JP6475323B2 - ヒドロゲルを用いた3次元的幹細胞骨分化誘導方法 - Google Patents
ヒドロゲルを用いた3次元的幹細胞骨分化誘導方法 Download PDFInfo
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Description
片側面にヒドロゲルが塗布された多孔性膜が非接触的に内部に位置した細胞培養容器で幹細胞を骨分化誘導する方法に関する。
多孔性膜を細胞培養容器の内部に非接触的に位置させ;
上記多孔性膜の片側表面にヒドロゲル溶液を塗布してゾルゲル相転移を通してヒドロゲルをコーティングし;
上記コーティングされたヒドロゲルの上に幹細胞を接種し;及び、
上記幹細胞を骨分化誘導培地で培養して幹細胞を骨分化誘導することができる。
細胞培養容器;
片側面に細胞が付着されるヒドロゲルが付着された多孔性膜;及び、
上記細胞培養容器の内部に上記ヒドロゲルが付着された多孔性膜が非接触的に配置されることを含む幹細胞分化装置に関する。
幹細胞の分化を促進させるために、3次元的に分化を行う。このために、細胞培養皿またはウェルの底に水平になるように非接触的に0.4μm乃至1μmの高分子膜(Corning,USA)を設け、生分解性合成バイオゲル(BASF,Germany)を滅菌された3次蒸留水に溶かして製造した多様な%の生分解性合成バイオゲル(BASF,Germany)を高分子膜の上に250μl/cm2塗布した後、1時間30分の間37℃でゲルに固めて分化用細胞培養器を製作した。ゾルゲル相転移後の生分解性合成バイオゲルの厚さは、最小1mmであり、平均2.5mmであった。脂肪由来間葉系幹細胞はCEFOgro ADMSC培地(CB-ADMSC-GM,CEFO,Korea)、骨髄由来間葉系幹細胞はCEFOgro BMMSC培地(CB-BMMSC-GM,CEFO,Korea)、胚性幹細胞由来間葉系幹細胞はCEFOgro ESMSC培地(CB-ESMSC-GM,CEFO,Korea)、臍帯由来間葉系幹細胞はCEFOgro UCMSC培地(CB-UCMSC-GM,CEFO,Korea)、歯周靱帯細胞(PDL)はCEFOgro PDL培地(CB-PDL-GM,CEFO,Korea)で37℃、CO2培養器にて3日乃至4日間培養した後、それぞれの幹細胞を上記バイオゲルの上に接種し、骨分化前処理培地(CB-DM-Osteo-PT,CEFO,Korea)を入れ、37℃、CO2培養器にて18時間培養した。その後、骨分化誘導培地(CB-DM-Osteo,CEFO,Korea)に培地を交換し37℃、CO2培養器にて5日間骨分化を誘導した(図1)。
従来の幹細胞分化方法である2次元的分化方法で幹細胞を分化した。具体的に、12ウェル細胞培養容器(皿)に細胞を接種し、骨分化誘導のための前処理培地(CB-DM-Osteo-PT,CEFO,Korea)で細胞密度が85乃至90%になるまで培養した後、骨分化誘導培地(CB-DM-Osteo,CEFO,Korea)に交換して14乃至21日間骨分化を誘導した(図2)。
上記実施例1で高分子膜の上に生分解性合成バイオゲルが0%である(高分子膜だけ存在)場合における幹細胞の分化を確認するために、高分子膜の上に幹細胞を接種した後、実施例1と同一に5日間培養した(図3)。
上記比較例1の方法で14日または5日間骨分化誘導した骨髄由来間葉系幹細胞、上記比較例2の方法(0%生分解性合成バイオゲル)で5日間骨分化誘導した骨髄由来間葉系幹細胞及び上記実施例の方法(5%、10%または15%生分解性合成バイオゲル)で骨分化誘導した骨髄由来間葉系幹細胞の骨分化を位相差顕微鏡を通して肉眼で確認した。また、骨分化した細胞をアリザリンレッド染色を通して確認するために各細胞をPBSで2回洗浄した後、70%エチルアルコールで10分間常温で固定し、3次蒸留水で2回洗浄した。その後、アリザリンレッド(Alizarin Red)染色キット(CB-SK-Osteo)のSol Iを処理し、常温で30分間反応させた。その後、Sol IIで3回きれいに洗浄した後、倒立顕微鏡(LEICA,Germany)でイメージ分析を行った。また、これを数値化するために、イメージ分析後、Sol IIIを処理して30分間常温で反応させ、染色された試薬を完全に溶かした。溶かした溶液を100μlずつとって96ウェルプレートに入れ、550nmで吸光度を測定した。その結果、比較例1の方法で5日間骨分化誘導した場合、及び比較例2の方法で5日間骨分化誘導した場合、骨分化がなされなかったが、実施例の方法で5日間骨分化を誘導した場合は、5日で十分な骨分化が誘導された。特に、実施例の方法で骨分化したとき、1乃至30%ヒドロゲルで骨分化が全てよく起き、特に10%濃度で骨分化がもっとも最適であることが分かった(図4)。
上記比較例1の方法で14日間骨分化誘導した脂肪由来間葉系幹細胞、上記比較例2の方法(0%生分解性合成バイオゲル)で5日間骨分化誘導した骨髄由来間葉系幹細胞及び上記実施例の方法(5%、10%または15%生分解性合成バイオゲル)で骨分化誘導した骨髄由来間葉系幹細胞の骨分化を位相差顕微鏡を通して肉眼で確認した。また、骨分化した細胞をアリザリンレッド染色を通して確認するために各細胞をPBSで2回洗浄した後、70%エチルアルコールで10分間常温で固定し、3次蒸留水で2回洗浄した。その後、アリザリンレッド(Alizarin Red)染色キット(CB-SK-Osteo)のSol Iを処理し、常温で30分間反応させた。その後、Sol IIで3回きれいに洗浄した後、倒立顕微鏡(LEICA,Germany)でイメージ分析を行った。その結果、比較例2の方法で5日間骨分化誘導した場合、骨分化が全くなされなかったが、実施例の方法で5日間骨分化したとき、1乃至30%生分解性合成バイオゲルで骨分化が全てよく起き、特に5乃至10%濃度で骨分化がもっとも最適であることが分かった(図5)。
上記比較例2の方法で5日間骨分化誘導した臍帯由来間葉系幹細胞、上記比較例2の方法で細胞を接種しBMP(bone morphogenic protein)2(peprotech,israel)100ng/mlまたはWnt3a(peprotech,israel)20ng/mlを処理して5日間骨分化を誘導した臍帯由来間葉系幹細胞及び上記実施例の方法(10%生分解性合成バイオゲル)で骨分化誘導した臍帯由来間葉系幹細胞の骨分化を位相差顕微鏡を通して肉眼で確認した。また、骨分化した細胞をアリザリンレッド染色を通して確認するために各細胞をPBSで2回洗浄した後、70%エチルアルコールで10分間常温で固定し、3次蒸留水で2回洗浄した。その後、アリザリンレッド(Alizarin Red)染色キット(CB-SK-Osteo)のSol Iを処理し、常温で30分間反応させた。その後、Sol IIで3回きれいに洗浄した後、倒立顕微鏡(LEICA,Germany)でイメージ分析を行った。また、これを数値化するために、イメージ分析後、Sol IIIを処理して30分間常温で反応させ、染色された試薬を完全に溶かした。溶かした溶液を100μlずつとって96ウェルプレートに入れ、550nmで吸光度を測定した。その結果、骨分化を促進させるものとして知られているBMPとWnt3aを処理し比較例2の方法で5日間骨分化誘導した場合よりも、本発明の実施例方法を用いた臍帯由来間葉系幹細胞の骨分化効率が著しく優れていた(図6)。
上記実施例の方法(10%生分解性合成バイオゲル)で骨分化を1日、3日、5日または7日間誘導した臍帯由来間葉系幹細胞につき、アリザリンレッド染色を通してイメージ分析を行った。また、これを数値化するために550nmで吸光度を測定した。その結果、骨分化誘導1日目から骨分化が誘導され始め、骨分化期間が長くなるほど骨分化が強く起きた。特に、臍帯由来間葉系幹細胞の場合は、従来の2次元方式で他の間葉系幹細胞に比べて骨分化がよく起きないものとして知られているが、本発明の実施例の3次元骨分化誘導方法を用いた場合は、他の間葉系幹細胞のように骨分化がよくなされていることが分かった(図7)。
骨髄由来間葉系幹細胞、脂肪由来間葉系幹細胞、臍帯由来間葉系幹細胞、胚性幹細胞由来間葉系幹細胞及び歯周靱帯細胞をそれぞれ上記比較例1の方法で14日間、上記比較例2の方法(0%生分解性合成バイオゲル)で5日間、または上記実施例の方法(10%生分解性合成バイオゲル)で5日間、骨分化誘導した後、アリザリンレッド染色を通して間葉系幹細胞の骨分化程度を確認した。
Claims (4)
- 0.1μm〜8μmの空隙を有する多孔性膜を、細胞培養容器の内部に対して非接触的に位置させること;
前記多孔性膜の片側表面に濃度5%〜15%のヒドロゲル溶液を塗布して、ゾルゲル相転移を通して、厚さ1mm〜4mmのヒドロゲルをコーティングすることであって、
前記ヒドロゲルは、細胞培養温度である37℃ではゲル状態であって1.E+00mPa・s〜1.E+06mPa・s(10 0 mPa・s〜10 6 mPa・s)の粘度を有し、前記細胞培養温度未満においてゾル状態に転移可能である、前記コーティングすること;
コーティングされた前記ヒドロゲルの上に幹細胞を接種すること;及び、
前記幹細胞を骨分化誘導培地で培養すること、を含み、
前記多孔性膜及び前記ヒドロゲルは、空気及び培地の透過を許容する、
7日以内に幹細胞からの骨分化を誘導する方法。 - 前記多孔性膜と前記細胞培養容器との間に培地が流入して幹細胞と培地の接触表面積を増加させることで骨分化誘導期間を短縮させることを特徴とする、請求項1に記載の幹細胞からの骨分化を誘導する方法。
- 前記幹細胞は、臍帯由来間葉系幹細胞(Umbilical cord mesenchymal stem cell)、脂肪由来間葉系幹細胞(adipose derived mesenchymal stem cell)、胚性幹細胞由来間葉系幹細胞(Embryonic stem cell derived mesenchymal stem cell)、歯周靱帯細胞(Periodontal ligament cell)又は骨髄由来間葉系幹細胞(Bone marrow-derived mesenchymal stem cell)であることを特徴とする、請求項1又は請求項2に記載の幹細胞からの骨分化を誘導する方法。
- 前記ゾルゲル相転移は、37℃で1時間〜2時間の間に行われることを特徴とする請求項1〜請求項3のいずれか1項に記載の幹細胞からの骨分化を誘導する方法。
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