JP2890447B2 - Animal cell culture method - Google Patents
Animal cell culture methodInfo
- Publication number
- JP2890447B2 JP2890447B2 JP1084948A JP8494889A JP2890447B2 JP 2890447 B2 JP2890447 B2 JP 2890447B2 JP 1084948 A JP1084948 A JP 1084948A JP 8494889 A JP8494889 A JP 8494889A JP 2890447 B2 JP2890447 B2 JP 2890447B2
- Authority
- JP
- Japan
- Prior art keywords
- cells
- cell
- animal cell
- voltage
- cell culture
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 210000004102 animal cell Anatomy 0.000 title claims description 14
- 238000004113 cell culture Methods 0.000 title 1
- 238000000034 method Methods 0.000 claims description 11
- 238000012258 culturing Methods 0.000 claims description 7
- 230000001419 dependent effect Effects 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 description 20
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 7
- 229910052697 platinum Inorganic materials 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000010408 film Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 229910021607 Silver chloride Inorganic materials 0.000 description 3
- 230000007910 cell fusion Effects 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 2
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001940 conductive polymer Polymers 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000029058 respiratory gaseous exchange Effects 0.000 description 2
- 239000010935 stainless steel Substances 0.000 description 2
- 229910001220 stainless steel Inorganic materials 0.000 description 2
- 239000010409 thin film Substances 0.000 description 2
- 102000012406 Carcinoembryonic Antigen Human genes 0.000 description 1
- 108010022366 Carcinoembryonic Antigen Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 229910045601 alloy Inorganic materials 0.000 description 1
- 239000000956 alloy Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 206010017758 gastric cancer Diseases 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 239000010931 gold Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 229910052741 iridium Inorganic materials 0.000 description 1
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 201000000050 myeloid neoplasm Diseases 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229920000767 polyaniline Polymers 0.000 description 1
- 229920000414 polyfuran Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920000128 polypyrrole Polymers 0.000 description 1
- 229920000123 polythiophene Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 （産業上の利用分野） 本発明は、動物細胞の培養方法に関する。Description: TECHNICAL FIELD The present invention relates to a method for culturing animal cells.
（従来の技術） 従来、細胞融合の分野においては、細胞の懸濁液の中
に、作用極・対極を設置し、１〜10KV/cmの高電圧を印
加することにより、細胞の融合を促進させる方法が知ら
れている。ところが、この方法は細胞融合を起こさせる
べく、１〜10KV/cmという高電圧をかけるため、単に細
胞の生体反応（増殖、代謝、呼吸、産生等）を促進する
という目的においてはその電圧は強すぎてしまい、細胞
自体が大きくダメージを受ける。(Prior art) Conventionally, in the field of cell fusion, a cell suspension is provided with a working electrode and a counter electrode, and a high voltage of 1 to 10 KV / cm is applied to promote cell fusion. A method for causing this to occur is known. However, in this method, a high voltage of 1 to 10 KV / cm is applied in order to cause cell fusion. Therefore, the voltage is high only for the purpose of promoting the biological reactions (proliferation, metabolism, respiration, production, etc.) of cells. Too much and the cells themselves are severely damaged.
又、微生物を電導性薄膜に固定化して、微弱電圧を印
加することにより、その生体反応を促進する方法が知ら
れているが、この方法は強固な細胞壁を有する微生物に
は可能であっても、動物細胞のように、細胞壁を持たな
い脆弱な細胞については極めて困難である。Further, a method of immobilizing microorganisms on a conductive thin film and applying a weak voltage to promote the biological reaction is known, but this method is possible even for microorganisms having strong cell walls. However, it is extremely difficult for a fragile cell having no cell wall, such as an animal cell.
（発明の目的） 本発明の目的は、付着依存性動物細胞を付着・増殖さ
せた足場に微弱電圧を印加することによりその生体反応
（細胞膜の透過、増殖、代謝、呼吸、産生等の複雑な機
能）を促進あるいは制御する方法を提供することにあ
る。(Object of the Invention) An object of the present invention is to apply a weak voltage to a scaffold to which attachment-dependent animal cells have been attached and proliferated, to thereby perform the biological reactions (complexities such as cell membrane permeation, proliferation, metabolism, respiration, and production). Function) is provided.
（発明の構成） 本発明は、付着依存性動物細胞を電導性を有する足場
に付着させ、該足場に微弱電圧を印加して該動物細胞を
培養する方法から成る。(Constitution of the Invention) The present invention comprises a method of adhering adhesion-dependent animal cells to a scaffold having electrical conductivity, and culturing the animal cells by applying a weak voltage to the scaffold.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
まず、本発明に係わる付着依存性動物細胞としては、
ヒト消化器系の癌胎児性抗原CEA（Carcino Embryonic A
ntigen）産生細胞、CHO、BHK、Vero、サルCV1、サルCO
S、マウスＬ、マウスC127、マウス3T3、HeLa、ヒト・ミ
エローマ等の細胞及びこれらの細胞から由来する細胞が
挙げられる。First, as the adhesion-dependent animal cell according to the present invention,
Human digestive system carcinoembryonic antigen CEA (Carcino Embryonic A
ntigen) Producing cells, CHO, BHK, Vero, monkey CV1, monkey CO
Cells such as S, mouse L, mouse C127, mouse 3T3, HeLa, and human myeloma, and cells derived from these cells.
付着・増殖させる足場としては、電導性を有するもの
であれば材質は特に限定されないが、一般には例えばガ
ラス又はプラスチック表面に白金、金、銀、イリジウム
等の金属及びそれらの合金を蒸着させたものや、アルミ
ニウム、ステンレスなどの金属表面に、基盤との接着性
の優れた強固で微生物に対して毒性のない電導性ポリマ
ー（例えば、ポリピロール、ポリチオフェン、ポリフラ
ン等の複素環式重合物やポリアニリン群）のモノマーを
電気化学重合法などの方法を用いて、コーティングを施
したものを用いればよい。The material of the scaffold to be attached and multiplied is not particularly limited as long as it has electrical conductivity, but generally, for example, a metal such as platinum, gold, silver, and iridium and an alloy thereof are deposited on a glass or plastic surface. Conductive polymers that have excellent adhesion to the substrate and are non-toxic to microorganisms, such as aluminum, stainless steel, and other metal surfaces (for example, heterocyclic polymers such as polypyrrole, polythiophene, and polyfuran, and polyanilines). What is necessary is just to use the thing which coated the monomer using the method, such as an electrochemical polymerization method.
微弱電圧は、例えば白金電極を作用極（足場の電導部
に接続）として、もう一方の白金電極を対極（培養液
中）として印加することが出来る。この場合、Ag−AgCl
電極を参照電極（蒸着膜やコーティング膜の極近傍に設
置）とすることにより、印加する微弱電圧を精密にコン
トロールできる。The weak voltage can be applied, for example, using a platinum electrode as a working electrode (connected to a conductive portion of a scaffold) and the other platinum electrode as a counter electrode (in a culture solution). In this case, Ag-AgCl
By using the electrode as a reference electrode (located very close to the deposited film or the coating film), the applied weak voltage can be precisely controlled.
印加する電圧は、０〜＋1V、好ましくは＋0.4〜＋0.6
V（vs/Ag−AgCl）とする。培養温度は、20〜50℃で好ま
しくは由来する動物の体温付近例えば35〜40℃とする。The applied voltage is 0 to +1 V, preferably +0.4 to +0.6
V (vs / Ag-AgCl). The culturing temperature is preferably 20 to 50 ° C., preferably around the body temperature of the animal from which it originated, for example, 35 to 40 ° C.
培養液（培地）は電解質を含むものであれば、天然培
地でも、合成培地でもよい。The culture solution (medium) may be a natural medium or a synthetic medium as long as it contains an electrolyte.
このように電圧を印加して培養を行なうと、電圧を印
加しない場合と比較して、一般には増殖速度を低下させ
ることができ、その結果細胞１ケ当たりの産生物の産生
速度を20〜30倍に増加させることが可能となる。When the culture is performed by applying a voltage in this manner, the growth rate can be generally reduced as compared with the case where no voltage is applied, and as a result, the production rate of the product per cell is reduced by 20 to 30. It is possible to increase by a factor of two.
（実施例） 以下に、実施例を示し、本発明の動物細胞の培養法に
ついて更に詳細に説明するが、本発明はその要旨を越え
ない限り以下の実施例に限定されるものではない。(Examples) Hereinafter, examples will be shown, and the method for culturing animal cells of the present invention will be described in more detail. However, the present invention is not limited to the following examples unless it exceeds the gist thereof.
実施例 白金を蒸着したプラスチックプレート（30m/mφ）
に、培養液（TS2培地＋10％FCS）を加え、CEA産生細胞
であるヒト胃癌細胞MKN45を分散させ、CO2インキュベー
ター内で、数時間培養し、細胞を十分プレートに接着さ
せた後、培地を交換して所定の電圧を印加しながら、さ
らに37℃で116時間培養した。途中、経時的に細胞の数
とCEA産生量を測定した（CEAの測定は、ダイナボット
（株）製のCEA・EIA（Enzyme Immuno Assay）ワンステ
ップ法による。） 実験装置の概略を図１に示す。Example Plastic plate with platinum deposited (30m / mφ)
Then, a culture solution (TS2 medium + 10% FCS) was added thereto, and human gastric cancer cells MKN45, which is a CEA-producing cell, were dispersed. The cells were cultured in a CO 2 incubator for several hours, and the cells were sufficiently adhered to a plate. The cells were further cultured at 37 ° C. for 116 hours while replacing and applying a predetermined voltage. The number of cells and the amount of CEA production were measured over time during the course of the measurement (the CEA was measured by the CEA-EIA (Enzyme Immuno Assay) one-step method manufactured by Dynabot Co., Ltd.). Show.
各々の電圧に対して、116時間後の細胞の数を図２に
示し、細胞１ケ当たりのCEA産生速度の印加時と無印加
時の比率（印加時での速度／無印加時での速度）を図３
に示す。プラス側の電位0.4〜0.5V（vs.Ag−AgCl）の範
囲で電気制御培養を行なった場合に、細胞の増殖が抑え
られ、反対に細胞１ケ当たりのCEA産生量は、無印加時
の20〜30倍と増加した。The number of cells after 116 hours for each voltage is shown in FIG. 2, and the ratio of the rate of CEA production per cell between the time of application and the time of no application (speed at the time of application / speed at the time of no application) 3)
Shown in When the electro-controlled culture is performed in the positive potential range of 0.4 to 0.5 V (vs. Ag-AgCl), cell growth is suppressed, and conversely, the amount of CEA produced per cell is Increased 20 to 30 times.
（発明の効果） 本発明によれば、白金等の金属蒸着膜や、アルミ、ス
テンレス等の金属基盤に、電導性ポリマーをコーティン
グした薄膜上に、付着依存性動物細胞を、微弱電圧を印
加しながら、培養することにより、動物細胞各々に均等
に電圧を印加することができ、産生物の産生を促進する
ことが可能である。この理由は、審かではないが、印加
電圧を調節することにより、動物細胞の複雑なメカニズ
ムを持つ生体反応に何らかの変化が生じるためと考えら
れる。(Effect of the Invention) According to the present invention, an adhesion-dependent animal cell is applied to a thin film obtained by coating a conductive polymer on a metal deposited film of platinum or the like, or a metal base such as aluminum or stainless steel. Meanwhile, by culturing, it is possible to apply a voltage evenly to each of the animal cells, and it is possible to promote the production of a product. The reason for this is not a question, but it is considered that adjusting the applied voltage causes some change in the biological reaction of the animal cell having a complicated mechanism.
図１は、実施例で使用した培養装置の概略を示す図であ
り、図中で１は作用極、２は対極、３は参照極、４は培
養液（培地）、５は白金蒸着膜および６は細胞を示す。 図２は、実施例における116時間後の細胞の数とその時
の印加電圧を示す図である。 図３は、実施例における116時間後の細胞１ケ当たりのC
EA産生速度比（印加時の速度／無印加時の速度）とその
時の印加電圧を示す図である。FIG. 1 is a diagram schematically showing a culture apparatus used in Examples, in which 1 is a working electrode, 2 is a counter electrode, 3 is a reference electrode, 4 is a culture solution (medium), 5 is a platinum-deposited film and 6 indicates cells. FIG. 2 is a diagram showing the number of cells after 116 hours and the applied voltage at that time in the example. FIG. 3 shows C per cell after 116 hours in Examples.
It is a figure which shows EA production speed ratio (speed at the time of application / speed at the time of no application), and the applied voltage at that time.
Claims (1)
場に付着させ、該足場に微弱電圧を印加して、該動物細
胞を培養することを特徴とする動物細胞培養方法。1. A method for culturing an animal cell, comprising attaching an adhesion-dependent animal cell to a scaffold having electrical conductivity, applying a weak voltage to the scaffold, and culturing the animal cell.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1084948A JP2890447B2 (en) | 1989-04-04 | 1989-04-04 | Animal cell culture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1084948A JP2890447B2 (en) | 1989-04-04 | 1989-04-04 | Animal cell culture method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02265476A JPH02265476A (en) | 1990-10-30 |
| JP2890447B2 true JP2890447B2 (en) | 1999-05-17 |
Family
ID=13844862
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1084948A Expired - Lifetime JP2890447B2 (en) | 1989-04-04 | 1989-04-04 | Animal cell culture method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2890447B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP5648453B2 (en) * | 2010-12-06 | 2015-01-07 | 大日本印刷株式会社 | Cell test substrate with auxiliary electrode |
| JP2016111980A (en) * | 2014-12-17 | 2016-06-23 | 大日本印刷株式会社 | Cell selection method |
-
1989
- 1989-04-04 JP JP1084948A patent/JP2890447B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02265476A (en) | 1990-10-30 |
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