JP2003038170A - Anterior eye part-associated cell sheet, three- dimensional structure and method for producing them - Google Patents
Anterior eye part-associated cell sheet, three- dimensional structure and method for producing themInfo
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- JP2003038170A JP2003038170A JP2001226141A JP2001226141A JP2003038170A JP 2003038170 A JP2003038170 A JP 2003038170A JP 2001226141 A JP2001226141 A JP 2001226141A JP 2001226141 A JP2001226141 A JP 2001226141A JP 2003038170 A JP2003038170 A JP 2003038170A
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- cell sheet
- cells
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Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、生物学、医学等の分野
における前眼部関連細胞シート、３次元構造体、及びそ
れらの製造法及びそれらを利用した治療法に関する。BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an anterior ocular segment-related cell sheet, a three-dimensional structure, a method for producing them, and a therapeutic method using them in the fields of biology, medicine and the like.
【０００２】[0002]
【従来の技術】医療技術の著しい発展により、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４０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２万人出てくるのに
対し、実際に移植治療が行える患者は約１／１０の２０
００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。2. Description of the Related Art With the remarkable development of medical technology, in recent years, organ transplantation in which an organ that has been difficult to treat has been replaced with another person's organ has become popular. The target organs are very diverse, such as the skin, cornea, kidney, liver, heart, etc., and the postoperative course has improved markedly, and it is already being established as a medical technique. As an example of corneal transplantation, about 40 years ago, an eye bank was established in Japan and transplantation activities started. However, the number of donors is still small, and about 20,000 patients need corneal transplants annually in Japan alone, while about 1/10 of the patients can actually undergo transplant treatment.
It is said that there are only about 00 people. Despite the almost established technique of corneal transplantation, there is a need for the next medical technique due to the problem of a shortage of donors.
【０００３】このような背景のもと、以前より、人工代
替物や細胞を培養して組織化させたものをそのまま移植
しようという技術が注目されている。その代表的な例と
して、人工皮膚及び培養皮膚があげられよう。ここで、
合成高分子を用いた人工皮膚は拒絶反応等が生じる可能
性があり、移植用皮膚としては好ましくない。一方、培
養皮膚は本人の正常な皮膚の一部を所望の大きさまで培
養したものであるため、これを使用しても拒絶反応等の
心配がなく、最も自然なマスキング剤と言える。Against this background, attention has long been paid to a technique for transplanting an artificial substitute or cells in which cells are cultured and organized as they are. Representative examples thereof include artificial skin and cultured skin. here,
Artificial skin using a synthetic polymer may cause rejection reactions or the like, and is not preferable as skin for transplantation. On the other hand, the cultured skin is obtained by culturing a part of normal skin of the person to a desired size, and therefore, even if it is used, there is no fear of rejection reaction and it can be said that it is the most natural masking agent.
【０００４】従来、そのような細胞培養は、ガラス表面
上あるいは種々の処理を行った合成高分子の表面上にて
行われていた。例えば、ポリスチレンを材料とする表面
処理、例えばγ線照射、シリコーンコーティング等を行
った種々の容器等が細胞培養用容器として普及してい
る。このような細胞培養用容器を用いて培養・増殖した
細胞は、トリプシンのような蛋白分解酵素や化学薬品に
より処理することで容器表面から剥離・回収される。Conventionally, such cell culture has been carried out on the surface of glass or on the surface of synthetic polymer subjected to various treatments. For example, various containers that have been subjected to surface treatment using polystyrene as a material, for example, γ-ray irradiation, silicone coating, and the like are widely used as cell culture containers. The cells cultivated and propagated using such a cell culture container are separated and collected from the container surface by treating with a protease or chemical such as trypsin.
【０００５】しかし、上述のような化学薬品処理を施し
て増殖した細胞を回収する場合、処理工程が煩雑にな
り、不純物混入の可能性が多くなること、及び増殖した
細胞が化学的処理により変成若しくは損傷し細胞本来の
機能が損なわれる例があること等の欠点が指摘されてい
た。かかる欠点を克服するために、これまでいくつかの
技術が提案されている。However, in the case of recovering the proliferated cells by the chemical treatment as described above, the treatment process becomes complicated and the possibility of contamination of impurities increases, and the proliferated cells are denatured by the chemical treatment. Or, it has been pointed out that there are some defects such as damage and loss of the original function of cells. Several techniques have been proposed so far in order to overcome such drawbacks.
【０００６】特公平２−２３１９１号公報には、ヒト新
生児由来角化表皮細胞を、ケラチン組織の膜が容器の表
面上に形成される条件下に、培養容器中で培養し、ケラ
チン組織の膜を酵素を用いて剥離させることを特徴とす
るケラチン組織の移植可能な膜を製造する方法、が記載
されている。具体的には、３Ｔ３細胞をフィーダーレイ
ヤーとして増殖、重層化させ、蛋白質分解酵素であるデ
ィスパーゼを用いて細胞シートを回収する技術が開示さ
れている。しかしながら、当該公報に記載されている方
法は次のような欠点を有していた。
（１）ディスパーゼは菌由来のものであり、回収された
細胞シートを十分に洗浄する必要性があること。
（２）培養された細胞ごとにディスパーゼ処理の条件が
異なり、その処理に熟練が必要であること。
（３）ディスパーゼ処理により培養された表皮細胞が病
理学的に活性化されること。
（４）ディスパーゼ処理により細胞外マトリックスが分
解されること。
（５）そのためその細胞シートを移植された患部は感染
され易いこと。[0006] Japanese Patent Publication No. 2-23191 discloses that human neonatal-derived keratinized epidermal cells are cultured in a culture container under the condition that a film of keratin tissue is formed on the surface of the container, and the keratin tissue film is formed. A method for producing an implantable membrane of keratinous tissue, which comprises peeling off the enzyme using an enzyme. Specifically, a technique is disclosed in which 3T3 cells are used as a feeder layer for growth and stratification, and a cell sheet is recovered using dispase, which is a proteolytic enzyme. However, the method described in this publication has the following drawbacks. (1) Dispase is derived from a bacterium, and it is necessary to thoroughly wash the recovered cell sheet. (2) The condition of dispase treatment differs depending on the cultured cells, and the treatment requires skill. (3) The epidermal cells cultured by the dispase treatment are pathologically activated. (4) The extracellular matrix is decomposed by the dispase treatment. (5) Therefore, the affected part where the cell sheet is transplanted is easily infected.
【０００７】しかしながら、本発明の対象とする角膜上
皮細胞、角膜内皮細胞、及び結膜上皮細胞などの前眼部
関連細胞は、皮膚細胞ほど細胞・細胞間の結合が強くな
く、上記ディスパーゼをもってしても、培養後、1枚の
シートとして剥離、回収することはできなかった。However, anterior ocular segment-related cells such as corneal epithelial cells, corneal endothelial cells, and conjunctival epithelial cells, which are the objects of the present invention, do not have as strong cell-cell bonds as skin cells, and have the above-mentioned dispase. However, after culturing, it could not be separated and collected as a single sheet.
【０００８】かかる課題を解決するために、最近、スポ
ンジ層と上皮層を除去した羊膜上で、角膜上皮細胞や結
膜上皮細胞を培養、組織化させ、その羊膜ごと移植用細
胞片とする技術が考案された（特開２００１−１６１３
５３号）。羊膜が十分な膜強度を持っていること、さら
に抗原性を持っていないことから、移植用細胞片の支持
体として好都合だが、羊膜というものがもともと眼内に
なく、より精密に眼内組織を構築していくためには、や
はり眼内の細胞だけで十分な強度を持ったシートの作成
が望まれていた。In order to solve such a problem, recently, a technique of culturing and organizing corneal epithelial cells and conjunctival epithelial cells on an amniotic membrane from which a sponge layer and an epithelial layer have been removed, and using the amniotic membrane as a cell fragment for transplantation has been proposed. Invented (Japanese Patent Laid-Open No. 2001-1613)
53). Since amniotic membrane has sufficient membrane strength and does not have antigenicity, it is convenient as a support for cell fragments for transplantation. In order to construct the sheet, it has been desired to create a sheet having sufficient strength only with cells in the eye.
【０００９】[0009]
【発明が解決しようとする課題】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、細胞、細胞間のデス
モソーム構造、及び細胞、基材間の基底膜様蛋白質が保
持された状態で回収される構造欠陥の少ない前眼部関連
細胞シートまたは３次元構造体を提供することを目的と
する。また、本発明は、ディスパーゼのような酵素で処
理することなく環境温度を変化させることにより、培養
・増殖させた細胞を容易にかつ１枚のシートとして十分
に強度を持った状態で支持体表面からの剥離・回収が可
能となる方法を提供することを目的とする。SUMMARY OF THE INVENTION The present invention has been made with the intention of solving the problems of the prior art as described above. That is, the present invention provides an anterior ocular segment-related cell sheet or a three-dimensional structure having few structural defects that are recovered in a state in which cells, a desmosome structure between cells, and a basement membrane-like protein between cells and a substrate are retained. The purpose is to provide. In addition, the present invention provides a method of changing the ambient temperature without treating with an enzyme such as dispase to allow the cultured and proliferated cells to be easily and sufficiently formed as one sheet on the surface of the support. It is an object of the present invention to provide a method that enables peeling / collection from an object.
【００１０】[0010]
【課題を解決するための手段】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、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前眼部関連細胞を培養
し、必要により培養細胞層を重層化させ、その後、培養
液温度を上限臨界溶解温度以上または下限臨界溶解温度
以下とし、培養した前眼部関連細胞シートまたは３次元
構造体を高分子膜に密着させ、そのまま高分子膜と共に
剥離することにより、構造欠陥の少ない前眼部関連細胞
シートまたは３次元構造体が得られることを見いだし
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。[Means for Solving the Problems] In order to solve the above-mentioned problems, the present inventors conducted research and development from various angles. As a result, the cells associated with the anterior segment of the eye were cultured on the cell culture support whose surface was coated with a temperature-responsive polymer, and the cultured cell layer was overlaid if necessary, and then the temperature of the culture solution was higher than the upper limit critical dissolution temperature. Alternatively, the anterior ocular segment-related cell sheet or the anterior ocular segment-related cell sheet having few structural defects can be obtained by bringing the cultured anterior ocular segment-related cell sheet or the three-dimensional structure into contact with the polymer film and peeling off together with the polymer film as it is, at or below the lower critical dissolution temperature. It has been found that a three-dimensional structure can be obtained. The present invention has been completed based on such findings.
【００１１】すなわち、本発明は、細胞、細胞間のデス
モソーム構造、及び細胞、基材間の基底膜様蛋白質が保
持された状態で回収される構造欠陥の少ない前眼部関連
細胞シートまたは３次元構造体を提供する。That is, the present invention provides a cell sheet or a three-dimensional anterior segment related cell sheet having few structural defects, which is recovered in a state in which cells, a desmosome structure between cells, and a basement membrane-like protein between cells and a substrate are retained. Provide a structure.
【００１２】また、本発明は、水に対する上限もしくは
下限臨界溶解温度が０〜８０℃である温度応答性ポリマ
ーで基材表面を被覆した細胞培養支持体上で細胞を培養
し、必要に応じて常法により培養細胞層を重層化させ、
その後（１）培養液温度を上限臨界溶解温度以上または
下限臨界溶解温度以下とし、（２）培養した前眼部関連
細胞シートまたは３次元構造体を高分子膜に密着させ、
及び（３）そのまま高分子膜と共に剥離することを特徴
とする前眼部関連細胞シートまたは３次元構造体の製造
法を提供する。The present invention also cultivates cells on a cell culture support having a substrate surface coated with a temperature-responsive polymer having an upper or lower critical solution temperature in water of 0 to 80 ° C., and if necessary, Layer the cultured cell layers by a conventional method,
After that, (1) the temperature of the culture solution is set to the upper limit critical melting temperature or higher or the lower limit critical melting temperature or lower, and (2) the cultured anterior ocular segment-related cell sheet or three-dimensional structure is adhered to the polymer membrane,
And (3) to provide a method for producing an anterior ocular segment-related cell sheet or a three-dimensional structure, which is characterized by being peeled off together with a polymer film.
【００１３】更に、本発明は、上記製造法で得られた高
分子膜に密着した前眼部関連細胞シートまたは３次元構
造体を再び細胞培養支持体、温度応答性ポリマーで表面
を被覆した細胞培養支持体、高分子膜、或いは他の細胞
シート等に付着させ、その後、密着した高分子膜を剥が
す操作を繰り返すことで重層化させることを特徴とする
３次元構造体の製造法を提供する。Further, the present invention provides the cell sheet or the three-dimensional structure associated with the anterior segment of the eye, which is adhered to the polymer membrane obtained by the above-mentioned production method, the surface of which is again coated with a cell culture support and a temperature-responsive polymer. Provided is a method for producing a three-dimensional structure, which is characterized in that it is attached to a culture support, a polymer membrane, or another cell sheet, and then the adhered polymer membrane is repeatedly peeled to form a multi-layer structure. .
【００１４】加えて、本発明は、深部まで欠損及び／ま
たは創傷した組織を治療するための上記前眼部関連細胞
シートまたは３次元構造体を提供する。更に加えて、本
発明は、深部まで欠損及び／または創傷した組織に対
し、上記前眼部関連細胞シートまたは３次元構造体を移
植することを特徴とする治療法を提供する。In addition, the present invention provides the anterior ocular segment-related cell sheet or three-dimensional structure for treating a tissue which is deeply damaged and / or injured. Furthermore, the present invention provides a therapeutic method characterized by transplanting the anterior ocular segment-related cell sheet or the three-dimensional structure to a tissue that is deeply defective and / or injured.
【００１５】更に、本発明は医療分野のみならず、化学
物質、毒物、或いは医薬品の安全性評価用細胞として有
用な前眼部関連細胞シートまたは３次元構造体を提供す
る。Further, the present invention provides an anterior ocular segment-related cell sheet or three-dimensional structure useful as cells for safety evaluation of chemical substances, poisons, or pharmaceuticals, not only in the medical field.
【００１６】[0016]
【発明の実施の形態】本発明の前眼部関連細胞シートま
たは３次元構造体の作製に使用される好適な細胞として
角膜上皮細胞、角膜内皮細胞、結膜上皮細胞等、及び上
皮幹細胞が挙げられるが、その種類は、何ら制約される
ものではない。本発明において、前眼部関連細胞シート
とは、上記したように生体における前眼部を形成する各
種細胞が培養支持体上で単層状に培養され、その後、支
持体よる剥離されたシートを意味し、その３次元構造体
とは、その各種表皮培養細胞シートが単独若しくは組み
合わされた状態で重層化されたシートを意味する。BEST MODE FOR CARRYING OUT THE INVENTION Suitable cells used for producing the anterior ocular segment-related cell sheet or three-dimensional structure of the present invention include corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells, and epithelial stem cells. However, the type is not restricted at all. In the present invention, the anterior ocular segment-related cell sheet means a sheet in which various cells forming the anterior ocular segment in the living body are cultured in a monolayer on a culture support as described above, and then peeled off by the support. The three-dimensional structure means a sheet in which the various epidermal culture cell sheets are laminated alone or in combination.
【００１７】本発明における前眼部関連細胞シートまた
は３次元構造体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損傷を受けていないもの
である。そのため、基材から剥離された前眼部関連細胞
シートまたは３次元構造体は、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、また強度の
高いものである。このことは、例えば、得られた前眼部
関連細胞シートまたは３次元構造体を移植等を目的に利
用した場合、本発明の前眼部関連細胞シートまたは３次
元構造体は十分に強度を持っているものであるため、患
部は外部と完全に隔離される。また、本発明のシートは
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いない。このことは、移植時にお
いて患部組織と良好に接着することができ、効率良い治
療を実施することができるようになる。以上のことを具
体的に説明すると、トリプシン等の通常の蛋白質分解酵
素を使用した場合、細胞、細胞間のデスモソーム構造及
び細胞、基材間の基底膜様蛋白質等は殆ど保持されてお
らず、従って、細胞は個々に分かれた状態となって剥離
される。その中で、蛋白質分解酵素であるディスパーゼ
に関しては、細胞、基材間の基底膜様蛋白質等を殆ど破
壊してしまうものの、デスモソーム構造については１０
〜６０％保持した状態で剥離させることができることで
知られているが、得られる細胞シートは強度の弱いもの
である。これに対して、本発明の細胞シートは、デスモ
ソーム構造、基底膜様蛋白質共に８０％以上残存された
状態のものであり、上述したような種々の効果を得るこ
とができるものである。The anterior ocular segment-related cell sheet or three-dimensional structure in the present invention is not damaged by proteolytic enzymes represented by dispase, trypsin and the like during culture. Therefore, the anterior ocular segment-related cell sheet or three-dimensional structure peeled from the base material retains the cells and the desmosome structure between the cells, has few structural defects, and has high strength. This means that, for example, when the obtained anterior segment-related cell sheet or three-dimensional structure is used for the purpose of transplantation, the anterior segment-related cell sheet or three-dimensional structure of the present invention has sufficient strength. The affected area is completely isolated from the outside. Further, in the sheet of the present invention, the basement membrane-like protein between cells and substrate formed during culture is not destroyed by the enzyme. This allows good adhesion to the affected tissue at the time of transplantation, and efficient treatment can be carried out. To explain the above specifically, when using a normal proteolytic enzyme such as trypsin, cells, desmosome structure between cells and cells, basement membrane-like protein between substrates, etc. are hardly retained, Therefore, the cells are separated and separated. Among them, dispase, which is a proteolytic enzyme, almost destroys basement membrane-like proteins between cells and substrates, but desmosome structure is 10
It is known that the cell sheet can be peeled while it is held at -60%, but the obtained cell sheet has weak strength. On the other hand, the cell sheet of the present invention has a desmosome structure and a basement membrane-like protein remaining in an amount of 80% or more, and can obtain various effects as described above.
【００１８】本発明における前眼部関連細胞シートまた
は３次元構造体は、以上に示すように、細胞、細胞間の
デスモソーム構造、及び、細胞、基材間の基底膜様蛋白
質双方を兼ね備え、しかも強度の高いシートであり、従
来技術からでは全く得られなかったものである。As described above, the anterior ocular segment-related cell sheet or three-dimensional structure of the present invention has both cells, a desmosome structure between cells, and a basement membrane-like protein between cells and a substrate, and It is a high-strength sheet, which has never been obtained from the prior art.
【００１９】細胞培養支持体において基材の被覆に用い
ら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。The temperature-responsive polymer used for coating the base material in the cell culture support 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 melting temperature or the lower critical melting temperature exceeds 80 ° C, cells may be killed, which is not preferable. Further, if the upper critical melting temperature or the lower critical melting temperature is lower than 0 ° C., the cell growth rate is generally extremely decreased or the cells are killed, which is also not preferable.
【００２０】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２−２１１８６
５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, for example, JP-A-2-21186.
The polymers described in Japanese Patent No. 5 can be mentioned. Specifically, for example, it can be obtained by homopolymerization or copolymerization of the following monomers. Examples of the monomer that can be used include (meth) acrylamide compounds, N- (or N, N-di) alkyl-substituted (meth) acrylamide derivatives, and vinyl ether derivatives, and in the case of copolymers, any of these can be used. 2 or more of can be used. Further, copolymerization with monomers other than the above monomers, grafting or copolymerization of polymers, or a mixture of polymers and copolymers may be used. Further, it is also possible to crosslink within a range that does not impair the original properties of the polymer.
【００２１】被覆を施される基材としては、通常細胞培
養に用いられるガラス、改質ガラス、ポリスチレン、ポ
リメチルメタクリレート等の化合物を初めとして、一般
に形態付与が可能である物質、例えば、上記以外の高分
子化合物、セラミックス類など全て用いることができ
る。The substrate to be coated is generally a substance that can be given a morphology such as glass, modified glass, polystyrene, polymethylmethacrylate, and other compounds that are commonly used for cell culture, for example, other than the above. All of the polymer compounds, ceramics, etc. can be used.
【００２２】温度応答性ポリマーの支持体への被覆方法
は、特に制限されないが、例えば、特開平２−２１１８
６５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。The method for coating the temperature-responsive polymer on the support is not particularly limited, but is, for example, JP-A-2-2118.
The method described in Japanese Patent Publication No. 65 may be followed. That is, such coating is carried out by electron beam irradiation (EB), γ ray irradiation, ultraviolet ray irradiation, plasma treatment, corona treatment, organic polymerization reaction, or physical coating such as coating or kneading of the base material and the above monomer or polymer. It can be performed by dynamic adsorption.
【００２３】本発明で示される支持体材料とは、温度応
答性高分子が被覆されたＡ領域と、（１）細胞と親和性
の低い高分子が被覆されている領域、（２）Ａ領域と異
なる量の温度応答性高分子が被覆されている領域、
（３）Ａ領域と異なる温度に応答する高分子が被覆され
ている領域のいずれか、または（１）〜（３）の任意の
２つもしくは３つの組み合わせからなるＢ領域の２領域
を表面に持つことを特徴とするものである。The support material shown in the present invention means an A region coated with a temperature responsive polymer, (1) a region coated with a polymer having a low affinity for cells, and (2) A region. An area coated with a different amount of temperature-responsive polymer,
(3) Any one of the regions coated with a polymer that responds to a temperature different from that of the A region, or two regions of the B region consisting of any two or three combinations of (1) to (3) on the surface. It is characterized by having.
【００２４】その製造法としては、最終的に上記の構造
を有するものであれば何ら制約されるものではないが、
例えば、基材表面上全体にまずＢ領域を作成し、その
後、最終的にＢ領域となる部分をマスクしてＡ領域を上
乗せする方法、或いはそのＡ，Ｂを逆にした方法、あ
らかじめＡ，Ｂの２層を被覆しておき、超音波或は走査
型機器によりどちらかの層を削り取る方法、被覆物質
をオフセット印刷する方法、等を単独または併用する方
法が挙げられる。The manufacturing method is not limited as long as it finally has the above structure,
For example, a method of first creating a B area on the entire surface of the base material, and then masking a part that finally becomes a B area and adding the A area, or a method in which A and B are reversed, Examples include a method in which two layers of B are coated and one of the layers is scraped off by ultrasonic waves or a scanning type device, a method in which the coating substance is offset printed, and the like are used alone or in combination.
【００２５】被覆領域の形態は、上部から観察して、例
えば、ラインとスペースからなるパターン、水玉模
様のパターン、格子状のパターン、その他特殊な形の
パターン、或いはこれらが混ざっている状態のパターン
が挙げられ何ら限定されるものではないが、眼内の各組
織の状態を考え、の円形状のパターンのものが好まし
い。The morphology of the covering region is, for example, a pattern consisting of lines and spaces, a polka dot pattern, a lattice pattern, a pattern of other special shape, or a pattern in which these are mixed when observed from above. Although not limited thereto, a circular pattern is preferable in consideration of the state of each tissue in the eye.
【００２６】被覆領域の大きさは何ら限定されるもので
はないが、眼内の各組織の大きさ、並びに培養した前眼
部関連細胞シートまたは３次元構造体を剥離した際、収
縮することを考え、円形状のパターンでその円内にある
細胞を使用する場合、その円の直径は５ｃｍ以下、好ま
しくは３ｃｍ以下、さらに２ｃｍ以下が好ましい。円の
外側を使う場合は、その円の直径は１ｍｍ以下、好まし
くは３ｍｍ以下、さらに５ｍｍ以下が好ましい。The size of the coated region is not limited, but it is not limited to the size of each tissue in the eye and the contraction when the cultured anterior ocular segment-related cell sheet or three-dimensional structure is peeled off. Considering that, when the cells within the circle are used in a circular pattern, the diameter of the circle is 5 cm or less, preferably 3 cm or less, and more preferably 2 cm or less. When the outside of the circle is used, the diameter of the circle is 1 mm or less, preferably 3 mm or less, and more preferably 5 mm or less.
【００２７】温度応答性高分子の被覆量は、０．３〜
６．０μｇ／ｃｍ2の範囲が良く、好ましくは０．５〜
３．５μｇ／ｃｍ2であり、さらに好ましくは０．８〜
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高分子上の細胞は
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６．０μｇ／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。The coating amount of the temperature responsive polymer is 0.3 to
The range of 6.0 μg / cm 2 is good, preferably 0.5-
3.5 μg / cm 2 , more preferably 0.8 to
It is 3.0 μg / cm 2 . When the coating amount is less than 0.2 μg / cm 2 , the cells on the polymer are difficult to be peeled off even if a stimulus is applied, and the working efficiency is significantly deteriorated, which is not preferable.
On the other hand, when it is 6.0 μg / cm 2 or more, it becomes difficult for the cells to adhere to the region, and it becomes difficult to sufficiently adhere the cells.
【００２８】本発明における細胞と親和性の低い高分子
とは、細胞が付着しないものならば何ら制約されるもの
ではないが、例えば、ポリアクリルアミド、ポリジメチ
ルアクリルアミド、ポリエチレングリコール、セルロー
ス等の親水性高分子、或いはシリコーン高分子、フッ素
高分子等の強疎水性高分子等が挙げられる。The polymer having a low affinity for cells in the present invention is not limited as long as it does not adhere to cells, but is hydrophilic such as polyacrylamide, polydimethylacrylamide, polyethylene glycol and cellulose. Examples thereof include polymers, and strongly hydrophobic polymers such as silicone polymers and fluoropolymers.
【００２９】本発明において、細胞の培養は上述のよう
にして製造された細胞培養支持体上（例えば、細胞培養
皿）で行われる。培地温度は、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, cell culture is performed on the cell culture support (eg, cell culture dish) produced as described above. The medium temperature is not particularly limited as long as the polymer coated on the surface of the base material has the upper limit critical dissolution temperature or lower, and if the polymer has the lower limit critical dissolution temperature or higher. But,
It goes without saying that culturing in a low temperature range where the cultured cells do not grow or in a high temperature range where the cultured cells die is inappropriate. Culture conditions other than temperature may be in accordance with a conventional method 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.
【００３０】本発明の方法においては、前記方法に従
い、前眼部関連細胞シートまたは３次元構造体の使用目
的に合わせて培養時間を設定すればよい。培養した細胞
を支持体材料から剥離回収するには、培養された前眼部
関連細胞シートまたは３次元構造体を高分子膜に密着さ
せ、細胞の付着した支持体材料の温度を支持体基材の被
覆ポリマーの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、そのまま高分子膜とと
もに剥離することができる。なお、シートを剥離するこ
とは細胞を培養していた培養液において行うことも、そ
の他の等張液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。前眼部関連細胞シートま
たは３次元構造体を密着させる際に使用する高分子膜と
しては、例えば、ポリビニリデンジフルオライド（ＰＶ
ＤＦ）、ポリプロピレン、ポリエチレン、セルロース及
びその誘導体、キチン、キトサン、コラーゲン、ウレタ
ン等を挙げることができる。In the method of the present invention, the culture time may be set according to the purpose of using the anterior ocular segment-related cell sheet or the three-dimensional structure according to the above method. In order to detach and collect the cultured cells from the support material, the cultured anterior ocular segment-related cell sheet or three-dimensional structure is brought into close contact with the polymer membrane, and the temperature of the support material with the cells attached is adjusted to the support base material. By controlling the coating polymer to have an upper critical solution temperature or higher or a lower critical solution temperature or lower, the polymer can be peeled off together with the polymer film. The sheet can be peeled off in the culture solution in which the cells are cultured or in another isotonic solution, and can be selected according to the purpose. Examples of the polymer film used for closely adhering the anterior ocular segment-related cell sheet or the three-dimensional structure include, for example, polyvinylidene difluoride (PV
DF), polypropylene, polyethylene, cellulose and derivatives thereof, chitin, chitosan, collagen, urethane and the like.
【００３１】本発明における３次元構造体の製造法は特
に限定されるものではないが、例えば、一般的に知られ
ている３Ｔ３細胞をフィーダーレイヤーとして増殖、重
層化させる方法、或いは上記の高分子膜に密着した表皮
培養細胞シートを利用することで製造する方法等を挙げ
ることができる。具体的には、次のような方法が例示さ
れる。
（１）高分子膜と密着した細胞シートを細胞培養支持体
に付着させ、その後培地を加えることで高分子膜を細胞
シートからはがし、そして更に別の高分子膜と密着した
細胞シートを付着させることを繰り返すことで細胞シー
トを重層化させる方法。
（２）高分子膜と密着した細胞シートを反転させ細胞培
養支持体上で高分子膜側で固定させ、細胞シート側に別
の細胞シートを付着させ、その後培地を加えることで高
分子膜を細胞シートからはがし、再び別の細胞シートを
付着させる操作を繰り返すことで細胞シートを重層化さ
せる方法。
（３）高分子膜と密着した細胞シート同士を細胞シート
側で密着させる方法。
（４）高分子膜と密着した細胞シートを生体の患部に当
て、細胞シートを生体組織に付着させた後、高分子膜を
はがし、再び別の細胞シートを重ねていく方法。The method for producing the three-dimensional structure in the present invention is not particularly limited, but, for example, a generally known method for proliferating and stacking 3T3 cells as a feeder layer, or the above-mentioned polymer. Examples include a method for producing by using an epidermal culture cell sheet that is in close contact with the membrane. Specifically, the following method is exemplified. (1) A cell sheet adhered to a polymer membrane is attached to a cell culture support, and then a medium is added to remove the polymer membrane from the cell sheet, and another cell sheet adhered to another polymer membrane is attached. A method of stacking cell sheets by repeating the above. (2) The cell sheet adhered to the polymer membrane is inverted and fixed on the polymer membrane side on the cell culture support, another cell sheet is attached to the cell sheet side, and then a medium is added to form the polymer membrane. A method of stacking cell sheets by repeating the procedure of peeling from the cell sheet and attaching another cell sheet again. (3) A method in which cell sheets that are in close contact with the polymer membrane are in close contact with each other on the cell sheet side. (4) A method in which a cell sheet in close contact with a polymer film is applied to an affected part of a living body, the cell sheet is attached to a living tissue, the polymer film is peeled off, and another cell sheet is stacked again.
【００３２】本発明における３次元構造体は、必ずしも
角膜上皮細胞だけからなるものでなくても良い。例え
ば、角膜上皮細胞からなる細胞シート或いは３次元構造
体に、同様に操作して作製した角膜内皮細胞シート及び
／または結膜上皮細胞シートを重ね合わせることも可能
である。生体内の前眼部組織により近いものとする上で
このような技術は極めて有効である。The three-dimensional structure in the present invention does not necessarily have to consist of corneal epithelial cells. For example, a corneal endothelial cell sheet and / or a conjunctival epithelial cell sheet produced by the same operation can be superposed on a cell sheet or a three-dimensional structure composed of corneal epithelial cells. Such a technique is extremely effective in making the tissue closer to the anterior segment tissue in the living body.
【００３３】前眼部関連細胞シートまたは３次元構造体
を高収率で剥離、回収する目的で、細胞培養支持体を軽
くたたいたり、ゆらしたりする方法、更にはピペットを
用いて培地を撹拌する方法等を単独で、あるいは併用し
て用いてもよい。加えて、必要に応じて培養細胞は等張
液等で洗浄して剥離回収してもよい。For the purpose of separating and collecting the anterior ocular segment-related cell sheet or three-dimensional structure with high yield, a method of gently tapping or shaking the cell culture support, and further stirring the medium with a pipette These methods may be used alone or in combination. In addition, the cultured cells may be washed with an isotonic solution or the like and separated and collected, if necessary.
【００３４】上述の方法により得られた前眼部関連細胞
シートまたは３次元構造体は、従来の方法により得られ
たものに比べて、剥離性の点でも非侵襲性の点でも極め
て優れており、移植用角膜等の臨床応用が強く期待され
る。特に、本発明の前眼部関連細胞の３次元構造体は従
来の移植シートとは異なり、基底膜様蛋白質をほじして
いるため、移植の際に患部組織を深く削っても、生着す
る。このことは、患部の治療効率の向上、更には患者の
負担の軽減もはかられ極めて有効な技術と考えられる。
なお、本発明の方法において使用される細胞培養支持体
は繰り返し使用が可能である。The anterior ocular segment-related cell sheet or three-dimensional structure obtained by the above-mentioned method is extremely superior in peelability and non-invasiveness as compared with those obtained by the conventional method. , Clinical applications such as cornea for transplantation are strongly expected. Particularly, since the three-dimensional structure of the anterior segment-related cells of the present invention is different from the conventional transplantation sheet and has the basement membrane-like protein, it is engrafted even if the affected tissue is deeply shaved during transplantation. . This is considered to be an extremely effective technique because it improves the treatment efficiency of the affected area and reduces the burden on the patient.
The cell culture support used in the method of the present invention can be repeatedly used.
【００３５】[0035]
【実施例】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。The present invention will be described in more detail based on the following examples, but they are not intended to limit the present invention.
【００３６】[0036]
【実施例１、２】市販のポリスチレン製細胞培養皿（ベ
クトン・ディッキンソン・ラブウェア（Ｂｅｃｔｏｎ
Ｄｉｃｋｉｎｓｏｎ Ｌａｂｗａｒｅ）社製 ファルコ
ン（ＦＡＬＣＯＮ）３００１ペトリディッシュ（直径
３．５ｃｍ）上に、Ｎ−イソプロピルアクリルアミドモ
ノマーを４０％(実施例１)、５０％（実施例２）になる
ようにイソプロピルアルコールに溶解させたものを０．
１０ｍｌ塗布した。その塗布面上に直径２ｃｍの孔を1
つ持つ直径３．５ｃｍの金属製マスクをのせ、そのまま
の状態で０．２５ＭＧｙの強度の電子線を照射し、培養
皿表面にＮ−イソプロピルアクリルアミドポリマー（Ｐ
ＩＰＡＡｍ）を円形状（島状の部分。マスク下の部分は
電子線が当たらず何も被覆されない海部分となる。）に
固定化した。次に、その金属製マスクをはずし、２０％
になるようにイソプロピルアルコールに溶解させたもの
を０．１０ｍｌ塗布した。そして、今度は、その円形部
分にのみ直径２ｃｍの円形の金属製マスクをおいて、そ
のままの状態で０．２５ＭＧｙの強度の電子線を照射す
ることで、円形ＰＩＰＡＡｍ層の外側にアクリルアミド
ポリマーを固定化した。照射後、金属製マスクをはず
し、イオン交換水により培養皿を洗浄し、残存モノマー
および培養皿に結合していないＰＩＰＡＡｍを取り除
き、クリーンベンチ内で乾燥し、エチレンオキサイドガ
スで滅菌することで細胞培養支持体材料を得た。ここで
島部分のＰＩＰＡＡｍの被覆量は、マスクを使わずに上
と全く同条件で作製した細胞培養支持体材料より求め
た。その結果、この条件下であれば、基材表面に温度応
答性高分子が１．６μｇ／ｃｍ2（実施例１）、２．２
μｇ／ｃｍ2（実施例２）被覆されることが分かった。
得られた細胞培養支持体材料上にて、常法により正常ウ
サギ角膜上皮細胞を培養した（使用培地：コルネパック
（クラボウ（株）製。３７℃、５％ＣＯ2下）。その結
果、何れの細胞培養支持体材料上の角膜上皮細胞におい
ても中心部の円形部分にのみ正常に付着し、増殖した。
培養１４日後、培養した細胞の上に直径２ｃｍのポリビ
ニリデンジフルオライド（ＰＶＤＦ）膜をかぶせ、培地
を静かに吸引し、細胞培養支持体材料ごと２０℃で３０
分インキュベートし冷却することで、何れの細胞培養支
持体材料上の細胞もそのかぶせた膜と共に剥離させられ
た。かぶせた膜は何れの細胞シートから容易に剥がすこ
とができた。また、剥離された細胞シートは、細胞、細
胞間のデスモソーム構造、及び細胞、基材間の基底膜様
蛋白質が保持され、１枚のシートとして十分に強度を持
ったものであった。Examples 1 and 2 Commercially available polystyrene cell culture dishes (Becton Dickinson Labware (Becton
Dissolve 40% (Example 1) and 50% (Example 2) of N-isopropylacrylamide monomer in isopropyl alcohol on a FALCON 3001 Petri dish (diameter 3.5 cm) manufactured by Dickinson Labware. What was made 0.
10 ml was applied. 1 hole with a diameter of 2 cm on the coated surface
A metal mask with a diameter of 3.5 cm is placed on the surface of the culture dish, and the surface of the culture dish is irradiated with an electron beam having an intensity of 0.25 MGy.
IPAAm) was immobilized in a circular shape (island-shaped portion. The portion under the mask is the sea portion where no electron beam hits and nothing is covered). Then, remove the metal mask, 20%
Then, 0.10 ml of a solution dissolved in isopropyl alcohol was applied. Then, a circular metal mask with a diameter of 2 cm was placed only on the circular portion, and the acrylamide polymer was fixed to the outside of the circular PIPAAm layer by irradiating an electron beam with an intensity of 0.25 MGy as it was. Turned into After irradiation, remove the metal mask, wash the culture dish with ion-exchanged water, remove the residual monomer and PIPAAm not bound to the culture dish, dry in a clean bench, and sterilize with ethylene oxide gas to perform cell culture. A support material was obtained. Here, the amount of PIPAAm coating on the island portion was determined from the cell culture support material prepared under exactly the same conditions as above without using a mask. As a result, under these conditions, the temperature-responsive polymer was 1.6 μg / cm 2 (Example 1) or 2.2 on the surface of the base material.
It was found that μg / cm 2 (Example 2) was coated.
Normal rabbit corneal epithelial cells were cultured on the obtained cell culture support material by a conventional method (use medium: Cornapak (Kurabo Co., Ltd., 37 ° C., under 5% CO 2 ). Also in the corneal epithelial cells on the cell culture support material, the cells were normally attached and proliferated only in the central circular portion.
After 14 days of culturing, a polyvinylidene difluoride (PVDF) membrane with a diameter of 2 cm is covered on the cultivated cells, the medium is gently aspirated, and the cell culture support material is kept at 20 ° C. for 30 minutes.
By incubating for minutes and cooling, the cells on any cell culture support material were detached with the overlaid membrane. The overlaid membrane could be easily peeled off from any cell sheet. The peeled cell sheet retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate, and had sufficient strength as one sheet.
【００３７】なお、上記各実施例において、「低温処
理」は２０℃で３０分インキュベートという条件下で行
われたが、本発明において「低温処理」はこれらの温度
及び時間に限定されない。本発明における「低温処理」
として好ましい温度条件は０℃〜３０℃であり、好まし
い処理時間は２分〜１時間である。In each of the above examples, the "low temperature treatment" was performed under the condition of incubation at 20 ° C for 30 minutes, but the "low temperature treatment" is not limited to these temperatures and times in the present invention. "Low temperature treatment" in the present invention
Is preferably 0 ° C. to 30 ° C., and a preferable treatment time is 2 minutes to 1 hour.
【００３８】[0038]
【実施例３】実施例１の細胞培養支持体上で、培地をマ
イトマイシンＣを含む通常のグリーンらの培地（ＤＭＥ
Ｍ＋ＡＢ（フィーダーレイヤー作製用）、ヒト新生児由
来角化表皮細胞用）。に変更する以外は同様な方法で正
常ウサギ角膜上皮細胞を培養させた。その結果、培養支
持体材料上の角膜上皮細胞は中心部の円形部分にのみ正
常に付着し、増殖し、さらに重層化した。培養１６日
後、細胞培養支持体材料ごと２０℃で３０分インキュベ
ートし冷却することで重層化角膜上皮細胞を剥離させら
れた。剥離された重層化角膜上皮細胞（３次元構造体）
は、円形で細胞、細胞間のデスモソーム構造、及び細
胞、基材間の基底膜様蛋白質が保持され、１枚のシート
として十分に強度を持ったものであった。[Example 3] On the cell culture support of Example 1, the medium was an ordinary medium containing mitomycin C (DME).
M + AB (for feeder layer preparation), for human neonatal-derived keratinized epidermal cells). Normal rabbit corneal epithelial cells were cultured in the same manner except that the procedure was changed to. As a result, the corneal epithelial cells on the culture support material were normally attached only to the circular portion in the central part, proliferated, and further stratified. After 16 days of culturing, the stratified corneal epithelial cells were detached by incubating the cell culture support material at 20 ° C. for 30 minutes and cooling. Exfoliated stratified corneal epithelial cells (three-dimensional structure)
The cells were circular and retained a cell, a desmosome structure between cells, and a basement membrane-like protein between cells and a substrate, and had sufficient strength as one sheet.
【００３９】[0039]
【比較例１、２】実施例１の細胞培養支持体を製造する
際のモノマー溶液を５％（比較例１）、６０％（比較例
２）とする以外は実施例１と同様に細胞培養支持体材料
を製造した。得られた細胞培養支持体上の被覆量は、そ
れぞれ０．１μｇ／ｃｍ2（比較例１）、６．２μｇ／
ｃｍ2（比較例２）であった。その後、実施例１と同様
な操作で正常ウサギ角膜上皮細胞を培養、剥離を試み
た。その結果、比較例１の支持体上の細胞は低温処理し
ても剥離し難く、逆に比較例２の支持体上に細胞が付着
し難く、細胞を十分に増殖させることが困難であり、何
れの細胞培養支持体も培養基材として好ましいものでは
なかった。[Comparative Examples 1 and 2] Cell culture was carried out in the same manner as in Example 1 except that the monomer solution for producing the cell culture support of Example 1 was 5% (Comparative Example 1) and 60% (Comparative Example 2). A support material was produced. The coating amounts on the obtained cell culture support were 0.1 μg / cm 2 (Comparative Example 1) and 6.2 μg / cm 2 , respectively.
It was cm 2 (Comparative Example 2). Then, normal rabbit corneal epithelial cells were cultured and peeled off in the same manner as in Example 1. As a result, the cells on the support of Comparative Example 1 were difficult to peel off even when subjected to low temperature treatment, on the contrary, the cells were difficult to attach to the support of Comparative Example 2, and it was difficult to sufficiently grow the cells. Neither cell culture support was preferred as a culture substrate.
【００４０】[0040]
【実施例４】ウサギ角膜より角膜内皮細胞を常法に従い
回収した。実施例１のポリイソプロピルアミド（ＰＩＰ
ＡＡｍ）をグラフト化培養皿上に２×１０6ｃｅｌｌｓ
／ｃｍ2の細胞密度で播種し、常法に従って培養した
（使用培地：１０％ウシ胎児血清を含むＤＭＥＭ、３７
℃、５％ＣＯ2下）。その結果、この角膜内皮細胞にお
いても中心部の円形部分にのみ正常に付着し、増殖し
た。１０日後、角膜内皮細胞がコンフルエントになった
ことを確認した後、実施例１と同様に培養した細胞の上
に直径２ｃｍのポリビニリデンジフルオライド（ＰＶＤ
Ｆ）膜をかぶせ、培地を静かに吸引し、細胞培養支持体
材料ごと２０℃で３０分インキュベートし冷却すること
で、細胞をそのかぶせた膜と共に剥離させられた。かぶ
せた膜は細胞シートから容易に剥がすことができた。ま
た、剥離された細胞シートは、細胞、細胞間のデスモソ
ーム構造、及び細胞、基材間の基底膜様蛋白質が保持さ
れ、１枚のシートとして十分に強度を持ったものであっ
た。Example 4 Corneal endothelial cells were collected from rabbit cornea according to a conventional method. Polyisopropylamide of Example 1 (PIP
2 × 10 6 cells on the grafted culture dish.
Cells were seeded at a cell density of / cm 2 and cultured according to a conventional method (use medium: DMEM containing 10% fetal bovine serum, 37
° C., under 5% CO 2). As a result, even in the corneal endothelial cells, they were normally attached and proliferated only in the central circular portion. After 10 days, after confirming that the corneal endothelial cells became confluent, polyvinylidene difluoride (PVD) having a diameter of 2 cm was placed on the cells cultured in the same manner as in Example 1.
F) The membrane was covered, the medium was gently aspirated, and the cells were detached together with the covered membrane by incubating together with the cell culture support material at 20 ° C. for 30 minutes and cooling. The overlaid membrane could be easily removed from the cell sheet. The peeled cell sheet retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate, and had sufficient strength as one sheet.
【００４１】[0041]
【実施例５】実施例２の細胞培養支持体材料の製造法に
おいて、まず直径２ｃｍの円形の金属製マスクを培養皿
の中心に置き、ＰＩＰＡＡｍを周囲に固定化し、次に直
径２ｃｍの円形の孔のあいた金属製マスクをかぶせるこ
とでポリアクリルアミドを中心部に固定化した細胞培養
支持体材料（実施例１の内側の高分子層と外側の高分子
層とが逆なものとなる。）を製造した。孔の外側のＰＩ
ＰＡＡｍの被覆量は２．１μｇ／ｃｍ2であった。次
に、ウサギの眼より結膜上皮細胞を常法に従い回収し
た。先のグラフト化培養皿上に２×１０5ｃｅｌｌｓ／
ｃｍ2の細胞密度で播種し、常法に従って培養した（使
用培地：１０％ウシ胎児血清を含むＭＥＭ、３７℃、５
％ＣＯ2下）。その結果、播種された結膜上皮細胞は中
心部の周囲のみに正常に付着し、増殖した。１０日後、
結膜上皮細胞がコンフルエントになったことを確認した
後、実施例１と同様に培養した細胞の上にポリビニリデ
ンジフルオライド（ＰＶＤＦ）膜をかぶせ、培地を静か
に吸引し、細胞培養支持体材料ごと２０℃で３０分イン
キュベートし冷却することで、細胞をそのかぶせた膜と
共に剥離させられた。かぶせた膜は細胞シートから容易
に剥がすことができた。また、剥離された結膜上皮細胞
シートは、細胞、細胞間のデスモソーム構造、及び細
胞、基材間の基底膜様蛋白質が保持され、１枚のシート
として十分に強度を持ったものであった。[Example 5] In the method for producing the cell culture support material of Example 2, first, a circular metal mask having a diameter of 2 cm was placed in the center of the culture dish, PIPAAm was immobilized on the periphery, and then a circular mask having a diameter of 2 cm was used. A cell culture support material having polyacrylamide immobilized in the center by covering with a metal mask having holes (the inner polymer layer and the outer polymer layer in Example 1 are reversed). Manufactured. PI outside the hole
The coverage of PAAm was 2.1 μg / cm 2 . Next, conjunctival epithelial cells were collected from the eyes of rabbits by a conventional method. 2 × 10 5 cells / on the above graft culture dish
The cells were seeded at a cell density of cm 2 and cultured according to a conventional method (use medium: MEM containing 10% fetal bovine serum, 37 ° C., 5
% CO 2 under). As a result, the seeded conjunctival epithelial cells were normally attached only to the periphery of the central part and proliferated. 10 days later,
After confirming that the conjunctival epithelial cells became confluent, the cells cultured in the same manner as in Example 1 were covered with a polyvinylidene difluoride (PVDF) membrane, the medium was gently aspirated, and the cell culture support material was used. Cells were detached together with the overlaid membrane by incubating and cooling at 20 ° C. for 30 minutes each. The overlaid membrane could be easily removed from the cell sheet. The peeled conjunctival epithelial cell sheet retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate, and had sufficient strength as one sheet.
【００４２】[0042]
【実施例６】冷却せずに静かに培地を除去した実施例２
の培養皿上の角膜上皮細胞シートに対し、実施例１で剥
離された角膜上皮細胞シートを直ちに重ねた。その後、
実施例３で用いた培地を静かに入れ、密着した高分子膜
を剥がした。この状態で２日間培養することで、角膜上
皮細胞の重層化シート（３次元構造体）を得た。得られ
た角膜上皮細胞の重層化シートは実施例３と同様に低温
処理を施すことにより支持体表面から剥離した。剥離さ
れた角膜上皮細胞の重層化シート（３次元構造体）は、
１枚のシートとして十分に強度を持ったものであった。Example 6 Example 2 in which the medium was gently removed without cooling
The corneal epithelial cell sheet peeled off in Example 1 was immediately overlaid on the corneal epithelial cell sheet on the culture dish of. afterwards,
The medium used in Example 3 was gently put in and the adhered polymer film was peeled off. By culturing in this state for 2 days, a stratified sheet of corneal epithelial cells (three-dimensional structure) was obtained. The obtained corneal epithelial cell multi-layered sheet was peeled from the surface of the support by the low temperature treatment as in Example 3. The exfoliated corneal epithelial cell stratification sheet (three-dimensional structure) is
The sheet was sufficiently strong as one sheet.
【００４３】[0043]
【実施例７】冷却せずに静かに培地を除去した実施例４
の培養皿上の角膜内皮細胞シートに対し、実施例３で剥
離された角膜上皮細胞の重層化シートを直ちに重ねた。
その後、実施例３で用いた培地を静かに入れ、密着した
高分子膜を剥がした。この状態で２日間培養すること
で、角膜内皮細胞層を有する角膜上皮細胞の重層化シー
ト（３次元構造体）を得た。得られた角膜上皮細胞の重
層化シートは実施例３と同様に低温処理を施すことによ
り支持体表面から剥離した。剥離された３次元構造体
は、細胞、細胞間のデスモソーム構造、及び細胞、基材
間の基底膜様蛋白質が保持され、１枚のシートとして十
分に強度を持ったものであった。[Example 7] Example 4 in which the medium was gently removed without cooling
The layered sheet of corneal epithelial cells peeled off in Example 3 was immediately overlaid on the corneal endothelial cell sheet on the culture dish.
Then, the medium used in Example 3 was gently put in, and the adhered polymer film was peeled off. By culturing for 2 days in this state, a stratified sheet (three-dimensional structure) of corneal epithelial cells having a corneal endothelial cell layer was obtained. The obtained corneal epithelial cell multi-layered sheet was peeled from the surface of the support by the low temperature treatment as in Example 3. The exfoliated three-dimensional structure retained the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate, and had sufficient strength as one sheet.
【００４４】[0044]
【実施例８】冷却せずに静かに培地を除去した実施例４
の培養皿上の角膜内皮細胞シートに対し、まず５％IV型
コラーゲン溶含有培地（コラーゲンが含まれること以外
は実施例４の培地と同一のもの。）を入れ、そのまま２
０分間静置させた。その後、再び冷却せずに静かに培地
を除去した。培養皿の上に残された角膜内皮細胞シート
に対し、実施例３で剥離された角膜上皮細胞の重層化シ
ートを直ちに重ねた。その後、実施例３で用いた培地を
静かに入れ、密着した高分子膜を剥がした。この状態で
２日間培養することで、角膜内皮細胞層を有する角膜上
皮細胞の重層化シート（３次元構造体）を得た。得られ
た角膜上皮細胞の重層化シートは実施例３と同様に低温
処理を施すことにより支持体表面から剥離した。剥離さ
れた３次元構造体は、１枚のシートとして十分に強度を
持ったものであった。[Example 8] Example 4 in which the medium was gently removed without cooling
First, a 5% IV type collagen lysis-containing medium (the same as the medium of Example 4 except that collagen was contained) was put into the corneal endothelial cell sheet on the culture dish of 1.
Let stand for 0 minutes. Then, the medium was gently removed without cooling again. The stratified sheet of corneal epithelial cells peeled off in Example 3 was immediately overlaid on the corneal endothelial cell sheet left on the culture dish. Then, the medium used in Example 3 was gently put in, and the adhered polymer film was peeled off. By culturing for 2 days in this state, a stratified sheet (three-dimensional structure) of corneal epithelial cells having a corneal endothelial cell layer was obtained. The obtained corneal epithelial cell multi-layered sheet was peeled from the surface of the support by the low temperature treatment as in Example 3. The peeled three-dimensional structure had sufficient strength as one sheet.
【００４５】[0045]
【実施例９】冷却せずに静かに培地を除去した実施例５
の培養皿上の孔のあいた結膜上皮細胞シートに対し、実
施例７で剥離された角膜内皮細胞層を有する角膜上皮細
胞の重層化シート（３次元構造体）を直ちに一部重ねる
ように置いた。その後、実施例３で用いた培地を静かに
入れ、密着した高分子膜を剥がした。この状態で２日間
培養することで、結膜上皮細胞シートが付着した角膜内
皮細胞層を有する角膜上皮細胞の重層化シート（３次元
構造体）を得た。得られた３次元構造体は実施例３と同
様に低温処理を施すことにより支持体表面から剥離し
た。剥離された３次元構造体は、１枚のシートとして十
分に強度を持ったものであった。[Example 9] Example 5 in which the medium was gently removed without cooling
The multilayered sheet of corneal epithelial cells (three-dimensional structure) having the corneal endothelial cell layer peeled off in Example 7 was immediately placed on the perforated conjunctival epithelial cell sheet on the culture dish of Example 3 so as to partially overlap it. . Then, the medium used in Example 3 was gently put in, and the adhered polymer film was peeled off. By culturing in this state for 2 days, a stratified sheet (three-dimensional structure) of corneal epithelial cells having a corneal endothelial cell layer to which the conjunctival epithelial cell sheet was attached was obtained. The obtained three-dimensional structure was subjected to low temperature treatment in the same manner as in Example 3 to be peeled from the surface of the support. The peeled three-dimensional structure had sufficient strength as one sheet.
【００４６】[0046]
【実施例１０】実施例３で得られた角膜上皮細胞の重層
化シート（３次元構造体）を角膜上皮細胞部を欠損させ
たウサギに常法に従い移植した。その際、角膜上皮細胞
の重層化シートを直接、創傷部へ縫合した。約3週間
後、抜糸した結果、角膜上皮細胞の重層化シートは眼球
に良好に生着していた。Example 10 The layered sheet of corneal epithelial cells (three-dimensional structure) obtained in Example 3 was transplanted to a rabbit deficient in the corneal epithelial cell part by a conventional method. At that time, a stratified sheet of corneal epithelial cells was directly sutured to the wound. Approximately 3 weeks later, as a result of thread removal, the stratified corneal epithelial cell sheet was successfully engrafted on the eyeball.
【００４７】以上の結果より、本技術によれば、本来、
眼内にある細胞だけで十分な強度を持ったシートの作成
が可能であることがわかった。このことは、治療の効率
化による患者の負担軽減、また、さらなる精密な組織の
構築化において極めて有効な技術と考えられる。From the above results, according to the present technology, originally,
It was found that it is possible to make a sheet having sufficient strength only with cells in the eye. This is considered to be an extremely effective technique for reducing the burden on the patient by improving the efficiency of treatment and for constructing a more precise tissue.
【００４８】[0048]
【発明の効果】本発明の前眼部関連細胞シートまたは３
次元構造体は、ディスパーゼ処理における場合のように
Ｅ−カドヘリン、ラミニン５を分解することがなく、し
かも構造欠陥が極めて少ないため、皮膚移植等の臨床応
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。EFFECT OF THE INVENTION Anterior ocular segment-related cell sheet or 3 of the present invention
The three-dimensional structure does not decompose E-cadherin and laminin-5 as in the case of dispase treatment, and has extremely few structural defects, and thus is strongly expected for clinical application such as skin transplantation. Therefore, the present invention relates to cell engineering,
It is an extremely useful invention in the fields of medicine and biology, such as medical engineering.
Claims (16)
細胞、基材間の基底膜様蛋白質が保持され、１枚のシー
トとして十分に強度を持った状態で回収される構造欠陥
の少ない前眼部関連細胞シートまたは３次元構造体。1. An anterior eye with few structural defects, which retains cells, desmosome structure between cells, and basement membrane-like protein between cells and substrate and is recovered as a single sheet with sufficient strength. Part-related cell sheet or three-dimensional structure.
となく基材から剥離された、請求項１記載の前眼部関連
細胞シートまたは３次元構造体。2. The anterior ocular segment-related cell sheet or three-dimensional structure according to claim 1, which is peeled from the substrate without being treated with a proteolytic enzyme.
内皮細胞、結膜上皮細胞及び上皮幹細胞であることを特
徴とする請求項１又は２記載の前眼部関連細胞シートま
たは３次元構造体。3. The anterior ocular segment-related cell sheet or the three-dimensional structure according to claim 1 or 2, wherein the anterior ocular segment-related cells are corneal epithelial cells, corneal endothelial cells, conjunctival epithelial cells and epithelial stem cells. body.
培養させたものである請求項１〜３のいずれか１項記載
の前眼部関連細胞シートまたは３次元構造体。4. The anterior ocular segment-related cell sheet or the three-dimensional structure according to any one of claims 1 to 3, wherein the three-dimensional structure is obtained by culturing stratified corneal epithelial cells.
胞シートもしくはその重層化物と角膜内皮細胞が組み合
わされたものである請求項１〜３のいずれか１項記載の
前眼部関連細胞シートまたは３次元構造体。5. The anterior ocular segment related cell sheet according to any one of claims 1 to 3, wherein the three-dimensional structure is a combination of at least a corneal epithelial cell sheet or a stratified product thereof and corneal endothelial cells. Three-dimensional structure.
体に、さらに結膜上皮細胞が組み合わされたものである
請求項１〜３のいずれか１項記載の前眼部関連細胞シー
トまたは３次元構造体。6. The anterior ocular segment-related cell sheet according to claim 1, wherein the three-dimensional structure is a combination of the three-dimensional structure of claim 5 and conjunctival epithelial cells. Or a three-dimensional structure.
度が０〜８０℃である温度応答性高分子で基材表面を被
覆した細胞培養支持体上で細胞を培養し、必要に応じて
常法により培養細胞層を重層化させ、その後、（１）培
養液温度を上限臨界溶解温度以上または下限臨界溶解温
度以下とし、さらに必要に応じ、（２）培養した前眼部
細胞シートまたはその重層化シートを高分子膜に密着さ
せ、（３）そのまま高分子膜と共に剥離することを特徴
とする前眼部関連細胞シートまたは３次元構造体の製造
法。7. The cells are cultured on a cell culture support having a substrate surface coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature in water of 0 to 80 ° C., and if necessary, by a conventional method. The cultured cell layer is overlaid, and then (1) the culture solution temperature is set to the upper limit critical melting temperature or higher or the lower limit critical melting temperature or lower, and, if necessary, (2) the cultured anterior segment cell sheet or its laminated sheet The method for producing a cell sheet or an anterior ocular segment-related cell sheet or three-dimensional structure, comprising:
が被覆されたＡ領域と、（１）細胞と親和性の低い高分
子が被覆されている領域、（２）Ａ領域と異なる量の温
度応答性高分子が被覆されている領域、（３）Ａ領域と
異なる温度に応答する高分子が被覆されている領域のい
ずれか、または（１）〜（３）の任意の２つもしくは３
つの組み合わせからなるＢ領域の２領域を表面に持つも
のを利用することを特徴とする請求項７記載の前眼部関
連細胞シートまたは３次元構造体の製造法。8. An amount different from an A region coated with a temperature responsive polymer as a cell culture support, (1) a region coated with a polymer having a low affinity for cells, and (2) an A region. Any of the region coated with the temperature-responsive polymer of (3) A region, and the region coated with a polymer that responds to a different temperature from A region, or any two of (1) to (3). Three
The method for producing an anterior ocular segment-related cell sheet or a three-dimensional structure according to claim 7, characterized in that one having two regions of B region consisting of one combination on the surface is used.
トまたは３次元構造体を再び細胞培養支持体、温度応答
性ポリマーで表面を被覆した細胞培養支持体、高分子膜
等に付着させ、重ね合わせていくこと、或いは他の細胞
シート等に一部、もしくは全部を付着させ、重ね合わせ
ていくことを含む請求項７又は８記載の３次元構造体の
製造法。9. The anterior ocular segment-related cell sheet or three-dimensional structure obtained in claim 7 is attached again to a cell culture support, a cell culture support whose surface is coated with a temperature-responsive polymer, a polymer membrane or the like. 9. The method for producing a three-dimensional structure according to claim 7 or 8, including stacking or stacking, or partially or entirely attaching to another cell sheet or the like and stacking.
されていない、請求項７〜９のいずれか１項記載の前眼
部関連細胞シートまたは３次元構造体の製造法。10. The method for producing an anterior ocular segment-related cell sheet or a three-dimensional structure according to any one of claims 7 to 9, wherein the exfoliation is not treated with a proteolytic enzyme.
ソプロピルアクリルアミド）である、請求項７〜９記載
の前眼部関連細胞シートまたは３次元構造体の製造法。11. The method for producing an anterior ocular segment-related cell sheet or a three-dimensional structure according to claim 7, wherein the temperature-responsive polymer is poly (N-isopropylacrylamide).
リビニリデンジフルオライドである、請求項７〜９のい
ずれか１項記載の前眼部関連細胞シートまたは３次元構
造体の製造法。12. The method for producing an anterior ocular segment-related cell sheet or a three-dimensional structure according to claim 7, wherein the polymer membrane is polyvinylidene difluoride subjected to a hydrophilic treatment. Law.
ト及びその重層化シート、角膜内皮細胞シート、及び結
膜上皮細胞シート、並びに請求項４記載の製造法で作製
した３次元構造体の中の１種もしくは２種以上のものか
らなる、請求項７〜９のいずれか１項記載の３次元構造
体の製造法。13. The other cell sheet includes a corneal epithelial cell sheet and a laminated sheet thereof, a corneal endothelial cell sheet, and a conjunctival epithelial cell sheet, and the three-dimensional structure produced by the production method according to claim 4. The method for producing a three-dimensional structure according to any one of claims 7 to 9, comprising one type or two or more types.
載の方法により製造される前眼部関連細胞シートまたは
３次元構造体。14. An anterior ocular segment-related cell sheet or three-dimensional structure produced by the method according to any one of claims 7 to 13.
しくは欠損した患部に対する治療用の請求項１〜６及び
１４のいずれか１項記載の前眼部関連細胞シートまたは
３次元構造体。15. The anterior ocular segment-related cell sheet or the three-dimensional structure according to any one of claims 1 to 6 and 14, which is used for treating an affected part in which a part or all of the anterior ocular segment tissue is damaged or defective.
しくは欠損した患部に対し、請求項１〜６及び１４のい
ずれか１項記載の前眼部関連細胞シートまたは３次元構
造体を移植することを特徴とする治療法。16. An anterior ocular segment-related cell sheet or a three-dimensional structure according to any one of claims 1 to 6 and 14 is transplanted to an affected part in which a part or all of the anterior ocular segment tissue is damaged or deleted. A treatment method characterized by:
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| JP2010031135A Division JP5264799B2 (en) | 2010-02-16 | 2010-02-16 | Anterior segment-related cell sheet, three-dimensional structure, and production method thereof |
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