JP2007105031A - 細胞の増殖基質としての粘膜下組織 - Google Patents
細胞の増殖基質としての粘膜下組織 Download PDFInfo
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Abstract
【解決手段】温血脊椎動物の粘膜下組織をin vitro細胞/組織増殖の基質として用いることにより細胞/組織培養の細胞増殖させる。また、粘膜下組織細胞増殖基質によって、in vivoで見いだされる環境と類似したコラーゲン性基質環境をin vitroで細胞に提供する。更に、該粘膜下組織は、真核細胞を、細胞増殖を誘導する条件下で粘膜下組織と接触させるとき、前記細胞の増殖及び分化を促進させる。
【選択図】なし
Description
[粘膜下組織の滅菌]
細胞培養技術は厳しい無菌状態で行われなければならないから、もしもその培養系が抗生物質を含まないならば、粘膜下組織を無菌状態に調製して細胞培養基質として使用しなければならない。粘膜下組織の生物向性に与える滅菌の影響を調べるために多数の滅菌法が研究された。組織の機械的強度及び生物向性を顕著には弱めない滅菌法が好ましい。腸粘膜下組織のための次のような滅菌法が評価された:過酢酸滅菌、2.5Mradガンマ−照射、1.0Mradガンマ−照射、エクスポル(Expor)(Alcide,Norfolk,CT)滅菌及びこれらの滅菌法の種々の組み合わせ。ガンマ照射は 60コバルト-ガンマチェンバーを用いて水和粘膜下組織で行われた。エクスポル滅菌は、メーカーの説明書により、滅菌剤容量(ml)対 腸粘膜下組織(g)比 10対1で行われた。
[過酢酸による粘膜下組織の滅菌]
粘膜下組織を室温で過酢酸/エタノール溶液に2時間浸す。過酢酸溶液(ml) 対 粘膜下組織(gram)比 10:1またはそれ以上を用いる。過酢酸/エタノール溶液は4%エタノール、0.1%(容量:容量)過酢酸、残りは水である。0.1%過酢酸成分は表1に明確に示される市販の35%過酢酸保存溶液を希釈したものである。好適には粘膜下組織は過酢酸溶液に浸しながら回転器で振動する。2時間後、過酢酸溶液を流し去り、その代わりに等量のラクテート加リンゲル溶液またはリン酸緩衝化食塩溶液(PBS)を加え、15分間(振動しながら)浸した。粘膜下組織をさらに4回ラクテート加リンゲル溶液またはPBSで洗い、その後さらに15分間滅菌水ですすいだ。
組成、重量%
過酢酸 35.5
過酸化水素 6.8
酢酸 39.3
硫酸 1.0
水 17.4
過酸化アセチル 0.0
安定化剤 500ppm
一般的活性酸素分析、重量%
過酸としての活性酸素 7.47
H2O2としての活性酸素 2.40
総活性酸素 10.67
[滅菌粘膜下組織上の種々の細胞型の増殖特性]
米国特許第4902508号に記載されているように、安楽死させたばかりの豚から小腸粘膜下組織を収穫し、調製した。種々の方法(ガンマ−照射、過酢酸、など)による滅菌後、粘膜下組織をポリプロピレン製枠内にクランプで止めて細胞増殖のための平らな表面領域(50mm2)を作る。枠を培養培地に浸し、培地栄養物がその粘膜下組織の両面に近づけるようにした。種々の細胞型を粘膜下組織に接種し(3×104細胞/粘膜下組織切片)、それから37℃の5%CO2、95%空気 インキュベーター中に置いた。種々の時間経過後、接種した粘膜下組織を10%中性緩衝化ホルマリン中で固定し、パラフィンに埋封し、切片にした(6μm)。種々の組織学的及び免疫組織化学的染色法を用いて細胞増殖特性を明らかにした。
細胞系 細胞系の説明
CHO チャイニーズハムスター卵細胞
3T3 スイスアルビノマウス胚線維芽細胞
C310T1/2 C3Hマウス胚、多型潜在性
FR ラット胎児皮膚(Sprague Dawley)
IMR90 ヒト胎児肺線維芽細胞
HT−29 ヒト結腸腺癌、中程度に分化している、II度
RPEC ラット肺内皮細胞
HUVEC ヒト臍静脈細胞
SCC−12 扁平上皮癌
細胞系
ラット心筋
ブタ平滑筋(大動脈)
ブタ内皮(大動脈)
ウサギ平滑筋(大動脈)
ウサギ内皮(大動脈)
ブタ平滑筋及び内皮(混合 & 同時培養)
ヒト骨芽細胞
ヒト内皮細胞
[腫瘍細胞増殖モデル系としての腸粘膜下組織細胞培養基質]
SCC−12として知られる顔のヒト扁平上皮癌からの確立された細胞系を invitro 培養したもの(W.グリーンリー(パードュ大学)から入手)の形態学及び侵襲特性を研究した。皮膚細胞(例えばスイス3T3マウス線維芽細胞のガンマ照射−またはマイトマイシンC−処理−フィーダー層)の標準細胞培養条件下で増殖させたとき、平らな細胞の単層が形成させる。しかしSCC−12細胞は、腸粘膜下組織の abluminal 面に接種するときは、組織学的検査で粘膜下基質コンポーネントの活発な分解及び腸粘膜下組織侵襲を示した。
これらの実験のために、小腸粘膜下組織を過酢酸で滅菌し、ポリプロピレン製枠に止めつけ、細胞増殖用の平らな表面領域(50mm2)を作り出した。その枠を培養培地に浸し、培地栄養を腸粘膜下組織の両側に近づけた。細胞を接種し(5×104 細胞/0.8cm2 腸粘膜下組織)、5%CO2、95%空気のインキュベーター、37℃、に入れた。3、7、10及び14日後、接種粘膜下組織を10%中性緩衝化ホルマリン中で固定し、パラフィンに埋封し、切片にした(5μm)。標準H&E組織染色を行い、形態学的に検査した。
種々の培養細胞の侵襲特性を侵襲チェンバーの使用により研究した。可溶性粘膜下組織被覆-ポリカルボネートフィルター上に増殖させた細胞と、マトリゲル被覆ポリカルボネートフィルター上に増殖させた細胞とを下記の方法により比較した。ポリカルボネートフィルター(13mm、8μm孔サイズ)を可溶性粘膜下組織またはマトリゲルで被覆し、層流フード中で空気乾燥し、無血清培地で再構成した。25μg/mlフィブロネクチンを含む無血清培地をブラインド ウェル チェンバーの下方ウェルに入れ、化学誘引物質として用いた。被覆フィルターを界面として侵襲チェンバーの上方及び下方ウェル間に置いた。0.1%BSAを含む無血清培地に懸濁した細胞(約2×105)を各侵襲チェンバー内の被覆フィルター上に接種し、そのチェンバーを5%CO2/95%空気中で37℃でインキュベートした。
[腸粘膜下組織は細胞分化を促進する]
FR上皮細胞は、腸粘膜下組織の内腔側(緻密層側)に培養したとき、層化多層を形成する。腸粘膜下組織に隣接する細胞は柱状の形をもち、多層表面に近づくとだんだん平らになる。14日後、デスモソームに似た構造が確認され、その細胞層を汎サイトケラチン抗体で染色するとサイトケラチンが陽性染色を示した。その上上皮細胞は、正常な健康状態で in vivo で見られるように、支持基質生成物(多分、基底膜)を生成するようにみえた。これらの研究結果は、腸粘膜下組織が上皮細胞の本来の成熟-及び分化過程を維持することを示唆する。
[ハムスター膵島の分離]
ハムスター膵島をゴトウ(Gotoh)らの既報(Transportation 43巻、725−730ページ(1987))の方法で分離した。つまり、齢6−8週のゴールデンハムスター(Harlan,Indianapolis,IN)をメトファン(メトキシフルラン;Pitman-Moore;Mundelein,IL)吸入により麻酔した。総胆管に立体顕微鏡下でポリエチレン製カテーテル(PE−10チューブ;CMS;ヒューストン、TX)を挿入し、それを介して、0.7mg/mlコラゲナーゼPを含む氷冷M−199培地(Gibco BRL から市販)約3−4mlを、膵臓全体が膨潤するまでゆっくりと注入した。膵臓を切除し、100μg/mlペニシリンG及び100μg/mlストレプトマイシン(付加的コラゲナーゼなし)を含むM−199培地中で37℃で約50分間消化した。消化物を氷冷M−199培地で3回洗い、滅菌500μmステンレス鋼メッシュ、その後100μmメッシュを次々と通過させた。フィコル密度勾配(1.045、1.075、1.085、1.100)による遠心分離800g、10分間、によって精製した後、膵島を最上の2界面から回収した。
ランゲルハンス島(島細胞)を、5%CO及び95%空気を補充したインキュベーター中の粘膜下組織細胞増殖基質上に37℃で培養した。島細胞は種々の形の腸粘膜下組織の存在下、次のような方法で培養された:
1.直接的接触:腸粘膜下組織と培養細胞とが物理的に互いに接触する。
2.間接的接触:腸粘膜下組織と培養細胞はステンレス鋼メッシュによって分離されている。
3.可溶性腸粘膜下組織を培養培地に加える。
4.細胞を、可溶性腸粘膜下組織被覆−培養プレート上に培養する。被覆は、可溶性腸粘膜下組織1mlを35mm培養プレートに置き、37℃で2時間加熱し、被覆プレートを吸引し、培養培地で一回洗うことによって、過剰の腸粘膜下組織液を除去する、という方法で行われる。
1.腸粘膜下組織由来細胞培養基質を数種類の方法で滅菌した:過酢酸処理またはガンマ照射。ガンマ照射した天然の(腸粘膜下組織分離後、その他の処理を一切しない)膜を―それらを同時培養前に培養培地で十分再水和した場合に限り―直接細胞培養基質として使用することができる(天然の膜は抗生物質の存在下で培養しなければならない)。過酢酸で滅菌した膜は、先ず最初に洗って残留過酢酸を除去し、それから培養する。なぜならば残留過酢酸は多分細胞毒だからである。一般的には過酢酸滅菌組織を大量の培地に24時間浸漬し、その後同培地でよく洗う。
対照群(粘膜下組織なしで培養した島)では7日間培養の検査の結果、線維芽細胞が島細胞におおいかぶさって増殖しているのが判明した。
Claims (14)
- 真核細胞のin vitro増殖及び分化を促進する方法であって、
前記細胞の増殖を誘導する条件下で、前記細胞と温血脊椎動物の粘膜下組織を含む細胞増殖基質とをin vitro接触させる段階を含む方法。 - 粘膜下組織が筋層から分離した粘膜下層、少なくとも筋層の内腔部分を含んでなる腸粘膜下組織である請求項1記載の方法。
- 細胞を細胞増殖基質と接触させる段階が、流動状粘膜下組織で被覆した培養器具上に細胞を培養することを含んでなる請求項1記載の方法。
- 細胞を細胞増殖基質と接触させる段階が、流動状粘膜下組織を液体細胞培養培地に加えることを含んでなる請求項1記載の方法。
- 細胞集団の増殖を促進する培養培地組成物であって、
温血脊椎動物の粘膜下組織と、前記細胞集団の増殖を促進する栄養とを含む培養培地組成物。 - 粘膜下組織が、筋層から分離した粘膜下層、少なくとも筋層の内腔部分からなる腸粘膜下組織である請求項5記載の組成物。
- 粘膜下組織が流動状粘膜下組織である請求項5記載の組成物。
- 粘膜下組織を、前記組織を可溶化するのに十分な時間プロテアーゼで消化し、実質上均質な溶液が提供される請求項5記載の組成物。
- 温血脊椎動物の粘膜下組織と、
前記細胞集団の増殖を促進する栄養と、
あらかじめ選択した細胞型の増殖集団とを含む細胞培養組成物。 - 粘膜下組織が腸粘膜下組織である請求項9記載の細胞培養組成物。
- あらかじめ選択した細胞型が、線維芽細胞過剰増殖を実質上伴わない島細胞である請求項9記載の細胞培養組成物。
- 温血脊椎動物の粘膜下組織の、移植または注入組織グラフト材料としての機能的特性を高める方法であって、
グラフト材料を宿主に移植または注入する前に、粘膜下組織にあらかじめ選択した細胞型を接種する段階を含む方法。 - グラフト材料を宿主に移植または注入する前に、そのグラフト材料を、あらかじめ選択した細胞型の増殖を誘導する条件にさらし、前記細胞を増殖させる段階をさらに含む請求項12記載の方法。
- In vitroで線維芽細胞の同時増殖なしに島細胞を増殖させる方法であって、
前記細胞の増殖を誘導する条件下で、島細胞を温血脊椎動物の粘膜下組織を含む細胞増殖基質とin vitro接触させる段階を含む方法。
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WO1996024661A1 (en) | 1996-08-15 |
EP1449916B1 (en) | 2011-05-04 |
EP1449916A3 (en) | 2004-12-22 |
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