JP2709479B2 - Callus immobilization method and biochemical reactor used for the same - Google Patents
Callus immobilization method and biochemical reactor used for the sameInfo
- Publication number
- JP2709479B2 JP2709479B2 JP63210439A JP21043988A JP2709479B2 JP 2709479 B2 JP2709479 B2 JP 2709479B2 JP 63210439 A JP63210439 A JP 63210439A JP 21043988 A JP21043988 A JP 21043988A JP 2709479 B2 JP2709479 B2 JP 2709479B2
- Authority
- JP
- Japan
- Prior art keywords
- culture solution
- cells
- callus
- air
- hollow fiber
- 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 - Lifetime
Links
- 206010020649 Hyperkeratosis Diseases 0.000 title claims description 14
- 238000000034 method Methods 0.000 title claims description 7
- 239000012510 hollow fiber Substances 0.000 claims description 35
- 238000006243 chemical reaction Methods 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 238000005842 biochemical reaction Methods 0.000 claims description 2
- 238000007599 discharging Methods 0.000 claims 2
- 241000196324 Embryophyta Species 0.000 description 8
- 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 4
- 239000007789 gas Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 239000012071 phase Substances 0.000 description 3
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 230000003100 immobilizing effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 229960000344 thiamine hydrochloride Drugs 0.000 description 2
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 2
- 239000011747 thiamine hydrochloride Substances 0.000 description 2
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 2
- OVSKIKFHRZPJSS-UHFFFAOYSA-N 2,4-D Chemical compound OC(=O)COC1=CC=C(Cl)C=C1Cl OVSKIKFHRZPJSS-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 244000000626 Daucus carota Species 0.000 description 1
- 235000002767 Daucus carota Nutrition 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- FAIXYKHYOGVFKA-UHFFFAOYSA-N Kinetin Natural products N=1C=NC=2N=CNC=2C=1N(C)C1=CC=CO1 FAIXYKHYOGVFKA-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000007792 gaseous phase Substances 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- QANMHLXAZMSUEX-UHFFFAOYSA-N kinetin Chemical compound N=1C=NC=2N=CNC=2C=1NCC1=CC=CO1 QANMHLXAZMSUEX-UHFFFAOYSA-N 0.000 description 1
- 229960001669 kinetin Drugs 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 229920002492 poly(sulfone) Polymers 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 229960004793 sucrose Drugs 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は中空糸表面への植物細胞の効率的な固定化を
促すカルスの固定化方法及びこれに用いる生物化学反応
装置に関する。Description: TECHNICAL FIELD The present invention relates to a method for immobilizing callus for promoting efficient immobilization of plant cells on the surface of a hollow fiber, and a biochemical reaction apparatus used for the method.
従来、中空糸を用いた培養法は、中空糸の外側に培養
液と遊離細胞を供給し、液体状態で増殖されるものであ
った。中空糸外側に空気を供給して気相でカルスを増殖
させるためには、細胞を中空糸表面に付着固定させる必
要がある。中空糸表面への植物細胞の固定化は固定化担
体を用いて行われている。Conventionally, in a culture method using a hollow fiber, a culture solution and free cells are supplied to the outside of the hollow fiber, and the cells are grown in a liquid state. In order to grow callus in the gas phase by supplying air to the outside of the hollow fiber, it is necessary to adhere and fix cells to the surface of the hollow fiber. Immobilization of plant cells on the surface of a hollow fiber is performed using an immobilization carrier.
固定化担体を使用する方法では充分な細胞が固定化さ
れず無駄になる細胞があった。また、固定化担体を中空
糸表面に固定させる操作が煩雑である上、固定化担体が
中空糸の目づまりの原因となり、培養液の連続供給がで
きなくなることもあった。In the method using the immobilizing carrier, sufficient cells were not immobilized and some cells were wasted. In addition, the operation of fixing the immobilized carrier on the surface of the hollow fiber is complicated, and the immobilized carrier may cause clogging of the hollow fiber, which may prevent continuous supply of the culture solution.
〔課題解決の手段〕 本発明は固定化担体を使用することなく中空糸表面に
細胞を付着させ、カルスを増殖させる方法を提供するこ
とを目的とし、その構成は、培養液を供給する多数の中
空糸を配設した反応管に、植物細胞と培養液を供給し、
空気を供給しながら一定期間培養した後、細胞を残して
培養液のみを排出して水位を低下させ、再び空気を供給
する操作を繰返すことを特徴とし、更に、多数の中空糸
を配設し、下部に空気供給口を開口し上部に空気排出口
を設けた反応管下部に、植物細胞を通過させないフィル
ターを設けた培養液排出装置を設けると共に、中空糸に
培養液を供給する培養液供給装置を設けたことを特徴と
する。[Means for Solving the Problems] An object of the present invention is to provide a method for adhering cells to the surface of a hollow fiber without using an immobilized carrier, and for growing callus. The plant cells and the culture solution are supplied to the reaction tube provided with the hollow fibers,
After culturing for a certain period of time while supplying air, it is characterized by repeating the operation of lowering the water level by draining only the culture solution while leaving the cells, and supplying air again, and further arranging a number of hollow fibers. In addition, a culture solution discharge device provided with a filter that does not allow plant cells to pass through is provided at the lower part of the reaction tube having an air supply port opened at the bottom and an air discharge port provided at the top, and a culture solution supply that supplies the culture solution to the hollow fibers. A device is provided.
本発明は、中空糸の間隙を充填した培養液と植物細胞
との間にフィルターでロ過した空気を供給することによ
り、細胞を撹拌し気相の空気を供給すると共に、中空糸
表面への細胞の付着を促進させる。ある程度付着した
後、フィルター付の培養液排出装置から培養液のみを抜
出して水位を低下させることにより、気液の界面に浮遊
している細胞を中空糸の表面に積極的に付着させる。し
かる後、エアーフィルターから空気の供給を続けて培養
する。この操作を数回繰返すことにより段をなしてカル
ス細胞が成長し、担体を用いなくとも効率的にカルス細
胞を中空糸に固定することができる。The present invention supplies air that has been filtered through a filter between a culture solution that fills the gaps between hollow fibers and plant cells, thereby stirring the cells and supplying gaseous phase air, Promotes cell attachment. After the cells adhere to some extent, only the culture solution is extracted from the culture solution discharge device with a filter to lower the water level, whereby the cells floating at the gas-liquid interface are positively attached to the surface of the hollow fiber. Thereafter, the culture is continued by supplying air from the air filter. By repeating this operation several times, the callus cells grow in steps, and the callus cells can be efficiently fixed to the hollow fibers without using a carrier.
また、カルス細胞の乾燥を防ぎ、かつ栄養供給のため
に培養液を中空糸の一方から供給する。水位調整のため
に排出した培養液は培養液槽に貯え、中空糸の一方から
中空糸に供給し、中空糸の他方から排出した栄養液も同
じく培養液槽に貯えて循環使用する。In addition, a culture solution is supplied from one of the hollow fibers for preventing callus cells from drying out and for supplying nutrients. The culture solution discharged for adjusting the water level is stored in a culture solution tank, one of the hollow fibers is supplied to the hollow fiber, and the nutrient solution discharged from the other of the hollow fibers is also stored in the culture solution tank and used for circulation.
第１図は本発明の１実施例を示す説明図である。１は
反応管であり、内部に多数の中空糸２を反応管１の長さ
方向に配設してある。中空糸は多孔質であれば特に限定
はないが、本実施例においてはバインダーとしてポリス
ルホンを用いこれに対し40重量％のセライトを配合した
中空糸を用いた。この中空糸は細胞との親和性が大き
く、特に優れた素材である。FIG. 1 is an explanatory view showing one embodiment of the present invention. Reference numeral 1 denotes a reaction tube in which a large number of hollow fibers 2 are arranged in the longitudinal direction of the reaction tube 1. The hollow fiber is not particularly limited as long as it is porous. In this example, a hollow fiber containing polysulfone as a binder and 40% by weight of celite was used. This hollow fiber has a high affinity for cells and is a particularly excellent material.
３は先端にエアーフィルター４を設けた空気供給管で
あり、ポンプ5aにより空気を供給される。エアーフィル
ター４は細菌は勿論、空気中の塵埃も通過させない連続
気泡を有する素材がよく、好ましくは連続気泡を有する
セラミックスが使用される。６は植物細胞を懸濁させた
培養液を入れる細胞槽であり、ポンプ5b、バルブ7aを介
して反応槽１内ににんじんカルス細胞の懸濁液300ml
（細胞濃度１×104cells/ml）を供給した。次いでポン
プ5aを駆動して反応管１内にフィルターを介して空気を
供給し、細胞を撹拌して中空糸２への吸着を促進させ
た。８は供給した空気の排出口であり、反応管の上部に
設けた。この状態で３〜４日培養すると撹拌されている
細胞の一部が中空糸表面に吸着し増殖し始めた。Reference numeral 3 denotes an air supply pipe provided with an air filter 4 at its tip, and is supplied with air by a pump 5a. The air filter 4 is preferably made of a material having continuous cells that do not allow passage of dust in the air as well as bacteria, and ceramics having continuous cells are preferably used. Reference numeral 6 denotes a cell tank for holding a culture solution in which plant cells are suspended, and 300 ml of a suspension of carrot callus cells is introduced into the reaction tank 1 via a pump 5b and a valve 7a.
(Cell concentration 1 × 10 4 cells / ml). Next, the pump 5a was driven to supply air into the reaction tube 1 through a filter, and the cells were stirred to promote adsorption to the hollow fiber 2. Reference numeral 8 denotes a discharge port of the supplied air, which is provided above the reaction tube. When the cells were cultured in this state for 3 to 4 days, a part of the stirred cells began to adsorb to the surface of the hollow fiber and proliferate.
９は培養液排出装置であり、反応管下部に設けた水位
調整口10、細胞フィルター11、バルブ7b及びポンプ5cが
順次連結し、細胞を残して培養液のみを排出して培養液
槽12に貯える。この操作を行うと水位が低下し、この
際、液体と気体の界面で浮遊している細胞が優先的に中
空糸２に吸着される。１回の水位を下げる操作でカルス
細胞は数mm成長した。この操作を数回繰返して効率的な
カルスの育成を行った。Reference numeral 9 denotes a culture medium discharge device, in which a water level adjusting port 10 provided at the lower part of the reaction tube, a cell filter 11, a valve 7b, and a pump 5c are sequentially connected, and only the culture medium is discharged while leaving cells, and the culture medium tank 12 is discharged. store. When this operation is performed, the water level decreases. At this time, cells floating at the interface between the liquid and the gas are preferentially adsorbed to the hollow fiber 2. Callus cells grew several mm by one operation of lowering the water level. This operation was repeated several times to efficiently grow calli.
13は中空糸に培養液を供給する培養液供給口であり、
反応管の最下端に設け、培養液槽12からポンプ5d、バル
ブ7cを介して中空糸に培養液を供給した。14は培養液排
出口であり、培養液槽12に連結し、培養液を循環して使
用した。中空糸１から浸出する培養液によりカルスに栄
養と適度の水分が供給された。13 is a culture solution supply port for supplying a culture solution to the hollow fiber,
The culture solution was provided at the lowermost end of the reaction tube, and the culture solution was supplied from the culture solution tank 12 to the hollow fiber via a pump 5d and a valve 7c. Reference numeral 14 denotes a culture solution outlet, which was connected to the culture solution tank 12 and used by circulating the culture solution. The culture solution leached from the hollow fiber 1 supplied nutrients and moderate water to the callus.
培養液としては特に限定はないが、Murashige ＆ Sko
og培地が一般に使用され、更に植物ホルモン、シューク
ロース、塩酸チアミンなどのカルス培養に一般に使用さ
れる成分を添加することが好ましい。本実施例において
は、上記Murashige ＆ Skoog培地に、 ミオイノシトール 100mg/l 塩酸チアミン 0.1mg/l グリシン 2mg/l シュークロース 30g/l 植物ホルモンとして、 2,4D（2,4ジクロロフェノキシ酢酸） 1mg/l カイネチン 1mg/l を添加した培養液を使用し、最初に供給する細胞懸濁液
にも同一培養液を用いた。Although there is no particular limitation on the culture solution, Murashige & Sko
An og medium is generally used, and it is preferable to add components commonly used for callus culture, such as plant hormones, sucrose, and thiamine hydrochloride. In the present Example, the above Murashige & Skoog medium was added to myo-inositol 100 mg / l thiamine hydrochloride 0.1 mg / l glycine 2 mg / l sucrose 30 g / l 2,4D (2,4 dichlorophenoxyacetic acid) 1 mg / l A culture solution supplemented with 1 mg / l of kinetin was used, and the same culture solution was used for the cell suspension to be supplied first.
カルス吸着後は反応管内の培養液を水位調整口10から
全部排出し、気相中でカルス培養を行なった。したがっ
て空気供給管３からの空気供給は不要であった。After the callus adsorption, all of the culture solution in the reaction tube was discharged from the water level adjusting port 10, and callus culture was performed in the gas phase. Therefore, air supply from the air supply pipe 3 was unnecessary.
本発明によるグルコースの消費速度を測定し、従来法
（中空糸を配設した本実施例と同一の反応管において細
胞を液相で培養する方法）におけるグルコース消費と比
較し第２図に示した。グルコース測定は酵素を用いた比
色定量により市販の測定機を用いて測定した。The glucose consumption rate according to the present invention was measured and compared with the glucose consumption in a conventional method (a method of culturing cells in a liquid phase in the same reaction tube as that of the present example in which hollow fibers were provided), as shown in FIG. . Glucose was measured by colorimetry using an enzyme with a commercially available measuring instrument.
本発明により担体を使用することなく植物細胞を中空
糸に効率的に付着させ、カル付着後は気相で高密度に植
物細胞を連続的に培養することができる。According to the present invention, plant cells can be efficiently adhered to hollow fibers without using a carrier, and plant cells can be continuously cultured at a high density in the gas phase after adhering cull.
第１図は本発明の実施例を示す説明図であり、第２図は
グルコースの消費速度を示すグラフである。 図面中、符号 １は反応管、２は中空糸、３は空気供給管、４はエアー
フィルター、５はポンプ、６は細胞槽、７はバルブ、８
は空気排出口、９は培養液排出装置、10は水位調整口、
11は細胞フィルター、12は培養液槽、13は培養液供給
口、14は培養液排出口である。FIG. 1 is an explanatory view showing an embodiment of the present invention, and FIG. 2 is a graph showing a consumption rate of glucose. In the drawings, reference numeral 1 denotes a reaction tube, 2 denotes a hollow fiber, 3 denotes an air supply tube, 4 denotes an air filter, 5 denotes a pump, 6 denotes a cell tank, 7 denotes a valve, 8
Is an air outlet, 9 is a culture solution outlet, 10 is a water level adjusting port,
11 is a cell filter, 12 is a culture solution tank, 13 is a culture solution supply port, and 14 is a culture solution discharge port.
Claims (2)
反応管に、植物細胞と培養液を供給し、空気を供給しな
がら一定期間培養した後、細胞を残して培養液のみを排
出して水位を低下させ、再び空気を供給する操作を繰返
すことを特徴とするカルスの固定化方法。Claims: 1. A plant cell and a culture solution are supplied to a reaction tube provided with a large number of hollow fibers for supplying the culture solution, and the culture is carried out for a certain period while supplying air. A method for fixing callus, comprising repeating the operation of discharging water to lower the water level and supplying air again.
を開口し上部に空気排出口を設けた反応管下部に、植物
細胞を通過させないフィルターを設けた培養液排出装置
を設けると共に、中空糸に培養液を供給する培養液供給
装置を設けたことを特徴とする生物化学反応装置。2. A culture solution discharging device provided with a number of hollow fibers, and provided with a filter that does not allow plant cells to pass therethrough at the lower portion of a reaction tube having an air supply opening at the lower portion and an air outlet at the upper portion. A biochemical reaction device further comprising a culture solution supply device for supplying a culture solution to the hollow fibers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63210439A JP2709479B2 (en) | 1988-08-26 | 1988-08-26 | Callus immobilization method and biochemical reactor used for the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63210439A JP2709479B2 (en) | 1988-08-26 | 1988-08-26 | Callus immobilization method and biochemical reactor used for the same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0260586A JPH0260586A (en) | 1990-03-01 |
| JP2709479B2 true JP2709479B2 (en) | 1998-02-04 |
Family
ID=16589349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63210439A Expired - Lifetime JP2709479B2 (en) | 1988-08-26 | 1988-08-26 | Callus immobilization method and biochemical reactor used for the same |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2709479B2 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4490624B2 (en) * | 2002-11-18 | 2010-06-30 | 日本碍子株式会社 | Air / water purifier with water drain |
| JP2011103882A (en) * | 2009-11-16 | 2011-06-02 | Shuhei Nakaji | Cell culture apparatus, and method and program for culturing cell |
-
1988
- 1988-08-26 JP JP63210439A patent/JP2709479B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0260586A (en) | 1990-03-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Etienne et al. | Improvement of somatic embryogenesis in Hevea brasiliensis (Müll. Arg.) using the temporary immersion technique | |
| JPH0547192B2 (en) | ||
| CZ298148B6 (en) | Disposable device for axenically culturing and harvesting cells and/or tissue, battery of such devices and corresponding method | |
| AU664596B2 (en) | Method and apparatus for growing biomass particles | |
| Styer | Bioreactor technology for plant propagation | |
| JPH03504925A (en) | Device to oxygenate the culture medium | |
| JPH0363348B2 (en) | ||
| JPH03992B2 (en) | ||
| JP2709479B2 (en) | Callus immobilization method and biochemical reactor used for the same | |
| Wöhler et al. | Large scale culturing of normal diploid cells on glass beads using a novel type of culture vessel | |
| JPH06165624A (en) | Cultivation of plant body and bioreactor | |
| Moorhouse et al. | A plant cell bioreactor with medium-perfusion for control of somatic embryogenesis in liquid cell suspensions | |
| Teng et al. | Regenerating lettuce from suspension culture in a 2-liter bioreactor | |
| CN204939495U (en) | A kind of high-throughput plant solid-liquid culture apparatus | |
| JPS61108371A (en) | Container for culturing cell on minute carrier or in capsule | |
| Woo et al. | Multiple shoot culture of Dianthus caryophyllus using mist culture system | |
| Gupta et al. | Conifer somatic embryo production from liquid culture | |
| CN223852609U (en) | Suction device for culture solution for cell culture | |
| CN216808827U (en) | Microbial reactor | |
| SU1242518A1 (en) | Device for growing cells or hybridoms | |
| JPH06319520A (en) | Photosynthetic bioreactor | |
| JPH0622753A (en) | Cell culture method and apparatus thereof | |
| JPH02245176A (en) | Oxygen feeding type bioreactor | |
| Wilson et al. | Plant cell bioreactors | |
| JPH06233639A (en) | Liquid culture process for scale of lily or the like and culture device therefor |