JP4475847B2 - Anterior segment-related cell sheet, three-dimensional structure, and production method thereof - Google Patents
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Description
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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２００１−１６１３５３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。
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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。
【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２−２１１８６５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。
【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。
【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２−２１１８６５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。
【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。
【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、▲１▼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、▲２▼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、▲３▼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。
【００２５】
被覆領域の形態は、上部から観察して、例えば、▲１▼ラインとスペースからなるパターン、▲２▼水玉模様のパターン、▲３▼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、▲２▼の円形状のパターンのものが好ましい。
【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。
【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。
【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。
【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。
【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（ＰＶＤＦ）、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。
【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。
【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。
【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。
【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。
【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。
【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（ＰＩＰＡＡｍ）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）、２．２μｇ／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。
【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。
【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（ＤＭＥＭ＋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。
【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。
【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（ＰＩＰＡＡｍ）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（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。
【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ＰＩＰＡＡｍ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。
【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。
【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。
【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２０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。
【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。
【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。
【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。
【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。[0001]
[Industrial application fields]
The present invention relates to an anterior ocular segment-related cell sheet, a three-dimensional structure, a manufacturing method thereof, and a therapeutic method using them in the fields of biology, medicine, and the like.
[0002]
[Prior art]
Due to the remarkable development of medical technology, in recent years, organ transplants that attempt to replace difficult-to-treat organs with other 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 40 years ago and transplantation activities began. However, the number of donors is still small, and there are about 20,000 patients who need corneal transplantation in Japan alone, whereas only about 1/10 patients who can actually undergo transplantation treatment are about 2000. It is said. 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.
[0003]
Against this background, techniques for transplanting artificial substitutes or cells that have been cultured and organized have attracted attention. Representative examples thereof include artificial skin and cultured skin. Here, artificial skin using a synthetic polymer may cause rejection and the like, and is not preferable as skin for transplantation. On the other hand, since the cultured skin is obtained by culturing a part of the normal skin of the person to a desired size, even if it is used, there is no concern about rejection or the like, and it can be said to be the most natural masking agent.
[0004]
Conventionally, such cell culture has been performed on the surface of a synthetic polymer subjected to various treatments. For example, various containers and the like 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. Cells cultured and proliferated using such a cell culture container are peeled and collected from the container surface by treatment with a protease such as trypsin or a chemical.
[0005]
However, when recovering cells grown by chemical treatment as described above, the treatment process becomes complicated, the possibility of contamination is increased, and the grown cells are altered or damaged by chemical treatment. Disadvantages have been pointed out that there are cases where the original function of cells is impaired. In order to overcome such drawbacks, several techniques have been proposed so far.
[0006]
In Japanese Patent Publication No. 2-23191, keratinized epidermis cells derived from human newborns are cultured in a culture container under the condition that a keratinous tissue film is formed on the surface of the container. A method for producing an implantable membrane of keratinous tissue, characterized in that it is exfoliated by use. Specifically, a technique is disclosed in which 3T3 cells are grown and layered as a feeder layer, and a cell sheet is recovered using dispase, which is a proteolytic enzyme. However, the method described in the publication has the following drawbacks.
(1) Dispase is derived from bacteria, and the collected cell sheet needs to be thoroughly washed.
(2) Dispase treatment conditions differ for each cultured cell, and skill is required for the treatment.
(3) The epidermal cells cultured by dispase treatment are pathologically activated.
(4) The extracellular matrix is degraded by dispase treatment.
(5) Therefore, the affected part transplanted with the cell sheet is easily infected.
[0007]
However, anterior ocular segment-related cells such as corneal epithelial cells, corneal endothelial cells, and conjunctival epithelial cells that are the subject of the present invention have less cell-cell binding than skin cells, and even with the above dispase, they can be cultured. Later, it could not be peeled off and collected as a single sheet.
[0008]
In order to solve this problem, recently, a technique has been devised in which corneal epithelial cells and conjunctival epithelial cells are cultured and organized on the amniotic membrane from which the sponge layer and the epithelial layer have been removed, and the amnion is used as a cell piece for transplantation. (Japanese Patent Laid-Open No. 2001-161353). Since amniotic membrane has sufficient membrane strength and is not antigenic, it is convenient as a support for transplanted cell debris. In order to construct the sheet, it was desired to create a sheet having sufficient strength with only cells in the eye.
[0009]
[Problems to be solved by 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 segment-related cell sheet or a three-dimensional structure with few structural defects that 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. The purpose is to provide. In addition, the present invention provides a surface of the support that is easily and sufficiently strong as a single sheet by changing the environmental temperature without treatment with an enzyme such as dispase. It is an object to provide a method that can be peeled off and collected.
[0010]
[Means for Solving the Problems]
In order to solve the above-mentioned problems, the present inventors have studied and developed from various angles. As a result, the anterior ocular segment-related cells are cultured on a cell culture support whose surface is coated with a temperature-responsive polymer, and if necessary, the cultured cell layer is layered, and then the temperature of the culture solution is higher than the upper critical solution temperature Alternatively, the anterior ocular segment-related cell sheet or three-dimensional structure cultured at a lower critical solution temperature or lower is brought into close contact with the polymer membrane, and peeled off together with the polymer membrane, so that We have found that a three-dimensional structure can be obtained. The present invention has been completed based on such findings.
[0011]
That is, the present invention provides an anterior segment-related cell sheet or a three-dimensional structure with few structural defects that 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.
[0012]
In the present invention, cells are cultured on a cell culture support having a substrate surface coated with a temperature-responsive polymer having an upper or lower critical solution temperature of 0 to 80 ° C., and if necessary, by a conventional method. The cultured cell layer is layered, and then (1) the temperature of the culture solution is set to the upper critical solution temperature or higher or the lower critical solution temperature or lower,
(2) The cultured anterior ocular segment-related cell sheet or three-dimensional structure is brought into close contact with the polymer membrane, and (3) is peeled off together with the polymer membrane as it is, Provide a method of manufacturing the body.
[0013]
Furthermore, the present invention provides a cell culture support, a cell culture support in which the anterior ocular segment-related cell sheet or three-dimensional structure adhered to the polymer membrane obtained by the above production method is coated with a temperature-responsive polymer. A method for producing a three-dimensional structure is provided, wherein the layer is formed by repeating an operation of attaching to a polymer film or another cell sheet and then peeling off the adhered polymer film.
[0014]
In addition, the present invention provides the anterior segment-related cell sheet or three-dimensional structure for treating tissue that has been deeply damaged and / or wounded.
In addition, the present invention provides a treatment method characterized by transplanting the anterior ocular segment-related cell sheet or three-dimensional structure to a tissue that has been deficient and / or wounded deeply.
[0015]
Furthermore, the present invention provides not only a medical field but also an anterior ocular segment-related cell sheet or three-dimensional structure useful as a cell for safety evaluation of chemical substances, poisons, or pharmaceuticals.
[0016]
DETAILED DESCRIPTION OF THE INVENTION
Examples of suitable cells used in the preparation of the anterior 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. It is not restricted. In the present invention, the anterior segment-related cell sheet means a sheet in which various cells forming the anterior segment in a living body are cultured in a single layer on a culture support as described above, and then peeled off by the support. The three-dimensional structure means a sheet that is layered in a state where the various epidermal cultured cell sheets are used alone or in combination.
[0017]
The anterior ocular segment-related cell sheet or three-dimensional structure in the present invention is not damaged by proteolytic enzymes typified by dispase, trypsin and the like during culture. Therefore, the anterior ocular segment-related cell sheet or three-dimensional structure peeled from the substrate retains the desmosome structure between cells and cells, has few structural defects, and has high strength. This is because, 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. In the sheet of the present invention, the cells formed during the culture and the basement membrane protein between the substrates are not damaged by the enzyme. This makes it possible to adhere well to the affected tissue at the time of transplantation, and to perform an efficient treatment. Specifically, when using a normal proteolytic enzyme such as trypsin, the cell, the desmosome structure between the cells and the cell, the basement membrane-like protein between the substrates is hardly retained, Therefore, the cells are separated and separated in an individual state. Among them, dispase, which is a proteolytic enzyme, almost destroys the basement membrane-like protein between cells and substrates, but the desmosome structure can be peeled off while maintaining 10-60%. As is known, the resulting cell sheet is weak. In contrast, the cell sheet of the present invention is in a state where 80% or more of the desmosome structure and the basement membrane-like protein remain, and can obtain various effects as described above.
[0018]
As described above, the anterior ocular segment-related cell sheet or three-dimensional structure in the present invention has both a cell, a desmosome structure between cells, and a basement membrane-like protein between cells and a substrate, and has high strength. It is a sheet that was not obtained at all from the prior art.
[0019]
The temperature-responsive polymer used for coating the substrate 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 lysis temperature or the lower critical lysis temperature exceeds 80 ° C., the cells may die, which is not preferable. Further, if 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 cells are killed, which is also not preferable.
[0020]
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.
[0021]
Examples of the base material to be coated include substances that can generally give form, such as glass, modified glass, polystyrene, polymethylmethacrylate, and the like, which are usually used for cell culture, such as polymers other than those described above. All compounds, ceramics, etc. can be used.
[0022]
The method for coating the support with the temperature-responsive polymer is not particularly limited, and may be, for example, the method described in JP-A-2-21865. 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.
[0023]
The support material shown in the present invention includes an A region coated with a temperature-responsive polymer,
(1) a region coated with a polymer having a low affinity for cells,
(2) a region coated with a temperature-responsive polymer in an amount different from that of region A;
(3) 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 composed of any two or three combinations of (1) to (3) on the surface It is characterized by having.
[0024]
The manufacturing method is not limited as long as it finally has the above structure. For example, (1) First, the region B is formed on the entire surface of the substrate, and then finally, A method of masking the portion to be the B region and adding the A region, or a method in which the A and B are reversed. (2) Two layers A and B are coated in advance, and an ultrasonic or scanning type device. The method of scraping off one of the layers by the above method, (3) the method of offset printing of the coating substance, etc. may be used alone or in combination.
[0025]
For example, (1) a pattern consisting of lines and spaces, (2) a polka dot pattern, (3) a lattice pattern, other specially shaped patterns, A mixed pattern is mentioned and is not limited at all. However, considering the state of each tissue in the eye, the circular pattern (2) is preferable.
[0026]
Although the size of the covering region is not limited at all, considering the size of each tissue in the eye, as well as the contraction when the cultured anterior segment-related cell sheet or three-dimensional structure is peeled off, When cells in the circle are used in the shape pattern, the circle has a diameter of 5 cm or less, preferably 3 cm or less, and more preferably 2 cm or less. When using the outside of a circle, the diameter of the circle is 1 mm or less, preferably 3 mm or less, and more preferably 5 mm or less.
[0027]
The coverage of the temperature responsive polymer may have a range of 0.3~6.0μg / cm 2, preferably 0.5~3.5μg / cm 2, more preferably 0.8~3.0μg / Cm 2 . When the coating amount is less than 0.2 μg / cm 2 , the cells on the polymer are difficult to peel off even when a stimulus is applied, and the working efficiency is remarkably deteriorated. On the other hand, when the concentration is 6.0 μg / cm 2 or more, it is difficult for cells to adhere to the region, and it becomes difficult to sufficiently attach the cells.
[0028]
The polymer having low affinity with cells in the present invention is not limited as long as cells do not adhere to it, for example, hydrophilic polymers such as polyacrylamide, polydimethylacrylamide, polyethylene glycol, and cellulose, Alternatively, strong hydrophobic polymers such as silicone polymers and fluorine polymers can be used.
[0029]
In the present invention, cells are cultured on a cell culture support (for example, a cell culture dish) produced as described above. The culture medium temperature is not particularly limited as long as the polymer coated on the substrate surface has an upper critical solution temperature or lower, and when the polymer has a lower critical solution temperature or higher, the temperature is not particularly 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.
[0030]
In the method of the present invention, the culture time may be set in accordance with the purpose of use of the anterior segment-related cell sheet or the three-dimensional structure in accordance with the above method. In order to peel and collect the cultured cells from the support material, the cultured anterior segment-related cell sheet or three-dimensional structure is closely attached to the polymer film, and the temperature of the support material to which the cells are attached is determined as the support base material. By setting the temperature to the upper critical solution temperature or lower or the lower critical solution temperature or lower of the coating polymer, it can be peeled off together with the polymer film. In addition, peeling of the sheet can be performed in a culture solution in which cells are cultured or in other isotonic solutions, and can be selected according to the purpose. Examples of the polymer membrane used when the anterior ocular segment-related cell sheet or three-dimensional structure is closely attached include, for example, polyvinylidene difluoride (PVDF), polypropylene, polyethylene, cellulose and derivatives thereof, chitin, chitosan, collagen, Examples thereof include urethane.
[0031]
The method for producing a three-dimensional structure in the present invention is not particularly limited. For example, a generally known method of growing and stratifying 3T3 cells as a feeder layer, or adhering to the above polymer membrane The method etc. which manufacture by using the prepared epidermal cultured cell sheet can be mentioned. Specifically, the following method is exemplified.
(1) A cell sheet in close contact with the polymer membrane is attached to the cell culture support, and then the polymer membrane is peeled off from the cell sheet by adding a medium, and a cell sheet in intimate contact with another polymer membrane is attached. A method of layering cell sheets by repeating this process.
(2) The cell sheet in intimate contact with the polymer membrane is inverted and fixed on the cell culture support on the polymer membrane side, another cell sheet is attached to the cell sheet side, and then the medium is added to the polymer membrane. A method of layering cell sheets by repeating the operation of peeling the cell sheet and attaching another cell sheet again.
(3) A method in which cell sheets in close contact with the polymer membrane are brought into close contact with each other on the cell sheet side.
(4) A method in which a cell sheet in close contact with a polymer membrane 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]
The three-dimensional structure in the present invention is not necessarily composed only 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 superimposed on a cell sheet or a three-dimensional structure composed of corneal epithelial cells. Such a technique is extremely effective in making it closer to the anterior ocular tissue in the living body.
[0033]
For the purpose of exfoliating and collecting the anterior segment cell sheet or three-dimensional structure with high yield, a method of tapping or shaking the cell culture support, a method of stirring the medium using a pipette, etc. May be used alone or in combination. In addition, the cultured cells may be separated and recovered by washing with an isotonic solution or the like as necessary.
[0034]
The anterior ocular segment-related cell sheet or three-dimensional structure obtained by the above-described method is extremely superior in both peelability and non-invasiveness compared to those obtained by the conventional method, and for transplantation. The clinical application of corneas is strongly expected. In particular, the three-dimensional structure of anterior segment-related cells of the present invention is free from a basement membrane-like protein, unlike conventional transplant sheets, so that it can be engrafted even if the affected tissue is deeply cut during transplantation. . This is considered to be an extremely effective technique that improves the treatment efficiency of the affected area and further reduces the burden on the patient. The cell culture support used in the method of the present invention can be used repeatedly.
[0035]
【Example】
Hereinafter, the present invention will be described in more detail based on examples, but these do not limit the present invention in any way.
[0036]
[Examples 1 and 2]
40% of N-isopropylacrylamide monomer on a commercially available polystyrene cell culture dish (Falcon 3001 Petri dish (diameter 3.5 cm) manufactured by Becton Dickinson Labware) (Example 1) Then, 0.10 ml of a solution dissolved in isopropyl alcohol was applied to 50% (Example 2), and a 3.5 cm diameter metal mask having one 2 cm diameter hole was placed on the coated surface, Irradiate an electron beam with an intensity of 0.25 MGy as it is, and form a circular shape (island-like part of the N-isopropylacrylamide polymer (PIPAAm) on the surface of the culture dish. And then the metal mask should be removed. Then, 0.10 ml of 20% dissolved in isopropyl alcohol was applied, and a circular metal mask with a diameter of 2 cm was placed only on the circular part, and 0.25 MGy was left as it was. The acrylamide polymer was immobilized on the outer side of the circular PIPAAm layer by irradiating with an electron beam with the intensity of 2. After irradiation, the metal mask was removed, the culture dish was washed with ion-exchanged water, and the remaining monomer and culture dish were bound. Untreated PIPAAm was removed, dried in a clean bench, and sterilized with ethylene oxide gas to obtain a cell culture support material, where the amount of PIPAAm covered on the islands was completely the same as above without using a mask. As a result, the temperature-responsive polymer was found on the surface of the substrate. .6μg / cm 2 (Example 1), 2.2μg / cm 2 (Example 2) was found to be coated. In the resulting cell culture support material, by a conventional method normal rabbit corneal epithelial cells (Culture medium used: Cornepack (Kurabo Co., Ltd., 37 ° C., 5% CO 2 ) As a result, the corneal epithelial cells on any cell culture support material only in the circular part at the center. After 14 days in culture, the cultured cells were covered with a 2 cm diameter polyvinylidene difluoride (PVDF) membrane, the medium was gently aspirated, and the whole cell culture support material was incubated at 20 ° C. for 30 minutes. By incubating for a minute and cooling, the cells on any cell culture support material were detached with the overlying membrane, which could be easily detached from any of the cell sheets. The peeled cell sheet was sufficiently strong as a single sheet, retaining cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
[0037]
In each of the above examples, “low temperature treatment” was performed under the condition of incubation at 20 ° C. for 30 minutes. However, in the present invention, “low temperature treatment” is not limited to these temperatures and times. A preferable temperature condition for the “low temperature treatment” in the present invention is 0 ° C. to 30 ° C., and a preferable treatment time is 2 minutes to 1 hour.
[0038]
[Example 3]
On the cell culture support of Example 1, a medium of normal Green et al. Containing mitomycin C (DMEM + AB (for feeder layer preparation), for human newborn-derived keratinized epidermal cells). Normal rabbit corneal epithelial cells were cultured in the same manner except that the above was changed. As a result, the corneal epithelial cells on the culture support material normally attached only to the circular part at the center, proliferated, and further stratified. After 16 days of culture, the stratified corneal epithelial cells were detached by incubating the whole cell culture support material at 20 ° C. for 30 minutes and cooling. The exfoliated stratified corneal epithelial cells (three-dimensional structure) are circular and retain the cell, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate. I had it.
[0039]
[Comparative Examples 1 and 2]
A cell culture support material was produced 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). The coating amounts on the obtained cell culture support were 0.1 μg / cm 2 (Comparative Example 1) and 6.2 μg / cm 2 (Comparative Example 2), respectively. Thereafter, normal rabbit corneal epithelial cells were cultured and detached in the same manner as in Example 1. As a result, the cells on the support of Comparative Example 1 are not easily detached even by low-temperature treatment, and conversely, the cells are difficult to adhere on the support of Comparative Example 2, and it is difficult to sufficiently proliferate the cells. None of the cell culture supports was preferred as a culture substrate.
[0040]
[Example 4]
Corneal endothelial cells were collected from rabbit cornea according to a conventional method. The polyisopropylamide (PIPAAm) of Example 1 was seeded on a grafted culture dish at a cell density of 2 × 10 6 cells / cm 2 and cultured according to a conventional method (medium used: DMEM containing 10% fetal bovine serum, 37 ° C., 5% CO 2 ). As a result, these corneal endothelial cells also adhered and proliferated normally only at the central circular portion. Ten days later, after confirming that the corneal endothelial cells had become confluent, a 2 cm diameter polyvinylidene difluoride (PVDF) membrane was placed on the cells cultured in the same manner as in Example 1, and the medium was gently aspirated. The cell culture support material was incubated at 20 ° C. for 30 minutes and cooled, so that the cells were detached together with the covered membrane. The covered membrane could be easily peeled from the cell sheet. The peeled cell sheet was sufficiently strong as a single sheet, retaining cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
[0041]
[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 is placed at the center of the culture dish, PIPAAm is fixed to the periphery, and then a metal having a circular hole having a diameter of 2 cm is formed. A cell culture support material (with the inner polymer layer and the outer polymer layer in Example 1 opposite to each other) in which polyacrylamide was immobilized at the center by covering with a mask was produced. The coating amount of PIPAAm outside the hole was 2.1 μg / cm 2 . Next, conjunctival epithelial cells were collected from rabbit eyes according to a conventional method. On previous grafted culture dish were seeded at a cell density of 2 × 10 5 cells / cm 2 , and cultured in a conventional manner (using medium: MEM, 37 ° C. containing 10% fetal bovine serum, 5% CO 2 below) . As a result, the seeded conjunctival epithelial cells normally attached and proliferated only around the center. Ten days later, after confirming that the conjunctival epithelial cells were confluent, a polyvinylidene difluoride (PVDF) membrane was placed on the cells cultured in the same manner as in Example 1, the medium was gently aspirated, and cell culture was performed. The whole support material was incubated at 20 ° C. for 30 minutes and cooled, so that the cells were detached together with the covered membrane. The covered membrane could be easily peeled from the cell sheet. The peeled conjunctival epithelial cell sheet was sufficiently strong as a single sheet, retaining cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
[0042]
[Example 6]
The corneal epithelial cell sheet peeled in Example 1 was immediately overlaid on the corneal epithelial cell sheet on the culture dish of Example 2 from which the medium was gently removed without cooling. Thereafter, the medium used in Example 3 was gently put, 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 was obtained. The obtained stratified sheet of corneal epithelial cells was peeled off from the support surface by performing a low temperature treatment in the same manner as in Example 3. The exfoliated layered sheet (three-dimensional structure) of corneal epithelial cells was sufficiently strong as a single sheet.
[0043]
[Example 7]
The layered sheet of corneal epithelial cells detached in Example 3 was immediately overlaid on the corneal endothelial cell sheet on the culture dish of Example 4 from which the medium was gently removed without cooling. Thereafter, the medium used in Example 3 was gently put, 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 was obtained. The obtained stratified sheet of corneal epithelial cells was peeled off from the support surface by performing a low temperature treatment in the same manner as in Example 3. The peeled three-dimensional structure was sufficiently strong as a single sheet, retaining the cells, the desmosome structure between the cells, and the basement membrane-like protein between the cells and the substrate.
[0044]
[Example 8]
For the corneal endothelial cell sheet on the culture dish of Example 4 where the medium was gently removed without cooling, first, a 5% type IV collagen solution-containing medium (same as the medium of Example 4 except that collagen was included). And left to stand for 20 minutes. Thereafter, the medium was gently removed without cooling again. The layered sheet of corneal epithelial cells peeled in Example 3 was immediately overlaid on the corneal endothelial cell sheet left on the culture dish. Thereafter, the medium used in Example 3 was gently put, 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 was obtained. The obtained stratified sheet of corneal epithelial cells was peeled off from the support surface by performing a low temperature treatment in the same manner as in Example 3. The peeled three-dimensional structure was sufficiently strong as a single sheet.
[0045]
[Example 9]
In contrast to the perforated conjunctival epithelial cell sheet on the culture dish of Example 5 in which the medium was gently removed without cooling, a stratified sheet of corneal epithelial cells having a corneal endothelial cell layer peeled in Example 7 (3 The dimensional structure was immediately placed partially overlapping. Thereafter, the medium used in Example 3 was gently put, 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 attached with a conjunctival epithelial cell sheet was obtained. The obtained three-dimensional structure was peeled off from the support surface by performing a low temperature treatment in the same manner as in Example 3. The peeled three-dimensional structure was sufficiently strong as a single sheet.
[0046]
[Example 10]
The stratified sheet (three-dimensional structure) of corneal epithelial cells obtained in Example 3 was transplanted to a rabbit lacking the corneal epithelial cell part according to a conventional method. At that time, the stratified sheet of corneal epithelial cells was sutured directly to the wound part. As a result of removing the thread after about 3 weeks, the stratified sheet of corneal epithelial cells was well engrafted in the eyeball.
[0047]
From the above results, it has been found that according to the present technology, it is possible to create a sheet having sufficient strength with only 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 the treatment and constructing a more precise tissue.
[0048]
【The invention's effect】
The anterior ocular segment-related cell sheet or three-dimensional structure of the present invention does not decompose E-cadherin and laminin 5 as in the case of dispase treatment, and has very few structural defects. Is strongly expected. Therefore, the present invention is extremely useful in the fields of medicine, biology, etc., such as cell engineering and medical engineering.
Claims (13)
（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）培養により形成させた、前眼部関連細胞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の移植用重層化シートの製造方法であって、
前眼部関連細胞が、角膜上皮細胞、結膜上皮細胞、上皮幹細胞、またはそれらの組み合わせであり、
細胞培養基材表面の温度応答性高分子の被覆量が、０．３〜６．０μｇ／ｃｍ ２ である、前記製造方法。An anterior ocular segment-related cell is cultured in the presence of feeder cells on a cell culture support in which the surface of the cell culture substrate is coated with a temperature-responsive polymer having an upper or lower critical dissolution temperature in water of 0 to 80 ° C. , Forming a layered sheet of anterior segment-related cells ,
(1) The culture temperature was above the upper or lower critical solution temperature or less,
(2) The layered sheet of anterior segment associated cells formed by culture is adhered to the polymer membrane,
(3) Peel off with the polymer film as it is,
A method for producing a stratified sheet for transplantation of anterior segment-related cells , characterized in that
The anterior segment-related cells are corneal epithelial cells, conjunctival epithelial cells, epithelial stem cells, or combinations thereof;
The said manufacturing method whose coating amount of the temperature-responsive polymer on the cell culture substrate surface is 0.3-6.0 microgram / cm < 2 > .
（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１に記載の製造方法。A cell culture support as a region A coated with a temperature-responsive polymer;
(1) a region coated with a polymer having a low affinity for cells,
(2) a region coated with a temperature-responsive polymer in an amount different from that of region A;
(3) 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 composed of any two or three combinations of (1) to (3) on the surface The manufacturing method according to claim 1, wherein what is possessed is used.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001226141A JP4475847B2 (en) | 2001-07-26 | 2001-07-26 | Anterior segment-related cell sheet, three-dimensional structure, and production method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2001226141A JP4475847B2 (en) | 2001-07-26 | 2001-07-26 | Anterior segment-related cell sheet, three-dimensional structure, and production method thereof |
Related Child Applications (2)
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