JP6005685B2 - 組織工学、細胞培養、および細胞送達用の多孔質足場の調製方法 - Google Patents
組織工学、細胞培養、および細胞送達用の多孔質足場の調製方法 Download PDFInfo
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- JP6005685B2 JP6005685B2 JP2014080564A JP2014080564A JP6005685B2 JP 6005685 B2 JP6005685 B2 JP 6005685B2 JP 2014080564 A JP2014080564 A JP 2014080564A JP 2014080564 A JP2014080564 A JP 2014080564A JP 6005685 B2 JP6005685 B2 JP 6005685B2
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Description
a)ある量の少なくとも1種の多糖、ある量の架橋剤、およびある量の孔形成剤を含むアルカリ性水溶液を調製するステップと、
b)前記溶液を、約4℃〜80℃の温度で、前記ある量の多糖が架橋結合するのに十分な時間置くことにより、前記溶液をヒドロゲルに変換するステップと、
c)前記ヒドロゲルを水溶液に浸すステップと、
d)ステップc)で得られた多孔質足場を洗浄するステップと
からなるステップを含む、多孔質足場の調製方法を提供することである。
本明細書で使用する「多糖」という用語は、2つ以上の単糖ユニットを含む分子を指す。
本発明の第1の目的は、
a)ある量の少なくとも1種の多糖、ある量の共有結合性架橋剤、およびある量の孔形成剤を含むアルカリ性水溶液を調製するステップと、
b)前記溶液を、約4℃〜約80℃で、前記ある量の多糖が架橋結合するのに十分な時間置くことにより、溶液をヒドロゲルに変換するステップと、および
c)前記ヒドロゲルを水溶液に浸すステップと、
d)ステップc)で得られた多孔質足場を洗浄するステップと
からなるステップを含む多孔質足場の調製方法に関する。
本発明の足場は、特に組織工学、組織修復、または組織再生に適している。多孔度の違いにより、足場の適当な領域への異なる細胞型の移入が促進されうる。他の実施形態において、多孔度の違いにより、組織の発生/修復/再生の適当な構造化に必要な、足場を構成する細胞型間における適当な細胞間連結の発生が促進されうる。例えば、細胞突起の伸長は、足場材料の多様な多孔度を介してより適当になるよう適応されうる。したがって、足場は任意の組織の細胞を含んでよい。
多糖ベースの足場の調製
プルラン/デキストラン75:25の混合物(プルラン、MW200,000、Hayashibara Inc.Okayama,Japan;デキストラン、MW500,000、Pharmacia)を用いて多糖ベースの足場を調製した。プルラン9gとデキストラン3gを蒸留水40mL中に溶解させることにより多糖溶液を調製した。次いでその多糖溶液に炭酸ナトリウム(8g)を加え、均質な混合物が得られるまで撹拌を続けた。架橋剤のトリメタリン酸三ナトリウムSTMP(Sigma,St Louis)をアルカリ性条件下で用いて多糖を化学的に架橋結合させた。簡単に言えば、10Mの水酸化ナトリウム1mlを多糖溶液10gに加え、続いてSTMP300mgを含む水1mlを加えた。次いでその混合物をペトリ皿(Nunclon(登録商標)、#150288)に注ぎ込み、50℃で15分間インキュベートした。得られたヒドロゲルをすぐに、20%酢酸溶液を含む大きいビーカーに浸漬し、少なくとも30分置いた。得られた足場をpH7.4のリン酸緩衝食塩水、次いで蒸留水を用いて、少なくとも2日間、広範囲に洗浄した。凍結乾燥ステップの後、多孔質足場を使用するまで室温で保管した。走査電子顕微鏡分析により、足場の多孔質を確認した(図1および2)。
多糖の種類
異なる種類の多糖を異なる比率で用い、多糖の総量は一定の値に保って、実施例1に記載の通りに多孔質足場を調製した。多糖はプルラン、デキストラン500、フコイダンLMW(低分子量)およびフコイダンHMW(高分子量)のいずれかであった。
孔形成剤の量
孔形成剤の量を変化させて、実施例1に記載の通りに多孔質足場を調製した。簡単に言えば、プルラン/デキストラン溶液に炭酸ナトリウムを2g、4g、または8g加えた。
架橋剤の濃度
架橋剤の量を200mg〜500mgに変化させて、実施例1に記載の通りに多孔質足場を調製した。
多孔質足場の細胞担持
ヒト骨髄間葉幹細胞(hMSC)を、実施例1の通りに調製した足場で培養した。円形の穿孔器を用いて、直径6mm、厚さ1mmの円形の多孔質足場を切り出した。培地は、10%のウシ胎児血清と1%のペニシリン/ストレプトマイシン(Sigma)を含む低グルコースDMEM(Gibco,Life Technology,New York)からなるものを使用した。細胞をトリプシン処理した後、細胞懸濁液20μLを用いて乾燥足場に再含水させた(足場あたり細胞106個)。次いで試料を培地1ml中で最大1週間維持した。細胞播種していない多孔質足場を培地中でインキュベートし、対照として用いた。
多孔質足場内における細胞の挙動の共焦点分析
多糖溶液に少量の(5mg)FITC−デキストランを加えることにより、実施例1の通りに蛍光足場を調製した。蛍光マーカー(PKH26、SIGMA P9691)を用いて、製造者の取扱説明書に従って標識したhMSCを、実施例5の通りに蛍光足場に播種した。共焦点イメージングにより、足場の多孔質構造を確認した。
生死アッセイによる細胞生存率
共焦点イメージングを使用して、細胞膜の透過性を測定する2つの蛍光プローブ、生細胞を染色する細胞透過性の緑色蛍光染料(カルセインAM)および死細胞を染色する細胞非透過性の赤色蛍光染料(ヨウ化プロピジウム)の使用に基づいて、生/死(live/dead)アッセイ(Calbiochem,San Diego,CA)による細胞生存率の評価を行った。7日目において、足場内にはほんの数個の死細胞が見られ、ほとんどの細胞は生細胞であった。
足場の多孔質に対する孔形成剤の影響
孔形成剤の量と性質を変化させて、実施例1に記載の通りに、多孔質足場を調製した。蛍光多孔質足場の共焦点分析のために、FITC−デキストラン5mgを多糖溶液に加えた。光学切片を、10×Plan−NeoFluar対物レンズ(開口数0.3)(Carl Zeiss)を備えたZeiss LSM 510共焦点顕微鏡(Carl Zeiss,Oberkochen,Germany)を用いて得た。FITC−デキストランを、アルゴンレーザーを用いて488nmで励起させ、その蛍光放射を505〜530nm帯域フィルターによって選択した。孔のサイズをImageJ(登録商標)ソフトウェアで評価した。空隙容量を、Amira(登録商標)ソフトウェアの統計学/容量測定モジュールを用いて計算し、結果を足場の容量%として表した。
正電荷を持つ多糖
DEAE−デキストランを唯一の多糖として用い、正電荷を持つ多孔質足場を調製した。簡単に言えば、DEAE−デキストラン溶液を、DEAE−デキストラン(Fluka参照#30461)1gを蒸留水1.5mL中に溶解させることにより調製した。次いでその多糖溶液に炭酸ナトリウム(100mg)を加え、均質な混合物が得られるまで撹拌を続けた。架橋剤のトリメタリン酸三ナトリウムSTMP(Sigma,St Louis)をアルカリ性条件下で用いて多糖を化学的に架橋結合させた。簡単に言えば、多糖溶液に10Mの水酸化ナトリウム150μLを加えた後、STMP45mgを含む水150μLを加えた。次いでその混合物をペトリ皿(Nunclon(登録商標),#150288)に注ぎ込み、50℃で15分間インキュベートした。得られたヒドロゲルをすぐに、20%酢酸溶液を含む大きいビーカーに浸漬し、少なくとも30分置いた。得られた足場をpH7.4のリン酸緩衝食塩水、次いで蒸留水を用いて、少なくとも2日間、広範囲に洗浄した。凍結乾燥ステップの後、多孔質足場を得、使用するまで室温で保管した。
負電荷を持つ多糖
フコイダン(Sigma 参照#F5631)をプルラン/デキストラン混合物に加えることにより、負電荷を持つ多孔質足場を調製した。簡単に言えば、プルラン9gおよびデキストラン3gを蒸留水40mLに溶解させることにより多糖溶液を調製し、次いでその多糖溶液にフコイダン1.2gを加えた。次いでその多糖溶液に炭酸ナトリウム(8g)を加え、実施例1の通りに架橋結合プロセスを行って負電荷を持つ多糖を含む三次元足場を得た。
三次元足場内におけるヒト間葉幹細胞の軟骨細胞様細胞への分化
ヒト骨髄間葉幹細胞(hMSC)を、血清を含まない軟骨細胞形成用培地中、実施例1の通りに調製した足場で培養した。軟骨細胞形成用培地は、10ng/mlのTGF−β3(Oncogene,Cambridge,MA)、100nMのデキサメタゾン(Sigma,St Louis,MO)、170μMのアスコルビン酸−2−リン酸(Sigma,St Louis,MO)、および5mLのITS−プラス(Collaborative Biomedical Products,Bedford,MA)を補充したDMEMからなっていた。3週間の培養後、細胞を播種した足場を10%のホルムアルデヒドで固定し、次いで凍結切片化した。凍結切片を、0.05%(w/v)のトルイジンブルーまたは0.1%のサフラニンO溶液のどちらかで染色した。細胞外マトリックス合成に対する強度に陽性の染色が観察され、それはMSCの軟骨細胞への分化を示す。
肝細胞の三次元培養
HepG2細胞、ヒト肝細胞癌細胞を、10%のウシ胎児血清と1%のペニシリン/ストレプトマイシン(Sigma)を含む低グルコースDMEM(Gibco,Life Technology,New York,USA)中、実施例1の通りに調製した足場で培養した。円形の穿孔器を用いて、直径6mm、厚さ1mmの円形の多孔性足場を切り出した。
Claims (4)
- トリメタリン酸三ナトリウムで架橋結合されたプルランとデキストランとを含む多孔質足場であって、足場の孔の平均サイズが1μm〜500μmで構成され、多孔度が4%〜75%の範囲である多孔質足場。
- 足場の孔の平均サイズが150μm〜350μmで構成されている、請求項1に記載の多孔質足場。
- 足場の孔の平均サイズが175μm〜300μmで構成されている、請求項1に記載の多孔質足場。
- 多孔度が4%〜50%の範囲である、請求項1から3のいずれか一項に記載の多孔質足場。
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CA2701858A1 (en) | 2009-04-16 |
US9522218B2 (en) | 2016-12-20 |
CN104725660A (zh) | 2015-06-24 |
EP2203194B1 (en) | 2013-04-10 |
JP2011500118A (ja) | 2011-01-06 |
US11511016B2 (en) | 2022-11-29 |
JP2014140381A (ja) | 2014-08-07 |
JP5579609B2 (ja) | 2014-08-27 |
CA2701858C (en) | 2016-05-24 |
WO2009047346A1 (en) | 2009-04-16 |
EP2203194A1 (en) | 2010-07-07 |
US20170080123A1 (en) | 2017-03-23 |
ES2408554T3 (es) | 2013-06-21 |
US20100221303A1 (en) | 2010-09-02 |
KR101474852B1 (ko) | 2014-12-23 |
SG185279A1 (en) | 2012-11-29 |
US20200016294A1 (en) | 2020-01-16 |
CN101848738A (zh) | 2010-09-29 |
HK1144182A1 (en) | 2011-02-02 |
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