JPH02114163A - Organism culture container - Google Patents
Organism culture containerInfo
- Publication number
- JPH02114163A JPH02114163A JP26713888A JP26713888A JPH02114163A JP H02114163 A JPH02114163 A JP H02114163A JP 26713888 A JP26713888 A JP 26713888A JP 26713888 A JP26713888 A JP 26713888A JP H02114163 A JPH02114163 A JP H02114163A
- Authority
- JP
- Japan
- Prior art keywords
- culture
- cells
- electrodes
- measurement
- container
- 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.)
- Granted
Links
- 239000011521 glass Substances 0.000 claims description 2
- 238000005259 measurement Methods 0.000 abstract description 19
- 241001465754 Metazoa Species 0.000 abstract description 4
- 239000007788 liquid Substances 0.000 abstract description 3
- 230000001066 destructive effect Effects 0.000 abstract 2
- 210000004027 cell Anatomy 0.000 description 48
- 230000001580 bacterial effect Effects 0.000 description 19
- 230000000694 effects Effects 0.000 description 11
- 238000000034 method Methods 0.000 description 11
- 244000005700 microbiome Species 0.000 description 9
- 230000012010 growth Effects 0.000 description 7
- 238000012258 culturing Methods 0.000 description 6
- 241000196324 Embryophyta Species 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 5
- 210000004102 animal cell Anatomy 0.000 description 5
- 239000002028 Biomass Substances 0.000 description 4
- 238000005070 sampling Methods 0.000 description 4
- 244000000231 Sesamum indicum Species 0.000 description 3
- 235000003434 Sesamum indicum Nutrition 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 208000025113 myeloid leukemia Diseases 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000003068 static effect Effects 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 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 1
- 102000008072 Lymphokines Human genes 0.000 description 1
- 108010074338 Lymphokines Proteins 0.000 description 1
- 101150042248 Mgmt gene Proteins 0.000 description 1
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- JEEWCODRWQBDAV-UHFFFAOYSA-K S(=O)(=O)([O-])[O-].[K+].P(=O)([O-])([O-])O.[Mg+2].[NH4+] Chemical compound S(=O)(=O)([O-])[O-].[K+].P(=O)([O-])([O-])O.[Mg+2].[NH4+] JEEWCODRWQBDAV-UHFFFAOYSA-K 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 238000004115 adherent culture Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010291 electrical method Methods 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000012091 fetal bovine serum Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- HRRDCWDFRIJIQZ-UHFFFAOYSA-N naphthalene-1,8-dicarboxylic acid Chemical compound C1=CC(C(O)=O)=C2C(C(=O)O)=CC=CC2=C1 HRRDCWDFRIJIQZ-UHFFFAOYSA-N 0.000 description 1
- 239000013630 prepared media Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 239000012138 yeast extract Substances 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
- C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
-
- 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
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/08—Flask, bottle or test tube
Abstract
Description
【発明の詳細な説明】
（産業上の利用分野）
本発明は、生物を培養するための容器に関するものであ
り、特に培養中の生物（微生物、動物細胞、植物細胞等
）ａ度のオンライン計測が可能となる生物培養容器に関
するものであり、さらに詳細には、１！気容量（誘電率
）を測定することにより生物濃度、生物の増殖活性を同
時に計測できる容器に関するものである。したがって本
発明は、バイオインダストリをはじめ、医療１食品工業
といった分野において非常に重要な役割を果たすもので
ある。Detailed Description of the Invention (Industrial Application Field) The present invention relates to a container for culturing living things, and in particular, to online measurement of the degree of living things (microorganisms, animal cells, plant cells, etc.) being cultured. This article relates to a biological culture container that enables 1! This invention relates to a container that can simultaneously measure biological concentration and biological growth activity by measuring air capacity (permittivity). Therefore, the present invention plays a very important role in fields such as bioindustry, medical industry, and food industry.
（従来の技術）
各種微生物、動・植物細胞等を用いて有用物質を生産す
るバイオリアクタや培養装置は、その内部の生物細胞濃
度が時々刻々変化するものであり、バイオリアクタ、培
養装置の制御を行ったり、発酵槽内部状態を知る上で生
物１度、生物増殖活性を１ｌｌｑ定することが非常に重
要である。(Prior art) Bioreactors and culture devices that produce useful substances using various microorganisms, animal and plant cells, etc., have biological cell concentrations inside them that change from time to time, and it is difficult to control the bioreactors and culture devices. It is very important to determine the biological growth activity of each organism once and to know the internal state of the fermenter.
バイオリアクタ等において、細胞の大きさが小さい各種
微生物の場合は、！＠濁濁液液した場合に限り、培養液
中の微生物の各種光学的性質に基づいて微生物濃度を測
定することが一応は可能である。しかし、ａ度が高くな
るとフロックを形成するカビや、微生物に比較して体積
が大きく、またフロックを形成する場合が多い植物細胞
や動物細胞では、乾燥重量や細胞の湿体績を求めたり、
懸濁液の一部を取り出し細胞や核を染色した後、顕微鏡
下で細胞数をカウントする等の方法がとられるのが通例
である。また光学的手法により計測可能な微生物におい
ても、濃度が高くなったり、また静置培養の場合のよう
に沈降した状態で増殖する際には、 １ｌ１１１定が困
難であった。In the case of various microorganisms with small cell sizes in bioreactors, etc.,! Only in the case of a turbid liquid, it is possible to measure the concentration of microorganisms based on various optical properties of the microorganisms in the culture solution. However, when the a degree is high, molds that form flocs, and plant cells and animal cells that have a larger volume than microorganisms and often form flocs, require the dry weight and wet weight of the cells to be determined.
It is customary to take out a portion of the suspension, stain the cells and nuclei, and then count the number of cells under a microscope. Furthermore, even for microorganisms that can be measured by optical methods, it is difficult to determine 1l111 when the concentration is high or when the microorganisms grow in a sedimented state as in the case of static culture.
特に、リンホカインその他方用な生理活性物質を産生ず
る動物細胞にあっては、培養容器の器壁に付着ししかも
単層でしか人工培養できない種類のものが多数存在する
が、このような細胞自体を測定するための有効な決定的
な方法すらなく、ましてやオンラインで測定しながら、
システマティックに培養を行うことのできる容器は全く
知られていない。In particular, among animal cells that produce physiologically active substances such as lymphokines, there are many types that adhere to the walls of culture vessels and can only be artificially cultured in a monolayer. There is no valid definitive way to measure it, let alone while measuring it online.
There are no known containers in which culture can be carried out systematically.
これら既知の培養容器を用いて濃度測定をしながら培養
するには、いずれのタイプの容器を採用しようとも、オ
ンライン計測することはできないためリアクタや培養装
置からその都度細胞をサンプリング法により採取しなけ
ればならず、培養系への雑菌汚染の危険性が大きく、雑
菌汚染のため高価な培養液を廃棄しなければならないこ
とが多く、培養効率の向上が望まれていたのである。ま
た生物濃度等の情報をリアクタや培養装置のオンライン
制御等に反映することは不可能であり、細胞をサンプリ
ングすることなく、オンラインで生物濃度を測定できる
方法の開発が重要視されてきたのである。To culture while measuring concentration using these known culture vessels, no matter which type of vessel is used, cells must be collected from the reactor or culture device each time using a sampling method, as online measurement is not possible. However, there is a high risk of bacterial contamination of the culture system, and expensive culture fluids often have to be discarded due to bacterial contamination, so there has been a desire to improve culture efficiency. Furthermore, it is impossible to reflect information such as biological concentration in the online control of reactors and culture equipment, so there has been an emphasis on the development of methods that can measure biological concentration online without sampling cells. .
上記の問題点の解決策として、本発明者により、電気容
量（誘電率）および／又は電気伝導度（導電率）を利用
して生物濃度を測定する方法が見いだされ、フロック状
になった菌体・細胞や固定化菌体・細胞を壊すことなく
測定することが一応は可能となった（特願昭６２−２２
４８１）。As a solution to the above problems, the present inventors discovered a method of measuring biological concentration using electrical capacitance (permittivity) and/or electrical conductivity (electrical conductivity). For the time being, it has become possible to measure without destroying the cells/immobilized bacterial bodies/cells.
481).
（発明が解決しようとする問題点）
しかし、従来のこの計測方法では一般に測定電極を相対
する位置に置き平行電極を形成して両電極間の電気容量
、電気伝導度を測定していた。しかしｉｌｌ’ｌ定対象
が培養器の底面付近に沈降した状態や、付着して増殖す
る場合にはａｊす定が困難であった。(Problems to be Solved by the Invention) However, in this conventional measurement method, the measurement electrodes are generally placed in opposing positions to form parallel electrodes, and the capacitance and electrical conductivity between the two electrodes are measured. However, it has been difficult to determine if the ill'l target has settled near the bottom of the incubator, or if it has adhered and proliferated.
特に、上記したように付着培養によって増殖する細胞に
はリンホカイン産生性等特に有用なものが多いが、この
ような細胞の培養にあっては、上記した電気的方法を利
用して細胞濃度を測定しながら培養することは非常に困
難であった。In particular, as mentioned above, cells grown by adherent culture have many particularly useful properties such as lymphokine production, but when culturing such cells, it is necessary to measure cell concentration using the electrical method described above. It was extremely difficult to cultivate the cells while using them.
（問題点を解決するための手段）
本発明は、上記の技術の現状に鑑みてなされたものであ
って、光学的測定システム等既知のシステムにとって代
る新しいシステムを備えた培養容器を開発する目的でな
されたものである。(Means for Solving the Problems) The present invention has been made in view of the current state of the technology described above, and aims to develop a culture vessel equipped with a new system to replace known systems such as an optical measurement system. It was done for a purpose.
そして特に本発明は、上記した電気的測定システムに着
目し、それを大巾に改良して、浮遊生物のみならず付着
ないし沈降生物にあっても、培養液を遂−サンプリング
することなく、生物濃度および生物の増殖活性を電気容
量により容易に測定するためになされたものである。In particular, the present invention focuses on the above-mentioned electrical measurement system and significantly improves it to detect not only floating organisms but also attached or sedimented organisms, without actually sampling the culture solution. This was done to easily measure concentration and growth activity of organisms by capacitance.
この目的を達成するために、上記した電気的測定システ
ムを利用した培養装置について各方面から鋭意検討した
結果、特に電極に注目すべきであるとの知見を得た。そ
して更に電極の種類、材料、設置数、設置位置、設置方
法等について広範な研究を行った。その結果、先ず、設
置方法が重要であるとの新知見を得た。In order to achieve this objective, as a result of intensive investigation from various aspects of a culture device using the above-mentioned electrical measurement system, it was found that particular attention should be paid to the electrodes. Furthermore, extensive research was conducted on the types of electrodes, materials, number of electrodes, locations, and methods of installation. As a result, we obtained new knowledge that the installation method is important.
そこで、この目的を達成するため電極の設置方法につい
て検討した。まず培養容器内に電極をとりつけたセンサ
を挿入する方法と、容器内面に電極を装着する方法とを
比較したが、電極間距離が短かくてよい場合には、電極
から３１ｇ定装置までの距離を短かくできる装着型が優
れている６また装着型は、容器壁面に電極を張り付けて
いるだけであるため、培養容器内の生物にダメージを与
える可能性は皆無である。これに対して、挿入型ではセ
ンサ部に細胞等が衝突することによって破壊される等の
ｉｉｆ能性がある。そのうえ、挿入型にあっては、挿入
されたセンサ部のために、培養容器内の空間部がせまく
なり、撹拌器を設けて撹拌するのが難しくなるし、温度
計の挿入等にも支障をきたす。したがって、電気的シス
テムを利用する場合であっても、これらの面では挿入型
は好ましくないことが判明した。Therefore, in order to achieve this objective, we investigated how to install the electrodes. First, we compared the method of inserting a sensor with an electrode attached into the culture container and the method of attaching the electrode to the inner surface of the container, but if the distance between the electrodes can be short, the distance from the electrode to the 31g The wearable type is superior because it can shorten the length of the culture vessel.6 Furthermore, since the wearable type only has electrodes attached to the wall of the container, there is no possibility of damaging the organisms inside the culture container. On the other hand, the insertion type has the possibility of being destroyed by cells etc. colliding with the sensor part. Furthermore, with the insertion type, the space inside the culture container is narrowed due to the inserted sensor part, making it difficult to provide a stirrer for stirring, and it also poses a problem when inserting a thermometer. Come. Therefore, even when using an electrical system, it has been found that the insertion type is not preferable in these respects.
そこで、装着型に着目して、電極の装着位置について検
討した。Therefore, we focused on the wearable type and considered the mounting position of the electrode.
電極を壁面に装着した場合に比較して、培養容器の底面
に装着することにより次のような利点があることが分っ
た。１）培養器と誘電測定装置とのアダプタの標準化が
可能となる。２）培養液が少量の場合にも測定できる。It was found that attaching the electrodes to the bottom of the culture container has the following advantages compared to attaching the electrodes to the wall. 1) Standardization of adapters between incubators and dielectric measuring devices becomes possible. 2) Measurement can be performed even when the culture solution is small.
３）生物が沈降した状態のままｉｌｌ！Ｉ定できる。と
くに２）、３）については、電極を壁面に装着するタイ
プのものでは劃定か非常に困難である。このようにして
、電極装着位置等の検討を加えた結果、複数の電極を底
面に装着した培養容器を用いることにより、生物濃度お
よび増殖活性を容易に測定することを可能にしたのであ
る。3) Ill ill with the living things in the settled state! I can determine. In particular, regarding 2) and 3), it is extremely difficult to determine if the electrode is attached to a wall. In this way, as a result of considering the electrode attachment position, etc., it became possible to easily measure biological concentration and proliferation activity by using a culture vessel with multiple electrodes attached to the bottom.
本発明においては、このように装着した電極を用いて、
誘電率、導電率等電気伝導度、電気容量を測定し、もっ
て菌体量を測定するのである。In the present invention, using the electrodes attached in this way,
The amount of bacterial cells is determined by measuring electrical conductivity such as dielectric constant and conductivity, and capacitance.
誘電率の測定から菌体量を算出するには、前記した本発
明者らの開発したシステムを利用するのがよい。例えば
、誘電率は、電極、リアクタ等の形状の影響を受けるた
め、予じめ菌体を含まない状態での周波数特性を求めて
おき、測定対象の周波数特性から減じることにより１周
波数変化に対する誘電率変化を求める。この際、菌体量
の変化に対して最も著しい変化を示す周波数での値を採
用してもよいし、適当な周波数帯域（数Ｈｚ〜数Ｍｌｌ
ｚ）の値から算出してもよい。In order to calculate the amount of bacterial cells from the measurement of the dielectric constant, it is preferable to use the system developed by the present inventors described above. For example, since the dielectric constant is affected by the shape of electrodes, reactors, etc., the frequency characteristics without bacterial cells are determined in advance, and the dielectric constant for one frequency change is calculated by subtracting it from the frequency characteristics of the measurement target. Find the rate change. At this time, the value at the frequency that shows the most significant change with respect to the change in the amount of bacterial cells may be adopted, or the value in an appropriate frequency band (several Hz to several milliliter) may be adopted.
It may be calculated from the value of z).
この場合子じめ、６１す定に用いる電極、リアクタにお
いて誘電率と菌体量との関係を求めておけば。In this case, the relationship between the dielectric constant and the amount of bacterial cells should be determined for the electrodes and reactor used in the test.
誘電率から容易に菌体量の算出が可能となる。The amount of bacterial cells can be easily calculated from the dielectric constant.
次に導電率は、電気の通り易さを示すものであるから、
溶液中のイオン濃度に大きく影響される。Next, conductivity indicates the ease with which electricity can pass, so
It is greatly influenced by the ion concentration in the solution.
そこで、菌体を含まない状態での導電率を求め、この値
で正規化した相対導電率を求めておく。この相対導電率
を求めることにより、測定系のイオン１度の影響を除去
して菌体量の測定をすることが可能となったのである。Therefore, the conductivity in a state that does not contain bacterial cells is determined, and the relative conductivity normalized by this value is determined. By determining this relative conductivity, it became possible to remove the influence of 1 degree ions in the measurement system and measure the amount of bacterial cells.
導電率測定においては、周波数を数ｌｌｚ〜数Ｍｌ＋７
゜まで変化させて測定すると周波数の変化に拘らずほぼ
一定の値を示す範囲が存在するので、この値を導電率と
して採用する。In conductivity measurement, the frequency is from several llz to several ml+7
When measured by changing the conductivity up to 100°, there is a range in which it shows a substantially constant value regardless of the change in frequency, so this value is adopted as the conductivity.
そして実際に菌体量を算出するには、まず１ｌｌｌ＋定
対象の導電率を求め、これから相対導電率を算出する。To actually calculate the amount of bacterial cells, first find the electrical conductivity of 1lll+a constant object, and then calculate the relative electrical conductivity.
このとき菌体を含まない場合の導電率を予じめ求めても
よく又は同時に求めてもよい。そして、これらの導電率
及び予じめ求めておいた菌体量を求めるための係数から
、実際の菌体量を算出するのである。At this time, the conductivity when no bacterial cells are included may be determined in advance or at the same time. Then, the actual amount of bacterial cells is calculated from these conductivities and a predetermined coefficient for determining the amount of bacterial cells.
このようにして、培養容器、リアクタ等を破壊すること
なく、オンラインで菌体量の測定をしながら培養ができ
るのである。In this way, it is possible to perform cultivation while measuring the amount of bacterial cells online without destroying the culture container, reactor, etc.
本発明の培養容器は、電気伝導度、電気容量を測定する
ための複数の電極を容器底部に装着してなるものであっ
て、例えば第１図に図示したような各種容器が例示され
る。The culture container of the present invention has a plurality of electrodes attached to the bottom of the container for measuring electrical conductivity and capacitance, and various containers such as those shown in FIG. 1 are exemplified.
第１図は１本発明に係る培養容器の実施例を図示したも
のであって、図中１は′ｒ！！極を表わし、２は培養容
器本体ないしリアクタを表オ〕す。いずれの実施例にお
いても、電極１は容器２の底部に複数個設置されている
（ただしくＣ）においては電極数は３個であり、他は２
個設置した実施例を図示した）。FIG. 1 illustrates an embodiment of a culture vessel according to the present invention, and 1 in the figure is 'r! ! 2 represents the main body of the culture vessel or the reactor. In any of the examples, a plurality of electrodes 1 are installed at the bottom of the container 2 (in C), the number of electrodes is 3, and in the other examples, 2 electrodes are installed.
)
第１図において、（ａ）は角型培養フラスコ、（ｂ）は
振どう培養等も可能な三角培養フラスコ、（Ｃ）はメリ
クロンローラ回転培養等も可能な試験管培養フラスコ、
（ｄ）はビン型培養フラスコ、（ｅ）は、細胞培養フラ
スコにそれぞれ電極を底面装着したものである。容器の
材料としては、ガラス、プラスチック、各種複合材等業
界既知の材料が適宜使用される。（ｆ）は、生物量の計
ｉ！＋１１を行う場合を図示したものであって、リアク
タ（培養容器：ここでは角型培養フラスコを図示）２に
は、その内部に培養液３を収容するとともに、その底部
には電極１を設置しておく。電極の設置数は１対又はそ
れ以上とする。図中、４は培養液３の液面を表わし、５
は生物（ここでは細胞）を表わす。In Figure 1, (a) is a rectangular culture flask, (b) is a triangular culture flask that can also be used for shaking culture, etc., (C) is a test tube culture flask that is also capable of mericron roller rotation culture, etc.
(d) is a bottle-shaped culture flask, and (e) is a cell culture flask with electrodes attached to the bottom. As the material for the container, materials known in the industry such as glass, plastic, various composite materials, etc. are used as appropriate. (f) is the total biomass i! This diagram shows the case of carrying out +11, in which a reactor (culture container: here a square culture flask is shown) contains a culture solution 3, and an electrode 1 is installed at the bottom of the reactor 2. I'll keep it. The number of electrodes installed shall be one or more pairs. In the figure, 4 represents the liquid level of culture solution 3, and 5
represents an organism (here, a cell).
測定装置６としては、測定周波数が固定の装置でも使用
可能であるが、複数の周波数で電気容量の測定ができる
タイプのものを使用するのが好ましい。測定結果は、ヒ
トが読み取りマニュアルによって生物量を算出してもよ
いし、インターフェースを介してコンピュータ（図示せ
ず）にデータを転送し、自動的に生物量を算出してもよ
い。Although a device with a fixed measurement frequency can be used as the measuring device 6, it is preferable to use a device that can measure capacitance at a plurality of frequencies. The measurement results may be read manually by a human to calculate the biomass, or the data may be transferred to a computer (not shown) via an interface to automatically calculate the biomass.
なお培養中の生物濃度を連続的に測定することが可能で
あるが、従来と同様、静置培養、振とう培養、メリクロ
ンローラ回転培養器等の方法で培養し、生物濃度、増殖
活性等をチエツクする必要のあるとき計測装置上にセッ
トして測定すれば、多数の試料について測定できる。第
１図（ｆ）においては、計測装置６として、ＬＣＲメー
タ等誘等率電率測定装置示した。Although it is possible to continuously measure the concentration of organisms during culture, it is possible to continuously measure the concentration of organisms, growth activity, etc. by culturing using methods such as static culture, shaking culture, and mericron roller rotary incubators. If you set it on a measuring device and measure it when you need to check it, you can measure a large number of samples. In FIG. 1(f), a dielectric constant electric constant measuring device such as an LCR meter is shown as the measuring device 6.
つぎに上記の培養容器を用いた実施例について述べるが
、これらは単なる例示であって、なんら本発明を制限す
るものではない。Next, examples using the above-mentioned culture vessels will be described, but these are merely illustrative and do not limit the present invention in any way.
実施例１
植物細胞の増殖培養に通常用いられる第１表の組成の基
本培地（植物細胞培養マニュアル；講談社）に、ナフタ
レン酸５ＸｌＯ−’Ｍ、ベンジルアデニンｌ Ｘ １０
−’Ｍを添加シタ培地１００ｍＱを、第１図（ｂ）ニ示
す電極を装着した５００Ｉ１１１２三角フラスコに分注
し１２０℃で１５分間殺菌した。これにあらかじめ培養
して得た、ごま（Ｓｅｓａｍｕｍ ｉｎｄｉｃｕｍ Ｌ
）の増殖細胞を１０１１１１移植して、２８℃、１２，
０００ルツクス、７５回転／毎分の撹拌装置の条件で２
週間培養した。なお同じ試料を複数個作製し、電気容量
を測定するとともに、湿重量を同時に求めた。Example 1 Naphthalic acid 5XlO-'M and benzyladenine l
100 mQ of Sita medium supplemented with -'M was dispensed into a 500I1112 Erlenmeyer flask equipped with the electrode shown in FIG. 1(b), and sterilized at 120° C. for 15 minutes. Sesame (Sesamum indicum L) obtained by culturing this in advance
) were transplanted into 101111 cells and incubated at 28°C for 12 days.
000 lux, 75 revolutions/minute under the conditions of a stirring device.
Cultured for a week. Note that a plurality of the same samples were prepared, and the capacitance was measured and the wet weight was determined at the same time.
第２図に培養中の電気容量値（８１１１定周波数１００
に１１Ｚ）および湿重量の変化をしめした。両者間に非
常に良い一致をみることができ、底面に電極を装着した
培養容器をもちいて細胞濃度、増殖活性を容易に測定で
きた。Figure 2 shows the capacitance value during culture (8111 constant frequency 100
11Z) and changes in wet weight are shown. Very good agreement was observed between the two, and cell concentration and proliferation activity could be easily measured using a culture vessel with electrodes attached to the bottom.
硝酸カルシウム
塩化カリウム
硫酸マグネシウム
リン酸第１カリウム
ホウ酸
硫酸マンガン
硫酸亜鉛
ヨーソカリウム
モリブデン酸ナトリウム
塩化コバルト
硫酸銅
エチレンジアミン４酢酸ナトリウム
硫酸第１鉄
ミオイノシトール
グリシン
塩酸ピリドキシン
ニコチン酸
塩酸チアミン
しよ糖
水
ｐＨ５，７
１、９００
／１４０
０．５
０．５
０．１
０ｇ
１、 ＯＯＯ＋ｓＱ
実施例２
動物細胞の培養に通常用いられている方法（細胞培養マ
ニュアル：ｍ談社）により、ヒト由来の骨髄性白血病絹
胞株に−５６２の細胞を培養した。すなわち通常用いら
れているＲＰＭＩ−１６４０培地（大日本製薬１）に１
０％の牛胎児血清を加えた培養液１０１を、第１図（ａ
）に示す一対の電極を装着した直径１０ｃ＋ｏの細胞培
養用プラスチックデイツシュに分注した。これに前記の
ヒト細胞を２ＸＩＯ’個／ＩＩＩＱになるようにして、
移植し、５％炭酸ガスインキュベータ中で３７℃にて４
日間静置培養した。なお実施例１と同様複数の試料を作
製し、電気容量を測定するとともに、測定後、ビリケル
チュールク血球計数板により各試料中のヒト細胞の数を
顕微鏡により測定した。なお電気容量から細胞濃度の算
出はあらかじめ求めておいたｉｌｌ’Ｊ定周波数３００
ＫＩＩｚにおける電気容量値と細胞濃度との関係から算
出した。1 , 900 /140 0.5 0.5 0.1 0g 1, OOO+sQ Example 2 Myeloid leukemia silk cells of human origin were cultured using a method commonly used for culturing animal cells (Cell Culture Manual: Mdansha). Cells of strain -562 were cultured. That is, 1 in the commonly used RPMI-1640 medium (Dainippon Pharmaceutical 1).
The culture solution 101 to which 0% fetal bovine serum was added was added to the culture solution 101 in Figure 1 (a).
) was dispensed into a plastic dish for cell culture with a diameter of 10c+o equipped with a pair of electrodes. To this, the human cells described above were added to 2XIO' cells/IIIQ,
Transplant and incubate at 37°C in a 5% carbon dioxide incubator for 4 days.
It was statically cultured for 1 day. A plurality of samples were prepared in the same manner as in Example 1, and the capacitance was measured. After the measurement, the number of human cells in each sample was measured using a microscope using a Billikerturk hemocytometer. Note that the cell concentration is calculated from the capacitance using the ill'J constant frequency 300 determined in advance.
It was calculated from the relationship between the capacitance value and cell concentration in KIIz.
第３図のように両者間に非常に良い一致をみることがで
き、底面に電極を装着した培養容器をもちいて細胞濃度
、増殖活性を容易に測定できた。As shown in Figure 3, there was very good agreement between the two, and cell concentration and proliferation activity could be easily measured using a culture vessel with electrodes attached to the bottom.
実施例３
第２表に示した組成の培地１０ｎ＋Ｕを試験管にとり、
常法により蒸気滅菌して培地を調製した。これに酵母（
Ｓａｃｃｈａｒｏｍｙｃｅｓ ｃｅｒｅｖｉｓｉａｅに
７）を移植した後、２８℃で約２４ｈｒ静置培養した。Example 3 10n+U of a medium having the composition shown in Table 2 was placed in a test tube,
A culture medium was prepared by steam sterilization using a conventional method. Add yeast (
After transplanting 7) to Saccharomyces cerevisiae, it was statically cultured at 28°C for about 24 hours.
次に第１図（ｂ）に示す電極を装着した５００ＩＩＩＮ
三角フラスコに別に調製した培地１５０ｍＱを分注し、
１２０℃で１５分間殺菌した培地に移植し約５０ｈｒ
振どう培養した。なお同じ試料を複数個作製し、電気容
量を測定するとともに、乾燥重量により菌体量を求めた
。Next, 500IIIN was equipped with the electrode shown in Figure 1(b).
Dispense 150 mQ of a separately prepared medium into an Erlenmeyer flask,
Transplant into a medium sterilized at 120℃ for 15 minutes and grow for about 50 hours.
Cultured in shaking. Note that a plurality of the same samples were prepared, and the capacitance was measured, and the amount of bacterial cells was determined from the dry weight.
第４図は菌体量（乾燥型：！ｉ）と測定周波数３００Ｋ
Ｈｚにおける電気容量値との関係であるが１両者間に非
常に高い直線関係（相関係数０．９９）のあることがわ
かった。第５図に培養中の電気容量（測定周波数３００
ＫＩＩｚ）の変化を示す。図のように誘導期、対数増殖
期をへて定常期にいたる増殖特性かえられ、底面に電極
を装着した培養容器をもちいて菌体濃度、増殖活性を容
易に測定できた。Figure 4 shows the amount of bacterial cells (dry type: !i) and the measurement frequency of 300K.
Regarding the relationship with the capacitance value in Hz, it was found that there was a very high linear relationship (correlation coefficient 0.99) between the two. Figure 5 shows the capacitance during culture (measurement frequency: 300
KIIz). As shown in the figure, the growth characteristics changed through the lag phase, the logarithmic growth phase, and then the stationary phase, and the cell concentration and growth activity could be easily measured using a culture container with an electrode attached to the bottom.
第２表
グルコース
酵母エキス
リン酸二水素カリウム
硫酸アンモニウム
硫酸マグネシウム
水
００ｇ
０．５
１００軸Ｑ
（発明の効果）
本発明は、従来サンプリング操作が必要であった生物濃
度や増殖活性を、電気容量（誘電率）を測定するという
全く新規な方法を採用することによって可能とするため
の培養容器において、培養液が少量の場合でも、また生
物が沈降した状態でもオンラインで測定可能とする従来
なしえなかった新規にして卓越した効果を有するもので
ある。Table 2 Glucose Yeast Extract Potassium Dihydrogen Phosphate Ammonium Magnesium Sulfate Water 00g 0.5 100 Axis Q (Effects of the Invention) The present invention is capable of measuring biological concentration and growth activity, which conventionally required sampling operations, by measuring electrical capacitance (permittivity). ) by adopting a completely new method of measuring the amount of microorganisms in the culture vessel.A new technology that was previously impossible to measure online, even when there is a small amount of culture solution or when organisms have settled. It has an outstanding effect.
したがって本発明によれば、動物細胞および植物細胞量
を非破壊的に測定することができ、バイオテクノロジー
、ワクチン製造、動物細胞および植物細胞を用いる実験
、研究の技術分野、その地番方面において広く本発明を
利用することができる。Therefore, according to the present invention, it is possible to non-destructively measure the amount of animal cells and plant cells, and it is widely used in the technical fields of biotechnology, vaccine production, experiments and research using animal and plant cells, and their lot numbers. The invention can be used.
第１図は、本発明に係る培養容器（ａ＝ｅ）、及び生物
量計測システム（ｆ）を図示したものである。
第２図は、ゴマ細胞の培養日数と、電気容量、湿重量と
の関係を図示したものであり、図中。
・−は電気容量、−〇−は湿重量をそれぞれ表わす。
第３図は、ヒト由来の骨髄性白血病細胞の培養１］数と
細胞数との関係を図示したものであり、図中、−・−は
ヘマサイトメータによる測定値、及び○−は電気容量に
よる測定値をそれぞれ表わす。
第４図は、酵母（サツカロミセス・セレビシェ）の菌体
濃度と電気容量との関係を図示したグラフであり、第５
図は、同酵母培養中の電気容量の変化を図示したグラフ
である。
第 １
［７１ｆｄｌ
ｆ１
了（〔）
代理人 弁理士 戸 １）親 男
第
図
焙表日数／
日
第
図
歯体濃度／ ｍｇｍｔ’FIG. 1 illustrates a culture container (a=e) and a biomass measuring system (f) according to the present invention. FIG. 2 illustrates the relationship between the number of days of culturing sesame cells, electric capacity, and wet weight.・− represents electric capacity, and −〇− represents wet weight, respectively. Figure 3 illustrates the relationship between the culture 1] number of human-derived myeloid leukemia cells and the number of cells. The measured values are shown respectively. FIG. 4 is a graph illustrating the relationship between bacterial cell concentration and electric capacity of yeast (Saccharomyces cerevisiae).
The figure is a graph illustrating changes in electric capacity during the cultivation of the same yeast. No. 1 [71fdl f1 了 ([) Agent Patent Attorney Door 1) Parent Male Diagram Number of Days / Day Diagram Tooth Body Concentration / mgmt'
Claims (1)
ガラスまたはプラスチック製生物培養容器。A biological culture container made of glass or plastic, characterized in that a plurality of electrodes are attached to the bottom of the container.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26713888A JPH02114163A (en) | 1988-10-25 | 1988-10-25 | Organism culture container |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP26713888A JPH02114163A (en) | 1988-10-25 | 1988-10-25 | Organism culture container |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02114163A true JPH02114163A (en) | 1990-04-26 |
| JPH0569463B2 JPH0569463B2 (en) | 1993-10-01 |
Family
ID=17440610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP26713888A Granted JPH02114163A (en) | 1988-10-25 | 1988-10-25 | Organism culture container |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH02114163A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07184686A (en) * | 1993-12-28 | 1995-07-25 | Nec Corp | Method for measuring cell activity |
| US6521190B1 (en) * | 2000-05-19 | 2003-02-18 | Digene Corporation | Cell collection apparatus |
| JP2006204123A (en) * | 2005-01-25 | 2006-08-10 | Seiko Instruments Inc | Culture vessel and culture unit |
| JP2016015891A (en) * | 2014-07-04 | 2016-02-01 | 大日本印刷株式会社 | Cell culture vessel |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56125652A (en) * | 1980-03-08 | 1981-10-02 | Nippon Tectron Co Ltd | Microorganism measuring method by impedance method |
-
1988
- 1988-10-25 JP JP26713888A patent/JPH02114163A/en active Granted
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS56125652A (en) * | 1980-03-08 | 1981-10-02 | Nippon Tectron Co Ltd | Microorganism measuring method by impedance method |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07184686A (en) * | 1993-12-28 | 1995-07-25 | Nec Corp | Method for measuring cell activity |
| US6521190B1 (en) * | 2000-05-19 | 2003-02-18 | Digene Corporation | Cell collection apparatus |
| JP2006204123A (en) * | 2005-01-25 | 2006-08-10 | Seiko Instruments Inc | Culture vessel and culture unit |
| JP2016015891A (en) * | 2014-07-04 | 2016-02-01 | 大日本印刷株式会社 | Cell culture vessel |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0569463B2 (en) | 1993-10-01 |
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