JPH10108673A - Culture of animal cell using hollow yarn type incubator - Google Patents

Culture of animal cell using hollow yarn type incubator

Info

Publication number
JPH10108673A
JPH10108673A JP8281545A JP28154596A JPH10108673A JP H10108673 A JPH10108673 A JP H10108673A JP 8281545 A JP8281545 A JP 8281545A JP 28154596 A JP28154596 A JP 28154596A JP H10108673 A JPH10108673 A JP H10108673A
Authority
JP
Japan
Prior art keywords
cells
culture
incubator
hollow fiber
culture solution
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
Application number
JP8281545A
Other languages
Japanese (ja)
Inventor
Hisako Kumazawa
久子 熊沢
Yasuo Tokushima
恭雄 徳島
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.)
Asahi Kasei Medical Co Ltd
Original Assignee
Asahi Medical 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 Asahi Medical Co Ltd filed Critical Asahi Medical Co Ltd
Priority to JP8281545A priority Critical patent/JPH10108673A/en
Publication of JPH10108673A publication Critical patent/JPH10108673A/en
Pending legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/10Hollow fibers or tubes

Abstract

PROBLEM TO BE SOLVED: To provide a method for carrying out culture at a prescribed flow rate of cultured medium in an animal cell culture by using a hollow yarn type incubator. SOLUTION: This method is to satisfy the formula, 1.5×N×(α/ c)<=(u)<=400×N×(α)/(c), wherein, flow rate of cultured medium fed to the hollow yarn type incubator is defined as (u)ml/min and live cell number of animal cells in the hollow yarn type incubator is defined as N (number), cell constant of the animal cells is defined as (α) and dissolved oxygen concentration of culture solution fed to the hollow yarn type incubator is defined as (c)g/ml.

Description

【発明の詳細な説明】DETAILED DESCRIPTION OF THE INVENTION

【0001】[0001]

【発明の属する技術分野】本発明は、動物細胞の培養方
法に関するものである。更に詳しくは、動物細胞を中空
糸型培養器で培養する方法に関するものである。
TECHNICAL FIELD The present invention relates to a method for culturing animal cells. More specifically, the present invention relates to a method for culturing animal cells in a hollow fiber type incubator.

【0002】[0002]

【従来の技術】動物細胞の培養法は、シャーレ等を用い
た静置培養法をはじめ、スピナーフラスコやタンク等を
用いた攪拌培養法、ローラーボトル法等、種々の方法が
利用されている。また、マイクロキャリア等の担体を用
いる培養方法もあり、改良、開発が盛んに行われてい
る。中空糸型培養器による動物細胞の培養方法は、動物
細胞を生体に近い環境で培養することを目的にKNAZ
EKらによって開発された。中空糸型培養器における中
空糸と培養細胞の関係は、生体内における血管とその周
囲に立体構造を形成する細胞との関係を模倣したもので
ある。中空糸型培養器では、細胞は本来の生体内での機
能を維持し易いといわれ、最近では膵細胞、肝細胞とい
った臓器細胞を中空糸型培養器で培養し、臓器本来の広
範囲な機能をもつハイブリッド型人工臓器を構築しよう
とする試みもある。また、中空糸型培養器を用いれば、
動物細胞を1〜5×108個/mlの高密度で培養する
ことが可能である。中空糸内側を流れる培養液と中空糸
外側に維持された細胞との間で中空糸壁面を介した物質
交換が連続的に行われるからであり、また、付着性細胞
にとっては細胞生育に必要不可欠な細胞の付着する面を
中空糸外膜面として、限られた容積内に多く確保するこ
とができるからである。通常、動物細胞の到達密度はシ
ャーレやフラスコを利用した静置培養では106個/m
lレベル、条件のよく検討された潅流培養等でも107
個/mlレベルに過ぎない。このように他の培養法には
ない利点を多く有すると考えられる中空糸型培養器を用
いた培養法であるが、その他の培養法と比較し、実際に
有用物質の工業生産に用いられる等、実用化にいたって
いる例は少ない。
2. Description of the Related Art Various methods are used for culturing animal cells, such as a stationary culture method using a petri dish or the like, a stirring culture method using a spinner flask or a tank, a roller bottle method, and the like. In addition, there is a culture method using a carrier such as a microcarrier, and improvements and developments have been actively conducted. The method of culturing animal cells using a hollow fiber type incubator uses the KAZZ method for culturing animal cells in an environment close to a living body.
Developed by EK et al. The relationship between the hollow fiber and the cultured cells in the hollow fiber type incubator simulates the relationship between a blood vessel in a living body and cells forming a three-dimensional structure around the blood vessel. In hollow fiber incubators, it is said that cells can easily maintain their original functions in vivo.In recent years, organ cells such as pancreatic cells and hepatocytes have been cultured in hollow fiber incubators to achieve the wide range of functions inherent in organs. Attempts have been made to construct hybrid artificial organs. Also, if you use a hollow fiber type incubator,
Animal cells can be cultured at a high density of 1 to 5 × 10 8 cells / ml. This is because mass exchange is continuously performed between the culture solution flowing inside the hollow fiber and the cells maintained outside the hollow fiber through the wall surface of the hollow fiber, and is essential for cell growth for adherent cells. This is because a large number of cells can be secured in a limited volume as the outer surface of the hollow fiber surface. Usually, the reached density of animal cells is 10 6 cells / m in stationary culture using a petri dish or a flask.
Even at l level, perfusion culture, etc., whose conditions have been well studied, 10 7
Only at the individual / ml level. As described above, it is a culture method using a hollow fiber type incubator that is considered to have many advantages not found in other culture methods, but compared to other culture methods, it is actually used for industrial production of useful substances, etc. There are few examples of practical use.

【0003】中空糸型培養器を用いた培養方法において
は、細胞に対する栄養分の供給と老廃物の除去を行うた
めに、培養器に適切な流速で培養液を送液しなければな
らない。低流速では、細胞の増殖、維持に必要な酸素、
グルコース、アミノ酸等の栄養分の供給が十分なされな
い。また、乳酸、アンモニアといった細胞の代謝産物の
除去が十分になされず、細胞の状態を悪化させてしま
う。逆に、流速を上げ過ぎてもまた、かえって細胞の状
態を悪化させるという事態が生じてくる。この原因は、
剪断力によって細胞が物理的ダメージを受けるからであ
ると考えられている。培養液には血清やアルブミンが添
加されることもある。培養液中の蛋白質には細胞を物理
的なダメージから保護する作用があるが、最近では動物
細胞培養においては、培養液として、再現性の向上、低
コスト化、物質生産を目的とする培養においては目的生
産物の精製が容易となる等の観点から、低血清培養液を
用いることが多くなってきた。無血清培養液や無蛋白培
養液が使用されることもある。中空糸型培養器を用いた
培養方法においては、フィンを設けた中空糸を用いる方
法(特開平3−60477)が開発されている。これに
よって細胞への栄養分の供給の効率は向上するが、培養
液の流速が不適切であれば、やはり細胞にダメージを与
えてしまうのである。中空糸型培養器を用いた動物細胞
の培養に関しては、その利点を十分に行かすことのでき
る技術が確立されていないのが現状である。
In a culture method using a hollow fiber type incubator, a culture solution must be supplied to the incubator at an appropriate flow rate in order to supply nutrients to cells and remove waste products. At low flow rates, the oxygen needed to grow and maintain cells,
The supply of nutrients such as glucose and amino acids is not sufficient. In addition, cellular metabolites such as lactic acid and ammonia are not sufficiently removed, which deteriorates the state of the cells. Conversely, if the flow rate is increased too much, a situation may occur in which the state of the cells is rather deteriorated. This is because
It is thought that the cells are physically damaged by the shearing force. Serum or albumin may be added to the culture solution. Proteins in culture media have the effect of protecting cells from physical damage, but recently, in animal cell culture, they have been used as culture media to improve reproducibility, reduce cost, and produce substances for the purpose of material production. From the viewpoint of easy purification of the target product, a low serum culture solution has been frequently used. A serum-free culture or a protein-free culture may be used. As a culture method using a hollow fiber type incubator, a method using hollow fibers provided with fins (Japanese Patent Laid-Open No. 3-60477) has been developed. This improves the efficiency of supplying nutrients to the cells, but also damages the cells if the flow rate of the culture solution is inappropriate. As for the cultivation of animal cells using a hollow fiber type incubator, at present, there is no established technique that can sufficiently achieve the advantages.

【0004】[0004]

【発明が解決しようとする課題】本発明は中空糸型培養
器を用いた動物細胞の有効な培養方法を提供することを
目的とする。更に具体的には、中空糸型培養器を用いた
動物細胞培養において、、中空糸型培養器に培養液を特
定の範囲の流速で送液しながら動物細胞を高密度で長期
培養できる培養方法を提供することを目的とする。
SUMMARY OF THE INVENTION An object of the present invention is to provide an effective method for culturing animal cells using a hollow fiber type incubator. More specifically, in a method of culturing animal cells using a hollow fiber type incubator, a culture method capable of long-term culturing animal cells at a high density while feeding a culture solution to a hollow fiber type incubator at a specific flow rate. The purpose is to provide.

【0005】[0005]

【課題を解決するための手段】即ち、本発明は、中空糸
型培養器を用いた動物細胞の培養方法であって、該中空
糸型培養器に送液される培養液の流速をu(ml/
分)、該中空糸型培養器内の該動物細胞の生細胞数をN
(個)、該動物細胞の細胞定数をα、該中空糸型培養器
に送液される該培養液の溶存酸素濃度をc(g/ml)
とするとき、1.5×N×α/c≦u≦400×N×α
/cであることを特徴とする培養方法である。本発明で
いう中空糸型培養器に用いられる中空糸は、物質透過性
を有する中空繊維状の膜であって、膜素材としては、通
常中空糸膜素材として用いられる、ポリエチレン、ポリ
プロピレン、セルロース、再生セルロース、セルロース
アセテート、ポリスルフォン、ポリアクリロニトリル、
ポリスチレン等が本発明においても用いられる。疎水性
材料、親水性材料いずれも使用可能である。膜そのもの
でも良く、親水性、細胞親和性、抗血栓性等をもつ物質
を塗布したりあるいは化学的結合等によって表面改質し
た膜でも良い。
That is, the present invention relates to a method for culturing animal cells using a hollow fiber type incubator, wherein the flow rate of the culture solution sent to the hollow fiber type incubator is u ( ml /
Min), the number of living cells of the animal cells in the hollow fiber type incubator is N
(Unit), the cell constant of the animal cell is α, and the dissolved oxygen concentration of the culture solution sent to the hollow fiber type incubator is c (g / ml).
Where 1.5 × N × α / c ≦ u ≦ 400 × N × α
/ C. The hollow fiber used in the hollow fiber type incubator according to the present invention is a hollow fiber membrane having material permeability, and as a membrane material, usually used as a hollow fiber membrane material, polyethylene, polypropylene, cellulose, Regenerated cellulose, cellulose acetate, polysulfone, polyacrylonitrile,
Polystyrene and the like are also used in the present invention. Either a hydrophobic material or a hydrophilic material can be used. The film itself may be used, or a film coated with a substance having hydrophilicity, cell affinity, antithrombotic property, or the like, or a surface modified by chemical bonding or the like may be used.

【0006】本発明でいう動物細胞は、付着性、浮遊
性、いずれも含む。例としては、Hela(ヒト子宮頸
部癌由来細胞)、Balb3T3(マウス胎仔由来細
胞)、L929(マウス総合組織由来細胞)、BGM
(アフリカミドリザル腎由来細胞)、Vero(アフリ
カミドリザル腎由来細胞)、BHK(新生仔シリアンハ
ムスター腎由来細胞)、CHO(チャイニーズハムスタ
ー卵巣由来細胞)、それらに遺伝子を導入した細胞やハ
イブリドーマ等が挙げられる。また、株化細胞には限ら
ず、肝細胞、膵細胞等の初代培養細胞、血液幹細胞、血
液前駆細胞、リンパ球、T細胞、B細胞等の血液関連細
胞も含む。これらの細胞に遺伝子を導入した細胞、適当
な液性因子を与えて賦活化した細胞等も含む。また、本
発明に係る培養液流速で動物細胞の培養を行う際、送液
する手段としては、所定の流速で送液できる手段であれ
ばいずれでもよい。培養期間中に、培養器内に維持され
た細胞の状態に応じて流速を変動できるよう流速が可変
なものが好ましい。例としてはチューブポンプ、ベロー
ズ型ポンプ、電磁式ポンプ等が挙げられる。また、重力
や圧力を利用して送液する手段も用いることができる。
The animal cells referred to in the present invention include both adherent and planktonic. Examples include Hela (human cervical cancer-derived cells), Balb3T3 (mouse embryo-derived cells), L929 (mouse synthetic tissue-derived cells), BGM
(African green monkey kidney-derived cells), Vero (African green monkey kidney-derived cells), BHK (newborn Syrian hamster kidney-derived cells), CHO (Chinese hamster ovary-derived cells), and cells or hybridomas into which genes have been introduced. . Not only cell lines but also primary cells such as hepatocytes and pancreatic cells, and blood-related cells such as blood stem cells, blood progenitor cells, lymphocytes, T cells, and B cells. These include cells in which a gene has been introduced into these cells, cells that have been activated by the application of an appropriate humoral factor, and the like. Further, when culturing animal cells at the culture solution flow rate according to the present invention, any means may be used as a means for sending the liquid, as long as it can send the liquid at a predetermined flow rate. It is preferable that the flow rate be variable so that the flow rate can be varied according to the state of the cells maintained in the incubator during the culture period. Examples include tube pumps, bellows type pumps, electromagnetic pumps and the like. In addition, a means for sending a liquid using gravity or pressure can also be used.

【0007】本発明でいう生細胞数とは、培養器内に維
持されている生細胞数をいうが、培養開始時にあって
は、培養器に添加する生細胞数でもよい。また、直接法
によって計数した生細胞数、間接法によって算出した生
細胞数、いずれも含む。ここでいう直接法とは、中空糸
型培養器内の細胞浮遊液の一部をサンプリングし、その
中に含まれる生細胞数を顕微鏡下等で数える方法であ
り、間接法とは、培養液中の酸素濃度、グルコース濃
度、乳酸濃度、アンモニア濃度等の、細胞の代謝の指標
となる物質の濃度の変化から、生細胞数を間接的に算出
する方法をいう。本発明でいう細胞定数αには、ハイブ
リドーマ等の浮遊性細胞にあっては0.027×10
-12、CHO等の付着性細胞にあっては、0.127×
10-12を用いる。細胞が単位時間あたりに消費する酸
素の消費量に基づく定数である。培養する動物細胞につ
いて単位時間あたりに消費する酸素の消費量(g/個/
分)が判っていればその値を用いても良い。本発明でい
う培養器に送液される培養液の溶存酸素濃度とは、培養
器に供給される直前の培養液中の溶存酸素濃度をいう。
溶存酸素濃度は、DO(dissolved oxyg
en)値ともいう。培養器の培養液入口側にDOセンサ
ーを設置し、培養器入口側の培養液の溶存酸素濃度を測
定することができる。また、培養液の入ったボトル中に
DOセンサーを設置し、適切ならばその値を培養器に送
液される培養液の溶存酸素濃度として用いてもよい。ま
た、培養系中に培養液に酸素付加をするための適切なガ
ス交換部があり、そこで十分なガス交換が行われている
ならば、培養器に送液される培養液の溶存酸素濃度とし
て平衡値(例えば、培養を37℃で実施する場合におい
ては、37℃で1気圧の大気と平衡状態にある純水中の
溶存酸素濃度6.86×10-6g/ml)を用いること
もできる。ガス交換部としては、通常の動物細胞の培養
に用いられるものが本発明においても使用可能である。
例としては、シリコン製のチューブや中空糸を用いたガ
ス交換器、テフロンチューブをコイル状に巻き培養液ボ
トル内に配したもの、培養液ボトル内の培養液に直接気
体を吹き込むもの、培養液ボトル内の培養液の液面を介
するもの等が挙げられる。
The number of viable cells in the present invention refers to the number of viable cells maintained in the incubator, but may be the number of viable cells added to the incubator at the start of culture. It also includes the number of living cells counted by the direct method and the number of living cells calculated by the indirect method. The direct method here is a method of sampling a part of the cell suspension in the hollow fiber type incubator and counting the number of viable cells contained therein under a microscope or the like. It refers to a method of indirectly calculating the number of living cells from a change in the concentration of a substance that is an index of cell metabolism, such as oxygen concentration, glucose concentration, lactate concentration, and ammonia concentration in the medium. The cell constant α in the present invention is 0.027 × 10 5 for floating cells such as hybridomas.
-12 , 0.127x for adherent cells such as CHO
Use 10-12 . It is a constant based on the amount of oxygen consumed by cells per unit time. The amount of oxygen consumed per unit time of animal cells to be cultured (g / cell /
If the value is known, the value may be used. The term “dissolved oxygen concentration of the culture solution sent to the incubator” as used in the present invention refers to the dissolved oxygen concentration in the culture solution immediately before being supplied to the incubator.
The dissolved oxygen concentration is determined by DO (dissolved oxyg).
en) Value. By installing a DO sensor at the culture solution inlet side of the incubator, the dissolved oxygen concentration of the culture solution at the incubator inlet side can be measured. Alternatively, a DO sensor may be provided in a bottle containing a culture solution, and if appropriate, the value may be used as the dissolved oxygen concentration of the culture solution sent to the incubator. Also, in the culture system, there is an appropriate gas exchange unit for adding oxygen to the culture solution, and if sufficient gas exchange is performed there, the dissolved oxygen concentration of the culture solution sent to the incubator An equilibrium value (for example, when culturing is performed at 37 ° C., the concentration of dissolved oxygen in pure water at 6.86 × 10 −6 g / ml in equilibrium with the atmosphere at 37 ° C. and 1 atm) may be used. it can. As the gas exchange unit, those used for normal animal cell culture can be used in the present invention.
Examples include a gas exchanger using a silicon tube or hollow fiber, a Teflon tube wound in a coil shape and placed in a culture solution bottle, a device that blows gas directly into the culture solution in the culture solution bottle, a culture solution Examples include those that pass through the liquid surface of the culture solution in the bottle.

【0008】本発明は、中空糸型培養器を用いて培養液
の流速をuとして動物細胞を培養する方法である。u
は、1.5×N×α/c≦u×400×N×α/cであ
る。細胞は培養液に溶解した酸素を得て生育維持、増殖
するが、この範囲未満の低流速では、培養器内に維持さ
れた細胞の数に応じた十分な量の酸素の供給がなされ
ず、効果的な培養が不可能になる。逆に、この範囲を超
えた高流速では、細胞にかかる剪断力が大きくなりすぎ
るため細胞が物理的なダメージを受け、よい培養結果が
得られない。特に、細胞を培養器に添加した直後(添加
の際、細胞は多少なりともダメージを受けており、更な
るダメージを与えることは好ましくない)や蛋白質含量
の低い培養液を用いる培養においては流速を上げすぎな
いことが重要である。好ましくは1.5×N×α/c≦
u≦300×N×α/cである。更に好ましくは1.5
×N×α/c≦u≦150×N×α/cである。本発明
による方法は、特に、流速による影響を受けやすくなる
無血清培養液を用いた動物細胞の培養に適し、特に抗体
の生産を目的としたハイブリドーマの無血清培養に適し
ている。
The present invention relates to a method for culturing animal cells using a hollow fiber type incubator at a flow rate of a culture solution of u. u
Is 1.5 × N × α / c ≦ u × 400 × N × α / c. Cells grow, maintain and proliferate by obtaining oxygen dissolved in the culture solution, but at a low flow rate less than this range, a sufficient amount of oxygen is not supplied according to the number of cells maintained in the incubator, Effective culture becomes impossible. Conversely, if the flow rate is higher than this range, the shearing force applied to the cells becomes too large, and the cells are physically damaged, and good culture results cannot be obtained. In particular, immediately after the cells are added to the incubator (at the time of addition, the cells are somewhat damaged and it is not desirable to cause further damage), or in culture using a culture solution with a low protein content, the flow rate should be increased. It is important not to raise too much. Preferably 1.5 × N × α / c ≦
u ≦ 300 × N × α / c. More preferably 1.5
× N × α / c ≦ u ≦ 150 × N × α / c. The method according to the present invention is particularly suitable for culturing animal cells using a serum-free culture solution that is susceptible to the flow rate, and particularly suitable for serum-free culturing of hybridomas for the purpose of producing antibodies.

【0009】[0009]

【発明の実施の形態】以下、実施例を用いて説明する。DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, description will be made with reference to embodiments.

【実施例】再生セルロース製中空糸を用い、中空糸外側
の膜面積が0.02m2かつ中空糸外容積2mlの培養
器(以下、培養器1)、及び中空糸外側の膜面積が1.
1m2かつ中空糸外容積60mlの中空糸型培養器(以
下、培養器2)を作製した。これらの培養器を用い、マ
ウス−マウスハイブリドーマHyGPD・YK−1−1
株(Riken Cell No.711)を培養し
た。培養液は極東E−RDF(極東製薬)を用いた。
尚、培養開始時の中空糸型培養器へ細胞を添加する際は
牛胎児血清を10%含む培養液に細胞を浮遊したが、以
後は血清の代わりに添加剤RD−1(極東製薬)を加え
た無血清培養液を使用した。
EXAMPLE Incubators (hereinafter referred to as incubator 1) having a membrane area of 0.02 m 2 on the outside of the hollow fiber and a volume of 2 ml of the outside of the hollow fiber using a hollow fiber made of regenerated cellulose, and having a membrane area of 1.
A hollow fiber type incubator (hereinafter, incubator 2) having a volume of 1 m 2 and an outer volume of the hollow fiber of 60 ml was prepared. Using these incubators, a mouse-mouse hybridoma HyGPD.YK-1-1 was used.
A strain (Riken Cell No. 711) was cultured. The culture solution used Far East E-RDF (Far East Pharmaceutical).
When the cells were added to the hollow fiber type incubator at the start of the culture, the cells were suspended in a culture solution containing 10% fetal bovine serum, but thereafter, the additive RD-1 (Kyokuto Pharmaceutical) was used instead of the serum. The added serum-free culture was used.

【0010】[0010]

【実施例1】培養器1、ガス交換器(内径2mm、膜厚
1mm、長さ2mのガス交換用シリコンチューブ)、ポ
ンプチューブ、培養液ボトル及びこれらを接続する回路
チューブからなる培養系を作製した。培養器に、細胞を
5×106個添加し、培養を開始した。この系において
培養器に送液される培養液の溶存酸素濃度は平衡値
(6.86×10-6g/ml)であると考え、培養液の
流速を2ml/分として培養を開始した。培養2日後、
培養器内の細胞の一部取りだし、トリパンブルー染色し
て顕微鏡下で観察してみたが、死細胞はほとんど見られ
ず、細胞の状態は良好であった。
Example 1 A culture system comprising an incubator 1, a gas exchanger (silicon tube for gas exchange having an inner diameter of 2 mm, a film thickness of 1 mm, and a length of 2 m), a pump tube, a culture solution bottle, and a circuit tube connecting these components is prepared. did. 5 × 10 6 cells were added to the incubator, and the culture was started. In this system, the concentration of dissolved oxygen in the culture solution sent to the incubator was considered to be an equilibrium value (6.86 × 10 −6 g / ml), and the culture was started at a flow rate of the culture solution of 2 ml / min. After 2 days of culture,
A portion of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. Almost no dead cells were found, and the cells were in good condition.

【0011】[0011]

【実施例2】実施例1と同様に培養器1に、細胞を5×
106個添加し、培養液の流速を2ml/分として培養
を開始した。培養液の流速は一定のまま、64日間培養
を継続した。培養終了時の生細胞数は、2×108個で
あった。64日間で産生されたIgG量は、ELISA
法で測定した培養液中のIgG濃度から算出したとこ
ろ、77.4mgであった。
Example 2 Cells were placed in an incubator 1 in the same manner as in Example 1,
10 6 cells were added, and the culture was started at a flow rate of the culture solution of 2 ml / min. The culture was continued for 64 days while the flow rate of the culture solution was kept constant. The number of viable cells at the end of the culture was 2 × 10 8 . The amount of IgG produced in 64 days was determined by ELISA
It was 77.4 mg when calculated from the IgG concentration in the culture solution measured by the method.

【0012】[0012]

【実施例3】培養器2、ガス交換器(シリコン製中空糸
膜を用いたもの、膜面積1.0m2)、ポンプチュー
ブ、培養液ボトル及びこれらを接続する回路チューブか
らなる培養系を作製した。細胞を5×108個添加し、
培養を開始した。この系において培養器に送液される培
養液の溶存酸素濃度は平衡値(6.86×10-6g/m
l)であると考え、培養液の流速を50ml/分として
培養を開始した。流速はその後、3日目100ml/
分、4日目以降150ml/分とし、28日間培養を継
続した。培養終了時の生細胞数は、2×1010個であっ
た。28日間で産生されたIgG量は、ELISA法で
測定した培養液中のIgG濃度から算出したところ、
1.0gであった。
EXAMPLE 3 A culture system comprising an incubator 2, a gas exchanger (using a hollow fiber membrane made of silicon, a membrane area of 1.0 m 2 ), a pump tube, a culture solution bottle and a circuit tube connecting these components was prepared. did. Add 5 × 10 8 cells,
Culture was started. In this system, the dissolved oxygen concentration of the culture solution sent to the incubator is an equilibrium value (6.86 × 10 −6 g / m 2).
Considering that 1), the culture was started at a flow rate of the culture solution of 50 ml / min. The flow rate was then 100 ml /
The culture was continued for 28 days at a rate of 150 ml / min from day 4 onward. The number of viable cells at the end of the culture was 2 × 10 10 . The amount of IgG produced in 28 days was calculated from the IgG concentration in the culture solution measured by the ELISA method,
1.0 g.

【0013】[0013]

【実施例4】培養器1に、細胞を5×106個添加し、
培養液の流速を6ml/分として培養を開始した。2日
後、培養器内の細胞を一部取りだし、トリパンブルー染
色して顕微鏡下で観察してみたところ、死細胞がやや見
られた。
Example 4 5 × 10 6 cells were added to the incubator 1
The culture was started at a flow rate of the culture solution of 6 ml / min. Two days later, some of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. As a result, some dead cells were found.

【0014】[0014]

【比較例1】培養器1に、細胞を5×106個添加し、
培養液の流速を8ml/分として培養を開始した。2日
後、培養器内の細胞を一部取りだし、トリパンブルー染
色して顕微鏡下で観察してみたところ、ほとんどが死細
胞であった。
Comparative Example 1 5 × 10 6 cells were added to the incubator 1
The culture was started at a flow rate of the culture solution of 8 ml / min. Two days later, some of the cells in the incubator were removed, stained with trypan blue, and observed under a microscope. Most of the cells were dead cells.

【0015】[0015]

【表1】 [Table 1]

【0016】[0016]

【発明の効果】本発明の培養方法によれば、動物細胞に
必要な物質の供給と老廃物の除去を適切に行うことがで
き、しかもその際、細胞に剪断力などの意図しないダメ
ージを与えることがない。従って、中空糸型培養器の利
点を十分に生かした高密度培養が可能で、しかもそれを
長期間維持することができるようになった。更に、抗体
の生産を目的としたハイブリドーマの培養においては、
高密度長期連続培養に加え、無血清培養液を使用した場
合でも抗体の生産が良好となった。
According to the culture method of the present invention, it is possible to appropriately supply a necessary substance to animal cells and remove waste products, and at the same time, unintended damage such as shearing force is given to the cells. Nothing. Therefore, high-density cultivation that makes full use of the advantages of the hollow fiber type incubator is possible, and can be maintained for a long period of time. Furthermore, in hybridoma culture for the purpose of antibody production,
In addition to the high-density long-term continuous culture, antibody production was improved even when a serum-free culture solution was used.

Claims (1)

【特許請求の範囲】[Claims] 【請求項1】 中空糸型培養器を用いた動物細胞の培養
方法であって、該中空糸型培養器に送液される培養液の
流速をu(ml/分)、該中空糸型培養器内にある該動
物細胞の生細胞数をN(個)、該動物細胞の細胞定数を
α、該中空糸型培養器に送液される該培養液の溶存酸素
濃度をc(g/ml)とするとき、1.5×N×α/c
≦u≦400×N×α/cであることを特徴とする培養
方法。
1. A method for culturing animal cells using a hollow fiber type incubator, wherein the flow rate of a culture solution sent to the hollow fiber type incubator is u (ml / min), The number of living cells of the animal cells in the vessel is N (cells), the cell constant of the animal cells is α, and the dissolved oxygen concentration of the culture solution sent to the hollow fiber type incubator is c (g / ml). ), 1.5 × N × α / c
≦ u ≦ 400 × N × α / c.
JP8281545A 1996-10-04 1996-10-04 Culture of animal cell using hollow yarn type incubator Pending JPH10108673A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP8281545A JPH10108673A (en) 1996-10-04 1996-10-04 Culture of animal cell using hollow yarn type incubator

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP8281545A JPH10108673A (en) 1996-10-04 1996-10-04 Culture of animal cell using hollow yarn type incubator

Publications (1)

Publication Number Publication Date
JPH10108673A true JPH10108673A (en) 1998-04-28

Family

ID=17640677

Family Applications (1)

Application Number Title Priority Date Filing Date
JP8281545A Pending JPH10108673A (en) 1996-10-04 1996-10-04 Culture of animal cell using hollow yarn type incubator

Country Status (1)

Country Link
JP (1) JPH10108673A (en)

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JPWO2004020614A1 (en) * 2002-08-28 2005-12-15 船津 和守 Modified cross-section hollow fiber membrane cell-containing device
JP2006109707A (en) * 2004-10-12 2006-04-27 Hiroshima Univ Apparatus for cell culture
JP2009543565A (en) * 2006-07-14 2009-12-10 ディーエスエム アイピー アセッツ ビー.ブイ. Improved method of culturing cells
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Publication number Priority date Publication date Assignee Title
JPWO2004020614A1 (en) * 2002-08-28 2005-12-15 船津 和守 Modified cross-section hollow fiber membrane cell-containing device
JP2006109707A (en) * 2004-10-12 2006-04-27 Hiroshima Univ Apparatus for cell culture
US9469865B2 (en) 2006-07-14 2016-10-18 Dpx Holdings B.V. Process for the culturing of cells
US8222001B2 (en) 2006-07-14 2012-07-17 Dsm Ip Assets B.V. Process for the culturing of cells
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JP2009543565A (en) * 2006-07-14 2009-12-10 ディーエスエム アイピー アセッツ ビー.ブイ. Improved method of culturing cells
US9670520B2 (en) 2006-07-14 2017-06-06 Patheon Holdings | B.V. Process for the culturing of cells
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