JP5124125B2 - 細胞培養基材、及び細胞培養基材の製造方法 - Google Patents
細胞培養基材、及び細胞培養基材の製造方法 Download PDFInfo
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Description
<キトサン溶液の調製>
脱アセチル化度93%のキトサン(北海道曹達株式会社製)1.6gをトリフルオロ酢酸(和光純薬工業株式会社製)20mlに50℃で12時間かけて溶解し、塩化メチレン5mlを加え、ガラスフィルター(3G型−2、フィルター径30mm、ポアサイズ40〜100μm;日本理化学器械株式会社製)でろ過してキトサントリフルオロ酢酸液を得た。
キトサントリフルオロ酢酸液を注射器(テルモシリンジSS−30ESZ;テルモ株式会社製)に入れて針(シェアフィールドSVセット22G;テルモ株式会社製)につなぎ、インフュージョンポンプ(11Plus;HARVARD APPRATUS製)にセットした。インフュージョンポンプの送液速度を2ml/時間とし、アルミニウムからなる電極板(5.5cm×5.5cm)と針との間に高圧直流電圧電源(HSP−30k−2;日本スタビライザー株式会社製)により27kVの電圧をかけて、1時間かけて針から電極板へキトサントリフルオロ酢酸液を噴出することで、電極板にキトサン繊維を得た。
キトサン繊維92mgに蒸留水を300ml加え、ホモジナイザー(T.K.HOMO MIXER;TOKUSYU KIKA KOGYO社製)により8000rpmで10分間ホモジナイズし、スラリー液を得た。なお、スラリー液は、乾燥減量(第14改正日本薬局方 乾燥減量試験法)の値から求めたキトサン濃度は約0.3mg/mlであった。得られたスラリー液をポリスチレン製24ウェル透明培養プレート(COSTAR3524;CORNING社製)の全24ウェルに1mlずつ分注し、50℃で1時間乾燥させることで、24ウェル透明培養プレートの底部上に白色でシート状の繊維体が一体となって設けられた実施例1の細胞培養基材を得た。写真を図1に示す。
実施例1と同様の方法により得たキトサン繊維239mgに蒸留水を200ml加え、ホモジナイザー(T.K.HOMO MIXER;TOKUSYU KIKA KOGYO社製)により8000rpmで25分間ホモジナイズし、スラリー液を得た。なお、スラリー液は、乾燥減量(第14改正日本薬局方 乾燥減量試験法)の値から求めたキトサン濃度は約1.2mg/mlであった。得られたスラリー液をポリスチレン製24ウェル透明培養プレート(COSTAR3524;CORNING社製)の全24ウェルに1mlずつ分注し、−25℃の冷凍庫に1晩入れて凍結させた。これを凍結乾燥機FDU−1100(東京理化器械株式会社製)にて−10℃で48時間かけて凍結乾燥することで、24ウェル透明培養プレートの底部上に白色で厚さ0.3cmの繊維体が一体となって設けられた実施例2の細胞培養基材を得た。写真を図2に示す。
実施例2と同様の方法により得たスラリー液を直径13mmの円形マイクロカバーグラス(MATSUNAMI製)の片側表面上に1枚当り0.13mlずつ滴下し、50℃で1時間乾燥させることで、マイクロカバーグラスの片側表面上に白色でシート状の繊維体が一体となって設けられた実施例3の細胞培養基材を得た。写真を図3に示す。
poly−L−lysine(Sigma社製;10μg/ml水溶液)をカバースリップに塗布させて比較例1の細胞培養基材を得た。
collagen type I(BD社製;10%水溶液)をカバースリップに塗布させて比較例2の細胞培養基材を得た。
実施例1〜3の細胞培養基材の繊維体を顕微鏡により観察した。写真をそれぞれ図4〜6に示す。
実施例3及び比較例1〜2の細胞培養基材に成体マウスから得られた後根神経節を移植片培養し、5%牛胎仔血清加DMEM培養液、5%CO2条件下で7日間培養した。これらを4%paraformaldehyde,methanolで固定したのち、ニューロンのマーカーであるneurofilament 160Kに対するマウスモノクロナル抗体(Sigma社製)とシュワン細胞のマーカーであるS100に対するウサギ・ポリクロナル抗体(DAKO社製)を用いて蛍光二重免疫染色を施行した。2次抗体として、Rhodamine標識ヤギ抗マウスIgG、FITC標識ヤギ抗ウサギIgGをそれぞれ用い、蛍光顕微鏡(オリンパス社製)で観察した。実施例3の細胞培養基材の結果を図7(a)〜(f)に示す。抗neurofilament 160K抗体を用いた蛍光染色の結果を図7(a)及び(d)、抗S100抗体を用いた蛍光染色の結果を図7(b)及び(e)、UV励起による結果を図7(c)及び(f)に示す。なお、図7(d)〜(f)は図7(a)〜(c)をさらに拡大した写真である。
Claims (7)
- エレクトロスピニングにより形成されたキトサン繊維を溶媒に分散させ、これをホモジナイズしたスラリー液を乾燥させることにより得た、繊維が絡み合った繊維体からなることを特徴とする細胞培養繊維体。
- 基板と、基板上に設けられた細胞培養繊維体とからなる細胞培養基材であって、前記細胞培養繊維体が、エレクトロスピニングにより形成されたキトサン繊維を溶媒に分散させ、これをホモジナイズしたスラリー液を前記基板に塗布して乾燥させることにより得られ、前記基板と一体となった、繊維が絡み合った繊維体からなることを特徴とする細胞培養基材。
- 請求項2に記載の細胞培養基材において、前記細胞培養繊維体がメッシュ状であることを特徴とする細胞培養基材。
- 請求項2又は3に記載の細胞培養基材において、前記細胞培養繊維体が、繊維径が5μm以下の繊維からなることを特徴とする細胞培養基材。
- 基板と、基板上に設けられた細胞培養繊維体とからなる細胞培養基材の製造方法であって、キトサン類を含むキトサン溶液をエレクトロスピニングにより繊維化してキトサン繊維を形成する工程と、前記キトサン繊維と溶媒とをホモジナイズすることによりスラリー液を形成する工程と、前記スラリー液を前記基板に塗布して乾燥させることにより当該基板と一体となった、繊維が絡み合った繊維体を形成する工程とを具備することを特徴とする細胞培養基材の製造方法。
- 請求項5に記載の細胞培養基材の製造方法において、前記乾燥が加熱乾燥であることを特徴とする細胞培養基材の製造方法。
- 請求項5に記載の細胞培養基材の製造方法において、前記乾燥が凍結乾燥であることを特徴とする細胞培養基材の製造方法。
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