JPWO2012137896A1 - シート状の膵島の製造方法 - Google Patents
シート状の膵島の製造方法 Download PDFInfo
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- JPWO2012137896A1 JPWO2012137896A1 JP2013508938A JP2013508938A JPWO2012137896A1 JP WO2012137896 A1 JPWO2012137896 A1 JP WO2012137896A1 JP 2013508938 A JP2013508938 A JP 2013508938A JP 2013508938 A JP2013508938 A JP 2013508938A JP WO2012137896 A1 JPWO2012137896 A1 JP WO2012137896A1
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Abstract
Description
[1]単離された膵島を、E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器中、該固相表面に接着した状態で、当該膵島がシート状の形態を呈するのに十分な期間培養することを含む、シート状の膵島の製造方法。
[2]当該ポリペプチドがE−カドヘリンの細胞外ドメインを含む、[1]記載の製造方法。
[3]当該ポリペプチドがE−カドヘリンの細胞外ドメイン及びイムノグロブリンのFc領域を含む融合ポリペプチドである、[1]記載の製造方法。
[4]E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器、及びシート状の膵島を含み、該シート状の膵島が該固相表面に接着した状態で培養可能となる、膵島培養物。
[5]E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器、及び単離された膵島を含む、シート状の膵島の製造用キット。
通常、体内であれば膵島組織内への血管誘導が生じるため、膵島が酸素不足に陥ることはない。しかしながら、移植した膵島の場合、移植部位における低酸素条件環境下では、酸素不足によって膵島の細胞死が誘導される懸念がある。本発明の方法により得られるシート状の膵島を用いれば、低酸素状態でも効率的にそれぞれの細胞へ酸素供給が可能となり、低酸素条件でも膵島の細胞死が抑制され、かつ膵島の形態をシート状にすることによってグルコース応答機能が増加することが考えられるので、移植医療に有利である。
膵島培養
体重20〜25g(9〜10週齢)の雄性マウス(C57BL/6J;日本チャールスリバー)から、collagenase from clostridium histdyticum Type V(GIBCO)を用いた消化法により膵島を分離した。この膵島に対してBiocoll Separating Solution(Biocheom AG)を用いた密度勾配法およびピペットマンを用いたハンドピックアップを行って、膵島を分離した。分離した膵島を3.5cm E−cad−Fcコートディッシュ(住友ベークライト株式会社)及び非処理ディッシュ(IWAKI)上で、10(v/v)% FBS(GIBCO)、抗生剤として1(v/v)% Anti−Anti(GIBCO)を添加したDulbecco’s Modified Eagle’s Medium(DMEM;SIGMA)中で培養した(37℃/CO2;5%)。膵島を培養器上に播種して7日後に培地交換を行い、以後3日毎に培地交換を行った。
膵島の形態観察
分離した膵島を3.5cm E−cad−Fcコートディッシュ(住友ベークライト)及び非処理ディッシュ(IWAKI)上に播種した。さらに、シングル化する膵島をトリプシン−EDTA(GIBCO)にて5分間、37℃にて処理することにより、シングルセルの状態に分散した膵島細胞を同様に播種した。膵島及びトリプシン-EDTA処理にてシングル化した膵島細胞は10% FBS(GIBCO)、抗生剤として1% Anti−Anti(GIBCO)を添加したDulbecco’s Modified Eagle’s Medium(DMEM; SIGMA)を用いて培養した(37℃ CO2;5%)。膵島を培養器上に播種して7日後に培地交換を行い、以後3日毎に培地交換を行った。
グルコース応答機能のStimulation Indexによる評価
実施例1と同一の培養条件下で、マウスより分離した膵島(40ピース)を、3.5cm E−cad−Fcコートディッシュ又は非処理ディッシュ上に播種し、培養した。培養開始後に培養液中のグルコース濃度によって膵島のインスリン分泌量を制御できるかを検討した。E−cad−Fcコートディッシュ又は非処理ディッシュ上で培養した膵島を低グルコース培地(1,000 mg/l DMEM)にて2回洗浄し、その後低グルコース培地(1,000 mg/l DMEM)中にて1時間培養し、上清中に分泌されたインスリン量を低濃度グルコースにおけるインスリン分泌量とした。その後、高グルコース培地(4,500 mg/l DMEM)にて2回洗浄し、その後高グルコース培地(4,500 mg/l DMEM)中にて1時間培養し、上清中に分泌されたインスリン量を高濃度グルコースにおけるインスリン分泌量とした。回収した上清を、ELISA(Enzyme−Linked ImmunoSorbent Assay)キットであるレビス インスリン−マウス((株)シバヤキ)を用いて非処理ディッシュ上およびE−cad−Fcコートディッシュ上における膵島のインスリン分泌量を測定し、膵島の機能を評価するためにStimulation Index (SI; 低濃度グルコース環境下でのインスリン分泌量に対する、高濃度グルコース環境下でのインスリン分泌量の比)を算出した(図2)。
グルコース応答機能に対するトリプシン処理の影響
マウス由来膵島(40個)を3.5cm E−cad−Fcコートディッシュもしくは非処理ディッシュ上に播種した。さらに、マウス由来膵島をトリプシン−EDTA(GIBCO)にて37℃、5分間インキュベートすることによりシングルセルの状態の膵島細胞を調製し、これを膵島と同様に播種した。培養開始日から11日後に、膵島及び膵島細胞のグルコース応答機能をSIによって検討した。ELISAによる評価は実施例3と同様の方法により行った。また、各培養日におけるSIを算出した。
低酸素条件における膵島内での細胞死誘導率の測定
実施例1と同一の培養条件下で、マウスより分離した膵島を、3.5cm E−cad−Fcコートディッシュ又は非処理ディッシュ上に播種し、1週間培養した。E−cad−Fcコートディッシュ上の膵島は1mM EDTA(エチレンジアミン四酢酸)を含むPBS(GIBCO)にて回収し、非処理ディッシュ上の膵島と同様に浮遊状態にて、低酸素条件(O2;5%)および高酸素条件(O2;20%)で1時間インキュベート(37℃)した。その後、DAPI Nucleic Acid Stain(DAPI;Lonza) 5μg/mlおよびPropidium iodide(PI;Roche) 0.5μg/mlを含むDMEM中で膵島を30分インキュベート(37℃)することで染色し、顕微鏡にて観察した(図4)。
細胞死率(%)=(PIで染色された細胞数/DAPIで染色された細胞数)×100
通常、体内であれば膵島組織内への血管誘導が生じるため、膵島が酸素不足に陥ることはない。しかしながら、移植した膵島の場合、移植部位における低酸素条件環境下では、酸素不足によって膵島の細胞死が誘導される懸念がある。本発明の方法により得られるシート状の膵島を用いれば、低酸素状態でも効率的にそれぞれの細胞へ酸素供給が可能となり、低酸素条件でも膵島の細胞死が抑制されるので、移植医療に有利である。
Claims (5)
- 単離された膵島を、E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器中、該固相表面に接着した状態で、当該膵島がシート状の形態を呈するのに十分な期間培養することを含む、シート状の膵島の製造方法。
- 当該ポリペプチドがE−カドヘリンの細胞外ドメインを含む、請求項1記載の製造方法。
- 当該ポリペプチドがE−カドヘリンの細胞外ドメイン及びイムノグロブリンのFc領域を含む融合ポリペプチドである、請求項1記載の製造方法。
- E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器、及びシート状の膵島を含み、該シート状の膵島が該ポリペプチドに接着した状態で培養可能となる、膵島培養物。
- E−カドヘリンのEC1ドメインを含み、且つ当該E−カドヘリンへの結合能を有するポリペプチドを固相表面に固定又はコーティングした培養容器、及び単離された膵島を含む、シート状の膵島の製造用キット。
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