JP2500137B2 - Incubator - Google Patents
IncubatorInfo
- Publication number
- JP2500137B2 JP2500137B2 JP3286291A JP28629191A JP2500137B2 JP 2500137 B2 JP2500137 B2 JP 2500137B2 JP 3286291 A JP3286291 A JP 3286291A JP 28629191 A JP28629191 A JP 28629191A JP 2500137 B2 JP2500137 B2 JP 2500137B2
- Authority
- JP
- Japan
- Prior art keywords
- medium
- pump
- dissolved oxygen
- reservoir
- bioreactor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 239000002609 medium Substances 0.000 claims abstract description 141
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims abstract description 62
- 239000001301 oxygen Substances 0.000 claims abstract description 62
- 229910052760 oxygen Inorganic materials 0.000 claims abstract description 62
- 239000001963 growth medium Substances 0.000 claims abstract description 10
- 238000007599 discharging Methods 0.000 claims abstract description 6
- 239000012510 hollow fiber Substances 0.000 claims description 20
- 239000007788 liquid Substances 0.000 claims description 15
- 239000012737 fresh medium Substances 0.000 claims description 14
- 238000005070 sampling Methods 0.000 claims description 9
- 238000000605 extraction Methods 0.000 claims description 3
- 239000000446 fuel Substances 0.000 claims 1
- 230000000050 nutritive effect Effects 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 description 16
- 230000036284 oxygen consumption Effects 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 238000001514 detection method Methods 0.000 description 7
- 230000002572 peristaltic effect Effects 0.000 description 5
- 238000012258 culturing Methods 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 239000000835 fiber Substances 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 235000006286 nutrient intake Nutrition 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000011796 hollow space material Substances 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS 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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/26—Conditioning fluids entering or exiting the reaction vessel
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/30—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration
- C12M41/32—Means for regulation, monitoring, measurement or control, e.g. flow regulation of concentration of substances in solution
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
- C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Sustainable Development (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Analytical Chemistry (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、動物細胞、微生物等を
培養する培養装置、特に、培地循環用ポンプにて培地
を、培地リザーバから、酸素を溶存させたのちホローフ
ァイバ式バイオリアクターに通過させ、再び前記培地リ
ザーバへ戻すように循環させる培養装置に関する。The present invention relates to a culture apparatus for culturing animal cells, microorganisms, etc., in particular, the medium at the medium circulation pump, after the medium reservoir, is dissolved oxygen Horofu
The present invention relates to a culturing device which is passed through a fiber type bioreactor and circulated so as to be returned to the medium reservoir again.
【０００２】[0002]
【従来の技術】この種の培養装置では、培地を収容した
培地リザーバと被培養物を収容したバイオリアクターと
に培地を循環させ、且つ、バイオリアクターへ流入する
培地に酸素を溶存させることで、バイオリアクター中の
被培養物に、その生命維持、生育、繁殖等に必要な各種
栄養源及び酸素を供給する。バイオリアクターへ供給さ
れる各種栄養源及び酸素の量は培地循環用ポンプによる
培地流量に依存する。2. Description of the Related Art In a culture device of this type, a medium is circulated in a medium reservoir containing a medium and a bioreactor containing a culture, and oxygen is dissolved in the medium flowing into the bioreactor. The culture material in the bioreactor is supplied with various nutrients and oxygen necessary for life support, growth, reproduction and the like. The amount of various nutrients and oxygen supplied to the bioreactor depends on the medium flow rate by the medium circulation pump.
【０００３】[0003]
【発明が解決しようとする課題】しかしながら、前述の
ように、この種の装置では、バイオリアクターへの栄養
源及び酸素の供給量が培地流量に依存し、且つ、その流
量は当初設定された流量に固定されるか、又は途中で変
更されるにしてもマニュアル操作で変更される。従っ
て、バイオリアクターにおいて必要とされる量の栄養源
及び溶存酸素が正確に供給される保証は無く、最悪の場
合、バイオリアクター内で栄養源や酸素が枯渇すること
もあり得る。However, as described above, in this type of device, the amounts of nutrient source and oxygen supplied to the bioreactor depend on the medium flow rate, and the flow rate is the initially set flow rate. It is fixed to or is changed manually even if it is changed on the way. Therefore, there is no guarantee that the required amount of nutrients and dissolved oxygen are accurately supplied in the bioreactor, and in the worst case, nutrients and oxygen may be depleted in the bioreactor.
【０００４】そこで本発明は、培地循環用ポンプにて培
地を、培地リザーバから、酸素を溶存させたのちホロー
ファイバ式バイオリアクターに通過させ、再び前記培地
リザーバへ戻すように循環させる培養装置において、バ
イオリアクター内の栄養源や酸素の消費量に応じた量の
栄養源及び酸素を従来より正確にバイオリアクターへ供
給できるようにすることを課題とする。Therefore, in the present invention, a medium circulation pump is used to dissolve oxygen in the medium from the medium reservoir and then the hollow medium is used.
In a culture device that circulates through a fiber type bioreactor and returns to the medium reservoir, the amount of nutrient source and oxygen in the bioreactor is adjusted to the bioreactor more accurately than before. The challenge is to be able to supply.
【０００５】[0005]
【課題を解決するための手段】本発明者は前記課題を解
決するため研究を重ねた結果、培地中の溶存酸素消費量
と栄養源の消費量が比例的に変動することに着目し、さ
らに溶存酸素消費量と栄養源消費量が比例的に変動する
ことから、ホローファイバ式バイオリアクターから出た
培地中の溶存酸素量を予め定めた量に維持するように培
地循環用ポンプの流量を制御する一方、培地リザーバか
ら培地を排出ポンプで排出するようにし、且つ、該排出
ポンプの流量を培地循環用ポンプの流量に比例変動さ
せ、該排出ポンプによる培地排出量を補うように培地リ
ザーバへ新鮮培地を供給すれば（換言すれば溶存酸素消
費量に比例又は略比例して培地リザーバ内の培地を交換
すれば）、培地中の溶存酸素量を所定のものに維持でき
るとともに確実に適切な栄養源の補給を行えることを見
出し、本発明を完成した。 The present inventor has solved the above-mentioned problems.
As a result of repeated research to determine the amount of dissolved oxygen consumed in the medium
And that the consumption of nutrients fluctuates proportionally,
In addition, the dissolved oxygen consumption and the consumption of nutrient sources fluctuate proportionally
Therefore, it came out of the hollow fiber type bioreactor.
Culture to maintain the amount of dissolved oxygen in the medium at a predetermined amount.
While controlling the flow rate of the ground circulation pump,
The medium with a discharge pump, and
The flow rate of the pump varies proportionally to the flow rate of the medium circulation pump.
Medium so that the amount of medium discharged by the discharge pump is supplemented.
If the fresh medium is supplied to the server (in other words, dissolved oxygen consumption is
Replace the medium in the medium reservoir in proportion to or approximately in proportion to the cost
The amount of dissolved oxygen in the medium can be maintained
As well as ensuring that the proper nutritional sources can be supplied.
Then, the present invention was completed.
【０００６】すなわち本発明は、培地循環用ポンプにて
培地を、培地リザーバから、酸素を溶存させたのちホロ
ーファイバ式バイオリアクターに通過させ、再び前記培
地リザーバへ戻すように循環させる培養装置において、
前記バイオリアクターを出た培地中の溶存酸素量を測定
する手段と、前記培地リザーバから培地を排出する培地
排出ポンプと、新鮮培地容器と、該容器から前記培地リ
ザーバへ新鮮培地を供給する培地供給ポンプと、前記ポ
ンプ動作を制御する制御部とを備え、前記制御部は、前
記培地循環用ポンプの流量を前記測定手段により測定さ
れる溶存酸素量が予め定めた溶存酸素量に維持されるよ
うに制御し、前記培地排出ポンプの流量を前記培地循環
用ポンプの流量に比例するように制御し、前記培地供給
ポンプを前記培地排出ポンプが停止すると始動し、前記
培地リザーバ内液面が所定のレベルに達すると停止する
ように制御することを特徴とする培養装置を提供するも
のである。この培養装置においては、培養した細胞によ
る生産物を取り出せるように、前記ホローファイバ式バ
イオリアクターのホローファイバ外空間の一端に生産物
取出し用培地ボトルを接続し、該ボトルを無菌フィルタ
を介して外気に連通させるとともに、前記ホローファイ
バ外空間の他端に生産物取出し用サンプリング配管を接
続することができる。 前記各ポンプの流量制御は、代表
的にはポンプの運転速度、例えば回転ポンプについては
その単位時間当たりの回転数をコントロールすることで
簡単に行える。 That is, the present invention provides a medium at a medium circulation pump, from the medium reservoir, holo mixture was allowed to dissolved oxygen
-In a culture device in which the fiber-type bioreactor is circulated so as to be returned to the medium reservoir again,
Means for measuring the amount of dissolved oxygen in the medium leaving the bioreactor, medium discharge pump for discharging the medium from the medium reservoir, fresh medium container, and medium supply for supplying fresh medium from the container to the medium reservoir comprising a pump, and a control unit for controlling the pump operation, wherein, prior to
Dissolved oxygen measured the flow rate of the serial medium circulation pump by said measuring means is maintained at a predetermined dissolved oxygen content
Control the flow rate of the medium discharge pump to circulate the medium.
The culture medium supply is controlled so as to be proportional to the flow rate of the pump
The pump is started when the medium discharge pump is stopped,
Stops when the liquid level in the medium reservoir reaches a predetermined level
The present invention provides a culture device characterized by being controlled as described above . In this culture device,
Hollow fiber type so that the product
Product at one end of the hollow fiber outer space of the Io reactor
Connect the extraction medium bottle and connect the bottle with a sterile filter.
To communicate with the outside air through the
Connect the sampling pipe for product extraction to the other end of the outside space.
You can continue. The flow rate control of each pump is representative
The operating speed of the pump, for example, for rotary pumps
By controlling the number of rotations per unit time
Easy to do.
【０００７】[0007]
【０００８】培地供給ポンプの前記の制御運転のため
に、例えば前記制御部に、前記培地リザーバ内の培地液
面が所定レベルに有るか否かを検出するレベル検出手段
及びツインタイマを含め、該ツインタイマで設定される
培地排出ポンプ用オン時間及びオフ時間に基づいて前記
排出ポンプを運転又は停止させ、該排出ポンプオフ時間
において、前記レベル検出手段による培地リザーバ内液
面の所定レベル到達検出まで前記培地供給ポンプを運転
するようにしてもよい。For the above-mentioned controlled operation of the medium supply pump
In, for example, the control unit includes a level detecting means and a twin timer for detecting whether the medium liquid level in the medium reservoir is at a predetermined level, the medium discharge pump ON time set by the twin timer, and The discharge pump may be operated or stopped based on the off time, and during the discharge pump off time, the culture medium supply pump may be operated until the level detection means detects that the liquid level in the culture medium reservoir reaches a predetermined level.
【０００９】[0009]
【作用】本発明培養装置によると、培地が、培地循環用
ポンプにて培地リザーバとバイオリアクターとに循環せ
しめられ、且つ、バイオリアクターに流入する培地に酸
素が溶存せしめられ、それによってバイオリアクター中
の被培養物に、その生命維持、生育、繁殖等に要求され
る各種栄養源及び酸素が供給される。According to the culture apparatus of the present invention, the medium is circulated between the medium reservoir and the bioreactor by the medium circulation pump, and oxygen is dissolved in the medium flowing into the bioreactor, whereby the medium in the bioreactor is dissolved. The various nutrient sources and oxygen required for life support, growth, reproduction, etc. are supplied to the culture.
【００１０】また、バイオリアクターを出た培地中の溶
存酸素量がその測定手段により測定される一方、前記循
環用ポンプ動作を制御する制御部が、該測定手段により
測定された溶存酸素量と予め定めた溶存酸素量とを比較
しつつ、測定溶存酸素量が予め定めた溶存酸素量より少
ないときは、培地流量を増加させるように、測定溶存酸
素量が予め定めた溶存酸素量より多いときには培地流量
を減少させるように前記ポンプ動作を制御する。Further, while the amount of dissolved oxygen in the medium leaving the bioreactor is measured by the measuring means, the control part for controlling the operation of the circulating pump operates in advance with the dissolved oxygen amount measured by the measuring means. While comparing with the determined dissolved oxygen amount, when the measured dissolved oxygen amount is less than the predetermined dissolved oxygen amount, the medium flow rate is increased so that the measured dissolved oxygen amount is greater than the predetermined dissolved oxygen amount, the medium is increased. Control the pump operation to reduce the flow rate.
【００１１】また、培地中の溶存酸素消費量と栄養源の
消費量は比例的に変動するから、そして培地排出ポンプ
はその流量が培地循環用ポンプの流量に比例するように
制御運転されるから、バイオリアクター内で消費される
溶存酸素量に比例又は略比例した量の培地交換が行わ
れ、それによって該培地リザーバ及びバイオリアクター
を含む培地循環系における栄養源の消費が補われる。前
述のように培養した細胞による生産物を取り出せるよう
にバイオリアクターのホローファイバ外空間に前記の生
産物取出し用の培地ボトル及びサンプリング配管を接続
してあるときは、該サンプリング配管から生産物を取り
出すことができ、それに伴って該培地ボトルからホロー
ファイバ外空間へ培地が補給される。 In addition, the amount of dissolved oxygen consumed in the medium and nutrients
Because the consumption varies proportionally, and the medium discharge pump
So that its flow rate is proportional to the flow rate of the medium circulation pump
Since the operation is controlled, the medium is exchanged in an amount proportional or approximately proportional to the amount of dissolved oxygen consumed in the bioreactor, thereby supplementing the consumption of nutrients in the medium circulation system including the medium reservoir and the bioreactor. Be seen. Before
Make it possible to take out the products produced by the cells cultured as described above.
In the hollow space outside the hollow fiber of the bioreactor,
Connect a culture medium bottle and sampling pipe for product removal
If so, remove the product from the sampling pipe.
The medium bottle can be
The medium is supplied to the space outside the fiber.
【００１２】[0012]
【００１３】また、該制御部が、前記培地リザーバ内の
培地液面が所定レベルに有るか否かを検出するレベル検
出手段及びツインタイマを含み、該ツインタイマで設定
される排出ポンプ用オン時間及びオフ時間に基づいて前
記排出ポンプを運転又は停止させ、該排出ポンプオフ時
間において、前記レベル検出手段による培地リザーバ内
液面の所定レベル到達検出まで前記供給ポンプを運転さ
せるものであるときは、前記オン時間の長さ及びオフ時
間の長さに応じて（新鮮培地供給量／溶存酸素消費量）
の割合が決定される。なお、該ツインタイマにおけるオ
ン時間及びオフ時間の設定は、所望の（新鮮培地供給量
／溶存酸素消費量）の割合を得られるように行うのであ
るが、該オン時間の長さ及びオフ時間の長さは、例えば
前記循環系を流れる培地中に含まれる栄養源であるグル
コースの量を測定し、その測定結果に基づいて定めるこ
とができる。Further, the control unit includes level detection means for detecting whether or not the medium liquid level in the medium reservoir is at a predetermined level and a twin timer, and the discharge pump ON time set by the twin timer. And when the discharge pump is operated or stopped based on the off time and the discharge pump is off until the predetermined level of the liquid level in the medium reservoir is detected by the level detection means, Depending on length of on-time and length of off-time (fresh medium supply / dissolved oxygen consumption)
Is determined. The on-time and off-time of the twin timer are set so that a desired ratio of (fresh medium supply amount / dissolved oxygen consumption amount) can be obtained. The length can be determined, for example, by measuring the amount of glucose, which is a nutrient source, contained in the medium flowing through the circulation system and based on the measurement result.
【００１４】[0014]
【実施例】以下本発明の実施例を図面を参照して説明す
る。図１は一実施例培養装置の全体の構成を示してい
る。この培養装置は細胞を培養するもので、主たる構成
要因はホローファイバカラムでできたバイオリアクター
１、液体培地Ｌを収容した培地リザーバ２、培地循環用
の回転速度可変のペリスタリックポンプ３、培地中に酸
素を溶存させるガス交換器４及び培地中の溶存酸素を検
出するための溶存酸素検出装置５を備えている。Embodiments of the present invention will be described below with reference to the drawings. FIG. 1 shows the overall configuration of the culture device of one embodiment. This culturing device is for culturing cells, and the main constituent factors are a bioreactor 1 made of a hollow fiber column, a medium reservoir 2 containing a liquid medium L, a peristaltic pump 3 with a variable rotation speed for circulating the medium, in the medium. Further, a gas exchanger 4 for dissolving oxygen and a dissolved oxygen detector 5 for detecting dissolved oxygen in the medium are provided.
【００１５】これらは配管６により、リザーバ２→ポン
プ３→ガス交換器４→バイオリアクター１→検出装置５
→リザーバ２の順序で直列にループ状に接続され、培地
もこの順序で流れる。図１において、バイオリアクター
１中のホローファイバの出入口端子１１、１２は、ホロ
ーファイバのキャピラリー内空間に通じている。These are connected by a pipe 6 to a reservoir 2 → pump 3 → gas exchanger 4 → bioreactor 1 → detector 5
→ The reservoirs 2 are connected in series in a loop and the medium flows in this order. In FIG. 1, the hollow fiber inlet / outlet terminals 11 and 12 in the bioreactor 1 communicate with the space inside the capillary of the hollow fiber.
【００１６】この装置によると、ポンプ３の運転により
リザーバ２より汲み出された培地は、ガス交換器４で大
気飽和になるまで酸素を溶存させ、バイオリアクター１
に送られ、リアクター内で消費し切れなかった溶存酸素
濃度が溶存酸素検出装置５で検出され、培地自体は再び
リザーバ２へ戻される。検出装置５で検出された溶存酸
素濃度Ｘは、電気信号に変換され、装置制御部９の一部
を構成するＰＩＤコントローラ９１に入力される。入力
された値に対し、ＰＩＤコントローラ９１は設定スイッ
チ９１１を通じて設定された値Ｘ0 と比較演算を行い、
次のような動作をする。According to this apparatus, the medium pumped out from the reservoir 2 by the operation of the pump 3 dissolves oxygen until it is saturated with air in the gas exchanger 4, and the bioreactor 1
The dissolved oxygen concentration, which was sent to the reactor and was not completely consumed in the reactor, is detected by the dissolved oxygen detector 5, and the medium itself is returned to the reservoir 2 again. The dissolved oxygen concentration X detected by the detection device 5 is converted into an electric signal and input to the PID controller 91 that constitutes a part of the device control unit 9. The PID controller 91 compares the input value with the value X 0 set through the setting switch 911,
It operates as follows.
【００１７】Ｘ＞Ｘ0 の場合、制御信号を通じてポンプ
３の回転数をおとし、循環流量を少なくする。Ｘ＜Ｘ0
の場合、ポンプ３の回転数を上げて循環流量を多くす
る。以上の動作の結果、溶存酸素濃度Ｘは、次第にＸ0
に近づく。かくしてバイオリアクター１内の溶存酸素濃
度は、細胞にとって必要とされる溶存酸素濃度Ｘ0 を保
ち続ける。When X> X 0 , the rotational speed of the pump 3 is controlled by the control signal to reduce the circulating flow rate. X <X 0
In the case of 1, the rotation speed of the pump 3 is increased to increase the circulation flow rate. As a result of the above operation, the dissolved oxygen concentration X gradually becomes X 0.
Approach. Thus, the dissolved oxygen concentration in the bioreactor 1 continues to maintain the dissolved oxygen concentration X 0 required for the cells.
【００１８】また、細胞の生育にともない、リザーバ２
にプールされる培地Ｌは、次第に老朽化して老廃物の蓄
積、栄養素の枯渇を生じる。そのため、定期的に培地を
交換する必要がある。この交換のために回転速度可変の
培地排出用ペリスタリックポンプ８及び廃液ボトル８
１、そして新鮮培地供給用ペリスタリックポンプ７及び
新鮮培地ボトル７１が設けられている。ボトル８１、７
１は、それぞれフィルタ８２、７２を通じて無菌的に大
気開放となっている。In addition, as the cells grow, the reservoir 2
The culture medium L pooled in step S1 gradually deteriorates, and waste products are accumulated and nutrients are depleted. Therefore, it is necessary to change the medium regularly. For this replacement, a peristaltic pump 8 for discharging the medium and a waste liquid bottle 8 with variable rotation speeds
1, and a peristaltic pump 7 for supplying fresh medium and a fresh medium bottle 71 are provided. Bottle 81, 7
1 is aseptically opened to the atmosphere through filters 82 and 72, respectively.
【００１９】培地交換の手順は以下のとおりである。ま
ず、電源１００から動作電力により、制御部９の構成要
素であるツインタイマ９３の内部接点９３２がオンのと
き、ペリスタリックポンプ８が動作し、リザーバ２内の
液を排出する。その際生ずるリザーバ２内の負圧はフィ
ルタ２１を通じて緩和される。ペリスタリックポンプ８
の回転数はＰＩＤコントローラ９１の支配下にあり、ポ
ンプ３の回転数と同じである。また、ツインタイマ９３
の内部接点９３３は、内部接点９３２と逆動作を行い、
接点９３２がオンのとき、接点９３３はオフであり、ポ
ンプ７は停止状態である。また、接点９３２、９３３の
それぞれのオン時間は設定用スイッチ９３１で設定され
る。The procedure for medium replacement is as follows. First, when the internal contact 932 of the twin timer 93, which is a component of the control unit 9, is turned on by operating power from the power supply 100, the peristaltic pump 8 operates to discharge the liquid in the reservoir 2. The negative pressure in the reservoir 2 generated at that time is relieved through the filter 21. Peristaltic pump 8
The rotation speed of is under the control of the PID controller 91 and is the same as the rotation speed of the pump 3. Also, the twin timer 93
Internal contact 933 of does the reverse operation of internal contact 932.
When the contact 932 is on, the contact 933 is off and the pump 7 is stopped. The ON time of each of the contacts 932 and 933 is set by the setting switch 931.
【００２０】次いで、ツインタイマ９３において、設定
用スイッチ９３１を通じて設定された接点９３２のオン
時間ｔ１がすぎると、接点９３２がオフとなると同時に
接点９３３がオンとなる。従って今度は、ポンプ８が停
止し、代わりにポンプ７が動作しリザーバ２内に培地を
供給する。そしてリザーバ２内の液面が上昇し、制御部
９の構成要素であるレベルセンサ９２の検出端９２１に
達すると、レベルセンサ９２内の内部接点９２２がオン
となり、ポンプ７も停止し、培地は供給されなくなる。Next, in the twin timer 93, when the ON time t1 of the contact 932 set through the setting switch 931 has passed, the contact 932 turns OFF and the contact 933 turns ON at the same time. Therefore, this time, the pump 8 is stopped and the pump 7 is operated instead to supply the medium into the reservoir 2. Then, when the liquid level in the reservoir 2 rises and reaches the detection end 921 of the level sensor 92 which is a component of the control unit 9, the internal contact 922 in the level sensor 92 is turned on, the pump 7 is also stopped, and the medium is removed. Will not be supplied.
【００２１】その後、ツインタイマ９３で設定用スイッ
チ９３１を通じて設定された接点９３３のオン時間ｔ２
が経過するとともに、最初の手順に戻る。このときのあ
りさまを示したのが、図２である。以上の一連の動作に
より、リザーバ２内培地は、ポンプ８により排出される
培地分だけ、新鮮培地と置換されることになる。Thereafter, the ON time t2 of the contact 933 set by the twin timer 93 through the setting switch 931 is set.
As time passes, the procedure returns to the first step. FIG. 2 shows the situation at this time. Through the above series of operations, the medium in the reservoir 2 is replaced with the fresh medium by the amount of the medium discharged by the pump 8.
【００２２】ところで、細胞の栄養消費量（特にグルコ
ース）は、酸素消費に比例すると考えられる。リアクタ
ー１内で消費される酸素量が増加すれば、溶存酸素検出
装置５に感知される溶存酸素濃度Ｘは下降気味になる。
そのため、Ｘ＜Ｘ0 の関係が生じ、ポンプ３の循環流量
は多くなる。ポンプ８で排出される時間当たりの培地量
は、ポンプ３の循環流量に等しい。従って、ツインタイ
マ９３で設定されるｔ１時間内にポンプ８で排出される
培地量も当然多くなり、結果として、栄養消費量に比例
して培地が交換される。その際の比例定数はＣ×ｔ１／
（ｔ１＋ｔ２）となるため、ツインタイマ９３での設定
時間ｔ１、ｔ２を設定スイッチ９３１を用いて任意に変
更することにより培地交換量、換言すれば（新鮮培地供
給量／溶存酸素消費量）の割合を決定することが可能で
ある。By the way, it is considered that the nutrient consumption of cells (particularly glucose) is proportional to the oxygen consumption. As the amount of oxygen consumed in the reactor 1 increases, the dissolved oxygen concentration X sensed by the dissolved oxygen detection device 5 tends to decrease.
Therefore, the relationship of X <X 0 occurs, and the circulation flow rate of the pump 3 increases. The amount of medium discharged per hour by the pump 8 is equal to the circulation flow rate of the pump 3. Therefore, the amount of the medium discharged by the pump 8 naturally increases within the time t1 set by the twin timer 93, and as a result, the medium is replaced in proportion to the nutrient consumption amount. The proportional constant at that time is C × t1 /
Since (t1 + t2), the set times t1 and t2 in the twin timer 93 are arbitrarily changed by using the setting switch 931 and the medium replacement amount, in other words, (fresh medium supply amount / dissolved oxygen consumption amount) ratio It is possible to determine
【００２３】また、この装置においては、細胞をバイオ
リアクター１のホローファイバ外空間に生育させるた
め、求める生産物は、ホローファイバ外空間に蓄積され
る。その生産物を取り出すための手段がサンプリング回
路１４及び培地ボトル１３であり、ボトル１３はフィル
タ１３１を通じて無菌的に大気開放となっている。サン
プリング回路１４からは、シリンジ等の手段を用いて無
菌的に外部に生産物が取り出されるようになっており、
その際、ホローファイバ外空間には、取り出された分だ
け培地ボトル１３より培地が供給される。Further, in this apparatus, since the cells are grown in the hollow fiber outer space of the bioreactor 1, the desired product is accumulated in the hollow fiber outer space. The means for taking out the product is the sampling circuit 14 and the culture medium bottle 13, and the bottle 13 is aseptically opened to the atmosphere through the filter 131. From the sampling circuit 14, the product is aseptically taken out to the outside by using a means such as a syringe,
At that time, the culture medium is supplied from the culture medium bottle 13 to the outer space of the hollow fiber in the amount taken out.
【００２４】なお、ｐＨセンサ２２は、リザーバ２内の
培地のｐＨが適正がどうか確認するためのセンサであ
る。以上説明した本発明に係る培養装置では、栄養源及
び酸素の供給にあたり、培地の状態を測定乃至検出する
手段としては培地中の溶存酸素量を測定する手段（上記
実施例では溶存酸素検出装置５）があれば足り、ｐＨセ
ンサ等の他のセンサ類は無くてもよく、それだけ構成を
簡素化できる。 また、一つの溶存酸素量測定手段５の測
定結果に基づいて培地循環用ポンプ３及び培地排出ポン
プ８、延いては培地供給ポンプ７も制御できるという簡
単な構成で、培地中の溶存酸素量及び栄養源を適切にコ
ントロールできる。また、培地供給ポンプ７は培地排出
ポンプ８が停止してから始動するので、培地交換が無駄
なく能率的に行われる。 さらに培地排出ポンプ８の流量
を培地循環用ポンプ３の流量に比例連携させるので、培
地排出ポンプ８を適当なオン・オフ時間でタイマ制御す
ることで培地排出量は酸素消費に比例的に変動し、結果
として培地交換量も比例的に変動するので、必要量の培
地交換のために培地リザーバ２内の液面設定レベルを変
更する必要がなく、この点でも構成が簡素化される。 さ
らに、採用するバイオリアクター１はホローファイバ式
バイオリアクターなので、従来のタンク式培養装置にお
いて設けられていた老廃物交換排出装置を別途設ける必
要がない。 また、培地中の溶存酸素量の測定はバイオリ
アクター１を出たところで行うので、特に問題となるバ
イオリアクター１の出口付近の細胞に対する溶存酸素量
を制御できるという利点もある。 また、ホローファイバ
式バイオリアクター１のホローファイバ外空間に蓄積さ
れる培養細胞による生産物をサンプリング回路１４から
容易に取り出すことができる。 The pH sensor 22 is a sensor for confirming whether the pH of the medium in the reservoir 2 is proper. In the culture device according to the present invention described above, the nutrient source and
Measuring and detecting the condition of the medium when supplying oxygen and oxygen
As a means, a means for measuring the amount of dissolved oxygen in the medium (the above
In the embodiment, it is sufficient to have a dissolved oxygen detector 5), and the pH
Sensor, etc.
Can be simplified. In addition, the measurement of one dissolved oxygen amount measuring means 5
Based on the determined results, the medium circulation pump 3 and the medium discharge pump
8 that the medium supply pump 7 can be controlled.
With a simple structure, the amount of dissolved oxygen and nutrients in the medium can be properly controlled.
You can control. Also, the medium supply pump 7 discharges the medium.
Since the pump 8 starts after it stops, there is no need to replace the medium.
It is done efficiently without Further, the flow rate of the medium discharge pump 8
Is proportional to the flow rate of the medium circulation pump 3,
Timer control of earth discharge pump 8 with appropriate on / off time
As a result, the amount of medium discharged fluctuates in proportion to oxygen consumption.
As the medium replacement amount also changes proportionally,
Change the liquid level setting level in the medium reservoir 2 for exchanging the ground.
There is no need to change it, and the configuration is simplified in this respect as well. Sa
In addition, the bioreactor 1 adopted is a hollow fiber type
Since it is a bioreactor, it can be installed in a conventional tank-type culture device.
Installed separately, it is necessary to install a separate waste exchange device.
No need. The amount of dissolved oxygen in the medium is
Since it will be performed after leaving Actor 1, there will be a particular problem.
Amount of dissolved oxygen for cells near the outlet of Io Reactor 1
There is also an advantage that can be controlled. Also hollow fiber
Accumulated in the hollow fiber outer space of the bioreactor 1
From the sampling circuit 14 the product of the cultured cells
It can be taken out easily.
【００２５】[0025]
【発明の効果】以上説明したように本発明によると、培
地循環用ポンプにて培地を、培地リザーバから、酸素を
溶存させたのちホローファイバ式バイオリアクターに通
過させ、再び前記培地リザーバへ戻すように循環させる
培養装置であって、バイオリアクター内の被培養物の活
動状態に応じた量の栄養源及び酸素を簡単な構成のもと
に、能率的に、正確にバイオリアクターへ供給すること
ができる培養装置が提供される。 また、ホローファイバ
式バイオリアクターのホローファイバ外空間の一端及び
他端に生産物取出し用の培地ボトル及びサンプリング配
管をそれぞれ接続し、該培地ボトルを無菌用フィルタを
介して外気に連通させることで、該サンプリング配管か
ら培養した細胞による生産物を容易に取り出せる。 As described above, according to the present invention, the medium circulation pump is used to dissolve oxygen in the medium reservoir and then pass the medium through the hollow fiber type bioreactor to return it to the medium reservoir again. It is a culture device that circulates into a bioreactor, and provides a nutrient source and oxygen in an amount suitable for the activity state of the culture in the bioreactor with a simple structure.
In addition, a culture device that can efficiently and accurately supply the bioreactor is provided. Also hollow fiber
End of the hollow fiber outer space of the bioreactor and
At the other end, a medium bottle for product removal and sampling
Connect the tubes to each other, and then attach the medium bottle to the filter for sterility.
By connecting to the outside air via the sampling pipe
The product of the cultured cells can be easily obtained.
【００２６】[0026]
【００２７】[0027]
【００２８】また、前記培地リザーバ内の培地液面が所
定レベルに有るか否かを検出するレベル検出手段及びツ
インタイマが設けられ、該ツインタイマで設定される排
出ポンプ用オン時間及びオフ時間に基づいて前記排出ポ
ンプが運転又は停止され、該排出ポンプオフ時間におい
て、前記レベル検出手段による培地リザーバ内液面の所
定レベル到達検出まで前記供給ポンプが運転されるとき
は、前記オン時間の長さ及びオフ時間の長さを任意に設
定して、所望の（新鮮培地供給量／溶存酸素消費量）の
割合を決定できる。Further, level detecting means for detecting whether or not the liquid surface of the medium in the medium reservoir is at a predetermined level and a twin timer are provided, and the discharge pump on-time and off-time set by the twin timer are set. When the discharge pump is operated or stopped based on the discharge pump off time, and the supply pump is operated until the discharge pump is off until the predetermined level of the liquid level in the medium reservoir is detected by the level detection means, the length of the on time and The length of the off-time can be set arbitrarily to determine the desired ratio of (fresh medium supply / dissolved oxygen consumption).
【図１】本発明の一実施例の構成を示す図である。FIG. 1 is a diagram showing a configuration of an exemplary embodiment of the present invention.
【図２】培地リザーバ内培地を交換するときのツインタ
イマ接点のオン時間及びオフ時間、それに関連する培地
供給ポンプ及び排出ポンプの動作タイミングを、リザー
バ内の液面高さ変化とともに示すタイミングチャートで
ある。FIG. 2 is a timing chart showing on-time and off-time of a twin timer contact when exchanging a medium in a medium reservoir, and associated operation timings of a medium supply pump and a discharge pump together with a change in liquid level in the reservoir. is there.
１ バイオリアクター ２ 培地リザーバ ３ 培地循環用ポンプ ４ ガス交換器 ５ 溶存酸素検出装置 ６ 配管 ７ 新鮮培地供給ポンプ ７１ 新鮮培地ボトル ８ 培地排出ポンプ ８１ 廃液ボトル ９ 制御部 ９１ ＰＩＤコントローラ ９２ レベルセンサ ９３ ツインタイマ ９３１ 時間設定スイッチ ９３２、９３３ タイマ接点 １００ 電源 1 Bioreactor 2 Medium Reservoir 3 Medium Circulation Pump 4 Gas Exchanger 5 Dissolved Oxygen Detector 6 Piping 7 Fresh Medium Supply Pump 71 Fresh Medium Bottle 8 Medium Discharge Pump 81 Waste Liquid Bottle 9 Control Unit 91 PID Controller 92 Level Sensor 93 Twin Timer 931 Time setting switch 932, 933 Timer contact 100 Power supply
Claims (2)
ーバから、酸素を溶存させたのちホローファイバ式バイ
オリアクターに通過させ、再び前記培地リザーバへ戻す
ように循環させる培養装置において、前記バイオリアク
ターを出た培地中の溶存酸素量を測定する手段と、前記
培地リザーバから培地を排出する培地排出ポンプと、新
鮮培地容器と、該容器から前記培地リザーバへ新鮮培地
を供給する培地供給ポンプと、前記ポンプ動作を制御す
る制御部とを備え、前記制御部は、前記培地循環用ポン
プの流量を前記測定手段により測定される溶存酸素量が
予め定めた溶存酸素量に維持されるように制御し、前記
培地排出ポンプの流量を前記培地循環用ポンプの流量に
比例するように制御し、前記培地供給ポンプを前記培地
排出ポンプが停止すると始動し、前記培地リザーバ内液
面が所定のレベルに達すると停止するように制御するこ
とを特徴とする培養装置。1. A hollow fiber type bi-fuel is prepared by dissolving oxygen in the medium from a medium reservoir with a medium circulation pump.
In a culture device which is passed through an o-reactor and circulated so as to be returned to the medium reservoir again, means for measuring the amount of dissolved oxygen in the medium leaving the bioreactor, and a medium discharge pump for discharging the medium from the medium reservoir A fresh medium container, a medium supply pump that supplies fresh medium from the container to the medium reservoir, and a control unit that controls the pump operation. The control unit includes a medium circulation pump.
Controlled to the amount of dissolved oxygen measured the flow rate of up by the measuring means is maintained at a dissolved oxygen of <br/> predetermined, the
Change the flow rate of the medium discharge pump to the flow rate of the medium circulation pump
Control so that the medium supply pump is proportional to the medium.
When the discharge pump is stopped, it starts and the liquid in the medium reservoir
Surface culture device comprising a this <br/> controlled to stop when it reaches a predetermined level.
のホローファイバ外空間の一端に接続された生産物取出
し用培地ボトルを設け、前記ボトルは無菌用フィルタを
介して外気に連通させ、前記ホローファイバ外空間の他
端には生産物取出し用サンプリング配管を接続したこと
を特徴とする請求項１記載の培養装置。 2. The hollow fiber type bioreactor.
Of the product connected to one end of the hollow fiber outer space
A culture medium bottle is provided, and the bottle has a sterile filter.
To communicate with the outside air through the outside of the hollow fiber outside space.
A sampling pipe for product extraction was connected to the end
The culture device according to claim 1, wherein
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3286291A JP2500137B2 (en) | 1991-10-31 | 1991-10-31 | Incubator |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP3286291A JP2500137B2 (en) | 1991-10-31 | 1991-10-31 | Incubator |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH05123156A JPH05123156A (en) | 1993-05-21 |
| JP2500137B2 true JP2500137B2 (en) | 1996-05-29 |
Family
ID=17702482
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP3286291A Expired - Fee Related JP2500137B2 (en) | 1991-10-31 | 1991-10-31 | Incubator |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2500137B2 (en) |
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| US7278528B2 (en) | 1999-02-19 | 2007-10-09 | Ronald H. Ball | Method of and apparatus for applying a film optionally including advertising or other visible material, to the surface of a handrail for an escalator or moving walkway |
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|---|---|---|---|---|
| JP4731987B2 (en) * | 2005-05-12 | 2011-07-27 | 株式会社カネカ | Automatic culture equipment |
| JP5691146B2 (en) * | 2009-08-12 | 2015-04-01 | 株式会社ニコン | Microscope system, control device, and control method |
| JP6095671B2 (en) * | 2011-10-07 | 2017-03-15 | ポール テクノロジー ユーケイ リミテッドＰａｌｌ Ｔｅｃｈｎｏｌｏｇｙ Ｕｋ Ｌｉｍｉｔｅｄ | Fluid treatment control system and corresponding method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4999298A (en) * | 1988-04-27 | 1991-03-12 | W. R. Grace & Co.-Conn. | Hollow fiber bioreactor culture system and method |
| JP2625990B2 (en) * | 1988-11-19 | 1997-07-02 | 株式会社島津製作所 | Cell culture device |
| JP3095464B2 (en) * | 1991-08-08 | 2000-10-03 | 宇宙開発事業団 | Cell culture device |
-
1991
- 1991-10-31 JP JP3286291A patent/JP2500137B2/en not_active Expired - Fee Related
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6682806B1 (en) | 1999-02-19 | 2004-01-27 | Ronald H. Ball | Method of applying a protective film, optionally including advertising or other visible material, to the surface of a handrail for an escalator or moving walkway |
| US7278528B2 (en) | 1999-02-19 | 2007-10-09 | Ronald H. Ball | Method of and apparatus for applying a film optionally including advertising or other visible material, to the surface of a handrail for an escalator or moving walkway |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH05123156A (en) | 1993-05-21 |
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