JP2015157146A - 細胞移植のための方法およびデバイス - Google Patents
細胞移植のための方法およびデバイス Download PDFInfo
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Abstract
Description
ここで、例が添付の図面に例示されている本開示の実施形態の詳細について述べる。すべての図面において、同じまたは同様の部品には、可能な限り同じ参照符号を使用する。本開示において、用語「細胞注入」と「細胞移植」は同義的に使用される。
細胞移植デバイスを、正常血糖の回復のためにLewisラットの同系膵島細胞の植え込みに使用した。植え込まれた細胞のグルコース反応を、ラットの門脈に直接投与された膵島細胞のグルコース反応と比較した。Lewisラットを、それぞれが9匹の3つの研究群に分けた。第1および第2の研究群では、それぞれ、デバイスを腹腔内および皮下空洞部に植え込んだ。第3の研究群では、膵島細胞を門脈に直接投与した。
100日目に植え込まれたデバイスを取り出してから、インスリンに対して特異的な一次抗体を用いてデバイス内でインスリンを検出した。図13Aは、皮下に植え込まれたデバイスの多孔質チャンバー内でのインスリン染色の結果を示している。チャンバー内でのインスリンの検出は、デバイス内に閉じ込められた膵島細胞が、移植後100日目に生存可能で機能的であったことを示唆した。
植え込み段階(デバイスの植え込みから膵島の生着までの時間)の適切な長さを決定するために、細胞移植デバイスを、8週齢のYorkshire−Landraceブタに2週間、4週間、および8週間、皮下に植え込んだ。それぞれの期間の植え込みの後、血管形成およびコラーゲン堆積のレベルを決定するためにデバイスを取り出して分析した。
血管および組織形成の肉眼的分析のための取り出されたデバイスの前面および背面の両方の写真を撮影した。1cm×1cmの格子を写真の上に載せて、微小血管および組織(細胞を含むコラーゲン)形成を定量化した。格子内の各1cm2のボックスを血管形成についてスコアを付け、取り出されたデバイスの全表面について全血管/cm2を計算できるようした。デバイスの内周および外周の平均厚みを測定して、コラーゲンの堆積量を求めた。図14は、異なる多孔質材料(メッシュ)を用いて形成された4つのデバイスについて計算した平均コラーゲン厚みおよび全血管/cm2の表を示している。植え込み後2週間で、4つすべての種類のメッシュで十分な微小血管および組織形成が観察された。結果はまた、微小血管形成およびコラーゲン堆積に必要な時間が、デバイスの材料(メッシュの空隙率、表面粗さなど)によって異なり得ることを示唆した。
ヘマトキシリンおよびエオシン(H&E)染色(図15A)およびvon Willebrand因子(図15B)を用いた内皮細胞の染色によって血管形成を決定した。図15Aは、植え込みの2週間後、4週間後、および8週間後の組織のデバイス内への組み込みを実証している。図15Bは、細胞移植の前のデバイスの様々な縁における血管形成を示している。組織のデバイス内への組み込みの評価は、膵島移植前に測定したすべての時点でデバイスがコラーゲンおよび微小血管を取り込むことを示した。
8週齢のYorkshire−Landraceブタに、細胞移植デバイスを4週間および8週間植え込んだ。動物を糖尿病にするために、90%の膵切除を行い、次いで手術の翌日に150mg/Kgのストレプトゾトシンを静脈投与した。膵切除を行う前に膵島を膵臓から単離した。回復および糖尿病の確認のための十分な時間が得られるように、移植片単離および膵切除の5日後に未成熟膵島移植片を動物に移植した。
9週間でデバイスを取り出した後、インスリンに対して特異的な一次抗体を用いてデバイス内でインスリンを検出した。図17Aは、取り出されたデバイスの多孔質チャンバー内でのインスリン染色の結果を示している。チャンバー内のインスリンの検出は、デバイス内に閉じ込められた膵島細胞が、移植後9週間で、生存可能で機能性であったことを示した。外植片の切片の免疫組織化学染色は、強い微小血管(図17B;矢印で示されている微小血管)によって囲まれた健常な十分に構成された膵島を実証した。
移植後の膵島移植片の機能をモニタリングするために、空腹時血糖値および非空腹時血糖値を毎週測定した。これらの測定値は、血糖値の長期コントロールにおける細胞移植デバイスの全体的有効性の決定に役立つ。空腹時血糖値は、移植片機能の制御された基準となる。簡単に述べると、レシピエント動物の静脈から1滴(数μl)の血液を採取し、Freestyle Lite glucometerまたは他のグルコース検査デバイスを用いて血糖値を測定する。
ブドウ糖負荷試験は、移植前のIVGTT結果と移植後のIVGTT結果との比較による膵島移植片の機能の評価において重要である。細胞移植デバイスの有効性を試験するために、IVGTTを膵切除の前(基準値)、デバイスへの膵島移植後の様々な時点、およびデバイスの取り出し後に行った。1回のデキストロースの投与によってIVGTTを行い、内因性インスリンが血糖値を基準値にするのにかかる時間を測定した。血糖値の測定に加えて、様々な時点で血液を採取して、インスリンがβ細胞によって産生されるときに生成される副産物であるC−ペプチド値を測定した。IVGTTの結果は、血糖値の絶対値(図19A)、血糖値の曲線下面積(AUC)(図19B)、およびC−ペプチド値における倍数変化(図19C)を用いて解明した。
Claims (26)
- 宿主の体内に細胞を植え込むためのデバイスであって、
近位端部および遠位端部を有する少なくとも1つのチャンバーを含む多孔質足場であって、前記多孔質足場は、血管および結合組織の前記少なくとも1つのチャンバー内への成長を助長する大きさの孔を有する、多孔質足場と、
前記少なくとも1つのチャンバー内、および/または、前記多孔質足場の少なくとも一部内に配置されるように構成された少なくとも1つの取り外し可能なプラグであって、前記多孔質足場の前記少なくとも一部は、前記足場の孔を一時的に塞ぐ生体適合性の生体分解性材料でコーティングされている、少なくとも1つの取り外し可能なプラグと
を含み、
前記多孔質足場は、ポリプロピレンメッシュを含み、
前記デバイスは、前記チャンバー内の細胞調製物をさらに含む、デバイス。 - 前記細胞調製物は、ランゲルハンス島細胞、セルトリ細胞、ドーパミン作動性ニューロン、幹細胞、間葉幹細胞、臍帯血細胞、胚性幹細胞、神経幹細胞のうちの1つ以上を含む、請求項1に記載のデバイス。
- 前記細胞調製物は、同種異系ドナー細胞、異種ドナー細胞、または同系ドナー細胞、遺伝子操作された細胞または細胞系、または患者由来の細胞を含む、請求項1または2に記載のデバイス。
- 前記細胞調製物は、カプセル化される、請求項1〜3のいずれか一項に記載のデバイス。
- 前記細胞調製物は、アルギネート、多糖ヒドロゲル、キトサン、カルシウム、またはアルギン酸バリウム、アルギネートおよびポリリシンの層状マトリックス、光重合ポリ(エチレングリコール)ポリマー、ポリアクリレート、ヒドロキシエチルメタクリレート、メチルメタクリレート、シリコンカプセル、シリコンナノカプセル、ポリメンブレン、アクリロニトリル−コ−塩化ビニル、またはこれらの組み合わせにおいてカプセル化される、請求項4に記載のデバイス。
- 前記細胞調製物は、幹細胞を含む、請求項4または5に記載のデバイス。
- 前記足場の前記近位端部および前記遠位端部のいずれかまたは両方における開口部と、前記近位端部および前記遠位端部のいずれかまたは両方における前記開口部を閉じる少なくとも1つのシールとをさらに含む、請求項1〜6のいずれか一項に記載のデバイス。
- 前記多孔質足場は、横方向に接続された複数のチャンバーを含む、請求項1〜7のいずれか一項に記載のデバイス。
- 横方向に接続された、3個のチャンバー、4個のチャンバー、5個のチャンバー、6個のチャンバー、7個のチャンバー、8個のチャンバー、9個のチャンバー、10個のチャンバー、11個のチャンバー、または12個のチャンバーを含む、請求項8に記載のデバイス。
- 前記複数のチャンバーは、超音波溶接によって形成されている、請求項8または9に記載のデバイス。
- 前記複数のチャンバー用の共通のシールをさらに含む、請求項8〜10のいずれか一項に記載のデバイス。
- 各チャンバー用のシールをさらに含む、請求項8〜10のいずれか一項に記載のデバイス。
- 前記取り外し可能なプラグは、前記少なくとも1つのチャンバー内に配置されるように構成された外側プラグおよび前記外側プラグ内に配置されるように構成された内側プラグを含む2プラグシステムを含む、請求項1〜12のいずれか一項に記載のデバイス。
- 前記外側プラグの内壁は、前記外側プラグの長さに沿った少なくとも1つの突出部を含む、請求項13に記載のデバイス。
- 前記多孔質足場の少なくとも一部は、1つ以上の成長因子、抗線維化剤、ポリマー、および/または、少なくとも1つの多孔質チャンバー内への血管形成または組織の組み込みを刺激する物質でコーティングされている、請求項1〜14のいずれか一項に記載のデバイス。
- 前記物質は、血管内皮成長因子(VEGF)を含む、請求項15に記載のデバイス。
- 前記ポリマーは、薬物溶出ポリマーである、請求項15に記載のデバイス。
- 前記多孔質足場の少なくとも一部は、前記多孔質足場の孔を塞ぐ1つ以上の生体適合性の生体分解性材料でコーティングされている、請求項1に記載のデバイス。
- 前記生体適合性の生体分解性材料は、コラーゲン、フィブロネクチン、細胞外マトリックスタンパク質、および膜細胞骨格タンパク質のうちの少なくとも1つを含む、請求項18に記載のデバイス。
- 前記多孔質足場の少なくとも一部は、前記少なくとも1つの多孔質チャンバー内への組織の組み込みを刺激するように粗面化されている、請求項1〜19のいずれか一項に記載のデバイス。
- 前記チャンバー内に配置されるように構成された少なくとも1つの細胞注入管を含む細胞送達デバイスをさらに含む、請求項1〜20のいずれか一項に記載のデバイス。
- 前記細胞調製物は、細胞型の組み合わせを含む、請求項2に記載のデバイス。
- 前記細胞調製物は、ランゲルハンス島細胞およびセルトリ細胞を含む、請求項2に記載のデバイス。
- 前記細胞調製物は、生体分解性ポリマーをさらに含む、請求項2に記載のデバイス。
- 前記生体分解性ポリマーは、ポリエチレン−イミンおよび硫酸デキストラン、ポリ(ビニルシロキサン)エコポリマーポリエチレンイミン、ホスホリルコリン、ポリ(エチレングリコール)、ポリ(乳酸−グリコール酸)、ポリ(乳酸)、ポリヒドロキシバレレートおよびコポリマー、ポリヒドロキシブチレートおよびコポリマー、ポリジアキサノン、ポリ無水物、ポリ(アミノ酸)、ポリ(オルトエステル)、ポリエステル、コラーゲン、ゼラチン、セルロースポリマー、キトサン、アルギネート、フィブロネクチン、細胞外マトリックスタンパク質、ビンキュリン、寒天、アガロース、ヒアルロン酸、マトリゲル、およびこれらの組み合わせから選択される、請求項24に記載のデバイス。
- 前記少なくとも1つのプラグは、ポリテトラフルオロエチレンを含む、請求項1に記載のデバイス。
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