JP6733126B2 - 三次元細胞集合体の作製方法 - Google Patents
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Description
ヒト臍帯動脈平滑筋細胞等の細胞をシャーレで単層培養したのち、トリプシンで処理して単層細胞を剥離し、細胞を10%ウシ血清入りDMEM培地に分散させた。遠心分離後に上清を除去して、100万個/mL〜数百万個/mLの適当な細胞密度に調整した。
ヒト平滑筋細胞を用いて、細胞収容容器内への細胞の追加と圧力印加を繰り返す方法(図5、下記(1)〜(3))で細胞集合体を作製した。細胞収容容器として、四角形又は円形のガラス製培養皿を用いた。培養皿にはフィブロネクチンによるコーティングを行なった。加圧パターンは、250秒間110kPa→250秒間180kPa(0.002Hz)のサイクルとした。インキュベータ内の温度は37℃とし、CO2濃度は圧力印加工程も含め5%で維持した。
(2) 50万個/mLの細胞懸濁液の0.4mLを円形ガラス製培養皿(0.7平方センチメートル)に加え、1日間大気圧で置いた後に2日間の加圧処理を行なった。細胞懸濁液0.4mLを培養皿に追加し、1日間大気圧で置いた後に1日間の加圧処理を行なった。
(3) 75万個/mLの細胞懸濁液の0.4mLを円形ガラス製培養皿(0.7平方センチメートル)に加え、1日間大気圧で置いた後に2日間の加圧処理を行なった。細胞懸濁液0.4mLを培養皿に追加し、1日大気圧で置いた後に1日間の加圧処理を行なった。さらに細胞懸濁液0.4mLを培養皿に追加し、1日間大気圧で置いた後に2日間の加圧処理を行なった。
ヒト平滑筋細胞を用いて、細胞追加と加圧処理を繰り返して細胞集合体を作製した場合と、加圧処理を行なわずに細胞の追加のみを行なって大気圧で放置することで細胞集合体を作製した場合との比較を行なった。細胞収容容器として、四角形ガラス製培養皿(0.8平方センチメートル)を用いた。培養皿にはフィブロネクチンによるコーティングを行なった。CO2濃度は5%一定、温度は37℃とした。
ラット新生児大動脈平滑筋細胞100万個をガラス製培養皿内で110-180kPa, 0.002Hzで24時間加圧し、翌日、培養皿内に細胞100万個を追加した。処理中のCO2濃度は5%一定、温度は37℃とした。この工程を15回繰り返し、1ヶ月後に15層積層した細胞集合体を得た。図12は得られた細胞集合体の切片をエラスチカワンギーソン染色した像である。スケールバーは100μmであり、細胞集合体の厚みは200μmを越えるほどとなった。生細胞をトリパンブルー染色でカウントしたところ、生細胞率は90%以上であった。また、弾性線維も層状に著明に形成されており、引っ張り試験で2倍以上の進展が見られた。これは、ヒトの小口径血管壁に匹敵する性状であり、得られた細胞集合体は極めて頑強性が高いことが確認された。
(1) シリンジポンプを使用してマイクロ流体チップ内で単層ヒト臍帯動脈平滑筋細胞に圧力印加(180kPa)し、細胞レベルで加圧の影響を調べた。F-アクチン及び細胞核を免疫蛍光染色して観察したところ、圧力印加しない場合(図9右)と比べて、圧力印加した細胞(図9左)ではアクチンフィラメントが劇的に増加することが確認された。
4 圧力容器
6 レギュレータ
8 コンプレッサ
10 インキュベータ
12 コンピュータ
14 細胞懸濁液
Claims (9)
- 細胞収容容器内に細胞懸濁液を収容する細胞収容工程、及び前記容器内の細胞に圧力を印加する圧力印加工程を含む、三次元細胞集合体の作製方法であって、前記細胞収容工程及び前記圧力印加工程が2回以上反復して行なわれ、2回目以降の細胞収容工程では、細胞収容容器内に収容した細胞へのさらなる細胞の追加が行われる、方法。
- 前記圧力印加工程は、多孔性の三次元足場材の非共存下で行われる、請求項1記載の方法。
- 印加する圧力が102kPa〜100MPaである、請求項1又は2記載の方法。
- 前記細胞が動物細胞である、請求項1〜3のいずれか1項に記載の方法。
- 細胞収容工程及び圧力印加工程の間に、細胞懸濁液を非加圧で維持する非加圧工程を含む、請求項1〜4のいずれか1項に記載の方法。
- 細胞懸濁液を収容する細胞収容容器を内部の気相中に収容するための圧力容器と、少なくとも該圧力容器を収容するためのインキュベータと、インキュベータ内の気相に連絡し、該圧力容器内に送り込む空気を圧縮するためのコンプレッサと、該コンプレッサ及び前記圧力容器内部に連結するレギュレータと、該レギュレータに接続し、圧力及び加圧パターンを制御する指令を発するコンピュータとを含み、コンプレッサで圧縮したインキュベータ内の空気を圧力容器内部に送ることにより、細胞収容容器内の細胞への圧力印加が行われる、三次元細胞集合体の作製装置。
- コンピュータによる指令が周期的加圧である、請求項6記載の装置。
- コンピュータによる指令が、数秒〜数分ないしは数百秒ごとに印加する圧力を変える周期的加圧である、請求項6又は7記載の装置。
- 細胞収容容器内に多孔性の三次元足場を含まないことを特徴とする、請求項6〜8のいずれか1項に記載の装置。
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