JP4452458B2 - Cytotoxicity test method - Google Patents
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- JP4452458B2 JP4452458B2 JP2003160007A JP2003160007A JP4452458B2 JP 4452458 B2 JP4452458 B2 JP 4452458B2 JP 2003160007 A JP2003160007 A JP 2003160007A JP 2003160007 A JP2003160007 A JP 2003160007A JP 4452458 B2 JP4452458 B2 JP 4452458B2
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Description
【０００１】
【発明の属する技術分野】
本発明は、化学物質や重金属などの各種の被検物質の細胞毒性試験方法に関する。
【０００２】
【従来の技術】
これまで、化学物質や重金属（亜鉛、カドミウム、銅、砒素、コバルト、モリブデン、ニッケル、鉛、セレン、クロム、錫、水銀など）などの各種の被検物質の毒性試験は動物個体を用いて行われてきたが、動物個体を用いた方法は、時間と費用がかかり過ぎるという問題だけでなく、動物愛護の観点からも問題がある。従って、近年、株化細胞（培養細胞）を用いた毒性試験方法、即ち、細胞毒性試験方法が検討されている。
【０００３】
【発明が解決しようとする課題】
水環境における被検物質の毒性試験は魚類由来の株化細胞を用いて行うことが望ましく、ニジマス卵巣由来の線維芽性株化細胞であるＲＴＧ−２細胞やファットヘッドミノー由来の上皮性株化細胞であるＦＨＭ細胞などを用いた毒性試験方法が既に検討されている。しかしながら、これらの方法は感受性などの面において十分なものではなく、また、ＲＴＧ−２細胞は、増殖速度が遅いといった問題や、増殖温度範囲が狭いといった問題があり、利便性の面においても必ずしも満足できるものではない。
そこで本発明は、新規な魚類株化細胞を用いた細胞毒性試験方法を提供することを目的とする。
【０００４】
【課題を解決するための手段】
上記の点に鑑みてなされた本発明は、請求項１記載の通り、チョウザメ由来の株化細胞としてベステルの虹彩色素上皮細胞由来の５０回以上の継代培養が可能な株化細胞であるＳＴＩＰ−１細胞（ＦＥＲＭ ＢＰ−８４２１）またはＳＴＩＰ−３細胞（ＦＥＲＭ ＢＰ−８４２２）に対する毒性に基づいて被検物質の毒性評価を行う細胞毒性試験方法である。
また、請求項２記載の細胞毒性試験方法は、被検物質の毒性評価にアラマーブルーアッセイ法を用いるものである。
【０００５】
【発明の実施の形態】
本発明の細胞毒性試験方法においては、被検物質の毒性評価を行うためにチョウザメ由来の株化細胞を用いる。チョウザメ（ｓｔｕｒｇｅｏｎ）としては、例えば、Ｈｕｓｏ属やＡｃｉｐｅｎｓｅｒ属に属するものが挙げられるが、好適にはＨｕｓｏ属に属するベルーガ（Ｈ．Ｈｕｓｏ）の雌とＡｃｉｐｅｎｓｅｒ属に属するステールリヤチ（Ａ．ｒｕｔｈｅｎｕｓ）の雄から作出された品種改良種であるベステル（Ｂｅｓｔｅｒ）が挙げられる。ベステルは交雑種であるため、ベステル由来の株化細胞は、各種の被検物質に対する感受性に関してＨｕｓｏ属チョウザメの細胞とＡｃｉｐｅｎｓｅｒ属チョウザメの細胞の双方の特性を兼ね備えていることが期待されるからである。
【０００６】
チョウザメ由来の株化細胞は、例えば、上皮細胞から樹立される。当該上皮細胞は、チョウザメのどの部位の組織のものであってもよいが、眼球のいずれかの組織の上皮細胞であることが望ましく、好適な上皮細胞としては、外界と非接触の状態で存在する虹彩色素上皮細胞や網膜色素上皮細胞などが挙げられる。これらの細胞は元来、微生物汚染の可能性がないので、無菌的に細胞を取出せば、その後の作業を無菌的に行うことで株化細胞の微生物汚染を確実に防ぐことができるからである。なお、株化細胞は、腎臓や卵巣を由来とする上皮細胞から樹立されたものであってもよいが、この場合、株化細胞を樹立するに当たっては、これらの組織から上皮細胞のみを選択して分離培養するためにはある程度の時間と労力を必要とするといった点や、微生物汚染の可能性が否定できないといった点を認識しておく必要がある。
【０００７】
株化細胞の樹立方法は、公知の方法に従って、初代培養細胞を継代培養することで行えばよい。培地は、魚類細胞の培養に通常用いられるＬ１５培地に牛胎児血清（ＦＢＳ）を加えたようなものでよい。
【０００８】
チョウザメ由来の株化細胞として好適なものは、例えば、ベステル眼球の虹彩色素上皮細胞由来の株化細胞であるＳＴＩＰ−１細胞（ＦＥＲＭ ＢＰ−８４２１）とＳＴＩＰ−３細胞（ＦＥＲＭ ＢＰ−８４２２）が挙げられる。いずれの細胞も、ＲＴＧ−２細胞やＦＨＭ細胞などよりも感受性が高いという利点を有する。また、いずれの細胞も、細胞外基質を添加することなしに継代培養が可能であること、５０回以上の継代培養が可能であることから、利便性の面において優れている。とりわけ、ＳＴＩＰ−１細胞は、２０℃での培養開始後２日目〜６日目のダブリングタイムが５０時間未満であるといった特性や、培養皿に添加してから接着するまでの着定率が１時間後に７５％以上であるといった特性を有するので、一度に大量の試験区を設定しても簡便かつ迅速に試験を行うことができる。
【０００９】
被検物質のチョウザメ由来の株化細胞に対する毒性は、例えば、アラマーブルーアッセイ法を用いて評価すればよい。
アラマーブルーアッセイ法は、動物細胞などの細胞代謝を測定するために開発されたバイオアッセイ法の一つである。アラマーブルーは、その還元に細胞への取込みを必要とする酸化還元色素であり、ミトコンドリア内で行われている呼吸代謝系の還元反応により、酸化型（無蛍光・青）から還元型（蛍光・赤）に変化する性質を持つ。細胞代謝が正常であると還元反応が進行する一方、細胞代謝に異常をきたすと酸化型のままであるので、色の変化を測定して細胞代謝の異常を調べることができる。また、細胞代謝の測定を蛍光や吸光に基づいて行ってもよい。この場合、蛍光は５３０ｎｍ〜５６０ｎｍの励起波長と５９０ｎｍの検出波長でモニターされ、吸光は５７０ｎｍと６００ｎｍでモニターされる。
アラマーブルーアッセイ法は、アラマーブルーが水溶性であり、ニュートラルレッドなどの他の色素を用いたバイオアッセイ法において必要とされる抽出操作や固定操作が不要なため、評価を簡便に行うことができるという点において望ましいものである。
なお、このアラマーブルーアッセイ法は、例えば、市販のキット（ＢＩＯＳＯＵＲＣＥ 社製） を用いて行うことができる。
【００１０】
【実施例】
以下に参考例と実施例を挙げ、本発明を具体的に説明する。
【００１１】
参考例：チョウザメ由来の株化細胞の樹立
１．ベステル眼球からの虹彩色素上皮細胞の分離
体長約１５ｃｍのベステル３０尾から眼球を摘出して７０％エタノール中で殺菌処理した後、殺菌処理した眼球をペニシリンとストレプトマイシンを添加したＰＢＳ（−）中でよく洗浄した。その後、眼球から角膜とレンズを取り除いて虹彩を切り出した。こうして得られた虹彩を０．０５％ＥＤＴＡで約４０分間処理し、虹彩色素上皮細胞と、虹彩のストローマや強膜などの結合組織との分離を容易にした後、これらの結合組織を取り除き、分離したシート状の虹彩色素上皮細胞を０．１２５％トリプシンで酵素処理してシングルセル状態の細胞（初代細胞）を得た。
【００１２】
２．初代培養
上記のようにして得られた初代細胞を、直径３．５ｃｍプラスチックディシュ（培養皿）に加えた、Ｌｅｉｂｏｖｉｔ’ｓ Ｌ１５培地（Ｇｉｂｃｏ社製）に１０％ＦＢＳ（Ｇｉｂｃｏ社製の牛胎児血清）とペニシリン（１０ｕｎｉｔ／ｍｌ）とストレプトマイシン（５０μｇ／ｍｌ）を添加した培地を用い、２０℃のＣＯ2インキュベータ内（但し大気雰囲気）で培養した。初代細胞の中から増殖性の優れた細胞を選択し、継代培養を繰り返した。
【００１３】
３．継代培養
培養皿が細胞で集密的（ｃｏｎｆｌｕｅｎｔ）な状態になったら、０．０５％ＥＤＴＡと０．１２５％トリプシンを含有する溶液で細胞を培養皿から剥離し遠心分離により細胞を回収し、別の培養皿に移し、上記の培地を用いて培養を継続した。これを繰り返すことによって、長期間培養可能な２種類の株化細胞（ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞）を得た。いずれの株化細胞も、継代培養の際、コラーゲンなどの細胞外基質を培養皿底面にコーティングするといったような添加をしなくても培養皿に着定した。なお、上記の２種類の株化細胞は独立行政法人産業技術総合研究所特許生物寄託センターに寄託されており、それらの受託番号は、ＳＴＩＰ−１細胞がＦＥＲＭ ＢＰ−８４２１、ＳＴＩＰ−３細胞がＦＥＲＭ ＢＰ−８４２２である。この特許出願の時点において、株化細胞ＳＴＩＰ−１の継代培養回数は１４０回を超え、株化細胞ＳＴＩＰ−３の継代培養回数は８０回を超える。
【００１４】
４．ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞の特性
（細胞の形態）
ＳＴＩＰ−１細胞は細長い細胞であるが上皮性の細胞であった（図１参照：培養開始後８日目の倍率１００倍の顕微鏡写真）。一方、ＳＴＩＰ−３細胞は典型的な敷石状の上皮性の細胞であった（図２参照：培養開始後１４日目の倍率１００倍の顕微鏡写真）。
【００１５】
（細胞の温度特性）
図３に示したように、ＳＴＩＰ−１細胞は１５℃〜３２℃の広い温度範囲で良好な増殖性を示し、特に２０℃での増殖性が優れていた。２０℃での培養開始後２日目〜６日目の細胞のダブリングタイム（指数関数的に細胞が増殖する時間）は３８．９時間であり、ＲＴＧ−２細胞と比較して約２倍の増殖速度を有していた。一方、図４に示したように、ＳＴＩＰ−３細胞は１５℃〜２０℃で良好な増殖性を示したが、３０℃以上ではその増殖性は阻害された。２０℃での培養開始後２日目〜６日目の細胞のダブリングタイムは７４．９時間であった。
【００１６】
（細胞増殖に対するＦＢＳの影響）
図５に示したように、ＳＴＩＰ−１細胞を増殖せしめるために必要なＬ１５培地に添加されるＦＢＳの濃度は４％で足りた。また、図６に示したように、ＳＴＩＰ−３細胞を増殖せしめるために必要なＬ１５培地に添加されるＦＢＳの濃度も４％で足りた。よって、これらの株化細胞を増殖せしめるために必要なＦＢＳは少量であることから、これらの株化細胞の大量培養は経済的に有利であることがわかった。
【００１７】
（細胞の染色体数）
ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞の染色体数を、継代培養８０回目の細胞について、培養６日目の対数増殖期の細胞にコルヒチン処理を行う常法にて調べた。具体的には、細胞に最終濃度が０．２０μｇ／ｍｌになるようにコルヒチンを加え、１８時間培養した後に培地を取り除いてから細胞をＰＢＳ（−）で洗浄した。次に、０．０５％ＥＤＴＡと０．１２５％トリプシンを含有する溶液で細胞を培養皿から剥離し遠心分離により細胞を回収した。こうして回収した細胞に０．０７５ＭのＫＣｌを添加して室温にて２０分間静置して低張処理を行った。低張処理した細胞懸濁液はカルノア液を用いて２０分間氷中で固定した後、フレームドライ法によって染色体標本とした。これをギムザ染色し、顕微鏡（倍率１０００倍）で染色体を計数した。その結果、ＳＴＩＰ−１細胞の染色体数は２ｎ＝１６６±７．６本、ＳＴＩＰ−３細胞の染色体数は２ｎ＝１２１±６．１本となり、いずれの細胞もベステルの染色体数である２ｎ＝１１７本と比較して増加していた（図７参照）。この結果は、いずれの細胞も株化細胞であることを特徴付けるものである。染色体を分類すると、いずれの細胞も基本的には２倍体であるが、ランダムに異数性を示していた。２ｎ＝１７３本のＳＴＩＰ−１細胞の一例の染色体標本を図８に、２ｎ＝１２６本のＳＴＩＰ−３細胞の一例の染色体標本を図９に示す。
【００１８】
（細胞の接着性）
ＳＴＩＰ−１細胞の培養皿への接着性を、細胞を培養皿に添加してから接着するまでの時間（着定率：Ｐｌａｔｉｎｇ Ｅｆｆｉｃｉｅｎｃｙ：一定時間後に培養皿に定着した細胞数を細胞皿に添加した全細胞数で割った値を百分率で表したもの）により調べた。結果を図１０に示す。図１０から明らかなように、ＳＴＩＰ−１細胞は細胞を培養皿に添加してから５分後には５１．４％の着定率を示し、１時間後には８３．５％、２４時間後には９４．８％と極めて高い数値を示した。
【００１９】
実施例１：ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞を用いた重金属類の細胞毒性試験
（Ａ）ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞に対するカドミウムの毒性評価を以下のようにして行った。
【００２０】
１．方法
細胞を９６穴マイクロプレート内で集密的（ｃｏｎｆｌｕｅｎｔ）な状態になるまで２０℃のＣＯ2インキュベータ内（但し大気雰囲気）で培養した。培養はＬｅｉｂｏｖｉｔ’ｓ Ｌ１５培地（Ｇｉｂｃｏ社製）に１０％ＦＢＳ（Ｇｉｂｃｏ社製の牛胎児血清）とペニシリン（１０ｕｎｉｔ／ｍｌ）とストレプトマイシン（５０μｇ／ｍｌ）を添加した培地を用いて行った。
被検物質（塩化カドミウム：ＣｄＣｌ2・２Ｈ2Ｏ）の０．２Ｍ水溶液を作成し、０．２μｍのフィルタで濾過滅菌してから４℃で保存した。
このカドミウム水溶液から０．０１ｍＭ，０．０２５ｍＭ，０．０５ｍＭ，０．０７５ｍＭ，０．１ｍＭ，０．２５ｍＭ，０．５ｍＭ，０．７５ｍＭ，１ｍＭの９段階の濃度のカドミウム水溶液を作成し、各ウェルに接種した。コントロールには培地のみを加えた。２４時間経過後、アラマーブルー色素を加えてさらに２４時間培養を続けた。
次に、マイクロプレートリーダを用いて２波長（５７０ｎｍと６００ｎｍ）で各ウェルの吸光度を測定した。カドミウム水溶液による細胞代謝の阻害値は、培地のみ加えたウェルをコントロールとして、カドミウム水溶液を接種したウェル（サンプル）のコントロールに対する減少率として求めた。計算式は以下に示す通りである。
【００２１】
阻害値（％）＝１００−（Ａ／Ｂ）×１００
※ Ａ＝サンプルウェル(A570nm-A600nm)-ブランクウェル(A570nm-A600nm)
Ｂ＝コントロールウェル(A570nm-A600nm)-ブランクウェル(A570nm-A600nm)
なお、（Ａ／Ｂ）×１００は細胞の生存率（％）を意味する。
【００２２】
以上の方法によって、５０％の細胞に影響が認められる毒性濃度ＥＣ50値でもってＳＴＩＰ−１細胞とＳＴＩＰ−３細胞に対するカドミウムの毒性評価を行った。
【００２３】
２．結果
ＳＴＩＰ−１細胞に対するカドミウムのＥＣ50値は０．０８９ｍＭであり、ＳＴＩＰ−３細胞に対するカドミウムのＥＣ50値は０．１ｍＭであった（図１１参照）。このＥＣ50値は、ＲＴＧ−２細胞を用いた場合やＦＨＭ細胞を用いた場合のＥＣ50値である０．１８ｍＭ〜０．３８ｍＭよりも低く、ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞は、ＲＴＧ−２細胞やＦＨＭ細胞よりも感受性が高いことがわかった。
【００２４】
（Ｂ）ＳＴＩＰ−１細胞に対するカドミウム以外の８種類の重金属類の毒性評価を（Ａ）に記載の方法と同様にして行った。結果を表１に示す。
【００２５】
【表１】
（Ｃ）実施例１のまとめ
ＳＴＩＰ−１細胞に対する９種類の重金属類の毒性評価を行った結果、トリブチルスズが最も強い毒性を示し、そのＥＣ50値は９×１０-4ｍＭであった。トリブチルスズは他の重金属類よりも約１００倍の低濃度で細胞に対して毒性を示した。また、ＳＴＩＰ−１細胞に対する重金属類を毒性の強い順に並べると、スズ＞水銀＞カドミウム＞亜鉛＝マンガン＞銅＞コバルト＞鉛＞ニッケルのようになり、既に報告されている他の魚類由来の培養細胞に対する毒性と同様の傾向であった。この中で、マンガンはＳＴＩＰ−１細胞に対して他の魚類由来の培養細胞よりも約１０倍以上強い毒性を示した。
【００２７】
実施例２：ＳＴＩＰ−１細胞を用いたフェノール類の細胞毒性試験
ＳＴＩＰ−１細胞に対する１２種類のフェノール類の毒性評価を実施例１の（Ａ）に記載の方法と同様にして行った。結果を表２に示す。
【００２８】
【表２】
表２から明らかなように、２，４，５−トリクロロフェノールがＳＴＩＰ−１細胞に対して最も強い毒性を示した。今回調べた１２種類のフェノール類のＥＣ50値は、他の魚類由来の培養細胞に対するＥＣ50値よりも２倍〜６倍低い値であった。これはＳＴＩＰ−１細胞がフェノール類の毒性を調べる上で有用な細胞であることを示唆するものであった。
【００３０】
【発明の効果】
本発明によれば、新規な魚類株化細胞、即ち、チョウザメ由来の株化細胞を用いた細胞毒性試験方法が提供される。
【図面の簡単な説明】
【図１】 ＳＴＩＰ−１細胞の顕微鏡写真。
【図２】 ＳＴＩＰ−３細胞の顕微鏡写真。
【図３】 各温度におけるＳＴＩＰ−１細胞の増殖曲線。
【図４】 各温度におけるＳＴＩＰ−３細胞の増殖曲線。
【図５】 ＳＴＩＰ−１細胞の増殖に対するＦＢＳの影響を示すグラフ。
【図６】 ＳＴＩＰ−３細胞の増殖に対するＦＢＳの影響を示すグラフ。
【図７】 ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞の染色体数を示すグラフ。
【図８】 ＳＴＩＰ−１細胞の染色体標本。
【図９】 ＳＴＩＰ−３細胞の染色体標本。
【図１０】 ＳＴＩＰ−１細胞の培養皿への接着性を示すグラフ。
【図１１】 ＳＴＩＰ−１細胞とＳＴＩＰ−３細胞に対するカドミウムの毒性を示すグラフ。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for testing cytotoxicity of various test substances such as chemical substances and heavy metals.
[0002]
[Prior art]
So far, toxicity tests on various test substances such as chemical substances and heavy metals (zinc, cadmium, copper, arsenic, cobalt, molybdenum, nickel, lead, selenium, chromium, tin, mercury, etc.) have been conducted using individual animals. However, the method using an animal individual has a problem from the viewpoint of animal welfare as well as the problem that it takes too much time and money. Therefore, in recent years, a toxicity test method using cell lines (cultured cells), that is, a cytotoxicity test method has been studied.
[0003]
[Problems to be solved by the invention]
The toxicity test of the test substance in the aquatic environment is preferably performed using a fish-derived cell line. RTG-2 cells, which are fibroblast cell lines derived from rainbow trout ovary, and epithelial cell lines derived from fathead minnow Toxicity test methods using FHM cells, which are cells, have already been studied. However, these methods are not sufficient in terms of sensitivity and the like, and RTG-2 cells have problems such as a slow growth rate and a narrow growth temperature range, and are not necessarily in terms of convenience. It is not satisfactory.
Accordingly, an object of the present invention is to provide a cytotoxicity test method using a novel fish cell line.
[0004]
[Means for Solving the Problems]
The present invention made in view of the above points, as described in
Also, the method of cytotoxicity testing according to
[0005]
DETAILED DESCRIPTION OF THE INVENTION
In the cytotoxicity test method of the present invention, a sturgeon-derived cell line is used to evaluate the toxicity of a test substance. Examples of the sturgeon include those belonging to the genus Huso and Acipenser, but preferably a female of Beluga ( H. Huso ) belonging to the genus Huso and a male of A. ruthenus belonging to the genus Acipenser. Bester, which is a breeding variety produced from Since Vester is a hybrid, Vestel-derived cell lines are expected to combine the characteristics of both Huso and Acipenser sturgeon cells with respect to sensitivity to various test substances. is there.
[0006]
The sturgeon-derived cell line is established from epithelial cells, for example. The epithelial cell may be from any tissue of the sturgeon, but is preferably an epithelial cell of any tissue of the eyeball, and suitable epithelial cells exist in a non-contact state with the outside world. And iris pigment epithelial cells and retinal pigment epithelial cells. Since these cells originally have no possibility of microbial contamination, if the cells are removed aseptically, microbial contamination of the established cells can be reliably prevented by performing the subsequent operations aseptically. . The established cell line may be established from an epithelial cell derived from the kidney or ovary. In this case, only the epithelial cell is selected from these tissues when establishing the established cell line. It is necessary to recognize that it takes a certain amount of time and labor to separate and culture, and that the possibility of microbial contamination cannot be denied.
[0007]
The established cell line may be established by subculturing the primary cultured cells according to a known method. The medium may be such that fetal bovine serum (FBS) is added to L15 medium usually used for culturing fish cells.
[0008]
Suitable cell lines derived from sturgeon include, for example, STIP-1 cells ( FERM BP-8421 ) and STIP-3 cells ( FERM BP-8422 ), which are cell lines derived from iris pigment epithelial cells of the vestel eyeball. Can be mentioned. Any cell has the advantage that it is more sensitive than RTG-2 cells, FHM cells, and the like. In addition, since any cell can be subcultured without adding an extracellular matrix and can be subcultured 50 times or more, it is excellent in terms of convenience. In particular, STIP-1 cells have a characteristic that the doubling time on the 2nd to 6th days after the start of the culture at 20 ° C. is less than 50 hours, and the settling rate from the addition to the culture dish to the adhesion is 1 Since it has a characteristic that it is 75% or more after time, even if a large number of test sections are set at once, the test can be performed easily and rapidly.
[0009]
The toxicity of the test substance to the sturgeon-derived cell line may be evaluated using, for example, the Alamar Blue assay.
The Alamar Blue assay is one of bioassays developed for measuring cellular metabolism of animal cells and the like. Alamar Blue is a redox dye that needs to be taken into cells for its reduction, and is reduced from oxidized (non-fluorescent, blue) to reduced (fluorescent) by the reductive reaction of the respiratory metabolic system in mitochondria.・ It has the property of changing to red. If the cell metabolism is normal, the reduction reaction proceeds. On the other hand, if the cell metabolism is abnormal, it remains in the oxidized form. Therefore, the color change can be measured to examine the abnormality of the cell metabolism. In addition, cell metabolism may be measured based on fluorescence or light absorption. In this case, fluorescence is monitored at an excitation wavelength of 530 nm to 560 nm and a detection wavelength of 590 nm, and absorbance is monitored at 570 nm and 600 nm.
The Alamar Blue assay method is easy to evaluate because Alamar Blue is water-soluble and does not require extraction or immobilization operations required in bioassays using other dyes such as neutral red. It is desirable in that it can.
This Alamar Blue assay can be performed using, for example, a commercially available kit (manufactured by Biosource).
[0010]
【Example】
Hereinafter, the present invention will be described in detail with reference to reference examples and examples.
[0011]
Reference example: establishment of sturgeon-derived cell lines Isolation of Iris Pigment Epithelial Cells from Vester Eyeballs Eyeballs were extracted from 30 tails of about 15 cm of vestel and sterilized in 70% ethanol. Washed well. Thereafter, the cornea and lens were removed from the eyeball, and the iris was cut out. The iris thus obtained was treated with 0.05% EDTA for about 40 minutes to facilitate separation of iris pigment epithelial cells from connective tissues such as iris stroma and sclera, and then these connective tissues were removed. The separated sheet-like iris pigment epithelial cells were treated with 0.125% trypsin to obtain single-cell cells (primary cells).
[0012]
2. Primary culture The primary cells obtained as described above were added to Leibovit's L15 medium (Gibco) added to a plastic dish (culture dish) with a diameter of 3.5 cm and 10% FBS (Gibco fetal calf serum). ), Penicillin (10 units / ml) and streptomycin (50 μg / ml) were added, and the cells were cultured in a CO 2 incubator (atmosphere) at 20 ° C. Cells having excellent growth ability were selected from the primary cells, and subculture was repeated.
[0013]
3. Subculture When the culture dish is confluent with cells, the cells are detached from the culture dish with a solution containing 0.05% EDTA and 0.125% trypsin, and the cells are collected by centrifugation. Then, it was transferred to another culture dish, and the culture was continued using the above medium. By repeating this, two types of cell lines (STIP-1 cells and STIP-3 cells) that can be cultured for a long period of time were obtained. All cell lines were settled on the culture dish during subculture without adding an extracellular matrix such as collagen to the bottom of the culture dish. The above two types of cell lines are deposited at the National Institute of Advanced Industrial Science and Technology Patent Organism Depositary. The deposit numbers are STIP-1 cells for FERM BP-8421 and STIP-3 cells. FERM BP-8422 . At the time of this patent application, the number of subcultures of the cell line STIP-1 exceeds 140 times, and the number of subcultures of the cell line STIP-3 exceeds 80 times.
[0014]
4). Characteristics of STIP-1 and STIP-3 cells (cell morphology)
STIP-1 cells were elongate cells but epithelial cells (see FIG. 1: micrograph at 100 × magnification on
[0015]
(Temperature characteristics of cells)
As shown in FIG. 3, STIP-1 cells showed good growth properties over a wide temperature range of 15 ° C. to 32 ° C., and were particularly excellent at 20 ° C. The doubling time (exponential cell growth time) of the second to sixth days after the start of culture at 20 ° C. is 38.9 hours, which is about twice that of RTG-2 cells. Has a growth rate. On the other hand, as shown in FIG. 4, STIP-3 cells showed good growth at 15 to 20 ° C., but the growth was inhibited at 30 ° C. or higher. The doubling time of the cells on the 2nd to 6th days after the start of culture at 20 ° C. was 74.9 hours.
[0016]
(Influence of FBS on cell proliferation)
As shown in FIG. 5, the concentration of FBS added to the L15 medium necessary for growing STIP-1 cells was sufficient to be 4%. As shown in FIG. 6, the concentration of FBS added to the L15 medium necessary for growing STIP-3 cells was also 4%. Therefore, since a small amount of FBS is required to grow these cell lines, it was found that mass culture of these cell lines is economically advantageous.
[0017]
(Number of cell chromosomes)
The number of chromosomes of STIP-1 cells and STIP-3 cells was examined by a conventional method in which cells in the logarithmic growth phase on the 6th day of culture were treated with colchicine for the 80th subcultured cells. Specifically, colchicine was added to the cells so that the final concentration was 0.20 μg / ml. After culturing for 18 hours, the medium was removed, and then the cells were washed with PBS (−). Next, the cells were detached from the culture dish with a solution containing 0.05% EDTA and 0.125% trypsin, and the cells were collected by centrifugation. Hypotonic treatment was performed by adding 0.075 M KCl to the collected cells and allowing to stand at room temperature for 20 minutes. The hypotonic cell suspension was fixed in ice for 20 minutes using Carnoy's solution, and then used as a chromosome specimen by the flame dry method. This was stained with Giemsa and the chromosomes were counted with a microscope (magnification 1000 times). As a result, the number of chromosomes of STIP-1 cells is 2n = 166 ± 7.6, the number of chromosomes of STIP-3 cells is 2n = 121 ± 6.1, and all cells have the number of chromosomes of
[0018]
(Cell adhesion)
The adhesion time of STIP-1 cells to the culture dish was the time from the addition of the cells to the culture dish until the cells were adhered (Settling Efficiency: Placing Efficiency: the number of cells fixed on the culture dish after a certain time was added to the cell dish. The value divided by the total number of cells was expressed as a percentage). The results are shown in FIG. As is clear from FIG. 10, STIP-1 cells showed a 51.4
[0019]
Example 1: Cytotoxicity test of heavy metals using STIP-1 cells and STIP-3 cells (A) Toxicity evaluation of cadmium to STIP-1 cells and STIP-3 cells was performed as follows.
[0020]
1. Method Cells were cultured in a 96-well microplate in a 20 ° C. CO 2 incubator (atmosphere) until confluent. The culture was performed using Leibovit's L15 medium (Gibco) supplemented with 10% FBS (Gibco fetal bovine serum), penicillin (10 units / ml) and streptomycin (50 μg / ml).
A 0.2M aqueous solution of a test substance (cadmium chloride: CdCl 2 .2H 2 O) was prepared, sterilized by filtration through a 0.2 μm filter, and stored at 4 ° C.
From this cadmium aqueous solution, nine levels of cadmium aqueous solutions of 0.01 mM, 0.025 mM, 0.05 mM, 0.075 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, and 1 mM were prepared. Wells were inoculated. Only the medium was added to the control. After 24 hours, Alamar Blue dye was added and the culture was continued for another 24 hours.
Next, the absorbance of each well was measured at two wavelengths (570 nm and 600 nm) using a microplate reader. The inhibition value of the cell metabolism by the cadmium aqueous solution was determined as a reduction rate with respect to the control of the well (sample) inoculated with the cadmium aqueous solution, with the well containing only the medium added as a control. The calculation formula is as follows.
[0021]
Inhibition value (%) = 100− (A / B) × 100
* A = Sample well (A570nm-A600nm)-Blank well (A570nm-A600nm)
B = Control well (A570nm-A600nm) -Blank well (A570nm-A600nm)
Note that (A / B) × 100 means cell survival rate (%).
[0022]
By the above method, toxicity evaluation of cadmium to STIP-1 cells and STIP-3 cells was performed with a toxic concentration EC 50 value at which 50% of the cells were affected.
[0023]
2. Results The EC 50 value of cadmium for STIP-1 cells was 0.089 mM, and the EC 50 value of cadmium for STIP-3 cells was 0.1 mM (see FIG. 11). This EC 50 value is lower than the EC 50 value of 0.18 mM to 0.38 mM when RTG-2 cells or FHM cells are used, and STIP-1 cells and STIP-3 cells -2 cells and FHM cells were found to be more sensitive.
[0024]
(B) Toxicity evaluation of 8 types of heavy metals other than cadmium to STIP-1 cells was performed in the same manner as described in (A). The results are shown in Table 1.
[0025]
[Table 1]
(C) Summary of Example 1 As a result of the toxicity evaluation of nine kinds of heavy metals on STIP-1 cells, tributyltin showed the strongest toxicity, and its EC 50 value was 9 × 10 −4 mM. Tributyltin was toxic to cells at a concentration about 100 times lower than other heavy metals. Moreover, when the heavy metals for STIP-1 cells are arranged in the order of strong toxicity, it becomes tin>mercury>cadmium> zinc = manganese>copper>cobalt>lead> nickel, and cultures derived from other fish already reported. The tendency was similar to the toxicity to cells. Among these, manganese was about 10 times more toxic to STIP-1 cells than cultured cells derived from other fish.
[0027]
Example 2: Cytotoxicity test of phenols using STIP-1 cells Toxicity evaluation of 12 types of phenols on STIP-1 cells was performed in the same manner as in the method described in (A) of Example 1. The results are shown in Table 2.
[0028]
[Table 2]
As is clear from Table 2, 2,4,5-trichlorophenol showed the strongest toxicity to STIP-1 cells. The EC 50 values of the 12 types of phenols examined this time were 2 to 6 times lower than the EC 50 values of cultured cells derived from other fish. This suggested that STIP-1 cells are useful for examining the toxicity of phenols.
[0030]
【The invention's effect】
According to the present invention, a cytotoxicity test method using a novel fish cell line, that is, a cell line derived from sturgeon is provided.
[Brief description of the drawings]
FIG. 1 is a micrograph of STIP-1 cells.
FIG. 2 is a micrograph of STIP-3 cells.
FIG. 3 is a growth curve of STIP-1 cells at each temperature.
FIG. 4 is a growth curve of STIP-3 cells at each temperature.
FIG. 5 is a graph showing the effect of FBS on the proliferation of STIP-1 cells.
FIG. 6 is a graph showing the effect of FBS on the growth of STIP-3 cells.
FIG. 7 is a graph showing the number of chromosomes in STIP-1 cells and STIP-3 cells.
FIG. 8 Chromosome specimen of STIP-1 cells.
FIG. 9 Chromosome specimen of STIP-3 cells.
FIG. 10 is a graph showing adhesion of STIP-1 cells to a culture dish.
FIG. 11 is a graph showing the toxicity of cadmium to STIP-1 cells and STIP-3 cells.
Claims (2)
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| JP2003160007A JP4452458B2 (en) | 2002-06-28 | 2003-06-04 | Cytotoxicity test method |
| US10/606,803 US7132230B2 (en) | 2002-06-28 | 2003-06-27 | Cytotoxic assay and new established cell line of sturgeon origin |
| US11/285,305 US7217564B2 (en) | 2002-06-28 | 2005-11-23 | Cytotoxic assay and new established cell line of sturgeon origin |
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