JP2021522831A - 組織脱細胞化の方法 - Google Patents
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Abstract
Description
ラット胸腺を、右総頸動脈、右鎖骨下動脈、右内乳動脈、右肋頸動脈幹、右大動脈弓、左大動脈弓、左肋頸動脈幹、左内乳動脈および左鎖骨下動脈を結紮することによって密封し(図1参照)、対象から摘出した。密封した胸腺を、溶液中で自由に浮遊させる間の左総頸動脈のカニューレ挿入によって潅流する以下の洗浄剤−酵素潅流処理(DET)を用いて並行して脱細胞化した:
a.MilliQH2Oで96時間、0.2mL/分、4℃で潅流;
b.4%SDCで0.2mL/分、RTで1.5時間潅流;
c.MilliQH2Oで24時間、0.2mL/分、RTで潅流;
d.0.1mg/mlのDNA分解酵素(温37℃)で0.2mL/分で30分間潅流;および
e.PBSで0.2mL/分で1時間、RTで潅流。
実施例1に概説したプロトコルに従って、ラット胸腺を抽出し、脱細胞化した。ラット重量は、173gであった。
実施例1のプロトコル概略に従って胸腺骨格を得た。次に、組織胸腺ドナーからのex vivo膨張に続いて、胸腺上皮細胞(TEC)および胸腺間葉細胞(TMC)の5:1(それぞれ)組合せを、直接針注射により同時に送達して、胸腺を再増殖させた。図5A〜Cに透明組織として示した胸腺葉に、針シリンジ(1ローブあたり1回注射、各40μl、2MのTEC:1回注射あたり0.4MのTMC)を細胞に注射した。胸腺外組織(図5A〜Cのより濃い部分)は、培養中に維持されたカニューレに接続することを可能にした。
上記実施例3に概説したプロトコルに従って、反復されたラット胸腺骨格を調製した。間質細胞を用いた4日間のin vitro培養後、再増殖した骨格を、8週間および11週間NSGマウスに皮下移植した。
上記実施例3に概説したプロトコルに従って、反復されたラット胸腺骨格を調製し、EpCAMおよびECアドヘリン陽性細胞によって同定されたヒト間質細胞で再増殖させた。間質細胞との4日間のin vitro培養後、再増殖した骨格を、11週間NSGマウスに皮下移植した。
in vivoでNSGマウスに再増殖および移植された胸腺骨格の一部を解離させ、FACS分析により分析したところ、ヒト細胞の高い生存率が示された(63.9%、中央パネル図9A);生細胞の大部分はCD45+であった(92.9%、右パネル図9A)。重要なことに、CD45+細胞の80%はCD3+でもあった(図9B、左パネル)ことから、胸腺骨格はリンパ系の誘導性微小環境であることが実証された。TCRabを発現する単一の陽性CD4+細胞およびCD8+細胞の存在は、成熟した機能性T細胞がin vivoで骨格内に産生されていることを実証している(図9B、中央および右パネル)。
胸腺骨格を、in vitroで間質細胞とともに再増殖させ、7日間培養に維持した後、未成熟トリプルネガティブ胸腺細胞(TN)を注射した。胸腺細胞を胸腺骨格に注射した7日後にFACSで分析したところ、CD4+細胞およびCD8+細胞の単一および二重陽性の両方に成熟した(図10B)。重要なことは、骨格における単一の陽性CD4/CD8の比率が天然の胸腺と類似していることである(図10A、B)。
Fan Y.,Tajima A.,Goh SK.,Geng X.,Gualtierottii G.,Grupillo M.,Coppola A.,Bertera S.,Rudert WA.,Banerjee I.,Bottino R.,Trucco M.Bioengineering Thymus Organoids to Restore Thymic Function and Induce Donor−Specific Immune Tolerance to Allografts,Molecular Therapy 2015;23:1262−1277
Goh SK.,Bertera S.,Olsen P.,Candiello J.,Halfter W.,Uechi G.,Balasubramani M.,Johnson S.,Sicari B.,Kollar E.,Badylak SF.,Banerjee I.Perfusion−decellularised pancreas as a natural 3D scaffold for pancreatic tissue and whole organ engineering.Biomaterials 2013;34(28):6760−6772
Katari R.,Peloso A.,Zambon JP.,Sokeer S.,Stratta RJ.,Atala A.,Orlando G.Renal Bioengineering with Scaffolds Generated from Human Kidneys,Nephron Exp.Nephrol.2014;126:119−124
Claims (21)
- 総頸動脈のない小葉臓器の少なくとも一部の脱細胞化細胞外マトリックス(ECM)骨格を作製する方法であって、前記方法が以下:
a)輸入血管を閉鎖して、非ヒトドナーまたは死/脳死ヒトドナー内の総頸動脈および/または主要動脈を伴わずに標的小葉臓器またはその一部を実質的に密封するステップ;
b)任意に:(i)前記閉鎖された輸入血管の少なくとも一部からの凝血および/もしくは血液を洗浄するステップ;ならびに/または(ii)前記臓器またはその一部を潅流して前記輸入血管の閉鎖を確認するステップ;
c)前記密封された臓器またはその一部を前記ドナーから除去するステップ;ならびに
d)前記密封された臓器またはその一部を洗浄剤および酵素溶液で潅流して脱細胞化されたECM骨格を得るステップ
を含む方法。 - 前記洗浄剤が、デオキシコール酸ナトリウム(SDC)、ドデシル硫酸ナトリウム(SDS)およびTriton X−100の1つ以上から選択される、請求項1に記載の方法。
- 前記SDC濃度が約1%〜約10%(w/v)であるか、または前記SDS濃度が約0.05%〜約1%(w/v)であるか、または前記Triton X−100濃度が約0.05%〜約5%(w/v)である、請求項1または2に記載の方法。
- 前記密封された臓器またはその一部を、約1〜約4時間洗浄剤で潅流する、請求項1〜3のいずれか一項に記載の方法。
- 前記密封された臓器またはその一部を、約0.2ml/分〜約1ml/分の速度で洗浄剤で潅流する、請求項1〜4のいずれか一項に記載の方法。
- 前記密封された臓器またはその一部が、単一サイクルの間潅流される、請求項1〜5のいずれか一項に記載の方法。
- 前記密封された臓器またはその一部が潅流中に溶液中に自由に浮遊している、請求項1〜6のいずれか一項に記載の方法。
- 総頸動脈を有しない前記小葉臓器またはその一部が、胸腺またはその葉、甲状腺またはその葉、副甲状腺または唾液腺である、請求項1〜7のいずれか一項に記載の方法。
- 前記胸腺を密封するために閉鎖された血管が、右または左総頸動脈、右鎖骨下動脈、右内乳動脈、右肋頸動脈、右大動脈弓、左大動脈弓、左肋頸動脈、左内乳動脈および左鎖骨下動脈、左内乳動脈、左肋頸動脈および左大動脈弓を含む、請求項8に記載の方法。
- 前記右総頸動脈が閉鎖され、前記胸腺が前記左総頸動脈を介して潅流されるか、または前記左総頸動脈が閉鎖され、前記胸腺が前記右総頸動脈を介して潅流される、請求項9に記載の方法。
- 前記甲状腺を密封するために閉鎖された血管が、上甲状腺動脈との接合後に前記右または左総頸動脈を含む、請求項8に記載の方法。
- 各甲状腺葉が前記右または左総頸動脈を介して潅流される、請求項11に記載の方法。
- 請求項1〜12のいずれか一項に記載の方法によって得られるかまたは得ることができる、脱細胞化されたECM骨格。
- 人工臓器を作製する方法であって、前記方法が、請求項13に記載の脱細胞化されたECM骨格を、間質細胞、例えば上皮細胞および間葉細胞の組合せとともに再増殖させ、前記再増殖した骨格を約4〜約7日間インビトロで培養し、その後、任意に前記再増殖した骨格をドナー細胞とともに播種するステップを含む方法。
- 前記脱細胞化されたECM骨格が、約2:1〜約5:1の比率で上皮細胞および間葉細胞で再増殖される、請求項14に記載の方法。
- 請求項13記載の脱細胞化されたECM骨格を含むか、または請求項14〜15のいずれか一項に記載の方法によって得られるかもしくは得ることができる、人工臓器。
- 療法に使用するための、請求項16に記載の人工臓器。
- 前記人工臓器が胸腺であり、ディジョージ症候群の処置に使用するためのものである、請求項16に記載の人工臓器。
- 化合物が薬理学的、免疫学的または毒物学的応答を誘発する能力を試験するためのin vitro方法であって、前記方法が請求項16に記載の人工臓器を前記化合物と接触させることを含む方法。
- 臓器移植の方法であって、前記方法が、請求項16に記載の人工臓器を患者に外科的に移植することを含む、方法。
- 疾患を処置する方法であって、前記方法が、請求項16に記載の人工臓器をそれを必要とする患者に外科的に移植することを含む、方法。
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EP3793356A1 (en) | 2021-03-24 |
CN112272517B (zh) | 2023-03-21 |
CN112272517A (zh) | 2021-01-26 |
JP2024063070A (ja) | 2024-05-10 |
WO2019220091A1 (en) | 2019-11-21 |
US12343447B2 (en) | 2025-07-01 |
US20210187164A1 (en) | 2021-06-24 |
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