JPH04148679A - Cell culture - Google Patents
Cell cultureInfo
- Publication number
- JPH04148679A JPH04148679A JP2270719A JP27071990A JPH04148679A JP H04148679 A JPH04148679 A JP H04148679A JP 2270719 A JP2270719 A JP 2270719A JP 27071990 A JP27071990 A JP 27071990A JP H04148679 A JPH04148679 A JP H04148679A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- amino acids
- serum
- cell culture
- cells
- 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.)
- Pending
Links
- 238000004113 cell culture Methods 0.000 title claims abstract description 13
- 235000001014 amino acid Nutrition 0.000 claims abstract description 21
- 150000001413 amino acids Chemical class 0.000 claims abstract description 21
- 210000002966 serum Anatomy 0.000 claims abstract description 19
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 claims abstract description 6
- 235000018417 cysteine Nutrition 0.000 claims abstract description 6
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229960003067 cystine Drugs 0.000 claims abstract description 6
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims abstract description 5
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims abstract description 5
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 claims abstract description 4
- 239000001963 growth medium Substances 0.000 claims description 5
- 210000004962 mammalian cell Anatomy 0.000 claims description 5
- 238000012258 culturing Methods 0.000 claims description 4
- 230000000694 effects Effects 0.000 abstract description 7
- 230000001988 toxicity Effects 0.000 abstract 1
- 231100000419 toxicity Toxicity 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 13
- 239000003797 essential amino acid Substances 0.000 description 10
- 235000020776 essential amino acid Nutrition 0.000 description 10
- 239000007758 minimum essential medium Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 231100000331 toxic Toxicity 0.000 description 6
- 230000002588 toxic effect Effects 0.000 description 6
- 239000000203 mixture Substances 0.000 description 5
- 235000013343 vitamin Nutrition 0.000 description 5
- 239000011782 vitamin Substances 0.000 description 5
- 229940088594 vitamin Drugs 0.000 description 5
- 229930003231 vitamin Natural products 0.000 description 5
- 206010052428 Wound Diseases 0.000 description 4
- 208000027418 Wounds and injury Diseases 0.000 description 4
- 206010003210 Arteriosclerosis Diseases 0.000 description 3
- 208000011775 arteriosclerosis disease Diseases 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000007792 addition Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 210000003556 vascular endothelial cell Anatomy 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 239000003145 cytotoxic factor Substances 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 230000002900 effect on cell Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 230000035876 healing Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Description
【発明の詳細な説明】
〔産業上の利用分野〕
本発明は、哺乳類の正常細胞を培養するための細胞培養
方法に関する。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a cell culture method for culturing normal mammalian cells.
〔従来の技術、発明が解決しようとする課題〕一般に、
哺乳類の正常細胞を培養するためには、１０〜２０％程
度の血清を培養液中に添加するのが通常である。しかし
ながら血清濃度を一定以上に上昇させると、培養液は細
胞に対して毒性を持つようになる。これは、血清中に細
胞に対する毒性因子が存在していることを示唆している
。[Prior art and problems to be solved by the invention] Generally,
In order to culture normal mammalian cells, approximately 10 to 20% serum is usually added to the culture medium. However, when the serum concentration increases beyond a certain level, the culture solution becomes toxic to cells. This suggests the presence of cytotoxic factors in the serum.
本発明は、かかる点に鑑み、一定量以上の血清を培養液
中に加えても培養効果を増進させることができるような
細胞培養方法を提供することを目的とする。In view of this, an object of the present invention is to provide a cell culture method that can enhance the culture effect even when a certain amount or more of serum is added to the culture medium.
そこで、本発明は哺乳類の正常細胞を培養する培養液中
に血清を添加し、この血清中にアミノ酸を添加するよう
に構成した。Therefore, the present invention is configured such that serum is added to a culture medium for culturing normal mammalian cells, and amino acids are added to this serum.
所定量の血清にアミノ酸を所定量添加して培養液中に添
加する。このアミノ酸としては、システィン、シスチン
が特に好ましく、トリプトファンでもよく、更にはこれ
らのアミノ酸の混合したものでもよい。A predetermined amount of amino acids is added to a predetermined amount of serum and added to the culture solution. As this amino acid, cysteine and cystine are particularly preferable, tryptophan may be used, and a mixture of these amino acids may also be used.
これらのアミノ酸を血清中に加えると、血清を培養液中
に加えても血清中に存在する細胞に対する毒性因子の毒
性効果が失われ、細胞培養が促進される。Addition of these amino acids to serum eliminates the toxic effects of toxic factors on cells present in serum and promotes cell culture even when serum is added to the culture medium.
この事実から、生体内の血管内皮細胞と動脈硬化につい
て次のような推論が成り立つ。血管内皮細胞は常に１０
０％血液に晒されながら機能している。これはこの細胞
だけが毒性因子に耐性を持つためと考えられる。動脈硬
化を起こした血管では、その進行にともない、ついには
内皮細胞がはがれて内部の結合組織が直接血流に接する
ことになる。この部分は修復不可能とされている。この
原因は、血清中の毒性因子によるものとおもわれる。こ
の際上記アミノ酸の一種、又は混合物を投与すれば、修
復が可能となるはずである。From this fact, the following inferences can be made regarding vascular endothelial cells and arteriosclerosis in vivo. Vascular endothelial cells are always 10
It functions while being exposed to 0% blood. This is thought to be because only these cells are resistant to toxic factors. As arteriosclerosis progresses, the endothelial cells of arteriosclerotic blood vessels eventually become detached and the connective tissue inside comes into direct contact with the blood flow. This part is considered irreparable. This is thought to be caused by toxic factors in the serum. At this time, repair should be possible by administering one or a mixture of the above amino acids.
また、傷創時には周囲の血管が破壊され、傷創部は上記
のような状態となる。従って血清中の毒性因子が傷創治
癒を遅延させる原因となっていることが予想される。上
記アミノ醸成いはその混合物を傷創部に投与すれば治癒
効果を著しく向上させることが期待される。Further, when a wound occurs, surrounding blood vessels are destroyed, and the wound area becomes in the above-mentioned state. Therefore, it is expected that toxic factors in serum are responsible for delaying wound healing. It is expected that administering the above amino acid mixture to the wound site will significantly improve the healing effect.
細胞培養のためにはＭＥＭ （最小限必要培地）が必要
であり、ＭＥＭ中においてアミノ酸、非必須アミノ酸、
ビタミン、ピルビン酸ナトリウム等の濃度を高くしたり
、また新たに添加して培養するときに以下に示すＭＥＭ
用補助培地が使用され、その例としては次頁の表１に示
すものが使用される。MEM (minimum essential medium) is required for cell culture, and in MEM, amino acids, non-essential amino acids,
When increasing the concentration of vitamins, sodium pyruvate, etc., or when culturing with new additions, use the MEM shown below.
Supplementary media are used, examples of which are shown in Table 1 on the next page.
なお、５０ｘＭＥＭ、１００１０Ｏｘは上記濃度の５０
倍量、１００倍量を含むことを意味する。Note that 50xMEM and 10010Ox are 50xMEM and 10010Ox at the above concentration.
It means double the amount and 100 times the amount.
第１図は、培養皿（ｗｅｌｌ）中に６０００個の細胞を
接種し必須アミノ酸、ビタミンおよびビタミンと必須ア
ミノ酸との混合液を血液９０％、残りＭＥＭからなる混
合液中にそれぞれの濃度（％）だけ加えたときの細胞の
増加状態を示すものであり、何も加えない場合には細胞
数が２５００位に減少するが、必須アミノ酸を加えた場
合には著しく増大することが判る。Figure 1 shows that 6,000 cells were inoculated into a culture dish (well), and essential amino acids, vitamins, and a mixture of vitamins and essential amino acids were added to a mixture of 90% blood and MEM at various concentrations (%). ) shows the state of increase in cells when nothing is added, and it can be seen that the number of cells decreases to 2,500 when nothing is added, but increases significantly when essential amino acids are added.
第２図は、必須アミノ酸液（ＦＡＡ）と非必須アミノ酸
液（Ｎ Ｅ Ａ Ａ） グルタミン液（Ｇ Ｌ Ｎ）
をそれぞれ濃度を異ならせて（０，１μｍ、２μｍ、４
μＩ）でＭＥＭに添加したときの細胞の培養状態を示す
ものであり、これによれば必須アミノ酸液（ＦＡＡ）の
４μｌ／ｗｅｌｌの場合に細胞数は著しく増大し、非必
須アミノ酸（ＮＥＡＡ）、グルタミンは細胞を死滅させ
ることが判る。Figure 2 shows essential amino acid solution (FAA), non-essential amino acid solution (NEAA), and glutamine solution (GLN).
with different concentrations (0, 1 μm, 2 μm, 4 μm).
This shows the culture state of cells when 4 μl/well of essential amino acid solution (FAA) was added to MEM. Glutamine is known to kill cells.
第３図は必須アミノ酸中のどのアミノ酸が細胞培養に寄
与するかを個別に測定したものである。FIG. 3 shows individual measurements of which amino acids among the essential amino acids contribute to cell culture.
細胞接種後（５０００個／ｗｅｌｌ）、血清を９０μｌ
／ｗｅｌｌで分注、そこへ、それぞれのアミノ酸をＤｕ
ｌｂｅｃｃｏｓ’ ＰＢＳ （リン酸緩衝液生理食塩
水）で希釈したものを、最終濃度がＯ，０，１０，５，
１，０，１０ｍＭとなるように１０μｌ／ｗｅｌｌ添加
した。その３日後に、ｗｅｌｌ中の細胞質の量を測定し
た結果である。ただし、シスチン、トリプトファン、チ
ロシンは溶解度の関係上、最終濃度を１０ｍＭにするこ
とが不可能であり、飽和濃度を１００ｍＭとし他のアミ
ノ酸と同様に扱った。これによれば、システィン（Ｃｙ
ｓＨ）、シスチン（Ｃｙｓ）は著しく細胞培養に効果が
あり、トリプトファン（Ｔｒｐ）は細胞の死滅を防止し
ていることが判る。After cell inoculation (5000 cells/well), add 90 μl of serum.
/well, and add each amino acid to it.
lbeccos' diluted with PBS (phosphate buffered saline) to a final concentration of O, 0, 10, 5,
10 μl/well was added at concentrations of 1, 0, and 10 mM. This is the result of measuring the amount of cytoplasm in the well 3 days later. However, cystine, tryptophan, and tyrosine could not have a final concentration of 10 mM due to their solubility, so they were treated in the same way as other amino acids with a saturation concentration of 100 mM. According to this, cysteine (Cy
sH) and cystine (Cys) are significantly effective for cell culture, and tryptophan (Trp) is found to prevent cell death.
第４図はＰＢＳで希釈することなく、直接４種類のアミ
ノ酸を血清中に最終濃度がグラフ上に示した値となるよ
うに加え、細胞を５千個接種して３日後にその細胞数を
測定した結果を示すものである。第４図によれば、シス
ティン、シスチンが培養効果が、特にシスティンを１．
０ｍＭ加えたときには細胞数が３倍にも増大することが
判る。Figure 4 shows that four types of amino acids were directly added to serum without dilution with PBS so that the final concentration would be the value shown on the graph, 5,000 cells were inoculated, and the number of cells was counted 3 days later. This shows the measured results. According to FIG. 4, cysteine and cystine have a culture effect, especially cysteine.
It can be seen that when 0mM was added, the number of cells increased three times.
本発明は、以上のように構成したので、哺乳類の正常細
胞を著しく培養増加させることができ、動脈硬化を防止
することができ、傷創時の血管の破壊の修復にも寄与し
うるものと思われる。Since the present invention is configured as described above, it is possible to significantly increase the culture of normal mammalian cells, prevent arteriosclerosis, and contribute to the repair of blood vessel destruction caused by wounds. Seem.
【図面の簡単な説明】
第１図は必須アミノ酸、ビタミンおよびビタミンとアミ
ノ酸を血清に加えたときにその加えた濃度による細胞培
養への影響を示すグラフ、第２図はＥＡＡ、ＮＥＡＡお
よびグルタミンのＭＥＭアミノ酸補助培地添加結果を示
すグラフ、第３図は各種アミノ酸の細胞培養への影響を
示すグラフ、第４図は４種類の必須アミノ酸の最終濃度
を変化させた場合の細胞培養に対する影響を示すグラフ
である。[Brief explanation of the drawings] Figure 1 is a graph showing the effect on cell culture of essential amino acids, vitamins, and the concentration of vitamins and amino acids added to serum. Graph showing the results of adding MEM amino acid supplementary medium, Figure 3 is a graph showing the effects of various amino acids on cell culture, and Figure 4 shows the effects on cell culture when the final concentrations of four essential amino acids were changed. It is a graph.
Claims (1)
し、この血清中にアミノ酸を添加することを特徴とする
細胞培養方法。 ２、前記アミノ酸はシステイン、シスチン又はトリプト
ファン又はこれらのアミノ酸の任意の組合せからなるこ
とを特徴とする請求項１記載の細胞培養方法。[Scope of Claims] 1. A cell culture method characterized by adding serum to a culture medium for culturing normal mammalian cells, and adding amino acids to this serum. 2. The cell culture method according to claim 1, wherein the amino acid consists of cysteine, cystine, tryptophan, or any combination of these amino acids.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2270719A JPH04148679A (en) | 1990-10-09 | 1990-10-09 | Cell culture |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2270719A JPH04148679A (en) | 1990-10-09 | 1990-10-09 | Cell culture |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH04148679A true JPH04148679A (en) | 1992-05-21 |
Family
ID=17490009
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2270719A Pending JPH04148679A (en) | 1990-10-09 | 1990-10-09 | Cell culture |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH04148679A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996005286A3 (en) * | 1994-08-16 | 1996-04-25 | Frontier Kk | Cell death accelerator and cell death inhibitor |
-
1990
- 1990-10-09 JP JP2270719A patent/JPH04148679A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996005286A3 (en) * | 1994-08-16 | 1996-04-25 | Frontier Kk | Cell death accelerator and cell death inhibitor |
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