JP5436205B2 - 足場 - Google Patents
足場 Download PDFInfo
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- JP5436205B2 JP5436205B2 JP2009508489A JP2009508489A JP5436205B2 JP 5436205 B2 JP5436205 B2 JP 5436205B2 JP 2009508489 A JP2009508489 A JP 2009508489A JP 2009508489 A JP2009508489 A JP 2009508489A JP 5436205 B2 JP5436205 B2 JP 5436205B2
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Description
別段の定めがない限り、「含んでいる」および「含む」という用語ならびにそれらの文法上の変形は、「開かれた」または「包含的な」表現を表すことを意図しており、それにより、それらは、列挙された要素を含むだけでなく、追加の列挙されていない要素の包含も可能にする。
Lakeshore Biomaterialsにより提供される1.24dL/gの固有粘度を有する1.729gのPGAを、Apollo Scientificにより提供される18.200gのヘキサフルオロイソプロパノール(HFIP)内に溶解し、9.5%w/wのPGA溶液を形成した。このポリマー溶液を、孔が10μmで直径が50mmのWhatmanのディスク状のフィルターを通して、0.03ml/分で分注するためのシリンジポンプセット内に存在する10mlのシリンジ内に濾過した。シリンジの出口を柔軟性のあるプラスチックチューブ(内経1.5mm)に取り付け、チューブの他方の端を、先細の部分を取り除くために水平にカットした18ゲージ針に取り付けた。針は、エレクトロスピニングの際の空気の動きを最小にするために用いる箱(内部寸法:幅55cm×高さ60cm×深さ60cm)の中の標的の15cm上に垂直に固定した。標的は、モーターに取り付けられているアルミニウムのマンドレル(直径5cm×長さ10cm)で構成されており、モーターにより標的が50rpmで回転し得、それにより繊維材料の一様な被覆が得られた。標的からの繊維足場の分離を容易にするために、交換可能なベーキングペーパー(12.7×17.5cm)で標的を覆った。
10mmの足場の円を切り出し、それらを10mmの金属の小片に接着し、切り出された足場を真空下で金/パラジウム合金でスパッタコーティングして表面の帯電を減少させることで、足場についての、図1に示すような走査型電子顕微鏡写真(SEM)が得られた。コーティングされた足場を、5kVの加速電圧、210μAのタングステンフィラメントの電流、および15mmの作動距離にセットされた走査型電子顕微鏡(JSM-6400)を用いて画像化した。
平均繊維直径は2.42μm±0.35であった。
正方形のサンプル(3×3cm2)を、はさみを用いて足場から切り出した。次に、サンプルをポリスチレン製のペトリ皿に置き、15mLのリン酸緩衝生理食塩水(PBS)で覆い、37℃のインキュベーターに24時間置いた。湿潤したサンプルを脱イオン水ですすいだ後、全てのサンプルを再び測定し、「縮小%=100×(最初の面積-最後の面積)/最初の面積」として定義される「収縮%」を表すことにより、サンプルの直径を最初の直径と比較した。各タイプの足場を3セット試験した。結果を表1に示す。
様々な構造を有する、エレクトロスピニングされた様々な足場の生物学的効果を、細胞に基づいた3回の独立したアッセイを用いて評価した。細胞の接着、増殖、および足場への移動を測定するためのアッセイを実施した。
エレクトロスピニングされた足場を様々な濃度のPGA溶液から調製し、約200nmから約3μmの様々な足場構造を得た。直径が約20μmの繊維を有するPGAフェルト(対照サンプル)を同時に評価した。足場/対照を、プレス型抜き機(Samco SB-25)を用いて直径が13mmのディスクに切断し、Minucellクリップ(Minutissue Vertriebs, GmbH)内に置き、Uvasol 400を用いて紫外線下で20分間滅菌した。ヒト真皮の線維芽細胞を13代継代およびそれ以降にわたって用いて、マイコプラズマの感染がないことをDAPI染色(Vector Labs)により確認した。
エレクトロスピニングされた足場を様々な濃度のPGA溶液から調製し、約200nmから約3μmの様々な足場構造を得た。直径が約20μmの繊維を有するPGAフェルト(対照サンプル)を同時に評価した。足場/対照を、プレス型抜き機(Samco SB-25)を用いて直径が13mmのディスクに切断し、Minucellクリップ(Minutissue Vertriebs, GmbH)内に置き、Uvasol 400を用いて紫外線下で20分間滅菌した。ヒト真皮の線維芽細胞を13代継代およびそれ以降にわたって用いて、マイコプラズマの感染がないことをDAPI染色(Vector Labs)により確認した。
細胞が試験サンプルの足場内に移動する能力、および前記足場を通って移動する能力を評価するためにアッセイを実施した。実験の機構を図2に示す。
ヒト真皮の線維芽(HDF)細胞を直径が13mmのナイロンディスク上に播種した。簡潔に説明すると、700万〜1200万個の細胞を、25個のナイロンディスクおよび50mLのDMEM+10%FCS(TCM)を含む、シリコン処理したTechneフラスコに添加し、次に15分ごとに24時間、45rpmで5分間撹拌した(37℃で5%CO2)。ナイロンディスクを、各々が2mLのTCMを含む、非組織培養用のプラスチック製の24ウェルプレートのウェル内に個々に移し、細胞がコンフルエントになるまで、5%CO2において37℃でさらに3日間インキュベートした。
エレクトロスピニングされた足場を様々な濃度のポリグリコール酸(PGA)溶液から調製し、約200nmから約3μmの様々な足場構造を得た。直径が約20μmの繊維を有するPGAフェルト(対照サンプル)を同時に評価した。足場/対照を、プレス型抜き機(Samco SB-25)を用いて直径が13mmのディスクに切断し、Uvasol 400を用いて紫外線下で20分間滅菌した。
無菌のナイロンディスク(直径13mm)をラット尾部のコラーゲンで無菌状態で被覆し、空気乾燥させた。コラーゲンで被覆されたナイロンディスクを図2に示す最上層として用いた。
上述した3つの層を13mmのMinucellクリップ(13mmのMinusheet、参照1300、Minucell and Minutissue Vertriebs, GmbH)内に共に挟んだ。minucellの組立品を、各々が2mLのDMEM/10%FCS(Sigma)を含む、非組織培養用のプラスチック製の24ウェルプレートのウェルに移した。プレートを37℃/5%CO2で2日間インキュベートした。この時間の後、minucellの組立品を解体し、層を分離して、400μLのDMEM/10%FCSを含む24ウェルプレートの個々のウェル内に移した。40μLのWST-1試薬(Roche diagnostics)を各ウェルに添加し、プレートを5%CO2において37℃で1時間インキュベートした。100μLの体積を3重で96ウェルプレートに移し、Multiskan Ascentプレートリーダーで、655nmの参照波長で、450nmでの吸光度を読み取った。細胞数に対する吸光度の標準較正曲線を、実施した各アッセイの試験プレートについて作成し、サンドイッチ構造の各層に存在する細胞数を概算するために用いた。全細胞数を各サンドイッチ構造について計算し(3つの層全てについての細胞数の合計)、中間層および最上層に存在する細胞を全細胞数に対するパーセンテージとして概算した。
図4を参照すると、送達室20は足場室40と通じており、足場室40は受容室70と通じている。粗フィルター30は送達室20内に位置している。足場支持構造体50は足場室40内に位置している。足場60は足場支持構造体50の最上部上に位置している。真空ポンプ80は受容室70に操作可能に接続している。細胞原料10は送達室20に導入され、真空により、粗フィルター30を通って足場室40内に引き込まれ、その後、足場60を通って引き込まれ、足場には細胞原料の一部が保持され、一部は通過して受容室70内に入る。
エレクトロスピニングされた足場を、ポリグリコリド(PGA)、ポリラクチドグリコリド共重合体(PLGA)、ポリグリコリドトリメチルカーボネート(PGA-co-TMC)、ポリカプロラクトン(PCL)、ポリ(D-ラクチド)(PDLA)、およびポリ(L-ラクチド)(PLLA)を用いたエレクトロスピニングのプロセスにより作製した。ポリマー溶液を、所望の速度でポリマーを分注するように設定されているシリンジポンプ内に存在する10mlのシリンジ内に入れた。次に、シリンジの先端をチューブ(内径1.5mm)に取り付け、チューブの他方の端を、先細の部分を取り除くために水平にカットした18ゲージ針に取り付けた。針は、エレクトロスピニングの際の空気の動きを最小にするために用いる箱(内部寸法:幅55cm×高さ60cm×深さ60cm)の中の標的の10から20cm上に垂直に固定した。標的は、モーターに取り付けられている、マンドレルとして知られている金属ローラーで構成されており、モーターを最大2000rpmで回転させ、それによりナノ繊維材料の一様な被覆を得た。金属表面からの足場の分離を容易にするために、交換可能なベーキングペーパーでマンドレルを覆った。エレクトロスピニングのプロセスを開始するために、針に電圧を加え、一方で標的をアースした。十分な電圧で、ポリマー溶液が針の先端から落ちるのが止まり、代わりに小さなポリマー噴流として、アースされた回転している標的に向かって離れて行き、そこでナノ繊維として堆積した。
足場の繊維の直径:
標準:2.41±0.25μm、2.41±0.22μm
極厚:2.43±0.60μm、2.40±0.19μm
サンプル採取の日に食肉処理場から入手した処分したばかりのブタの2つの大腿骨頭から、骨梁を取り除いた。大腿骨を膝から取り外し、全ての筋肉、軟骨、腱、および、骨髄サンプルを汚染し得る他のあらゆる組織を除去した。大腿骨頭を糸のこぎりを用いて細かくして骨梁を露出させ、それを無菌条件下で穿孔機を用いて除去した。
大腿骨頭の骨髄から単離した細胞は、最初はメッシュを通過しない固体画分であるため、細胞を希釈して流体画分とした。骨髄を、リン酸緩衝生理食塩水(PBS)、血漿、または新鮮なブタ血液で、1ml当たり1×107個の白血球(WBC)の濃度まで希釈した。細胞懸濁液の100μlのアリコートを取り、Coulterカウンター(Beckman Coulter Ac.T 5 diff. Counter)で計数した。Beckman Coulterカウンターは、サンプルに含まれている白血球および赤血球の両方のカテゴリーの数を、サイズおよび粒度の両方に基づいて計数し、それにより、様々な細胞の最初のおよび最終的な細胞濃度の測定を行うことが可能となり、その結果、足場上に捕捉された単核細胞のパーセンテージを計算することが可能となった。
細胞播種機器は、ヒト骨髄、ブタ骨髄、ブタ骨髄間質細胞、および真皮の線維芽細胞を含む、様々な異なる細胞原料で用いられている。ヒト骨髄を、骨髄穿刺液としてヒトドナーから直接得た。ブタ骨髄間質細胞およびヒト真皮の線維芽細胞を、組織培養で増殖させた源から得た。これらの細胞源は流体として得、実施例3で記載した固体骨髄画分のように希釈した。結果は、異なる細胞原料の間で同等かつ再現性のあるものであり、類似の単核細胞画分が、エレクトロスピニングされた足場上に捕捉された。
5mlの前記細胞懸濁液を、細胞播種機器の最上部にある送達室内に真空下で添加した。この室には、あらゆる大きな粒子、例えば骨の小片を捕捉するための、最初の70μmのフィルターが存在する。流体のほとんどは、ステンレス鋼製のHollanderメッシュ上に支持されているエレクトロスピニングされた足場を含む足場室内に素通りした。大きい単核細胞画分のほとんどは、エレクトロスピニングされた足場の上または内部に捕捉され、小さい画分はメッシュを通って受容室内に引き込まれた。細胞が播種された足場を取り除き、受容室内の不要な細胞を廃棄した。
エレクトロスピニングされた足場は、7〜15μmの孔径を有する場合に、有核細胞の選択的な捕捉について最も効果的であった。
PGAおよびPLGAのエレクトロスピニングされた繊維の両方で同一の結果が得られた。
エレクトロスピニングされた足場をまた、支持メッシュの収量と組み合わせて、他の不織メッシュと比較し、データは、これらのエレクトロスピニングされた足場により、特に単核細胞群の捕捉に関して良好な結果が得られることを示す。
PGA足場は標準で100μmであり、極厚の深さのもので300μmである。
ボランティアからのインフォームドコンセントを得て、約17mlの新鮮なヒト血液を、静脈穿刺により、2つのACD-Aバキュテナー内に回収した。あるいは、ブタ血液を、4%(w/v)のクエン酸3ナトリウム溶液を1:9の割合(クエン酸ナトリウム対血液)で含む瓶内に回収した。白血球、血小板、および赤血球の数を、Beckman Coulter Ac.T 5 diff.カウンターを用いて測定した。
方法
深さが100μm(標準)および300μm(極厚)のPGA足場を、エレクトロスピニングし、かつ上述のように30mmのディスクに切断することで作製した。3つの異なるドナーから得た、4%(w/v)のクエン酸3ナトリウム溶液を1:9の割合(クエン酸ナトリウム対血液)で含む、5mlのブタ血液の3セットを、機器と、大気圧よりも100mBar低い真空とを用いて、足場を通して濾過したが、足場は、250μmの金属メッシュにより支持されており、(i)5mlの水、(ii)5mlのPBS、(iii)5mlのDMEM+10%FCSのいずれかを濾過することで事前に湿潤させているか、または(iv)乾燥したままのものである。濾液内の白血球、赤血球、および血小板の数をBeckman Coulter Ac.T 5 diff.カウンターを用いて測定し、元の血液サンプル内の細胞数と比較した。
(参考文献)
20 送達室
30 粗フィルター
40 足場室
50 足場支持構造体
60 足場
70 受容室
80 真空ポンプ
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