JP4074970B2 - Cell concentration and / or physiological activity measuring device - Google Patents
Cell concentration and / or physiological activity measuring device Download PDFInfo
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- JP4074970B2 JP4074970B2 JP28041698A JP28041698A JP4074970B2 JP 4074970 B2 JP4074970 B2 JP 4074970B2 JP 28041698 A JP28041698 A JP 28041698A JP 28041698 A JP28041698 A JP 28041698A JP 4074970 B2 JP4074970 B2 JP 4074970B2
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- 230000001766 physiological effect Effects 0.000 title claims description 30
- 230000008859 change Effects 0.000 claims description 25
- 239000007787 solid Substances 0.000 claims description 14
- 238000005259 measurement Methods 0.000 claims description 13
- 230000007246 mechanism Effects 0.000 claims description 9
- 238000010521 absorption reaction Methods 0.000 claims description 2
- 239000007789 gas Substances 0.000 description 67
- 210000004027 cell Anatomy 0.000 description 25
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 5
- 239000001301 oxygen Substances 0.000 description 5
- 229910052760 oxygen Inorganic materials 0.000 description 5
- 239000000758 substrate Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 238000009630 liquid culture Methods 0.000 description 4
- 230000000694 effects Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000005273 aeration Methods 0.000 description 2
- 210000004102 animal cell Anatomy 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000013557 nattō Nutrition 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000036978 cell physiology Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Description
【０００１】
【産業上の利用分野】
本発明は動物細胞、植物細胞や微生物の培養、特に麹や納豆の固体培養及び細胞を用いた各種の分析や解析における細胞の濃度及び／または生理活性の測定に利用される。
【０００２】
【従来の技術】
従来培養時の細胞の濃度や生理活性を測定する場合基質の濃度変化、培養槽へ供給するガスの供給濃度と排出濃度の差や通気を停止した際の溶存酸素の濃度変化から求めていたが細胞の濃度や生理活性が小さい場合それらの濃度変化が小さく正確な測定ができなかった。特に固体培養では細胞の濃度や生理活性を自動的に計測する手段が無く発酵の適切な制御に支障を来していた。また食品や水質検査で細菌検査が一般に行われており、シャーレ上で培養してコロニー数をカウントする方法が一般的であるが培養に時間がかかり短時間での計測ができなかった。
【０００３】
【解決しようとする課題】
本発明は従来の欠点に鑑みて細胞濃度や生理活性が小さい場合でもこれを容易に短時間にかつ無菌状態を保って計測する手段を提供する。
【０００４】
【課題を解決する手段】
本発明者は上記課題を解決する手段を検討し、ガスを閉鎖したまま循環することによってガス濃度の変化を増幅することができること、更にガスを閉鎖循環状態で培養するとガスが吸収されまたは発生して気相が陰圧または陽圧になって培養条件が変動することがあるのでガス循環閉鎖流路中に可撓部分を含めることによってこれを解決出来ること等を見いだし本発明を完成させた。請求項１に記載の発明の細胞濃度及び／または生理活性測定装置はガス循環閉鎖流路を有する培養槽及び該ガス循環閉鎖流路中のガス成分濃度を測定する手段を有し、該ガス成分濃度を測定する手段により測定した該ガス成分濃度の変化から該ガス成分濃度の変化速度を計測することにより細胞濃度及び／または生理活性を測定する細胞濃度及び／または生理活性測定装置において、該ガス循環閉鎖流路に、その体積変化によって圧力変動無くガスの体積変化を吸収できる可撓部分を有することを特徴とする。
【０００５】
請求項２に記載の発明の細胞濃度及び／または生理活性測定装置は上記細胞濃度及び／または生理活性測定装置がさらに上記ガスを間欠的に交換する機構を有することを特徴とする。請求項３に記載の発明の細胞濃度及び／または生理活性測定装置は上記ガスを間欠的に交換する機構が上記ガス成分濃度が設定値に達したら自動的に上記ガスを交換する機構を含むことを特徴とする。請求項４に記載の発明の細胞濃度及び／または生理活性測定装置は上記培養槽が固体培養槽であることを特徴とする。
【０００７】
【作用】
培養槽中のガスを閉鎖循環することによってガスは再利用され培養槽中で消費または発生するガスは積算される。従ってワンパスでは変化量が少なかったガス成分も増幅して計測できる。ガスの循環流路を閉鎖系にしておくとガスの吸収や発生によって循環流路の圧力が変動し循環流路や培養槽に損傷を与える場合がありまた培養に影響を与える場合もある。そこで本発明は循環流路中に可撓部分を含むことによってこの問題を解決した。可撓部分の体積変化によって圧力変動無くガスの体積変化を吸収できる。循環を続けているとガス組成が変化し培養に適切ではなくなることもあるので間欠的にガスを交換する機構を備えることが望ましい。
【０００８】
上記の間欠的にガスを交換する機構が上記ガス濃度が設定値に達したら自動的にガスを交換する機構を含むことによって供給ガス濃度を培養に適切な濃度範囲に保っことが出来、細胞濃度及び／または生理活性の測定精度が向上する。本発明は液体培養でも固体培養でも適用可能であるが、液体培養では液中の成分濃度の変化や溶存酸素濃度の変化等計測対象が種々有るが固体発酵ではこれに相当する計測は難しく本発明は特に固体培養に有効である。
【００１０】
【本発明の実施の形態】
次に本発明の実施の形態を説明する。本発明は液体培養及び固体培養、好気発酵及び嫌気発酵、微生物、動物細胞及び植物細胞の培養やこれらの組み合わせに利用できる。また必ずしも培養時の細胞の濃度及び／または生理活性だけでなく、細胞の生理活性を測定することによって間接的に各種成分例えば抗生物質や毒物を分析や解析する系にも利用でき、本発明ではこれらも含めて細胞に影響を与える系を培養と記している。
【００１１】
本発明を例えば好気性液体培養に応用する場合、酸素を含むガスを液体中に散気供給するが培養槽の排気口にコンデンサーを設けて水分を還流し、乾燥ガスをポンプで無菌フィルターを介して培養槽に再び散気する。このガス循環閉鎖流路中のガス成分例えば酸素または炭酸ガスを計測するとそれらのガス濃度が変化する。その変化速度を計測することによって細胞の濃度及び／または生理活性を測定できる。この測定から呼吸活性や呼吸商も測定でき生理活性測定が可能になる。測定するガス成分としては上記酸素、炭酸ガスのほか水素、アンモニアが有用で嫌気発酵ではメタンも有効な測定対象となる。
【００１２】
納豆、麹等の基質が固体の固体培養でもガスを閉鎖循環するとガス濃度が変化する。その変化速度から細胞濃度、生理活性さらに培養過程や培養の終点の推定等が可能になる。
【００１３】
培養液に抗生物質や毒物を添加した場合、添加しない場合との上記のガス濃度の変化速度の違いから添加物の細胞生理に与える影響が推測でき添加物の効果や安全性が評価できる。
【００１４】
【実施例１】
本発明をさらに詳細に実施例に従って説明する。図１は本発明の実施例１の模式フロー図を示す。培養槽１に液体培地２が張ってあり散気ユニット３から培養液中に散気する。散気されたガスはコンデンサー４で水分を還流しポンプ５、可撓性容器６、ガス濃度測定装置７さらに無菌フィルター８を通って再び散気する閉鎖循環流路を形成する。攪拌装置を備える事も多いが図示しない。
【００１５】
上記実施例の実施の態様を以下に説明する。散気されたガスはポンプ５で循環して使用される。ガス成分が培養によって消費されまたは発生するとその変化はワンパスの場合に比べて増幅されるので細胞濃度及び／または生理活性が小さい場合でも単位時間あたりの変化量を測定することによって大きな変化量として測定でき精度のよい測定が可能となる。培養中発生するまたは吸収されるガスによる循環ガスの体積変化は可撓性容器６によって吸収され培養槽には圧力変動は生じない。
【００１６】
【参考例】
図２は参考例の模式フロー図を示す。図中の符号は実施例１と同じ名称及び機能のものは同じ符号を用いた。培養槽１に固体基質１１があり固体培養をしている。ガスはポンプ５、ガス濃度測定装置７、無菌フィルター８を通って循環される。ガス濃度測定装置７はガス濃度設定装置１２と比較され設定値を超えるまたは下回ると一定時間電磁弁１３及び１４が開いてガス供給源１５から新しいガスが供給される。破線及び矢印１６はその制御系統を示す。電磁弁１３及び１４が開いている間はポンプ５は停止している。破線１７はガス濃度測定装置７とガス濃度設定装置１２を比較している状態を示す。
【００１７】
参考例のように間欠的に新しいガスを導入すると培養条件を一定に保つことが出来る。この時培養中のガス濃度の変化速度から細胞濃度及び／または生理活性を測定できる。設定ガス濃度は時間と共に変化させてもよい。ガス濃度測定装置としては連続的な測定が望ましいが間欠的にサンプリングして測定しても機能を果たす場合もある。連続的にガスを循環することが望ましくない場合はポンプはガス濃度測定時だけ作動させてもよい。また通常は新鮮なガスを培養槽に供給し排気口を開放していて細胞濃度及び／または生理活性測定時に上記のようにガス循環閉鎖流路を形成してもよい。
【００１８】
【発明の効果】
以上本発明を実施例を挙げて説明したが本発明はこれらに限定されるものではない。本発明により細胞濃度や生理活性が低くても増幅して測定できるので精度よく短時間にかつ無菌を保って測定が可能になった。さらに培養液や固体基質をサンプリングしなくてもまた培養系に影響を与えることなく測定が可能になった。
【図面の簡単な説明】
【図１】本発明の実施例１の模式フロー図を示す。
【図２】参考例の模式フロー図を示す。
【符号の説明】
１ 培養槽
２ 培養液
３ 散気ユニット
４ コンデンサー
５ ポンプ
６ 可撓性容器
７ ガス濃度測定装置
８ 無菌フィルター
１１ 固体基質
１２ ガス濃度設定装置
１３ 電磁弁
１４ 電磁弁
１５ ガス供給源
１６ 制御系統[0001]
[Industrial application fields]
The present invention is used for culturing animal cells, plant cells, and microorganisms, particularly solid culture of straw and natto, and measuring cell concentrations and / or physiological activities in various analyzes and analyzes using cells.
[0002]
[Prior art]
Conventionally, when measuring cell concentration and physiological activity during culture, it was obtained from the change in substrate concentration, the difference between the supply and discharge concentrations of gas supplied to the culture tank, and the change in dissolved oxygen concentration when aeration was stopped. When the cell concentration and physiological activity were small, their concentration change was small and accurate measurement could not be performed. In particular, in solid culture, there is no means for automatically measuring cell concentration and physiological activity, which hinders proper control of fermentation. In addition, bacterial tests are generally performed in food and water quality tests, and a method of counting the number of colonies by culturing on a petri dish is generally used, but it takes a long time to culture and cannot be measured in a short time.
[0003]
[Problems to be solved]
The present invention provides means for easily measuring even in a short time and in a sterile state even when the cell concentration and physiological activity are small in view of the conventional drawbacks.
[0004]
[Means for solving the problems]
The present inventor has studied a means for solving the above-mentioned problems, can amplify a change in gas concentration by circulating the gas in a closed state, and further, if the gas is cultured in a closed circulation state, the gas is absorbed or generated. Thus, the present invention has been completed by finding that this can be solved by including a flexible portion in the gas circulation closed channel because the gas phase may become negative pressure or positive pressure and the culture conditions may fluctuate. Cell concentration and / or physiological activity measurement equipment of the first aspect of the present invention have a means for measuring the gas component concentration of the culture tank and in the gas circulation closed flow path having a gas circulation closed passage, the In a cell concentration and / or physiological activity measuring apparatus for measuring a cell concentration and / or physiological activity by measuring a change rate of the gas component concentration from a change in the gas component concentration measured by means for measuring a gas component concentration, to the gas circulation closed flow path, characterized in that it have a flexible portion capable of absorbing a volume change of the pressure fluctuations without gas by the volume change.
[0005]
Cell concentration and / or physiological activity measuring device of the invention described in
[0007]
[Action]
By closing and circulating the gas in the culture tank, the gas is reused and the gas consumed or generated in the culture tank is integrated. Therefore, it is possible to amplify and measure a gas component whose amount of change is small in one pass . If the gas circulation channel is kept in a closed system, the pressure of the circulation channel fluctuates due to gas absorption and generation, which may damage the circulation channel and the culture tank, and may affect the culture. The present invention solves this problem by including a flexible portion in the circulation flow path. The volume change of the gas can be absorbed without a pressure fluctuation by the volume change of the flexible part. When continues to recycling the gas composition sometimes it becomes not suitable to change the culture to a mechanism to exchange intermittently gas is desirable.
[0008]
The mechanism for exchanging the gas intermittently includes a mechanism for automatically exchanging the gas when the gas concentration reaches the set value, so that the supply gas concentration can be kept in a concentration range suitable for culture, and the cell concentration And / or the measurement accuracy of physiological activity improves. The present invention can be applied to both liquid culture and solid culture. In liquid culture, there are various measurement objects such as changes in component concentration and dissolved oxygen concentration in the liquid, but in solid fermentation, measurement corresponding to this is difficult. Is particularly effective for solid culture.
[0010]
[Embodiments of the Invention]
Next, an embodiment of the present invention will be described. The present invention can be used for liquid culture and solid culture, aerobic fermentation and anaerobic fermentation, culture of microorganisms, animal cells and plant cells, and combinations thereof. In addition, the present invention can be used not only in the concentration and / or physiological activity of cells during culture but also indirectly in a system for analyzing and analyzing various components such as antibiotics and toxins by measuring the physiological activity of cells. A system that affects cells including these is described as culture.
[0011]
When the present invention is applied to, for example, aerobic liquid culture, oxygen-containing gas is diffused and supplied to the liquid, but a condenser is provided at the exhaust port of the culture tank to recirculate moisture, and the dry gas is pumped through a sterile filter. Aerate again in the culture tank. When gas components such as oxygen or carbon dioxide in the gas circulation closed channel are measured, their gas concentrations change. By measuring the rate of change, cell concentration and / or physiological activity can be measured. From this measurement, respiratory activity and respiratory quotient can be measured, and physiological activity can be measured. As gas components to be measured, hydrogen and ammonia are useful in addition to the above oxygen and carbon dioxide, and methane is also an effective measurement target in anaerobic fermentation.
[0012]
Even in solid culture where the substrates such as natto and koji are solid, the gas concentration changes when the gas is closed and circulated. From the rate of change, it is possible to estimate the cell concentration, physiological activity, culture process and end point of culture.
[0013]
When antibiotics or poisons are added to the culture solution, the effect on the cell physiology of the additive can be estimated from the difference in the gas concentration change rate from the case where it is not added, and the effect and safety of the additive can be evaluated.
[0014]
[Example 1]
The invention will be described in more detail according to examples. FIG. 1 shows a schematic flow diagram of Embodiment 1 of the present invention. A
[0015]
Embodiments of the above embodiment will be described below. The diffused gas is circulated by the
[0016]
[Reference example]
FIG. 2 shows a schematic flow diagram of a reference example . In the figure, the same reference numerals are used for the same names and functions as those in the first embodiment. There is a
[0017]
When new gas is introduced intermittently as in the reference example, the culture conditions can be kept constant. At this time, the cell concentration and / or physiological activity can be measured from the change rate of the gas concentration during the culture. The set gas concentration may be changed with time. As a gas concentration measuring device, continuous measurement is desirable, but there are cases where it functions even if it is measured intermittently by sampling. If it is not desirable to circulate the gas continuously, the pump may be activated only during gas concentration measurement. Ordinarily, fresh gas may be supplied to the culture tank and the exhaust port may be opened to form the gas circulation closed channel as described above when measuring the cell concentration and / or physiological activity.
[0018]
【The invention's effect】
Although the present invention has been described with reference to examples, the present invention is not limited to these examples. According to the present invention, even if the cell concentration or physiological activity is low, it can be amplified and measured, so that measurement can be performed accurately and in a short time while maintaining sterility. Furthermore, measurement can be performed without sampling the culture medium or solid substrate and without affecting the culture system.
[Brief description of the drawings]
FIG. 1 shows a schematic flow diagram of Example 1 of the present invention.
FIG. 2 shows a schematic flow diagram of a reference example .
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28041698A JP4074970B2 (en) | 1998-08-26 | 1998-08-26 | Cell concentration and / or physiological activity measuring device |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP28041698A JP4074970B2 (en) | 1998-08-26 | 1998-08-26 | Cell concentration and / or physiological activity measuring device |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2000060596A JP2000060596A (en) | 2000-02-29 |
| JP4074970B2 true JP4074970B2 (en) | 2008-04-16 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP28041698A Expired - Fee Related JP4074970B2 (en) | 1998-08-26 | 1998-08-26 | Cell concentration and / or physiological activity measuring device |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4074970B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP4036208A1 (en) | 2012-10-26 | 2022-08-03 | Massachusetts Institute Of Technology | Humidity control in chemical reactors |
| CA3173080A1 (en) | 2013-08-23 | 2015-02-26 | Sanofi | Small volume bioreactors with substantially constant working volumes and associated systems and methods |
-
1998
- 1998-08-26 JP JP28041698A patent/JP4074970B2/en not_active Expired - Fee Related
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| Publication number | Publication date |
|---|---|
| JP2000060596A (en) | 2000-02-29 |
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