JP6676719B2 - 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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Description
特定の実施形態では、例えば以下が提供される:
(項目1)
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。
(項目2)
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、項目1に記載のデバイス。
(項目3)
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、項目1または2に記載のデバイス。
(項目4)
前記細胞調製物は、カプセル化される、項目1〜3のいずれか一項に記載のデバイス。
(項目5)
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、項目4に記載のデバイス。
(項目6)
前記細胞調製物は、幹細胞を含む、項目4または5に記載のデバイス。
(項目7)
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、項目1〜6のいずれか一項に記載のデバイス。
(項目8)
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、項目1〜7のいずれか一項に記載のデバイス。
(項目9)
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、項目8に記載のデバイス。
(項目10)
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、項目8または9に記載のデバイス。
(項目11)
前記複数のチャンバー用の共通のシールをさらに含む、項目8〜10のいずれか一項に記載のデバイス。
(項目12)
各チャンバー用のシールをさらに含む、項目8〜10のいずれか一項に記載のデバイス。
(項目13)
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、項目1〜12のいずれか一項に記載のデバイス。
(項目14)
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、項目13に記載のデバイス。
(項目15)
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、項目1〜14のいずれか一項に記載のデバイス。
(項目16)
前記物質は、血管内皮成長因子(VEGF)を含む、項目15に記載のデバイス。
(項目17)
前記ポリマーは、薬物溶出ポリマーである、項目15に記載のデバイス。
(項目18)
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、項目1に記載のデバイス。
(項目19)
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、項目18に記載のデバイス。
(項目20)
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、項目1〜19のいずれか一項に記載のデバイス。
(項目21)
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、項目1〜20のいずれか一項に記載のデバイス。
(項目23)
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、項目2に記載のデバイス。
(項目24)
前記細胞調製物は、生体分解性ポリマーをさらに含む、項目2に記載のデバイス。
(項目25)
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、項目24に記載のデバイス。
(項目26)
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、項目1に記載のデバイス。
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 (17)
- 糖尿病を処置するために、糖尿病の処置を必要とする患者において細胞を移植するためのデバイスであって、該デバイスは、
少なくとも1つのチャンバーの壁を形成する免疫学的に適合性のポリマーメッシュを含む多孔質足場であって、該チャンバーが、該チャンバーの近位端部および遠位端部のいずれかまたは両方に開口部を備え、該近位端部と該遠位端部とが、該壁が境界を成すルーメンによって離れており、該多孔質足場は、該少なくとも1つのチャンバーの該壁の周囲でのおよび該壁を通る血管および結合組織の成長を助長する大きさの孔を有する、多孔質足場と;
該少なくとも1つのチャンバーの該ルーメン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非多孔質プラグであって、該チャンバーの該ルーメンに沿って延びている、プラグと;
該チャンバーの該近位端部および該遠位端部のいずれかまたは両方を閉じるように構成された少なくとも1つのシールと;
を備え、
該デバイスは、該患者の体内に植え込まれるように構成され;
該デバイスは、該デバイスに血管および結合組織が浸潤するまで、該患者の体内に維持されるように構成され;
植え込まれた該デバイスは、アクセス可能であり、かつ、該少なくとも1つのシールを開けるように構成され;
該プラグは、引き抜かれるように構成され;
該チャンバーは、細胞が注入されるように構成され;
該少なくとも1つのシールは、再び閉じられるように構成され;
該細胞は、糖尿病の処置を提供し;そして
該細胞は、ランゲルハンス島細胞を含む、
デバイス。 - 前記多孔質足場が、細胞送達前にイメージングされ得る、請求項1に記載のデバイス。
- 細胞が、ランゲルハンス島細胞と、セルトリ細胞、間葉幹細胞、分化した幹細胞および遺伝子操作された細胞のうちの1つ以上とを含む、請求項1に記載のデバイス。
- 前記細胞が幹細胞をさらに含む、請求項1に記載のデバイス。
- 前記細胞が、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、または患者由来の細胞をさらに含む、請求項1に記載のデバイス。
- 前記細胞が、遺伝子操作された細胞または細胞系をさらに含む、請求項1に記載のデバイス。
- 前記細胞が、分化した幹細胞またはカプセル化された細胞をさらに含む、請求項1に記載のデバイス。
- 前記細胞が、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、ヒドロゲルメタクリレート、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、またはアクリロニトリル−コ−塩化ビニルにおいてカプセル化される、請求項1に記載のデバイス。
- 前記少なくとも1つのシールが、前記多孔質足場に超音波溶接されるポリマーフィルムである、請求項1に記載のデバイス。
- 前記デバイスが、前記多孔質足場の少なくとも一部をコーティングする材料をさらに含むように構成され、該材料が、組織の組み込みおよび血管形成を刺激する、請求項1に記載のデバイス。
- 前記材料が、成長因子、抗線維化剤、ポリマー、血管内皮成長因子(VEGF)、コラーゲン、フィブロネクチン、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および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−コ−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)、ゼラチン、セルロースポリマー、キトサン、アルギネート、ビンキュリン、寒天、アガロース、ヒアルロン酸、およびマトリゲルのうちの1つ以上を含む、請求項10に記載のデバイス。
- 前記多孔質足場が、多孔質の前記チャンバー内への血管および結合組織の成長を助長して、血管新生コラーゲンマトリックス中で前記プラグを被包するように構成され、該プラグは、該血管新生コラーゲンマトリックス中で被包された該チャンバー内に空間を作るように、該チャンバーから引き抜くことができる、請求項1に記載のデバイス。
- 前記多孔質足場が、1つのチャンバー、2つのチャンバー、3つのチャンバー、4つのチャンバー、5つのチャンバー、6つのチャンバー、7つのチャンバー、8つのチャンバー、10のチャンバー、12のチャンバー、またはそれ以上のチャンバーを含むように構成される、請求項1に記載のデバイス。
- 多孔質足場が、横方向に接続された複数のチャンバーを含むように構成される、請求項1に記載のデバイス。
- 前記植え込まれた細胞がインスリンを産生し、前記糖尿病がインスリン依存糖尿病である、請求項1に記載のデバイス。
- 前記デバイスが、前記患者を正常血糖に回復させるように構成される、請求項1に記載のデバイス。
- 前記患者が無自覚性低血糖を有する、請求項16に記載のデバイス。
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