JP3994159B2 - Animal-derived component-free culture medium and cell line production method using the same - Google Patents

Description

【００３２】
そこで、約３０代継代目までは、その増殖が不安定であることを考慮して、４〜７日間隔で無血清培養液を新鮮な無血清培養液に交換し、細胞の継代間隔は、その増殖性の相違によって２１〜２８日とした。
その結果、良好な増殖を示す細胞が得られ、さらに約５０代継代を重ねたのち、安定した増殖を示すようになり、約１１０代継代を経過したのちも安定かつ良好な増殖性を示し、完全無血清培養液に順化した細胞を得た。
【００３３】
次に、上記の組成からなる無血清培養液において、Tryptose Phosphate Brothの添加量を順次減量した他は同様にして調製した培養液を用いて、同様に継代培養を行った。その結果、Tryptose Phosphate Brothの添加量を約０．１８ｇ／Ｌまで減少させても、ＭＤＢＫ細胞の増殖性を維持することができたが、Tryptose Phosphate Brothを全く添加しない場合は、細胞の増殖は甚だ減弱した。
【００３４】
そこで、上記の組成からなる無血清培養液において、Tryptose Phosphate Brothを段階的に減少させ、約１２０代継代目からは下記の組成からなる完全動物由来成分無添加培養液に移行して継代培養を続けた。継代４日目に培養液を新鮮な培養液に交換し、細胞の継代間隔は、その増殖性の相違をみて７〜１０日とした。
【００３５】

【００３６】
その結果、安定した増殖を示す細胞が得られた。さらに、約１５代継代を経過したものも安定かつ良好な増殖性を示していた。そこで、この細胞を動物由来成分無添加培養液順化細胞株として、ＭＤＢＫ−ＮＳＴ細胞と命名した。
【００３７】
実施例２（ＭＤＢＫ−ＮＳＴ細胞の増殖性および増殖形態）
実施例１で得たＭＤＢＫ−ＮＳＴ細胞の前記動物由来成分無添加培養液における増殖性を調べた。すなわち、ＭＤＢＫ−ＮＳＴ細胞のまきこみ数を約２００万個／２５ｃｍ^２ボトルとして上記の組成からなる完全動物由来成分無添加培養液で７日間培養した。その結果得られた動物由来成分無添加培養液におけるＭＤＢＫ−ＮＳＴ細胞の増殖曲線を、図１に示す。尚、図１中の▲は、培養液を７日間通して用いた場合の結果を、●は培養液を４日目に更新した場合の結果を示す。
図１から明らかなとおり、培養液を７日間通して用いた場合、３日後には細胞数は約６００万個／２５ｃｍ^２ボトルに増加したが、細胞の増殖は５日目から減少する傾向にあった。しかしながら、一方、培養液を３〜４日後に交換した場合、細胞の増殖は経時的に増加し、７日後には約８００万個／２５ｃｍ^２ボトルに達した。
この結果から、完全動物由来成分無添加培養液を用いてＭＤＢＫ−ＮＳＴ細胞を培養するにあたり、培養液を３〜４日後に更新することにより、細胞の増殖性が良好となることが明らかとなった。
さらに、ＭＤＢＫ−ＮＳＴ細胞の増殖形態の経時的な変化を観察したところ、ＭＤＢＫ−ＮＳＴ細胞は約４日目に単層を形成した。約５〜６日後に細胞室内に小空胞が観察されたが、４日目の培養液の更新により、小空胞の出現は消失した。
【００３８】
実施例３（ＭＤＢＫ−ＮＳＴ細胞の性質）
（１）ＭＤＢＫ−ＮＳＴ細胞の染色体数
ＭＤＢＫ−ＮＳＴ細胞の染色体数の分布率を調べた。結果を図２に示す。
図２から明らかなとおり、ＭＤＢＫ−ＮＳＴ細胞の染色体数は、４３（２ｎ）を中心に分布していることが解明された。牛の正常染色体数は６０（２ｎ）、親細胞であるＭＤＢＫ細胞のそれは５１（２ｎ）であることから、ＭＤＢＫ−ＮＳＴ細胞においては、多数の染色体の脱落が生じているものと考えられる。
【００３９】
（２）ＭＤＢＫ−ＮＳＴ細胞の酵素活性等
また、組織化学的検査によって、ＭＤＢＫ−ＮＳＴ細胞の酵素活性等について調べた。
【００４０】
▲１▼ アルカリ性フォスファターゼ活性（Burston法）
培養細胞をアセトン固定した後に、Vectore Red Alkaline Phosphatase Substrate Kit I（VECTORE LABORATORIES）を用いて検出した。
その結果、アルカリ性フォスファターゼ活性が検出され、ＭＤＢＫ−ＮＳＴ細胞と同様であった。
【００４１】
▲２▼ 上皮系細胞骨格であるサイトケラチン
培養細胞をアセトン固定した後に、マウスモノクローナル抗体：Mab to Cytokeratin 5/8 Pan Epitherial Cytokeratin（Progen）を一次抗体とし、ヒストファインSAB-PO（M）キット（ニチレイ）を用いたSAB法で検出した。
その結果、ＭＤＢＫ−ＮＳＴ細胞にはサイトケラチンが検出され、親細胞であるＭＤＢＫ細胞と同様であった。
【００４２】
▲３▼ γ−グルタミルトランスペプチターゼ（γ−ＧＴＰ）活性
培養細胞をアセトン固定した後に、N-γ-Glutamyl-α-NaphtylamideとFastgarnet GBC塩を含有する基質液に浸漬し、０．１Ｍ ＣｕＳＯ_４液で処理して検出した。
その結果、ＭＤＢＫ−ＮＳＴ細胞には、γ−グルタミルトランスペプチターゼ活性が検出されず、親細胞であるＭＤＢＫ細胞と同様であった。
【００４３】
（３）ＭＤＢＫ−ＮＳＴ細胞の病原体による汚染の有無
さらに、ＭＤＢＫ−ＮＳＴ細胞の病原体の汚染について調べた。
▲１▼ ペスチウイルスの汚染
ペスチウイルスは、牛由来培養細胞に高頻度に迷入している。そこで、ペスチウイルスの遺伝子を増幅する逆転写酵素−ポリメラーゼ連鎖反応法およびウイルス非構造蛋白質ＮＳ３を検出する間接蛍光抗体法を行ってペスチウイルスの汚染を調べた。
【００４４】
逆転写酵素−ポリメラーゼ連鎖反応法は、Vileekら（Vileek，S.，Herring，A．J.，Herring，J．A.，Nettleton，P．F.，Lowings，J．P.，Paton，D．J.，1994．Pestiviruses isolated from pigs，cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis．Arch．Virol．136，309-323）のプライマーを用い、寶酒造のRNA PCR Kit（AMV）Ver．2.1を使用して実施した。
ウイルス非構造蛋白質ＮＳ３を検出する間接蛍光抗体法は、アセトン固定した培養細胞に、ペスチウイルスのＮＳ３に対するモノクローナル抗体JCU/BVD/CF10（JCU TropBio）を一次抗体として反応させ、ついで、FITC標識抗マウスIgG抗体を二次抗体として反応させ、蛍光顕微鏡で観察し、特異蛍光を示す細胞をペスチウイルス感染細胞と認定した。
その結果、ペスチウイルスの汚染は否定された。
【００４５】
▲２▼ マイコプラズマの汚染
Kojimaらの報告（Akemi Kojima，Toshio Takahashi，Mayumi Kijima，Yasuaki Ogikubo，Makoto Nishimura，Shinzo Nishimura，Ryo Harasawa and Yutaka Tamura，1997，Detection of Mycoplasma in Avian Live Virus Vaccines by Polymerase Chain Reaction．Biologicals 25，365-371）に従い、マイコプラズマの遺伝子を増幅するポリメラーゼ連鎖反応法を行った。尚、ポリメラーゼ連鎖反応法には、タカラ酒造のＤＮＡポリメラーゼEx Taqを用いた。
その結果マイコプラズマの汚染は否定された。
【００４６】
▲３▼ レトロウイルスの汚染
レトロウイルスは、培養細胞に高頻度に迷入している。そこで、実施例１と同様に、完全動物由来成分無添加培養液を用いてＭＤＢＫ−ＮＳＴ細胞を培養し、培養２日目および９日目の培養細胞上清を高速遠心法により濃縮した試料について、「逆転写酵素活性−非放射性」（ロシュ）のキットを用いて、レトロウイルスが有する逆転写酵素活性について調べた。
また、レトロウイルスが迷入している豚由来ＰＫ−１５細胞を陽性対照試料、逆転写酵素０．０６２５ｎｇを陽性対照、逆転写酵素０ｇを陰性対照として、同様に逆転写酵素活性の有無について調べた。結果を図３に示す。
図３から明らかなように、ＭＤＢＫ−ＮＳＴ細胞の試料に、いずれも逆転写酵素活性が認められない。このことから、ＭＤＢＫ−ＮＳＴ細胞には、レトロウイルスの迷入がないことが明らかである。
【００４７】
実施例４（ＭＤＢＫ−ＮＳＴ細胞におけるＢＶＤＶの増殖性）
ＭＤＢＫ−ＮＳＴ細胞における牛ウイルス性下痢ウイルス（ＢＶＤＶ）のＮｏｓｅ株の増殖性を調べた。
すなわち、培養フラスコに培養したＭＤＢＫ−ＮＳＴ細胞に、ＢＶＤＶのＮｏｓｅ株をＭＯＩ（multiplicity of infection）＝０．０１で接種した。ウイルスを細胞に１時間吸着させた後に、ウイルス液を取り除き、新鮮な動物由来成分無添加培養液を添加して３７℃に静置した。ウイルスを接種してから経時的に培養上清液および細胞を改修し、それらに含まれるウイルス量をウシ精巣細胞を用いて測定した。尚、ウイルス量の測定には、一般的な１０倍階段希釈法を用い、細胞変性効果（ＣＰＥ）を示す細胞をＢＶＤＶのＮｏｓｅ株が感染したとみなして、Kerburの算出法に従ってウイルス量を算出した。
【００４８】
その結果、ウイルスを接種した後５〜７日で、培養上清に放出される感染ウイルスが１０^３〜１０^４ＴＣＩＤ_５０／ｍｌ、培養細胞内に存在する感染ウイルスが１０^７ＴＣＩＤ_５０／ｍｌのレベルで増殖することが解明された。なお、培養液に牛血清を５％の割合で添加すると、ウイルスを接種した後５日で、培養上清に放出される感染ウイルスは、１０^７ＴＣＩＤ_５０／ｍｌレベルに達することが明らかとなった。
【００４９】
実施例５（ＭＤＢＫ−ＮＳＴ細胞におけるＢＶＤＶの感受性）
ＭＤＢＫ−ＮＳＴ細胞における各種ウイルスの感受性を調べた。即ち、ＭＤＢＫ−ＮＳＴ細胞とＭＤＢＫ細胞でウイルス液の感染価をそれぞれ測定し、その差を見ることによりウイルスの細胞への感染性の程度を評価した。
【００５０】
▲１▼ＢＶＤＶの感受性
各ＢＶＤＶウイルス液を１０倍階段希釈し、各希釈培養液に浮遊させたＭＤＢＫ−ＮＳＴ細胞およびＭＤＢＫ細胞のそれぞれを培養プレートに同時に入れて、３７℃、５％二酸化炭素の条件下で７日間静置培養した。細胞病原性ＢＶＤＶ（Ｎｏｓｅ株、Ｏｓｌｏｓｓ株、ＫＺ９１株、Ｓｉｎｇｅｒ株および５９１２株）については、ＣＰＥをＢＶＤＶ陽性の指標とし、非細胞病原性ＢＶＤＶ（Ｎｏ．１２−４２株、関東東山株、Ｎｅｗ Ｙｏｒｋ−１株、Ｉｎｄｉａｎａ−４６株およびＧＢＫ株）については、抗ＮＳ３モノクローナル抗体を用いたPeroxidase Linked Assay法を用い、Kaerberの算出法に従ってウイルス感染価を評価した。
結果を図４に示す。尚、図４中、黒色部はＭＤＢＫ−ＮＳＴ細胞における感受性を、白色部はＭＤＢＫ細胞における感受性を示す。
【００５１】
図４から明らかなとおり、ＭＤＢＫ−ＮＳＴ細胞は、細胞病原性株および非細胞病原性株のいずれにおいても、ウイルスに対する感受性が高く、親細胞であるＭＤＢＫ細胞と同等の感受性を示すことが確認された。
【００５２】
▲２▼ウシ伝染性鼻気管炎、ウシパラインフルエンザ、ウシＲＳウイルス感染症およびオーエスキー病のワクチン株または病原性株の感受性
ウシ伝染性鼻気管炎ウイルス（ＩＢＲＶ）、ウシパラインフルエンザウイルス（ＰＩ３Ｖ）、ウシＲＳウイルス感染症ウイルス（ＢＲＳＶ）およびオーエスキー病ウイルス（ＡＤＶ）の各ウイルス液を１０倍階段希釈し、各希釈培養液に浮遊させたＭＤＢＫ−ＮＳＴ細胞およびＭＤＢＫ細胞のそれぞれを同時に培養プレートもしくは培養チューブに同時に入れて、３７℃、５％二酸化炭素の条件下で７日間静置培養した。なお、ウシＲＳウイルス感染症ウイルス（ＢＲＳＶ）については、３４℃で１４日間回転培養した。培養後にＣＰＥをウイルス陽性の指標とし、Kaerberの算出法に従ってウイルス感染価を評価した。
結果を図５に示す。尚、図５中、黒色部はＭＤＢＫ−ＮＳＴ細胞における感受性を、白色部はＭＤＢＫ細胞における感受性を示す。
【００５３】
図５から明らかな通り、ＭＤＢＫ−ＮＳＴ細胞は、いずれのウイルスに対しても親細胞であるＭＤＢＫ細胞と同等の感受性を示すことが確認された。
このことから、ＭＤＢＫ−ＮＳＴ細胞がワクチンおよび診断抗原製造用の細胞として有用であること考えられる。さらに、当該細胞に感受性のあるウイルスによる疾病のワクチンの製造用細胞としても有用と考えられる。
【００５４】
【発明の効果】
上記の課題を解決すべく検討を重ねた。すなわち、既知の無血清培養液成分から、病原体の迷入の原因となりうる血清やTryptose Phosphate Broth等の動物由来成分を削除し、緩衝剤の添加を検討した。その結果、基礎培養液としてのEagle MEMと、Bacto-PeptoneおよびＢＥＳとを、それぞれ所定量含有する動物由来成分無添加培養液は、病原体の迷入の危険がなく、従来動物由来成分を要求するとされていた細胞系の作出に利用できることを見出した。
また、この動物由来成分無添加培養液により、病原体の迷入がない細胞系を作出できることを見出した。
更に、この病原体の迷入がない細胞系は、上記の動物由来成分が削除された動物由来成分無添加培養液で生育可能なので、疾病の診断用抗原、ワクチン、医薬品等の生物学的製剤の製造に用いることができることを見出した。
本発明の動物由来成分無添加培養液は、病原体の迷入の原因となりうる動物由来成分を含まない上に、従来動物由来成分を要求するとされていた細胞系の作出に利用できる。
また、本発明の動物由来成分無添加培養液によれば、哺乳類由来細胞から既知の病原体の迷入の恐れのない清浄な細胞系を作出することが可能となり、その利用性を広げることができる。
また、本発明の作出方法によれば、病原体の迷入がない細胞系を効率よく得ることができる。特に、反芻動物由来の細胞を対象とした場合には、近年のＢＳＥ問題への懸念も回避できる。
更に、本発明の作出方法により得られる病原体の迷入がない細胞系は、上記の動物由来成分が削除された動物由来成分無添加培養液で生育可能なので、疾病の診断用抗原、ワクチン、医薬品等の生物学的製剤の製造に用いることにより、ウイルス検査は勿論のこと、抗体検査の技術的改善、生物学的製剤の製造の均一化、安全性の確保、省力化、大量生産による低コスト化等に飛躍的な技術を提供することができる。
【００５５】
特に、本発明の作出方法により得られるＭＤＢＫ−ＮＳＴ細胞は、レトロウイルス、ペスチウイルス、マイコプラズマ等の病原体の迷入がなく、しかも、上記の動物由来成分が削除された動物由来成分無添加培養液で生育可能である。従って、例えば、ＭＤＢＫ−ＮＳＴ細胞を用いたウイルス検査法や抗体検査法は、動物由来成分無添加培養液を用いて実施できるため、高頻度に検出される牛ウイルス性下痢ウイルス（ＢＶＤＶ）に対する抗体やその他の牛に感染する各種ウイルスに対する抗体を事前に検査する必要がないといったメリットを有する。
また、牛ウイルス性下痢・粘膜病、牛伝染性鼻気管炎、牛パラインフルエンザ、牛ＲＳウイルス感染症等のウシの疾病や、オーエスキー病等のウシ以外の動物の疾病の診断用抗原、ワクチン、医薬品など生物学的製剤の製造に使用することにより、製品の均一化、安全性の確保、省力化、大量生産による製造コストの低減を図ることができる。
【図面の簡単な説明】
【図１】 動物由来成分無添加培養液におけるＭＤＢＫ−ＮＳＴ細胞の増殖曲線である。
【符号の説明】
図１中の▲は、培養液を７日間通して用いた場合の結果を、●は培養液を４日目に更新した場合の結果を示す。
【図２】 ＭＤＢＫ−ＮＳＴ細胞の染色体数の分布率を示す図である。
【図３】 ＭＤＢＫ−ＮＳＴ細胞の培養上清の逆転写酵素活性を示す図である。
【図４】 ＢＶＤＶの感受性を示す図である。
【図５】 ＢＶＤＶ以外の各種ウイルスの感受性を示す図である。
【符号の説明】
図４および図５中、黒色部はＭＤＢＫ−ＮＳＴ細胞における感受性を、白色部はＭＤＢＫ細胞における感受性を示す。[0001]
BACKGROUND OF THE INVENTION
  The present invention relates to an animal-derived component-free culture solution and a cell line production method using the same, and more specifically, an animal-derived component-free culture solution for mammalian cells that does not contain animal-derived components, and the animal-derived component No added cultureUsing animalsA method to create a cell line that can grow without the addition of derived components,The cell lineThe present invention relates to a method for producing an antigen for diagnosis of a disease, a vaccine, and a pharmaceutical product.
[0002]
[Prior art]
In culturing and examining viruses, components extracted and extracted from animals, that is, animal-derived components are frequently used. For example, primary culture cells and cell lines derived from animals are necessary as one of the materials for producing biological preparations such as diagnostic antigens, vaccines, and pharmaceuticals for diseases, and in order to prepare such cells In addition, a culture solution to which serum is added is essential, and addition of Tryptose Phosphate Broth and the like has also been performed on this culture solution.
However, animal-derived components such as tissue cells, serum, and Tryptose Phosphate Broth of these animals have the risk of entering a known or unknown pathogen and the presence of antibodies. For example, in kidney-derived cells, pestiviruses, mycoplasmas, and the like can be considered (see Non-Patent Document 1).
Therefore, when producing biological preparations such as diagnostic antigens, vaccines, and pharmaceuticals during virus culture and testing, sufficient testing is required before use to avoid the risk of pathogen invasion. Yes.
[0003]
In addition, animal-derived components such as serum and Tryptose Phosphate Broth used in the culture solution, especially those derived from ruminants such as bovine serum, are affected by the recent bovine spongiform encephalopathy (BSE) problem. ing. In other words, when animal-derived components derived from ruminants such as bovine serum are used in the production of vaccines, it is necessary to obtain one that has been proven to be safe and without the risk of pathogen invasion. It is forced.
Conventionally, serum-free culture solutions to which various growth factors and the like are added in place of bovine serum are commercially available according to the purpose of many biological studies. Such a serum-free culture solution avoids the risk of invading pathogens derived from bovine serum, but is not practical for the production of large quantities of vaccines and diagnostic antigens because of its high selling price.
[0004]
On the other hand, cultured cells such as porcine renal tubular epithelium-derived cell lines can be grown in known serum-free media, but as described above, such known serum-free media include Tryptose Phosphate. Due to the addition of animal-derived components such as Broth, the risk of pathogen invasion cannot be completely ruled out.
[0005]
As described in Non-patent Document 2, bovine kidney epithelium-derived cell lines (MDBK cells) formed a cell monolayer in a plastic culture bottle, and the invasion of bovine viral diarrhea virus (BVDV) was denied. It is a known cell having the characteristics of MDBK cells are well-established cultured cells that are easy to handle, and are sensitive to many viruses, and thus have been conventionally used for disease diagnosis and production of biological products such as vaccines.
When this MDBK cell is subcultured as a parent cell, it was conventionally used to add Tryptose Phosphate Broth to Eagle MEM as a basic culture medium, and further with bovine serum, so that retrovirus, pestivirus, mycoplasma, etc. There was a problem that the risk of invasion of pathogens could not be completely denied.
[0006]
[Non-Patent Document 1]
Ryo Harazawa, Hiroshi Mizusawa, Masao Takeuchi: “Simple detection of“ contamination ”in animal cell culture”, Protein / Nucleic Acid / Enzyme, Vol.40 No.15, p2361-2368 (1995)
[Non-Patent Document 2]
Madin, S.H. and Darby, N.B. 1958. Established kidney cell lines of normal adult bovine. Proc. Soc. Exp. Biol. Med. 98, p574
[0007]
[Problems to be solved by the invention]
The object of the present invention is to provide a low-cost animal-derived component-free culture solution that does not contain animal-derived components such as serum and Tryptose Phosphate Broth, and other animal-derived auxiliary components. The purpose is to establish a method for differentiating mammalian cells using an additive-free culture solution to create a cell line free from pathogen invasion.
[0008]
[Means for Solving the Problems]
The study was repeated to solve the above problems. That is, in order to solve the adverse effects on the culture solution and cultured cells caused by the deletion of serum and Tryptose Phosphate Broth and other animal-derived components that may cause pathogen invasion from known serum-free culture solution components The addition of buffer was considered. As a result, an animal-derived component-free culture solution containing a predetermined amount of Eagle MEM, Bacto-Peptone and BES as a basic culture solution has no risk of pathogen invasion and requires a conventional animal-derived component. It was found that it can be used for the production of cell lines.
In addition, the present inventors have found that a cell line free from pathogen invasion can be produced using this animal-derived component-free culture solution.
Furthermore, since the cell line without the pathogen invasion can be grown in an animal-derived component-free culture solution in which the above-mentioned animal-derived components are deleted, in addition to virus culture and testing, antigens for diagnosis of diseases, vaccines, pharmaceuticals It has been found that it can be used for the production of biological products such as
[0009]
In addition, the present inventors have succeeded in producing cells that can be grown in the above-described culture solution without addition of animal origin using MDBK cells as a material. The produced MDBK-NST cells not only exhibit the same properties as the parental MDBK cells, but also the invasion of various pathogens such as retroviruses, pestiviruses and mycoplasmas in addition to BVDV. It has also been found that it can be suitably used for the production of biological preparations such as antigens for diagnosis of diseases, vaccines, and pharmaceuticals, in addition to culture and testing.
The present invention has been completed based on such findings.
[0010]
  The present invention described in claim 1 includes Eagle's Minimum Essential Medium [Eagle MEM] as a basic culture medium, Bacto-Peptone 3.00 to 12.00 g / L, and N, N-Bis (2-hydroxyetyl) -2- aminoethanesulfonic acid [BES] 1.00-5.00 g / LConsist ofIt is characterized byBovine kidney epitheliumIt is an animal-derived component-free culture solution for cells.
  Claim 2The invention described isBovine kidney epitheliumCells were treated with Eagle's Minimum Essential Medium [Eagle MEM] as the basic culture medium, Bacto-Peptone 3.00 to 12.00 g / L and N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid [BES] 1 After acclimatization to a serum-free culture solution containing 0.000-5.00 g / L and an animal-derived component and not containing serum, the content of the animal-derived component in the serum-free culture solution is sequentially increased Lower the culture and finallyClaim 1Culturing using the described culture medium,Claim 1Method for producing a cell line capable of growing in the described culture medium and free of pathogen invasionIt is.
[0011]
  Claim 3The invention described isClaim 2Using the cell line obtained by the production method described in
It is a method for producing a disease diagnosis antigen, vaccine or pharmaceutical product.
[0012]
DETAILED DESCRIPTION OF THE INVENTION
  Hereinafter, the present invention will be described in detail.
  The present invention according to claim 1Bovine kidney epitheliumThe animal-free component culture medium for cells is Eagle's Minimum Essential Medium [Eagle MEM] as a basic culture medium, Bacto-Peptone 3.00 to 12.00 g / L, and N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid [BES] 1.00-5.00 g / LConsist ofIt is characterized by this.
[0013]
That is, first, the culture solution of the present invention according to claim 1 is characterized in that it contains a predetermined amount of Eagle MEM, Bacto-Peptone, and BES as a basic culture solution.
Here, Eagle MEM as the basic culture medium refers to Eagle MEM and its modified culture medium. Examples of the modified culture medium include Dulbecco's Modified Eagle Medium (DMEM), DMEM / Ham's F-12 medium, Ham's F-12 medium, and Ham's F-10 medium. Eagle MEM can be prepared by mixing a predetermined amount of a predetermined component, but it is easy to use various commercially available products (for example, manufactured by Nissui Pharmaceutical).
[0014]
The culture solution of the present invention according to claim 1 needs to contain such Eagle MEM. The content of Eagle MEM can be appropriately adjusted according to the growth status of mammalian cells, but is usually from 5.00 to 15.00 g / L, preferably from 7.00 to 13.00 g / L. Preferably it is 9.00 to 10.00 g / L.
[0015]
Moreover, in the culture solution of this invention which concerns on Claim 1, Bacto-Peptone needs to contain 3.00-12.00g / L, Preferably it is 5.00-10.00g / L. When it is less than 3.00 g / L or more than 12.00 g / L, mammalian-derived cells cannot grow and the object of the present invention cannot be achieved.
[0016]
Furthermore, in the culture solution of the present invention according to claim 1, such BES [N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid] is 1.00 to 5.00 g / L, preferably 1 It is necessary to contain from .50 to 4.00 g / L, particularly preferably from 2.00 to 3.00 g / L. When the amount is less than 1.00 g / L or exceeds 5.00 g / L, mammalian-derived cells cannot grow and the object of the present invention cannot be achieved.
[0017]
  The culture solution of the present invention according to claim 1 is characterized by not containing any animal-derived component.
  Here, the animal-derived component is a component / extract extracted / extracted from an animal, for example, serum such as bovine serum (FBS: Fetal Bovine Serum), plasma, tissue extract, bovine amniotic fluid, bovine Examples include fetal extracts (Bovine Embryo Extract: BEE), chicken fetal extracts (Chicken Embryo Extract: CEE) and other amniotic fluid, ascites and other biological solutions, Tryptose Phosphate Broth (for example, manufactured by DIFCO), and lactose. In the case of biological solutions, in the present invention, ruminant-derived ones can be achieved in that the object of the present invention can be achieved, which is to eliminate concerns about bovine spongiform encephalopathy (BSE) problem.Does not containIt is preferable.
  The culture solution of the present invention according to claim 1 may contain other components such as normal MEM, Bacto-Peptone, and BES, as long as they contain a predetermined amount and do not contain animal-derived components. It may contain an antibiotic (penicillin, streptomycin, kanamycin, etc.), an antifungal agent (amphotericin B, etc.), etc., which are components added to the culture solution.
[0018]
  The culture solution of the present invention according to claim 1 comprises:Bovine kidney epitheliumUsed for cell cultureIt is done.
  like thisBovine kidney epitheliumAs cells, MDBK used in the examplesCells, etc.Of established cell lines.
[0019]
  Among these cell lines, MDBK cells are stored as ATCC No. CCL-22 in the American Type Culture Collection in the US and are available upon application.it can.
[0020]
  Since the culture solution of the present invention according to claim 1 does not contain any animal-derived components, there is no risk of pathogen invasion, and therefore it can be used to create a cell line free from pathogen invasion. Providing a method for creating cell lines that are free of pathogen invasionClaim 2It is this invention concerning.
  That is,Claim 2The present invention according toBovine kidney epitheliumCells were treated with Eagle's Minimum Essential Medium [Eagle MEM] as the basic culture medium, Bacto-Peptone 3.00 to 12.00 g / L and N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid [BES] 1 After acclimatization to a serum-free culture solution containing 0.000-5.00 g / L and an animal-derived component and not containing serum, the content of the animal-derived component in the serum-free culture solution is sequentially increased Lower the culture and finallyClaim 1Culturing using the described culture medium,Claim 1The present invention provides a method for producing a cell line that can be grown in the culture medium described above and that is free from pathogen invasion.
[0021]
  Claim 2The production method of the present invention according toBovine kidney epitheliumThe cells were acclimated to a serum-free culture solution containing a predetermined amount of Eagle MEM, Bacto-Peptone, BES, and an animal-derived component as a basic culture solution, but no serum, and then serum-free. Cultivate by lowering the content of animal-derived components in the culture solution in order, and finallyClaim 1It culture | cultivates using the culture solution of description.
[0022]
  Claim 2In the production method according to the present invention,Bovine kidney epitheliumCells are acclimated to a predetermined serum-free culture. Here, the serum-free culture medium refers to a culture medium that contains Eagle MEM, Bacto-Peptone, BES, and animal-derived components as a basic culture medium in a predetermined amount, but does not contain serum.
  That is, the serum-free medium isClaim 1Compared to the animal-derived component-free culture solution of the present invention, the difference is that the animal-derived component is contained. The content of animal-derived components isBovine kidney epitheliumIt can be suitably prepared according to the growth state of the cells. For example, in the case of Tryptose Phosphate Broth, it can be 1.00 to 5.00 g / L, preferably 2.00 to 4.00 g / L, more preferably 2.50 to 3.50 g / L.
[0023]
  The definition of animal-derived components and the contents of Eagle MEM, Bacto-Peptone, and BES are as detailed in the description of the present invention according to claim 1.
  Since the serum-free culture solution does not contain serum, it does not contain serum such as bovine serum (FBS) among animal-derived components. The serum-free culture medium is as described above as long as it contains a predetermined amount of Eagle MEM, Bacto-Peptone, BES, and animal-derived components as a basic culture medium but does not contain serum. It may contain other components such as antibiotics and antifungal agents.
  Such a serum-free culture solution is a materialBovine kidney epitheliumA composition suitable for the cell can be appropriately prepared and used, or a commercially available product can be used, but other than those containing animal-derived components are used later.Claim 1The same culture medium as described is preferable from the viewpoint of cell proliferation and economy.
[0024]
  Claim 2In the present invention according toBovine kidney epitheliumCells are acclimated to serum free media as described above. Acclimatization means using a serum-free medium as described above, maintaining the exchange and passage of the serum-free medium according to the degree of cell proliferation, and acclimatizing to a complete serum-free medium (serum It will no longer be required).Bovine kidney epitheliumThe definition of the cell is as described in detail in the description of the present invention according to claim 1.
  The acclimatization of the serum-free culture solution may be performed while observing the degree of cell proliferation, and it differs depending on the composition of the mammal-derived cells and the serum-free culture solution used as the material. When acclimatizing MDBK cells, the serum-free culture medium is usually replaced with a fresh serum-free culture medium at intervals of 3 to 10 days, preferably at intervals of 4 to 7 days. Moreover, although it is difficult to define similarly about the subculture space | interval of a cell, for example, in the case of MDBK cell, it is 18-30 days, Preferably, it is 21-28 days.
  Claim 2In the production method of the present invention according to the present invention, the culture is further carried out by successively lowering the content of animal-derived components in the serum-free culture solution.Claim 1It culture | cultivates using the culture solution of description.
  That is, the passage is maintained by sequentially exchanging with a culture solution in which the content of animal-derived components in the serum-free culture solution is lowered, and finally no animal-derived components are contained.Claim 1It is to culture | cultivate with the culture solution of description. The animal-derived components are as described in detail in the description of the present invention according to claim 1.
  For example, when MDBK cells are used, the culture solution in which the content of the animal-derived component is lowered by about ½ amount is changed 3 to 4 times in a 4 to 7 day cycle, and finally 0 is obtained.
[0025]
  Claim 2According to the production method of the present invention according to the present invention, it is possible to produce a cell line free from pathogen invasion that can grow in an animal-derived component-free culture solution.
  For example,Bovine kidney epitheliumWhen MDBK cells are used as the cells, MDBK-NST cells can be produced. MDBK-NST cells are deposited with the RIKEN Cell Bank. MDBK-NST cells have the same activity as MDBK cells for alkaline phosphatase activity, cytokeratin and γ-glutamyl transpeptidase activity which are epithelial cytoskeleton, and there is no invasion of pathogens such as retrovirus, pestivirus, mycoplasma, Moreover, the sensitivity to viruses is the same as that of MDBK cells, which are parent cells.
[0026]
  Claim 2The cell line obtained by the production method described in 1.Claim 1Since it can grow in the described animal-derived component-free culture medium, it can be used for virus culture and testing. In particular, when MDBK-NST cells are used in, for example, virus testing or antibody testing,Claim 1Since it can be carried out using the described animal-derived component-free culture broth, it has the advantage that it is not necessary to test in advance for antibodies to BVDV that are usually detected at high frequency and antibodies against other viruses that infect cattle. ,preferable.
[0027]
  Also,Claim 2The cell line obtained by the production method described in 1.Claim 1Since it can grow in the described animal-derived component-free culture medium, it can be used for the production of biological preparations such as antigens for diagnosis of diseases, vaccines, and pharmaceuticals. In particular, MDBK-NST cells can be used for the production of biological preparations such as antigens for diagnosis of bovine and porcine diseases, vaccines, pharmaceuticals and the like. Providing such a manufacturing methodClaim 3It is this invention concerning.
[0028]
  That is,Claim 3The invention described isClaim 2A method for producing an antigen, a vaccine or a pharmaceutical for diagnosis of a disease using the cell line obtained by the production method described in 1. above.
[0029]
  Claim 3Examples of diseases targeted for diagnostic antigens, vaccines, and pharmaceuticals produced by the method of the present invention described include all diseases of mammals including humans.
  Claim 3In the described method of the present invention, bovine viral diarrhea / mucosal disease, bovine infectious nose can be used as diagnostic antigens, vaccines and pharmaceuticals produced when MDBK-NST cells are used as a cell line. Examples include tracheitis, bovine parainfluenza, and bovine RS virus infection. Examples of swine diseases include Aujeszky's disease.
  Also,Claim 3The pharmaceutical produced by the method of the present invention described herein includes a physiologically active substance (interferon or the like) using a recombinant or the like in addition to a usual so-called pharmaceutical preparation.
  For the production of such diagnostic antigens, vaccines and pharmaceuticals, a known method may be applied. For example, a virus is grown using a cell culture method or a floating cell culture method using a roller bottle, and a virus stock solution is prepared. It is possible to apply the manufacturing method.
[0030]
【Example】
Next, an Example is given and this invention is demonstrated in detail.
Example 1 (Production of MDBK-NST cells by acclimation to an animal-derived component-free culture medium)
In subculture of MDBK cells (ATCC No. CCL-22) which are parental cells, when the culture medium was changed to a serum-free culture medium having the following composition, the number of cells was reduced and the cell proliferation was remarkably reduced.
[0031]

[0032]
Therefore, up to about the 30th passage, considering that the growth is unstable, the serum-free culture medium is replaced with a fresh serum-free culture medium at intervals of 4 to 7 days, and the cell passage interval is Depending on the proliferative difference, the period was 21 to 28 days.
As a result, cells showing good growth were obtained, and after about 50 passages, stable growth was achieved. After about 110 passages, stable and good growth was achieved. Cells that were shown and acclimated to a complete serum-free culture were obtained.
[0033]
Next, subculture was similarly performed using a culture solution prepared in the same manner except that the addition amount of Tryptose Phosphate Broth was sequentially reduced in the serum-free culture solution having the above composition. As a result, even when the addition amount of Tryptose Phosphate Broth was reduced to about 0.18 g / L, the proliferation of MDBK cells could be maintained. However, when Tryptose Phosphate Broth was not added at all, cell proliferation was It was very weak.
[0034]
Therefore, Tryptose Phosphate Broth is reduced stepwise in the serum-free culture solution composed of the above composition, and from about the 120th passage, it is transferred to a complete animal-derived component-free culture solution composed of the following composition. Continued. The culture medium was replaced with a fresh culture medium on the fourth day of passage, and the cell passage interval was set to 7 to 10 days in view of the difference in proliferation.
[0035]

[0036]
As a result, cells showing stable growth were obtained. Furthermore, even those after about 15 passages showed stable and good growth properties. Therefore, this cell was named MDBK-NST cell as an animal-derived component-free culture medium-acclimated cell line.
[0037]
Example 2 (Proliferation and growth form of MDBK-NST cells)
The growth of the MDBK-NST cells obtained in Example 1 in the above-mentioned animal-derived component-free culture medium was examined. That is, the number of MDBK-NST cells is about 2 million / 25 cm.²The bottles were cultured for 7 days in a complete animal-derived component-free culture solution having the above composition. The resulting MDBK-NST cell growth curve in the animal-derived component-free culture medium is shown in FIG. In FIG. 1, ▲ indicates the result when the culture solution was used for 7 days, and ● indicates the result when the culture solution was updated on the fourth day.
As is apparent from FIG. 1, when the culture solution was used for 7 days, the number of cells was about 6 million cells / 25 cm after 3 days.²Although increased in bottles, cell proliferation tended to decrease from day 5. However, when the culture medium is changed after 3 to 4 days, the proliferation of cells increases with time, and after 7 days, about 8 million cells / 25 cm.²Reached the bottle.
From this result, when MDBK-NST cells are cultured using a complete animal-derived component-free culture solution, it becomes clear that the cell growth is improved by renewing the culture solution after 3 to 4 days. It was.
Furthermore, when the time-dependent change of the proliferation form of MDBK-NST cell was observed, the MDBK-NST cell formed a monolayer on about the 4th day. Small vacuoles were observed in the cell chambers after about 5 to 6 days, but the appearance of the small vacuoles disappeared by renewal of the culture medium on the 4th day.
[0038]
Example 3 (Properties of MDBK-NST cells)
(1) Number of chromosomes in MDBK-NST cells
The distribution rate of the number of chromosomes of MDBK-NST cells was examined. The results are shown in FIG.
As is clear from FIG. 2, it was elucidated that the number of chromosomes in MDBK-NST cells was distributed centering on 43 (2n). Since the number of normal chromosomes in cattle is 60 (2n) and that of MDBK cells, which are parental cells, is 51 (2n), it is considered that many chromosomes are lost in MDBK-NST cells.
[0039]
(2) MDBK-NST cell enzyme activity, etc.
Further, the enzyme activity of MDBK-NST cells was examined by histochemical examination.
[0040]
(1) Alkaline phosphatase activity (Burston method)
The cultured cells were fixed with acetone and then detected using Vectore Red Alkaline Phosphatase Substrate Kit I (VECTORE LABORATORIES).
As a result, alkaline phosphatase activity was detected, which was the same as that of MDBK-NST cells.
[0041]
(2) Cytokeratin, an epithelial cytoskeleton
After the cultured cells were fixed with acetone, the mouse monoclonal antibody: Mab to Cytokeratin 5/8 Pan Epitherial Cytokeratin (Progen) was used as a primary antibody, and detection was performed by the SAB method using a Histofine SAB-PO (M) kit (Nichirei).
As a result, cytokeratin was detected in MDBK-NST cells, which was the same as that of the parental MDBK cells.
[0042]
(3) γ-glutamyl transpeptidase (γ-GTP) activity
The cultured cells were fixed with acetone, and then immersed in a substrate solution containing N-γ-Glutamyl-α-Naphtylamide and Fastgarnet GBC salt, and 0.1 M CuSO₄It detected by processing with a liquid.
As a result, γ-glutamyl transpeptidase activity was not detected in MDBK-NST cells, which was the same as that of parental MDBK cells.
[0043]
(3) MDBK-NST cell contamination by pathogens
Furthermore, it investigated about the contamination of the pathogen of MDBK-NST cell.
▲ 1 pestivirus contamination
Pestivirus frequently invades bovine-derived cultured cells. Therefore, the reverse transcriptase-polymerase chain reaction method for amplifying the pestivirus gene and the indirect fluorescent antibody method for detecting the virus nonstructural protein NS3 were used to examine the contamination of the pestivirus.
[0044]
The reverse transcriptase-polymerase chain reaction method is described by Vileek et al. (Vileek, S., Herring, AJ, Herring, JA, Nettleton, PF, Lowings, JP, Paton, D. et al. J., 1994. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. (AMV) Ver. Performed using 2.1.
The indirect fluorescent antibody method for detecting the virus nonstructural protein NS3 is a method in which cultured cells fixed with acetone are reacted with a monoclonal antibody JCU / BVD / CF10 (JCU TropBio) against NS3 of pestivirus as a primary antibody, and then FITC-labeled anti-mouse IgG. The antibody was reacted as a secondary antibody, observed with a fluorescence microscope, and cells showing specific fluorescence were identified as pestivirus-infected cells.
As a result, pestivirus contamination was denied.
[0045]
(2) Mycoplasma contamination
Kojima et al. (Akemi Kojima, Toshio Takahashi, Mayumi Kijima, Yasuaki Ogikubo, Makoto Nishimura, Shinzo Nishimura, Ryo Harasawa and Yutaka Tamura, 1997, Detection of Mycoplasma in Avian Live Virus Vaccines by Polymerase Chain Reaction. Biologicals 25, 365-371 The polymerase chain reaction method for amplifying the mycoplasma gene was performed. For the polymerase chain reaction, DNA polymerase Ex Taq from Takara Shuzo was used.
As a result, mycoplasma contamination was denied.
[0046]
▲ 3 ▼ Retrovirus contamination
Retroviruses frequently infiltrate cultured cells. Thus, in the same manner as in Example 1, MDBK-NST cells were cultured using a complete animal-derived component-free culture solution, and the cultured cell supernatants on day 2 and day 9 were concentrated by high-speed centrifugation. The reverse transcriptase activity of retroviruses was examined using a kit "Reverse transcriptase activity-non-radioactive" (Roche).
In addition, the presence or absence of reverse transcriptase activity was similarly examined using PK-15 cells derived from pigs with retroviruses as a positive control sample, 0.0625 ng of reverse transcriptase as a positive control, and 0 g of reverse transcriptase as a negative control. . The results are shown in FIG.
As is clear from FIG. 3, no reverse transcriptase activity is observed in the MDBK-NST cell samples. From this, it is clear that MDBK-NST cells are free of retroviruses.
[0047]
Example 4 (Proliferation of BVDV in MDBK-NST cells)
The growth of the Nose strain of bovine viral diarrhea virus (BVDV) in MDBK-NST cells was examined.
That is, MDBK-NST cells cultured in a culture flask were inoculated with BVDV Nose strain at MOI (multiplicity of infection) = 0.01. After the virus was adsorbed to the cells for 1 hour, the virus solution was removed, fresh animal-derived component-free culture solution was added, and the mixture was allowed to stand at 37 ° C. The culture supernatant and cells were repaired over time after inoculation with the virus, and the amount of virus contained in them was measured using bovine testis cells. For the measurement of viral load, a general 10-fold serial dilution method was used, and cells showing cytopathic effect (CPE) were regarded as infected with BVDV Nose strain, and the viral load was calculated according to the Kerbur calculation method. did.
[0048]
As a result, 5 to 7 days after virus inoculation, 10 infectious viruses were released into the culture supernatant.³-10⁴TCID₅₀/ Ml, 10 infectious viruses present in cultured cells⁷TCID₅₀It was elucidated to grow at the level of / ml. In addition, when bovine serum is added at a rate of 5% to the culture solution, the infectious virus released into the culture supernatant is 10 days after the virus inoculation.⁷TCID₅₀/ Ml level was found to be reached.
[0049]
Example 5 (sensitivity of BVDV in MDBK-NST cells)
The sensitivity of various viruses in MDBK-NST cells was examined. That is, the infectivity of the virus solution was measured for each of MDBK-NST cells and MDBK cells, and the degree of infectivity of the virus cells was evaluated by observing the difference.
[0050]
(1) Sensitivity of BVDV
Each BVDV virus solution was serially diluted 10-fold, and each of MDBK-NST cells and MDBK cells suspended in each diluted culture solution was simultaneously placed in a culture plate and allowed to stand for 7 days under conditions of 37 ° C. and 5% carbon dioxide. Incubated. For cytopathogenic BVDV (Nose strain, Osloss strain, KZ91 strain, Singer strain and 5912 strain), CPE was used as a BVDV positive index, and non-cytopathogenic BVDV (No. 12-42 strain, Kanto Higashiyama strain, New York strain). -1 strain, Indiana-46 strain and GBK strain) were evaluated for viral infectivity according to Kaerber's calculation method using the Peroxidase Linked Assay method using anti-NS3 monoclonal antibody.
The results are shown in FIG. In addition, in FIG. 4, a black part shows the sensitivity in MDBK-NST cell, and a white part shows the sensitivity in MDBK cell.
[0051]
As is clear from FIG. 4, it was confirmed that MDBK-NST cells are highly sensitive to viruses in both cytopathogenic strains and non-cytopathogenic strains, and have the same sensitivity as the parental MDBK cells. It was.
[0052]
(2) Susceptibility of vaccine strains or pathogenic strains of bovine infectious rhinotracheitis, bovine parainfluenza, bovine RS virus infection and Aujeszky's disease
Bovine infectious rhinotracheitis virus (IBRV), bovine parainfluenza virus (PI3V), bovine RS virus infectious disease virus (BRSV) and Aujeszky's disease virus (ADV) are diluted 10-fold in a stepwise manner and each diluted culture. Each of the MDBK-NST cells and MDBK cells suspended in the liquid was simultaneously placed in a culture plate or a culture tube, and statically cultured at 37 ° C. under 5% carbon dioxide for 7 days. The bovine RS virus infectious disease virus (BRSV) was rotationally cultured at 34 ° C. for 14 days. After culturing, virus infectivity was evaluated according to Kaerber's calculation method using CPE as a virus positive index.
The results are shown in FIG. In FIG. 5, the black part shows sensitivity in MDBK-NST cells, and the white part shows sensitivity in MDBK cells.
[0053]
As is clear from FIG. 5, it was confirmed that MDBK-NST cells showed the same sensitivity to any virus as the parental MDBK cells.
From this, it is considered that MDBK-NST cells are useful as cells for producing vaccines and diagnostic antigens. Furthermore, it is considered useful as a cell for producing a vaccine for disease caused by a virus sensitive to the cell.
[0054]
【The invention's effect】
The study was repeated to solve the above problems. That is, from the known serum-free culture fluid components, serum-derived components that could cause pathogen invasion and animal-derived components such as Tryptose Phosphate Broth were deleted, and addition of a buffer was examined. As a result, an animal-derived component-free culture solution containing a predetermined amount of Eagle MEM, Bacto-Peptone and BES as a basic culture solution has no risk of pathogen invasion and requires a conventional animal-derived component. It was found that it can be used for the production of cell lines.
In addition, the present inventors have found that a cell line free from pathogen invasion can be produced using this animal-derived component-free culture solution.
Furthermore, since this cell line without pathogen invasion can be grown in an animal-derived component-free culture solution from which the above-mentioned animal-derived components have been deleted, production of biological preparations such as disease diagnosis antigens, vaccines, and pharmaceuticals It was found that it can be used.
The animal-derived component-free culture medium of the present invention does not contain animal-derived components that can cause pathogen invasion, and can be used to produce cell lines that have been conventionally required to have animal-derived components.
In addition, according to the culture solution without an animal-derived component of the present invention, it is possible to create a clean cell line from a mammal-derived cell without fear of entering a known pathogen, and the utility can be expanded.
Moreover, according to the production method of the present invention, a cell line free from pathogen invasion can be obtained efficiently. In particular, when a ruminant-derived cell is used as a target, concerns about the recent BSE problem can be avoided.
Furthermore, since the cell line free from pathogen invasion obtained by the production method of the present invention can be grown in an animal-derived component-free culture solution from which the animal-derived component has been deleted, antigens for diagnosis of diseases, vaccines, pharmaceuticals, etc. It can be used for the manufacture of biologics of this type, as well as virus testing, technical improvements in antibody testing, uniform manufacturing of biologics, ensuring safety, labor saving, and cost reduction through mass production. And so on.
[0055]
In particular, the MDBK-NST cells obtained by the production method of the present invention are grown in an animal-free component-free culture solution that is free from the invasion of pathogens such as retroviruses, pestiviruses, and mycoplasmas, and in which the aforementioned animal-derived components are deleted. Is possible. Therefore, for example, since the virus test method and antibody test method using MDBK-NST cells can be carried out using an animal-derived component-free culture solution, an antibody against bovine viral diarrhea virus (BVDV) detected at high frequency There is an advantage that it is not necessary to examine in advance antibodies against various viruses that infect cattle.
In addition, bovine viral diarrhea / mucosal disease, bovine infectious rhinotracheitis, bovine parainfluenza, bovine RS virus infection and other bovine diseases, and antigens and vaccines for diagnosis of diseases of animals other than cattle such as Auerski disease By using it for the production of biological preparations such as pharmaceuticals, it is possible to make products uniform, ensure safety, save labor, and reduce manufacturing costs by mass production.
[Brief description of the drawings]
FIG. 1 is a proliferation curve of MDBK-NST cells in an animal-derived component-free culture medium.
[Explanation of symbols]
In FIG. 1, ▲ indicates the result when the culture solution is used for 7 days, and ● indicates the result when the culture solution is renewed on the fourth day.
FIG. 2 is a graph showing the distribution rate of the number of chromosomes in MDBK-NST cells.
FIG. 3 is a diagram showing the reverse transcriptase activity of the culture supernatant of MDBK-NST cells.
FIG. 4 is a diagram showing the sensitivity of BVDV.
FIG. 5 is a graph showing the sensitivity of various viruses other than BVDV.
[Explanation of symbols]
In FIG. 4 and FIG. 5, the black part indicates sensitivity in MDBK-NST cells, and the white part indicates sensitivity in MDBK cells.

Claims (3)

Eagle's Minimum Essential Medium [Eagle MEM] as basal medium, Bacto-Peptone 3.00 to 12.00 g / L and N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid [BES] 1.00 An animal-derived component-free culture solution for bovine kidney epithelial cells , characterized by comprising 5.00 g / L. Bovine kidney epithelial cells were treated with Eagle's Minimum Essential Medium [Eagle MEM] as a basic culture solution, Bacto-Peptone 3.00 to 12.00 g / L, and N, N-Bis (2-hydroxyetyl) -2-aminoethanesulfonic acid [ BES] 1.00 to 5.00 g / L and an animal-derived component, and after acclimatization to a serum-free culture solution containing no serum, the inclusion of the animal-derived component in the serum-free culture solution The culture medium according to claim 1 , wherein the culture is carried out at a reduced amount in order, and finally cultured using the culture medium according to claim 1. How to create cell lines without A method for producing an antigen, a vaccine or a pharmaceutical for diagnosing a disease, using the cell line obtained by the production method according to claim 2 .

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