JP2014140384A - Implement for transferring cultured cell, and method for using the same - Google Patents
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Abstract
Description
本発明は、生物学、医学等の分野における培養細胞の移動方法及びその利用方法に関する。 The present invention relates to a method for moving cultured cells in the fields of biology, medicine, and the like, and a method for using the method.
今日、動物細胞培養技術が著しく進歩し、動物細胞を対象とした研究開発もさまざまな分野に広がって実施されるようになってきた。対象となる動物細胞の使われ方も、開発当初の細胞そのものを製品化したり、その産生物を製品化するだけでなく、今日では細胞やその表層蛋白質を分析することで有用な医薬品を設計したり、患者本人の細胞を生体外で増殖させたり、或いはその機能を高めてから生体内へ戻して治療するということも実施されるようになった。現在、動物細胞を培養する技術、並びに評価、解析、利用する技術は、研究者が注目している一分野である。ところで、ヒト細胞を含め動物細胞の多くは付着依存性のものである。すなわち、動物細胞を生体外で培養しようとするときは、それらを一度、基材表面に付着させる必要性がある。そして、培養した細胞をばらばらに剥離させず、その基材表面上で培養した形態を保持したまま剥離させる必要性も出てきている。 Today, animal cell culture technology has made significant progress, and research and development on animal cells has been extended to various fields. The target animal cells are used not only to commercialize the original cells and products of their products, but also to design useful drugs by analyzing the cells and their surface proteins. In some cases, the patient's own cells are proliferated outside the living body, or the function of the patient is increased and then returned to the living body for treatment. Currently, techniques for culturing animal cells and techniques for evaluation, analysis, and utilization are one field that researchers are paying attention to. By the way, many animal cells including human cells are adhesion-dependent. That is, when culturing animal cells in vitro, it is necessary to attach them to the substrate surface once. And it is necessary to peel the cultured cells while keeping the cultured form on the surface of the base material without peeling them apart.
特に患者本人の細胞を生体外で再生する技術について言えば、近年、治療困難となった臓器を他人の臓器と置き換えようとする臓器移植が一般化してきた。対象となる臓器も皮膚、角膜、腎臓、肝臓、心臓等と実に多様で、また、術後の経過も格段に良くなり、医療の一技術としてすでに確立されつつある。一例として角膜移植をあげると、約５０年前に日本にもアイバンクが設立され移植活動が始められた。しかしながら、未だにドナー数が少なく、国内だけでも角膜移植の必要な患者が年間約２万人いるのに対し、実際に移植治療が行える患者は約１／１０の２０００人程度でしかないといわれている。角膜移植というほぼ確立された技術があるにもかかわらず、ドナー不足という問題のため、次なる医療技術が求められているのが現状である。このような背景のもと、患者本人の正常な細胞を所望の大きさまで培養し移植しようとする技術が開発された。 In particular, with regard to a technique for regenerating a patient's own cells in vitro, organ transplants that attempt to replace difficult-to-treat organs with other people's organs have become common. The target organs are very diverse, such as skin, cornea, kidney, liver, heart, etc., and the progress after surgery has improved remarkably, and has already been established as a medical technology. Taking corneal transplantation as an example, an eye bank was established in Japan about 50 years ago and transplantation activities began. However, the number of donors is still small, and there are about 20,000 patients who need corneal transplants in Japan alone, whereas it is said that there are only about 1/10 of the 2000 patients who can actually perform transplantation treatment. Yes. Despite the almost established technology of corneal transplantation, the current situation is that the next medical technology is required due to the shortage of donors. Under such circumstances, a technique for culturing and transplanting normal cells of a patient to a desired size has been developed.
一方で、例えば、特開平０５−１９２１３８号公報には、水に対する上限若しくは下限臨界溶解温度が０〜８０℃であるポリマーで基材表面を被覆した細胞培養支持体上にて、皮膚細胞を上限臨界溶解温度以下又は下限臨界溶解温度以上で培養し、その後上限臨界溶解温度以上又は下限臨界溶解温度以下にすることにより培養皮膚細胞が剥離されることを特徴とする皮膚細胞培養法が記載されている。この方法においては、温度応答性ポリマーを被覆した培養基材から温度により細胞を剥離させているが、この方法では剥離性が悪く、得られた細胞シートは構造欠陥の多いものであった。したがって、特開平０５−１９２１３８号公報に記載の方法をｉｎ ｖｉｔｒｏでの心筋様組織構築に適用することも困難であった。 On the other hand, for example, Japanese Patent Application Laid-Open No. 05-192138 discloses an upper limit of skin cells on a cell culture support in which a substrate surface is coated with a polymer having an upper or lower critical solution temperature of 0 to 80 ° C. A skin cell culture method characterized by culturing at or below the critical dissolution temperature or above the lower critical dissolution temperature and then peeling the cultured skin cells above the upper critical solution temperature or below the lower critical solution temperature is described. Yes. In this method, cells are detached from the culture substrate coated with the temperature-responsive polymer by temperature. However, this method has poor releasability, and the obtained cell sheet has many structural defects. Therefore, it has been difficult to apply the method described in JP-A No. 05-192138 to the construction of myocardial tissue in vitro.
さらに国際出願公開公報ＷＯ０２／０８３８７号では温度応答性ポリマーで基材表面を被覆した細胞培養支持体上で心筋組織の細胞を培養し、心筋様細胞シートを得、その後、培養液温度を上限臨界溶解温度以上又は下限臨界溶解温度以下とし、培養した重層化細胞シートをポリマー膜に密着させ、そのままポリマー膜と共に剥離させること、及びそれを所定の方法で３次元構造化させることにより、構造欠陥の少ない、ｉｎ ｖｉｔｒｏでの心筋様組織として幾つかの機能を備えた細胞シート、及び３次元構造が構築されることを見いだした。しかしながら、この方法でも心筋様細胞シートの積層化は簡便な操作で行えるものではなく、より簡便で正確に積層化できる技術が強く望まれていた。 Furthermore, in International Publication No. WO02 / 08387, myocardial tissue cells are cultured on a cell culture support whose surface is coated with a temperature-responsive polymer to obtain a myocardial cell sheet. By bringing the cultured multi-layered cell sheet into close contact with the polymer membrane and peeling it with the polymer membrane as it is, and making it three-dimensionally structured by a predetermined method. It has been found that a cell sheet having several functions and a three-dimensional structure are constructed as a few in vitro myocardial tissue. However, even with this method, lamination of myocardial cell sheets is not possible with a simple operation, and a technique that can be more simply and accurately laminated has been strongly desired.
以上のような課題を開発するために、特開２００５−１７６８１２号公報には細胞接着部を有する培養細胞移動治具に関する技術が示されており、この治具を用いることにより細胞培養基材上の培養細胞を剥離させ、その後、その剥離させた培養細胞を再び付着させることができるようになった。しかしながら、ここで示される細胞接着部とは平面状の形態のものだけであって、必ずしも広範囲な細胞培養基材表面上の培養細胞と均一に接することができず、移動させたい細胞を全て移動させることが困難なものであった。 In order to develop the problems as described above, Japanese Patent Application Laid-Open No. 2005-176812 discloses a technique relating to a cultured cell transfer jig having a cell adhesion portion. The cultured cells can be detached, and then the detached cultured cells can be attached again. However, the cell adhesion part shown here is only in a planar form, and it cannot necessarily contact the cultured cells on the surface of a wide range of cell culture substrate uniformly, and all the cells to be moved are moved. It was difficult to do.
本発明は、上記のような従来技術の問題点を解決することを意図してなされたものである。すなわち、本発明は、従来技術と全く異なった発想からの新規な培養細胞移動治具を提供することを目的とする。また、本発明は、その利用方法を提供することを目的とする。 The present invention has been made with the intention of solving the problems of the prior art as described above. That is, an object of the present invention is to provide a novel cultured cell transfer jig based on a completely different idea from the prior art. Moreover, an object of this invention is to provide the utilization method.
本発明者らは、上記課題を解決するために、種々の角度から検討を加えて、研究開発を行った。その結果、培養細胞移動治具の細胞接着部を凸状にすることによって細胞培養基材上の任意の表面全体と均一に接触することができるようになることを見出した。また、その培養細胞移動治具の細胞接着部と培養細胞との付着力を弱めることで、剥離させた培養細胞を特定の場所へ再び付着させることを見出した。本発明はかかる知見に基づいて完成されたものである。 In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, it has been found that by making the cell adhesion part of the cultured cell transfer jig convex, it is possible to uniformly contact the entire arbitrary surface on the cell culture substrate. Moreover, it discovered that the peeled cultured cell was made to adhere again to a specific place by weakening the adhesive force of the cell adhesion part and cultured cell of the cultured cell movement jig | tool. The present invention has been completed based on such findings.
すなわち、本発明は、細胞培養基材表面上に培養させた細胞を剥離させ、その後、その剥離させた培養細胞を再び他の場所へ付着させるための非荷重下で凸状の形態の細胞接着部を有する培養細胞移動治具であって、細胞培養基材表面と接する際、当該細胞接着部の凸部先端部から接し始め、荷重により凸状の形態の当該細胞接着部が変形し、最終的に細胞培養基材の任意の表面全体と接触することのできる、培養細胞移動治具を提供する。
また、本発明は、その培養細胞移動治具に設けた細胞接着部に細胞培養基材上の培養細胞を付着させることで培養細胞を細胞培養基材上から剥離させ、その後、その培養細胞移動治具の細胞接着部と培養細胞との付着力を弱めることで、剥離させた培養細胞を特定の場所へ再び付着させることを特徴とする培養細胞移動方法を提供する。
さらに、本発明は、組織の一部或いは全部を損傷もしくは欠損した患部に対し、シート状の培養細胞を生体組織内に移植することを特徴とする治療法を提供する。
That is, the present invention provides a cell adhesion in a convex shape under non-loading for detaching cells cultured on the surface of the cell culture substrate and then attaching the detached cultured cells to other places again. A cell transfer jig having a portion, and when contacting the cell culture substrate surface, the cell adhesion portion begins to contact from the tip of the convex portion of the cell adhesion portion, the convex shape of the cell adhesion portion is deformed by the load, and finally In particular, a cultured cell transfer jig capable of contacting an entire surface of a cell culture substrate is provided.
In addition, the present invention allows the cultured cells to be detached from the cell culture substrate by attaching the cultured cells on the cell culture substrate to the cell adhesion part provided in the cultured cell transfer jig, and then the cultured cell transfer Provided is a method for moving cultured cells, characterized in that the peeled cultured cells are attached again to a specific location by weakening the adhesion between the cell adhesion part of the jig and the cultured cells.
Furthermore, the present invention provides a treatment method characterized by transplanting a sheet-like cultured cell into a living tissue for an affected part in which a part or all of the tissue is damaged or missing.
本発明に記載される凸状の細胞接着部を有する培養細胞移動治具を用いれば、細胞培養基材上の任意の範囲内の培養細胞を効率良く剥離させられ、その剥離させた培養細胞を再び簡便に付着させるようになる。そのため培養細胞を移動させたい場所へ簡便に移動させられ、しかも正確に移動できるようになる。 By using the cultured cell moving jig having a convex cell adhesion part described in the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells are removed. It comes to adhere easily again. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately.
本発明は、細胞培養基材上の培養細胞を剥離させ、その後、その細胞を再び付着させるための凸状の細胞接着部を有する培養細胞移動治具を提供する。細胞接着部が凸状であれば、細胞培養基材表面の培養細胞に移動治具の細胞接着部を近づけていくと、まず、細胞接着部の最も凸になった部分が接することができる。そしてさらに細胞接着部を近づけていくと、最も凸になった部分を中心に細胞接着部が剥離したい細胞培養面全体に広がって接触することとなる。このような状態で細胞接着部と培養細胞とを接触させることで、細胞接着部と培養細胞との間に気泡が入ることがなく細胞接着部と培養細胞間を密着させられ、結果として効率よく培養細胞を剥離させられるようになる。その際、細胞接着部の凸部の形状、曲率は特に限定されるものではなく、細胞接着部のいずれかの場所がその他のところに比べ凸になっていれば良いが、細胞培養基材上の培養細胞を広範囲に剥離させるには、細胞接着部の最も凸な部分は細胞接着部の中心にあった方が良い。ここでの細胞接着部の中心とは、最も凸な部分が点状もしくはそれを中心とした面状のものであれば細胞接着部の中心部を意味し、最も凸な部分が線状もしくはそれを中心とした面状のものであれば細胞接着部の中心を含む中心線部を意味する。 The present invention provides a cultured cell moving jig having a convex cell adhesion part for peeling cultured cells on a cell culture substrate and then attaching the cells again. If the cell adhesion part is convex, when the cell adhesion part of the moving jig is brought closer to the cultured cells on the surface of the cell culture substrate, first, the most convex part of the cell adhesion part can contact. When the cell adhesion part is further brought closer, the cell adhesion part spreads and contacts the entire cell culture surface to be peeled around the most convex part. By contacting the cell adhesion part and the cultured cell in such a state, air bubbles do not enter between the cell adhesion part and the cultured cell, and the cell adhesion part and the cultured cell are brought into close contact with each other. The cultured cells can be detached. At that time, the shape and curvature of the convex part of the cell adhesion part are not particularly limited, and any part of the cell adhesion part may be convex as compared with other parts. In order to peel the cultured cells in a wide range, it is better that the most convex part of the cell adhesion part is at the center of the cell adhesion part. Here, the center of the cell adhesion part means the center part of the cell adhesion part if the most convex part is a dot or a surface having the center as the center, and the most convex part is linear or it. If it is a planar shape centering on, it means the center line part including the center of the cell adhesion part.
本発明は、凸になった細胞接着部を培養細胞面に凸部の方から接触させるものである。その際、その凸部の全部を利用しても、その一部だけを利用しても良い。いずれにせよ、まず凸部の細胞接着部が培養細胞面に接し、さらに細胞接着部が培養細胞面に近づくことで、細胞接着部が平面状に変わり接していくことになる。その際、凸部の寸法は、細胞接着に利用する範囲において最も凸な部分の高さとして、０．５ｍｍ〜５ｍｍの範囲が良く、好ましくは０．８ｍｍ〜３ｍｍの範囲が良く、さらに好ましくは１．０ｍｍ〜２．５ｍｍの範囲が良く、最も好ましくは１．２ｍｍ〜２．０ｍｍの範囲が良い。凸部の高さが０．５ｍｍ以下の場合、細胞接着部が平面のときと変わらず、移動させたい細胞を必ずしも全てを剥離できず好ましくなく、また、凸部の高さが５ｍｍ以上のとき、その凸状の細胞接着部が最終的に平面に変わるときの歪や、場合によっては凸状の細胞接着部を平面状にするための圧力が培養細胞への負荷となり好ましくない。 In the present invention, the convex cell adhesion part is brought into contact with the cultured cell surface from the convex part. In that case, you may utilize all the convex parts, or only a part. In any case, the cell adhesion portion of the convex portion first comes into contact with the cultured cell surface, and further the cell adhesion portion approaches the cultured cell surface, so that the cell adhesion portion changes into a flat shape and comes into contact. In that case, the dimension of the convex part is preferably in the range of 0.5 mm to 5 mm, preferably in the range of 0.8 mm to 3 mm, more preferably as the height of the most convex part in the range used for cell adhesion. The range of 1.0 mm to 2.5 mm is good, and the range of 1.2 mm to 2.0 mm is most preferred. When the height of the convex part is 0.5 mm or less, it is not preferable because the cell adhesion part is not flat, and it is not always preferable to peel off all the cells to be moved, and when the height of the convex part is 5 mm or more. The strain when the convex cell adhesion portion finally changes to a flat surface, or the pressure for making the convex cell adhesion portion flat according to circumstances, is unfavorable as a load on the cultured cells.
また、本発明においては、細胞接着部全体に対する凸になっている面積の割合は特に限定されるものではないが、凸な部分の面積の割合として、４０％〜１００％の範囲が良く、好ましくは５０％〜１００％の範囲が良く、さらに好ましくは７０％〜１００％の範囲が良く、最も好ましくは８０％〜１００％の範囲が良い。種々検討した結果、凸部の面積の割合が４０％以下の場合、細胞接着部と培養細胞との間に気泡が入る場合が多く本発明として好ましくない。 In the present invention, the ratio of the convex area to the whole cell adhesion part is not particularly limited, but the ratio of the area of the convex part is preferably in the range of 40% to 100%, preferably Is preferably in the range of 50% to 100%, more preferably in the range of 70% to 100%, and most preferably in the range of 80% to 100%. As a result of various investigations, when the ratio of the area of the convex portion is 40% or less, there are many cases where air bubbles enter between the cell adhesion portion and the cultured cell, which is not preferable as the present invention.
さらに、本発明における凸部の形状は、細胞接着部側から観察したときの形状、並びに細胞接着部に対し垂直に観察したときの形状の何れも特に限定されるものではなく、垂直にしたときの形状を例にとると、利用する細胞接着部全体が連続的、もしくは段階的に徐々に凸状になっていても良い。 Furthermore, the shape of the convex part in the present invention is not particularly limited to the shape when observed from the cell adhesion part side and the shape when observed perpendicular to the cell adhesion part side, and when it is vertical For example, the entire cell adhesion part to be used may be convex continuously or stepwise.
本発明における細胞接着部は培養細胞に細胞接着部の凸部より接し始め、最終的に剥離させたい培養細胞の範囲全体に細胞接着部が平坦になる。本発明においては、培養細胞剥離時に細胞接着部の形態を前述のように変えられれば特にその変化の作用機構、細胞接着部の材質等は特に限定されるものではないが、例えば、非荷重下で凸状の形態となる柔軟性のある材質からなる細胞接着部が挙げられる。このような材質の細胞接着部であれば大きな負荷を必要とせずに変形し、最終的に細胞培養基材の任意の表面全体と接触することができる。このような器材の材質は特に限定されるものではないが、例えばシリコーンゴム、アクリルゴム、アクリロニトリルブタジエンゴム、イソプレンゴム、ウレタンゴム、エチレンプロピレンゴム、エピクロルヒドリンゴム、クロロプレンゴム、スチレンブタジエンゴム、ブタジエンゴム、フッ素ゴム、ポリイソブチレン、アラビアゴム、グアーガム、等のゴム類、ポリウレタン類、ウレタンフォーム類、ポリアクリルアミド、ポリジメチルアクリルアミド、ポリアクリル酸及びその塩、ポリヒドロキシエチルメタクリレート、ポリヒドロキシエチルアクリレート、ポリビニルアルコール、ポリビニルピロリドン、セルロース、カルボキシメチルセルロース、フィブリンゲル、コラーゲン、ゼラチン等の含水ゲル等のいずれか、或いは２種類以上の混合物等が挙げられる。また、このような材質の膜が袋状になっており、その中に気体や液体が満たされたようなものでも良い。 The cell adhesion part in the present invention starts to contact the cultured cell from the convex part of the cell adhesion part, and the cell adhesion part becomes flat over the entire range of the cultured cell to be finally peeled off. In the present invention, the action mechanism of the change, the material of the cell adhesion part, etc. are not particularly limited as long as the form of the cell adhesion part can be changed as described above at the time of culture cell detachment. And a cell adhesion portion made of a flexible material having a convex shape. If it is a cell adhesion part of such a material, it will deform | transform without requiring a big load and can finally contact with the whole arbitrary surface of a cell culture substratum. The material of such equipment is not particularly limited. For example, silicone rubber, acrylic rubber, acrylonitrile butadiene rubber, isoprene rubber, urethane rubber, ethylene propylene rubber, epichlorohydrin rubber, chloroprene rubber, styrene butadiene rubber, butadiene rubber, Rubbers such as fluoro rubber, polyisobutylene, gum arabic and guar gum, polyurethanes, urethane foams, polyacrylamide, polydimethylacrylamide, polyacrylic acid and salts thereof, polyhydroxyethyl methacrylate, polyhydroxyethyl acrylate, polyvinyl alcohol, Polyvinylpyrrolidone, cellulose, carboxymethyl cellulose, fibrin gel, collagen, water-containing gel such as gelatin, or more Mixtures thereof, and the like. Moreover, the film | membrane of such a material may be a bag shape, and the thing filled with gas and liquid may be sufficient as it.
本発明における細胞接着部の表面には、さらに、例えば、細胞接着性タンパク質、細胞接着性ペプチド、或いは温度応答性ポリマーの１種、もしくは２種以上からなるものが被覆されていても良い。その中の細胞接着性タンパク質としては、フィブリンゲル、フィブロネクチン、ラミニン、コラーゲン、ゼラチンなどの１種、もしくは２種以上からなるものが挙げられる。また、細胞接着性ペプチドとしては、ＲＧＤペプチド、ＲＧＤＳペプチド、ＧＲＧＤペプチド、ＧＲＧＤＳペプチドなどの１種、もしくは２種以上からなるものが挙げられる。細胞接着部における細胞接着性タンパク質、細胞接着性ペプチドの固定化方法は特に限定されないが、常法として知られる細胞接着性タンパク質、細胞接着性ペプチド水溶液の塗布による物理的吸着などを行えば良い。細胞接着部における細胞接着性タンパク質、細胞接着性ペプチドの固定化量は移動させたい細胞を付着させられるに十分な量が固定化されていれば良く特に限定されるものではないが、その固定化量は０．００５μｇ／ｃｍ２以上、好ましくは０．０１μｇ／ｃｍ２以上、さらに好ましくは０．０２μｇ／ｃｍ２以上である。細胞接着性タンパク質、細胞接着性ペプチドの固定化量の測定は常法に従えば良く、例えばＦＴ−ＩＲ−ＡＴＲを用いて細胞接着部を直接測る方法、あらかじめラベル化した細胞接着性タンパク質、細胞接着性ペプチドを同様な方法で固定化し細胞接着部に固定化されたラベル化細胞接着性タンパク質、細胞接着性ペプチド量より推測する方法などが挙げられるがいずれの方法を用いても良い。 The surface of the cell adhesion part in the present invention may be further coated with, for example, one or more of cell adhesion protein, cell adhesion peptide, or temperature responsive polymer. Among them, examples of the cell adhesion protein include one or more of fibrin gel, fibronectin, laminin, collagen, gelatin and the like. Examples of the cell adhesion peptide include one or more of RGD peptide, RGDS peptide, GRGD peptide, GRGDS peptide and the like. The method for immobilizing the cell adhesion protein and the cell adhesion peptide in the cell adhesion part is not particularly limited, and physical adsorption or the like may be performed by applying a cell adhesion protein or a cell adhesion peptide aqueous solution known as a conventional method. The amount of the cell adhesion protein and cell adhesion peptide immobilized in the cell adhesion part is not particularly limited as long as it is sufficient to immobilize the cells to be moved. the amount is 0.005 / cm 2 or more, preferably 0.01 [mu] g / cm 2 or more, more preferably 0.02 .mu.g / cm 2 or more. Measurement of the amount of immobilized cell adhesion protein and cell adhesion peptide may be in accordance with a conventional method. For example, a method of directly measuring a cell adhesion part using FT-IR-ATR, a cell adhesion protein labeled in advance, a cell Examples include a method of inferring from the amount of the cell-adhesive peptide, a labeled cell-adhesive protein immobilized on the cell-adhesive portion by immobilizing the adhesive peptide by the same method, and the method of inferring from the amount of the cell-adhesive peptide.
本発明における細胞接着部の表面には温度応答性ポリマーが被覆されているものであっても良い。その温度応答性ポリマーはホモポリマー、コポリマーのいずれであっても良く、このようなポリマーとしては、例えば、特開平２−２１１８６５号公報に記載されているポリマーが挙げられる。具体的には、例えば、以下のモノマーの単独重合または共重合
によって得られる。使用し得るモノマーとしては、例えば、（メタ）アクリルアミド化合物、Ｎ−（若しくはＮ，Ｎ−ジ）アルキル置換（メタ）アクリルアミド誘導体、またはビニルエーテル誘導体が挙げられ、コポリマーの場合は、これらの中で任意の２種以上を使用することができる。更には、上記モノマー以外のモノマー類との共重合、ポリマー同士のグラフトまたは共重合、あるいはポリマー、コポリマーの混合物を用いてもよい。また、ポリマー本来の性質を損なわない範囲で架橋することも可能である。各種ポリマーの基材表面への被覆方法は、特に制限されないが、例えば、特開平２−２１１８６５号公報に記載されている方法に従ってよい。すなわち、かかる被覆は、基材と上記モノマーまたはポリマーを、電子線照射（ＥＢ）、γ線照射、紫外線照射、プラズマ処理、コロナ処理、有機重合反応のいずれかにより、または塗布、混練等の物理的吸着等により行うことができる。細胞接着部における親水性ポリマーの固定化量は移動させたい細胞を付着させられるに十分な量が固定化されていれば良く特に限定されるものではないが、その固定化量は０．５μｇ／ｃｍ２以上、好ましくは１．０μｇ／ｃｍ２以上、さらに好ましくは１．５μｇ／ｃｍ２以上である。温度応答性ポリマーの固定化量の測定は常法に従えば良く、例えばＦＴ−ＩＲ−ＡＴＲを用いて細胞接着部を直接測る方法、あらかじめラベル化した温度応答性ポリマーを同様な方法で固定化し細胞接着部に固定化されたラベル化温度応答性ポリマー量より推測する方法などが挙げられるがいずれの方法を用いても良い。
The surface of the cell adhesion part in the present invention may be coated with a temperature-responsive polymer. The temperature-responsive polymer may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer. The method for coating the surface of the base material with various polymers is not particularly limited. For example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption. The amount of the hydrophilic polymer immobilized in the cell adhesion part is not particularly limited as long as the amount sufficient to attach the cells to be moved is immobilized, but the amount immobilized is 0.5 μg / cm 2 or more, preferably 1.0 μg / cm 2 or more, more preferably 1.5 μg / cm 2 or more. The amount of the temperature-responsive polymer immobilized may be measured according to a conventional method. For example, the cell adhesion part is directly measured using FT-IR-ATR, and the temperature-responsive polymer previously labeled is immobilized by the same method. A method inferred from the amount of the labeling temperature-responsive polymer immobilized on the cell adhesion part may be mentioned, and any method may be used.
本発明に用いられる培養細胞移動治具内の細胞接着部は移動させたい培養細胞、培養細胞塊、培養細胞シートの大きさに合わせて随時決めれば良く何ら限定されるものではない。また、細胞接着部を有する培養細胞移動治具においても細胞接着部の大きさに合わせて随時決めていけば良い。さらに培養細胞移動治具の形状も特に限定されるものではなく、治具を移動させるために必要なグリップや他の装置を接合できるような仕組みを設けても良い。 The cell adhesion part in the cultured cell moving jig used in the present invention is not limited at all as long as it is determined at any time according to the size of the cultured cell, cultured cell mass, and cultured cell sheet to be moved. Moreover, what is necessary is just to determine at any time according to the magnitude | size of a cell adhesion part also in the culture cell movement jig | tool which has a cell adhesion part. Furthermore, the shape of the cultured cell moving jig is not particularly limited, and a mechanism that can join a grip and other devices necessary for moving the jig may be provided.
本発明に使用される細胞は、例えば角膜上皮細胞、角膜内皮細胞、網膜色素細胞、表皮角化細胞、口腔粘膜細胞、結膜上皮細胞、心筋細胞、線維芽細胞、血管内皮細胞、肝実質細胞、骨格筋筋芽細胞、間葉系幹細胞、肺胞上皮細胞、中皮細胞、軟骨細胞、滑膜細胞、骨細胞、歯根膜細胞、その他の幹細胞等のいずれかもしくは２者以上の混合物が挙げられるが、その種類は、何ら制約されるものではない。また、その細胞の由来は特に制約されるものではないが、たとえばヒト、イヌ、ネコ、ウサギ、ラット、ブタ、ヒツジなどが挙げられるが、本発明の培養細胞をヒトの治療に用いる場合はヒト由来の細胞を用いる方が望ましい。 Examples of cells used in the present invention include corneal epithelial cells, corneal endothelial cells, retinal pigment cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatocytes, Examples include skeletal muscle myoblasts, mesenchymal stem cells, alveolar epithelial cells, mesothelial cells, chondrocytes, synoviocytes, bone cells, periodontal ligament cells, other stem cells, or a mixture of two or more. However, the type is not restricted at all. The origin of the cells is not particularly limited, and examples include humans, dogs, cats, rabbits, rats, pigs, sheep, and the like. When the cultured cells of the present invention are used for human therapy, humans are used. It is desirable to use cells derived from them.
本発明における細胞培養のための培地は培養される細胞に対し通常用いられるものを用いれば特に制約されるものではないが、得られた培養細胞をヒトの治療に用いる場合は用いる培地の成分は由来が明確なもの、もしくは医薬品として認められているものが望ましい。 The culture medium for cell culture in the present invention is not particularly limited as long as it is a commonly used medium for cells to be cultured. However, when the obtained cultured cells are used for human therapy, the components of the medium to be used are Those with a clear origin or those recognized as pharmaceuticals are desirable.
本発明における培養基材の形状は特に制約されるものではないが、例えばディッシュ、マルチプレート、フラスコ、セルインサートのような形態のもの、或いは平膜状のものなどが挙げられる。 The shape of the culture substrate in the present invention is not particularly limited, and examples thereof include dishes, multiplates, flasks, cell inserts, and flat membranes.
本発明は、培養細胞移動治具に設けた細胞接着部に細胞培養基材上の培養細胞を付着させることで培養細胞を細胞培養基材上から剥離させ、その後、その培養細胞移動治具の細胞接着部と培養細胞との付着力を弱めることで、剥離させた培養細胞を特定の場所へ再び付着させることを特徴とする培養細胞移動方法を提供する。この方法に従えば、細胞培養基材上の培養細胞を簡便に剥離させられ、その剥離させた培養細胞を再び簡便に付着させるようになる。そのため培養細胞を移動させたい場所へ簡便に移動させられ、しかも正確に移動できるようになることを見出した。さらに、培養細胞の細胞培養基材上からの剥離工程、剥離させた培養細胞を特定の場所へ再び付着させる工程のいずれか、もしくは双方の工程を自動化することで、なお一層培養細胞の移動が簡便に正確に行われるようになることを見出した。 In the present invention, the cultured cells are detached from the cell culture substrate by attaching the cultured cells on the cell culture substrate to the cell adhesion part provided in the cultured cell transfer jig, and then the cultured cell transfer jig Provided is a method for moving cultured cells, characterized in that the peeled cultured cells are attached again to a specific location by weakening the adhesion between the cell adhesion part and the cultured cells. According to this method, the cultured cells on the cell culture substrate can be easily detached, and the detached cultured cells are simply attached again. For this reason, it has been found that the cultured cells can be easily moved to a desired location and can be moved accurately. Furthermore, by either automating either or both of the steps of detaching cultured cells from the cell culture substrate and reattaching the detached cultured cells to a specific location, the cultured cells can be moved further. It has been found that it can be carried out easily and accurately.
本発明において培養細胞を移動させるには、まず培養細胞移動治具に設けた細胞接着部に細胞培養基材上の培養細胞を付着させる必要がある。その付着させる方法は何ら限定されるものではないが、本発明における培養細胞移動治具には細胞接着部が設けてあるため、その部分を移動させたい培養細胞上に乗せ、静置させるだけで良い。本発明の細胞接着部に上述した含水ゲルを用いた場合、物理的な吸着の他にそれ自身の吸水性を利用して培養細胞を吸い上げて補足することができる。後者の場合、あらかじめ含水ゲルに吸水させる量を変えることでその補足力を変えられ好都合である。本発明の細胞接着部の表面に細胞接着性タンパク質、細胞接着性ペプチドを被覆した場合、培養細胞は培養細胞移動治具に対しその細胞接着性タンパク質、細胞接着性ペプチドを介して付着する。また、細胞接着部の表面に温度応答性ポリマーを被覆した場合、細胞接着部ポリマー層表面の親疎水性の性質に培養細胞が物理的に付着する。その際、付着を促進させる目的で培養細胞に負担がかからない程度に荷重を負荷させたり、或いは付着するまで十分に時間をかけることなどを行っても良い。さらに、培地量を増減、培養温度を変化させるなど培養細胞の付着を促進する操作を併用しても良い。また、その付着操作を上下方向に稼動できるＺ−ステージを利用して自動で行っても良い。 In order to move the cultured cells in the present invention, it is necessary to first attach the cultured cells on the cell culture substrate to the cell adhesion portion provided in the cultured cell moving jig. The attachment method is not limited in any way, but the cultured cell transfer jig in the present invention is provided with a cell adhesion part, so that the part is placed on the cultured cell to be moved and left to stand. good. When the above-mentioned hydrogel is used for the cell adhesion part of the present invention, the cultured cells can be sucked up and supplemented using its own water absorption in addition to physical adsorption. In the latter case, it is convenient to change the supplemental force by changing the amount of water absorbed by the hydrogel in advance. When the cell adhesion protein or cell adhesion peptide is coated on the surface of the cell adhesion part of the present invention, the cultured cells adhere to the cultured cell migration jig via the cell adhesion protein and cell adhesion peptide. Further, when the surface of the cell adhesion part is coated with a temperature-responsive polymer, the cultured cells physically adhere to the hydrophilicity / hydrophobicity of the cell adhesion part polymer layer surface. At that time, for the purpose of promoting the adhesion, a load may be applied to the extent that the cultured cells are not burdened, or a sufficient time may be taken until the cells adhere. Furthermore, an operation for promoting adhesion of cultured cells, such as increasing or decreasing the amount of medium and changing the culture temperature, may be used in combination. Moreover, you may perform automatically the adhesion operation using the Z-stage which can operate | move up and down.
上述した方法により培養細胞移動治具に付着した培養細胞は、その培養細胞移動治具とともに移動することで自由に希望する場所へ移動させることができる。その際、培養細胞が汚染されることを防ぐ意味で、培養細胞の移動は無菌的に行われる方が良い。また、付着した細胞が乾燥しないように移動操作を加湿下で行っても良い。さらに、その付着操作を上下、左右方向に稼動できるステージを利用して自動で行っても良い。 The cultured cells attached to the cultured cell moving jig by the above-described method can be freely moved to a desired place by moving together with the cultured cell moving jig. At that time, it is better that the cultured cells are moved aseptically in order to prevent the cultured cells from being contaminated. Further, the moving operation may be performed under humidification so that the attached cells are not dried. Further, the attaching operation may be automatically performed using a stage that can be operated in the vertical and horizontal directions.
本発明では、上述した方法で移動させた培養細胞を再び付着させたい場所に乗せ、再付着させる技術である。その再付着させる方法は特に限定されるものではないが、通常、移動してきた培養細胞を再付着させたい場所へ付着させた後、培養細胞移動治具の細胞接着部と培養細胞との付着を弱め、培養細胞移動治具を培養細胞から離すことで操作を完了する。その際、培養細胞移動治具の細胞接着部が残存しても特に問題にならなければ、培養細胞移動治具の細胞接着部で細胞接着部と培養細胞を一緒に剥がす方法でも良い。この細胞接着部と培養細胞を一緒に剥がす方法の場合、細胞接着部が時間と共に溶解しその溶解物が細胞に影響を与えないものが良く、このようなものとしてコラーゲンやゼラチン等が好都合だが、本発明ではこれらに何ら限定されるものではない。或いは、上述したように細胞接着部に含水ゲルを用いた場合、含水ゲルが十分に吸水した状態であれば細胞接着部に付着している細胞の接着力が弱く、細胞接着部から細胞を容易に剥離させることができる。同様に、含水ゲルの吸水力で培養細胞を吸着させた場合においても、含水ゲルに十分吸水させることで細胞接着部に付着している細胞の接着力が弱められる。培養細胞移動治具の細胞接着部の表面と培養細胞との付着を弱めるために、例えば細胞接着部の表面に細胞接着性タンパク質、細胞接着性ペプチドが被覆されている場合、それらと細胞との付着性より強く付着するアミノ酸、ペプチド、タンパク質などを添加する方法、十分に培地を投入する方法などの方法が挙げられる。また、培養細胞移動治具の細胞接着部の表面に温度応答性ポリマーが被覆されている場合、細胞接着部ポリマー層表面の十分に親水性に変えることによって培養細胞を剥離させられる。一方で、再付着させたい場所へ付着させることを促進させる目的で培養細胞に負担がかからない程度に荷重をかけたり、付着するまで十分な時間をかけること、さらには培養温度を変えることなどを併用しても良い。また、その付着操作を上下方向に稼動できるＺ−ステージを利用して自動で行っても良い。 In the present invention, the cultured cells that have been moved by the above-described method are placed on a place where they are to be reattached and reattached. The reattachment method is not particularly limited, but usually, after the cultured cells that have migrated are attached to the place where they want to be reattached, the adhesion between the cell adhesion part of the cultured cell transfer jig and the cultured cells is performed. The operation is completed by weakening and separating the cultured cell moving jig from the cultured cells. At that time, if there is no particular problem even if the cell adhesion portion of the cultured cell migration jig remains, a method of peeling the cell adhesion portion and the cultured cell together at the cell adhesion portion of the culture cell migration jig may be used. In the case of the method of peeling the cell adhesion part and the cultured cell together, it is preferable that the cell adhesion part dissolves with time and the lysate does not affect the cell, and as such, collagen or gelatin is convenient. The present invention is not limited to these. Alternatively, as described above, when a water-containing gel is used for the cell adhesion part, if the water-containing gel has sufficiently absorbed water, the adhesion force of the cells adhering to the cell adhesion part is weak, and the cells can be easily removed from the cell adhesion part. Can be peeled off. Similarly, even when cultured cells are adsorbed by the water-absorbing power of the water-containing gel, the adhesive force of the cells attached to the cell adhesion portion is weakened by sufficiently absorbing the water-containing gel. In order to weaken the adhesion between the surface of the cell adhesion part of the cultured cell migration jig and the cultured cell, for example, when the surface of the cell adhesion part is coated with a cell adhesion protein or a cell adhesion peptide, Examples thereof include a method of adding an amino acid, a peptide, a protein, or the like that adheres more strongly than an adhesive property, and a method of sufficiently adding a medium. Further, when the temperature-responsive polymer is coated on the surface of the cell adhesion part of the cultured cell migration jig, the cultured cell can be peeled off by sufficiently changing the surface of the cell adhesion part polymer layer. On the other hand, in order to promote the attachment to the place where it wants to reattach, it is used in combination with applying a load to the extent that the cultured cells are not burdened, allowing sufficient time to attach, and changing the culture temperature. You may do it. Moreover, you may perform automatically the adhesion operation using the Z-stage which can operate | move up and down.
本発明で言うところの再び付着させる場所とは特に制約されるものではなく、例えば培養基材表面、生体内組織表面、生体外組織表面、別の培養細胞、或いは後で述べる別の培養細胞シート上でも良い。ここでいう生体内組織表面、生体外組織表面とはたとえばヒト、イヌ、ネコ、ウサギ、ラット、ブタ、ヒツジなどが挙げられるが由来に限定されるものではない。また、別の培養細胞とは角膜上皮細胞、表皮角化細胞、口腔粘膜細胞、結膜上皮細胞、心筋細胞、線維芽細胞、血管内皮細胞、肝実質細胞のいずれかもしくは２者以上の混合物が挙げられるが、その種類は、何ら制約されるものではないが、本発明の培養細胞をヒトの治療に用いる場合はヒト由来の細胞を用いる方が望ましい。 The place to be reattached in the present invention is not particularly limited. For example, the surface of the culture substrate, the surface of the in vivo tissue, the surface of the in vitro tissue, another cultured cell, or another cultured cell sheet described later. It ’s fine. Examples of the in vivo tissue surface and in vitro tissue surface herein include, but are not limited to, humans, dogs, cats, rabbits, rats, pigs, and sheep. Another cultured cell includes corneal epithelial cells, epidermal keratinocytes, oral mucosal cells, conjunctival epithelial cells, cardiomyocytes, fibroblasts, vascular endothelial cells, hepatic parenchymal cells, or a mixture of two or more. However, the type is not limited at all, but when the cultured cells of the present invention are used for human therapy, it is desirable to use human-derived cells.
細胞培養基材表面に温度応答性ポリマーが被覆されていれば、国際出願公開公報ＷＯ０２／０８３８７号に示す通り培養温度を変化させるだけで培養細胞をシート状に細胞培養基材表面から剥離させられ、本発明の技術を用いることでその剥離操作、移動操作、さらに再付着操作が簡便に正確に行えるようになる。その場合、基材表面に被覆される温度応答性ポリマーは、水溶液中で上限臨界溶解温度または下限臨界溶解温度０℃〜８０℃、より好ましくは２０℃〜５０℃を有する。上限臨界溶解温度または下限臨界溶解温度が８０℃を越えると細胞が死滅する可能性があるので好ましくない。また、上限臨界溶解温度または下限臨界溶解温度が０℃より低いと一般に細胞増殖速度が極度に低下するか、または細胞が死滅してしまうため、やはり好ましくない。 If the temperature-responsive polymer is coated on the surface of the cell culture substrate, the cultured cells can be peeled off from the surface of the cell culture substrate just by changing the culture temperature as shown in International Application Publication No. WO02 / 08387. By using the technique of the present invention, the peeling operation, moving operation, and reattachment operation can be performed easily and accurately. In that case, the temperature-responsive polymer coated on the substrate surface has an upper critical solution temperature or a lower critical solution temperature of 0 ° C. to 80 ° C., more preferably 20 ° C. to 50 ° C. in an aqueous solution. If the upper critical lysis temperature or the lower critical lysis temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, when the upper critical lysis temperature or the lower critical lysis temperature is lower than 0 ° C., the cell growth rate is generally extremely reduced or the cells are killed, which is also not preferable.
本発明に用いる温度応答性ポリマーはホモポリマー、コポリマーのいずれであってもよい。このようなポリマーとしては、例えば、特開平２−２１１８６５号公報に記載されているポリマーが挙げられる。具体的には、例えば、以下のモノマーの単独重合または共重合によって得られる。使用し得るモノマーとしては、例えば、（メタ）アクリルアミド化合物、Ｎ−（若しくはＮ，Ｎ−ジ）アルキル置換（メタ）アクリルアミド誘導体、またはビニルエーテル誘導体が挙げられ、コポリマーの場合は、これらの中で任意の２種以上を使用することができる。更には、上記モノマー以外のモノマー類との共重合、ポリマー同士のグラフトまたは共重合、あるいはポリマー、コポリマーの混合物を用いてもよい。また、ポリマー本来の性質を損なわない範囲で架橋することも可能である。 The temperature-responsive polymer used in the present invention may be either a homopolymer or a copolymer. Examples of such a polymer include polymers described in JP-A-2-21865. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include a (meth) acrylamide compound, an N- (or N, N-di) alkyl-substituted (meth) acrylamide derivative, or a vinyl ether derivative. Two or more of these can be used. Furthermore, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Moreover, it is also possible to crosslink within a range that does not impair the original properties of the polymer.
温度応答性ポリマーの基材表面への被覆方法は、特に制限されないが、例えば、特開平２−２１１８６５号公報に記載されている方法に従ってよい。すなわち、かかる被覆は、基材と上記モノマーまたはポリマーを、電子線照射（ＥＢ）、γ線照射、紫外線照射、プラズマ処理、コロナ処理、有機重合反応のいずれかにより、または塗布、混練等の物理的吸着等により行うことができる。温度応答性ポリマーの被覆量は、０．４〜４．５μｇ／ｃｍ２の範囲が良く、好ましくは０．７〜３．５μｇ／ｃｍ２であり、さらに好ましくは０．９〜３．０μｇ／ｃｍ２である。０．２μｇ／ｃｍ２より少ない被覆量のとき、刺激を与えても当該ポリマー上の細胞は剥離し難く、作業効率が著しく悪くなり好ましくない。逆に４．５μｇ／ｃｍ２以上であると、その領域に細胞が付着し難く、細胞を十分に付着させることが困難となる。 A method for coating the surface of the base material with the temperature-responsive polymer is not particularly limited. For example, the method described in JP-A-2-21865 may be used. That is, such coating is performed by applying a substrate and the above monomer or polymer to one of electron beam irradiation (EB), γ-ray irradiation, ultraviolet irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical application such as coating and kneading. It can be performed by, for example, mechanical adsorption. The coating amount of the temperature-responsive polymer is preferably in the range of 0.4 to 4.5 μg / cm 2 , preferably 0.7 to 3.5 μg / cm 2 , more preferably 0.9 to 3.0 μg / cm 2. cm 2 . When the coating amount is less than 0.2 μg / cm 2 , the cells on the polymer are hardly detached even when a stimulus is applied, and the working efficiency is remarkably deteriorated. On the other hand, if it is 4.5 μg / cm 2 or more, it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells.
本発明において、培養細胞をシート状で剥離させ、培養細胞移動治具を用いて培養細胞シート同士を積層化させたり、培養細胞シートを生体内組織や生体外組織に移植させたりするためには、温度応答性ポリマーが被覆された細胞培養基材上で細胞を培養し、培養細胞をシート状に剥離させなければならない。その際、培地の温度は、培養基材表面に被覆された前記ポリマーが上限臨界溶解温度を有する場合はその温度以下、また前記ポリマーが下限臨界溶解温度を有する場合はその温度以上であれば特に制限されない。しかし、培養細胞が増殖しないような低温域、あるいは培養細胞が死滅するような高温域における培養が不適切であることは言うまでもない。温度以外の培養条件は、常法に従えばよく、特に制限されるものではない。例えば、使用する培地については、公知のウシ胎児血清（ＦＣＳ）等の血清が添加されている培地でもよく、また、このような血清が添加されていない無血清培地でもよい。 In the present invention, in order to exfoliate cultured cells in a sheet form and stack the cultured cell sheets using a cultured cell moving jig, or to transplant the cultured cell sheets to in vivo tissues or in vitro tissues. The cells must be cultured on a cell culture substrate coated with a temperature-responsive polymer, and the cultured cells must be peeled into a sheet. At that time, the temperature of the medium is not more than that temperature when the polymer coated on the surface of the culture substrate has an upper critical solution temperature, and particularly if it is more than that temperature if the polymer has a lower critical solution temperature. Not limited. However, it goes without saying that culturing in a low temperature range where cultured cells do not proliferate or in a high temperature range where cultured cells die is inappropriate. The culture conditions other than the temperature may be in accordance with conventional methods and are not particularly limited. For example, the medium to be used may be a medium to which serum such as known fetal calf serum (FCS) is added, or a serum-free medium to which such serum is not added.
本発明の方法において、培養細胞シートを温度応答性ポリマーが被覆された細胞培養基材上から剥離回収するには、培養細胞シートを培養細胞移動治具に付着させ、培養基材表面の温度を上限臨界溶解温度以上若しくは下限臨界溶解温度以下にすることによって剥離させることができる。なお、培養細胞シートを剥離することは細胞を培養していた培養液中において行うことも、その他の等張液中において行うことも可能であり、目的に合わせて選択することができる。 In the method of the present invention, in order to peel and recover the cultured cell sheet from the cell culture substrate coated with the temperature-responsive polymer, the cultured cell sheet is attached to the cultured cell moving jig, and the temperature of the culture substrate surface is adjusted. Separation can be achieved by setting the temperature to the upper critical solution temperature or higher or the lower critical solution temperature or lower. It should be noted that peeling the cultured cell sheet can be performed in a culture solution in which cells are cultured or in another isotonic solution, and can be selected according to the purpose.
本発明における温度応答性ポリマーが被覆された細胞培養基材上から剥離され、培養細胞移動治具を用いることで得られた培養細胞シートは、培養時にディスパーゼ、トリプシン等で代表される蛋白質分解酵素による損傷を受けておらず、培養時に形成される細胞−基材間の基底膜様蛋白質も酵素による破壊を受けておらず、また、細胞−細胞間のデスモソーム構造が保持され、構造的欠陥が少なく強度の高いものである。さらに、培養細胞移動治具を用いることで正確に培養細胞シート同士を積層化させたり、患部組織へ正確に移動させることが可能となる。これらのことにより、例えば培養細胞シートの移植時においては患部組織と良好に正確に接着させることができ、効率良い治療を実施することができるようになる。 The cultured cell sheet peeled from the cell culture substrate coated with the temperature-responsive polymer in the present invention and obtained using a cultured cell transfer jig is a proteolytic enzyme represented by dispase, trypsin, etc. The cell-substrate basement membrane-like protein formed during culture is not damaged by the enzyme, and the cell-cell desmosome structure is retained, resulting in structural defects. There is little and high strength. Furthermore, by using the cultured cell moving jig, it is possible to accurately stack the cultured cell sheets or move them accurately to the affected tissue. For these reasons, for example, at the time of transplantation of a cultured cell sheet, it can be adhered to the affected tissue well and accurately, and an efficient treatment can be performed.
本発明で示すところの培養細胞シートと生体組織との固定方法は特に限定されるものではなく、培養細胞シートと生体組織を縫合しても良く、或いは本発明で示すところの培養細胞シートは生体組織と速やかに生着するため、患部に付着させた培養細胞シートは生体側と縫合しなくても良い。 The method for fixing the cultured cell sheet and the living tissue shown in the present invention is not particularly limited, and the cultured cell sheet and the living tissue may be sutured, or the cultured cell sheet shown in the present invention is a living body. The cultured cell sheet attached to the affected part does not need to be sutured to the living body side so as to be engrafted with the tissue quickly.
本発明の凸状の細胞接着部を有する培養細胞移動治具を用いれば、細胞培養基材上の任意の範囲内の培養細胞を効率良く剥離させられ、その剥離させた培養細胞を再び簡便に付着できるようになる。そのため培養細胞を移動させたい場所へ簡便に移動させられ、しかも正確に移動できるようになる。 By using the cultured cell migration jig having the convex cell adhesion part of the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells can be simply and easily removed again. It becomes possible to adhere. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately.
以下に、本発明を実施例に基づいて更に詳しく説明するが、これらは本発明を何ら限定するものではない。
［実施例１］
Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
[Example 1]
図１に示すようなシリコーンシートを重ね合わせた型を用い、本発明であるＴ字形状の柄付き土台（部品Ａ）と下向きに凸状に成型された吸水性を有する柔軟な素材（部品Ｂ）からなる培養細胞移動治具（図２）を作製した。各部品の材質としては土台部分にポリカーボネートを、柔軟性素材にゼラチンゲルを選択した。そのときの細胞接着部のようすを図３に示す。これらの各部品を組み合わせることで、培養細胞移動治具を得た。温度により細胞の接着、非接着が制御できるポリ−Ｎ−イソプロピルアクリルアミドが被覆された温度応答性培養皿の培養面一杯となるコンフルエントになるまでヒト骨格筋筋芽細胞を増殖させた。培養方法は常法に従った（細胞播種数１×１０５個／ｃｍ２、３７℃、５％ＣＯ２、培養期間４日間）。また、そのポリ−Ｎ−イソプロピルアクリルアミドの被覆量は１．９μｇ／ｃｍ２とした。次に、培養細胞移動治具を静かに培養細胞面に接触させ、培養細胞移動治具を培養細胞面上に置いた。さらに培養温度を下げ、培養皿表面を非接着状態にして培養細胞移動治具に細胞を補足させたところ、細胞は培養細胞移動治具の細胞接着部であるゼラチンゲルに付着した。骨格筋筋芽細胞は細胞接着部であるゼラチンゲルの密着した部分のみ剥離された培養皿の写真を図４に示す。図４における培養皿に残された細胞は染色により可視化して確認される。培養皿を３枚準備し、連続して培養細胞移動治具を付着させていくことにより３層に積層化したシート状骨格筋筋芽細胞がゼラチンゲル上に得られた。シート状骨格筋筋芽細胞が付着した培養細胞移動治具は別の培養皿上に一旦密着させシート状の積層化細胞の周辺部をピンセットで押さえながら培養細胞移動治具を離すことでシート状骨格筋筋芽細胞だけを剥離させることができた（図５）。
［実施例２］
Using a mold in which silicone sheets are overlapped as shown in FIG. 1, a T-shaped base with a handle (part A) according to the present invention and a flexible material (part B) having a water-absorbing shape that is formed in a downward convex shape. A cultured cell transfer jig (FIG. 2) was prepared. As the material of each part, polycarbonate was selected for the base part and gelatin gel was selected for the flexible material. The appearance of the cell adhesion part at that time is shown in FIG. By combining these parts, a cultured cell transfer jig was obtained. Human skeletal muscle myoblasts were grown until the culture surface of the temperature-responsive culture dish coated with poly-N-isopropylacrylamide, which can control the adhesion and non-adhesion of cells depending on the temperature, became full. The culture method was in accordance with a conventional method (cell seeding number 1 × 10 5 cells / cm 2 , 37 ° C., 5% CO 2, culture period 4 days). The coating amount of the poly-N-isopropylacrylamide was 1.9 μg / cm 2 . Next, the cultured cell moving jig was gently brought into contact with the cultured cell surface, and the cultured cell moving jig was placed on the cultured cell surface. When the culture temperature was further lowered and the surface of the culture dish was made non-adhered to supplement the cells with the cultured cell migration jig, the cells adhered to the gelatin gel which is the cell adhesion part of the culture cell migration jig. Fig. 4 shows a photograph of the culture dish in which only the portion where the gelatin gel, which is the cell adhesion portion, is adhered to the skeletal muscle myoblasts is peeled off. The cells left in the culture dish in FIG. 4 are confirmed by visualization by staining. Three culture dishes were prepared, and sheet-shaped skeletal myoblasts laminated in three layers were obtained on a gelatin gel by continuously attaching a culture cell migration jig. The cultured cell transfer jig to which the sheet-shaped skeletal myoblasts are attached is temporarily brought into close contact with another culture dish, and the cultured cell transfer jig is released while holding the periphery of the sheet-shaped laminated cells with tweezers. Only skeletal muscle myoblasts could be detached (FIG. 5).
[Example 2]
実施例１と同様に、本発明であるＴ字形状の柄付き土台（部品Ａ）と下向きに凸状に成型された吸水性を有する柔軟な素材（部品Ｂ）からなる培養細胞移動治具（図２）を作製した。各部品の材質としては土台部分にポリカーボネートを、柔軟性素材にコラーゲンゲルを選択し、コラーゲンゲル表面にポリグリコール酸製不織布を被覆した。実施例１と同様に、温度により細胞の接着、非接着が制御できるポリ−Ｎ−イソプロピルアクリルアミドが被覆された温度応答性培養皿の培養面一杯となるコンフルエントになるまでイヌ歯根膜細胞を増殖させた。培養方法は常法に従った（細胞播種数１×１０５個／ｃｍ２、３７℃、５％ＣＯ２、培養期間４日間）。また、そのポリ−Ｎ−イソプロピルアクリルアミドの被覆量は２．０μｇ／ｃｍ２とした。次に、培養細胞移動治具を静かに培養細胞面に接触させ、培養細胞移動治具を培養細胞面上に置いた。さらに、培養温度を下げ、培養皿表面を非接着状態にして培養細胞移動治具に細胞を補足させたところ、細胞は培養細胞移動治具の細胞接着部の表面であるポリグリコール酸製不織布に付着した。培養皿を３枚準備し、連続して培養細胞移動治具を付着させていくことにより３層に積層化したシート状歯根膜細胞がポリグリコール酸製不織布上に得られた。シート状歯根膜細胞が付着した培養細胞移動治具は別の培養皿上に一旦密着させポリグリコール酸製不織布の周辺部をピンセットで押さえながら培養細胞移動治具を離すことでシート状歯根膜細胞をポリグリコール酸製不織布と共に剥離させることができた。
［比較例１］
In the same manner as in Example 1, a cultured cell moving jig (T-shaped handle base (part A) according to the present invention and a flexible material (part B) having water absorption formed in a downward convex shape) FIG. 2) was produced. As the material of each part, polycarbonate was selected for the base part, collagen gel was selected for the flexible material, and the surface of the collagen gel was covered with a non-woven fabric made of polyglycolic acid. As in Example 1, canine periodontal ligament cells were allowed to grow until the culture surface of the temperature-responsive culture dish coated with poly-N-isopropylacrylamide, which can control the adhesion and non-adhesion of cells depending on the temperature, became full. It was. The culture method followed a conventional method (cell seeding number 1 × 10 5 cells / cm 2 , 37 ° C., 5% CO 2 , culture period 4 days). Moreover, the coating amount of the poly-N-isopropylacrylamide was 2.0 μg / cm 2. Next, the cultured cell moving jig was gently brought into contact with the cultured cell surface, and the cultured cell moving jig was placed on the cultured cell surface. Furthermore, when the culture temperature was lowered and the culture dish surface was made non-adhered to supplement the cells with the cultured cell migration jig, the cells were placed on the polyglycolic acid nonwoven fabric that is the surface of the cell adhesion part of the cultured cell migration jig. Attached. Three culture dishes were prepared, and sheet-like periodontal ligament cells laminated in three layers were obtained on a non-woven fabric made of polyglycolic acid by continuously attaching a culture cell migration jig. The cultured cell transfer jig to which the sheet-like periodontal ligament cells have adhered is temporarily brought into close contact with another culture dish, and the culture cell transfer jig is released while holding the periphery of the non-woven fabric made of polyglycolic acid with tweezers. Can be peeled off together with the polyglycolic acid nonwoven fabric.
[Comparative Example 1]
培養細胞を回収するために、図６に示すようなアクリル製の培養細胞移動治具を作製した。図６で右の部品の下の円盤部分が細胞接着部である。このものを図６左の部品の筒状のところに図７の右部品の軸を挿入して使った。細胞培養方法、使用した温度応答性ポリマーが被覆した細胞培養基材は実施例１と同様な方法、基材を使用した。培養４日後、培養基材上の骨格筋筋芽細胞がコンフルエントになったことを確認した後、培養細胞上に静かに培養細胞移動治具を載せた。細胞培養基材を２０℃下で１５分間処理した後、静かに培養細胞移動治具を培養基材から離していった。その結果、培養細胞は２０％程度しか剥離しなかった。培養細胞移動治具の細胞接着部が平面なため培養細胞の付着している部分全体と均一に接することができなかったためと考えられる。 In order to collect the cultured cells, an acrylic cultured cell transfer jig as shown in FIG. 6 was prepared. In FIG. 6, the disk part below the right part is a cell adhesion part. This was used by inserting the shaft of the right part of FIG. 7 into the cylindrical part of the left part of FIG. As the cell culture method and the cell culture substrate coated with the temperature-responsive polymer used, the same method and substrate as in Example 1 were used. After 4 days of culture, after confirming that the skeletal myoblasts on the culture substrate had become confluent, the cultured cell transfer jig was gently placed on the cultured cells. After the cell culture substrate was treated at 20 ° C. for 15 minutes, the cultured cell transfer jig was gently separated from the culture substrate. As a result, only about 20% of cultured cells were detached. This is probably because the cell adhesion part of the cultured cell transfer jig was flat and could not uniformly contact the entire part to which the cultured cells adhered.
本発明に記載される凸状の細胞接着部を有する培養細胞移動治具を用いれば、細胞培養基材上の任意の範囲内の培養細胞を効率良く剥離させられ、その剥離させた培養細胞を再び簡便に付着させるようになる。そのため培養細胞を移動させたい場所へ簡便に移動させられ、しかも正確に移動できるようになる。さらに、細胞培養基材表面に温度応答性ポリマーを被覆したものを用いれば、生体組織への生着性が極めて高い培養細胞シートが得られるようになる。この方法で得られる培養細胞シートは、たとえば角膜移植、皮膚移植、角膜疾患治療、虚血性心疾患治療等の臨床応用が強く期待される。したがって、本発明は細胞工学、医用工学、などの医学、生物学等の分野における極めて有用な発明である。 By using the cultured cell moving jig having a convex cell adhesion part described in the present invention, the cultured cells in an arbitrary range on the cell culture substrate can be efficiently detached, and the detached cultured cells are removed. It comes to adhere easily again. Therefore, the cultured cells can be easily moved to a place where the cells are desired to move, and can be moved accurately. Furthermore, if a cell culture substrate surface coated with a temperature-responsive polymer is used, a cultured cell sheet with extremely high engraftment on living tissue can be obtained. The cultured cell sheet obtained by this method is strongly expected to have clinical applications such as corneal transplantation, skin transplantation, corneal disease treatment, ischemic heart disease treatment and the like. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.
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