JP3060384B2 - Cell culture method connected with adsorption column and apparatus therefor - Google Patents
Cell culture method connected with adsorption column and apparatus thereforInfo
- Publication number
- JP3060384B2 JP3060384B2 JP63271238A JP27123888A JP3060384B2 JP 3060384 B2 JP3060384 B2 JP 3060384B2 JP 63271238 A JP63271238 A JP 63271238A JP 27123888 A JP27123888 A JP 27123888A JP 3060384 B2 JP3060384 B2 JP 3060384B2
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- Prior art keywords
- culture
- tank
- animal cells
- medium
- useful substance
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Enzymes And Modification Thereof (AREA)
Description
【発明の詳細な説明】 産業上の利用分野 本発明は、動物細胞を培養して有用物質、特に生理活
性物質を連続的に有利に生産し得る方法およびそのため
の装置に関する。Description: FIELD OF THE INVENTION The present invention relates to a method and apparatus for continuously and advantageously producing useful substances, particularly physiologically active substances, by culturing animal cells.
従来技術とその背景 動物細胞を培養して生理活性物質のような有用物質を
生産しようとする試みが広く普及している。また、近年
においては、動物細胞の大量培養技術が開発され、特に
ヒト組織由来の正常細胞の培養には、10tを超えるよう
な容積の巨大な培養装置も開発されている。このような
大型の装置を用いる培養は、ホモロ−ガスな培養として
一般に称せられている浮遊培養やマイクロキャリア−付
着細胞の培養に用いられていた。一方、最近では、これ
に対してヘテロ−ガスな培養としてホロ−フアイバ−、
セラミックス、多孔性ポリマ−等の表面に細胞を増殖さ
せ、これを培養容器中に密封し、いわゆる動物細胞固定
化リアクタ−として利用する方法が開発されている
〔「バイオテクノロジ−」６巻784（1988）〕。2. Description of the Related Art Attempts to produce useful substances such as physiologically active substances by culturing animal cells have become widespread. In recent years, a technique for mass-culturing animal cells has been developed, and particularly for culturing normal cells derived from human tissues, a huge culture device having a volume exceeding 10 t has been developed. Culture using such a large-sized apparatus has been used for suspension culture and culture of microcarrier-adherent cells which are generally called homologous culture. On the other hand, recently, a hetero-gas culture such as a hollow fiber,
A method has been developed in which cells are grown on the surface of ceramics, a porous polymer, or the like, and the cells are sealed in a culture vessel and used as a so-called animal cell-immobilized reactor [“Biotechnology”, Vol. 6, 784 ( 1988)].
最近、このヘテロ−ガスな培養方法が有用物質の生産
に適しているといわれ、アルギン酸カルシウム中に細胞
を固定化して利用する方法（特開昭62−163688号）、ま
たホロフアイバ−等の中空繊維に細胞を固定して培養す
る方法（特表昭62−500356号）、さらには多孔性の多数
の貫通孔を有する円筒形セラミックを使用した細胞培養
法（特開昭59−154984号）等が開示されている。Recently, this hetero-gas culture method is said to be suitable for the production of useful substances. A method in which cells are immobilized in calcium alginate and used (Japanese Patent Laid-Open No. 62-163688), and hollow fibers such as holofibers are used. And a cell culture method using a cylindrical ceramic having a large number of porous through-holes (JP-A-59-154984). It has been disclosed.
叙上のような装置を用いて動物性細胞、特に付着性を
有する正常細胞を培養して有用物質を生産する方法が行
われてきた。A method of producing useful substances by culturing animal cells, particularly normal cells having adherence, using the above-described apparatus has been performed.
しかして、一般的にはホモロ−ガスの培養では、長期
に培養を行つて物質を生産しようとする例は比較的少
く、インタ−フェロンの例にみられるように単回の培養
が多かつた。しかし、これに対し、ヘテロ−ガスな培養
では、動物細胞を固定化してリアクタ−として用いるこ
とにより、長期間に亘つて培養液を連続的に回収するこ
とが可能となつたことから、目的物質を連続的に回収す
ることが製造工程上の効率化の観点から必要となつた。However, in general, in homologous gas cultivation, there are relatively few examples of producing a substance by culturing over a long period of time, and a large number of single cultivations as in the case of interferon. . However, in the case of hetero-gas culture, by immobilizing animal cells and using them as a reactor, it is possible to continuously recover a culture solution over a long period of time. It has become necessary to continuously recover the wastewater from the viewpoint of improving efficiency in the manufacturing process.
従来法では、培養後の培地を回収し、この培地を精製
処理に付し、一方培養槽には新鮮な培地を補給する方法
が一般的であつた。しかし、組織化された細胞は増殖時
と異なり、物質生産時には培養液中の栄養物質の消費も
少ないため、培地を循環して物質を繰返し生産させる方
法が生産コスト上から有利である。In the conventional method, a method of recovering a culture medium after culturing and subjecting the culture medium to a purification treatment, while replenishing the culture tank with a fresh culture medium has been common. However, since the organized cells consume less nutrients in the culture medium during the production of the substance, unlike the proliferation, the method of circulating the medium and repeatedly producing the substance is advantageous from the viewpoint of production cost.
ところで、上述の方法では培養液中の目的物質の濃度
を上昇させて回収し得るが、細胞によつては細胞自身の
調節機能として細胞表面に自身の生産物質のレセプタ−
を有し、フィ−ドバック機構により生産を停止したり、
抑制する細胞がある。特に、がん化されていない正常細
胞ではこの特性が顕著である。したがつて、従来、この
ような細胞では培地を循環させる方法は採用できなかつ
た。By the way, in the above-mentioned method, the concentration of the target substance in the culture solution can be increased and the target substance can be collected.
To stop production by the feedback mechanism,
There are cells to suppress. In particular, this property is remarkable in non-cancerous normal cells. Therefore, conventionally, a method of circulating the culture medium cannot be adopted for such cells.
発明が解決しようとする課題 本発明は、動物細胞、特に有限増殖性があり、細胞表
面にレセプターを有する動物細胞を培養して有用物質を
生産するにあたり、培地を循環させて用いることによ
り、細胞の生産物質による生産阻害を防止して、目的物
質を連続方式で有利に生産するための方法およびそのた
めの装置を提供することを課題とする。PROBLEM TO BE SOLVED BY THE INVENTION The present invention provides an animal cell, especially a finitely proliferating animal cell having a receptor on the cell surface, to produce a useful substance by circulating a culture medium and using it in a cell. It is an object of the present invention to provide a method and an apparatus for effectively producing a target substance in a continuous manner by preventing production inhibition by a production substance.
以下本発明を詳しく説明する。 Hereinafter, the present invention will be described in detail.
課題を解決するための手段 本発明は、動物細胞をセラミッツス、多孔性ポリマ
−、ホロ−フアイバ−等を培養支持体として封入した培
養槽で増殖させたリアクタ−に、目的物質に親和性を有
する酵素阻害物質、蛋白質又は抗体を固定した多孔性ポ
リマ−、例えばセファデックス、アクリルアミド樹脂等
を充填した吸着カラムを連結させて、動物細胞により生
産された目的物質を含有した培養液を常時循環させるこ
とにより、目的物質を上記カラムに吸着させてその生産
をフィ−ドバック阻害しないようにするための、動物細
胞の培養による有用物質の生産方法およびその生産装置
に係るものである。Means for Solving the Problems The present invention has an affinity for a target substance in a reactor in which animal cells are grown in a culture vessel in which a ceramics, a porous polymer, a hollow fiber or the like is enclosed as a culture support. A culture medium containing a target substance produced by animal cells is constantly circulated by connecting an adsorption column filled with a porous polymer on which an enzyme inhibitor, a protein or an antibody is immobilized, such as Sephadex or acrylamide resin. Accordingly, the present invention relates to a method for producing a useful substance by culturing animal cells and an apparatus for producing the same, in order to adsorb the target substance onto the column so as not to inhibit the production thereof from being fed back.
すなわち、本発明は、有限増殖性且つ細胞表面に生産
する有用物質に対するレセプターを有する動物細胞を培
養して有用物質を生産する方法において、該細胞の培養
液を培地循環槽を介し、該有用物質と親和性を有する酵
素阻害物質、蛋白質、又は抗体を固定化した多孔性ポリ
マーを充填した少なくとも１個以上の吸着カラムに通液
して目的とする該有用物質を吸着させ生産物質によるフ
ィードバック阻害を解除し、一方カラムからの培養液を
上記培地循環槽を介して動物細胞による有用物質の生産
を連続的に行うことを特徴とする、動物細胞の培養によ
る有用物質の生産方法及びこのような方法を実施するた
めの装置に関する。That is, the present invention relates to a method for producing a useful substance by culturing animal cells having a finite-proliferation and a receptor for a useful substance produced on a cell surface, wherein the culture solution of the cells is passed through a culture medium circulation tank. Is passed through at least one or more adsorption columns packed with a porous polymer immobilized with an enzyme inhibitor, protein, or antibody having an affinity for and adsorbs the useful substance of interest, thereby inhibiting feedback inhibition by the produced substance. Releasing the culture solution from one column through the medium circulation tank to continuously produce a useful substance by animal cells, and producing a useful substance by culturing animal cells and such a method. To an apparatus for implementing the method.
本発明では、上記吸着カラムに充填する物質としては
種々の物質が用いられるが、実際には、培地成分を吸着
せずに目的の生産物質のみに選択的に結合する物質を用
いる。In the present invention, various substances are used as the substance to be packed in the adsorption column. In practice, however, a substance that does not adsorb the medium components and selectively binds only to the target production substance is used.
このような物質としては、目的の生産物質が酵素の場
合には特異的な酵素阻害剤を、また該生産物質が蛋白質
の場合には抗体、モノクロ−ナル抗体を、さらに目的物
質が糖蛋白質の場合にはレクチンなどの蛋白質などが挙
げられる。Examples of such a substance include a specific enzyme inhibitor when the target substance is an enzyme, an antibody and a monoclonal antibody when the target substance is a protein, and a glycoprotein when the target substance is a protein. In such cases, proteins such as lectins and the like can be mentioned.
これらの物質を、多孔性ポリマ−の種類に応じて共有
結合やスペンサ−を介した結合をさせてカラムに充填
し、目的の生産物質の吸着に用いる。Depending on the type of the porous polymer, these substances are packed into a column through a covalent bond or a bond via a spencer, and used for adsorption of a target product.
この吸着カラムは１個の培養槽について少くとも１
本、好ましくは２本以上をバルブを介して連結し、１本
のカラムが目的の生産物質で飽和された場合、バルブの
切替えにより新しいカラムと交換されるように構成して
おくことが好ましい。This adsorption column has at least one adsorption column per culture tank.
It is preferable that two or more, preferably two or more, be connected via a valve so that when one column is saturated with the target product, the column is replaced with a new column by switching the valve.
上記カラムの切替え交換により、培養槽における生産
物質の濃度が上昇するのを防ぎ、延いてはフィ−ドバッ
ク阻害が起ることを防止し、かつ微生物の汚染も防止で
きる。By switching the columns, it is possible to prevent the concentration of the product in the culture tank from increasing, thereby preventing feedback inhibition, and preventing the contamination of microorganisms.
本発明における細胞培養槽は、培地のpH、CO2及びO2
濃度を調整するための培地循環槽を介して培養液供給タ
ンクと連結しており、生産物質を含む循環培養液はその
栄養成分が常時モニタ−されていて、その培養条件が劣
化した場合、新鮮な培地が供給されるように構成されて
いる。In the present invention, the cell culture tank is provided with a medium pH, CO 2 and O 2.
It is connected to the culture medium supply tank via a medium circulation tank for adjusting the concentration, and the nutrients of the circulating culture medium containing the product are constantly monitored, and if the culture conditions deteriorate, fresh The medium is configured to be supplied.
このような培養槽を用いて動物細胞を培養するには公
知の装置を利用して行い得るが、好ましくは連続的に培
地のみを交換できるものが望ましく、マイクロキャリヤ
−を使用した浮遊培養のための装置、セラミックス充填
固定式リアクタ−、多孔管式細胞培養装置等を例示でき
る。Culture of animal cells using such a culture tank can be carried out using a known apparatus, but it is preferable that the medium can be continuously changed, and it is preferable that the medium be used for suspension culture using a microcarrier. , A ceramic-filled fixed reactor, a perforated tube cell culture device, and the like.
本発明に従つて動物細胞を培養して有用物質を生産す
るに際しては、まず細胞生育用培地、一般には血清含有
培地を用い、細胞を培養槽内に培養液1ml当り105〜109
個、好ましくは106〜108個の密度になるまで生育させた
後、培養槽内および装置全体を無血清培地などを用いて
洗浄し、次いでバルブを切替え、吸着カラムとラインを
接続させた後、培地を生産培地とし吸着カラムと細胞の
培養槽の間で培地循環槽を介して循環させて細胞による
物質生産を行う。When producing useful substances by culturing animal cells according to the present invention, first, a cell growth medium, generally a serum-containing medium, is used, and cells are placed in a culture tank at 10 5 to 10 9 per 1 ml of culture solution.
After the cells were grown to a density of preferably 10 6 to 10 8 cells, the inside of the culture tank and the entire apparatus were washed using a serum-free medium or the like, and then the valves were switched to connect the adsorption column and the line. Thereafter, the medium is used as a production medium and circulated through the medium circulation tank between the adsorption column and the cell culture tank to produce a substance by the cells.
以下実施例により、本発明に係る動物細胞の培養によ
る有用物質の生産方法およびその生産装置を具体的に説
明する。Hereinafter, a method for producing a useful substance by culturing animal cells and an apparatus for producing the same according to the present invention will be specifically described with reference to examples.
実施例１ 本例は、吸着カラムを連結させた装置を用いて細胞培
養によるヒト組織プラスミノ−ゲンアクチベ−タ−（以
下ｔ−PAと称する）の生産について示したものである。Example 1 This example illustrates the production of human tissue plasminogen activator (hereinafter referred to as t-PA) by cell culture using an apparatus connected with an adsorption column.
ここで用いた装置の配列は添付の図１に示した経路図
で表わされる。The arrangement of the apparatus used here is represented by the route diagram shown in FIG.
図において、は細胞の培養槽（３容）であつて、
該培養槽は、特開昭62−236480号公報に開示されている
アルミナを主成分としたセラミックスを充填し、細胞培
養と物質の生産を同時的に行い得る機能を有している。In the figure, indicates a cell culture tank (3 volumes).
The culture tank is filled with ceramics containing alumina as a main component disclosed in Japanese Patent Application Laid-Open No. Sho 62-236480, and has a function of simultaneously performing cell culture and production of a substance.
本装置においては、細胞は充填されたセラミックスの
表面に付着して生育する。ｔ−PAの生産にあたつては、
IMR−90細胞（ATCC CCL−186）をＴ−フラスコで培養
し、コンフルエントになつた状態で採取し、105/mlの細
胞密度で培養槽の底部P1より、培養槽内のリアクタ−
内に接種し、10％FCS含有DMEM培地を満たし、培地の循
環を行いつつ１週間培養を行い増殖させた。その後生産
用培地に切換えて、毎日半量ずつ培地を新鮮培地に交換
して生産を行つた。In this device, cells grow on the surface of the filled ceramics. For the production of t-PA,
Culturing IMR-90 cells (ATCC CCL-186) in T- flasks confluent were taken at Natsuta state, 10 5 / ml from the bottom P 1 of the cell density in the culture vessel, the reactor of the culture tank -
Then, the cells were inoculated into a DMEM medium containing 10% FCS, and cultured for one week while circulating the medium to grow. Thereafter, the medium was switched to a production medium, and the medium was replaced with a fresh medium by half each day for production.
図中は、生産物質であるｔ−PA吸着カラムであつ
て、抗ｔ−PAモノクロ−ナル抗体を固定化したアフィゲ
ル10（BoRAD社製）5mlを充填した内径0.9cmのガラス製
カラムである。抗ｔ−PAモノクロ−ナル抗体は、特開昭
61−221128号公報に開示された方法で得た。The figure shows a t-PA adsorption column as a product, which is a glass column having an inner diameter of 0.9 cm and packed with 5 ml of Affigel 10 (BoRAD) on which an anti-t-PA monoclonal antibody is immobilized. Anti-t-PA monoclonal antibody is disclosed in
Obtained by the method disclosed in JP-A-61-221128.
また図のは、培地循環槽であり、培養槽で生産さ
れたｔ−PAを含んだ培養液と、吸着カラムにより、ｔ
−PAを選択的に吸着した後の培地を回収し、培地循環槽
において、pH、CO2及びO2濃度を調整し、再度、細胞
培養槽へ戻した。Also shown in the figure is a culture medium circulation tank, in which a culture solution containing t-PA produced in the culture tank and t
Medium was collected after selectively adsorbs -PA, the medium circulation bath, pH, and adjust the CO 2 and O 2 concentration, again, it was returned to the cell culture vessel.
図１のP1、P2、P3でのｔ−PA濃度をモニタ−して本装
置でのｔ−PA生産効率を、単位時間当りの生産量として の式で表現される。The t-PA concentration at P 1 , P 2 , and P 3 in FIG. 1 is monitored, and the t-PA production efficiency of this apparatus is defined as the production amount per unit time. It is expressed by the following expression.
コントロ−ルとして、図１の吸着カラムを除いた装置
で、同条件でｔ−PAの生産を行つて効果を比較した。培
地の循環は、１日に約半量の培地が交換されるような速
さで培地を循環させ、カラムに吸着されたｔ−PA量、お
よび、培地槽内ｔ−PA槽を測定した。ｔ−PAの測定は、
抗ｔ−PA抗体を使用した酵素抗体法により測定を行つ
た。As a control, t-PA was produced under the same conditions using an apparatus excluding the adsorption column in FIG. 1 to compare the effects. The medium was circulated at such a rate that about half of the medium was exchanged per day, and the amount of t-PA adsorbed on the column and the t-PA tank in the medium tank were measured. The measurement of t-PA is
The measurement was performed by an enzyme antibody method using an anti-t-PA antibody.
物質生産時の培地は、特開昭62−205784号公報に開示
されたプロテオ−スペプトン等のｔ−PA生産誘導物質を
含むDMEM培地を使用した。As a medium for producing the substance, a DMEM medium containing a t-PA production inducer such as proteo-speptone disclosed in JP-A-62-205784 was used.
上述の通り、細胞増殖後、培地を生産培地に切り替え
た後、P1、P2、P3各場所での経時的なｔ−PA生産をモニ
タ−するとともに、生産されたｔ−PA総量を測定した。As described above, after cell proliferation, after switching the media production medium, monitored over time t-PA production in P 1, P 2, P 3 each location - as well as, the produced t-PA total It was measured.
図２に示す通り、本発明では明らかに、吸着カラムに
よりｔ−PAが吸着されている。As shown in FIG. 2, t-PA is clearly adsorbed by the adsorption column in the present invention.
図３には、回収されたｔ−PAの総量を示す。吸着カラ
ムを装着していない装置に比較して、吸着カラムを装着
した本発明の装置では、７時間目までのｔ−PA総回収量
は約３倍であつた。FIG. 3 shows the total amount of recovered t-PA. In the apparatus of the present invention equipped with an adsorption column, the total recovered amount of t-PA by 7 hours was about three times that of the apparatus equipped with no adsorption column.
実施例２ 本例は、図４に例示した本発明による連続生産装置を
用いて細胞を連続的に培養する態様を示したものであ
る。Example 2 This example shows an embodiment in which cells are continuously cultured using the continuous production apparatus according to the present invention illustrated in FIG.
図において、（１）は細胞培養槽であつて、（16）よ
り培地が供給され、（17）より培養後の培地が排出され
る。（13）はポンプであり、培養後の培地のモニタ−も
行う。（18）のバルブを切り替えることにより、（18）
の洗浄後、殺菌液、又は不用の培養液は（11）を介し、
（５）の回収槽へ回収される。培養液はライン（７）を
通り、（20）のバルブより吸着カラム（２）に供給され
る。吸着カラムは、並列に設置されており、バルブ（2
0）の切り替えにより、いずれのカラムへも流路を切り
替えることが可能である。また取りはずしも自由であ
る。細胞の生育時は、バルブ（19）を切り替えることに
よりライン（８）を介して、カラムをバイパスすること
ができる。カラム通過後の培養液は、（21）のモニタ−
により目的物質の吸着をモニタ−される。その後ライン
（９）を介し（３）の培地循環槽へ回収される。培地槽
には、（15）の攪拌羽根を回転させることにより、培地
を攪拌し、ライン（10）を介して新鮮培地槽（４）より
供給された新鮮培地と培地循環槽（３）で混合される。
また同時にpH、CO2、O2濃度が調整される。調整された
培地は、ライン（６）を介し、ポンプ（12）を通し（1
6）より培養槽（１）へ循環される。In the figure, (1) is a cell culture tank, in which a medium is supplied from (16), and a cultured medium is discharged from (17). (13) is a pump, which also monitors the medium after culturing. By switching the valve of (18), (18)
After washing, the germicidal solution or unnecessary culture solution is passed through (11),
It is collected in the collection tank of (5). The culture solution is supplied to the adsorption column (2) from the valve (20) through the line (7). The adsorption columns are installed in parallel and a valve (2
By switching 0), it is possible to switch the flow path to any column. It is also free to remove. During cell growth, the column can be bypassed via line (8) by switching valve (19). The culture solution after passing through the column is monitored by the monitor in (21).
Monitor the adsorption of the target substance. Thereafter, it is collected in the medium circulation tank of (3) via the line (9). In the medium tank, the medium is stirred by rotating the stirring blade of (15), and mixed with the fresh medium supplied from the fresh medium tank (4) via the line (10) in the medium circulation tank (3). Is done.
At the same time, the pH, CO 2 and O 2 concentrations are adjusted. The conditioned medium passes through line (6) and through pump (12) (1
It is circulated from 6) to the culture tank (1).
（14）は新鮮培地槽（４）より、新鮮培地を、培地循
環槽（３）へ移槽するためのポンプである。(14) is a pump for transferring the fresh medium from the fresh medium tank (4) to the medium circulation tank (3).
発明の効果 以上述べたとおり、本発明によると、培養によつて生
産された目的物質は吸着カラムにより選択的に吸着され
るので、目的物質が生産細胞のレセプタ−に結合して起
るフィ−ドバック阻害が解除され、生産効率が向上する
ようになる。また、物質生産に用いた培地の再使用が可
能となり、さらには吸着カラムに目的物質が吸着される
ことから、従来は独立した各工程であつた培養工程と回
収工程を連続的に実施することが可能となる。Effect of the Invention As described above, according to the present invention, the target substance produced by the culture is selectively adsorbed by the adsorption column, so that the target substance is bound to the receptor of the production cell. The inhibition of deback is released, and the production efficiency is improved. In addition, since the medium used for the substance production can be reused and the target substance is adsorbed on the adsorption column, it is necessary to continuously perform the culture step and the recovery step, which were conventionally independent steps. Becomes possible.
図１は本発明装置に用いて本発明の方法を実施するため
の工程経路を示したものであり、図２は図１のP1、P2、
P3でのｔ−PA濃度をモニタ−して経時的なｔ−PAの生産
をモニタ−すると共に生産されたｔ−PAの総量を示した
ものであり、図３は回収されたｔ−PAの総量を示したも
のである。図４は、本発明による連続生産装置の概略を
示したものである。 図１において、 ……培養槽 ……吸着カラム ……培地循環槽 図４において、 （１）……培養槽 （２）……吸着カラム （３）……培地循環槽 （４）……新鮮培地槽 （６）……循環ラインFIG. 1 shows a process route for carrying out the method of the present invention using the apparatus of the present invention, and FIG. 2 shows P 1 , P 2 ,
Monitoring the t-PA concentration in P 3 - monitoring the production of over time t-PA by - and shows the total amount of the produced t-PA as well as, t-PA Figure 3 is recovered It shows the total amount of. FIG. 4 schematically shows a continuous production apparatus according to the present invention. In FIG. 1, a culture tank ... an adsorption column ... a medium circulation tank In FIG. 4, (1) ... a culture tank (2) ... an adsorption column (3) ... a medium circulation tank (4) ... a fresh medium Vessel (6) ... circulation line
───────────────────────────────────────────────────── フロントページの続き (56)参考文献 特開 昭62−269697（ＪＰ，Ａ) 特公 昭46−10910（ＪＰ，Ｂ１) (58)調査した分野(Int.Cl.7，ＤＢ名) C12P 1/00 C12M 3/00 C12N 9/00 ──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-62-269697 (JP, A) JP-B-46-10910 (JP, B1) (58) Fields investigated (Int. Cl. 7 , DB name) C12P 1/00 C12M 3/00 C12N 9/00
Claims (7)
質に対するレセプターを有する動物細胞を培養して有用
物質を生産する方法において、該細胞の培養液を培地循
環槽を介し、該有用物質と親和性を有する酵素阻害物
質、蛋白質、又は抗体を固定化した多孔性ポリマーを充
填した少なくとも１個以上の吸着カラムに通液して目的
とする該有用物質を吸着させ生産物質によるフィードバ
ック阻害を解除し、一方カラムからの培養液を上記培地
循環槽を介して動物細胞による有用物質の生産を連続的
に行うことを特徴とする、動物細胞の培養による有用物
質の生産方法。1. A method for producing a useful substance by culturing animal cells having a finite-proliferation and a receptor for a useful substance produced on a cell surface, the method comprising the steps of: Pass the liquid through at least one adsorption column filled with a porous polymer to which an enzyme inhibitor, protein, or antibody having affinity is immobilized to adsorb the target useful substance and release feedback inhibition by the production substance And a method for producing a useful substance by culturing animal cells, comprising continuously producing a useful substance from animal cells using the culture solution from one column through the medium circulation tank.
し、新鮮な培地と混合し調整した後、動物細胞を培養す
るための培養槽に循環させることを特徴とする、請求項
１記載の動物細胞の培養による有用物質の生産方法。2. The method according to claim 1, wherein the culture solution from the column is introduced into a medium circulation tank, mixed with fresh medium and adjusted, and then circulated to a culture tank for culturing animal cells. A method for producing a useful substance by culturing animal cells.
又は２記載の動物細胞の培養による有用物質の生産方
法。3. The animal cell of claim 1, wherein the animal cell is human IMR-90.
Or a method for producing a useful substance by culturing the animal cell according to 2;
ーゲンアクチベーターである、請求項１〜３のいずれか
に記載の動物細胞の培養による有用物質の生産方法。4. The method for producing a useful substance by culturing animal cells according to claim 1, wherein the target useful substance is human tissue plasminogen activator.
ラムと連結させると共に、吸着カラムからの培養液を上
記培地循環槽を介して培養槽へ循環させるように構成す
ることを特徴とする、請求項１又は２記載の動物細胞の
培養による有用物質を生産するための装置。5. A culture tank for culturing animal cells is connected to an adsorption column, and a culture solution from the adsorption column is circulated to the culture tank through the medium circulation tank. An apparatus for producing a useful substance by culturing animal cells according to claim 1 or 2.
ラムと連結させ、且つ該培地循環槽を上記培養槽と連結
させて吸着カラムからの培養液を培地循環槽へ循環させ
るように構成し、必要に応じて、吸着カラムからの培養
液を直接培地循環槽へ給送するためのバイパスを配置す
ることを特徴とする、請求項５記載の装置。6. A structure in which a culture tank for culturing animal cells is connected to an adsorption column, and the medium circulation tank is connected to the culture tank to circulate a culture solution from the adsorption column to the medium circulation tank. 6. The apparatus according to claim 5, wherein a bypass for feeding the culture solution from the adsorption column directly to the medium circulation tank is arranged as necessary.
ムのいずれかに選択的に切り換えて連結し得るように構
成すると共に、培養液が通液していないカラムを着脱自
在にしてなる、請求項５又は６記載の装置。7. A structure in which two or more culture tanks can be selectively switched and connected to one of two or more adsorption columns arranged in parallel, and a column through which a culture solution does not flow is made detachable. An apparatus according to claim 5 or claim 6.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63271238A JP3060384B2 (en) | 1988-10-27 | 1988-10-27 | Cell culture method connected with adsorption column and apparatus therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63271238A JP3060384B2 (en) | 1988-10-27 | 1988-10-27 | Cell culture method connected with adsorption column and apparatus therefor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02117388A JPH02117388A (en) | 1990-05-01 |
| JP3060384B2 true JP3060384B2 (en) | 2000-07-10 |
Family
ID=17497280
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63271238A Expired - Lifetime JP3060384B2 (en) | 1988-10-27 | 1988-10-27 | Cell culture method connected with adsorption column and apparatus therefor |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP3060384B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0416183A (en) * | 1990-05-07 | 1992-01-21 | Ngk Insulators Ltd | Method and device for culturing animal cell |
| MY180565A (en) * | 2011-04-14 | 2020-12-02 | Gs Caltex Corp | Apparatus and method for separating and refining product manufactured by microbial fermentation by using adsorbent |
| JP7376887B2 (en) * | 2019-08-20 | 2023-11-09 | Ｊｆｅエンジニアリング株式会社 | Animal cell growth promotion method, continuous culture method, and continuous culture device |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS6125483A (en) * | 1984-07-17 | 1986-02-04 | Hitachi Ltd | Method and apparatus for cell culture |
-
1988
- 1988-10-27 JP JP63271238A patent/JP3060384B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02117388A (en) | 1990-05-01 |
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