JP6490869B2 - 心筋組織再生用3次元構造体およびその製造方法 - Google Patents
心筋組織再生用3次元構造体およびその製造方法 Download PDFInfo
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Description
(a)脱細胞化された細胞外基質および架橋剤を含む組織工学用構造体形成溶液および心臓前駆細胞を含む第1バイオプリンティング用組成物と、前記組織工学用構造体形成溶液、間葉系幹細胞および血管内皮成長因子を含む第2バイオプリンティング用組成物で印刷および架橋化して第1バイオプリント層および第2バイオプリント層を交互に配置して3次元構造体を形成する段階;および
(b)前記架橋された3次元構造体を熱ゲル化して架橋−ゲル化3次元構造体を得る段階;
を含む心筋組織再生用3次元構造体の製造方法に関する。
血管内皮前駆細胞(endothelial progenitor cells)、血管内皮細胞(endothelial cells)および心筋細胞(cardiomyocytes)からなる細胞群より選択された1種以上の細胞;および
線維芽細胞成長因子(fibroblast growth factor、FGF)、血小板由来成長因子(platelet−derived growth factor、PDGF)、アンジオポエチン−1(angiopoietin−1)、形質転換成長因子−ベータ(transforming growth factor beta、TGF−β)、エリスロポエチン(erythropoietin、EPO)、幹細胞因子(stem cell factor、SCF)、表皮成長因子(epidermal growth factor、EGF)およびコロニー刺激因子(colony stimulating factor、CSF)からなる成長因子群より選択された1種以上の成長因子;
をさらに含み得る。
基質金属蛋白分解酵素(matrix metalloproteinase、MMP)および基質金属蛋白分解阻害酵素(tissue inhibitor matrix metalloproteinase、TIMP)からなる酵素群より選択された1種以上の酵素をさらに含み得る。
を含む製造方法によって製造され得る。
前記心筋組織再生用3次元構造体は、脱細胞化された細胞外基質および架橋剤を含む組織工学用構造体形成溶液、および心臓前駆細胞を含む第1バイオプリンティング用組成物と、前記組織工学用構造体形成溶液、間葉系幹細胞および血管内皮成長因子を含む第2バイオプリンティング用組成物を印刷および架橋化によって交互に積層された構造を1以上含み得る。
1−1:脱細胞化された細胞外基質の製造
脱細胞化された細胞外基質は、豚の心臓組織を用いてFalguni Pati, et al., Nat Commun.5, 3935 (2014)に開示された方法により製造した(以下、「hdECM」)。前記製造されたhdECMは、最終的に凍結乾燥して使用前まで冷凍保管した。前記製造されたhdECMの光学顕微鏡写真および組織染色写真を図2に示した。
前記得られた凍結乾燥されたhdECMに液体窒素を注ぎ、モルタルおよび乳棒で粉砕した。前記得られたhdECM粉末(330mg)を0.5M酢酸水溶液(10ml)に添加した後、ペブシン(33mg)(P7125、Sigma−Aldrich)を加えた後、常温で48時間攪拌した。得られた溶液の温度を10℃以下に維持し、リボフラビン(2mg)を加えて10℃以下に冷却させた10M NaOH溶液を滴加してpHを約pH7に調節した。前記得られたプレゲル(pre−gel)状態の組織工学用構造体製造溶液は、約4℃で冷蔵保管した。
プリンティング工程を行う直前に前記プレゲル状態の組織工学用構造体製造溶液に心臓前駆細胞5X106cells/mlを添加して第1バイオプリンティング用組成物を製造した。前記心臓前駆細胞は、人間心筋組織由来の心臓前駆細胞、韓国釜山大学校医学専門大学院、生理学教室から得たものである。
2−1:脱細胞化された細胞外基質の製造
脱細胞化された細胞外基質は、豚の心臓組織を用いてFalguni Pati, et al., Nat Commun.5, 3935 (2014)に開示された方法により製造した(以下、「hdECM」)。前記製造されたhdECMは、最終的に凍結乾燥して使用前まで冷凍保管した。前記製造されたhdECMの光学顕微鏡写真および組織染色写真を図2に示した。
前記得られた凍結乾燥されたhdECMに液剤窒素を注ぎ、モルタルおよび乳棒で粉砕した。0.5M酢酸水溶液(10ml)にhdECM粉末(330mg)を添加した後、ペブシン(33mg)(P7125、Sigma−Aldrich)を加えた後、常温で48時間攪拌した。得られた溶液の温度を10℃以下に維持し、リボフラビン(2mg)を加えて10℃以下に冷却させた10M NaOH溶液を滴加してpHを約pH7に調節した。前記得られたプレゲル(pre−gel)状態の組織工学用構造体溶液は、約4℃で冷蔵保管した。
プリンティング工程を行う直前に前記プレゲル状態の組織工学用構造体溶液に間葉系幹細胞5X106cells/mlと血管内皮成長因子(製品番号:293−VE−10、R&D systems社)(100ng/ml)をそれぞれ添加して第2バイオプリンティング用組成物を製造した。前記間葉系幹細胞は(下鼻甲介 (inferior turbinate)由来間葉系幹細胞、韓国カトリック大学校耳鼻咽喉科学教室)から得たものである。
製造例1で得られた第1バイオプリンティング用組成物を用いてFalguni Pati, et al., Nat Commun.5, 3935 (2014)に開示された方法により3次元構造体を製作した。
製造例1および2で得られた第1バイオプリンティング用組成物および第2バイオプリンティング用組成物を用いて3次元構造体を製作した。
具体的には、PCLフレームワーク(polycaprolactone(PCL)framework)を多軸組織臓器プリンティングシステム(Jin−Hyung Shim et al., J. Micromech. Microeng. 22 085014 (2012))のシリンジ(第1シリンジ)にロードし、約80℃で加熱して重合体を溶融させた。製造例1で得られたプレゲル形態の第1バイオプリンティング用組成物および製造例2で得られた第2バイオプリンティング用組成物を他のシリンジ(それぞれ、第2、3シリンジ)にロードし、温度を約10℃以下に維持させた。第1シリンジに約600kPaの空気圧を加えて約100um以下の線幅を有して、約300umの空隙を有する120um厚さの薄いPCLフレームワークを製作し、PCLフレームワークの上に第2シリンジの内容物を噴射した後、約360nmのUVAを3分間照射して架橋化した。その後、第2、3シリンジの内容物の噴射および前記架橋化による積層工程(layer−by−layer process)を行い、3次元構造体形状を形成した。
製造例1で得られたプレゲル形態の溶液を約360nmのUVAを3分間照射して架橋化した後、約37℃のインキュベータ(humid incubator)に入れて30分間維持して熱ゲル化を誘導してヒドロゲルを形成させた(架橋ヒドロゲルA)。また、リボフラビンを用いないことを除いては製造例1と同様な方法で製造したプレゲル状態の組織工学用構造体製造溶液を約37℃のインキュベータ(humid incubator)に入れて30分間維持して熱ゲル化を誘導してヒドロゲルを形成させた(ヒドロゲルB)。
4ないし5週齢のラット(重量:300±30g)の胸部を切開して、左前下行枝(Left Anterior Descending artery、LAD)の永久的な結紮(permanent ligation)を行い心筋梗塞を誘発した。
Claims (23)
- (a)脱細胞化された細胞外基質を含む組織工学用構造体形成溶液、および心臓前駆細胞を含む第1バイオプリンティング用組成物と、前記組織工学用構造体形成溶液、間葉幹細胞および血管内皮成長因子を含む第2バイオプリンティング用組成物を交互に一方向で一つの層に印刷して第1バイオプリント層および第2バイオプリント層を製造し、それらを交互に積層して配置して3次元構造体を形成する段階;および
(b)前記3次元構造体を熱的ゲル化してゲル化3次元構造体を得る段階を含み、
前記(a)段階は、熱的ゲル化が起こらない温度で行い、
前記(b)段階で熱的ゲル化を起こす温度で行う、
心筋組織再生用3次元構造体の製造方法。 - 前記脱細胞化された細胞外基質は、心臓組織から由来した請求項1に記載の製造方法。
- 前記第1バイオプリンティング用組成物および前記第2バイオプリンティング用組成物中の脱細胞化された細胞外基質は、独立してそれぞれの総重量に対して、1ないし4重量%の含有量で存在する請求項1に記載の製造方法。
- 前記心臓前駆細胞は、第1バイオプリンティング用組成物中、105ないし108cells/mlの範囲で存在する請求項1に記載の製造方法。
- 前記間葉幹細胞は、第2バイオプリンティング用組成物中、105ないし108cells/mlの範囲で存在する請求項1に記載の製造方法。
- 前記血管内皮成長因子は、第2バイオプリンティング用組成物中、50ないし1000ng/mlの範囲で存在する請求項1に記載の製造方法。
- 前記組織工学用構造体形成溶液は、pH6.5ないし7.5の範囲を有し、酸および蛋白質分解酵素をさらに含む請求項1に記載の製造方法。
- 前記組織工学用構造体形成溶液は、
バイオプリンティング組成物の総重量に対して、
酸0.03ないし30重量%;および蛋白質分解酵素0.1ないし0.4重量%;
を含む請求項7に記載の製造方法。 - 前記組織工学用構造体形成溶液は、
酢酸および塩酸からなる群から選択された1種以上の酸;
ペブシンおよびマトリックスメタロプロテアーゼ(MMP)からなる群から選択された1種以上の蛋白質分解酵素;および
pH調節剤;をさらに含む請求項7に記載の製造方法。 - 第1バイオプリンティング用組成物および第2バイオプリンティング用組成物は、独立して、
血管内皮前駆細胞(endothelial progenitor cells)、血管内皮細胞(endothelial cells)および心筋細胞(cardiomyocytes)からなる細胞群より選択された1種以上の細胞;および
線維芽細胞成長因子(fibroblast growth factor、FGF)、血小板由来成長因子(platelet−derived growth factor、PDGF)、アンジオポエチン−1(angiopoietin−1)、形質転換成長因子−ベータ(transforming growth factor beta、TGF−β)、エリスロポエチン(erythropoietin、EPO)、幹細胞因子(stem cell factor、SCF)、表皮成長因子(epidermal growth factor、EGF)およびコロニー刺激因子(colony stimulating factor、CSF)からなる成長因子群より選択された1種以上の成長因子;
をさらに含む請求項1に記載の製造方法。 - 第1バイオプリンティング用組成物および第2バイオプリンティング用組成物は、独立して、
基質金属蛋白分解酵素(matrix metalloproteinase、MMP)および基質金属蛋白分解阻害酵素(tissue inhibitor matrix metalloproteinase、TIMP)からなる酵素群より選択された1種以上の酵素をさらに含む請求項10に記載の製造方法。 - 第1バイオプリンティング用組成物および第2バイオプリンティング用組成物は、独立して、15℃で測定されたせん断率(shear rate)1 s−1での粘度が1ないし30Pa・Sの範囲である請求項1に記載の製造方法。
- 前記第1バイオプリンティング用組成物および第2バイオプリンティング用組成物は、独立して、架橋剤をさらに含む請求項1に記載の製造方法。
- 前記架橋剤は、第1バイオプリンティング用組成物および第2バイオプリンティング用組成物に独立してそれぞれの組成物の総重量に対して、0.001ないし0.1重量%の含有量で含まれる請求項13に記載の製造方法。
- 前記製造方法は、架橋化反応をさらに含む請求項1に記載の製造方法。
- 前記架橋化は、15℃以下で1ないし10分間行われることを特徴とする請求項15に記載の製造方法。
- 前記熱的ゲル化は、25℃ないし37℃で行う請求項1に記載の製造方法。
- 前記心筋組織再生用3次元構造体は、50ないし1000μmの厚さを有する請求項1に記載の製造方法。
- 第1バイオプリンティング用組成物と、第2バイオプリンティング用組成物を印刷して製造される第1バイオプリント層および第2バイオプリント層を熱的ゲル化して形成された心筋組織再生用3次元構造体であって、
前記第1バイオプリンティング用組成物は、脱細胞化された細胞外基質を含む組織工学用構造体形成溶液および心臓前駆細胞を含み、
前記第2バイオプリンティング用組成物は、前記組織工学用構造体形成溶液、間葉幹細胞および血管内皮成長因子を含み、
前記第1バイオプリント層および第2バイオプリント層は、独立して、第1バイオプリンティング用組成物と第2バイオプリンティング用組成物を交互に一方向で一つの層に印刷して製造される、心筋組織再生用3次元構造体。 - 前記第1バイオプリント層および第2バイオプリント層を形成するために印刷されたバイオプリンティング用組成物は繊維、テープ、または織物形態である請求項19に記載の心筋組織再生用3次元構造体。
- 前記第1バイオプリント層および第2バイオプリント層が、それぞれ一方向繊維形態であり、前記第1バイオプリント層および第2バイオプリント層が互いに一定の交差角を有するように積層される請求項19に記載の心筋組織再生用3次元構造体。
- 前記心筋組織再生用3次元構造体は、50ないし1000μmの厚さを有する請求項19に記載の心筋組織再生用3次元構造体。
- 前記心筋組織再生用3次元構造体は、1rad/sでのモデュラスが1ないし100kPaである請求項19に記載の心筋組織再生用3次元構造体。
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