surface was 60% or less. Cells suspended in 20% (v / v) FBS-containing MCDB131 medium were seeded in a dish at 9.3 × 10 6 cells / plate. After culturing the cells at 37 ° C. and 5% CO 2 for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 2, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained.
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の径となるように格子点状にプロットし、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
On the adhesive surface of a multiwell plate for cell culture (BD, code: 353046), a non-adhesive polymer (poly-HEMA) is plotted in a lattice point shape at an interval of 4 mm so as to have a diameter of about 3 mm. It was dried for about 1 hour. The ratio of the adhesive surface to the entire culture surface was 60% or less. Cells suspended in 20% (v / v) FBS-containing MCDB131 medium were seeded in a dish at 9.3 × 10 6 cells / plate. After culturing the cells at 37 ° C. and 5% CO 2 for 6 hours, the cell culture formed into a sheet by pipetting was detached from the plate.
As shown in FIG. 2, those cultured for 6 hours can be easily detached by pipetting, so that a high quality sheet-shaped cell culture with few wrinkles was obtained.
実施例３
上記実施例１の方法を以下のとおり改変して行った。培養基材は、浮遊系細胞用のペトリディッシュ（BD製、code:35-1008）に代えてIWAKIペトリディッシュ（ＡＧＣテクノグラス製、３．５ｃｍ径）を用いた。培養基材表面の細胞接着性領域の形成は、以下の手順で行った。
（１）シリコンシートを３５ｍｍ径に打ち抜いて円状のシリコンシートを作製し、この円状シリコンシートに４ｍｍ径の穴を４ｍｍ間隔で格子点状に開けた。
（２）（１）により得たシリコンシートを上記ペトリディッシュの培養表面上に置き、ＵＶ照射装置（センエンジニアリング製：PL16-110）内に設置した。
（３）照射高を１５ｍｍ、照射紫外線波長を１８４．９ｎｍに設定し、紫外線を１５秒間照射して、島状に親水化処理をしたディッシュを得た。
図３に示す通り、６時間の培養によりシート状細胞培養物の形成が確認され、約１分間のピペッティングにより容易に剥離可能であり、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 3
The method of Example 1 was modified as follows. As a culture substrate, IWAKI Petri dish (manufactured by AGC Techno Glass, 3.5 cm diameter) was used instead of Petri dish for floating cells (BD, code: 35-1008). Formation of the cell adhesion region on the surface of the culture substrate was performed according to the following procedure.
(1) A silicon sheet was punched out to a diameter of 35 mm to produce a circular silicon sheet, and holes having a diameter of 4 mm were formed in the circular silicon sheet in lattice points at intervals of 4 mm.
(2) The silicon sheet obtained by (1) was placed on the culture surface of the Petri dish and placed in a UV irradiation apparatus (manufactured by Sen Engineering: PL16-110).
(3) Irradiation height was set to 15 mm, irradiation ultraviolet wavelength was set to 184.9 nm, and ultraviolet irradiation was performed for 15 seconds to obtain a dish that was hydrophilized into an island shape.
As shown in FIG. 3, the formation of a sheet-like cell culture was confirmed by culturing for 6 hours, and can be easily detached by pipetting for about 1 minute, resulting in a high-quality sheet-like cell culture with few wrinkles. It was.
上記実施例１の方法を以下のとおり改変して行った。培養基材は、浮遊系細胞用のペトリディッシュ（BD製、code:35-1008）に代えてIWAKIペトリディッシュ（ＡＧＣテクノグラス製、３．５ｃｍ径）を用いた。培養基材表面の細胞接着性領域の形成は、以下の手順で行った。
（１）シリコンシートを３５ｍｍ径に打ち抜いて円状のシリコンシートを作製し、この円状シリコンシートに４ｍｍ径の穴を４ｍｍ間隔で格子点状に開けた。
（２）（１）により得たシリコンシートを上記ペトリディッシュの培養表面上に置き、ＵＶ照射装置（センエンジニアリング製：PL16-110）内に設置した。
（３）照射高を１５ｍｍ、照射紫外線波長を１８４．９ｎｍに設定し、紫外線を１５秒間照射して、島状に親水化処理をしたディッシュを得た。
図３に示す通り、６時間の培養によりシート状細胞培養物の形成が確認され、約１分間のピペッティングにより容易に剥離可能であり、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 3
The method of Example 1 was modified as follows. As a culture substrate, IWAKI Petri dish (manufactured by AGC Techno Glass, 3.5 cm diameter) was used instead of Petri dish for floating cells (BD, code: 35-1008). Formation of the cell adhesion region on the surface of the culture substrate was performed according to the following procedure.
(1) A silicon sheet was punched out to a diameter of 35 mm to produce a circular silicon sheet, and holes having a diameter of 4 mm were formed in the circular silicon sheet in lattice points at intervals of 4 mm.
(2) The silicon sheet obtained by (1) was placed on the culture surface of the Petri dish and placed in a UV irradiation apparatus (manufactured by Sen Engineering: PL16-110).
(3) Irradiation height was set to 15 mm, irradiation ultraviolet wavelength was set to 184.9 nm, and ultraviolet irradiation was performed for 15 seconds to obtain a dish that was hydrophilized into an island shape.
As shown in FIG. 3, the formation of a sheet-like cell culture was confirmed by culturing for 6 hours, and can be easily detached by pipetting for about 1 minute, resulting in a high-quality sheet-like cell culture with few wrinkles. It was.
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。さらに本発明の細胞培養容器は、市販の細胞培養器具に対して生体由来物質を用いた処理かまたは他の物質を用いることのない処理を行うことで製造することが可能であるため、シート状細胞培養物の剥離を容易にするための従来の表面処理と比較して非常に安全性が高く、したがってヒト医療および獣医療において多大な貢献が期待できる。
According to the present invention, it is possible to easily and quickly produce a high-quality sheet-shaped cell culture having sufficient physical strength that can withstand a washing operation such as immersion stirring and a transplanting operation such as removal, holding, and transfer. It becomes. In addition, when the method of the present invention is used, the peeling process that is most likely to cause damage to the sheet-shaped cell culture in the normal manufacturing process can be performed very easily or does not need to be performed. Cell culture can be produced. Furthermore, since the cell culture container of the present invention can be manufactured by performing a treatment using a biological substance on a commercially available cell culture instrument or a treatment without using other substances, a sheet shape Compared with conventional surface treatments for facilitating the detachment of cell cultures, it is very safe and can therefore be expected to make a significant contribution in human and veterinary medicine.
Claims (9)
- シート状細胞培養物を製造するための細胞培養器具であって、シート状細胞培養物を形成するための略平面状の細胞非接着性の培養基材表面を有し、該培養基材表面は細胞接着性処理された領域を複数有し、該領域が前記培養基材表面に均一に分散して存在している、前記細胞培養器具。 A cell culture instrument for producing a sheet-shaped cell culture, which has a substantially planar cell non-adhesive culture substrate surface for forming a sheet-shaped cell culture, The cell culture instrument, comprising a plurality of cell-adhesive treated regions, wherein the regions are uniformly dispersed on the culture substrate surface.
- 細胞接着性処理が、紫外線照射、プラズマ放電またはコロナ放電により行われる、請求項１に記載の細胞培養器具。 The cell culture instrument according to claim 1, wherein the cell adhesion treatment is performed by ultraviolet irradiation, plasma discharge or corona discharge.
- 細胞接着性処理が、紫外線照射により行われる、請求項２に記載の細胞培養器具。 The cell culture instrument according to claim 2, wherein the cell adhesion treatment is performed by ultraviolet irradiation.
- 細胞接着性処理された領域が、２～４ｍｍ間隔で存在する直径１～４ｍｍの点状の領域である、請求項１～３のいずれか一項に記載の細胞培養器具。 The cell culture device according to any one of claims 1 to 3, wherein the cell-adhesive-treated region is a dotted region having a diameter of 1 to 4 mm that exists at intervals of 2 to 4 mm.
- 細胞接着性処理された領域が、培養基材表面全体の４％～６５％である、請求項１～４のいずれか一項に記載の細胞培養器具。 The cell culture instrument according to any one of claims 1 to 4, wherein the area subjected to the cell adhesion treatment is 4% to 65% of the entire culture substrate surface.
- シート状細胞培養物の製造方法であって、請求項１～５のいずれか一項に記載の細胞培養器具の培養基材表面に細胞を播種し、インキュベートすることを含む、前記方法。 A method for producing a sheet-shaped cell culture, which comprises seeding and incubating cells on the surface of a culture substrate of the cell culture device according to any one of claims 1 to 5.
- 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、請求項６に記載の方法。 The method according to claim 6, further comprising peeling the sheet-shaped cell culture formed on the culture substrate from the substrate.
- 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、請求項６または７に記載の方法。 The method according to claim 6 or 7, wherein the cells are seeded at a density capable of forming a sheet-shaped cell culture without substantially growing.
- 細胞が、骨格筋芽細胞である、請求項６～８のいずれか一項に記載の方法。 The method according to any one of claims 6 to 8, wherein the cell is a skeletal myoblast.
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