JP4997451B2 - Culture solution and method for avian-derived cells - Google Patents
Culture solution and method for avian-derived cells Download PDFInfo
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Description
本発明は、鳥類由来細胞用の培養液及び培養方法に係り、より詳細には、鳥類由来細胞の多数の継代培養が可能な培養液及び培養方法に関する。 The present invention relates to a culture solution and a culture method for avian-derived cells, and more particularly to a culture solution and a culture method capable of performing many subcultures of avian-derived cells.
培養細胞を用いた実験研究では、試験管や細胞培養フラスコ内で容易に長期培養できる株化細胞を用いると実験が容易に行えることが知られている。基礎免疫学の実験では、実験動物由来株化細胞を用い、その同種の実験動物で実験研究が行われている。
従来から、哺乳動物の組織由来の細胞の培養に適した多種の細胞培養液が、提供されている。In experimental research using cultured cells, it is known that experiments can be easily performed using cell lines that can be cultured for a long period of time in a test tube or cell culture flask. In basic immunology experiments, experimental animal-derived cell lines are used, and experimental studies are conducted on the same type of experimental animals.
Conventionally, various cell culture media suitable for culturing cells derived from mammalian tissues have been provided.
しかしながら、従来提供されている各種の細胞培養液は、哺乳動物の組織由来の細胞の培養には適しているが、鳥類組織由来の細胞の培養には適しておらず、鳥類由来細胞についての多数の継代培養は実現されていなかった。
そこで、本発明者は、鳥類由来細胞の多数の継代培養を実現することができる培養液及び培養方法を提供することを目的とする。However, various types of cell culture media that have been conventionally provided are suitable for culturing cells derived from mammalian tissues, but are not suitable for culturing cells derived from avian tissues. The subculture of was not realized.
Then, this inventor aims at providing the culture solution and culture | cultivation method which can implement | achieve many subcultures of avian origin cells.
本発明者は、上記事情に鑑み、鋭意研究を重ねた結果、L-オルニチンを少なくとも含む、鳥類由来細胞用の培養液を用いて培養することにより、鳥類由来細胞を極めて優位に培養することができることを見出し、本発明を完成させるに至った。 In light of the above circumstances, the present inventor has intensively studied and, as a result, can cultivate avian-derived cells extremely advantageously by culturing using a culture solution for avian-derived cells containing at least L-ornithine. The present inventors have found that the present invention can be accomplished and have completed the present invention.
すなわち、本発明は、
（１）L-オルニチンを少なくとも含む、鳥類由来細胞用の培養液；
（２）前記L-オルニチンの含有量が、１７０〜２５０ｍｇ／Ｌである、前記（１）記載の培養液；
（３）前記培養液が、加熱不活化したウシ胎児血清、又は健常なヒナ鶏血清をさらに含む、前記（１）又は（２）記載の培養液；
（４）L-アラニン、L-アルギニン、L-システイン、L-グルタミン、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-オルニチン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-タイロシン、及びL-バリン、を含む、前記（１）〜（３）の何れかに記載の培養液；
（５）鳥類由来細胞の培養方法であって、（ａ）鳥類由来細胞を準備する工程と、（ｂ）培養液中にて前記細胞を培養する工程と、を含み、前記培養液が、L-オルニチンを少なくとも含む、培養方法；
（６）前記培養液が、加熱不活化したウシ胎児血清、又は健常なヒナ鶏血清をさらに含む、前記（５）記載の培養方法；
（７）前記培養工程は、ホウケイ酸ガラス容器又は細胞培養用プラスチック容器内で行われる、前記（５）又は（６）記載の培養方法；を提供する。That is, the present invention
(1) A culture solution for avian-derived cells containing at least L-ornithine;
(2) The culture solution according to (1), wherein the content of L-ornithine is 170 to 250 mg / L;
(3) The culture solution according to (1) or (2), wherein the culture solution further comprises heat-inactivated fetal bovine serum or healthy chick chicken serum;
(4) L-alanine, L-arginine, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine, L -The culture solution according to any one of (1) to (3) above, which contains proline, L-serine, L-threonine, L-tryptophan, L-tylosin, and L-valine;
(5) A method for culturing an avian-derived cell, comprising: (a) a step of preparing an avian-derived cell; and (b) a step of culturing the cell in a culture solution, wherein the culture solution is L A culture method comprising at least ornithine;
(6) The culture method according to (5), wherein the culture solution further comprises heat-inactivated fetal bovine serum or healthy chick chicken serum;
(7) The culture method according to (5) or (6), wherein the culture step is performed in a borosilicate glass container or a plastic container for cell culture.
本発明によれば、鳥類由来細胞の多数の継代培養が可能な培養液及び培養方法が提供される。 ADVANTAGE OF THE INVENTION According to this invention, the culture solution and culture | cultivation method in which many subculture of avian origin cells are possible are provided.
次に、本発明の実施の形態について、詳細に説明する。以下の実施形態は、本発明を説
明するための例示であり、本発明をこの実施形態にのみ限定する趣旨ではない、本発明は、その要旨を逸脱しない限り、さまざまな形態で実施することできる。Next, embodiments of the present invention will be described in detail. The following embodiments are exemplifications for explaining the present invention, and are not intended to limit the present invention only to the embodiments. The present invention can be implemented in various forms without departing from the gist thereof. .
〔培養液〕
本発明の培養液は、鳥類由来細胞用の培養液であって、l-オルニチンを少なくとも含むものである。
このような培養液を用いれば、鳥類由来細胞をフラスコ容器内で容易に優位に培養することができ、鳥類由来細胞の多数の継代培養が可能となる。[Culture medium]
The culture solution of the present invention is a culture solution for birds-derived cells and contains at least l-ornithine.
By using such a culture solution, avian-derived cells can be easily and advantageously cultured in a flask container, and a large number of subcultures of avian-derived cells are possible.
本発明の目的を一層達成するために、L-オルニチンの溶媒１リットル中における含有量は、１７０〜２５０ｍｇ／Ｌであることが好ましく、特に、１９０〜２２０ｍｇ／Ｌであることが好ましい。 In order to further achieve the object of the present invention, the content of L-ornithine in 1 liter of solvent is preferably 170 to 250 mg / L, and particularly preferably 190 to 220 mg / L.
上記「鳥類由来細胞」とは、ニワトリ（鶏）、七面鳥、アヒル、鴨、チャボ、ダチョウ等の鳥類に由来する細胞という意味である。 The above “bird-derived cells” mean cells derived from birds such as chickens (chicken), turkeys, ducks, duck, chabos and ostriches.
上記培養液は、溶媒として再蒸留水を用いることが好ましく、例えば、脱イオン化し、２回蒸留後に、オートクレープ処理した水が好ましい。培養液の無菌化は、メンブランフィルター処理により行われる。 The culture medium preferably uses double-distilled water as a solvent. For example, water that has been deionized and autoclaved after double distillation is preferred. Sterilization of the culture solution is performed by membrane filter treatment.
上記培養液は、加熱不活化（例えば、５６℃、３０分）したウシ胎児血清、又は健常なヒナ鶏血清をさらに含むことが好ましい。これにより、本発明の目的を一層達成することができる。
前記のごとく加熱不活化したウシ胎児血清、又は健常なヒナ鶏血清は、培養液に対して５〜１０容量％添加することが好ましい。It is preferable that the culture medium further contains heat-inactivated fetal bovine serum (for example, 56 ° C., 30 minutes) or healthy chick chicken serum. Thereby, the object of the present invention can be further achieved.
It is preferable to add 5 to 10% by volume of the fetal bovine serum or the healthy chick chicken serum inactivated by heating as described above.
上記培養液は、L-アラニン、L-アルギニン、L-システイン、L-グルタミン、グリシン、L-ヒスチジン、L-イソロイシン、L-ロイシン、L-リジン、L-メチオニン、L-オルニチン、L-フェニルアラニン、L-プロリン、L-セリン、L-スレオニン、L-トリプトファン、L-タイロシン、及びL-バリン、を含むことが好ましい。これにより、本発明の目的を一層達成することができる。 The culture broth is L-alanine, L-arginine, L-cysteine, L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-ornithine, L-phenylalanine , L-proline, L-serine, L-threonine, L-tryptophan, L-tylosin, and L-valine. Thereby, the object of the present invention can be further achieved.
上記培養液は、培養液作製時に炭酸ナトリウムを含んだ状態で作製してもよい。また、培養液作製時に炭酸ナトリウムを不含の状態で作製しておき、使用時に血清添加と同時に、別に濾過滅菌しておいた７．５％の炭酸ナトリウム液を添加してもよい。その場合、７．５％の炭酸ナトリウム液の添加量は、１０００ｍＬの培養液当たり２０〜３０ｍＬが好ましい。 The culture solution may be prepared in a state containing sodium carbonate at the time of preparation of the culture solution. Alternatively, it may be prepared in a state free of sodium carbonate at the time of preparation of the culture solution, and 7.5% sodium carbonate solution that has been separately sterilized by filtration may be added simultaneously with the addition of serum during use. In that case, the addition amount of the 7.5% sodium carbonate solution is preferably 20 to 30 mL per 1000 mL of the culture solution.
上記培養液は、塩類、糖、アミノ酸、ビタミン類、抗生物質等を含んでもよい。
上記培養液には、通常の細胞培養液に添加される各種抗生物質（例えば、ペニシリン、ストレプトマイシン、カナマイシン、ゲンタマイシン、ナイスタチン等）を添加してもよい。
上記培養液は、表１に示す組成を有することが好ましい。The culture solution may contain salts, sugars, amino acids, vitamins, antibiotics and the like.
Various antibiotics (for example, penicillin, streptomycin, kanamycin, gentamicin, nystatin, etc.) added to normal cell culture medium may be added to the culture medium.
The culture solution preferably has the composition shown in Table 1.
上記培養液は乾燥させ、粉末状にして保存することもできる。この場合、２〜８℃で乾燥状態にて保存することが好ましい。粉末が変色、顆粒状／凝集状態、不溶解性を示した場合は、使用不可とする。
The culture broth can be dried and stored in powder form. In this case, it is preferable to store in a dry state at 2 to 8 ° C. If the powder shows discoloration, granular / agglomerated state, or insolubility, it cannot be used.
〔培養方法〕
本発明の培養方法は、鳥類由来細胞の培養方法であって、（ａ）鳥類由来細胞を準備する工程と、（ｂ）培養液中にて前記細胞を培養する工程と、を含み、前記培養液が、L-オルニチンを少なくとも含むものである。
このような培養方法によれば、鳥類由来細胞をフラスコ内で容易に優位に培養することができ、鳥類由来細胞の多数の継代培養が可能となる。[Culture method]
The culture method of the present invention is a method for culturing avian-derived cells, comprising: (a) preparing avian-derived cells; and (b) culturing the cells in a culture solution, The liquid contains at least L-ornithine.
According to such a culture method, avian-derived cells can be easily and advantageously cultured in a flask, and a large number of subcultures of avian-derived cells are possible.
本発明の培養方法においては、上記の培養液を用いることができる。
本発明の培養方法においては、培養液は、加熱不活化（例えば、５６℃、３０分）したウシ胎児血清、又は健常なヒナ鶏血清をさらに含むことが好ましい。In the culture method of the present invention, the above culture solution can be used.
In the culture method of the present invention, the culture solution preferably further contains heat-inactivated fetal bovine serum (eg, 56 ° C., 30 minutes) or healthy chick chicken serum.
本発明の培養方法においては、前記培養工程は、ホウケイ酸ガラス容器又は細胞培養用プラスチック容器内で行われることが好ましい。
具体的には、前記培養工程では、容器として、洗浄・滅菌した中性ガラス製細胞培養瓶、又は市販の滅菌済み細胞培養用プラスチックフラスコ（例えば、デンマークのNunc社製品、米国のCorning社製品、米国のFalcon社製品、日本の住友ベークライト社製品）を用いることが好ましい。In the culture method of the present invention, the culture step is preferably performed in a borosilicate glass container or a cell culture plastic container.
Specifically, in the culturing step, as a container, a neutral glass cell culture bottle that has been washed and sterilized, or a commercially available plastic flask for cell culture (for example, Nunc product from Denmark, Corning product from the United States, It is preferable to use Falcon products from the United States and Sumitomo Bakelite products from Japan.
以下に、本発明を実施例により詳細に説明するが、本発明はこれらに限定されるものではない。 EXAMPLES The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.
〔実施例１〕
上記表１に示す組成を、脱イオン化した後、２回蒸留し、高圧滅菌処理した水、又は超純水装置（米国のミリポア社製）で作製した水であって、室温（２０〜２４℃）下で、約９０％容量分の上記精製水に培養液組成を添加し、完全に溶解し透明になるまで撹拌した。この溶液約９０００ｍＬに対して１N-HCl液を１ｍＬ加えた後、さらに精製水を加えて１００容量分とした。
次に、２００〜２２０ｎｍの細孔のメンブランフィルターで濾過滅菌した。そして、この溶液に、前記のごとく加熱不活化したウシ胎児血清を１０容量％添加して細胞増殖液（実務上の細胞培養液）を得た。[Example 1]
The composition shown in Table 1 was deionized, then distilled twice, and autoclaved, or water produced with an ultrapure water device (Millipore, USA) at room temperature (20-24 ° C. ), The culture solution composition was added to about 90% volume of the purified water and stirred until it was completely dissolved and transparent. After adding 1 mL of 1N HCl solution to about 9000 mL of this solution, purified water was further added to make up 100 volumes.
Next, the solution was sterilized by filtration through a membrane filter having pores of 200 to 220 nm. Then, 10% by volume of fetal bovine serum inactivated as described above was added to the solution to obtain a cell growth solution (practical cell culture solution).
得られた上記細胞培養液を用いて、ホウケイ酸ガラス製フラスコ、及び細胞培養用プラスチック製フラスコ内で、孵化直前の鶏胎児及びヒナ鶏の、肺臓、腎臓、脾臓、及び骨髄の細胞を培養した。
組織細切片を０．１〜０．０５％ＥＤＴＡ（エチレンジアミン四酢酸／リン酸緩衝液）液中で単個に分離し、その細胞を遠心沈殿して集め、細胞増殖液中に浮遊させ、フラスコに分注して３８．５〜３９．０℃に培養した。７日程度の培養（初代培養細胞）で充分の細胞増殖が得られた。
この初代培養細胞をさらにＥＤＴＡ液で単個に分散し、細胞増殖液中に浮遊したものを９６穴のマイクロプレート（Flow laboratories, Inc社製）に、限界希釈法（Limited Dilution Method）によって培養し、クローン細胞株を樹立した。各クローン細胞株は、フラスコ培養に移し、継代することで、株化細胞を樹立した。鶏胎児の肺臓及び腎臓由来の培養細胞から、２つの株化細胞を樹立した。これらいずれの細胞株も、３８．５〜３９．０℃の培養温度条件で容易に増殖し、少なくとも４０代までの継代に成功した。Using the obtained cell culture solution, the cells of lung, kidney, spleen, and bone marrow of chicken embryos and chicks just before hatching were cultured in a borosilicate glass flask and a plastic flask for cell culture. .
Tissue subsections are separated into single pieces in 0.1-0.05% EDTA (ethylenediaminetetraacetic acid / phosphate buffer) solution, and the cells are collected by centrifugation, suspended in cell growth solution, and flasks. The solution was dispensed into 38.5 to 39.0 ° C. Sufficient cell growth was obtained by culturing for about 7 days (primary cultured cells).
These primary cultured cells are further dispersed into single pieces with EDTA solution, and the cells suspended in the cell growth solution are cultured in a 96-well microplate (manufactured by Flow laboratories, Inc.) by the Limited Dilution Method. A clonal cell line was established. Each clonal cell line was transferred to a flask culture and subcultured to establish a cell line. Two cell lines were established from cultured cells derived from the lungs and kidneys of chicken fetuses. All of these cell lines proliferated easily at a culture temperature of 38.5 to 39.0 ° C., and succeeded in passage up to at least 40 generations.
これより、鶏胎児の肺臓及び腎臓からの培養細胞が、in vitroで極めて優位に培養できることが分かった。なお、鶏胎児の肺由来の細胞は、繊維芽細胞形態を示し、鶏胎児腎臓由来の細胞は、上皮細胞形態を示した。 From this, it was found that cultured cells from the lungs and kidneys of chicken fetuses can be cultured extremely in vitro. In addition, the cell derived from a chicken fetus lung showed a fibroblast form, and the cell derived from a chicken embryo kidney showed an epithelial cell form.
次に、細胞における、非特異的エステラーゼ及び酸ホスファターゼの生産性を、常法により検査した。その結果、鳥胎児の肺由来細胞について両酵素の生産が認められたことから、これら細胞は単球−マクロファージ系統であることが分かった。 Next, the productivity of non-specific esterase and acid phosphatase in the cells was examined by a conventional method. As a result, production of both enzymes was observed in avian fetal lung-derived cells, indicating that these cells are a monocyte-macrophage lineage.
次に、発ガン性との関連を調べる目的で、細胞の浮遊液（リン酸緩衝液１ｍＬ当たり２，０００，０００個の細胞を含む）０．５ｍＬを、生後３日目の白色レグホーン種のヒナ鶏１０体の皮下にそれぞれ接種し、１ケ月後に剖検した結果、発ガン性に関する病理学的な所見は認められなかった。 Next, for the purpose of investigating the relationship with carcinogenicity, 0.5 mL of cell suspension (containing 2,000,000 cells per 1 mL of phosphate buffer) was added to 3 days old white leghorn species. As a result of inoculating 10 chicks subcutaneously and autopsying one month later, no pathological findings regarding carcinogenicity were found.
また、これらの培養細胞を、鶏白血病の共通抗原として知られるＰ２７抗原の検出キット（米国のＩＤＥＸＸ社製）を用いて調べたところ、明確な陽性反応は認められなかった。 Further, when these cultured cells were examined using a P27 antigen detection kit (manufactured by IDEXX, USA) known as a common antigen of chicken leukemia, no clear positive reaction was observed.
さらに、これらの細胞培養に、鶏ニューカッスル病ウイルス（宮寺株）を感染重度０．０１で感染させたところ、結果として、ウイルスは細胞内で増殖し、強い細胞病変を惹起し、ウイルス感染後、２〜４日の培養過程で、大部分の細胞は死滅した。また、接種ウイルスの有意の複製（感染させたウイルスの増殖）も認められた。 Furthermore, when these cell cultures were infected with chicken Newcastle disease virus (Miyadera strain) at an infection severity of 0.01, as a result, the virus proliferated in the cells and caused strong cell lesions. Most cells died during the 2-4 days of culture. In addition, a significant replication of the inoculated virus (growth of the infected virus) was also observed.
〔実施例２〕
加熱不活化したウシ胎児血清の代わりに、健常なヒナ鶏血清を１０容量％添加した培養液を用いた場合であっても、上記の細胞株は極めて良く増殖し、少なくとも４０代までのフラスコ培養での継代に成功した。[Example 2]
Even when a culture solution supplemented with 10% by volume of healthy chick chicken serum is used in place of heat-inactivated fetal bovine serum, the above cell lines proliferate very well, and flask culture up to at least 40 generations. Succeeded in the passage.
〔比較例１〕
L-オルニチンを含まない、市販の各種哺乳動物用細胞培養液に、加熱不活化したウシ胎児血清を１０容量％添加した培養液を用いて、上記鶏由来の細胞を培養したところ、５〜６代までしか継代できなかった。[Comparative Example 1]
The above chicken-derived cells were cultured using a culture solution in which 10% by volume of heat-inactivated fetal bovine serum was added to various commercially available mammalian cell culture solutions that did not contain L-ornithine. It was only possible to pass until the generation.
以上の結果から、本発明の培養液を用いれば、鳥類由来細胞のフラスコ内培養が極めて容易にできることが分かった。特に、培養液に加熱不活化したウシ胎児血清、又は健常なヒナ鶏血清を添加して用いることにより、鳥類細胞の単個培養や骨髄細胞の培養も可能となることが分かった。
これにより、本発明の培養液を用いれば、鳥類由来の種々の細胞が容易に培養できたことから、それら培養細胞で増殖可能な鳥類由来のウイルスの培養が可能となる。すなわち、細胞培養で増殖が可能となったウイルスを抗原として用いれば、ワクチン開発も可能となる。また、本発明の培養液により培養される細胞は、鳥類由来細胞のウイルス学、免疫学、生化学、薬理学、病理学に多様な応用の可能性がある。例えば、本発明の培養液は、鳥インフルエンザウイルスの研究に有用である。
From the above results, it was found that the use of the culture solution of the present invention makes it possible to culture avian-derived cells in a flask very easily. In particular, it has been found that single avian cell culture and bone marrow cell culture can be achieved by adding heat-inactivated fetal bovine serum or healthy chick chicken serum to the culture medium.
Thereby, if the culture solution of the present invention is used, various birds-derived cells can be easily cultured, and thus it is possible to culture a bird-derived virus that can be grown on these cultured cells. In other words, vaccines can be developed by using, as antigens, viruses that can be grown in cell culture. In addition, cells cultured with the culture medium of the present invention have various applications in avian-derived cell virology, immunology, biochemistry, pharmacology, and pathology. For example, the culture solution of the present invention is useful for research on avian influenza viruses.
Claims (5)
(a)鳥類由来細胞を準備する工程と、
(b)培養液中にて前記細胞を培養する工程と、を含み、
前記培養液が、L-オルニチンおよび加熱不活化したウシ胎児血清を含む、培養方法。A method for culturing avian-derived cells,
(a) preparing avian-derived cells;
(b) culturing the cells in a culture solution,
The culture method, wherein the culture solution contains L-ornithine and heat-inactivated fetal bovine serum.
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