JPH07203945A - Apparatus for culturing cell of living body - Google Patents

Apparatus for culturing cell of living body

Info

Publication number
JPH07203945A
JPH07203945A JP149294A JP149294A JPH07203945A JP H07203945 A JPH07203945 A JP H07203945A JP 149294 A JP149294 A JP 149294A JP 149294 A JP149294 A JP 149294A JP H07203945 A JPH07203945 A JP H07203945A
Authority
JP
Japan
Prior art keywords
culture
oxygen
tank
cells
microcarriers
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
JP149294A
Other languages
Japanese (ja)
Inventor
Michiyo Nitsuta
三知代 新田
Ryoichi Haga
良一 芳賀
Sei Murakami
聖 村上
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.)
Hitachi Ltd
Original Assignee
Hitachi 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 Hitachi Ltd filed Critical Hitachi Ltd
Priority to JP149294A priority Critical patent/JPH07203945A/en
Publication of JPH07203945A publication Critical patent/JPH07203945A/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
    • C12M27/00Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/16Vibrating; Shaking; Tilting
    • 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
    • C12M25/16Particles; Beads; Granular material; Encapsulation
    • 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
    • C12M27/00Means for mixing, agitating or circulating fluids in the vessel
    • C12M27/02Stirrer or mobile mixing elements
    • 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
    • C12M29/00Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/06Nozzles; Sprayers; Spargers; Diffusers
    • 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
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/34Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of gas
    • 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
    • C12M41/00Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/30Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
    • C12M41/36Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of biomass, e.g. colony counters or by turbidity measurements

Abstract

PURPOSE:To provide an apparatus for culturing a cell of a living body capable of embodying the high-density culture of the cell of the living body for a long period using a porous microcarrier. CONSTITUTION:This apparatus for culturing a cell of a living body is provided by installing a screen (3a) in a culture tank 1, preventing a microcarrier from leaking out of a culture solution, dissolving oxygen in the culture solution in a tank 2 for submerged aeration by the submerged aeration, circulating the resultant oxygen-enriched culture solution, efficiently supplying oxygen to the cell, simultaneously monitoring the turbidity and dissolved oxygen concentration (DO) in the culture solution, judging the concentration distribution of the microcarrier in the culture tank and oxygen consumption by the cell in the culture tank, changing the internal pressure in the tank 2 for the submerged aeration, oxygen concentration and aeration gas flow rate of the aeration gas based on the resultant information, balancing the sedimenting rate of the microcarrier with the circulation rate of the culture solution, supplying the oxygen required for the cell and further reducing the vibrating operation with a vibrator 11 and the existing concentration of the microcarrier near the screen (3a).

Description

【発明の詳細な説明】Detailed Description of the Invention

【0001】[0001]

【産業上の利用分野】本発明は、生体の細胞の培養装置
及び方法に関し、特に、生体の細胞を生体外で増殖せし
め有用物質を生産させるのに有効に用いられるマイクロ
キャリア等の固体粒子を用いた液中通気方式による生体
の細胞の培養装置及び方法に関する。
BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus and method for culturing living cells, and particularly to solid particles such as microcarriers which are effectively used to grow living cells in vitro to produce useful substances. The present invention relates to an apparatus and method for culturing living cells by submerged aeration.

【0002】[0002]

【従来の技術】生体の細胞の培養装置における酸素供給
方法として、いわゆる液中通気方式が最も酸素供給能力
に優れているのは周知である。しかし、従来の液中通気
方式では、細胞が多数凝集して細胞塊を形成する場合
や、マイクロキャリアなどの固体粒子を用いて付着性細
胞を培養する場合に適用できないことがあった。これ
は、液中通気によって生じる気泡にマイクロキャリアや
細胞塊が同伴されて上昇し、培養液面上に形成される泡
沫層に捕捉されて、培養液中に戻らなくなってしまうた
めである。これらの問題を改善するための公知例は、培
養槽内で液中通気する方法としては、サイトテクノロジ
ー、1990年第3巻39〜42ページ(Cytotechinol
ogy,,p39〜42,1990)、特開昭61−25418
2 号公報に記載されている。これらは、マイクロキャリ
ア等の固体粒子が混入しないような細孔径の多孔質部材
で仕切られた液中通気部を培養槽内に設置したものであ
る。また、培養槽外で液中通気する方法としては、特開
平3−505041 号公報,特開昭62−244380号公報等に記載
されている。これらは、培養槽とは別個に培地貯槽内を
設け、該貯槽内で液中通気して酸素を培地に溶解させ、
該酸素富化培地を培養槽に供給するものである。
2. Description of the Related Art It is well known that the so-called submerged aeration method has the highest oxygen supply capacity as an oxygen supply method in a cell culture apparatus of a living body. However, the conventional submerged aeration method may not be applicable when a large number of cells are aggregated to form a cell mass or when adherent cells are cultured using solid particles such as microcarriers. This is because air bubbles generated by aeration in the liquid are accompanied by microcarriers and cell aggregates and ascend, and are trapped by the foam layer formed on the surface of the culture liquid and cannot return to the culture liquid. A known example for ameliorating these problems is a method of aerating liquid in a culture tank, see Cytotechnology, Vol. 3, 1990, pp. 39-42 (Cytotechinol).
ogy, 3 , p39-42, 1990), JP-A-61-25418.
It is described in Publication No. 2. These are those in which a submerged aeration section partitioned by a porous member having a pore size such that solid particles such as microcarriers are not mixed is installed in the culture tank. Further, a method for ventilating in the liquid outside the culture tank is described in JP-A-3-505041 and JP-A-62-244380. These are provided in the medium storage tank separately from the culture tank, aeration in the liquid in the storage tank to dissolve oxygen in the medium,
The oxygen-enriched medium is supplied to the culture tank.

【0003】[0003]

【発明が解決しようとする課題】前記の公知例を大別す
ると、1)培養槽内に多孔質フィルタで構成された酸素
供給モジュールを設置し該フィルタ内から通気すること
で気泡とマイクロキャリアの接触を防ぐ方法、2)培養
槽外で液中通気し酸素富化した培地を供給する方法があ
る。しかし、1)では酸素供給能力はフィルタの面積に
依存するが、培養槽内に設置可能なフィルタ面積には限
界があり、培養後期におけるフィルタの目詰りによる酸
素供給量の減少が問題となる。また、1)2)ともに酸
素や栄養源が枯渇した培地のみを抜き出すための、マイ
クロキャリアと液との特別の分離手段が別途必要とな
る。
The above-mentioned known examples are broadly divided into 1) by installing an oxygen supply module composed of a porous filter in the culture tank and venting air from the inside of the filter to remove bubbles and microcarriers. There are methods of preventing contact, 2) a method of supplying oxygen-enriched medium by aerating in the liquid outside the culture tank. However, in 1), although the oxygen supply capacity depends on the area of the filter, there is a limit to the filter area that can be installed in the culture tank, and there is a problem that the oxygen supply amount decreases due to clogging of the filter in the latter stage of the culture. In addition, both 1) and 2) require a separate means for separating the microcarrier and the liquid in order to extract only the medium depleted of oxygen and nutrients.

【0004】更に、これら従来技術は、表面型マイクロ
キャリアを用いた到達細胞濃度培養液1mlあたりの細
胞数106 個レベルに対応して開発されたものであり、
最近開発された多孔質マイクロキャリア(例、特開昭64
−43530 号公報)を用いた場合に達成し得る107 個レ
ベルの高密度細胞培養に見合う十分な酸素を供給するこ
とはできない。
Further, these prior arts were developed corresponding to the level of 10 6 cells per 1 ml of culture medium having reached cell concentration using surface type microcarriers,
Recently developed porous microcarriers (eg JP-A-64)
-43530) cannot supply sufficient oxygen commensurate with the high density cell culture at the level of 10 7 cells that can be achieved.

【0005】本発明は、従来技術の問題点を解決し、多
孔質マイクロキャリアを用いての生体の細胞の高密度培
養に対応可能な、液中通気による効率的な酸素供給を実
施する培養装置を提供することにある。
The present invention solves the problems of the prior art and is capable of coping with high density culturing of cells of a living body using porous microcarriers, and a culturing apparatus for efficiently supplying oxygen by submerged aeration. To provide.

【0006】本発明の更なる目的は、培養液の濁度変化
をモニタして、培養液中に浮遊している細胞数及び/ま
たはマイクロキャリア数を監視し、該結果をもとに、培
養液の撹拌方法及び/または撹拌速度、培養液の循環速
度等を変化させて、細胞の生存,増殖及び/または物質
生産に適正な酸素供給を実施し、効率的な培養及び有用
物質の生産を実現することのできる生体の細胞の培養装
置を提供することにある。
[0006] A further object of the present invention is to monitor changes in the turbidity of the culture solution to monitor the number of cells and / or microcarriers suspended in the culture solution, and based on the results, culture By changing the stirring method and / or stirring speed of the liquid, the circulation speed of the culture liquid, etc., oxygen supply is carried out appropriately for cell survival, growth and / or substance production, and efficient culture and production of useful substances are carried out. An object is to provide a device for culturing living cells that can be realized.

【0007】[0007]

【課題を解決するための手段】前記目的を達成するた
め、本発明になる生体の細胞の培養装置は、基本的に以
下に述べる構成となっている。
In order to achieve the above-mentioned object, an apparatus for culturing cells of a living body according to the present invention is basically constructed as described below.

【0008】マイクロキャリア等の固体粒子に細胞を付
着させて浮遊状態で培養するための培養槽があり、該培
養槽内には、マイクロキャリア等の固体粒子の通過を阻
止しかつ培養液を通過させる細孔を有するスクリーンが
設置されている。そして、該培養槽とは別に、液中通気
により培養液に酸素を効率的に溶解させるための液中通
気槽を有している。これら培養槽と液中通気槽は少なく
とも2つの接続管によって相互に接続されており、該接
続管には、液中通気槽で酸素富化された培養液を培養槽
へ循環させるための循環手段が装備されている。また、
培養槽には該培養槽内の培養液中のマイクロキャリア等
の固体粒子を浮遊させ、混合するための撹拌手段と、該
培養槽内に設置されたスクリーンを振動させるための振
動手段とを有している。さらに、液中通気槽から培養槽
への培養液の循環方向は、培養液下部から上部への上向
流となるように、前記接続管及び該培養液の循環手段が
設置されていることを特徴とする。
There is a culture tank for adhering cells to solid particles such as microcarriers and culturing them in a floating state. Inside the culture tank, solid particles such as microcarriers are prevented from passing and a culture solution is passed. A screen having fine pores is installed. In addition to the culture tank, it has a submerged aeration tank for efficiently dissolving oxygen in the culture solution by submerged aeration. The culture tank and the submerged aeration tank are connected to each other by at least two connecting pipes, and a circulation means for circulating the oxygen-enriched culture liquid in the submerged aeration tank to the culture tank. Is equipped with. Also,
The culture tank has a stirring means for suspending and mixing solid particles such as microcarriers in the culture solution in the culture tank, and a vibrating means for vibrating a screen installed in the culture tank. is doing. Further, the connecting pipe and the circulation means for the culture solution are installed so that the circulation direction of the culture solution from the submerged aeration tank to the culture tank is an upward flow from the lower part to the upper part of the culture solution. Characterize.

【0009】スクリーンを培養槽上部に水平に設置し
て、培養槽底部と前記スクリーン間をマイクロキャリア
等の固体粒子の存在する培養域とする。これにより、撹
拌されて浮遊し、培養液の循環流と共に上昇しても、ス
クリーンにトラップされて、マイクロキャリア等の固体
粒子が培養液中から逸失するのを有効に阻止することが
できる。
The screen is installed horizontally on the upper part of the culture tank, and the space between the bottom of the culture tank and the screen is a culture area where solid particles such as microcarriers are present. This makes it possible to effectively prevent the solid particles such as microcarriers from being lost from the culture solution by being trapped in the screen even if the solution is stirred and floats and rises along with the circulation flow of the culture solution.

【0010】生体の細胞の高密度培養が長期に及んだ場
合、スクリーンの細孔内に細胞の断片等が付着し、培養
液の上向流ろ過を妨げて酸素供給量が減少する恐れがあ
る。高密度培養の場合、酸素供給量の減少は、細胞の生
存率の低下や物質生産能の低下などの悪影響を引き起こ
す。本発明の好ましい様態においては、その対策とし
て、スクリーンの目詰りを防ぐ手段を更に有する。この
手段は、スクリーンに振動を生じさせる振動発生手段、
前記培養液の循環量を急激に変化させてスクリーンに圧
力変動を与えて洗浄する手段等が望ましい。前記振動発
生手段としては、内部の空気圧変化により形状を変形し
うるように成型された伸縮性を有する、例えばべローズ
のような部材による手段,ソレノイドによる往復手段等
であって良い。スクリーンは、培養液のろ過効率を高め
るために、培養液の循環流賂に面しかつ循環方向に対し
て垂直となるように設置するのが好ましい。また、スク
リーンを構成する部材は、特に限定するものではない
が、オートクレーブ滅菌やスチーム滅菌の操作条件に耐
え、腐食性,細胞毒性を示さず、物理的強度の高い部材
で構成されているのが望ましい。
When a high-density culture of living cells is carried out for a long period of time, cell fragments or the like may adhere to the pores of the screen, obstructing the upward flow filtration of the culture medium and reducing the oxygen supply. is there. In high-density culture, a decrease in oxygen supply causes adverse effects such as a decrease in cell viability and a decrease in substance productivity. In a preferred embodiment of the present invention, as a countermeasure, a means for preventing clogging of the screen is further provided. This means is a vibration generating means for causing the screen to vibrate,
A means for washing the screen by changing the circulating amount of the culture solution rapidly to change the pressure of the screen is preferable. The vibration generating means may be, for example, a means such as a bellows-like member having elasticity that is molded so that its shape can be deformed by a change in internal air pressure, and a reciprocating means such as a solenoid. In order to enhance the filtration efficiency of the culture solution, the screen is preferably installed so as to face the circulation tub of the culture solution and be perpendicular to the circulation direction. In addition, the member constituting the screen is not particularly limited, but it is composed of a member having high physical strength that withstands the operating conditions of autoclave sterilization and steam sterilization, does not show corrosiveness and cytotoxicity. desirable.

【0011】スクリーンを構成する部材の細孔径は、使
用するマイクロキャリア等の固体粒子の大きさにより、
培養域から漏出することのない大きさが選択される。好
ましくは、該当する細孔径のステンレス製の金網を成型
加工したものを用いる。
The pore size of the member constituting the screen depends on the size of solid particles such as microcarriers used.
A size that does not leak from the culture area is selected. It is preferable to use a stainless steel wire net having a corresponding pore diameter, which is molded and processed.

【0012】液中通気槽の形状は特に限定するものでは
ないが、耐圧構造で、十分な酸素溶解速度係数を持つ必
要がある。また、通気ノズルは、気泡の滞留時間が長く
なり、酸素が効率良く溶解するように液中通気槽下部に
設置するのが望ましい。更に、液中通気槽と酸素溶解槽
は2つの接続管によって相互に接続されている。接続管
路中には送液手段が設置され、液中通気槽から培養槽へ
酸素が富化された培養液を循環させる。培養液の循環方
向は、培養槽内で上向流となるように該接続管及び送液
手段は配置されている。更に該接続管路内には、培養液
の溶存酸素濃度を測定する手段を設けるのが好ましい。
The shape of the submerged aeration tank is not particularly limited, but it is required to have a pressure resistant structure and a sufficient oxygen dissolution rate coefficient. Further, it is desirable that the aeration nozzle be installed in the lower part of the submerged aeration tank so that the residence time of bubbles becomes long and oxygen is efficiently dissolved. Further, the submerged aeration tank and the oxygen dissolution tank are connected to each other by two connecting pipes. A liquid feeding means is installed in the connecting conduit to circulate the oxygen-enriched culture solution from the submerged aeration tank to the culture tank. The connection pipe and the liquid feeding means are arranged so that the circulation direction of the culture solution is an upward flow in the culture tank. Further, it is preferable to provide a means for measuring the dissolved oxygen concentration of the culture solution in the connecting conduit.

【0013】培養槽内には、マイクロキャリア等の固体
粒子及び生体の細胞に損傷を与えずにかつ均一に混合す
ることのできる撹拌手段を設置する。さらに好ましく
は、培養液の溶存酸素濃度,pH,濁度を測定する手段
を設置する。
In the culture tank, a stirring means capable of uniformly mixing solid particles such as microcarriers and living cells without damaging them is installed. More preferably, a means for measuring the dissolved oxygen concentration, pH and turbidity of the culture solution is installed.

【0014】本発明になる生体の細胞の培養装置におい
ては、これら培養槽内の溶存酸素濃度,pH及び濁度、
さらに、接続管路内の培養液の溶存酸素濃度を測定する
手段によって得た情報により、培養条件を生体の細胞の
生存及び/または増殖に適するように制御し得る。本制
御方法について更に詳細に説明する。
In the apparatus for culturing cells of a living body according to the present invention, the dissolved oxygen concentration, pH and turbidity in these culture tanks,
Furthermore, the information obtained by the means for measuring the dissolved oxygen concentration of the culture medium in the connecting conduit can control the culture conditions to be suitable for the survival and / or proliferation of the cells of the living body. This control method will be described in more detail.

【0015】マイクロキャリア等の固体粒子を用いた付
着性動物細胞の浮遊撹拌培養においては、まず、種細胞
をマイクロキャリア等の固体粒子に付着させるための操
作が必要となる。本発明では、予め常法により調製した
マイクロキャリアと最終培養量の1/4〜1/2量の培
地を培養槽内に注入する。ここに、さらに常法により調
製しておいた種細胞液を接種し、撹拌して、細胞をマイ
クロキャリアに付着させる。撹拌は、増殖期での撹拌速
度の1/10〜1/4程度で非常にゆっくりと連続撹拌
するか、または、静置と撹拌を一定周期で繰り返す、間
歇撹拌するのが望ましい。この付着操作は、通常、数時
間〜24時間程度で終了する。本発明では、この付着操
作中、適時、培養液上清の濁度を測定する。最初は、ほ
とんどの細胞が浮遊しているので濁度が高く、細胞がマ
イクロキャリアに付着するにつれて濁度は減少してい
く。この濁度の経時変化をモニタし、さらに、マイクロ
キャリアに付着しないで浮遊している細胞数を算出し、
細胞の付着状態を監視する。濁度の減少がおこらない場
合は、細胞が付着していないと判断できるので、撹拌速
度を減少させたり、静置時間を延長するといった操作に
より、細胞の付着を促進する。濁度の変化が止まりかつ
濁度から必要量の細胞がマイクロキャリアに付着したと
判定されたら、細胞の付着操作を終了して、通常の培養
に移る。培地を最終培養量までフィルアップし、全部の
マイクロキャリアが浮遊する速度まで撹拌速度を上昇さ
せて、培養を継続する。この通常の培養においても濁度
を適時測定する。酸素や栄養分の供給からいえば、物質
移動速度が大きくなるので撹拌速度は高いほうが良い。
しかし、撹拌速度を上げ過ぎると、細胞が撹拌の剪断力
によりダメージをうけて死滅する場合がある。細胞が死
滅して、マイクロキャリアから剥離して浮遊し、培養液
の濁度が上昇した場合は、濁度の上昇がなくなるまで撹
拌速度を低下させる。本発明では、以上述べた方法によ
り細胞のマイクロキャリアへの付着状態を培養期間中良
好な状態に保つことができる。
In suspension culture of adherent animal cells using solid particles such as microcarriers, first, an operation for attaching seed cells to solid particles such as microcarriers is required. In the present invention, microcarriers prepared by a conventional method and 1/4 to 1/2 the amount of the final culture amount of the medium are injected into the culture tank. The seed cell solution prepared by a conventional method is further inoculated therein, and stirred to allow the cells to adhere to the microcarrier. It is desirable that the stirring be carried out very slowly and continuously at a rate of about 1/10 to 1/4 of the stirring rate in the growth phase, or intermittent stirring in which standing and stirring are repeated at a constant cycle. This attaching operation is normally completed in about several hours to 24 hours. In the present invention, the turbidity of the culture supernatant is measured at appropriate times during this attaching operation. Initially, most of the cells are suspended, so the turbidity is high, and the turbidity decreases as the cells attach to the microcarriers. This change in turbidity over time is monitored, and the number of floating cells that do not adhere to the microcarriers is calculated,
Monitor the adherence of cells. If the turbidity does not decrease, it can be determined that the cells are not attached, and therefore the attachment of the cells is promoted by an operation such as reducing the stirring speed or extending the standing time. When the change in turbidity stops and it is determined from the turbidity that the required amount of cells have adhered to the microcarriers, the cell attachment operation is terminated and normal culture is started. The medium is filled up to the final culture amount, the stirring speed is increased to a speed at which all the microcarriers float, and the culture is continued. Even in this normal culture, the turbidity is appropriately measured. From the viewpoint of supplying oxygen and nutrients, the mass transfer rate is high, so the stirring speed should be high.
However, if the stirring speed is increased too much, the cells may be damaged by the shearing force of stirring and die. When the cells die and are detached from the microcarriers and floated to increase the turbidity of the culture solution, the stirring speed is reduced until the turbidity does not increase. In the present invention, the adhered state of cells to the microcarriers can be maintained in a good state during the culture period by the method described above.

【0016】更に、筆者等は鋭意誠心実験した結果、特
に多孔質マイクロキャリアにおいて細胞濃度107 レベ
ルの高密度培養時に、培養当初に比較してマイクロキャ
リアが非常に重くなることを見出した。マイクロキャリ
アが重くなると、培養槽上部のマイクロキャリア濃度は
薄くなり、培養槽下部のマイクロキャリア濃度は濃くな
る。場合によっては一部マイクロキャリアが培養槽底部
に堆積する。これにより、マイクロキャリアを培養液中
で均一に浮遊させるためには、高密度培養時には撹拌速
度を上昇させることが必要であることが判明した。しか
しながら、撹拌速度の過度の上昇は、前述したように細
胞の良好な付着状態を阻害する。この相反する二つの現
象をクリアする方法として、筆者等は次のような培養方
法を見出した。培養初期の増殖期においては、すべての
マイクロキャリアにおいて良好に細胞が増殖するために
マイクロキャリアの均一な撹拌が必要であるが、高密度
化して定常期に入った培養後期においては、多少の不均
一が生じても、増殖した細胞が剥離せず、有用物質生産
能が高い状態を維持することが効率的であると考えられ
る。培養槽内において、A:スクリーン下近傍、B:中
心部及びC:下部の少なくとも3点の濁度を測定する。
また、測定した濁度の値から、測定地点近傍でのマイク
ロキャリアの濃度(存在する粒子数)を算出する。マイ
クロキャリアが均一に浮遊している場合、3点の濁度は
同じになる。細胞が増殖してマイクロキャリアが重くな
ってくると、3点の濁度はC>B>Aとなる。3点の濁
度差から、マイクロキャリアが培養槽底部に堆積せずに
培養槽内で濃度差はあるがすべて浮遊しておりかつスク
リーン近傍にはほとんどマイクロキャリアが存在せず、
かつ、細胞の剥離がおこらないように撹拌速度を制御す
る。細胞の剥離はスクリーンより上の位置のマイクロキ
ャリアが存在しない培養液中の濁度を測定して、細胞数
を算出して監視する。これより、培地中の酸素及び栄養
分がすべての細胞に供給され、かつ、スクリーン近傍に
マイクロキャリアが存在しないので、培養液の上向流ろ
過がマイクロキャリアによって妨げられることなく、ス
クリーンの目詰りも効果的に抑制することができる。よ
って、培養初期のマイクロキャリアが均一に浮遊してい
る時は、頻繁にスクリーンを振動させ、高密度培養時に
おいて、マイクロキャリアがスクリーン近傍にほとんど
存在しないようにした場合は、振動させる頻度を減少さ
せるのが望ましい。
Furthermore, as a result of earnest experiments, the authors have found that the microcarriers become extremely heavier than those at the beginning of the culturing, especially in the high-density culturing with a cell concentration of 10 7 level in the porous microcarriers. As the microcarrier becomes heavier, the concentration of microcarriers in the upper part of the culture tank becomes thinner and the concentration of microcarriers in the lower part of the culture tank becomes higher. In some cases, some microcarriers are deposited on the bottom of the culture tank. From this, it was found that in order to uniformly suspend the microcarriers in the culture medium, it is necessary to increase the stirring speed during high-density culture. However, an excessive increase in the stirring speed hinders the good adherence of cells as described above. As a method of clearing these two contradictory phenomena, the authors have found the following culture method. In the early growth phase of the culture, uniform agitation of the microcarriers is necessary for good cell growth in all the microcarriers, but in the latter phase of the culture, where the densification is high and the stationary phase is reached, there are some problems. Even if homogeneity occurs, it is considered effective to maintain the state in which the proliferated cells are not detached and the useful substance-producing ability is high. In the culture tank, turbidity is measured at least at three points: A: under the screen, B: central part, and C: lower part.
Further, the concentration of microcarriers (the number of existing particles) near the measurement point is calculated from the measured turbidity value. If the microcarriers are uniformly suspended, the turbidity at the three points will be the same. When the cells grow and the microcarrier becomes heavier, the turbidity at three points becomes C>B> A. From the turbidity difference at three points, microcarriers did not deposit at the bottom of the culture tank, but there were differences in concentration within the culture tank, but all were floating and there were almost no microcarriers near the screen,
At the same time, the stirring speed is controlled so that cell detachment does not occur. The detachment of cells is monitored by measuring the turbidity in the culture solution in which the microcarriers above the screen are not present and calculating the cell number. As a result, oxygen and nutrients in the medium are supplied to all cells, and since there is no microcarrier in the vicinity of the screen, upflow filtration of the culture solution is not hindered by the microcarrier and the screen is not clogged. It can be effectively suppressed. Therefore, when the microcarriers at the beginning of the culture are uniformly suspended, the screen is vibrated frequently, and when the microcarriers are hardly present near the screen during high-density culture, the frequency of vibrating is reduced. It is desirable to let

【0017】本発明において濁度とは、マイクロキャリ
アを含む培養液及び/または培養液上清の吸光度を波長
が600〜900nmの範囲の光で測定したときの値を
いう。筆者等は、既に、マイクロキャリア等の培養担体
を用いる生体の細胞の浮遊撹拌培養において、該濁度測
定が細胞の剥離操作及び細胞の付着操作の監視及び制御
の有効な手段となりうることを発見し、特開平5−16847
1 号公報として出願済みである。本発明では、これをさ
らに応用して、培養状態を監視,制御するものである。
600〜900nmの範囲の光を用いることで、培地や
血清自体の吸光を低く押さえ、細胞やマイクロキャリア
の濃度変化を濁度変化として精度良く測定することが可
能である。
In the present invention, the term "turbidity" refers to a value obtained by measuring the absorbance of a culture solution containing a microcarrier and / or a culture solution supernatant with light having a wavelength in the range of 600 to 900 nm. The authors have already discovered that the measurement of turbidity can be an effective means for monitoring and controlling detachment of cells and attachment of cells in suspension stirring culture of living cells using culture carriers such as microcarriers. Japanese Patent Laid-Open No. 5-16847
It has been filed as Publication No. 1. In the present invention, this is further applied to monitor and control the culture state.
By using the light in the range of 600 to 900 nm, it is possible to suppress the absorption of the medium or serum itself to be low, and it is possible to accurately measure the change in concentration of cells or microcarriers as the change in turbidity.

【0018】高密度培養時に、前述したようなマイクロ
キャリアの浮遊状態を保つには、撹拌速度以外に、液中
通気槽から培養槽へ循環させる酸素が富化された培養液
の循環量(循環速度)も関係する。液の循環速度が速い
とマイクロキャリアは上向流により培養槽上部に集まり
やすくなる。よって、撹拌速度と培養液の循環速度の両
方を制御する必要があり、培養液の循環速度は、マイク
ロキャリアの沈降速度と等しいのがより望ましい。この
場合の培養液の循環速度は、培養液の培養槽断面での空
塔速度であり、次式で表される。培養液の循環速度(cm
/s)=培養液の循環量(ml/min.)/60/培養
槽の底面積(cm2) 一方、細胞への酸素供給量は、酸素富化培養液の循環速
度と酸素富化培地中の酸素濃度に比例する。培養状態を
良好に保つには、細胞に必要充分量の酸素を供給しなけ
ればならない。本発明においては、接続管路内の培養液
の溶存酸素濃度を測定する手段によって得た情報、すな
わち培養槽に流入する酸素が富化された培養液中の溶存
酸素濃度と、培養槽から流出する酸素が消費された培養
液中の溶存酸素濃度を測定する。測定結果より、培養槽
内の細胞の酸素消費速度,酸素消費量および細胞数を算
出する。酸素消費速度及び酸素消費量より、必要な酸素
供給量が算出される。また、細胞数よりマイクロキャリ
アの沈降速度が算出される。これらの、必要な酸素供給
量とマイクロキャリアの沈降速度から、マイクロキャリ
アの沈降速度≧培養液の循環速度でありかつ必要とする
酸素を供給できるように、液中通気槽に通気する酸素含
有ガスの酸素濃度,通気量及び内圧を変化させる。
In order to maintain the above-mentioned floating state of the microcarriers during high-density culture, in addition to the stirring speed, the circulation amount (circulation) of the oxygen-enriched culture solution to be circulated from the submerged aeration tank to the culture tank. Speed) is also relevant. If the circulation speed of the liquid is high, the microcarriers are likely to collect in the upper part of the culture tank due to the upward flow. Therefore, it is necessary to control both the stirring rate and the circulation rate of the culture solution, and it is more desirable that the circulation rate of the culture solution be equal to the sedimentation rate of the microcarriers. The circulation speed of the culture solution in this case is the superficial velocity of the culture solution in the cross section of the culture tank, and is represented by the following equation. Circulation rate of culture solution (cm
/ S) = circulation amount of culture medium (ml / min.) / 60 / bottom area of culture tank (cm 2 ) On the other hand, the amount of oxygen supplied to cells is determined by the circulation rate of the oxygen-enriched culture medium and the oxygen-enriched medium. It is proportional to the oxygen concentration inside. To maintain a good culture condition, the cells must be supplied with a sufficient amount of oxygen. In the present invention, the information obtained by the means for measuring the dissolved oxygen concentration of the culture solution in the connecting conduit, that is, the dissolved oxygen concentration in the oxygen-enriched culture solution flowing into the culture tank, and the outflow from the culture tank The dissolved oxygen concentration in the culture solution in which the oxygen to be consumed is measured. From the measurement results, the oxygen consumption rate of cells in the culture tank, the oxygen consumption amount, and the number of cells are calculated. The required oxygen supply amount is calculated from the oxygen consumption rate and the oxygen consumption amount. Further, the sedimentation rate of microcarriers is calculated from the number of cells. Based on the required oxygen supply rate and the sedimentation rate of the microcarriers, the sedimentation rate of the microcarriers is equal to or higher than the circulation rate of the culture solution, and the oxygen-containing gas aerated in the submerged aeration tank so that the required oxygen can be supplied. Change the oxygen concentration, aeration rate and internal pressure of.

【0019】液中通気槽での酸素含有ガスの通気によ
り、培養液が発泡する事態が考えられる場合には、液中
通気槽気相部に破泡手段を設ける。破泡手段としては、
特に限定するものではなく、回転翼等により泡沫に機械
的衝撃を与える方法,破泡機能を有する固体表面に泡沫
を接触させる方法,消泡機能を有する物質を添加剤とし
て用いる等、公知の方法を用いることができる。
When it is considered that the culture solution foams due to aeration of the oxygen-containing gas in the submerged aeration tank, a bubble breaking means is provided in the gas phase portion of the submerged aeration tank. As means for breaking bubbles,
The method is not particularly limited, and a known method such as a method in which a foam is mechanically impacted by a rotary blade or the like, a method in which the foam is brought into contact with a solid surface having a foam breaking function, a substance having a defoaming function is used as an additive, etc. Can be used.

【0020】[0020]

【作用】本発明による生体の細胞の培養装置の好ましい
態様においては、培養槽内にマイクロキャリアは通過さ
せないが液成分は通過させる細孔径を有したスクリーン
を培養槽内上部に水平に設置し、マイクロキャリア等の
固体粒子が培養液面上部の気相部に形成される泡沫層に
取り込まれるのを阻止する。培養槽内のスクリーンより
下の部分が実際に細胞が増殖する培養域となる。
In a preferred embodiment of the apparatus for culturing cells of a living body according to the present invention, a screen having a pore diameter that does not allow passage of microcarriers but allows passage of liquid components is placed horizontally in the upper part of the culture tank, It prevents solid particles such as microcarriers from being incorporated into the foam layer formed in the gas phase portion above the culture solution surface. The area below the screen in the culture tank is the culture area where cells actually grow.

【0021】液中通気槽には酸素を溶解させるべき培養
液が貯留されている。該溶液は、新しい培養液、または
培養液からマイクロキャリア等の固形分を除去した溶
液、または両者の混合液である。
A culture solution for dissolving oxygen is stored in the submerged aeration tank. The solution is a fresh culture solution, a solution obtained by removing solid contents such as microcarriers from the culture solution, or a mixed solution of both.

【0022】本発明において、液中通気による培養槽内
の細胞への酸素の供給は以下のようにして行われる。ま
ず、液中通気槽下部に設置した酸素含有ガス通気用ノズ
ルから酸素含有ガスを通気し、内部の培養液に酸素を溶
解させる。充分に酸素が溶解して酸素富化された培養液
を、第1の接続管を通して、該接続管に設置された送液
手段により培養槽下部から流入させる。酸素富化された
培養液は、培養槽内を上昇し、その過程で培養槽内の培
養液と接触,混合されて、培養槽内の細胞に酸素を供給
する。ついで、スクリーンを通過し、第2の接続管に流
入し、該接続管に設置された送液手段によって液中通気
に戻り、再び酸素富化される。
In the present invention, oxygen is supplied to cells in the culture tank by submerged aeration in the following manner. First, an oxygen-containing gas is aerated through an oxygen-containing gas aeration nozzle installed in the lower part of the submerged aeration tank to dissolve oxygen in the culture solution inside. A culture solution in which oxygen is sufficiently dissolved and oxygen-enriched is caused to flow from the lower part of the culture tank through the first connecting pipe by the liquid feeding means installed in the connecting pipe. The oxygen-enriched culture solution rises in the culture tank, and in the process of being contacted with and mixed with the culture solution in the culture tank, oxygen is supplied to the cells in the culture tank. Then, it passes through the screen, flows into the second connecting pipe, returns to submerged aeration by the liquid feeding means installed in the connecting pipe, and is enriched with oxygen again.

【0023】液中通気槽に貯留されている酸素溶解用の
培養液は以上述べた経路を循環し、酸素富化された培養
液と培養領域内の培養液が接触すると、両液間で液中の
成分の拡散がおこり、細胞への酸素供給及び栄養分の供
給と、老廃成分及び二酸化炭素ガスの抜き出しが行われ
る。培養領域を形成するスクリーンはマイクロキャリア
の培養領域からの漏出を防ぎ、培養液面上の気相部に形
成される泡沫層への取り込みを防止する。これにより、
液中通気による効率的な酸素供給を可能にし、多孔質マ
イクロキャリアによる付着性動物細胞の細胞濃度107
レベルの高密度培養を実現する。
The oxygen-dissolving culture solution stored in the submerged aeration tank circulates through the above-mentioned route, and when the oxygen-enriched culture solution and the culture solution in the culture area come into contact with each other, the solution is dissolved between the two solutions. Diffusion of internal components occurs, oxygen is supplied to cells, nutrients are supplied, and waste components and carbon dioxide gas are extracted. The screen forming the culture area prevents leakage of the microcarriers from the culture area and prevents the microcarriers from being taken up by the foam layer formed in the gas phase portion on the culture solution surface. This allows
It enables efficient oxygen supply by aeration in the liquid, and the cell concentration of adherent animal cells by the porous microcarrier is 10 7
Achieve high-level high-density culture.

【0024】また、本発明になる生体の細胞の培養装置
では、細胞のマイクロキャリアへの付着操作は、培養槽
内の濁度を制御因子として、以下のように行われる。ま
ず、種細胞が接種される前の培養液上清の濁度を測定す
る。次に、種細胞を接種した後の濁度を測定する。後者
から前者を差し引いた値が浮遊している細胞の与える濁
度である。接種した細胞量は、別途、接種前に測定して
おく。濁度は細胞数に正比例するので、これから、細胞
数と濁度の関係式を導くことができる。培養液を低速で
連続撹拌するか、間歇撹拌することにより、細胞をマイ
クロキャリアに付着させる。この付着操作の際の培養量
は、通常の培養量の1/4〜1/2とするのがより望ま
しい。細胞がマイクロキャリアに付着すると、培養液の
濁度は、付着した細胞に反比例して減少する。よって、
濁度の減少量により細胞の付着数がわかり、また、浮遊
したままの細胞数も算出することができる。濁度の減少
がおこらない場合は、細胞がマイクロキャリアに付着し
ていないので、撹拌速度を低下させたり、静置時間を延
長したりして、付着状態を改善する。このように濁度の
経時変化をモニタすることで、細胞の付着状態を、培養
液をサンプリングして顕微鏡観察することなく、オンラ
インで監視することができる。濁度の測定結果から、必
要量の細胞がマイクロキャリアに付着した時点で、付着
操作を終了し、培養液量を通常の培養量まで増加し、培
養を継続する。
Further, in the apparatus for culturing cells of a living body according to the present invention, the operation of attaching the cells to the microcarriers is carried out as follows with the turbidity in the culture tank as a controlling factor. First, the turbidity of the culture solution supernatant before inoculation of seed cells is measured. Next, the turbidity after seeding with the seed cells is measured. The value obtained by subtracting the former from the latter is the turbidity given by the floating cells. The amount of cells inoculated is measured separately before inoculation. Since the turbidity is directly proportional to the cell number, a relational expression between the cell number and the turbidity can be derived from this. The cells are attached to the microcarriers by continuously stirring the culture medium at low speed or by intermittently stirring it. It is more desirable that the culture amount in the attaching operation is 1/4 to 1/2 of the normal culture amount. When cells attach to the microcarriers, the turbidity of the culture decreases inversely with the attached cells. Therefore,
The number of attached cells can be known from the amount of decrease in turbidity, and the number of cells still floating can be calculated. When the turbidity does not decrease, the cells are not attached to the microcarriers, and therefore the agitation speed is reduced or the standing time is extended to improve the attachment state. By monitoring the change in turbidity with time in this manner, the adhered state of cells can be monitored online without sampling the culture solution and observing it under a microscope. From the measurement result of turbidity, when a required amount of cells adheres to the microcarriers, the attaching operation is terminated, the amount of culture solution is increased to a normal culture amount, and the culture is continued.

【0025】さらに、本発明では、以下に詳細に説明す
る方法により、培養液の濁度と同時に培養液の溶存酸素
濃度をモニタすることで、細胞が対数的に増殖する増殖
期および高密度になってからの定常期を通して、細胞の
生存,増殖及び有用物質生産に適した培養状態を保つこ
とができる。培養槽内のA:下部,B:中央部,C:ス
クリーン下近傍、及び、D:スクリーン上近傍の少なく
とも4ヶ所地点で培養液の濁度を測定する。A〜Cでの
濁度はマイクロキャリアと細胞が懸濁された培養液の濁
度を測定している。Dでは、マイクロキャリアは存在し
ないので、細胞のみが懸濁された培養液の濁度を測定す
ることになる。細胞の増殖期においては、マイクロキャ
リアが均一に混合されていることが必要である。マイク
ロキャリアが均一に撹拌されていれば、培養槽内の濁度
も一定になる。よって、濁度の値が、A=B=Cとなる
まで撹拌速度を上昇させればよい。ただし、撹拌速度の
上昇によってDの値が上昇する、すなわち、マイクロキ
ャリアから細胞が剥離してくるような場合には、剥離が
おこらなくなるまで撹拌速度を低下させる。すなわち、
A=B=CかつD=一定の条件を満たすように撹拌速度
を設定,制御する。細胞が高密度になると、マイクロキ
ャリアが重くなって沈降しやすくなる。この時、培養槽
内での濁度はA>B>Cという状態になる。つまり、培
養槽上部では、マイクロキャリアの濃度が低くなり、底
部の方へ行くに従って濃度が高くなる。培養槽上部でマ
イクロキャリアの濃度が低くなるということは、すなわ
ち、スクリーンの目詰りが抑制されるという良い効果が
生じる。これは、本発明の特徴の1つである培養槽上部
にスクリーンを設置し、培養液の流れを上向流とした場
合にのみに生まれる効果である。高密度培養時にスクリ
ーンの目詰りが生じると、急速に酸素供給量が低下し、
細胞に致命的なダメージを与えてしまう。よって、多
少、マイクロキャリアの濃度が培養槽内で不均一になっ
ても、スクリーンの目詰りが発生せず、充分な酸素が供
給できる方が望ましい。各ヶ所で測定した培養液の濁度
から、各地点でのマイクロキャリアの濃度を算出し、ス
クリーン近傍にはマイクロキャリアがほとんど存在せ
ず、かつ、底部でマイクロキャリアの堆積がおこらない
ように撹拌速度を設定,制御する。すなわち、マイクロ
キャリアの濃度と濁度の関係式を予め求めて、さらに、
マイクロキャリアの堆積がおこり始める濁度(A′,
B′)を把握しておく。これより、A≦A′,B≦B′
かつD=一定となるように、撹拌速度を設定,制御す
る。
Further, in the present invention, by monitoring the turbidity of the culture solution and the dissolved oxygen concentration of the culture solution by the method described in detail below, the growth phase and the high density in which the cells logarithmically grow are increased. Through the stationary phase after that, it is possible to maintain a culture state suitable for cell survival, growth and production of useful substances. The turbidity of the culture solution is measured at least at four points in the culture tank: A: lower part, B: central part, C: near the screen, and D: near the screen. For the turbidity in A to C, the turbidity of the culture solution in which the microcarriers and cells are suspended is measured. In D, since no microcarriers are present, the turbidity of the culture solution in which only cells are suspended will be measured. During the cell growth phase, it is necessary that the microcarriers are uniformly mixed. If the microcarriers are uniformly stirred, the turbidity in the culture tank will also be constant. Therefore, the stirring speed may be increased until the turbidity value becomes A = B = C. However, when the value of D increases as the stirring speed increases, that is, when cells are detached from the microcarriers, the stirring speed is reduced until detachment does not occur. That is,
The stirring speed is set and controlled so that A = B = C and D = constant conditions are satisfied. As the cells become denser, the microcarriers become heavier and more likely to settle. At this time, the turbidity in the culture tank is A>B> C. That is, the concentration of the microcarriers becomes lower in the upper part of the culture tank, and becomes higher toward the bottom part. The lower concentration of the microcarriers in the upper part of the culture tank has a good effect that the clogging of the screen is suppressed. This is one of the features of the present invention, which is an effect produced only when a screen is installed above the culture tank and the flow of the culture solution is an upward flow. If the screen becomes clogged during high-density culture, the oxygen supply will decrease rapidly,
Causes fatal damage to cells. Therefore, it is desirable that the screen is not clogged and sufficient oxygen can be supplied even if the concentration of the microcarriers becomes slightly uneven in the culture tank. Calculate the concentration of microcarriers at each point from the turbidity of the culture solution measured at each point, and stir so that there are few microcarriers near the screen and microcarriers do not accumulate at the bottom. Set and control the speed. That is, the relational expression between the concentration of microcarriers and turbidity is obtained in advance, and
Turbidity (A ′,
Understand B '). From this, A ≦ A ′, B ≦ B ′
And the stirring speed is set and controlled so that D = constant.

【0026】さらに、上述したマイクロキャリアの培養
槽内での存在状態、すなわち浮遊状態は、撹拌速度のほ
かに、液中通気槽から培養槽への培養液の循環速度によ
っても影響される。培養液の液循環速度が大き過ぎる
と、マイクロキャリアは培養槽の上部に集まりやすくな
る。マイクロキャリアが培養槽の上部に集積すると、マ
イクロキャリアが大きな抵抗となって培養液の上向流を
妨げ、同時にスクリーンの目詰りを促進するといった好
ましくない状態になる。よって、循環速度は、マイクロ
キャリアが培養槽上部に集積しないような速度、すなわ
ち、培養液の上向流速度(培養槽断面での培地の空塔速
度)≦マイクロキャリアの沈降速度を満たすのがより望
ましい。一方、細胞に供給される酸素量は、該上向流速
度と培養液の酸素濃度によって決まる。しかし、上向流
速度は前記のように上限が設定されているので、培養液
中の酸素濃度を変化させて、細胞に充分な酸素を供給す
る。培養液中の酸素濃度は、通気する酸素含有ガス中の
酸素濃度及びその通気量、または酸素供給槽の内圧によ
って変化させることができる。培養液中の酸素濃度の上
限は、液中通気槽の構造及び化学工学的特性により決ま
る。
Further, the above-mentioned existing state of the microcarriers in the culture tank, that is, the floating state, is influenced by the circulation speed of the culture solution from the submerged aeration tank to the culture tank, in addition to the stirring speed. If the liquid circulation speed of the culture solution is too high, the microcarriers are likely to collect in the upper part of the culture tank. When the microcarriers are accumulated on the upper part of the culture tank, the microcarriers become a large resistance and hinder the upward flow of the culture solution, and at the same time, the clogging of the screen is promoted, which is an undesirable state. Therefore, the circulation speed is such that the microcarriers do not accumulate in the upper part of the culture tank, that is, the upward flow speed of the culture solution (superficial velocity of the medium in the cross section of the culture tank) ≦ the sedimentation speed of the microcarriers. More desirable. On the other hand, the amount of oxygen supplied to the cells is determined by the upward flow velocity and the oxygen concentration of the culture solution. However, since the upper limit of the upward flow velocity is set as described above, the oxygen concentration in the culture solution is changed to supply sufficient oxygen to the cells. The oxygen concentration in the culture solution can be changed by the oxygen concentration in the oxygen-containing gas to be aerated and the amount of the aeration, or the internal pressure of the oxygen supply tank. The upper limit of the oxygen concentration in the culture solution is determined by the structure and chemical engineering characteristics of the submerged aeration tank.

【0027】本発明になる生体の細胞の培養装置を構成
する培養槽と液中通気槽は、酸素富化した培養液の上向
流速度≦マイクロキャリアの沈降速度の条件下で、高密
度培養時においても細胞に充分な酸素を供給できるよう
な構造及び化学工学的な特性を持つのがより好ましい態
様である。本発明の培養槽と液中通気槽の構造と化学工
学的特性について、簡便なモデルを用いて以下に説明す
る。
The culture tank and the submerged aeration tank which compose the apparatus for culturing cells of a living body according to the present invention are high-density culture under the conditions of upward flow velocity of oxygen-enriched culture liquid ≤ sedimentation velocity of microcarriers. It is a more preferred embodiment to have a structural and chemical engineering property that can supply sufficient oxygen to cells even at times. The structures and chemical engineering characteristics of the culture tank and submerged aeration tank of the present invention will be described below using a simple model.

【0028】今、培養槽、液中通気槽の容量を1リット
ルとし、循環する酸素富化培養液は、培養槽内で瞬間的
に均一に混合されるものとする。培養槽の底面積:S(c
m2),液深:H(cm),培地循環量:Q(cm3/s)、液
中通気槽の酸素溶解速度計数:KLa(h-1)、マイクロ
キャリアの沈降速度:Vc(cm/s)、培地の上向流速
度:Vf(cm/s)とし、培養槽内で維持する細胞濃度=
4×107cells/ml、溶存酸素濃度は2ppm 、培養温
度は37℃とする。液中通気槽には純酸素を通気するも
のとし、37℃での培養液中の飽和酸素濃度:C*(pp
m)=34,細胞に供給する培養液の酸素濃度:C(pp
m)とする。細胞1個あたりの酸素消費速度は1、筆者等
が実験により測定した値である2×10-13(mol/cell
・h)を、及び、マイクロキャリアの沈降速度:Vc
は、筆者等が実験に用いた多孔質マイクロキャリアの沈
降速度であるVc=0.02 を用いることとする。
Now, it is assumed that the capacity of the culture tank and the submerged aeration tank is 1 liter, and the circulating oxygen-enriched culture solution is instantaneously and uniformly mixed in the culture tank. Bottom area of culture tank: S (c
m 2 ), liquid depth: H (cm), medium circulation amount: Q (cm 3 / s), oxygen dissolution rate coefficient in submerged aeration tank: KLa (h -1 ), sedimentation rate of microcarriers: Vc (cm) / S), the upward flow velocity of the medium: Vf (cm / s), and the cell concentration to be maintained in the culture tank =
4 × 10 7 cells / ml, dissolved oxygen concentration 2 ppm, culture temperature 37 ° C. Pure oxygen should be ventilated in the submerged aeration tank, and the saturated oxygen concentration in the culture solution at 37 ° C: C * (pp
m) = 34, oxygen concentration of the culture solution supplied to the cells: C (pp
m). The oxygen consumption rate per cell is 1, which is the value measured by the authors through experiments, 2 × 10 -13 (mol / cell
H) and sedimentation rate of microcarriers: Vc
Is to use Vc = 0.02, which is the sedimentation rate of the porous microcarriers used by the authors in the experiments.

【0029】(I)常圧通気で、KLa=18の場合 培養槽内の細胞の酸素消費速度は、細胞一個当たりの酸
素消費速度と細胞数の積で表されるので、 (2×10-13)×(4×107×1000)=8×10-3(mol/h) 8×10-3(mol/h)=256(mg−酸素/h) …………(1) KLaの関係式から、dC/dT=KLa(C*−C) …………(2) よって、(2)式より、C=C*−1/KLa・dC/dT…………(3) 前提条件より、dC/dT=細胞の酸素消費速度である
から、KLa=18,C* =34を代入して、供給する
培地の酸素濃度を求めると C=34−256/18=19.8 供給される酸素は、培養槽内の溶存酸素濃度を維持する
分と細胞が消費する分との和なので、細胞の消費分は、
溶存酸素濃度を2ppm に維持すると仮定しているので、
19.8−2=17.8(mg/l) すなわち、 5.6×10-4(mol/l) ……………(4) となる。細胞に供給される酸素量=酸素濃度(4)×循
環量Qであり、これが細胞の消費する酸素量(1)と等
しいので、 (5.6×10-4)・Q=8×10-3、よって、Q=1
4.3(l/h)、すなわち、Q=4.0(cm3/s) この時、前提条件よりVf=Vcで、Vf=Q/Sなの
で、S=Q/Vfより、 S=4.0/0.02=200(cm2) ……………(5) H=1000/200=5(cm) ……………(6) となり、培養槽が円柱形であるとすると内径が約16cm
となる。
(I) When KLa = 18 under normal pressure aeration Since the oxygen consumption rate of cells in the culture tank is represented by the product of the oxygen consumption rate per cell and the number of cells, (2 × 10 − 13 ) × (4 × 10 7 × 1000) = 8 × 10 −3 (mol / h) 8 × 10 −3 (mol / h) = 256 (mg-oxygen / h) ………… (1) KLa From the relational expression, dC / dT = KLa (C * −C) ………… (2) Therefore, from the expression (2), C = C * −1 / KLa · dC / dT ………… (3) From the conditions, dC / dT = cell oxygen consumption rate, so KLa = 18 and C * = 34 are substituted to determine the oxygen concentration of the medium to be supplied, C = 34-256 / 18 = 19.8. The amount of oxygen consumed is the sum of the amount maintained by the dissolved oxygen concentration in the culture tank and the amount consumed by the cells.
Assuming that the dissolved oxygen concentration is maintained at 2ppm,
19.8-2 = 17.8 (mg / l) That is, 5.6 × 10 −4 (mol / l) ... (4) The amount of oxygen supplied to cells = oxygen concentration (4) × circulation amount Q, which is equal to the amount of oxygen consumed by cells (1), so that (5.6 × 10 −4 ) · Q = 8 × 10 − 3 , so Q = 1
4.3 (l / h), that is, Q = 4.0 (cm 3 / s) At this time, since Vf = Vc and Vf = Q / S from the preconditions, S = 4 from S = Q / Vf. 0.0 / 0.02 = 200 (cm 2 ) ………… (5) H = 1000/200 = 5 (cm) ………… (6), and assuming that the culture tank is cylindrical, the inner diameter Is about 16 cm
Becomes

【0030】このような形状の培養槽は実現不可能であ
る。よって、液中通気槽のKLa =18では、常圧純酸
素通気による通気による酸素供給では、酸素供給能が不
足しており、すくなくともKLa>18 でなければなら
ないことがわかる。しかし、この場合も、0.5〜1.0
kg/cm2 で純酸素を加圧通気すれば、充分な酸素を供
給することが計算上可能である。
A culture tank having such a shape cannot be realized. Therefore, it is understood that when KLa = 18 in the submerged aeration tank, the oxygen supply ability is insufficient in the oxygen supply by the aeration by the normal pressure pure oxygen aeration, and at least KLa> 18 is required. However, in this case as well, 0.5-1.0
If pure oxygen is pressurized and ventilated at kg / cm 2 , sufficient oxygen can be calculated.

【0031】ところで、筆者等は、多孔質マイクロキャ
リアを用いた付着性動物細胞の培養において、細胞濃度
が高密度になるとマイクロキャリアが重くなり、沈降速
度が大きくなることを発見した。筆者等の実験に用いた
多孔質マイクロキャリアの沈降速度は、上述したように
細胞が付着していない状態では0.02(cm/s)であっ
たが、細胞濃度が107レベルになると0.04(cm/
s)と2倍に増加した。これにより、上述の(5)及び
(6)式は、 S′=4.0/0.04=100(cm2) ……………(5′) H′=1000/100=10(cm) ……………(6′) となり、培養槽の内径は約11cmで、実際的な形状とな
る。よって、この場合は、液中通気槽を加圧することな
く、常圧通気で必要な酸素を供給することができる。よ
って、マイクロキャリアの沈降速度の変化は、酸素供給
方法に大きな影響を与えることがわかる。上述の計算
は、液中通気槽の容量や使用するマイクロキャリアの比
重等の前提条件を変えることによって、実現できる培養
槽の形状は様々なものが与えられる。
By the way, the authors have found that in the culture of adherent animal cells using a porous microcarrier, the microcarrier becomes heavier and the sedimentation rate becomes higher as the cell density becomes higher. The sedimentation rate of the porous microcarriers used in the experiments by the authors was 0.02 (cm / s) in the state where cells were not attached as described above, but it was 0 when the cell concentration reached 10 7 level. .04 (cm /
s) and doubled. As a result, the above equations (5) and (6) are as follows: S ′ = 4.0 / 0.04 = 100 (cm 2 ) ... (5 ′) H ′ = 1000/100 = 10 (cm ) ……………… (6 ′), and the inner diameter of the culture tank is about 11 cm, which is a practical shape. Therefore, in this case, the required oxygen can be supplied by atmospheric aeration without pressurizing the submerged aeration tank. Therefore, it is understood that the change in the sedimentation rate of the microcarriers has a great influence on the oxygen supply method. In the above calculation, various shapes of the culture tank can be realized by changing the preconditions such as the capacity of the submerged aeration tank and the specific gravity of the microcarriers used.

【0032】本発明において、より好ましくは、液中通
気槽から培養槽へ培養液が流入するケ所と、流出するケ
所の2ヶ所において、培養液中の溶存酸素濃度を測定す
るのが望ましい。これにより、培養槽中の細胞の酸素消
費量を算出し、さらに、その時の細胞数及びマイクロキ
ャリアの沈降速度を求める。これらのデータから、細胞
が必要とする酸素を、酸素富化培養液の循環速度とマイ
クロキャリアの沈降速度とが等しくなるような条件で供
給できるように、液中通気槽の内圧、通気ガスの通気量
及び通気ガス中の酸素濃度を変化させる。
In the present invention, it is more preferable to measure the dissolved oxygen concentration in the culture solution at two points, that is, the point where the culture solution flows from the submerged aeration tank to the culture tank and the point where the culture solution flows out. Thereby, the oxygen consumption of the cells in the culture tank is calculated, and the cell number and the sedimentation rate of the microcarriers at that time are calculated. From these data, the oxygen required by the cells can be supplied under conditions such that the circulation rate of the oxygen-enriched culture solution and the sedimentation rate of the microcarriers are equal, so that the internal pressure of the submerged aeration tank and the aeration gas Change the amount of oxygen and the oxygen concentration in the gas.

【0033】さらに好ましくは、あわせて、培養槽内の
少なくとも上部,中部,下部の3点において、培養液の
濁度を測定する。これらの濁度変化からも、マイクロキ
ャリアの沈降速度を算出し、あわせて、酸素供給制御方
法決定のデータとして使用する。
More preferably, in addition, the turbidity of the culture solution is measured at least at three points of the upper part, the middle part and the lower part in the culture tank. From these changes in turbidity, the sedimentation rate of microcarriers is also calculated and used together as data for determining the oxygen supply control method.

【0034】本発明においては、培養槽内においても、
培養液の溶存酸素濃度とpHを測定し、培養槽内の溶存
酸素濃度及びpHが細胞の生存,増殖に適した条件に制
御される。pH低下の場合は、アルカリ溶液を添加する
か、培養液の灌流率を上昇させる。
In the present invention, even in the culture tank,
The dissolved oxygen concentration and pH of the culture solution are measured, and the dissolved oxygen concentration and pH in the culture tank are controlled to conditions suitable for cell survival and growth. When the pH is lowered, an alkaline solution is added or the perfusion rate of the culture solution is increased.

【0035】このように、培養液の灌流は培養液のpH
が低下し始めた時点で開始し、以後細胞濃度の上昇に合
わせて灌流率を増加させていくのが良い。ポンプ等を用
いて酸素や栄養分が枯渇しかわりに細胞が生産した有用
物質を含む培養液を、培養槽外に所定量抜き出し、同量
の酸素と栄養分が豊富な培地を培養槽に注入し、培養槽
内の液量を一定に保って灌流培養を行う。本発明におい
ては、培養槽上部のスクリーンより上の液部分から液を
抜き出すことで、マイクロキャリアを含まない液のみを
容易に培養槽外に取りだすことができ、特別な他の分離
手段を必要としない。
As described above, the perfusion of the culture medium depends on the pH of the culture medium.
It is better to start at the time when the permeation rate starts to decrease, and thereafter increase the perfusion rate as the cell concentration increases. A predetermined amount of a culture solution containing useful substances produced by cells instead of depletion of oxygen and nutrients using a pump or the like is extracted outside the culture tank, and the same amount of oxygen and nutrient-rich medium is injected into the culture tank. Perfusion culture is performed while keeping the liquid volume in the culture tank constant. In the present invention, by extracting the liquid from the liquid portion above the screen in the upper part of the culture tank, it is possible to easily take out only the liquid containing no microcarriers outside the culture tank, and a special separation means is required. do not do.

【0036】以上の説明から明らかなように、本発明に
よる培養装置は、高い酸素供給能力により、多孔質マイ
クロキャリアによる生体の細胞の高密度培養が可能であ
り、構造が簡単であるので工業レベルまでのスケールア
ップが容易である。
As is clear from the above description, the culture device according to the present invention is capable of high-density culture of living cells using porous microcarriers due to its high oxygen supply capacity, and has a simple structure, so that it is on an industrial level. It is easy to scale up.

【0037】[0037]

【実施例】図1は、本発明になる培養装置の一実施例で
ある。
EXAMPLE FIG. 1 shows an example of a culture apparatus according to the present invention.

【0038】本培養装置は、ステンレス鋼性の内容積1
0リットル,培養容積8リットルの培養槽1,ステンレ
ス鋼製の内容積10リットル,液容積8リットルの液中
通気槽2、図示していないが、新鮮培地貯槽,培地貯
槽,ガス供給系として酸素源,炭酸ガス源,窒素源、及
び培養状況を監視し制御するための制御部から構成され
ている。
The main culturing apparatus has a stainless steel internal volume of 1
0 liter, culture volume 8 liter culture vessel 1, stainless steel inner volume 10 liter, liquid volume 8 liter submerged aeration vessel 2, fresh medium storage tank, medium storage tank, oxygen as gas supply system (not shown) Source, carbon dioxide gas source, nitrogen source, and a control unit for monitoring and controlling the culture condition.

【0039】培養槽1には、マイクロキャリアが懸濁さ
れた培養液が貯留され、細孔径100μmのステンス製フ
ィルタで構成されたスクリーン3aが設置されている。
さらに、培養槽1内にはレベルセンサ12a,温度セン
サ13,pHセンサ8,DOセンサ7b,濁度計4a〜
4d,撹拌器5,撹拌モータ6、及び加振器11が設置
されている。
A culture solution in which microcarriers are suspended is stored in the culture tank 1, and a screen 3a composed of a stainless steel filter having a pore diameter of 100 μm is installed.
Furthermore, in the culture tank 1, a level sensor 12a, a temperature sensor 13, a pH sensor 8, a DO sensor 7b, and a turbidity meter 4a-
4d, the stirrer 5, the stirring motor 6, and the vibrator 11 are installed.

【0040】液中通気槽2には、レベルセンサ12b,
DOセンサ7d,酸素含有ガス通気用ノズル15と、通
気により発生する泡沫を破泡するための消泡層14が設
置されている。
The submerged aeration tank 2 includes a level sensor 12b,
The DO sensor 7d, the oxygen-containing gas ventilation nozzle 15 and the defoaming layer 14 for breaking the foam generated by ventilation are provided.

【0041】培養槽1と液中通気槽2とは、それぞれ、
DOセンサ7aとポンプ9a,DOセンサ7cとポンプ
9bが設置された2本の接続管で接続されている。DO
センサ7aとポンプ9aが設置された接続管は、培養槽
1ではスクリーン3aよりも上の液相部分と液中通気槽
2の上部の液相部分との両端で開口している。DOセン
サ7cとポンプ9bが設置された接続管には、さらに培
養槽1側に、逆止弁10cが設置されており、培養槽1
内のマイクロキャリアが液中通気槽2に混入しないよう
になっており、培養槽1と液中通気槽2の下部の液相部
分に両端が開口している。
The culture tank 1 and the submerged aeration tank 2 are respectively
The DO sensor 7a and the pump 9a and the DO sensor 7c and the pump 9b are connected by two connecting pipes. DO
In the culture tank 1, the connection pipe in which the sensor 7a and the pump 9a are installed is open at both ends of the liquid phase portion above the screen 3a and the liquid phase portion above the submerged aeration tank 2. A check valve 10c is further installed on the side of the culture tank 1 in the connecting pipe provided with the DO sensor 7c and the pump 9b.
The microcarriers therein are not mixed in the submerged aeration tank 2, and both ends are opened to the liquid phase portions below the culture tank 1 and the submerged aeration tank 2.

【0042】まず、あらかじめ、培養槽1,液中通気槽
2,新鮮培地貯槽,培地貯槽及びこれらを接続する配管
を高圧蒸気滅菌する。マイクロキャリアを、あらかじめ
常法により調製した後、培養槽内のスクリーン3aで仕
切られた培養領域に注入して滅菌する。別途、マイクロ
キャリア調製槽を設置して、マイクロキャリアを調製,
滅菌,平衡化した後、培養槽1に無菌的に供給する手段
をとっても良い。
First, the culture tank 1, the submerged aeration tank 2, the fresh medium storage tank, the medium storage tank and the pipes connecting them are sterilized by high pressure steam. Microcarriers are prepared in advance by a conventional method, and then injected into the culture area partitioned by the screen 3a in the culture tank and sterilized. Separately, a microcarrier preparation tank is installed to prepare microcarriers.
After sterilization and equilibration, a means for aseptically supplying to the culture tank 1 may be used.

【0043】次いで、培養する生体の細胞の生育に必要
な栄養分を含んだ培地を調製し、滅菌処理して新鮮培地
貯留槽及び液中通気槽2に保管する。保管した新鮮培地
のうち必要量をポンプ9bにより培養槽1に移送する。
この時の培地量は、通常の培養量の1/4〜1/2であ
る。
Next, a medium containing the nutrients necessary for the growth of the cells of the living organism to be cultured is prepared, sterilized, and stored in the fresh medium storage tank and the submerged aeration tank 2. A necessary amount of the stored fresh medium is transferred to the culture tank 1 by the pump 9b.
The amount of medium at this time is 1/4 to 1/2 of the usual culture amount.

【0044】次に、培養槽1外部に設けたウォータジャ
ケットに温水を通じて、培養槽内を培養する生体の細胞
の生育に適した温度に調節する。培養槽内の温度状況は
温度センサ13によって制御部におくられ、その情報を
もとに制御部は培養槽内の温度を一定に保つ。
Next, warm water is passed through a water jacket provided outside the culture tank 1 to adjust the temperature in the culture tank to a temperature suitable for the growth of cells of a living organism. The temperature condition in the culture tank is sent to the control unit by the temperature sensor 13, and the control unit keeps the temperature in the culture tank constant based on the information.

【0045】温度が安定したら、別途調製した種細胞液
を培養槽1に接種する。接種と同時に撹拌モータ6によ
り撹拌器5を稼働させて培養液を所定の撹拌速度で撹拌
し、浮遊している種細胞をマイクロキャリアに付着させ
る。この付着操作の撹拌速度は、通常の撹拌速度の1/
10〜1/4の低速撹拌とする。付着操作終了の目安を
撹拌開始から24時間とし、この間、濁度計4dにより
適時培養液の濁度を測定する。濁度測定時は、撹拌を停
止してマイクロキャリアを沈降させ、培養液上清の濁度
を測定する。濁度の測定結果は制御部におくられ、その
結果をもとに制御部は、マイクロキャリアに付着しない
で浮遊している細胞数及び/またはマイクロキャリアに
付着した細胞数を算出し、細胞の付着状況を判定する。
細胞の付着状態が不良と判定された場合には、撹拌速度
を低下させる、または、培養液の静置時間を延長すると
いった操作を行い、細胞の付着を促進する。濁度の情報
により、制御部が所定量の細胞がマイクロキャリアに付
着したと判定した時点で付着操作を終了し、通常の培養
に移る。
After the temperature is stabilized, the seed cell solution prepared separately is inoculated into the culture tank 1. Simultaneously with the inoculation, the stirrer 5 is operated by the stirrer motor 6 to stir the culture solution at a predetermined stirrer speed to attach the floating seed cells to the microcarriers. The stirring speed of this adhesion operation is 1 / g of the normal stirring speed.
Low speed stirring of 10 to 1/4. The standard of completion of the attachment operation is set to 24 hours from the start of stirring, and during this period, the turbidity of the culture solution is measured at an appropriate time by the turbidimeter 4d. When measuring turbidity, stirring is stopped and the microcarriers are allowed to settle, and the turbidity of the culture supernatant is measured. The turbidity measurement result is sent to the control unit, and based on the result, the control unit calculates the number of cells floating without adhering to the microcarrier and / or the number of cells adhering to the microcarrier, Determine the adhesion status.
When it is determined that the adhered state of the cells is poor, the operation of lowering the stirring speed or extending the standing time of the culture solution is performed to promote the adherence of the cells. When the control unit determines that a predetermined amount of cells have adhered to the microcarrier based on the turbidity information, the adhesion operation is terminated, and normal culture is started.

【0046】ポンプ9bを稼働させて必要量の新鮮培地
を培養槽1に注入し、撹拌器5の撹拌速度を上昇させ
る。撹拌速度は、濁度計4a〜4dで濁度を測定し、4
aの濁度が増加せず一定で、かつ、4b〜4dの濁度が
一定となるまで上昇させる。制御部は、これらの濁度情
報により、培養液上清中の浮遊細胞濃度と、マイクロキ
ャリアの浮遊濃度を算出し、培養槽内の培養液の混合状
態、細胞の付着状態を監視する。
The pump 9b is operated to inject a required amount of fresh medium into the culture tank 1, and the stirring speed of the stirrer 5 is increased. The stirring speed was measured by measuring the turbidity with the turbidimeters 4a to 4d.
The turbidity of a is not increased and is constant, and the turbidity of 4b to 4d is increased until it is constant. The control unit calculates the floating cell concentration in the culture medium supernatant and the floating concentration of the microcarriers based on the turbidity information, and monitors the mixed state of the culture medium and the cell adhesion state in the culture tank.

【0047】細胞の増殖により、培養液中のpH,DO
の変化や、栄養成分の枯渇,老廃物の蓄積が起こる。こ
のため、培養槽1には、pHセンサ8,DOセンサ7
b,2本の接続管にはDOセンサ7a,7cが設置さ
れ、pH,DO,濁度を計測する。これらの計測情報は
制御部に伝送され、培養状況を判定する。
Depending on the growth of cells, pH and DO in the culture solution
Changes, depletion of nutrients, and accumulation of waste products. Therefore, the pH sensor 8 and the DO sensor 7 are provided in the culture tank 1.
b, the DO sensors 7a and 7c are installed in the two connecting pipes to measure pH, DO, and turbidity. These pieces of measurement information are transmitted to the control unit to determine the culture status.

【0048】生体の細胞への酸素の供給は以下の方法に
よって行われる。液中通気槽において通気ノズル15か
ら酸素含有ガスを通気し培地中に酸素を溶解させ、液中
通気槽の培地内のDOをDOセンサ7dによって測定
し、制御部は液中通気槽内のDOを監視する。生体の細
胞により培養液中の酸素が消費されて溶存酸素濃度が低
下したことをDOセンサ7bの情報により制御部が判断
すると、ポンプ9a及び9bを稼働させて、酸素が溶解
した培地を循環させる。液中通気槽内で酸素富化された
培地は、ポンプ9bにより培養槽1下部から流入し、培
養槽1内を上昇していく過程で、培養槽1内の培養液と
接触,混合されて、細胞へ酸素と栄養分を供給し、老廃
成分と二酸化炭素を受け取って、ポンプ9aにより液中
通気槽2に戻される。さらに、DOセンサ7aと7cに
より接続管内の培養液のDOを測定し、培養槽入口と出
口のDOを監視して、これらの情報により制御部は培養
槽内の細胞の酸素消費速度を算出する。制御部は、細胞
が必要とする酸素を供給できるように、液中通気槽2で
の酸素含有ガスの通気量及び酸素含有ガス中の酸素濃
度,液中通気槽及び培養槽の内圧、及び酸素富化培養液
の循環量を変化させる。
Oxygen is supplied to cells of a living body by the following method. In the submerged aeration tank, an oxygen-containing gas is vented from the aeration nozzle 15 to dissolve oxygen in the medium, DO in the medium in the submerged aeration tank is measured by the DO sensor 7d, and the control unit DO in the submerged aeration tank. To monitor. When the control unit judges from the information of the DO sensor 7b that oxygen in the culture solution has been consumed by living cells and the dissolved oxygen concentration has decreased, the pumps 9a and 9b are operated to circulate the medium in which oxygen is dissolved. . The medium enriched with oxygen in the submerged aeration tank is contacted with and mixed with the culture solution in the culture tank 1 in the process of flowing from the lower part of the culture tank 1 by the pump 9b and rising in the culture tank 1. , Oxygen and nutrients are supplied to the cells, waste components and carbon dioxide are received, and returned to the submerged aeration tank 2 by the pump 9a. Furthermore, the DO sensors 7a and 7c measure the DO of the culture solution in the connecting tube, monitor the DO at the inlet and the outlet of the culture tank, and the controller calculates the oxygen consumption rate of the cells in the culture tank based on these information. . The control unit supplies the oxygen required by the cells so that the aeration amount of the oxygen-containing gas in the submerged aeration tank 2 and the oxygen concentration in the oxygen-containing gas, the internal pressure of the submerged aeration tank and the culture tank, and the oxygen. Vary the circulating volume of enriched medium.

【0049】さらに、酸素富化培養液の循環量は、その
循環速度がマイクロキャリアの沈降速度とバランスする
ように制御される。細胞が高密度になってくると、マイ
クロキャリアが重くなり、その沈降速度が増加し、培養
槽1底部に堆積しやすくなる。この現象を、濁度計4b
〜4dからの情報で制御部が察知する。具体的には、培
養槽上部(スクリーン3aより下)に設置されている濁
度計4b付近にはマイクロキャリアがほとんど存在しな
くなり濁度は急激に低下する。逆に4c及び4dでは、
マイクロキャリアが集積してくるので増加する。マイク
ロキャリアが培養槽1下部の方に集積してスクリーン3
a付近のマイクロキャリアが減少し、かつ培養槽下部に
堆積しないで浮遊している状態を保持するように、制御
部は撹拌速度及び液循環量を制御する。マイクロキャリ
アの培養槽内での濃度分布は、濁度計4b〜4dの情報
により制御部が判定する。濁度計4b,4cからの情報
を、設定してあるそれぞれの上限値と比較し、上限値以
上とならないように、撹拌速度及び循環量を制御する。
Further, the circulation amount of the oxygen-enriched culture medium is controlled so that the circulation rate is balanced with the sedimentation rate of the microcarriers. As the cells become denser, the microcarriers become heavier, their sedimentation rate increases, and they are more likely to deposit on the bottom of the culture tank 1. This phenomenon is measured by the turbidimeter 4b.
The information from 4d is detected by the control unit. Specifically, the microcarriers hardly exist near the turbidimeter 4b installed above the culture tank (below the screen 3a), and the turbidity sharply decreases. On the contrary, in 4c and 4d,
Increases as microcarriers accumulate. The microcarriers are accumulated in the lower part of the culture tank 1 and the screen 3
The control unit controls the stirring speed and the liquid circulation amount so that the amount of microcarriers in the vicinity of a decreases and the floating state is maintained without depositing in the lower part of the culture tank. The control unit determines the concentration distribution of the microcarriers in the culture tank based on the information from the turbidimeters 4b to 4d. The information from the turbidimeters 4b and 4c is compared with the respective set upper limits, and the stirring speed and the circulation amount are controlled so as not to exceed the upper limits.

【0050】液中通気により液中通気槽2に生じる泡沫
層は、消泡層14により破泡する。消泡層14は、表面
をポリキシロン酸で疎水化したステンレス製金網で構成
されており、該表面に泡沫が接触すると破泡する。
The foam layer generated in the submerged aeration tank 2 by the submerged aeration is broken by the defoaming layer 14. The defoaming layer 14 is composed of a stainless wire mesh whose surface is hydrophobized with polyxylonic acid. When the foam comes into contact with the surface, the foam is broken.

【0051】長期培養において、スクリーン3aの細孔
の目詰りによる酸素供給量の減少が生じないように、培
養中適時に目詰り防止操作を行う。目詰り防止操作は以
下のように行われる。培養期間中定期的に、加振器11
によりスクリーン3aを振動させ、重度の目詰りが起き
ることのないように、軽い目詰り状態の状態で目詰り物
質を除去する。加振器11は空気圧変化によって上端が
培養槽のふたに固定されているベローズ11aを伸縮さ
せることで、スクリーン3aを振動させる。弁11bを
開け空気を通気すると加圧されベローズ11aは伸び、
弁11cを開けて減圧すると縮む。その伸縮でシャフト
11dが上下し、スクリーンが振動する。図示していな
いが差圧計により、スクリーン3a前後での差圧を測定
する。この情報により制御部がスクリーン3aの目詰り
状態を判定し、上記方法による振動操作を行う。別の目
詰り除去操作としては、瞬間的に培養液の循環量を急激
に変化させて、スクリーンに圧力変化を与えるのも有効
である。以上の操作を培養期間中適宜行うことで、スク
リーンの目詰りによる酸素供給量の減少を防ぐことがで
きる。
In the long-term culture, a clogging prevention operation is performed at a suitable time during the culture so that the oxygen supply amount is not decreased due to the clogging of the pores of the screen 3a. The clogging prevention operation is performed as follows. During the culture period, shaker 11
By virtue of this, the screen 3a is vibrated, and the clogging substance is removed in the state of the light clogging so that the serious clogging does not occur. The vibrator 11 vibrates the screen 3a by expanding and contracting the bellows 11a, the upper end of which is fixed to the lid of the culture tank, by changing the air pressure. When the valve 11b is opened and air is ventilated, it is pressurized and the bellows 11a expands,
When the valve 11c is opened and the pressure is reduced, it contracts. The expansion and contraction causes the shaft 11d to move up and down, and the screen vibrates. Although not shown, the differential pressure across the screen 3a is measured by a differential pressure gauge. The control unit determines the clogging state of the screen 3a based on this information, and performs the vibration operation by the above method. As another clogging removal operation, it is also effective to instantaneously and rapidly change the circulating amount of the culture solution to change the pressure on the screen. By appropriately performing the above operation during the culture period, it is possible to prevent the decrease in the oxygen supply amount due to the clogging of the screen.

【0052】本実施例による実際の培養結果の一例を示
す。培養には、旭化成マイクロキャリア(旭化成工業
製)を4g/lの濃度で用い、培地は新生子牛血清(大
日本製薬より購入)を5%(V/V)添加したERDF
培地(極東製薬工業製)を使用してCHO−K1細胞を
培養した。
An example of an actual culture result according to this example is shown. For culturing, Asahi Kasei Microcarriers (manufactured by Asahi Kasei Corporation) was used at a concentration of 4 g / l, and the medium was ERDF supplemented with 5% (V / V) of newborn calf serum (purchased from Dainippon Pharmaceutical).
The CHO-K1 cells were cultured using a medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.).

【0053】図2に付着操作時に濁度計4dにより培養
液上清の濁度の経時変化をモニタした結果を示す。培養
液量は通常の培養量の1/2の4リットルとした。図2
からわかるように、培養開始後、濁度は順調に減少し、
0.6 日目以降一定となった。種細胞を接種したときの
細胞濃度は2×106個/mlであり、0.6日で90%
以上の細胞がマイクロキャリアに付着したと判定され
た。
FIG. 2 shows the results of monitoring the change over time in the turbidity of the culture supernatant with the turbidimeter 4d during the attachment operation. The culture liquid volume was 4 liters, which is 1/2 of the normal culture volume. Figure 2
As you can see, the turbidity decreased steadily after the start of culture,
It has been constant since the 0.6th day. When seed cells were inoculated, the cell concentration was 2 × 10 6 cells / ml, 90% in 0.6 days.
It was determined that the above cells were attached to the microcarrier.

【0054】図3は、付着操作終了後のマイクロキャリ
アに付着した細胞の増殖曲線と培養液の濁度変化につい
て示したグラフである。細胞濃度は、細胞の酸素消費速
度から算出すると同時に、適時培養液をサンプリング
し、常法によりマイクロキャリアに付着している細胞数
を測定した。細胞濃度とは、マイクロキャリアに付着し
ている細胞数を培養液1ml当りの細胞数に換算した値
である。
FIG. 3 is a graph showing the growth curve of cells attached to microcarriers and the change in turbidity of the culture medium after completion of the attachment operation. The cell concentration was calculated from the oxygen consumption rate of the cells, at the same time, the culture solution was sampled at appropriate times, and the number of cells attached to the microcarriers was measured by a conventional method. The cell concentration is a value obtained by converting the number of cells attached to the microcarrier into the number of cells per 1 ml of the culture solution.

【0055】細胞は、培養日数14日目、細胞濃度=約
5×107 個/mlまで対数的に増殖し、以後、定常期
となり、その細胞濃度を維持した。この間、細胞濃度が
3×106 個/mlとなり、培養液のpHが低下し始め
た培養4日目から培地の灌流を開始し、以後、細胞の増
殖にあわせて灌流率を4まで増加させた。
The cells were logarithmically grown to a cell concentration of about 5 × 10 7 cells / ml on the 14th day of culture, and thereafter, the stationary phase was reached and the cell concentration was maintained. During this period, the perfusion of the medium was started from the 4th day of the culture when the cell concentration became 3 × 10 6 cells / ml and the pH of the culture solution began to decrease, and thereafter, the perfusion rate was increased to 4 in accordance with the growth of the cells. It was

【0056】培養液の濁度の測定結果を示すグラフ中
の、4a〜4dの記号は、図1中の濁度計の記号を示
す。4aはスクリーン3aより上の部分の培養液上清中
の濁度の測定結果を示す。培養期間中、上清の濁度は
0.1 以下であり、マイクロキャリアからの細胞の剥離
はほとんど起こらなかったことがわかる。4b,4c,
4dの値は、それぞれ、培養槽下部,中央部,上部(ス
クリーン3aより下)の濁度を表す。培養10日目まで
3点の濁度はほぼ一致し、一定であった。以降、細胞濃
度が1×107 以上になると、濁度は4bでは減少し、
逆に、4c,4dでは増加した。これより、スクリーン
3a下近傍にはマイクロキャリアがほとんど存在せずか
つマイクロキャリアの堆積が起こらない状態、すなわ
ち、4d<2.0,4c<1.5 の状態を保つように、
撹拌速度及び培養液の循環量を制御した。この結果、培
養期間中、培養槽内の培養液を、1.0≦DO≦2.0
,6.8≦pH≦7.2 の状態に維持することができ
た。本実施例では、35日目で培養を停止しているが、
さらに継続することは可能であった。
The symbols 4a to 4d in the graph showing the results of measuring the turbidity of the culture solution are the symbols of the turbidimeter in FIG. 4a shows the measurement result of the turbidity in the culture supernatant of the portion above the screen 3a. During the culturing period, the turbidity of the supernatant was 0.1 or less, which means that the cells were hardly detached from the microcarriers. 4b, 4c,
The value of 4d represents the turbidity of the lower part, the center part, and the upper part (below the screen 3a) of the culture tank. By the 10th day of culture, the turbidity at 3 points was almost the same and constant. After that, when the cell concentration became 1 × 10 7 or more, the turbidity decreased at 4b,
On the contrary, it increased in 4c and 4d. As a result, in order to maintain a state where there are almost no microcarriers near the bottom of the screen 3a and deposition of microcarriers does not occur, that is, 4d <2.0, 4c <1.5,
The stirring speed and the circulation amount of the culture solution were controlled. As a result, during the culturing period, the culture solution in the culture tank was maintained at 1.0 ≦ DO ≦ 2.0.
, 6.8 ≦ pH ≦ 7.2 could be maintained. In this example, the culture was stopped on the 35th day,
It was possible to continue further.

【0057】以上のように、本培養装置によれば、マイ
クロキャリア等の固形物を含有する場合においても泡沫
の発生を考慮することなく、液中通気による酸素供給が
可能となり、また、培養液の濁度測定によりマイクロキ
ャリアの沈降速度を判定し、これとバランスする循環速
度で酸素富化培地を循環させ、スクリーンの目詰りを効
果的に抑制し、生体の細胞の細胞濃度107 レベルでの
高密度培養が可能になる。
As described above, according to the main culture apparatus, oxygen can be supplied by submerged aeration without considering generation of bubbles even when a solid substance such as a microcarrier is contained, and the culture medium can be supplied. The sedimentation rate of the microcarriers is determined by measuring the turbidity of the cells, and the oxygen-enriched medium is circulated at a circulation rate that is in balance with this to effectively prevent clogging of the screen and to achieve a cell concentration of 10 7 cells in the living body. It enables high-density culture.

【0058】[0058]

【発明の効果】本培養装置によれば、多孔質マイクロキ
ャリアを用いた生体の細胞の細胞濃度107 レベルでの
長期高密度培養が可能であり、これにより従来の10倍
以上の効率で、生体の細胞による有用物質の効率的な生
産を実現することができる。
EFFECTS OF THE INVENTION According to the present culturing apparatus, long-term high-density culturing of living cells using a porous microcarrier at a cell concentration level of 10 7 is possible. Efficient production of useful substances by living cells can be realized.

【図面の簡単な説明】[Brief description of drawings]

【図1】本発明になる培養装置の一実施例を示す図であ
る。
FIG. 1 is a diagram showing an embodiment of a culture device according to the present invention.

【図2】本発明による生体の細胞の培養において、培養
初期の培養液の濁度変化の一実施例を示す図である。
FIG. 2 is a diagram showing an example of changes in the turbidity of a culture solution at the initial stage of culture in culturing living cells according to the present invention.

【図3】本発明による生体の細胞の培養結果の一実施例
を示す図である。
FIG. 3 is a diagram showing an example of the result of culturing living cells according to the present invention.

【符号の説明】[Explanation of symbols]

1…培養槽、2…液中通気槽、3a…スクリーン、4a
〜4d…濁度計、5…撹拌器、6…撹拌モータ、7a〜
7d…DOセンサ、8…pHセンサ、9a〜9d…ポン
プ、10a,10b…制御弁、10c…逆止弁、11…
加振器、11a…ベローズ、11b,11c…制御弁、1
1d…シャフト、12a,12b…レベルセンサ、13
…温度センサ、14…消泡層、15…通気ノズル。
1 ... Culture tank, 2 ... Submerged aeration tank, 3a ... Screen, 4a
~ 4d ... Turbidity meter, 5 ... Stirrer, 6 ... Stirring motor, 7a ...
7d ... DO sensor, 8 ... pH sensor, 9a-9d ... Pump, 10a, 10b ... Control valve, 10c ... Check valve, 11 ...
Shaker, 11a ... Bellows, 11b, 11c ... Control valve, 1
1d ... Shaft, 12a, 12b ... Level sensor, 13
... temperature sensor, 14 ... defoaming layer, 15 ... ventilation nozzle.

Claims (5)

【特許請求の範囲】[Claims] 【請求項1】マイクロキャリア等の固体粒子に細胞を付
着させて浮遊状態で培養する培養槽と、該培養槽内に設
置して前記固体粒子の通過を阻止しかつ培養液を通過せ
しめる細孔を有するスクリーンと、前記培養液に酸素を
溶解させるために前記培養槽とは別に配置した液中通気
槽と、酸素が溶解した前記培養液を前記培養槽と前記液
中通気槽との間で循環させる手段と、前記培養液を撹拌
する撹拌手段と、前記スクリーンを振動させる振動手段
を有し、前記培養液の循環方向が前記培養槽内では上向
流であることを特徴とする生体の細胞の培養装置。
1. A culture tank in which cells are adhered to solid particles such as microcarriers and cultured in a floating state, and pores which are installed in the culture tank to prevent passage of the solid particles and allow passage of a culture solution. A screen having, a submerged aeration tank arranged separately from the culture tank for dissolving oxygen in the culture solution, the oxygen-dissolved culture solution between the culture tank and the submerged aeration tank A living body characterized by having a means for circulating, a stirring means for stirring the culture solution, and a vibrating means for vibrating the screen, wherein the circulation direction of the culture solution is an upward flow in the culture tank. Cell culture device.
【請求項2】前記培養液の濁度を測定する該手段を具備
し、前記測定結果から前記培養液中に浮遊している細胞
数を算出し、該算出結果により、前記培養液の撹拌速度
及び/又は前記培養液の循環速度を変化させて前記生体
の細胞を培養することを特徴とする請求項1記載の生体
の細胞の培養装置。
2. The means for measuring the turbidity of the culture medium, the number of cells floating in the culture medium is calculated from the measurement result, and the stirring speed of the culture medium is calculated from the calculation result. And / or culturing the cells of the living body by changing the circulation speed of the culture solution, The apparatus for culturing cells of the living body according to claim 1.
【請求項3】前記培養液の濁度を測定する該手段を具備
し、該測定結果から前記培養液中に浮遊しているマイク
ロキャリア等の固体粒子数を算出し、前記算出結果によ
り、前記培養液の撹拌速度及び/又は前記培養液の循環
速度及び/又は前記スクリーンの振動回数を変化させて
前記生体の細胞を培養することを特徴とする請求項1又
は2記載の生体の細胞の培養装置。
3. A method for measuring the turbidity of the culture medium, the method comprising: measuring the turbidity of the culture medium; calculating the number of solid particles such as microcarriers suspended in the culture medium from the measurement result; 3. The cell culture of the living body according to claim 1 or 2, wherein the cell culture of the living body is performed by changing the stirring speed of the culture solution and / or the circulation speed of the culture solution and / or the frequency of vibration of the screen. apparatus.
【請求項4】請求項1,2又は3において、前記培養液
の濁度と溶存酸素濃度を測定する該手段を具備し、前記
培養液の濁度及び又は溶存酸素濃度の測定結果から、前
記生体の細胞が付着したマイクロキャリア等固体粒子の
沈降速度を算出し、該算出結果より、前記酸素が溶解し
た培養液の上向流速度を前記マイクロキャリア等の固体
粒子の沈降速度以下となるように制御して培養すること
を特徴とする生体の細胞の培養方法。
4. The method according to claim 1, wherein the means for measuring the turbidity and the dissolved oxygen concentration of the culture medium is provided, and the turbidity and / or the dissolved oxygen concentration of the culture medium are measured to obtain the Calculating the sedimentation velocity of solid particles such as microcarriers to which cells of a living body are attached, and based on the calculation result, the upward flow velocity of the culture solution in which the oxygen is dissolved is equal to or less than the sedimentation velocity of solid particles such as the microcarriers A method for culturing cells of a living body, which comprises culturing under controlled conditions.
【請求項5】請求項1,2又は3において、更に前記培
養槽へ前記培養液が流入する所と流出する所の2ヶ所に
おいて溶存酸素濃度を測定する該手段を装備し、前記溶
存酸素濃度の測定結果から前記培養槽内の細胞の酸素消
費量を算出し、前記算出結果及び前記酸素溶解槽の酸素
溶解速度係数及び前記マイクロキャリア等の固体粒子の
沈降速度から、前記生体の細胞が必要とする酸素を供給
できかつ前記酸素が溶解した培養液の上向流速度が前記
マイクロキャリア等の固体粒子の沈降速度以下となるよ
うに、前記酸素溶解槽及び/又は前記培養槽の内圧及び
/又は前記培養液の液循環量を変化させることを特徴と
する生体の細胞の培養装置。
5. The dissolved oxygen concentration according to claim 1, further comprising the means for measuring the dissolved oxygen concentration at two places, where the culture solution flows into and out of the culture tank. The oxygen consumption of the cells in the culture tank is calculated from the measurement result, and the biological cells are required from the calculation result and the oxygen dissolution rate coefficient of the oxygen dissolution tank and the sedimentation speed of solid particles such as the microcarriers. So that the oxygen can be supplied and the upward flow velocity of the culture solution in which the oxygen is dissolved is equal to or lower than the sedimentation velocity of solid particles such as the microcarriers, and / or the internal pressure of the oxygen dissolution tank and / or the culture tank. Alternatively, an apparatus for culturing cells of a living body, characterized in that the liquid circulation amount of the culture solution is changed.
JP149294A 1994-01-12 1994-01-12 Apparatus for culturing cell of living body Pending JPH07203945A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP149294A JPH07203945A (en) 1994-01-12 1994-01-12 Apparatus for culturing cell of living body

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP149294A JPH07203945A (en) 1994-01-12 1994-01-12 Apparatus for culturing cell of living body

Publications (1)

Publication Number Publication Date
JPH07203945A true JPH07203945A (en) 1995-08-08

Family

ID=11502958

Family Applications (1)

Application Number Title Priority Date Filing Date
JP149294A Pending JPH07203945A (en) 1994-01-12 1994-01-12 Apparatus for culturing cell of living body

Country Status (1)

Country Link
JP (1) JPH07203945A (en)

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10148610A (en) * 1996-11-18 1998-06-02 Nitto Denko Corp Method for measuring number of particles in liquid, and method for operating ultrafiltration membrane module utilizing the same
JP2003235544A (en) * 2002-02-20 2003-08-26 Hitachi Ltd Method for controlling culture of biological cell, control device for controlling culture apparatus and culture apparatus
JP2009195162A (en) * 2008-02-21 2009-09-03 Ccs Inc Culture apparatus for algae
JP2011083263A (en) * 2009-10-19 2011-04-28 Jgc Corp System and method for culturing cell
JP2011092117A (en) * 2009-10-30 2011-05-12 Hitachi Plant Technologies Ltd Method and device for culturing biological cell
JP2012152188A (en) * 2011-01-28 2012-08-16 Terumo Corp Method for assessing sheet formation
JP2018000130A (en) * 2016-07-05 2018-01-11 株式会社Ihi Cell culture device
JP2018050550A (en) * 2016-09-29 2018-04-05 佐竹化学機械工業株式会社 Spinner culture device
JP2018126104A (en) * 2017-02-09 2018-08-16 株式会社ティ・アンド・シー・テクニカル Cell density measurement method and cell density change tracking method
CN109943479A (en) * 2017-12-20 2019-06-28 内蒙古久科康瑞环保科技有限公司 A kind of automatic enrichment culture device and method of resistance to bacterium with high salt
JP2020124172A (en) * 2019-02-06 2020-08-20 株式会社日立製作所 Culture system, culture method, and method for producing cultured product
WO2022065400A1 (en) * 2020-09-25 2022-03-31 昭和電工マテリアルズ株式会社 Cell culture apparatus and method for producing cell groups
WO2022065401A1 (en) * 2020-09-25 2022-03-31 昭和電工マテリアルズ株式会社 Cell culture apparatus and method for producing cell groups
US20230348837A1 (en) * 2021-08-17 2023-11-02 Jgc Japan Corporation Calculation device, control device, culture system, and method for designing culture system

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH10148610A (en) * 1996-11-18 1998-06-02 Nitto Denko Corp Method for measuring number of particles in liquid, and method for operating ultrafiltration membrane module utilizing the same
JP2003235544A (en) * 2002-02-20 2003-08-26 Hitachi Ltd Method for controlling culture of biological cell, control device for controlling culture apparatus and culture apparatus
JP2009195162A (en) * 2008-02-21 2009-09-03 Ccs Inc Culture apparatus for algae
JP2011083263A (en) * 2009-10-19 2011-04-28 Jgc Corp System and method for culturing cell
JP2011092117A (en) * 2009-10-30 2011-05-12 Hitachi Plant Technologies Ltd Method and device for culturing biological cell
JP2012152188A (en) * 2011-01-28 2012-08-16 Terumo Corp Method for assessing sheet formation
JP2018000130A (en) * 2016-07-05 2018-01-11 株式会社Ihi Cell culture device
JP2018050550A (en) * 2016-09-29 2018-04-05 佐竹化学機械工業株式会社 Spinner culture device
JP2018126104A (en) * 2017-02-09 2018-08-16 株式会社ティ・アンド・シー・テクニカル Cell density measurement method and cell density change tracking method
CN109943479A (en) * 2017-12-20 2019-06-28 内蒙古久科康瑞环保科技有限公司 A kind of automatic enrichment culture device and method of resistance to bacterium with high salt
JP2020124172A (en) * 2019-02-06 2020-08-20 株式会社日立製作所 Culture system, culture method, and method for producing cultured product
WO2022065400A1 (en) * 2020-09-25 2022-03-31 昭和電工マテリアルズ株式会社 Cell culture apparatus and method for producing cell groups
WO2022065401A1 (en) * 2020-09-25 2022-03-31 昭和電工マテリアルズ株式会社 Cell culture apparatus and method for producing cell groups
US20230348837A1 (en) * 2021-08-17 2023-11-02 Jgc Japan Corporation Calculation device, control device, culture system, and method for designing culture system
US11859166B2 (en) * 2021-08-17 2024-01-02 Jgc Japan Corporation Calculation device, control device, culture system, and method for designing culture system

Similar Documents

Publication Publication Date Title
JPH07203945A (en) Apparatus for culturing cell of living body
JP3244701B2 (en) Method and apparatus for growing biomass particles
JP2777385B2 (en) Biological cell culture method, culture system and culture device
JP5460241B2 (en) Biological cell culture method and culture apparatus
JPH09500818A (en) Particle sedimentation tank used for cell culture
JPS63133978A (en) Cell cultivation device
Emery et al. Oxygenation of intensive cell-culture system
JPH0584063A (en) Reactor for causing biological reaction by biological catalyst
CA2175485C (en) Continuous settling apparatus
JP2011177046A (en) Method and apparatus for culturing cell
AU7078791A (en) Method for culturing cells
JP3289984B2 (en) Apparatus and method for culturing living cells
WO1986000636A1 (en) Cell growth
JPH0416153B2 (en)
JP2613892B2 (en) Animal cell culture method and culture device
JPH0347074A (en) Method and device for culturing animal cell with lactic acid as indicator
JPH06141850A (en) Culture system for animal cell and method for culture of animal cell
US5151362A (en) Apparatus containing a septum which impedes cell permeation for cell culture and method of use
JPS5921388A (en) Method for cell culture
JPH0775549A (en) Living body cell culture arrangement
JPH06133765A (en) Method for supplying oxygen and culture device used therefor
JPH078230B2 (en) Cell culture device
JPS63164879A (en) Bubble-tower perfusion culture and apparatus therefor
JPH0428352B2 (en)
JP2810140B2 (en) Cell culture method and device