JP2016052270A - Sheet-like cell culture recovery system and method - Google Patents
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Abstract
Description
本発明は、シート状細胞培養物を回収するための方法および該方法を用いるためのシステムに関する。 The present invention relates to a method for recovering a sheet cell culture and a system for using the method.
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 a severe decrease in 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.
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１に記載の温度応答性培養皿は、培養皿からの細胞培養物の剥離を容易にしたものの、完全に剥離させ得るものではなく、完全な剥離のためにはピペッティングなどの人為的操作が必要であることから、コンタミネーションや皺、破れ、破損などが生じる原因となり、とくに脆弱な細胞培養物を剥離する際には慎重を要するものである。 Although the temperature-responsive culture dish described in Patent Document 1 facilitates the exfoliation of the cell culture from the culture dish, it cannot be completely exfoliated, and for complete exfoliation, an artificial process such as pipetting is required. Because manipulation is necessary, it can cause contamination, wrinkles, tears, breakage, etc., and is especially cautious when peeling fragile cell cultures.
本発明の目的は、培養面からシート状細胞培養物を簡便、迅速かつ破損させることなく回収する方法およびかかる方法を実行するためのシステムを提供することにある。 An object of the present invention is to provide a method for recovering a sheet-shaped cell culture from a culture surface in a simple, rapid and without damaging manner and a system for executing such a method.
剥離の際に問題となる前記の人為的操作を減らすことを目的として、剥離作業を自動化するシステムが提供された（特許文献２および３）。しかしながら、これらのシステムもまた、とくに脆弱なシート状細胞培養物を扱う際には、剥離に時間がかかり、剥離時に皺、破れ、破損などが生じる可能性が依然として残るものであった。 For the purpose of reducing the above-mentioned artificial operation which becomes a problem at the time of peeling, a system for automating the peeling work has been provided (Patent Documents 2 and 3). However, these systems also take a long time to peel off, particularly when handling fragile sheet-like cell cultures, and there is still a possibility that wrinkles, tears, breakage, etc. will occur at the time of peeling.
そこで本発明者らは、さらなるシート状細胞培養物の剥離方法を研究する中で、液体中のシート状細胞培養物を振動させる際に、往復振動を与えることで、回転振動を与えるよりも迅速に剥離することができ、振動を与える時間を短時間にすることが可能となるために振動による破損を生じるリスクを低減できることを見出した。さらに、剥離の際にシート状細胞培養物にしわやよれが生じた場合、液体中のシート状細胞培養物を中心として回転振動を与えることにより、シートを伸展し、しわやよれを改善することができることを見出し、鋭意研究を重ねた結果、本発明を完成するに至った。 Accordingly, the present inventors studied a further method of peeling a sheet-shaped cell culture, and when vibrating the sheet-shaped cell culture in a liquid, by applying a reciprocating vibration, the present invention was quicker than a rotational vibration. It has been found that the risk of breakage due to vibration can be reduced because it can be peeled off and the time for applying vibration can be shortened. Furthermore, when wrinkling or twisting occurs in the sheet-shaped cell culture during peeling, the sheet is stretched around the sheet-shaped cell culture in the liquid to extend the sheet and improve wrinkling or twisting. As a result of finding out that it can be performed and intensive research, 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を接着培養面から剥離する方法であって、接着培養面に接着したシート状細胞培養物が容器内の液体中に浸漬した状態で、該液体を振動させること（容器を回転または円振動させることによって振動させることを除く）を含む、前記方法。
〔２〕シート状細胞培養物が、脆弱なシート状細胞培養物である、〔１〕の方法。
〔３〕液体の振動が、容器を往復振動することによって生じるものであり、該往復振動の振動数が一定であり、振幅がシート状細胞培養物と接着培養面との接着面積の減少に従って増大する、〔１〕または〔２〕の方法。
〔４〕振動数が、２００ｒｐｍ以下である、〔３〕の方法。
〔５〕液体の往復振動が、容器を往復振動することによって生じるものであり、該振動の振幅が一定であり、振動数がシート状細胞培養物と接着培養面との接着面積の減少に従って増大する、〔１〕または〔２〕の方法。
〔６〕液体が、シート状細胞培養物がちょうど浸漬する量の液体である、〔１〕〜〔５〕の方法。
〔７〕液体がハンクス平衡塩溶液である、〔６〕の方法。
〔８〕接着培養面が、周辺環境に依存して性状が変化するものである、〔１〕〜〔７〕の方法。
That is, the present invention relates to the following.
[1] A method of peeling a sheet-like cell culture from an adhesion culture surface, wherein the liquid is vibrated in a state where the sheet-like cell culture adhered to the adhesion culture surface is immersed in a liquid in a container ( Oscillating the container by rotating or circularly oscillating).
[2] The method according to [1], wherein the sheet-shaped cell culture is a fragile sheet-shaped cell culture.
[3] The vibration of the liquid is caused by reciprocating vibration of the container, the frequency of the reciprocating vibration is constant, and the amplitude increases as the adhesion area between the sheet-like cell culture and the adhesion culture surface decreases. The method of [1] or [2].
[4] The method of [3], wherein the frequency is 200 rpm or less.
[5] The reciprocating vibration of the liquid is caused by reciprocating vibration of the container, the amplitude of the vibration is constant, and the frequency increases as the adhesion area between the sheet-like cell culture and the adhesion culture surface decreases. The method of [1] or [2].
[6] The method of [1] to [5], wherein the liquid is an amount of liquid in which the sheet-like cell culture is just immersed.
[7] The method according to [6], wherein the liquid is a Hanks balanced salt solution.
[8] The method of [1] to [7], wherein the adhesion culture surface changes in properties depending on the surrounding environment.
〔９〕シート状細胞培養物を伸展させる方法であって、シート状細胞培養物が容器内の液体中に浸漬した状態で、前記容器内の液体を回転させることを含む、前記方法。
〔１０〕シート状細胞培養物が、脆弱なシート状細胞培養物である、〔９〕の方法。
〔１１〕容器内の液体の回転が、容器を回転振動させることにより生じるものである、〔９〕または〔１０〕の方法。
〔１２〕液体が、ハンクス平衡塩液である、〔９〕〜〔１１〕の方法。
[9] A method for extending a sheet-shaped cell culture, the method comprising rotating the liquid in the container while the sheet-shaped cell culture is immersed in the liquid in the container.
[10] The method according to [9], wherein the sheet-shaped cell culture is a fragile sheet-shaped cell culture.
[11] The method according to [9] or [10], wherein the rotation of the liquid in the container is caused by rotating and vibrating the container.
[12] The method according to [9] to [11], wherein the liquid is Hanks balanced salt solution.
本発明によれば、従来よりも簡便、迅速に、かつ破損のリスクを低減して、シート状細胞培養物を接着培養面から剥離することが可能となる。本発明により、シート状細胞培養物の製造において律速工程かつ品質低下のリスクを有する工程であったシート状細胞培養物の剥離工程が改善される。また、よれやしわが生じた場合であっても、剥離作業に引き続き迅速に伸展作業を行うことが可能となるため、破損のリスク低減と相俟って、一層品質を向上できる。したがって、シート状細胞培養物の生産効率向上および品質改善に寄与することができる。 According to the present invention, it is possible to peel a sheet-like cell culture from an adhesion culture surface more easily and quickly than before and with reduced risk of breakage. According to the present invention, the sheet cell culture exfoliation process, which is a rate-limiting process and a process having a risk of quality deterioration in the production of the sheet cell culture, is improved. In addition, even when wrinkles or wrinkles occur, the extension work can be performed quickly following the peeling work, so that the quality can be further improved in combination with the risk of damage. Therefore, it can contribute to the production efficiency improvement and quality improvement of a sheet-like cell culture.
本発明は、簡便、迅速かつ破損のリスクを抑えたシート状細胞培養物の剥離方法に関する。また、しわ、よれの生じたシート状細胞培養物を伸展し、しわ、よれを解消するための、シート状細胞培養物の伸展方法に関する。さらに、前記剥離方法を用いる剥離工程および前記伸展方法を用いる伸展工程を含む、シート状細胞培養物の回収方法、並びにかかる方法を実現するためのシステムに関する。
以下、本発明の好適な実施態様に基づき、本発明を説明する。
（１）剥離方法
本発明のシート状細胞培養物の回収方法は、シート状細胞培養物を接着培養面から剥離する工程を包含する。したがって本発明は、１つの側面において、接着培養面に接着したシート状細胞培養物が容器内の液体中に浸漬した状態で、該液体に接着培養面に並行に単一軸の往復振動をさせることを含む、シート状細胞培養物を接着培養面から剥離する方法に関する。
The present invention relates to a method for exfoliating a sheet-shaped cell culture that is simple, quick and suppresses the risk of breakage. Further, the present invention relates to a method for extending a sheet-shaped cell culture for extending the wrinkled and kinked sheet-shaped cell culture and eliminating wrinkles and kinking. Furthermore, it is related with the collection | recovery method of a sheet-like cell culture including the peeling process using the said peeling method, and the extending process using the said extending method, and the system for implement | achieving this method.
Hereinafter, the present invention will be described based on preferred embodiments of the present invention.
(1) Peeling method The method for recovering a sheet-like cell culture of the present invention includes a step of peeling the sheet-like cell culture from the adhesion culture surface. Accordingly, in one aspect, the present invention allows a single-axis reciprocal vibration to be performed in parallel to the adhesion culture surface in a state where the sheet-like cell culture adhered to the adhesion culture surface is immersed in the liquid in the container. The present invention relates to a method of peeling a sheet-shaped cell culture from an adhesion culture surface.
本発明において「シート状細胞培養物」は、典型的には哺乳動物、例えば、ヒトや家畜等の疾病、傷病の治療等に用いられるものであるが、これに限定されるものではない。細胞培養物とは、典型的には培養した細胞および／またはその産生物で構成されるが、生体の所定部（例えば患部等）を補填および／または支持するための各種の材料（補填材料や支持材料）なども含む。 In the present invention, “sheet-like cell culture” is typically used for the treatment of diseases and injuries of mammals such as humans and domestic animals, but is not limited thereto. The cell culture is typically composed of cultured cells and / or products thereof, but various materials (such as supplementary materials and supplementary materials) for supplementing and / or supporting a predetermined part (for example, an affected part) of a living body. Support material).
シート状細胞培養物を構成する細胞は、シート状細胞培養物を形成し得るものであれば特に限定されず、例えば、接着細胞（付着性細胞）を含む。接着細胞は、例えば、接着性の体細胞（例えば、心筋細胞、線維芽細胞、上皮細胞、内皮細胞、肝細胞、膵細胞、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞、滑膜細胞、軟骨細胞など）および幹細胞（例えば、筋芽細胞、心臓幹細胞などの組織幹細胞、胚性幹細胞、ｉＰＳ（induced pluripotent stem）細胞などの多能性幹細胞、間葉系幹細胞等）などを含む。体細胞は、幹細胞、特にｉＰＳ細胞から分化させたものであってもよい。シート状細胞培養物を構成する細胞の例としては、限定されずに、筋芽細胞（例えば、骨格筋芽細胞）、間葉系幹細胞（例えば、骨髄、脂肪組織、末梢血、皮膚、毛根、筋組織、子宮内膜、胎盤、臍帯血由来のものなど）、心筋細胞、線維芽細胞、心臓幹細胞、胚性幹細胞、ｉＰＳ細胞、滑膜細胞、軟骨細胞、上皮細胞（例えば、口腔粘膜上皮細胞、網膜色素上皮細胞、鼻粘膜上皮細胞など）、内皮細胞（例えば、血管内皮細胞など）、肝細胞（例えば、肝実質細胞など）、膵細胞（例えば、膵島細胞など）、腎細胞、副腎細胞、歯根膜細胞、歯肉細胞、骨膜細胞、皮膚細胞等が挙げられる。シート状細胞培養物を構成する細胞が由来する動物も特に限定されず、例えば、ヒト、非ヒト霊長類、イヌ、ネコ、ブタ、ウマ、ヤギ、ヒツジなどが含まれる。 The cells constituting the sheet-shaped cell culture are not particularly limited as long as they can form a sheet-shaped cell culture, and include, for example, adherent cells (adherent cells). Adherent cells include, for example, adherent somatic cells (eg, cardiomyocytes, fibroblasts, epithelial cells, endothelial cells, hepatocytes, pancreatic cells, kidney cells, adrenal cells, periodontal cells, gingival cells, periosteum cells, skin Cells, synovial cells, chondrocytes, etc.) and stem cells (eg tissue stem cells such as myoblasts, cardiac stem cells, embryonic stem cells, pluripotent stem cells such as iPS (induced pluripotent stem) cells, mesenchymal stem cells, etc.) Etc. Somatic cells may be differentiated from stem cells, particularly iPS cells. Examples of cells constituting the sheet-shaped cell culture include, but are not limited to, myoblasts (for example, skeletal myoblasts), mesenchymal stem cells (for example, bone marrow, adipose tissue, peripheral blood, skin, hair root, Muscle tissue, endometrium, placenta, umbilical cord blood-derived), cardiomyocytes, fibroblasts, cardiac stem cells, embryonic stem cells, iPS cells, synovial cells, chondrocytes, epithelial cells (eg, oral mucosal epithelial cells) Retinal pigment epithelial cells, nasal mucosal epithelial cells, etc.), endothelial cells (eg, vascular endothelial cells), hepatocytes (eg, liver parenchymal cells), pancreatic cells (eg, islet cells), kidney cells, adrenal cells , Periodontal ligament cells, gingival cells, periosteum cells, skin cells and the like. The animal from which the cells constituting the sheet-shaped cell culture are derived is not particularly limited, and includes, for example, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep and the like.
本発明において、「接着培養面」（単に「培養面」と記載することもある）とは、シートを形成する細胞が接着する面を意味する。通常は接着培養面に接着する形でシート状細胞培養物が形成される。接着培養面としては、典型的には細胞培養容器の培養面、細胞培養担体の表面などが挙げられるが、これに限定されるものではない。接着培養面は、液体を入れた容器に必ずしも固定されている必要はないが、好ましくは固定されている。 In the present invention, “adhesion culture surface” (sometimes simply referred to as “culture surface”) means a surface to which cells forming a sheet adhere. Usually, a sheet-like cell culture is formed so as to adhere to the adhesion culture surface. The adhesion culture surface typically includes the culture surface of a cell culture container, the surface of a cell culture carrier, and the like, but is not limited thereto. The adhesion culture surface does not necessarily have to be fixed to the container containing the liquid, but is preferably fixed.
本発明の剥離方法は、シート状細胞培養物が液体中に浸漬した状態で実行される。本発明において「液体中に浸漬」された状態とは、静置時にシート全体が液体に浸かっている状態をいう。すなわち、静置時にシート状細胞培養物が液体に覆われた状態であれば「液体中に浸漬」された状態であり、その状態から振動を与えた場合にシートの一部が一時的に液体の外部に露出することがあったとしても、再び静置した場合に液体に覆われた状態に戻れば、一時的に外部に露出した状態もまた「液体中に浸漬」された状態であるといえる。 The peeling method of the present invention is performed in a state where the sheet-shaped cell culture is immersed in a liquid. In the present invention, the state of being “immersed in the liquid” means a state in which the entire sheet is immersed in the liquid at the time of standing. That is, if the sheet-like cell culture is in a state of being covered with liquid at the time of standing, it is in a state of being “immersed in the liquid”, and a part of the sheet is temporarily liquid when subjected to vibration from that state. Even if it is exposed to the outside, if it returns to the state covered with the liquid when it is left standing again, the state of being temporarily exposed to the outside is also a state of being “immersed in the liquid” I can say that.
本発明の剥離方法においては、シート状細胞培養物を浸漬させた液体を振動させることで、液体とシート状細胞培養物との間に摩擦力を生じさせ、かかる摩擦力がシート状細胞培養物を接着培養面から剥離する力となって剥離が可能となるものと考えられる。したがって、シート状細胞培養物と接着培養面とが接着している面の外縁に対して垂直に剥離力が作用するのが最も効率よく剥離が進行する。逆に該外縁に対して平行に近い方向に剥離力が作用するのが最も効率が悪いことになる。したがって容器内の液体が回転する方向に運動する円振動や回転は好ましくない。 In the peeling method of the present invention, the liquid in which the sheet-shaped cell culture is immersed is vibrated to generate a frictional force between the liquid and the sheet-shaped cell culture, and the frictional force is generated by the sheet-shaped cell culture. It is considered that peeling becomes possible due to the force of peeling from the adhesion culture surface. Therefore, the peeling progresses most efficiently when the peeling force acts perpendicularly to the outer edge of the surface where the sheet-shaped cell culture and the adhesion culture surface are bonded. On the contrary, it is the most inefficient that the peeling force acts in a direction nearly parallel to the outer edge. Therefore, circular vibration or rotation that moves in the direction in which the liquid in the container rotates is not preferable.
本発明の剥離方法は、接着培養面に接着したシート状細胞培養物が容器内の液体中に浸漬した状態で、該液体を振動させることを含む。かかる振動は液体とシート状細胞培養物との間に生じる摩擦力が、シート状細胞培養物を接着培養面から剥離する力となる限りいかなる振動でもよい。典型的には、例えば接着培養面に平行な単一軸の往復振動などが挙げられる。ここで、「接着培養面に平行に単一軸の往復振動をする」とは、往復振動の軸が接着培養面と平行な平面上に存在し、一つの軸（「振動軸」ともいう）に沿って行き来することをいう。このとき、振動軸自体が固定されている必要は必ずしもなく、任意に変化してもよい。ただし、上述のとおり、剥離力の作用効率の観点から、振動軸の変化と往復振動とが組み合わさって全体の動きが円振動となる場合は望ましくない。したがって好ましくは、本発明の剥離方法において、容器を回転または円振動させて、液体が容器内で回転するように運動する場合を含まない。 The peeling method of the present invention includes vibrating the liquid in a state where the sheet-like cell culture adhered to the adhesion culture surface is immersed in the liquid in the container. Such vibration may be any vibration as long as the frictional force generated between the liquid and the sheet-shaped cell culture becomes a force for peeling the sheet-shaped cell culture from the adhesion culture surface. Typically, for example, a single axis reciprocating vibration parallel to the adhesion culture surface can be mentioned. Here, “single axis reciprocating vibration parallel to the adhesion culture surface” means that the axis of reciprocation vibration exists on a plane parallel to the adhesion culture surface, and one axis (also referred to as “vibration axis”). To go back and forth. At this time, the vibration shaft itself does not necessarily have to be fixed and may be arbitrarily changed. However, as described above, from the viewpoint of the working efficiency of the peeling force, it is not desirable when the change of the vibration axis and the reciprocating vibration combine to make the entire movement a circular vibration. Therefore, preferably, the peeling method of the present invention does not include a case where the liquid is rotated or circularly oscillated so that the liquid moves in the container.
液体に振動をさせる方法としては、液体中に存在するシート状細胞培養物を破損させない限りにおいて、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できる。具体的には、これに限定するものではないが、例えば液体の入った容器を往復運動させて振動する、板状の攪拌子などを液体中で往復させる、などの方法が挙げられる。シート状細胞培養物に直接的にダメージを与えるリスクが少ない、コンタミネーションのリスクが少ない、などの観点から、液体に直接触れずに容器を運動させることにより、液体を振動させる方法が好ましい。容器の運動としては、例えば水平振動、揺動、回転、円振動、水平偏芯震動などが挙げられ、剥離力の作用効率などの観点から、好ましくは水平振動、揺動、水平偏芯震動などが挙げられる。 As a method for vibrating the liquid, any method known in the art can be used as long as the sheet-shaped cell culture existing in the liquid is not damaged. Specific examples include, but are not limited to, a method in which a container containing liquid is reciprocated to vibrate, or a plate-like stirrer is reciprocated in the liquid. From the viewpoint of reducing the risk of directly damaging the sheet-shaped cell culture and reducing the risk of contamination, a method in which the liquid is vibrated by moving the container without directly touching the liquid is preferable. Examples of the movement of the container include horizontal vibration, rocking, rotation, circular vibration, horizontal eccentric vibration, and the like. From the viewpoint of the action efficiency of the peeling force, preferably horizontal vibration, rocking, horizontal eccentric vibration, etc. Is mentioned.
本発明の剥離方法は、シート状細胞培養物を浸漬した液体とシート状細胞培養物との間の摩擦力を剥離力とするものであるため、剥離力が極端に強くなることや偏って負荷がかかることがない。したがって、とくにシート状細胞培養物が脆弱なシート状細胞培養物である場合に好適に用いることができる。「脆弱」なシートとは、例えば液体外でシートのつかみ具への固定がなされる通常の引張試験機（例えば、ＪＩＳ Ｋ ７１６１等に記載のもの）での引張特性の評価が、その脆弱性のために困難であるか、実質的に不可能であるシートを指す。かかるシートとしては、例えば、引張特性の各数値が小さく、通常の引張試験機では正確に測定することが困難な試料などが挙げられる。かかる脆弱なシートとしては、引張試験において、例えば、１０Ｎ（ニュートン）未満、５Ｎ未満、２Ｎ未満、１Ｎ未満、０．５Ｎ未満、０．１Ｎ未満、０．０５Ｎ未満の破断荷重を示すものが挙げられる。また、通常の引張試験の測定限界は、破断荷重として一般に１Ｎ程度であるため、好ましくは、これを下回る破断荷重（例えば、０．５Ｎ未満）を示すものが挙げられる。 The peeling method of the present invention uses the frictional force between the liquid in which the sheet-shaped cell culture is immersed and the sheet-shaped cell culture as the peeling force, so that the peeling force becomes extremely strong or unevenly loaded. Will not take. Therefore, it can be suitably used particularly when the sheet-shaped cell culture is a fragile sheet-shaped cell culture. “Vulnerable” sheet refers to, for example, the evaluation of tensile properties with a normal tensile tester (for example, as described in JIS K 7161) in which the sheet is fixed to the gripping tool outside the liquid. Refers to a sheet that is difficult or substantially impossible. Examples of such a sheet include a sample that has a small numerical value of tensile properties and is difficult to measure accurately with a normal tensile testing machine. Examples of such fragile sheets include those exhibiting a breaking load of less than 10N (Newton), less than 5N, less than 2N, less than 1N, less than 1N, less than 0.5N, less than 0.1N, and less than 0.05N in a tensile test. It is done. Moreover, since the measurement limit of a normal tensile test is generally about 1 N as the breaking load, preferably, a breaking load lower than this (for example, less than 0.5 N) is exemplified.
剥離対象であるシート状細胞培養物が脆弱なシートである場合、ピンセットなどの器具でつまむことが困難であることや、剥離する際に破損が生じるリスクが高いことなどの理由から、細胞培養面からの剥離が格段に困難となる。しかし本態様の剥離方法を用いることにより、迅速かつ簡便に剥離することが可能となるため、脆弱なシートであっても破損するリスクが最小限に抑えられる。 When the cell culture to be peeled is a fragile sheet, it is difficult to pinch with an instrument such as tweezers, or because there is a high risk of breakage when peeling, the cell culture surface Detachment from the plate becomes extremely difficult. However, by using the peeling method of this aspect, it becomes possible to peel quickly and easily, so that even a fragile sheet can be minimized.
本方法に用いることができる細胞は、シート状細胞培養物を形成できる細胞（「シート形成細胞」ともいう）であればとくに限定されず、当該技術分野において知られたあらゆる細胞を用いることができる。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る。本発明の剥離方法は、治療上の有用性や、脆弱性に由来する慣用の技法によるハンドリングの難しさなどの観点から、骨格筋芽細胞からなるシート状細胞培養物に対して用いるのがとくに好ましい。 The cells that can be used in the present method are not particularly limited as long as they can form a sheet-like cell culture (also referred to as “sheet-forming cells”), and any cells known in the art can be used. . Specific examples 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. The exfoliation method of the present invention is particularly used for sheet-like cell cultures composed of skeletal myoblasts from the viewpoint of therapeutic utility and difficulty in handling by conventional techniques derived from vulnerability. preferable.
上述のとおり、本発明の好ましい一態様において、液体の振動は、容器を往復振動することにより生じるものである。本発明において「往復振動」という語は、単一軸方向の往復運動を意味し、好ましくは水平振動（水平横振とう）である。以下別段の記載のない限り、かかる好適な態様を用いて本発明の剥離方法を説明するものとする。
かかる態様において、振動数が一定である場合、振幅の大きさを変更することにより、シート状細胞培養物に作用する剥離力を変更することができる。また逆に、振幅が一定である場合、振動数を変更することにより、シート状細胞培養物に作用する剥離力を変更することができる。ここで本明細書において「剥離力」とは、シート状細胞培養物に作用する、接着培養面からシート状細胞培養物を剥がそうとする力を意味する。シート状細胞培養物に剥離力を作用させた場合、シート状細胞培養物と接着培養面との接着力が抵抗力となり、その結果シート状細胞培養物に剪断力が生じる、すなわち破断荷重がかかることとなる。
As described above, in a preferred embodiment of the present invention, the vibration of the liquid is caused by reciprocating the container. In the present invention, the term “reciprocating vibration” means a reciprocating motion in a single axial direction, preferably horizontal vibration (horizontal lateral vibration). Hereinafter, unless otherwise specified, the peeling method of the present invention will be described using this preferred embodiment.
In such an embodiment, when the frequency is constant, the peel force acting on the sheet-shaped cell culture can be changed by changing the amplitude. Conversely, when the amplitude is constant, the peeling force acting on the sheet-shaped cell culture can be changed by changing the frequency. As used herein, “peeling force” means a force acting on a sheet-like cell culture to peel the sheet-like cell culture from the adhesion culture surface. When a peeling force is applied to a sheet-like cell culture, the adhesion force between the sheet-like cell culture and the adhesion culture surface becomes a resistance force, and as a result, a shearing force is generated on the sheet-like cell culture, that is, a breaking load is applied. It will be.
剥離に際しては、シート状細胞培養物と接着培養面との接着面積に比例して、シート状細胞培養物と接着培養面との接着力が大きくなると考えられる。したがって剥離開始段階における接着力が最も大きい。接着力が大きい場合、大きな剥離力を作用させる、すなわち大きな振幅で振動すると、シート状細胞培養物が破損するリスクが高まることになる。したがって剥離開始段階においては小さな剥離力を作用させる、すなわち小さな振幅で振動することが好ましい。しかしながら剥離が進むにつれ、接着力も低下してくると考えられるため、大きな剥離力を作用させた場合であってもシート状細胞培養物の破損のリスクは低下し、逆に剥離力が大きい方が剥離の進行が速くなる。したがって剥離の進行、すなわち接着面積の減少に従って振幅を大きくするのが好ましい。 At the time of peeling, it is considered that the adhesive force between the sheet-like cell culture and the adhesion culture surface increases in proportion to the adhesion area between the sheet-like cell culture and the adhesion culture surface. Therefore, the adhesive strength at the start of peeling is the largest. When the adhesive force is large, applying a large peeling force, that is, vibrating with a large amplitude increases the risk of damaging the sheet-like cell culture. Therefore, it is preferable to apply a small peeling force at the start of peeling, that is, to vibrate with a small amplitude. However, as peeling progresses, the adhesive force is also expected to decrease, so even if a large peeling force is applied, the risk of breakage of the sheet-shaped cell culture is reduced. Peeling progresses faster. Therefore, it is preferable to increase the amplitude as the peeling progresses, that is, as the adhesion area decreases.
振幅の制御は、いかなる方法で行ってもよい。振幅制御の例としては、これに限定するものではないが、例えば剥離の進行状況を、例えば画像解析、光学センサー、目視などにより随時光学的に観察し、剥離の進行に合わせて振幅を増大させていってもよい。また、剥離の進行は、同一条件下においては剥離開始からの経過時間と相関すると考えられるため、剥離開始からの時間経過とともに振幅を増大させてもよい。
振幅の大きさは、振動する容器の形状や種類、シート状細胞培養物の脆弱さ、液体の量や種類などにより変化し得、当業者であれば適切な数値を適宜決定できる。例えば、シート状細胞培養物が骨格筋芽細胞である場合、およそ０．１ｃｍ〜１０ｃｍ程度の振幅が適切である。
The amplitude may be controlled by any method. An example of amplitude control is not limited to this. For example, the progress of peeling is optically observed, for example, by image analysis, an optical sensor, visual observation, etc., and the amplitude is increased in accordance with the progress of peeling. May be. Further, since the progress of peeling is considered to correlate with the elapsed time from the start of peeling under the same conditions, the amplitude may be increased with the passage of time from the start of peeling.
The magnitude of the amplitude can vary depending on the shape and type of the vibrating container, the fragility of the sheet-shaped cell culture, the amount and type of the liquid, and a person skilled in the art can appropriately determine an appropriate numerical value. For example, when the sheet-like cell culture is a skeletal myoblast, an amplitude of about 0.1 cm to 10 cm is appropriate.
振動数が一定である場合は振幅が大きければ剥離力が大きいと考えられるが、逆に振幅が一定である場合であれば、振動数が大きければ剥離力が大きいと考えられる。したがって大きな振動数での振動は、シート状細胞培養物を破損するリスクを高めることとなる。振動数も振幅と同様、振動する容器の形状や種類、シート状細胞培養物の脆弱さ、液体の量や種類などにより変化し得、当業者であれば適切な数値を適宜決定できる。例えば、シート状細胞培養物が骨格筋芽細胞である場合、およそ５ｒｐｍ〜３００ｒｐｍ程度の振動数が適切である。 When the frequency is constant, it is considered that the peeling force is large when the amplitude is large. Conversely, when the amplitude is constant, the peeling force is considered large when the frequency is large. Therefore, vibration at a large frequency increases the risk of damaging the sheet cell culture. Similarly to the amplitude, the vibration frequency can vary depending on the shape and type of the vibrating container, the weakness of the sheet-shaped cell culture, the amount and type of the liquid, and those skilled in the art can appropriately determine an appropriate numerical value. For example, when the sheet-like cell culture is a skeletal myoblast, a frequency of about 5 rpm to about 300 rpm is appropriate.
シート状細胞培養物を浸漬する液体は、シート状細胞培養物の品質を低下させる（例えば細胞毒性を有する、製造工程不純物を含む、など）でなければとくに限定されない。液体を入れ替える工程が不要となることから、好ましい一態様において、液体として培養培地を用いる。他の液体の例としては、これに限定するものではないが、例えばハンクス平衡塩溶液（ＨＢＳＳ）、アール平衡塩液（ＥＢＳＳ）、ＨＥＰＥＳ、リン酸緩衝生理食塩水（ＰＢＳ）、電解質輸液（大塚生食注）などが挙げられる。また、シート状細胞培養物を補強する目的のため、シート状細胞培養物の支持体とともに振動させてもよい。かかる支持体としては、これに限定するものではないが、例えばコラーゲン、フィブリノーゲンなどの生体材料、ポリ乳酸などの生分解性ポリマーなどから製造された薄膜シートなどが挙げられる。 The liquid in which the sheet-shaped cell culture is immersed is not particularly limited as long as the quality of the sheet-shaped cell culture is not deteriorated (for example, it has cytotoxicity, contains manufacturing process impurities, etc.). In a preferred embodiment, a culture medium is used as the liquid because the step of replacing the liquid becomes unnecessary. Examples of other liquids include, but are not limited to, Hanks balanced salt solution (HBSS), Earl balanced salt solution (EBSS), HEPES, phosphate buffered saline (PBS), electrolyte infusion (Otsuka Raw food injection). Moreover, you may vibrate with the support body of a sheet-like cell culture for the purpose of reinforcing a sheet-like cell culture. Examples of such a support include, but are not limited to, a biomaterial such as collagen and fibrinogen, and a thin film sheet manufactured from a biodegradable polymer such as polylactic acid.
容器に入れる液体の量は、シート状細胞培養物を浸漬可能な量であればとくに限定されない。シート状細胞培養物を浸積可能な量は、容器の大きさやシート状細胞培養物の厚み、シート形成細胞の種類などにより変化し得、当業者であれば適切な量をすぐに決定できる。一般に容器中の液体が占める範囲が、シート状細胞培養物が自由に移動できる範囲であると考えられるが、シートのしわ、よれの発生を抑制する、シートの破損のリスクを低減させる、などの観点から、液体の量はなるべく少ない方が好ましい。したがって、好ましい態様において、液体の量は、シート状細胞培養物がちょうど浸漬する量である。具体的には例えば、UpCell３．５ｃｍ（株式会社セルシード、型番：CS3007）の場合は、約１．５５ｍｌであるが、これに限定するものではない。また、剥離終了後に後続の工程に最適化させるために液量を調整してもよい。 The amount of the liquid to be placed in the container is not particularly limited as long as it is an amount capable of immersing the sheet-shaped cell culture. The amount that can be immersed in the sheet-shaped cell culture can vary depending on the size of the container, the thickness of the sheet-shaped cell culture, the type of sheet-forming cells, etc., and those skilled in the art can readily determine an appropriate amount. In general, the range occupied by the liquid in the container is considered to be the range in which the sheet-like cell culture can move freely, but it suppresses the occurrence of wrinkling and kinking of the sheet, reduces the risk of damage to the sheet, etc. From the viewpoint, the amount of liquid is preferably as small as possible. Thus, in a preferred embodiment, the amount of liquid is the amount that the sheet-like cell culture is just immersed. Specifically, for example, in the case of UpCell 3.5 cm (CELLSEED Co., Ltd., model number: CS3007), it is about 1.55 ml, but is not limited thereto. In addition, the amount of liquid may be adjusted after the end of peeling in order to optimize the subsequent process.
接着培養面は、シート状細胞培養物を剥離しやすいように、その周辺環境に依存して性状が変化するものであってよい。本態様において接着培養面の「周辺環境」には、典型的には容器内の液体の温度、ｐＨ、接着角（親水性または疎水性）などが挙げられる。接着培養面の「性状が変化する」とは、接着培養面の有する物理化学的性質が変化することをいい、好ましくは接着培養面とシート状細胞培養物の接着性に関与する性状が変化する。性状変化の例としては、これに限定するものではないが、例えば親水性が疎水性に変化する（または疎水性が親水性に変化する）などの変化が挙げられる。「周辺環境に依存して性状が変化する」とは、物理化学的性質の変化が周辺環境の変化によって引き起こされることを意味し、典型的には温度変化に応答して親水性が変化する、ｐＨ変化に応答して吸水性が変化する、などが挙げられる。 The adhesion culture surface may change its properties depending on the surrounding environment so that the sheet-like cell culture can be easily peeled off. In this embodiment, the “ambient environment” of the adhesion culture surface typically includes the temperature, pH, adhesion angle (hydrophilic or hydrophobic) of the liquid in the container. “Change in properties” of an adhesion culture surface means a change in the physicochemical properties of the adhesion culture surface, and preferably the properties related to the adhesion between the adhesion culture surface and the sheet-like cell culture change. . Examples of property changes include, but are not limited to, changes such as changes in hydrophilicity to hydrophobicity (or changes in hydrophobicity to hydrophilicity). “The property changes depending on the surrounding environment” means that a change in physicochemical properties is caused by a change in the surrounding environment, and the hydrophilicity typically changes in response to a temperature change. The water absorption changes in response to a change in pH.
上記剥離方法による剥離の後、シート状細胞培養物によれやしわが生じている場合、続けてかかるよれやしわを解消するための伸展工程が行われてよい。 After peeling by the above peeling method, when wrinkles or wrinkles are generated in the sheet-like cell culture, an extension step for eliminating the wrinkles or wrinkles may be performed.
（２）伸展方法
本発明のシート状細胞培養物の回収方法は、接着培養面から剥離したシート状細胞培養物にしわやよれが生じていた場合、シート状細胞培養物を伸展してしわやよれを解消する工程を包含する。したがって本発明は、１つの側面において、シート状細胞培養物が容器内の液体中に浸漬した状態で、前記容器内の液体を回転させることを含む、シート状細胞培養物を伸展する方法に関する。
(2) Extension method When the sheet-like cell culture recovery method of the present invention is wrinkled or wrinkled on the sheet-like cell culture peeled from the adhesion culture surface, the sheet-like cell culture is extended to cause wrinkles. It includes a step of eliminating the twist. Therefore, in one aspect, the present invention relates to a method for extending a sheet-shaped cell culture, comprising rotating the liquid in the container while the sheet-shaped cell culture is immersed in the liquid in the container.
本発明において、シート状細胞培養物を「伸展」するとは、シート状細胞培養物に生じたしわやよれを、伸ばして解消することを意味する。
本発明の伸展方法は、シート状細胞培養物を浸漬した液体を容器内で回転させることにより、液体中に遠心力を生じさせ、かかる遠心力およびシート状細胞培養物と液体との間の摩擦力によりシート状細胞培養物が外側に牽引され、結果として伸展できるものと考えられる。したがって容器内の液体は、容器の中央を中心として回転することが好ましい。
In the present invention, “stretching” a sheet-shaped cell culture means extending and eliminating wrinkles and kinks generated in the sheet-shaped cell culture.
The stretching method of the present invention causes a centrifugal force to be generated in the liquid by rotating the liquid in which the sheet-shaped cell culture is immersed in the container, and the centrifugal force and friction between the sheet-shaped cell culture and the liquid are generated. It is considered that the sheet-shaped cell culture is pulled outward by the force and can be extended as a result. Therefore, the liquid in the container is preferably rotated around the center of the container.
液体を回転させる方法としては、液体中に存在するシート状細胞培養物を破損させない限りにおいて、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できる。具体的には、これに限定するものではないが、例えば液体の入った容器を回転または回転振動させる、攪拌子などを液体中で回転させる、などの方法が挙げられる。シート状細胞培養物に直接的にダメージを与えるリスクが少ない、コンタミネーションのリスクが少ない、などの観点から、液体に直接触れずに容器を回転または回転振動させることにより、内部の液体を回転させる方法が好ましい。力の伝達効率を考えると、容器を回転振動させるのが最も好ましい。なお、本発明において、容器を「回転」させるとは、容器自体を自転させることをいい、容器を「回転振動」させるとは、容器を円運動させて振動することをいう。 As a method for rotating the liquid, any method known in the art can be used as long as the sheet-shaped cell culture existing in the liquid is not damaged. Specific examples include, but are not limited to, methods such as rotating or rotationally vibrating a container containing a liquid, and rotating a stirrer or the like in the liquid. From the standpoint of reducing the risk of directly damaging the sheet-like cell culture and reducing the risk of contamination, the container can be rotated or oscillated without direct contact with the liquid to rotate the liquid inside. The method is preferred. Considering the force transmission efficiency, it is most preferable to vibrate the container. In the present invention, “rotating” the container means rotating the container itself, and “rotating and vibrating” the container means vibrating the container by circular motion.
本発明の伸展方法は、シート状細胞培養物を浸漬した液体を回転させた際に生じる遠心力および、該液体とシート状細胞培養物との間の摩擦力を伸展させる力とするものであるため、力が極端に強くなることやシート状細胞培養物に偏って負荷がかかることがない。したがって、とくにシート状細胞培養物が脆弱なシート状細胞培養物である場合に好適に用いることができる。 The stretching method of the present invention is a force for stretching the centrifugal force generated when the liquid in which the sheet-shaped cell culture is immersed is rotated and the frictional force between the liquid and the sheet-shaped cell culture. Therefore, the force does not become extremely strong and the load is not applied to the sheet-shaped cell culture. Therefore, it can be suitably used particularly when the sheet-shaped cell culture is a fragile sheet-shaped cell culture.
伸展対象であるシート状細胞培養物が脆弱なシートである場合、ピンセットなどの器具でつまむことが困難であることや、伸展させる際に破損が生じるリスクが高いことなどの理由から、しわやよれが生じた場合にそれを解消することが格段に困難となる。しかし本態様の伸展方法を用いることにより、迅速、簡便かつ破損のリスクも低く伸展することが可能になる。 If the sheet-like cell culture to be stretched is a fragile sheet, it is difficult to pinch with an instrument such as tweezers or because it has a high risk of breakage when stretched. If this occurs, it will be much more difficult to eliminate it. However, by using the extending method of this embodiment, it is possible to extend quickly, simply and with a low risk of breakage.
本方法に用いることができる細胞は、シート形成細胞であればとくに限定されず、当該技術分野において知られたあらゆる細胞を用いることができる。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る。本発明の伸展方法は、治療上の有用性や、脆弱性に由来する慣用の技法によるハンドリングの難しさなどの観点から、骨格筋芽細胞からなるシート状細胞培養物に対して用いるのがとくに好ましい。 The cell that can be used in the present method is not particularly limited as long as it is a sheet-forming cell, and any cell known in the art can be used. Specific examples 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. The stretching method of the present invention is particularly used for a sheet-like cell culture composed of skeletal myoblasts from the viewpoint of therapeutic utility and difficulty in handling by conventional techniques derived from vulnerability. preferable.
シート状細胞培養物を浸漬する液体は、シート状細胞培養物の品質を低下させる（例えば細胞毒性を有する、製造工程不純物を含む、など）でなければとくに限定されず、上記（１）の剥離方法に用いられる液体と同様の液体を用い得る。本発明の伸展方法は、通常シート状細胞培養物を剥離した後、必要に応じて行われるものであることから、好ましい一態様において、前記剥離の際に用いた液体をそのまま使う。 The liquid in which the sheet-shaped cell culture is immersed is not particularly limited as long as it does not deteriorate the quality of the sheet-shaped cell culture (for example, it has cytotoxicity, contains manufacturing process impurities, etc.). Liquids similar to those used in the method can be used. Since the extending method of the present invention is usually performed as needed after exfoliating the sheet-shaped cell culture, in a preferred embodiment, the liquid used in the exfoliation is used as it is.
容器に入れる液体の量は、シート状細胞培養物を浸漬可能な量であればとくに限定されない。シート状細胞培養物を浸積可能な量は、容器の大きさやシート状細胞培養物の厚み、シート形成細胞の種類などにより変化し得、当業者であれば適切な量をすぐに決定できる。一般に容器中の液体が占める範囲が、シート状細胞培養物が自由に移動できる範囲であると考えられるが、シートのしわ、よれを伸展して解消する必要があることから、可動域は広い方がよいため、液体の量はなるべく多い方が好ましい。具体的には例えば、UpCell 3.5cm（株式会社セルシード、型番：CS3007）の場合は、約１．５５ｍｌであるが、これに限定するものではない。また、別の好ましい態様において、剥離工程の後、液量の調整を行うことなくそのまま用いられる。 The amount of the liquid to be placed in the container is not particularly limited as long as it is an amount capable of immersing the sheet-shaped cell culture. The amount that can be immersed in the sheet-shaped cell culture can vary depending on the size of the container, the thickness of the sheet-shaped cell culture, the type of sheet-forming cells, etc., and those skilled in the art can readily determine an appropriate amount. In general, the range occupied by the liquid in the container is considered to be the range in which the sheet-like cell culture can move freely, but it is necessary to extend and eliminate the wrinkles and kinks of the sheet. Therefore, the amount of liquid is preferably as large as possible. Specifically, for example, in the case of UpCell 3.5cm (CELLSEED Co., Ltd., model number: CS3007), it is about 1.55 ml, but is not limited thereto. Moreover, in another preferable aspect, it uses as it is, without adjusting a liquid quantity after a peeling process.
以上のとおり、本発明の剥離方法を行う工程および、必要に応じて、続けて本発明の伸展方法を行う工程により、シート状細胞培養物を簡便、迅速かつ破損のリスクを低減し、しわ、よれのない高品質な状態で回収可能となる。
なお、本発明の回収方法に係る好適な態様として本発明の剥離方法および伸展方法を説明してきたが、当然ながらこれらの剥離方法および伸展方法は、それぞれ独立した方法として用いることも可能である。したがって本発明の剥離方法および伸展方法は、それぞれ単独で用いられても良いし、別の方法と組み合わせて用いられてもよい。
As described above, the step of performing the peeling method of the present invention and, if necessary, the step of continuously performing the extending method of the present invention, the sheet-like cell culture is easily and quickly reduced in risk of damage, wrinkles, It can be collected in a high-quality state without dripping.
In addition, although the peeling method and extension method of the present invention have been described as preferred embodiments relating to the recovery method of the present invention, it is of course possible to use these peeling methods and extension methods as independent methods. Therefore, the peeling method and the extending method of the present invention may be used alone or in combination with another method.
（３）システム
本発明は、一側面において、上記回収方法を実行するためのシステムに関する。
本発明のシステムは、少なくとも
（ｉ）シート状細胞培養物を培養している培養容器を収納する収納部
（ｉｉ）収納部内の培養容器に振動を与えて、シート状細胞培養物を剥離および／または伸展させる振動部
を有している。
(3) System In one aspect, the present invention relates to a system for executing the recovery method.
The system of the present invention includes at least (i) a storage unit that stores a culture container in which a sheet-shaped cell culture is cultured, (ii) a vibration is applied to the culture container in the storage unit, and the sheet-shaped cell culture is separated and / or Or it has a vibrating part to extend.
培養容器の収納部の形状は限定されず、例えば倉庫状のものであってもよく、培養容器を振動台に固定するだけのものであってもよい。倉庫状とは収納部として温度や湿度を制御可能な密閉された空間に１つ以上の配置用棚部材を設け、棚部材の上に１つ以上の培養容器を配置できることを意味する。 The shape of the storage part of the culture vessel is not limited, and may be, for example, a warehouse-like shape, or may be only one that fixes the culture vessel to the shaking table. The warehouse shape means that one or more placement shelf members can be provided in a sealed space where the temperature and humidity can be controlled as a storage section, and one or more culture vessels can be placed on the shelf members.
収納部は、一態様において、培養容器の周辺環境を制御し得る制御部を有している。本態様における周辺環境とは、これに限定されるものではないが、例えば温度、圧力、湿度、ＣＯ２濃度などが挙げられる。制御部は、制御すべき設定を入力する入力部や、設定値を記録しておく記録部、現在の周辺環境の状態を測定する測定部、測定部にて測定した情報などを出力する出力部などを有していてもよい。例えば、本発明の１つの態様において、培養容器として周辺環境に依存して性状が変化する接着培養面を有する培養皿（例えば温度応答性培養皿）を用いることができ、培養容器の周辺環境を剥離に適した範囲に制御することで、剥離効率を上げることが可能となる。 In one embodiment, the storage unit has a control unit that can control the surrounding environment of the culture vessel. The peripheral environment in the present embodiment, but are not limited to, such as temperature, pressure, humidity, etc. CO 2 concentration and the like. The control unit is an input unit that inputs settings to be controlled, a recording unit that records setting values, a measurement unit that measures the current state of the surrounding environment, and an output unit that outputs information measured by the measurement unit, etc. And so on. For example, in one embodiment of the present invention, a culture dish (for example, a temperature-responsive culture dish) having an adhesion culture surface whose properties change depending on the surrounding environment can be used as the culture container. By controlling to a range suitable for peeling, the peeling efficiency can be increased.
また、本発明のシステムの一態様において、該収納部は、培養容器周辺を細胞培養に適した環境に保つことが可能である。したがって、かかる態様における収納部は、細胞培養インキュベーターとして機能することができる。この態様において、収納部をインキュベーターとして用いてシート形成細胞を培養し、シート形成完了後、シート状細胞培養物を剥離するステップへとそのまま移行することも可能である。さらに、本発明の収納部は、一態様において剥離前および／または剥離後のシート状細胞培養物を一定温度で保存しておくことが可能である。 Further, in one embodiment of the system of the present invention, the storage unit can keep the periphery of the culture container in an environment suitable for cell culture. Therefore, the storage part in this aspect can function as a cell culture incubator. In this embodiment, it is also possible to culture the sheet-forming cells using the storage portion as an incubator, and to proceed to the step of peeling the sheet-shaped cell culture after completion of the sheet formation. Furthermore, the storage unit of the present invention can store the sheet-shaped cell culture before and / or after peeling at a constant temperature in one embodiment.
振動部は、収納部の内部にあっても外部にあってもよく、培養容器のみを振動させるものであっても収納部ごと振動させるものであってもよい。また、収納部が倉庫状である場合には、棚部材を振動させるものであってもよい。振動の種類は少なくとも往復振動が可能である必要があり、好ましい態様においては、さらに回転振動が可能である。本発明に用いることができる振動機としては、例えばTAITEC社製ダブルシェーカーNR-3などが挙げられる。
シートの損傷の軽減という観点から、振動は必要以上に与えないことが好ましい。したがって振動部は、一定時間後に振動が停止するような機構を備えていてもよいし、前記制御部によって振動および／または振動時間を制御されていてもよい。
The vibration unit may be inside or outside the storage unit, and may vibrate only the culture vessel or may vibrate the entire storage unit. Moreover, when a storage part is warehouse shape, a shelf member may be vibrated. The type of vibration must be capable of at least reciprocating vibration, and in a preferred embodiment, further rotational vibration is possible. Examples of the vibrator that can be used in the present invention include a double shaker NR-3 manufactured by TAITEC.
From the viewpoint of reducing damage to the sheet, it is preferable not to apply vibration more than necessary. Therefore, the vibration unit may be provided with a mechanism that stops the vibration after a certain time, and the vibration and / or vibration time may be controlled by the control unit.
上述のように、制御部は周辺環境の制御のみならず、振動の制御にも関与し得る。また、他の部材において何らかの制御（例えば入力部による入力値や測定部による測定値の出力制御など）が必要な場合においても、上記制御部が兼用可能である。したがって、本発明のシステムにおけるあらゆる制御を制御部が担ってよい。制御部、入力部、記録部、測定部および出力部は当該技術分野において知られた部材を用いることができる。典型的には、制御部としてはＣＰＵなど、入力部としては、キーボード、タッチパネルなど、記録部としては光磁気ディスク、フラッシュメモリなど、測定部としてはセンサー、タイマーなど、出力部としてはモニタ、プリンタなどが挙げられるが、これに限定するものではない。 As described above, the control unit can be involved not only in the control of the surrounding environment but also in the control of vibration. In addition, the control unit can also be used when other members require some control (for example, input value control by the input unit or output control of the measurement value by the measurement unit). Therefore, the control unit may be responsible for any control in the system of the present invention. As the control unit, the input unit, the recording unit, the measurement unit, and the output unit, members known in the technical field can be used. Typically, the control unit is a CPU, the input unit is a keyboard, a touch panel, the recording unit is a magneto-optical disk, a flash memory, the measurement unit is a sensor, a timer, the output unit is a monitor, a printer, etc. However, it is not limited to this.
以上、本発明におけるシステムを説明したが、各部について、様々な形態を考えることができる。したがって、本発明の目的を達成する範囲において、本発明の改変した態様もまた本発明の範囲に包含され、かかる改変は当業者にとっては容易に理解可能である。 As mentioned above, although the system in this invention was demonstrated, various forms can be considered about each part. Therefore, within the scope of achieving the object of the present invention, modified embodiments of the present invention are also included in the scope of the present invention, and such modifications can be easily understood by those skilled in the art.
以下に本発明の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。 Hereinafter, the present invention will be described in more detail with reference to specific embodiments of the present invention, but the present invention is not limited to these specific examples.
例１．振動剥離
（１）シート状細胞培養物の調製
シート状骨格筋芽細胞培養物は以下の通りに作製した。
骨格筋芽細胞としては、ヒト骨格筋試料より単離したヒト骨格筋芽細胞を、ＦＢＳを含むＭＣＤＢ１３１培地（GIBCO製）で増殖させた後、回収し、ＤＭＳＯを含む凍結保存液内で凍結保存したものを用いた。
凍結保存したヒト骨格筋芽細胞を約３７℃に設定したウォーターバス内で解凍し、ヒト血清アルブミンを含む緩衝液で洗浄した。その後、細胞を、ヒト血清を含有するＤＭＥＭ／Ｆ１２培地（GIBCO製）を入れた温度応答性培養皿（UpCell(R)３．５ｃｍディッシュ、セルシード製）に６×１０７個／ｃｍ２の密度で播種し、３７℃、５％ＣＯ２で２４時間培養し、単層のシートを形成させた
Example 1. Vibration peeling (1) Preparation of sheet-like cell culture A sheet-like skeletal myoblast culture was prepared as follows.
As skeletal myoblasts, human skeletal myoblasts isolated from human skeletal muscle samples were grown in MCDB131 medium (GIBCO) containing FBS, recovered, and cryopreserved in a cryopreservation solution containing DMSO. What was done was used.
The cryopreserved human skeletal myoblasts were thawed in a water bath set at about 37 ° C. and washed with a buffer containing human serum albumin. Thereafter, the density of cells, DMEM / F12 medium containing human serum (GIBCO Co.) temperature-responsive culture dishes containing (UpCell (R) 3.5cm dish, made Cellseed) to 6 × 10 7 cells / cm 2 And cultivated at 37 ° C. and 5% CO 2 for 24 hours to form a single-layer sheet.
（２）水平振動剥離試験
（１）で調製したシート状細胞培養物の培養培地を廃棄し、４℃に冷却したハンクス平衡塩液（ＨＢＳＳ）１．５５ｍＬを添加した。恒温機（EYELA LTI-600SD）を２５℃に設定し、その中で、TAITEC社製ダブルシェーカーNR-3を用いて１００ｒｐｍ、振幅４ｃｍで横方向に水平往復振動させた。比較例として、振動を７４ｒｐｍの回転振動にしたもの、振動をさせなかったもの、２００ｒｐｍ、振幅４ｃｍの横方向水平往復振動にしたもの、培地をＨＢＳＳに置換せず、７４ｒｐｍの回転振動にしたもの、液量を２．５ｍＬとし、１００ｒｐｍ、振幅４ｃｍの回転振動にしたものについても観察した。結果を表１および図１に示す。
(2) Horizontal vibration peeling test The culture medium of the sheet-like cell culture prepared in (1) was discarded, and 1.55 mL of Hanks balanced salt solution (HBSS) cooled to 4 ° C was added. A thermostat (EYELA LTI-600SD) was set at 25 ° C., and a horizontal shaker was horizontally reciprocated at 100 rpm and an amplitude of 4 cm using a double shaker NR-3 manufactured by TAITEC. As a comparative example, the vibration is a vibration of 74 rpm, the vibration is not performed, the vibration is a horizontal reciprocating horizontal vibration of 200 rpm and an amplitude of 4 cm, the medium is not replaced with HBSS, and the vibration is a rotation of 74 rpm. The liquid volume was 2.5 mL, and the rotation vibration of 100 rpm and amplitude 4 cm was also observed. The results are shown in Table 1 and FIG.
１００ｒｐｍ、振幅４ｃｍの水平横往復で振動した場合、振動開始から３０分で剥離が完了した（図１ａ）。それに対し、７４ｒｐｍの回転振動の場合（比較例１）は、３時間でシート周縁の剥離が観察できたが、中心部は剥離せず、４時間で剥離が完了した（図１ｂ）。振動を加えなかった場合（比較例２）では４時間で剥離が観察され、４時間半で剥離が完了した（図１ｃ）。２００ｒｐｍ、振幅４ｃｍの水平横往復で振動した場合は、シートに破損が生じた。冷ＨＢＳＳに置換せずに７４ｒｐｍで回転振動を行った場合は、７時間が経過しても剥離しなかった（図１ｄ）。液量を２．５ｍＬとし、１００ｒｐｍで回転振動を行った場合は、シート周辺部分がぼろぼろに破損した（図１ｅ）。
以上のことから、水平横方向の往復振動により、シートの剥離時間が大幅に短縮でき、破損も生じないことが分かった。
In the case of vibration at a horizontal horizontal reciprocation of 100 rpm and amplitude of 4 cm, peeling was completed in 30 minutes from the start of vibration (FIG. 1a). In contrast, in the case of rotational vibration at 74 rpm (Comparative Example 1), peeling of the sheet peripheral edge was observed in 3 hours, but the central portion was not peeled off, and peeling was completed in 4 hours (FIG. 1b). When vibration was not applied (Comparative Example 2), peeling was observed in 4 hours, and peeling was completed in 4 and a half hours (FIG. 1c). In the case of vibration at 200 rpm and horizontal horizontal reciprocation with an amplitude of 4 cm, the sheet was damaged. When rotation vibration was performed at 74 rpm without replacing with cold HBSS, peeling did not occur even after 7 hours (FIG. 1d). When the liquid volume was 2.5 mL and rotational vibration was performed at 100 rpm, the peripheral portion of the sheet was damaged in a shabby manner (FIG. 1e).
From the above, it was found that the sheet peeling time can be greatly shortened and no damage is caused by the horizontal reciprocating vibration.
（３）他の振動による剥離試験
（１）で調製したシート状細胞培養物の培養培地を廃棄し、４℃に冷却したハンクス平衡塩液（ＨＢＳＳ）１．５５ｍＬを添加した。恒温機（EYELA LTI-600SD）を４℃に設定し、その中で、TAITEC社製ダブルシェーカーNR-3を用いて３０ｒｐｍ、振幅４ｃｍで横方向に水平往復振動させた。他の例として、アズワン社製Hot Rocking Mixerを用いて２．８秒／往復の速度で揺動させたもの、バイオサン社製PST-60HL Plusを用いて２５０ｒｐｍで水平偏芯震動させたものについても観察した。結果を表２および図２に示す。
(3) Peeling test by other vibrations The culture medium of the sheet-like cell culture prepared in (1) was discarded, and 1.55 mL of Hanks balanced salt solution (HBSS) cooled to 4 ° C. was added. A thermostat (EYELA LTI-600SD) was set at 4 ° C., and a horizontal shaker was horizontally oscillated at 30 rpm and an amplitude of 4 cm using a double shaker NR-3 manufactured by TAITEC. As another example, what was rocked at a speed of 2.8 seconds / reciprocating using Hot Rocking Mixer manufactured by ASONE, and what was subjected to horizontal eccentric vibration at 250 rpm using BioSTAN PST-60HL Plus Also observed. The results are shown in Table 2 and FIG.
３０ｒｐｍ、振幅４ｃｍの水平横振とうした場合（実施例２）、振とう開始から約２時間で剥離が完了した（図２ａ）。水平横振とうに代えて揺動により液体を往復振動させた場合（実施例３）、揺動開始から約４時間で剥離が完了した（図２ｂ）。また、水平偏芯震動により、振幅を減少させる代わりに振動数を大きくした場合（実施例４）、震動開始から約１時間で剥離が完了した（図２ｃ）。いずれの場合もシート状細胞培養物の破損は観察されなかった。 In the case of horizontal shaking with 30 rpm and amplitude of 4 cm (Example 2), peeling was completed in about 2 hours from the start of shaking (FIG. 2a). When the liquid was reciprocally vibrated by rocking instead of horizontal horizontal shaking (Example 3), peeling was completed in about 4 hours from the start of rocking (FIG. 2b). Further, when the frequency was increased instead of decreasing the amplitude by horizontal eccentric vibration (Example 4), separation was completed in about 1 hour from the start of the vibration (FIG. 2c). In any case, no breakage of the sheet-like cell culture was observed.
例２．伸展実験
例１と同様にシート状細胞培養物を調製し、剥離した。剥離の際によれ、しわが生じたシート状細胞培養物を伸展実験に用いた。
恒温機（EYELA LTI-600SD）を２５℃に設定し、その中でTAITEC社製ダブルシェーカーNR-3の中心に容器を設置し、７０ｒｐｍで回転振動させた。比較例として、同一の条件で回転振とう機の端に容器を設置したもの、および１００ｒｐｍ、振幅４ｃｍの水平横方向の往復振動を与えたものについても観察を行った。結果を図３に示す。
振とう機の中心に設置した場合、２分間の振動でシートの伸展が完了した（図３ａ）。振とう機の端に設置した場合は、シートが片側に寄ってしまった（図３ｂ）。水平横方向の往復振動の場合は、伸展しなかった。
Example 2. A sheet-shaped cell culture was prepared and exfoliated in the same manner as in extension experiment example 1. A sheet-like cell culture in which wrinkles were generated during peeling was used for the extension experiment.
A thermostat (EYELA LTI-600SD) was set at 25 ° C., and a container was placed in the center of a double shaker NR-3 manufactured by TAITEC, and rotated at 70 rpm. As comparative examples, observations were also made on a case where a container was installed at the end of a rotary shaker under the same conditions and a case where a horizontal reciprocating vibration of 100 rpm and an amplitude of 4 cm was given. The results are shown in FIG.
When installed in the center of the shaker, the extension of the sheet was completed with 2 minutes of vibration (FIG. 3a). When installed at the end of the shaker, the sheet has moved to one side (FIG. 3b). In the case of horizontal horizontal reciprocating vibration, it did not extend.
本発明の方法によれば、形成したシート状細胞培養物を短時間で、簡便に、破損することなく剥離可能である。また、剥離の際に多少よれやしわが生じた場合であっても、簡便に伸展することが可能となり、高品質なシート状細胞培養物を提供可能となる。 According to the method of the present invention, the formed sheet-shaped cell culture can be easily peeled off in a short time without being damaged. In addition, even if some wrinkles or wrinkles are generated at the time of peeling, it can be easily extended, and a high-quality sheet-shaped cell culture can be provided.
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