JP2016119875A - Cell activator - Google Patents
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- JP2016119875A JP2016119875A JP2014262073A JP2014262073A JP2016119875A JP 2016119875 A JP2016119875 A JP 2016119875A JP 2014262073 A JP2014262073 A JP 2014262073A JP 2014262073 A JP2014262073 A JP 2014262073A JP 2016119875 A JP2016119875 A JP 2016119875A
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- DFPOZTRSOAQFIK-UHFFFAOYSA-N S,S-dimethyl-beta-propiothetin Chemical compound C[S+](C)CCC([O-])=O DFPOZTRSOAQFIK-UHFFFAOYSA-N 0.000 claims abstract description 40
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
本発明は、細胞賦活剤に関する。 The present invention relates to a cell activator.
現在、培養細胞は、医薬品（例えば、抗体、細胞成長因子、免疫活性物質など）の製造、医薬品研究用のツール、化粧品の評価などのように多分野に使用されている。細胞培地中には、細胞の成長・分裂を制御するために、各種の血清由来成分（例えば、アルブミン、インスリン、細胞成長因子など）を添加することが多い。このとき、添加される成分が少ないほど、培養細胞の成長・分裂のコントロール、未知成分由来の因子を制御できる可能性が高まるので、そのような代替物質を得ることが求められている（例えば、特許文献１、２）。
一方、ジメチルスルフォニオプロピオナート（dimethylsulfoniopropionate、以下、DMSPと省略する）は、スルホニウムの一種である有機硫黄化合物であり、ミズゴケやプランクトンが生産することが知られている。DMSPは、化学的に不安定であるため、その特性を調べた研究は少ないが、ニワトリ、金魚などに与えると、生体賦活作用を示すことが知られている（非特許文献１）。
Currently, cultured cells are used in many fields such as production of pharmaceuticals (for example, antibodies, cell growth factors, immunologically active substances, etc.), pharmaceutical research tools, and cosmetics evaluation. In order to control cell growth and division, various serum-derived components (eg, albumin, insulin, cell growth factor, etc.) are often added to the cell culture medium. At this time, the smaller the added component, the higher the possibility of controlling the growth and division of cultured cells and the control of factors derived from unknown components, so it is required to obtain such an alternative substance (for example, Patent Documents 1 and 2).
On the other hand, dimethylsulfoniopropionate (hereinafter abbreviated as DMSP) is an organic sulfur compound that is a kind of sulfonium, and it is known that sphagnum and plankton are produced. Since DMSP is chemically unstable, there are few studies investigating its characteristics, but it is known that when it is applied to chickens, goldfish, etc., it exhibits a biostimulatory effect (Non-patent Document 1).
しかしながら、DMSPが培養細胞に与える影響を調べた研究は認められず、その知見は不明であった。
本発明は、上記した事情に鑑みてなされたものであり、その目的は、細胞賦活剤、特に培養細胞の培地中に添加することで、細胞活性を増強させられるものを提供することである。
However, there were no studies examining the effect of DMSP on cultured cells, and the findings were unknown.
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a cell activator, particularly one that can enhance cell activity by being added to a culture medium of cultured cells.
本発明者は、鋭意検討の結果、ジメチルスルフォニオプロピオナート（dimethylsulfoniopropionate：DMSP）を培養細胞の培地中に添加することで、細胞活性を強力に増加させることを見出し、基本的には本発明を完成するに至った。
こうして、上記目的を達成するための発明に係る細胞賦活剤は、DMSPを主要成分として含むことを特徴とする。
DMSPは、示性式が(CH3)2S+CH2CH2COO-で示され、分子量が130.04の化合物である。海や湿地帯の植物やプランクトンが生産する。化学的に不安定で、硫酸塩エアロゾルと有機エアロゾルとに変わり、雲を発生させることが知られている。
本発明に係る細胞賦活剤は、培養細胞の培地中に添加することにより、その効果を発揮する。
As a result of intensive studies, the present inventor has found that adding dimethylsulfoniopropionate (DMSP) to the culture medium of cultured cells strongly increases the cell activity, and basically the present invention. It came to complete.
Thus, the cell activator according to the invention for achieving the above object includes DMSP as a main component.
DMSP is a compound having a molecular formula of (CH 3 ) 2 S + CH 2 CH 2 COO − and a molecular weight of 130.04. Produced by plants and plankton from the sea and wetlands. It is known that it is chemically unstable and generates a cloud instead of sulfate aerosol and organic aerosol.
The cell activator according to the present invention exhibits its effect when added to the culture medium of cultured cells.
また、他の発明に係る細胞の培養方法は、培地中にDMSPを添加して培養することを特徴とする。
このとき、培地中に添加させるDMSPの最終濃度は、0.4μg/ml以上であることが好ましい。本実施例によれば、436μg/mlまで濃度を上昇させても、細胞増殖を大きく阻害するような要因は認められなかった。但し、DMSPは不安定であることから、その機能は時間的に効果が減少する可能性がある。このため、DMSPを培地に添加するときには、一定時間毎（例えば、48時間〜72時間毎）に添加することが好ましい。
培養される細胞としては、特に限定されないが、例えば初代培養細胞、培養細胞株、組換培養細胞株などが例示される。また、由来としては、特に限定されないが、哺乳類（ヒト、チンパンジー、サル（カニクイザル、アカゲザル、アフリカミドリザル、ニホンザルを含む）、ウシ、ウマ、ブタ、ラクダ、シカ、イヌ、ネコ、ウサギ、ラット、マウス、チャイニーズハムスター、シリアンハムスターなど）、鳥類（ニワトリなど）、昆虫細胞などが例示される。なお、種の異なる２つ以上の細胞をハイブリッドさせて作製された細胞（例えば、抗体産生用ハイブリドーマなど）を用いることもできる。
また、細胞が由来する器官・組織としては、特に限定されないが、血球・リンパ系、血管系、脳・神経系、骨髄、筋組織、胸腺、唾液腺、口腔、食道、胃、肝臓、胆嚢、脾臓、小腸、大腸、直腸、皮膚、角膜、肺、甲状腺、哺乳器、子宮、子宮頸部、卵巣、精巣、膵臓、腎臓、副腎皮質、膀胱、胎盤、臍帯、胎仔、胎子、尾、間葉系幹細胞、ガン細胞（メラノーマ、テラトカルシノーマ、腹水、肉腫など）などが例示される。
Another cell culture method according to another invention is characterized in that DMSP is added to a medium and cultured.
At this time, the final concentration of DMSP added to the medium is preferably 0.4 μg / ml or more. According to this example, even when the concentration was increased to 436 μg / ml, no factor that greatly inhibited cell proliferation was observed. However, since DMSP is unstable, its function may be less effective over time. For this reason, when adding DMSP to a culture medium, it is preferable to add it for every fixed time (for example, every 48 hours-72 hours).
Although it does not specifically limit as a cell cultured, For example, a primary cultured cell, a cultured cell line, a recombinant cultured cell line etc. are illustrated. The origin is not particularly limited, but mammals (humans, chimpanzees, monkeys (including cynomolgus monkeys, rhesus monkeys, African green monkeys, Japanese monkeys), cattle, horses, pigs, camels, deer, dogs, cats, rabbits, rats, mice , Chinese hamsters, Syrian hamsters, etc.), birds (chicken etc.), insect cells and the like. A cell produced by hybridizing two or more cells of different species (for example, a hybridoma for producing an antibody) can also be used.
In addition, the organ / tissue from which the cell is derived is not particularly limited. Small intestine, large intestine, rectum, skin, cornea, lung, thyroid, mammal, uterus, cervix, ovary, testis, pancreas, kidney, adrenal cortex, bladder, placenta, umbilical cord, fetus, fetus, tail, mesenchymal system Examples include stem cells and cancer cells (melanoma, teratocarcinoma, ascites, sarcoma, etc.).
本発明によれば、細胞賦活剤、特に培養細胞の培地中に添加することで、細胞活性を増強させられるものを提供することが可能となる。 ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to provide the thing which can enhance cell activity by adding in a cell activator, especially the culture medium of a cultured cell.
次に、本発明の実施形態について、図表を参照しつつ説明するが、本発明の技術的範囲は、これらの実施形態によって限定されるものではなく、発明の要旨を変更することなく様々な形態で実施することができる。 Next, embodiments of the present invention will be described with reference to the drawings. However, the technical scope of the present invention is not limited by these embodiments, and various forms can be made without changing the gist of the invention. Can be implemented.
＜DMSPの効果確認試験１＞
１．試験方法
(1)被験細胞の培養
被験細胞として、ATCCから購入したCHO-DP12（抗ヒトインターロイキン8抗体を産生するCHO由来細胞）を使用した。この細胞を無血清培地A（HyClone CDM4CHO （サーモフィッシャーサイエンティフィック株式会社）、glutamine 4mM 及びメトトレキサレート（MTX、シグマアルドリッチ）200nMを含む）に馴化して用いた。
<DMSP effect confirmation test 1>
1. Test method
(1) Culture of test cells CHO-DP12 (CHO-derived cells producing anti-human interleukin 8 antibody) purchased from ATCC was used as test cells. The cells were used after acclimation to serum-free medium A (containing HyClone CDM4CHO (Thermo Fisher Scientific Co., Ltd.), glutamine 4 mM and methotrexate (MTX, Sigma Aldrich) 200 nM).
(2)MTTアッセイ
MTT細胞数測定キット（ナカライテスク）を用いて、細胞数を測定した。具体的には、次の方法に従った。
(i)96 well（ウエル）培養プレート（住友ベークライト）に、7000 cells/well (90μl)で細胞懸濁液を播種した。
(ii)各ウェルに、ろ過滅菌済みDMSP溶液（溶媒：超純水）を10μl添加し、ボルテックスにて混合した。DMSPは、最終濃度として、0, 0.436, 1.45, 4.36, 14.5, 43.6, 145及び436μg/mlとなるように添加した。各DMSP濃度について、サンプル数n=5とした。
(iii)培養プレート中の細胞を炭酸ガスインキュベーター（CO2 5.0%、36.5℃）内で培養した。
(iv)培養開始から、24時間後、48時間後及び72時間後に、インキュベーターから培養プレートを取り出し、各ウェルにMTT試薬（ナカライテスク）10μl添加してボルテックス混合した。
(v)炭酸ガスインキュベーター内で、２〜４時間保温した。
(vi)培養プレートを取り出し、各ウェルにイソプロピルアルコール（ナカライテスク）100μl添加し、フィルムでウェルを覆い、炭酸ガスインキュベーター内で16-20時間保温した。
(vii)マイクロプレートリーダー（サーモフィッシャーサイエンティフィック）で570 nmにおける吸光度を測定した。
(2) MTT assay
The number of cells was measured using an MTT cell count kit (Nacalai Tesque). Specifically, the following method was followed.
(i) The cell suspension was seeded on a 96-well culture plate (Sumitomo Bakelite) at 7000 cells / well (90 μl).
(ii) 10 μl of filter sterilized DMSP solution (solvent: ultrapure water) was added to each well and mixed by vortexing. DMSP was added to final concentrations of 0, 0.436, 1.45, 4.36, 14.5, 43.6, 145 and 436 μg / ml. For each DMSP concentration, the number of samples was n = 5.
(iii) The cells in the culture plate were cultured in a carbon dioxide incubator (CO2 5.0%, 36.5 ° C.).
(iv) 24 hours, 48 hours and 72 hours after the start of the culture, the culture plate was taken out from the incubator, and 10 μl of MTT reagent (Nacalai Tesque) was added to each well and mixed by vortexing.
(v) Insulated for 2 to 4 hours in a carbon dioxide incubator.
(vi) The culture plate was taken out, 100 μl of isopropyl alcohol (Nacalai Tesque) was added to each well, the well was covered with a film, and incubated for 16-20 hours in a carbon dioxide incubator.
(vii) Absorbance at 570 nm was measured with a microplate reader (Thermo Fisher Scientific).
２．試験結果
結果を表１〜表４及び図１〜図３に示した。
表１〜表３には、培養開始から24時間後、48時間後及び72時間後におけるDMSP濃度と570nmにおける吸光度を測定した結果（n=5）を示した。また、表４には、コントロール（DMSP添加なし（0 μg/ml））の吸光度を基準（1.000）とした場合の各DMSP濃度における吸光度（平均値）を相対値として示した。
図１〜図３には、培養開始から24時間後、48時間後及び72時間後におけるDMSP濃度と570nmにおける吸光度との関係をそれぞれ示した。
図１に示すように、DMSP添加なしを基準とした場合、DMSPを添加したサンプルの吸光度値は、全て高かった。また、Student's t検定を行った結果、培養時間に関わらず、濃度0.436μg/mlの時に有意差（P＜0.05）が確認された。
培養日数が経過するにつれて、DMSP添加したサンプルの吸光度値は、DMSP添加なしの吸光度値と比較して時の増加率が大きくなっていた。
以上の結果から、DMSP添加によりCHO-DP12の細胞増殖または代謝が活性化されたと考えられた。DMSPによる細胞賦活化は、少なくとも今回検討した濃度範囲では有効であり、培養72時間は有効であると考えられた。
Tables 1 to 3 show the results of measuring the DMSP concentration and the absorbance at 570 nm (n = 5) after 24 hours, 48 hours and 72 hours from the start of culture. In Table 4, the absorbance (average value) at each DMSP concentration when the absorbance of the control (no DMSP added (0 μg / ml)) was used as a reference (1.000) was shown as a relative value.
1 to 3 show the relationship between the DMSP concentration and the absorbance at 570 nm after 24 hours, 48 hours and 72 hours from the start of the culture, respectively.
As shown in FIG. 1, when no DMSP was added as a reference, the absorbance values of the samples to which DMSP was added were all high. In addition, as a result of Student's t test, a significant difference (P <0.05) was confirmed at a concentration of 0.436 μg / ml regardless of the culture time.
As the number of days of culture passed, the absorbance value of the sample added with DMSP increased with time compared to the absorbance value without DMSP added.
From the above results, it was considered that cell growth or metabolism of CHO-DP12 was activated by the addition of DMSP. Cell activation by DMSP was effective at least in the concentration range examined this time, and it was considered effective for 72 hours in culture.
このように、本実施形態によれば、培養細胞の培地中に添加することで、細胞活性を増強させられる細胞賦活剤を提供できた。 Thus, according to this embodiment, the cell activator which can enhance cell activity was able to be provided by adding in the culture medium of a cultured cell.
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