JPH0543349B2 - - Google Patents

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Publication number
JPH0543349B2
JPH0543349B2 JP58050184A JP5018483A JPH0543349B2 JP H0543349 B2 JPH0543349 B2 JP H0543349B2 JP 58050184 A JP58050184 A JP 58050184A JP 5018483 A JP5018483 A JP 5018483A JP H0543349 B2 JPH0543349 B2 JP H0543349B2
Authority
JP
Japan
Prior art keywords
fraction
medium
molecular weight
cells
royal jelly
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
Application number
JP58050184A
Other languages
Japanese (ja)
Other versions
JPS59175876A (en
Inventor
Shuichi Hashizume
Kazuhiko Kuroda
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Morinaga and Co Ltd
Original Assignee
Morinaga and Co Ltd
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Morinaga and Co Ltd filed Critical Morinaga and Co Ltd
Priority to JP58050184A priority Critical patent/JPS59175876A/en
Publication of JPS59175876A publication Critical patent/JPS59175876A/en
Publication of JPH0543349B2 publication Critical patent/JPH0543349B2/ja
Granted legal-status Critical Current

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Description

【発明の詳細な説明】[Detailed description of the invention]

本発明は、動物細胞または動物細胞由来の細胞
を培養する低タンパク質濃度の培地に関するもの
であり、胎盤血の分子量5−8万の画分を含有す
ることを特徴とする細胞培養用培地に関するもの
である。 動物細胞および動物細胞由来の細胞には、抗
体、インターフエロン、インシユリン、酵素など
の有効物質を産生するものが知られており、その
細胞を培養することによつてこれらの有効物質を
得ることの実施または試みがなされている。従来
よりこれらの細胞を培養するためには、ほとんど
の場合、培地に血清を添加することが必要であ
る。血清としては一般に牛胎児血清、小牛血清な
どが用いられており、添加量は通常10%程度であ
る。しかし、これらの血清は高価であること、品
質が一定しないこと、供給に制限があることなど
の難点がある。 本発明者らは、これらのことを解決すべく研究
を重ねた結果、低タンパク質濃度の培地で細胞増
殖が可能なことを見いだし、本発明を完成したも
のである。 胎盤はヒト、家畜(牛、馬など)からのもので
ある。胎盤血の分子量5−8万の画分(以下胎盤
血画分と略す)は、例えば胎盤から自然にしみ出
た血液を2万×gで遠心し、その上清を更にSe
−phadex G−200(Pharmacia Fine
Chemicals)、Cellulofine GC−700(生化学工業
株式会社)などを用いるゲルろ過法により分子量
分画して得ることができる。そのゲルろ過に用い
る平衡化および溶出用溶液は、例えばリン酸緩衝
化生理食塩水、100mM Na−Hepes緩衝液(PH
7.4)、水などである。本発明の細胞培養用培地
は、この胎盤血画分を通常の動物細胞の培養に用
いられる基礎培地(血清を含まない)に添加して
用いる。 また、これに乳および/または乳の成分、ロー
ヤルゼリーおよび/またはローヤルゼリーの成分
の中より1種以上を基礎培地に添加して用いるこ
ともできる。 乳の成分は、以下のようにして得る。乳を例え
ば2万×gで遠心して溶液画分(以下乳の遠心溶
液画分と略す)を得る。乳の分子量20万以上の画
分および分子量4−9万の画分は、胎盤血の分子
量分画と同様の方法で得ることができる。ラクト
フエリンは例えばLo¨nnerdalらの方法
(Lo¨nnerdal、B.、Carlsson、J.、and Porath、
J(1977)FEBS Lett.75、89−92)で精製する。 また、ローヤルゼリーの成分は、以下のように
して得る。ローヤルゼリーの分子量20万以上の画
分および分子量5−8万の画分は、胎盤血の分子
量分画と同様の方法で得ることができる。ローヤ
ルゼリーの分子量約58000のタンパク質(以下ロ
ーヤルゼリータンパク質と略す)は、例えば以下
のようにして精製する。ローヤルゼリーをリン酸
緩衝化生理食塩水で2倍に希釈後、それに対し透
析する。透析後、2万×gで遠心し、得られる上
清を胎盤血の場合と同様の方法で分子量分画し、
5−8万の分子量画分を得る。この画分を1mM
Tris−HCl緩衝液(PH8.0)に透析後、同様の
緩衝液で平衡化しBlue Sepharose(Pharmacia
Fine Chemicals)にかけ、素通り画分すなわち
ローヤルゼリータンパク質を得る。 本発明培地の基礎培地としては、一般の動物細
胞の培養に用いられるものであればいかなるもの
も用いることができる。例えば、Dulbecco′s
Modified Eagle medium(以下D′MEMと略す)、
F−12、RPMI−1640などの基礎培地、また、こ
れら基礎培地にインシユリン(2−10μg/ml)、
セレニウム(10-11−10-8M)、トランスフエリン
(10−35μg/ml)、核酸前駆体(例えばチミジ
ン、ヒポキサンチンなど、10-6M程度)、血清ア
ルブミン(50−400μg/ml)などを添加した培
地も基礎培地として用いることができる。本発明
の培地に添加する胎盤血画分の最終タンパク質濃
度は50−300μg/mlであることが望ましい。そ
の他の乳および乳の成分については0.5−350μ
g/ml、ローヤルゼリーおよびローヤルゼリーの
成分については10−200μg/mlが望ましい。 本発明培地は、繊維芽細胞、リンパ芽球細胞な
どの動物細胞の培養に用いることもできるが、リ
ンパ球由来細胞などの動物細胞由来の細胞の培養
に最も好適に用いられる。本発明の培地を用いれ
ば血清を大量に添加した従来の培地にほぼ匹敵す
る細胞増殖を得ることができ、しかも従来の培地
と比べコストダウンが期待できる。また、血清を
10%添加した場合の培地中のタンパク質濃度が3
mg/ml以上であるのに比べ、本発明培地は0.4
mg/ml以下と低タンパク質濃度であることから、
有効物質産生に本発明培地を用いることにより有
効物質の分離・精製が容易となる。更に、本発明
培地は分子量が5−8万の画分を添加することか
ら、有効物質の分子量が5−8万の範囲外の場合
には、有効物質の分離・精製が容易となる。例え
ば抗体(分子量15万以上)を産生する細胞を培養
する場合には、胎盤血画分と、ラクトフエリンお
よび/または乳の分子量4−9万の画分、ローヤ
ルゼリータンパク質および/またはローヤルゼリ
ーの分子量5−8万の画分を必要に応じ加え、用
いるなどのように行う。更に、ヒトの胎盤血画分
と、要すればヒトラクトフエリンなどの人乳成分
および/または人乳を添加した培地でヒトの細胞
を培養する場合には、培地中の成分がすべてヒト
由来であることから、培地中に産生した有効物質
を完全に精製しなくともヒトに投与可能であると
いう利点を有する。 次に実施例により本発明を説明する。 実施例 1 健康人の末梢リンパ球株細胞(Bir7)の105
細胞を、D′MEM/F−12(1:1)に第1表に
示す添加物を添加した培地2mlに、植え込む。5
%CO2、37℃の条件下で3日間培養を行う。得ら
れた結果は第1表に示すとおりである。
The present invention relates to a medium with a low protein concentration for culturing animal cells or cells derived from animal cells, and relates to a cell culture medium characterized by containing a fraction of placental blood with a molecular weight of 50,000 to 80,000. It is. Animal cells and cells derived from animal cells are known to produce effective substances such as antibodies, interferon, insulin, and enzymes, and it is possible to obtain these effective substances by culturing these cells. being carried out or attempted. Traditionally, in order to culture these cells, it is almost always necessary to add serum to the culture medium. Fetal bovine serum, calf serum, etc. are generally used as serum, and the amount added is usually about 10%. However, these serums have drawbacks such as being expensive, having inconsistent quality, and limited supply. As a result of repeated research to solve these problems, the present inventors discovered that cell growth is possible in a medium with a low protein concentration, and completed the present invention. Placentas can come from humans or livestock (cows, horses, etc.). Placental blood fraction with a molecular weight of 50,000 to 80,000 (hereinafter referred to as placental blood fraction) can be obtained by, for example, centrifuging blood naturally exuded from the placenta at 20,000 x g, and then subjecting the supernatant to further Separation.
-phadex G-200 (Pharmacia Fine
Chemicals), Cellulofine GC-700 (Seikagaku Corporation), etc. by gel filtration method to obtain molecular weight fractionation. Equilibration and elution solutions used for gel filtration include, for example, phosphate buffered saline, 100mM Na-Hepes buffer (PH
7.4), water, etc. The cell culture medium of the present invention is used by adding this placental blood fraction to a basal medium (not containing serum) used for normal animal cell culture. Furthermore, one or more of milk and/or milk components, royal jelly and/or royal jelly components may be added to the basal medium. The milk components are obtained as follows. Milk is centrifuged at, for example, 20,000×g to obtain a solution fraction (hereinafter referred to as milk centrifuged solution fraction). The fraction of milk with a molecular weight of 200,000 or more and the fraction with a molecular weight of 40,000 to 90,000 can be obtained in the same manner as the molecular weight fraction of placental blood. Lactoferrin can be obtained, for example, by the method of Lo¨nnerdal et al. (Lo¨nnerdal, B., Carlsson, J., and Porath,
J (1977) FEBS Lett. 75, 89-92). Moreover, the components of royal jelly can be obtained as follows. The fraction of royal jelly with a molecular weight of 200,000 or more and the fraction with a molecular weight of 50,000 to 80,000 can be obtained in the same manner as the molecular weight fraction of placental blood. Royal jelly protein having a molecular weight of about 58,000 (hereinafter abbreviated as royal jelly protein) is purified, for example, as follows. Royal jelly is diluted twice with phosphate buffered saline and then dialyzed against it. After dialysis, it was centrifuged at 20,000 x g, and the resulting supernatant was subjected to molecular weight fractionation in the same manner as for placental blood.
A molecular weight fraction of 50,000 to 80,000 is obtained. This fraction was added to 1mM
After dialysis against Tris-HCl buffer (PH8.0), equilibration with the same buffer and Blue Sepharose (Pharmacia
Fine Chemicals) to obtain a flow-through fraction, that is, royal jelly protein. As the basal medium of the culture medium of the present invention, any medium used for culturing general animal cells can be used. For example, Dulbecco's
Modified Eagle medium (hereinafter abbreviated as D′MEM),
Basal media such as F-12, RPMI-1640, insulin (2-10 μg/ml),
Selenium (10 -11 -10 -8 M), transferrin (10-35 μg/ml), nucleic acid precursors (e.g. thymidine, hypoxanthine, etc., about 10 -6 M), serum albumin (50-400 μg/ml), etc. A medium supplemented with can also be used as a basal medium. The final protein concentration of the placental blood fraction added to the culture medium of the present invention is preferably 50-300 μg/ml. 0.5−350μ for other milk and milk components
g/ml, 10-200 μg/ml for royal jelly and royal jelly components. Although the culture medium of the present invention can be used for culturing animal cells such as fibroblasts and lymphoblastoid cells, it is most preferably used for culturing animal cell-derived cells such as lymphocyte-derived cells. By using the medium of the present invention, it is possible to obtain cell proliferation that is almost comparable to that of a conventional medium containing a large amount of serum, and moreover, it is expected that the cost will be lower than that of the conventional medium. Also, serum
The protein concentration in the medium when adding 10% is 3
mg/ml or more, compared to 0.4 mg/ml for the culture medium of the present invention.
Due to its low protein concentration of less than mg/ml,
By using the culture medium of the present invention for producing an effective substance, it becomes easy to separate and purify the effective substance. Furthermore, since a fraction having a molecular weight of 50,000 to 80,000 is added to the culture medium of the present invention, if the molecular weight of the effective substance is outside the range of 50,000 to 80,000, the effective substance can be easily separated and purified. For example, when culturing cells that produce antibodies (molecular weight of 150,000 or more), placental blood fraction, lactoferrin and/or milk fraction with molecular weight of 40,000 to 90,000, royal jelly protein and/or royal jelly with molecular weight of 5 to 90,000 are used. 80,000 fractions are added and used as needed, and so on. Furthermore, when culturing human cells in a medium supplemented with a human placental blood fraction and, if necessary, human milk components such as human lactoferrin and/or human milk, it is necessary to ensure that all components in the medium are of human origin. Therefore, it has the advantage that the effective substance produced in the medium can be administered to humans without being completely purified. Next, the present invention will be explained with reference to examples. Example 1 10 5 cells of a peripheral lymphocyte cell line (Bir7) from a healthy person are implanted in 2 ml of a medium containing D'MEM/F-12 (1:1) and the additives shown in Table 1. 5
Culture is carried out under conditions of % CO 2 and 37° C. for 3 days. The results obtained are shown in Table 1.

【表】 なおD′MEM/F−12に添加物としてヒト胎盤
血画分単独、ヒト胎盤血画分およびラクトフエリ
ン、ヒト胎盤血画分とラクトフエリンおよびロー
ヤルゼリータンパク質の3種をそれぞれ加えた培
地で30日間以上継代培養してもそれぞれの増殖速
度に変化がみられなかつた。このことは永続的に
継代培養することが可能であることを示してい
る。 実施例 2 マウスのミエローマ(NS−1)とマウスB−
リンパ球(BALB/c)が融合して生成したハ
イブリドーマについて植え込みの細胞数を0.5×
105個にへらし、実施例1と同様にして培養を行
う。得られた結果は第2表に示すとおりである。
[Table] In addition, D'MEM/F-12 was supplemented with three types of additives: human placental blood fraction alone, human placental blood fraction and lactoferrin, human placental blood fraction and lactoferrin, and royal jelly protein. No change was observed in the respective growth rates even after subculturing for more than one day. This indicates that continuous subculture is possible. Example 2 Mouse myeloma (NS-1) and mouse B-
For hybridomas generated by fusion of lymphocytes (BALB/c), the number of implanted cells is 0.5
Cut into 10 5 pieces and culture in the same manner as in Example 1. The results obtained are shown in Table 2.

【表】 %
【table】 %

Claims (1)

【特許請求の範囲】[Claims] 1 胎盤血の分子量5−8万の画分含有すること
を特徴とする細胞培養用培地。
1. A cell culture medium characterized by containing a fraction of placental blood with a molecular weight of 50,000 to 80,000.
JP58050184A 1983-03-25 1983-03-25 Medium for cell culture Granted JPS59175876A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP58050184A JPS59175876A (en) 1983-03-25 1983-03-25 Medium for cell culture

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP58050184A JPS59175876A (en) 1983-03-25 1983-03-25 Medium for cell culture

Publications (2)

Publication Number Publication Date
JPS59175876A JPS59175876A (en) 1984-10-04
JPH0543349B2 true JPH0543349B2 (en) 1993-07-01

Family

ID=12852096

Family Applications (1)

Application Number Title Priority Date Filing Date
JP58050184A Granted JPS59175876A (en) 1983-03-25 1983-03-25 Medium for cell culture

Country Status (1)

Country Link
JP (1) JPS59175876A (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59166079A (en) * 1983-03-14 1984-09-19 Morinaga & Co Ltd Medium for cell culture
JPS59173078A (en) * 1983-03-22 1984-09-29 Morinaga & Co Ltd Medium for cell culture

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS59166079A (en) * 1983-03-14 1984-09-19 Morinaga & Co Ltd Medium for cell culture
JPS59173078A (en) * 1983-03-22 1984-09-29 Morinaga & Co Ltd Medium for cell culture

Also Published As

Publication number Publication date
JPS59175876A (en) 1984-10-04

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