JPH0322972A - Serum-free culture medium - Google Patents

Abstract

PURPOSE:To obtain the novel title culture medium having good proliferation of gene recombinant Chinese hamster ovary-derived cell by adding a peptide like cell growth factor, hormone, lipid and binding factor to a serum-free culture medium for culturing animal cell. CONSTITUTION:The aimed culture medium obtained by adding a peptide cell growth factor (preferably fibroblast growth factor), hormone (preferably dexamethasone), lipid (preferably low-density lipoprotein) and/or binding factor (preferably fibronectin) to a serum-free culture medium for culturing animal cell containing no serum.

Description

Translated fromJapanese

【発明の詳細な説明】く産業上の利用分野〉本発明は、遺伝子組換えチャイニーズハムスター卵巣由
来細胞（以下ＣＨＯ細胞と略する）を培養する培地に関
するものである。DETAILED DESCRIPTION OF THE INVENTION Industrial Application Field The present invention relates to a medium for culturing genetically modified Chinese hamster ovary-derived cells (hereinafter abbreviated as CHO cells).

く従来の技術〉近年、インターフェロン等の有用な生理活性物質が数多
く発見され、医薬品としての応用が期待されている．こ
れらの複雑な構造の生理活性蛋白質を取得する手段とし
て動物細胞培養が注目されるようになってきた。そして
大量の物質を得るためには、遺伝子工学の手法を用い、
目的の外来遺伝子を導入した遺伝子組換え細胞の培養が
重要となってきた。現在、この遺伝子組換えの宿主とな
る動物細胞として最も広く用いられている細胞の１つに
４ＣＨＯ細胞テのジヒドロ葉酸還元酵素（ＤＨＰＲ）欠
損株（Ｃ　Ｈ　Ｏｄｈｆｒ−）がある。しかしこのＣ　
Ｈ　Ｏｄｈｆｒ一細胞を宿主とした遺伝子組換え細胞は
、培地に牛胎児血清〈以下ＦＣＳと略す〉等の血清を１
０％程度添加する必要があった。これらの血清は、非常
に高価である上原因不明のロソト差があり、大量かつ多
種類の異種蛋白を含んでいるため培養上清中の生理活性
蛋白を精製する際において大変困難を生じる。従って培
地中より血清を除く必要がある。現在、血清の代替とし
てインシュリン、トランスフエリン等のホルモンや血清
アルブミン等既知の物質を加えた無血清培地が開発され
、市販の無血清培地も数多く存在する．しかしながら、
これらの無血清培地において遺伝子組換えＣ　Ｈ　Ｏ細
胞は良好な増殖が得られず、今までに目的の無血清培地
は存在していない。Prior Art> In recent years, many useful physiologically active substances such as interferon have been discovered, and their application as pharmaceuticals is expected. Animal cell culture has been attracting attention as a means to obtain physiologically active proteins with these complex structures. In order to obtain large amounts of substances, genetic engineering techniques are used,
Culture of genetically modified cells into which a desired foreign gene has been introduced has become important. Currently, one of the most widely used animal cells as hosts for genetic recombination is the dihydrofolate reductase (DHPR)-deficient strain of 4CHO cells (C H Odhfr-). But this C
Genetically modified cells using H Odhfr cells as hosts are prepared by adding serum such as fetal bovine serum (hereinafter abbreviated as FCS) to the culture medium.
It was necessary to add about 0%. These sera are very expensive, have unexplained differences, and contain a large amount and many types of foreign proteins, making it very difficult to purify physiologically active proteins in the culture supernatant. Therefore, it is necessary to remove serum from the medium. Currently, serum-free media containing hormones such as insulin and transferrin, and known substances such as serum albumin have been developed as an alternative to serum, and there are many commercially available serum-free media. however,
Genetically modified C H O cells do not proliferate well in these serum-free media, and to date, no serum-free media of interest exists.

〈本発明の解決しようとする課題〉本発明の目的は、遺伝子組換えＣＨＯ細胞の良好に増殖
しうる新規な無血清培地を得ることにある。<Problems to be Solved by the Present Invention> An object of the present invention is to obtain a novel serum-free medium in which genetically modified CHO cells can proliferate favorably.

く課題を解決するための手段〉本発明者らは、前述の課題を解決すべ＜　Ｃ　Ｈ　０細
胞の増殖を支持する物質を探索した結果、目的の無血清
培地を調製することができた。Means for Solving the Problems> As a result of searching for a substance that supports the proliferation of C H 0 cells, the present inventors were able to prepare the desired serum-free medium.

本発明は、通常の実質的に血清を含まない動物細胞培養
用無血清培地に、１）ベプヂド性細胞戒長因子、２）ホ
ルモン、３）脂質、及び４）接着因子の内いずれかｌつ
以上を添加してなる、ｉｆｆ（ｉ子組換えＣＨＯ細胞培
養用無血清培地である。The present invention provides a method for adding any one of 1) veptidic cell lengthening factors, 2) hormones, 3) lipids, and 4) adhesion factors to a normal serum-free medium for animal cell culture that does not substantially contain serum. IF (serum-free medium for culturing recombinant CHO cells) is prepared by adding the above ingredients.

さて、本発明において実質的に血清を含まない培地とは
、通常のローズウエル・パーク・メモリアル・インステ
ィテユート１６４０　（　Ｒ　Ｐ　Ｍ　Ｉ　１６４０）
培地を単独または混合した培地に、インシュリン、トラ
ンスフェリンや、アルブミン等の血清蛋白威業基盤技術
チシンポジウム予稿集５３〜６６頁，（１９８Ｂ））、
ＨＢ１０２培地（Ｈａｎａ　Ｂｉｏｌｏｇｉｃｓ社製）
など及びこれらを改変したものである。Now, in the present invention, the substantially serum-free medium is the usual Rosewell Park Memorial Institute 1640 (RPMI 1640).
Serum proteins such as insulin, transferrin, and albumin may be added to the culture medium alone or in a mixed medium.
HB102 medium (manufactured by Hana Biologics)
etc. and modifications thereof.

また添加するベプチド性威長因子であるＦＧＦ濃度は、
培地あたり０．　１　　１　０　０ｎｇ／＠Ｊの範囲が
好ましい。In addition, the concentration of FGF, which is a peptide prestige factor, is
0 per medium. A range of 1 100 ng/@J is preferred.

ホルモンとして用いるＤＸＩＩ度は、培地あたり０．０
　５　　１　０　０ｎｇ／ｍｌの範囲が、Ｔ３の濃度は
、０．　１　　１　０　０ｎｇ／＋ｎ１の範囲が好まし
い。The DXII degree used as a hormone is 0.0 per medium.
The concentration of T3 is in the range of 0.5 100 ng/ml. A range of 1 100 ng/+n1 is preferred.

脂質として用いるＬＤＬの濃度は、培地あたり０．　１
−２　０　０ｎｇ／ｍ１の範囲が好ましい。The concentration of LDL used as lipid was 0.0% per medium. 1
A range of -200 ng/ml is preferred.

接着因子として用いるファイブロネクチンの濃度は、培
地あたり１５０μｇ　／　ｖａ　１の範囲が望ましい。The concentration of fibronectin used as an adhesion factor is preferably in the range of 150 μg/va 1 per medium.

さて、本発明のＣＨＯ細胞培養用無血清培地は、上記の
物質の内１種類以上を含有することを特徴とするもので
ある。Now, the serum-free medium for CHO cell culture of the present invention is characterized by containing one or more of the above-mentioned substances.

本発明の遺伝子組換えＣＨＯ細胞とは、何らかの外来遺
伝子を導入した遺伝子組換えＣＨＯ細胞で、Ｃ　Ｈ　Ｏ
ｄｈｆｒ一株を宿主とした細胞を含む。The genetically modified CHO cells of the present invention are genetically modified CHO cells into which some foreign gene has been introduced,
Contains cells hosted by one strain of dhfr.

〈本発明の効果〉本発明の無血清培地を用いて遺伝子組換えＣＨＯ細胞を
培養すると充分な細胞増殖が認められる。<Effects of the present invention> When genetically modified CHO cells are cultured using the serum-free medium of the present invention, sufficient cell proliferation is observed.

もちろん、本発明に係る無血清培地はＣＨＯ細胞以外の
動物細胞の培養にも使用できる。以下、本発明を実施例
に従って説明する。Of course, the serum-free medium according to the present invention can also be used for culturing animal cells other than CHO cells. Hereinafter, the present invention will be explained according to examples.

実施例１）表１の無血清培地（Ａ）を基礎培地として、ＦＮ（ヒト
由来ファイブロネクチン）５μｇ　／　＋ｎ　ｌを添加
し、無血清培地（Ｂ）をつくり培養を実施した。細胞は
、Ｃ　Ｈ　Ｏｄｈｆｒ−を宿主とし、Ｅｒｙ　ｔｈｒｏ
ｉｄ　Ｄｉｆｆｅｒｅｎｔｉａｉｔｉｏｎ　Ｆａｃｔｏ
ｒ　（以下ＥＤＦと略す）遺伝子を導入した遺伝子組換
えＣＨＯ綱胞（以下ＥＤＦ産生ＣＨＯ細胞、ＬＦＤ−５
　０　１　４　６）（Ｉｌｉｏｃｈｅｍｉｃａｌ　ａｎ
ｄ　Ｂｉｏｐｈｙｓｉｃａｌ　ＲｅｓｅａｒｃｈＣｏｎ
ｉｍｕｎｉｃａｔｉｏｎｓ，　　１　５　１　（　１　
）　２　３　０　）を使用した。初発細胞密度Ｉ　Ｘ　
１　０’　／ｃ４にてシャーレに播種し、付着増殖させ
４日後にトリブシン処理にて細胞を剥し検鏡により生細
胞数を測定した。無添加区（無血清培地（Ａ）〉及び対
照培地として１０％ＦＣＳ添加したアルファ一最小基本
培地（肝門−α）（以下単に１０％ＦＣＳ添加培地と略
す）において同時に培養を行った。その結果を１０％Ｆ
ＣＳ添加培地における細胞数を１００％として比較し示
した（第１図）。この結果よりＦＮ添加培地において無
添加区の約５％の細胞数の増加、また、明かな付着性の
向上が認められた。Example 1) Using the serum-free medium (A) in Table 1 as a basal medium, 5 μg/+nl of FN (human-derived fibronectin) was added to prepare a serum-free medium (B) and culture was performed. The cells host C H Odhfr- and Ery thro.
id Differentiation Facto
r (hereinafter abbreviated as EDF) gene introduced into genetically recombinant CHO cells (hereinafter referred to as EDF-producing CHO cells, LFD-5)
0 1 4 6) (Iliochemical an
dBiophysical ResearchCon
communication, 1 5 1 ( 1
)230) was used. Initial cell density I
The cells were seeded in a petri dish at 10'/c4, allowed to adhere and proliferate, and after 4 days, the cells were peeled off by treatment with tribucin and the number of viable cells was measured using a microscope. Cultivation was carried out simultaneously in an additive-free group (serum-free medium (A)) and as a control medium, alpha-1 minimal basic medium (hepatic portal-α) supplemented with 10% FCS (hereinafter simply referred to as 10% FCS-supplemented medium). 10% F on the result
The comparison is shown with the number of cells in the CS-added medium taken as 100% (Fig. 1). The results showed that in the FN-added medium, the number of cells increased by about 5% compared to the non-added group, and a clear improvement in adhesion was observed.

第ｌ表　無血清培地（Ａ）Ｍｉ成実施例２）前述の実施例１）の無血清培地（Ｂ）に、ＦＧＦ（ウシ
脳由来Ｆ　Ｇ　Ｆ　：　Ｊｕｒｎａｌ　ｏｆ　Ｂｉｏｌ
ｏｇｉｃａｌＣｈｅｍｉｓｔｒｙ，　２　５　３　．　
３　７　３　６　）を１０ｎｇ／ｍｊ！の濃度で添加し
、実施例１）と同様の方法にて培養を実施した。５日間
培養した後の生細胞数測定結果を１０％ＦＣＳ添加培地
を１００％として表した（第２図）。Table 1 Serum-free medium (A) Mi formation Example 2) FGF (Bovine brain-derived FGF: Journal of Biol) was added to the serum-free medium (B) of the above-mentioned Example 1).
logicalchemistry, 2 5 3.
3 7 3 6 ) at 10ng/mj! was added at a concentration of , and culture was carried out in the same manner as in Example 1). The results of measuring the number of living cells after culturing for 5 days are expressed with the 10% FCS-added medium as 100% (Fig. 2).

この結果より、培地（Ｂ）と比較すると、ＦＧＦ添加で
は約２２％の細胞数の増加が認められた。From this result, when compared with medium (B), an approximately 22% increase in cell number was observed with the addition of FGF.

実施例３）無血清培地（Ｂ）に、ＤＸを５ｎｇ／ｍｌ．及び、Ｔ３
を０．２ｎｇ／僧ｌ０）濃度で各々添加し実施例１）と
同様の方法で培養を行った。５日間培養を行った後、生
細胞数測定結果を実施例２）と同扛に比較した（第３図
）。Example 3) 5 ng/ml of DX was added to serum-free medium (B). and T3
were added at a concentration of 0.2 ng/lO) and cultured in the same manner as in Example 1). After culturing for 5 days, the results of measuring the number of living cells were compared with those in Example 2) (Fig. 3).

この結果より、ＤＸ添加で約１２％、］゛３添力■で１
２％の細胞数の増加が認められた。From this result, about 12% with DX addition, 1 with ゛3 addition■
A 2% increase in cell number was observed.

実施例４）無血清培地（Ｂ）に、ＬＤＬ（鶏卯黄山来ＬＤＬ　：ｊ
Ｕ織培養研究，６．７０）５０μｇ／ｍＡの濃度で添加
し実施例１）と同様に培養な行い４日間培畏後、生細胞
を測定した。測定結果を実施例２）と同様に比較した結
果、２６％の細胞数の増加がｌ２、められた（第４図）
。Example 4) In serum-free medium (B), LDL (Kiuo Huangshanrai LDL:j
U tissue culture study, 6.70) The cells were added at a concentration of 50 μg/mA and cultured in the same manner as in Example 1). After 4 days of incubation, viable cells were measured. As a result of comparing the measurement results in the same manner as in Example 2), a 26% increase in cell number was observed (Figure 4).
.

実施例５）ＦＮ５ｔＪＦ！，／ｍｌ，ＦＧＦ　１　０ｎｇ／ｍｊＬ
　ＤＸ５ｎｇ／ｎｊ！，　Ｔ３　０．　２ｎｇ／ｍｊｌ
！，　Ｌ　Ｄ　Ｌ　５　０　１ｔＢ／ｍｌ！、の全てを
同時に添加した無血清培地（Ｃ）をつくり、実施例１）
と同様の方法にて４日間培養を行った。その結果、この
培地において無添加区（無血清培地（Ａ））の約５３％
の細胞数の増加が認められその数は１０％ＦＣＳ添加培
地に匹敵するも第１図は遺伝子組換えＣＨ○細胞の増殖
に及ぼすＦＮの効果を調べたものである。Example 5) FN5tJF! ,/ml, FGF 10ng/mjL
DX5ng/nj! , T3 0. 2ng/mjl
! , LDL 50 1tB/ml! A serum-free medium (C) was prepared by adding all of , Example 1)
Culture was performed for 4 days in the same manner as above. As a result, in this medium, approximately 53% of the non-additive group (serum-free medium (A))
An increase in the number of cells was observed, and the number was comparable to that in the medium supplemented with 10% FCS. Figure 1 shows an investigation of the effect of FN on the proliferation of genetically modified CH○ cells.

第２図は遺伝子組換えＣ　Ｈ○細胞の増殖に及ぼすＦＧ
Ｆの効果を調べたものである。Figure 2 shows the effect of FG on the proliferation of genetically modified CH○ cells.
This study investigated the effect of F.

第３図は遺伝子Ｍｉ換えＣＨＯ細胞の増殖に及ぼすＤＸ
およびＴ，の効果を調べたものである。Figure 3 shows the effect of DX on the proliferation of transgenic Mi-modified CHO cells.
This study investigated the effects of and T.

第４図は遺伝子組換えＣ　Ｈ　Ｏ細胞の増殖に及ぼずＬ
ＤＬの効果を調べたものである。Figure 4 shows that the proliferation of genetically modified CHO cells was not achieved.
This study investigated the effects of DL.

第５図は遺伝子ｍ換えＣＩ−１０１）１胞用無血清培地
（無血清培地（Ｃ））を用いたときの遺伝子組換えＣＨ
○細胞の増殖を調べたものである。Figure 5 shows the recombinant CH when using the recombinant CI-101) serum-free medium for one cell (serum-free medium (C)).
○This is a study of cell proliferation.

Claims (5)

Translated fromJapanese

【特許請求の範囲】[Claims]（１）実質的に血清を含まない動物細胞培養用無血清培
地に１）ペプチド性細胞成長因子、２）ホルモン、３）
脂質、及び４）接着因子のうちいずれか１つ以上を添加
してなる遺伝子組換えＣＨＯ細胞培養用無血清培地。(1) In a serum-free medium for animal cell culture that does not substantially contain serum, 1) peptide cell growth factors, 2) hormones, 3)
A serum-free medium for culturing recombinant CHO cells, which contains one or more of lipids and 4) adhesion factors.（２）ペプチド性細胞成長因子が、ファイブロブラスト
・グロース・ファクター（以下ＦＧＦと略す）である、
請求項（１）記載の遺伝子組換えＣＨＯ細胞培養用培地
。(2) the peptide cell growth factor is fibroblast growth factor (hereinafter abbreviated as FGF),
The recombinant CHO cell culture medium according to claim (1).（３）ホルモンがデキサメサゾン（以下ＤＸと略す）、
トリヨウドチロニン（以下Ｔ＿３と略す）である請求項
（１）記載の遺伝子組換えＣＨＯ細胞培養用培地。(3) The hormone is dexamethasone (hereinafter abbreviated as DX),
The recombinant CHO cell culture medium according to claim 1, which is triiodothyronine (hereinafter abbreviated as T_3).（４）脂質がローデンシティー・リポプロテイン（以下
ＬＤＬと略す）である請求項（１）記載の遺伝子組換え
ＣＨＯ細胞培養用培地。(4) The medium for culturing recombinant CHO cells according to claim (1), wherein the lipid is low density lipoprotein (hereinafter abbreviated as LDL).（５）接着因子が、ファイブロネクチン（以下ＦＮと略
す）である請求項（１）記載の遺伝子組換えＣＨＯ細胞
培養用培地。(5) The recombinant CHO cell culture medium according to claim (1), wherein the adhesion factor is fibronectin (hereinafter abbreviated as FN).