JP5140829B2 - Medium addition factor - Google Patents
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本発明は、動物細胞培養のための基礎培地に添加して、動物細胞の増殖の促進および細胞の有用性の向上を可能とする、培地添加因子に関する。 The present invention relates to a medium addition factor that can be added to a basal medium for animal cell culture to promote the growth of animal cells and improve the usefulness of the cells.
今日、生命科学の分野では、細胞培養あるいは組織培養の技術が盛んに用いられ、有用物質の大量生産には目的物質を産生する動物細胞の大量培養が行われている。しかしながら、このような細胞・組織は一般に培養が困難で、これらの動物細胞を培養する為には、通常、アミノ酸・ビタミン・無機塩および糖類からなる基礎培地に、動物細胞増殖因子を添加する必要がある。この動物細胞増殖因子としては通常、ウシ胎仔血清または仔ウシ血清が使用される。ウシ胎仔血清または仔ウシ血清は基礎培地に通常２〜２０容量％添加される（非特許文献１）。 Today, in the field of life science, cell culture or tissue culture techniques are actively used, and mass production of animal cells producing target substances is performed for mass production of useful substances. However, such cells and tissues are generally difficult to cultivate, and in order to culture these animal cells, it is usually necessary to add animal cell growth factor to a basic medium consisting of amino acids, vitamins, inorganic salts and sugars. There is. As this animal cell growth factor, fetal bovine serum or calf serum is usually used. Fetal calf serum or calf serum is usually added to the basal medium in an amount of 2 to 20% by volume (Non-patent Document 1).
しかしウシ胎仔血清及び仔ウシ血清は、その供給に制限があり、非常に高価で、さらにロットによりその特質に差がある（非特許文献２）。また動物由来の血清には、ヤコブ病を導くことが危惧されている狂牛病・ヒツジのスクレイピーといったプリオンに加え、ウイルス感染の危険がある（非特許文献３）。 However, fetal bovine serum and calf serum are limited in supply, are very expensive, and have different characteristics depending on the lot (Non-patent Document 2). Moreover, in addition to prions such as mad cow disease and sheep scrapie which are feared to lead to Jacob disease, animal-derived sera have a risk of virus infection (Non-patent Document 3).
また血清はあらゆる種類の成分から構成される物質である為、生命科学の分野で実験に用いた場合、複雑な実験系となり、原因と結果の関係を論じる際に混乱が生じるという問題がある（非特許文献３）。 In addition, since serum is a substance composed of all kinds of components, when used in experiments in the field of life science, it becomes a complicated experimental system, and there is a problem that confusion occurs when discussing the relationship between causes and results ( Non-patent document 3).
最近では、実験系をより単純な系にする目的で、ウシ胎仔血清及び仔ウシ血清に代えて既知の細胞成長因子類やホルモン類が細胞培養に用いる培地の添加因子として利用されるようになってきている（非特許文献４）。しかしこれらの因子自体、天然における存在量が微量であり、ウシ胎仔血清または仔ウシ血清にもまして高価である事から、使用には制限があった。
本発明の課題は、動物細胞の培養に際し、非常に有効であるが問題の多いウシ胎仔または仔ウシ血清の使用量を極力減らす事を可能にすると共に、通常より優れた細胞増殖効果および目的有用産物の産生促進効果をもたらす添加因子（添加剤）を提供する事にある。 It is an object of the present invention to reduce the amount of fetal calf serum or calf serum that is very effective but problematic in culturing animal cells, and to achieve a cell proliferation effect superior to usual and useful for the purpose. It is to provide an additive factor (additive) that brings about the production promotion effect of the product.
本発明者は、上記課題を解決するために鋭意研究した結果、植物由来のフルクタンを低濃度で培地に添加する事により、細胞の増殖や機能が活性化される事を見出し、本発明を完成するに至った。
即ち、本発明は次の通りである。
〔１〕フルクタンを有効成分として含有する、培地添加因子。
〔２〕フルクタンが植物の根または根茎由来である、上記〔１〕記載の添加因子。
〔３〕フルクタンがネギ属植物、アヤメ科植物、イネ科植物、キク科植物、ユリ科植物、ラン科植物からなる群より選ばれる植物の根または根茎由来である、上記〔１〕または〔２〕記載の添加因子。
〔４〕フルクタンがラッキョウ、ニンニク、タマネギからなる群より選ばれるネギ属植物の根または根茎由来である、上記〔１〕〜〔３〕のいずれかに記載の添加因子。
〔５〕動物細胞培養用である、上記〔１〕〜〔４〕のいずれかに記載の添加因子。
〔６〕動物細胞が哺乳動物細胞である、上記〔５〕記載の添加因子。
〔７〕細胞増殖促進剤である、上記〔１〕〜〔６〕のいずれかに記載の添加因子。
〔８〕細胞機能および／または生存率の向上剤である、上記〔１〕〜〔６〕のいずれかに記載の添加因子。
〔９〕凍結保護剤である、上記〔１〕〜〔４〕のいずれかに記載の添加因子。
〔１０〕上記〔１〕〜〔９〕のいずれかに記載の添加因子を含む、培養用培地。
〔１１〕添加因子を０．０００１〜１０重量％含有する、上記〔１０〕記載の培地。
〔１２〕動物細胞を上記〔１０〕または〔１１〕記載の培地にて培養することを特徴とする、動物細胞の培養方法。
〔１３〕動物細胞が哺乳動物細胞である、上記〔１２〕記載の方法。
As a result of diligent research to solve the above problems, the present inventor has found that cell growth and functions are activated by adding plant-derived fructan to the medium at a low concentration, and the present invention has been completed. It came to do.
That is, the present invention is as follows.
[1] A medium addition factor containing fructan as an active ingredient.
[2] The additive factor according to [1], wherein the fructan is derived from a plant root or rhizome.
[3] The above [1] or [2], wherein the fructan is derived from a root or rhizome of a plant selected from the group consisting of an Allium plant, Iridaceae plant, Gramineae plant, Asteraceae plant, Lily family plant, Orchidaceae plant ] Additive factor of description.
[4] The additive factor according to any one of [1] to [3] above, wherein the fructan is derived from the roots or rhizomes of a genus Leek plant selected from the group consisting of Rakkyo, garlic, and onion.
[5] The additive factor according to any one of [1] to [4], which is for animal cell culture.
[6] The additive factor according to [5] above, wherein the animal cell is a mammalian cell.
[7] The additive factor according to any one of [1] to [6], which is a cell growth promoter.
[8] The additive factor according to any one of [1] to [6] above, which is an agent for improving cell function and / or survival rate.
[9] The additive factor according to any one of [1] to [4], which is a cryoprotectant.
[10] A culture medium containing the additive factor according to any one of [1] to [9].
[11] The medium described in [10] above, containing an additive factor of 0.0001 to 10% by weight.
[12] A method for culturing animal cells, comprising culturing animal cells in the medium described in [10] or [11] above.
[13] The method described in [12] above, wherein the animal cell is a mammalian cell.
本発明によれば、フルクタンを有効成分とする安価で安全性の高い優れた細胞培養用培地添加因子を提供することができる。本発明の培地添加因子は、培養培地に添加することで、細胞の形態や種類を問題にせず増殖を促進させ、さらにタンパク質生産などの細胞機能を発揮させる事ができる。また、本発明の培地添加因子を培地に添加すると、細胞の生存率や冷凍保存性も高めることもできる。さらに、フルクタンは天然物由来であり人体への安全性が高いので、本発明の培地添加因子を添加した培地で培養した細胞は、臨床診断薬（抗体）生産、タンパク質医薬生産、再生医療・細胞治療への応用が可能である。 According to the present invention, it is possible to provide an excellent cell culture medium addition factor that is inexpensive and highly safe, which contains fructan as an active ingredient. By adding the medium addition factor of the present invention to the culture medium, growth can be promoted without causing any problem in the form and type of cells, and cell functions such as protein production can be exhibited. Moreover, when the culture medium addition factor of this invention is added to a culture medium, the survival rate of a cell and frozen preservation property can also be improved. Furthermore, since fructan is derived from natural products and highly safe to the human body, cells cultured in a medium supplemented with the medium addition factor of the present invention are used for clinical diagnostic (antibody) production, protein drug production, regenerative medicine / cells. Application to treatment is possible.
本発明は、フルクタンを有効成分として含有する培地添加因子を提供する。 The present invention provides a medium addition factor containing fructan as an active ingredient.
本発明の有効成分であるフルクタンは、フルクトースのみからなるホモ多糖であり、主に微生物および植物内に存在している。フルクタンには、禾本科植物の葉や茎などに存在し、また、細菌の作用により蔗糖から生成される細菌分泌多糖であるレバン（Ｄ−フラクトフラノースがβ２→６結合）、キク科、ユリ科、アヤメ科、ラン科植物の根、根茎、殻物などに存在するイヌリン（Ｄ−フラクトフラノースがβ２→１結合）、ラッキョウ、ニンニク、タマネギなどのネギ属植物の球根に含まれるフルクタン（特許第３１１１３７８号公報参照）などが知られているが、本発明においてフルクタンとは、レバン、イヌリン、ネギ属植物由来フルクタンの何れをも含む。なお、本発明においてフルクタンとは、フルクタンの加水分解物をも含む意である、ここでフルクタンの加水分解物とは、細胞の増殖や機能を活性化する限り特に限定されないが、例えば、重合度２０〜１０００（分子量３〜２００ｋＤａ（好ましくは分子量６〜１００ｋＤａ））のフルクタンの加水分解物などが挙げられる。 Fructan, which is an active ingredient of the present invention, is a homopolysaccharide consisting only of fructose and is mainly present in microorganisms and plants. Fructan is present in the leaves and stems of scallops, and is a bacterially secreted polysaccharide that is produced from sucrose by the action of bacteria. , Fructans contained in bulbs of genus plants such as inulin (D-fructofuranose is β2 → 1 linked), rootfish, garlic, onion, etc. present in roots, rhizomes, shells, etc. In the present invention, fructan includes all of levan, inulin, and leek plant-derived fructans. In the present invention, fructan is meant to include a hydrolyzate of fructan, and the hydrolyzate of fructan is not particularly limited as long as it activates cell growth and function. Examples thereof include hydrolysates of fructan having a molecular weight of 20 to 1000 (molecular weight of 3 to 200 kDa (preferably molecular weight of 6 to 100 kDa)).
本発明で用いるフルクタンとしては、合成物、天然物の何れであってもよいが、微生物由来、植物由来などの天然物であることが好ましい。フルクタンは、好ましくは植物由来、より好ましくは植物の根または根茎由来である。このような植物の例としては、ラッキョウ、ニンニク、タマネギなどのネギ属植物、アヤメ科、イネ科、キク科、ユリ科、ラン科植物などが挙げられ、ラッキョウ、ニンニク、タマネギなどのネギ属植物の根または根茎由来のフルクタンが、β（２→６）結合とβ（２→１）結合が混合しているために構造がより複雑であり、様々な生理活性をしめし得るという理由で特に好ましい。 The fructan used in the present invention may be either a synthetic product or a natural product, but is preferably a natural product such as a microorganism or a plant. The fructan is preferably derived from a plant, more preferably from a plant root or rhizome. Examples of such plants include leeks, garlic, onions and other leeks, irises, gramineae, asteraceae, liliaceae, orchids, and leeks, garlic, onions and other genus plants. Fructans derived from roots or rhizomes are particularly preferred because they have a more complex structure due to the mixture of β (2 → 6) and β (2 → 1) linkages and can exhibit various physiological activities .
本発明で用いるフルクタンは、具体的には、熱水を用いて抽出し、その後、液体クロマトグラフィなどの技法により調製することができる。また、特許第３１１１３７８号公報に記載の方法により調製してもよい。さらに、フルクタンは市販のものを用いてもよく、例えば、三里浜特産農業協同組合製のフルクタンなどが挙げられる。 Specifically, the fructan used in the present invention can be extracted by using hot water and then prepared by a technique such as liquid chromatography. Further, it may be prepared by the method described in Japanese Patent No. 311378. Further, commercially available fructans may be used, and examples thereof include fructans manufactured by Sanrihama Special Agriculture Cooperative.
本発明の培地添加因子は、フルクタンのみから構成されていてもよい。また、培地に添加することで細胞の増殖や機能を活性化する限り本発明の培地添加因子中のフルクタンの純度は特に限定されず、例えば、フルクトースやその他の糖類、タンパク質、核酸、脂質、無機塩類などを含んでいてもよい。 The medium addition factor of this invention may be comprised only from fructan. Further, the purity of fructan in the medium addition factor of the present invention is not particularly limited as long as cell growth and function are activated by adding to the medium. For example, fructose and other saccharides, proteins, nucleic acids, lipids, inorganics It may contain salts.
本発明の培地添加因子は、必要に応じて滅菌することができる。滅菌方法としては、濾過滅菌、高圧蒸気滅菌などが挙げられる。特に、本発明で有効成分として用いられるフルクタンは、熱に強いため、より確実性の高い滅菌法である高圧蒸気滅菌が適用できるという点で優れている。 The medium addition factor of the present invention can be sterilized as necessary. Examples of the sterilization method include filtration sterilization and high-pressure steam sterilization. In particular, since fructan used as an active ingredient in the present invention is resistant to heat, it is excellent in that high-pressure steam sterilization, which is a more reliable sterilization method, can be applied.
本発明の培地添加因子は、培養培地に添加することができる。添加方法としては、特に限定されないが、培地に溶解させて添加する方法が望ましい。 The medium addition factor of the present invention can be added to the culture medium. The addition method is not particularly limited, but a method of adding it by dissolving in a medium is desirable.
培地は、動物細胞の培養に用いられる培地を基礎培地とし、本発明の培地添加因子を添加することにより調製することができる。基礎培地としては、例えば、ＥａｇｌｅＭＥＭ培地、αＭＥＭ培地、ＤＭＥＭ培地、ハム培地、ＲＰＭＩ１６４０培地、Ｆｉｓｃｈｅｒ’ｓ培地、およびこれらの混合培地が挙げられる。培地は、例えば、脂肪酸または脂質、アミノ酸、ビタミン、増殖因子、抗酸化剤、２−メルカプトエタノール、ピルビン酸、緩衝剤、無機塩類等を含むことができる。好ましくは、培地はＤＭＥＭ培地、ＲＰＭＩ１６４０培地がよい。 The medium can be prepared by using a medium used for culturing animal cells as a basal medium and adding the medium addition factor of the present invention. Examples of the basal medium include Eagle MEM medium, αMEM medium, DMEM medium, ham medium, RPMI 1640 medium, Fischer's medium, and mixed media thereof. The medium can contain, for example, fatty acids or lipids, amino acids, vitamins, growth factors, antioxidants, 2-mercaptoethanol, pyruvic acid, buffers, inorganic salts, and the like. Preferably, the medium is DMEM medium or RPMI 1640 medium.
培地中の本発明の培地添加因子の含有量は、通常、有効成分（フルクタン）量として、０．０００１〜１０重量％、好ましくは、０．０００５〜１重量％、より好ましくは、０．０００５〜０．１重量％である。なお、本発明の培地添加因子は少量でも充分な効果を示すが、水溶性に優れ且つ安全性も高い為、必要に応じて多量に添加することも可能である。 The content of the medium addition factor of the present invention in the medium is usually 0.0001 to 10% by weight, preferably 0.0005 to 1% by weight, more preferably 0.0005 as the amount of active ingredient (fructan). ~ 0.1 wt%. In addition, although the medium addition factor of the present invention shows a sufficient effect even in a small amount, it is excellent in water solubility and high safety, so that it can be added in a large amount as necessary.
本発明の培地添加因子を含有する培地には、培地添加因子の他に、通常動物細胞の培養培地に使用されるタンパク質やポリペプチド、ホルモン、アミノ酸、ビタミン類、脂質因子、糖類、浸透圧調節剤、ｐＨ緩衝剤、界面活性剤などの各成分が含まれていてもよい。 In the medium containing the medium addition factor of the present invention, in addition to the medium addition factor, proteins, polypeptides, hormones, amino acids, vitamins, lipid factors, saccharides, osmotic pressure regulation, which are usually used in culture media for animal cells Each component such as an agent, a pH buffer, and a surfactant may be included.
本発明の培地添加因子を含有する培地は、動物細胞の培養に用いることができる。該培地で培養可能な細胞の種類は、特に限定されず、例えば、ヒトを含む哺乳動物（マウス、ラット、モルモット、ハムスター、ウサギ、ネコ、イヌ、ヒツジ、ブタ、ウシ、ウマ、ヤギ、サル、ヒトなど）、鳥類（ニワトリ、ダチョウなど）、は虫類（ワニなど）、両生類（カエルなど）、魚類（ゼブラフィッシュ、メダカなど）などの脊椎動物、昆虫（蚕、蛾、ショウジョウバエなど）などの非脊椎動物などの細胞が挙げられる。より好ましくは、本発明で対象とされる細胞は、哺乳動物細胞である。 The medium containing the medium addition factor of the present invention can be used for culturing animal cells. The types of cells that can be cultured in the medium are not particularly limited. For example, mammals including humans (mouse, rat, guinea pig, hamster, rabbit, cat, dog, sheep, pig, cow, horse, goat, monkey, Vertebrates such as humans), birds such as chickens and ostriches, reptiles such as crocodiles, amphibians such as frogs, fishes such as zebrafish and medaka, and non-vertebrates such as insects such as moths, moths, and fruit flies Examples include cells such as animals. More preferably, the cells targeted by the present invention are mammalian cells.
当該細胞は、例えば、ＢＨＫ細胞、ＨｅｐＧ２細胞、ＭＧ６３細胞、ＣＨＯ細胞、ハイブリドーマ細胞などの癌細胞を含む培養細胞株であっても、線維芽細胞などの個体や組織より単離された細胞、さらには膵ランゲルハンス島のような細胞集合体・組織もしくは組織片の細胞であってもよい。 The cell may be, for example, a cultured cell line containing cancer cells such as BHK cells, HepG2 cells, MG63 cells, CHO cells, hybridoma cells, cells isolated from individuals or tissues such as fibroblasts, May be cells of a cell aggregate / tissue or tissue fragment such as pancreatic islets of Langerhans.
培養温度、ＣＯ２濃度、培養時間等の他の培養条件は、培養する細胞の種類によって異なり、当業者であれば適宜設定することができる。一般的には、培養温度は、特に限定されるものではないが、例えば約３０〜４０℃、好ましくは約３７℃である。また、ＣＯ２濃度は、例えば約１〜１０％、好ましくは約５％である。培養時間は、例えば約１日〜１４日間である。 Other culture conditions such as culture temperature, CO 2 concentration, and culture time vary depending on the type of cells to be cultured, and can be appropriately set by those skilled in the art. In general, the culture temperature is not particularly limited, but is, for example, about 30 to 40 ° C, preferably about 37 ° C. The CO 2 concentration is, for example, about 1 to 10%, preferably about 5%. The culture time is, for example, about 1 day to 14 days.
本発明の培地添加因子を含有する培地で細胞を培養すると、後述の実施例から明らかなように細胞増殖が促進され、および／または生存率が向上する。また、抗体や組み換えタンパク質などのタンパク質生産能などの細胞機能が向上する。従って、本発明の培地添加因子は、細胞増殖促進剤、細胞機能および／または生存率の向上剤としても用いることができる。 When cells are cultured in a medium containing the medium-added factor of the present invention, cell growth is promoted and / or the survival rate is improved, as will be apparent from Examples described later. In addition, cell functions such as the ability to produce proteins such as antibodies and recombinant proteins are improved. Therefore, the medium-added factor of the present invention can also be used as a cell growth promoter, a cell function and / or a viability improver.
さらに、動物細胞を凍結保存する際、本発明の培地添加因子を含む細胞凍結液を用いれば、凍結保存中の細胞の死滅および凍結操作に伴う細胞死滅を抑制し、細胞の生存率を高めることができる。従って、本発明の培地添加因子は、凍結保護剤としても用いることができる。 Furthermore, when cryopreserving animal cells, the use of a cell freezing solution containing the medium-added factor of the present invention suppresses cell death during cryopreservation and cell death associated with freezing operation, and increases cell survival rate. Can do. Therefore, the medium addition factor of the present invention can also be used as a cryoprotectant.
本発明の培地添加因子を凍結保護剤として用いる場合、ＰＢＳなどの緩衝液、培養に用いられる培地、血清などのいずれか、あるいはこれらに塩や糖類、アミノ酸などを加えて改良したものをベースとし、これらにさらに５〜２０容積％のＤＭＳＯや、グリセリンなどの因子を添加したものにフルクタンを添加する。細胞凍結液中の本発明の培地添加因子の含有量は、通常、有効成分量として、０．０００１〜１０重量％、好ましくは、０．０００５〜１重量％、より好ましくは、０．００１〜０．１重量％である。 When the medium addition factor of the present invention is used as a cryoprotectant, it is based on a buffer solution such as PBS, a culture medium used for culture, serum, or the like, which is improved by adding salts, saccharides, amino acids, etc. Further, fructan is added to those added with factors such as 5 to 20% by volume of DMSO and glycerin. The content of the medium addition factor of the present invention in the cell frozen solution is usually 0.0001 to 10% by weight, preferably 0.0005 to 1% by weight, more preferably 0.001 to 1, as the amount of active ingredient. 0.1% by weight.
また、凍結細胞の回復時の培養液に本発明の培地添加因子を含む培地を用いれば、解凍操作における細胞死の抑制および速やかな細胞状態の回復を達成することができる。凍結細胞の回復時の培養液に含める本発明の培地添加因子の濃度は、上述の細胞培養に用いる培地中の濃度と同様でよい。 In addition, if a medium containing the medium addition factor of the present invention is used as a culture solution when frozen cells are recovered, cell death can be suppressed and rapid cell state recovery can be achieved in the thawing operation. The concentration of the medium-added factor of the present invention included in the culture solution at the time of recovery of the frozen cells may be the same as the concentration in the medium used for the cell culture described above.
本明細書中で挙げられた特許および特許出願明細書を含む全ての刊行物に記載された内容は、本明細書での引用により、その全てが明示されたと同程度に本明細書に組み込まれるものである。 The contents of all publications, including patents and patent application specifications cited in this specification, are hereby incorporated by reference herein to the same extent as if all were explicitly stated. Is.
以下に実施例を用いて本発明を詳述するが、本発明は以下の実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail using examples, but the present invention is not limited to the following examples.
〔試験例１〕ハイブリドーマ細胞の増殖促進効果
フルクタン（ラッキョウより熱水処理により抽出したフルクタン、あるいは特許第３１１１３７８号公報記載の方法によりラッキョウより調製されたフルクタン）はあらかじめ指定の濃度（０．００１重量％（１０μｇ/ｍｌ））になるようＲＰＭＩ１６４０培地に溶解させて添加し、抗体産生マウスハイブリドーマ細胞（東京大学の牧島房夫らによって樹立された）を２．６×１０４（cells/ml）の細胞密度で２４ウェル培養プレートに播種した。細胞は５％容量ＣＯ２−９５％空気の雰囲気下、３７℃で２日間培養した。細胞密度は通常の血球計算盤で計測した。
[Test Example 1] Hybridoma cell growth promoting effect Fructan (fructan extracted from Rakkyo by hot water treatment or fructan prepared from Rakkyo by the method described in Japanese Patent No. 311378) has a predetermined concentration (0.001 wt. % (10 μg / ml) and dissolved in RPMI 1640 medium, and antibody-producing mouse hybridoma cells (established by Fukuo Makishima et al., University of Tokyo) of 2.6 × 10 4 (cells / ml) Seeded in 24-well culture plates at cell density. The cells were cultured for 2 days at 37 ° C. in an atmosphere of 5% CO 2 -95% air. Cell density was measured with a normal hemocytometer.
この結果、フルクタン添加によって浮遊性細胞であるハイブリドーマ細胞の増殖が強く促進される事が明らかとなった。 As a result, it has been clarified that the addition of fructan strongly promotes the growth of hybridoma cells, which are suspension cells.
〔試験例２〕ＨｅｐＧ２細胞の増殖促進効果
フルクタンはあらかじめ指定の濃度（０．００１重量％（１０μｇ/ｍｌ））になるようＤＭＥＭ培地に溶解させて添加し、ヒトの肝由来の細胞で、バイオ人工肝臓にも利用されているＨｅｐＧ２細胞（理研細胞バンク）を３．１×１０４（cells/ml）の細胞密度で２４ウェル培養プレートに播種した。細胞は５％容量ＣＯ２−９５％空気の雰囲気下、３７℃で４日間培養した。細胞密度は、トリプシン処理によって培養皿より剥離した後に、血球計算盤で計測した。
[Test Example 2] Growth-promoting effect of HepG2 cells Fructan was dissolved in DMEM medium at a specified concentration (0.001% by weight (10 μg / ml)) in advance and added to cells derived from human liver. HepG2 cells (RIKEN Cell Bank), which is also used for artificial livers, were seeded in 24-well culture plates at a cell density of 3.1 × 10 4 (cells / ml). The cells were cultured for 4 days at 37 ° C. in an atmosphere of 5% CO 2 -95% air. The cell density was measured with a hemocytometer after peeling from the culture dish by trypsin treatment.
また、１×１０５個のＨｅｐＧ２細胞に対して、無添加、０．０００５重量％、０．００１重量％、０．００２重量％のフルクタンを添加して２日間培養したところ、それぞれ、３．９８×１０５個、４．３４×１０５個、４．４２×１０５個、４．１５×１０５個の細胞数が得られた。 Further, 1 × 10 5 HepG2 cells were added with no addition, 0.0005 wt%, 0.001 wt% and 0.002 wt% fructan and cultured for 2 days. A cell number of 98 × 10 5 cells, 4.34 × 10 5 cells, 4.42 × 10 5 cells, and 4.15 × 10 5 cells was obtained.
この結果、フルクタンは培地に添加する事で、付着性細胞である、ＨｅｐＧ２の増殖を促進する事が明らかとなった。 As a result, it was clarified that fructan promotes the growth of HepG2, which is an adherent cell, when added to the medium.
〔試験例３〕ＢＨＫ細胞の増殖
フルクタンはあらかじめ指定の濃度（０．００１重量％（１０μｇ/ｍｌ））になるようＤＭＥＭ培地に溶解させて添加し、ベービーハムスターの腎臓由来の細胞で、医薬品など生理活性の高い組み換えタンパク質の生産にひろく利用されているＢＨＫ細胞（American Type Culture Collection (ATCC)）を２．４×１０４（cells/ml）の細胞密度で２４ウェル培養プレートに播種した。細胞は５％容量ＣＯ２−９５％空気の雰囲気下、３７℃で３日間培養した。細胞密度は、トリプシン処理によって培養皿より剥離した後に、血球計算盤で計測した。
[Test Example 3] Proliferation of BHK cells Fructan is dissolved in DMEM medium at a specified concentration (0.001% by weight (10 μg / ml)) in advance, and is a cell derived from the kidney of a baby hamster. BHK cells (American Type Culture Collection (ATCC)) widely used for the production of recombinant proteins with high biological activity were seeded in a 24-well culture plate at a cell density of 2.4 × 10 4 (cells / ml). The cells were cultured for 3 days at 37 ° C. in an atmosphere of 5% volume CO 2 -95% air. The cell density was measured with a hemocytometer after peeling from the culture dish by trypsin treatment.
この結果、フルクタンを添加した培地は、有用物生産に利用される細胞の増殖を促進した。 As a result, the medium supplemented with fructan promoted the growth of cells used for production of useful substances.
〔試験例４〕冷凍保護効果
動物細胞を冷凍保存する際、凍結保存中での細胞死滅に対するフルクタンの効果を調査したところ、細胞生存の改善が認められた。
通常動物細胞を冷凍保存する方法は一般的であるが、その際に凍結保存中での細胞死滅、および凍結操作・解凍操作に伴う細胞死滅が問題になる。
ＰＢＳに、終濃度１０％になるようＤＭＳＯを添加し、さらにフルクタンを０．０１％（１００μｇ/ｍｌ）になるよう添加したものを凍結液としてハイブリドーマ細胞を−８０℃で１週間凍結保存した。解凍し培養したところ、フルクタン無添加の対照凍結液で凍結・解凍した細胞と比較すると、フルクタン凍結液を用いた場合には、解凍３日後には、３倍の生細胞数が得られた。
本試験によりフルクタンは培地だけでなく細胞凍結液に加える事で、細胞の生存率を高め、冷凍保護効果を示す事が明らかとなった。
また、解凍後の培養液にフルクタンを添加することも、解凍操作における細胞死を抑制し、速やかに細胞の状態が回復する効果が認められた。
[Test Example 4] Cryoprotective effect When cryopreserving animal cells, the effect of fructan on cell killing during cryopreservation was investigated, and an improvement in cell survival was observed.
Usually, a method for cryopreserving animal cells is common, but at that time, cell death during cryopreservation and cell death associated with freezing and thawing operations become problems.
DMSO was added to PBS to a final concentration of 10%, and fructan was further added to 0.01% (100 μg / ml), and the hybridoma cells were frozen and stored at −80 ° C. for 1 week. When thawed and cultured, compared to cells frozen and thawed with a control frozen solution without addition of fructan, three times as many viable cells were obtained after 3 days of thawing when the fructan frozen solution was used.
This test revealed that fructan was added not only to the medium but also to the cell frozen solution to increase the cell viability and show a cryoprotective effect.
In addition, addition of fructan to the culture solution after thawing was also effective in suppressing cell death in the thawing operation and quickly recovering the cell state.
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