JPS6328588B2 - - Google Patents
Info
- Publication number
- JPS6328588B2 JPS6328588B2 JP1484382A JP1484382A JPS6328588B2 JP S6328588 B2 JPS6328588 B2 JP S6328588B2 JP 1484382 A JP1484382 A JP 1484382A JP 1484382 A JP1484382 A JP 1484382A JP S6328588 B2 JPS6328588 B2 JP S6328588B2
- Authority
- JP
- Japan
- Prior art keywords
- culture
- culture tank
- cylindrical culture
- cylindrical
- tank
- 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
Links
- 238000007789 sealing Methods 0.000 claims description 26
- 238000007664 blowing Methods 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 210000004027 cell Anatomy 0.000 description 37
- 239000007789 gas Substances 0.000 description 31
- 241000196324 Embryophyta Species 0.000 description 20
- 238000000034 method Methods 0.000 description 20
- 238000012258 culturing Methods 0.000 description 15
- 229910052760 oxygen Inorganic materials 0.000 description 11
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 10
- 239000001301 oxygen Substances 0.000 description 10
- 238000003756 stirring Methods 0.000 description 9
- 239000002609 medium Substances 0.000 description 8
- 238000010586 diagram Methods 0.000 description 4
- 229930000044 secondary metabolite Natural products 0.000 description 4
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000007599 discharging Methods 0.000 description 3
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 238000011437 continuous method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 239000000049 pigment Substances 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- NEZONWMXZKDMKF-JTQLQIEISA-N Alkannin Chemical compound C1=CC(O)=C2C(=O)C([C@@H](O)CC=C(C)C)=CC(=O)C2=C1O NEZONWMXZKDMKF-JTQLQIEISA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- MYMOFIZGZYHOMD-UHFFFAOYSA-N Dioxygen Chemical compound O=O MYMOFIZGZYHOMD-UHFFFAOYSA-N 0.000 description 1
- -1 H3BO4 Chemical compound 0.000 description 1
- 241001071917 Lithospermum Species 0.000 description 1
- 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 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000005273 aeration Methods 0.000 description 1
- UNNKKUDWEASWDN-UHFFFAOYSA-N alkannin Natural products CC(=CCC(O)c1cc(O)c2C(=O)C=CC(=O)c2c1O)C UNNKKUDWEASWDN-UHFFFAOYSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000498 cooling water Substances 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 229910000366 copper(II) sulfate Inorganic materials 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 229910001882 dioxygen Inorganic materials 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 239000003617 indole-3-acetic acid Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019799 monosodium phosphate Nutrition 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- MKWYFZFMAMBPQK-UHFFFAOYSA-J sodium feredetate Chemical compound [Na+].[Fe+3].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O MKWYFZFMAMBPQK-UHFFFAOYSA-J 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
【発明の詳細な説明】
本発明は、とくに植物の細胞および／または組
織（以下、単に細胞等と略記する）を大量に効率
よく培養することのできる回転式円筒型培養装置
に関する。DETAILED DESCRIPTION OF THE INVENTION The present invention particularly relates to a rotary cylindrical culture device capable of efficiently culturing a large amount of plant cells and/or tissues (hereinafter simply referred to as cells, etc.).
植物の細胞や組織からは医薬などの有用な物質
が得られる場合があり、これらの植物の細胞や組
織を工業的に培養し、有用な二次代謝産物を得よ
うとする試みがなされている。 Useful substances such as medicines may be obtained from plant cells and tissues, and attempts are being made to industrially culture these plant cells and tissues to obtain useful secondary metabolites. .
従来、発酵分野等におけるように微生物を大量
に培養する装置としては通常撹拌羽根を備えた通
気培養槽が使用されている。また、植物の細胞等
を大量に培養する場合にも同様に撹拌装置を備え
た通気培養槽を使用する試みがなされている。こ
の場合の撹拌は主として細胞等の沈澱を防ぎ、細
胞等が増殖するために必要な栄養源や酸素と充分
に接触させるために行われるものである。植物の
細胞等を培養する場合には、微生物の培養の場合
と異なり、その増殖には比較的撹拌強度の影響を
受け易く、撹拌強度が強すぎると増殖の低下や停
止が起こる場合が多く、その結果、二次代謝産物
の生産を効率良く行えない場合が多い。 BACKGROUND ART Conventionally, an aerated culture tank equipped with a stirring blade has been used as an apparatus for culturing microorganisms in large quantities, such as in the field of fermentation. Furthermore, attempts have also been made to use an aerated culture tank equipped with a stirring device in the case of culturing large amounts of plant cells and the like. The stirring in this case is mainly performed to prevent cells from settling and to bring them into sufficient contact with nutrients and oxygen necessary for proliferation of cells. When culturing plant cells, etc., unlike the case of culturing microorganisms, their growth is relatively susceptible to the influence of agitation intensity, and if the agitation intensity is too strong, growth often decreases or stops. As a result, it is often difficult to efficiently produce secondary metabolites.
前述の通気培養槽にみられる欠点を改善する培
養装置として、回転式円筒型培養装置を植物の細
胞等の培養に使用する方法も提案されている。こ
の型の装置では、槽の容積が大きくなると酸素移
動容量係数が小さくなるので、それを高めるため
には回転速度を著しく速くすることが必要とな
る。ところが、植物の細胞等の培養の際にはこれ
らの細胞等は撹拌の衝撃に弱いために、円筒型培
養槽の回転速度を上げる方法によつて酸素移動容
量係数を大きくすることは不可能であり、たとえ
ば100以上の通常の回転式円筒型培養槽を使用
して植物の細胞等を大量にかつ効率良く培養する
ことは困難である。たとえば、日本醗酵工学会講
演要旨集、昭和56年247頁（1981年）には、撹拌
効果を向上させるために内部に邪魔板を備えた回
転式円筒型培養槽の液面の内部に空気を供給し、
回転させながら植物のカルス細胞を培養する方法
が記載されている。この方法でも植物の細胞等は
衝撃によつて壊れ易く、増殖を効率良く行うこと
はできず、前述の欠点は改善されていない。 As a culture device that improves the drawbacks of the above-mentioned aerated culture tank, a method has also been proposed in which a rotary cylindrical culture device is used for culturing plant cells and the like. In this type of device, as the volume of the tank increases, the oxygen transfer capacity coefficient decreases, so in order to increase it, it is necessary to significantly increase the rotation speed. However, when culturing plant cells, etc., it is impossible to increase the oxygen transfer capacity coefficient by increasing the rotation speed of the cylindrical culture tank because these cells are susceptible to the shock of stirring. However, it is difficult to efficiently culture plant cells in large quantities using, for example, 100 or more conventional rotating cylindrical culture tanks. For example, the Japanese Society of Fermentation Engineering Abstracts, 1981, p. 247 (1981) states that air is introduced into the liquid surface of a rotating cylindrical culture tank equipped with internal baffles to improve the stirring effect. supply,
A method for culturing plant callus cells while rotating is described. Even with this method, plant cells etc. are easily destroyed by impact, and efficient proliferation cannot be achieved, so the above-mentioned drawbacks have not been improved.
本発明者らは、培養装置とくに回転式円筒型培
養装置の前述の欠点を改善し、植物の細胞等を大
規模にかつ効率良く製造することのできる培養装
置を検当した結果、前記公知文献のように培養槽
の内部に邪魔板等を設置しなくとも、回転式円筒
型培養槽の培養液の内部に酸素含有気体の供給装
置として多孔中空体を設置し、該多孔中空体を通
して酸素含有気体を供給することにより、前記目
的が充分に達成できることを見出し、本発明に到
達した。本発明の培養装置を使用することによ
り、植物の細胞等を工業的規模で効率よく培養で
きるという特徴があり、また増殖したこれらの細
胞等から効率よく二次代謝産物を生産できるとい
う特徴を有している。さらには、従来から使用さ
れている通気撹拌槽においては、長時間の培養を
継続すると、培養槽の内壁の液面付近に大量の細
胞等が付着し、培養の円滑な進行を妨害したり、
培養終了後の細胞等の回収に支障をきたしたりす
るという欠点があつたが、本発明の培養装置では
これらの欠点をも克服することができる。また、
本発明の回転式円筒型培養装置を使用すると、植
物の細胞等の培養の際の増殖速度が低下したりま
たは停止することがなく、増殖した細胞等による
二次代謝産物の生産を円滑に行うことができると
いう特徴がある。 The present inventors have investigated a culture device that can improve the above-mentioned drawbacks of culture devices, particularly rotary cylindrical culture devices, and can efficiently produce plant cells on a large scale. Instead of installing a baffle plate or the like inside the culture tank, a porous hollow body is installed as a supply device for oxygen-containing gas inside the culture medium of a rotating cylindrical culture tank, and oxygen-containing gas is supplied through the porous hollow body. It has been discovered that the above object can be fully achieved by supplying gas, and the present invention has been achieved. By using the culture device of the present invention, plant cells, etc. can be efficiently cultured on an industrial scale, and secondary metabolites can be efficiently produced from these proliferated cells, etc. are doing. Furthermore, in conventionally used aerated agitation tanks, if culture is continued for a long time, a large amount of cells etc. will adhere to the inner wall of the culture tank near the liquid level, interfering with the smooth progress of culture.
Although there was a drawback that recovery of cells etc. after completion of culture was hindered, the culture device of the present invention can overcome these drawbacks. Also,
When the rotary cylindrical culture device of the present invention is used, the growth rate during culturing of plant cells, etc. does not decrease or stop, and the proliferated cells, etc. can smoothly produce secondary metabolites. It has the characteristic of being able to
本発明を概説すれば、本発明は、
(a) 水平軸または水平軸から45゜以内の傾斜角を
有する中心軸のまわりに回転できるように設置
され、かつ少なくとも一端の中心部に円形また
は円筒型の開口部を有する回転式円筒型培養
槽、
(b) 該開口部に摺接し、かつ回転しないように該
回転式円筒型培養槽の該中心軸上に固定設置さ
れた封止装置、
(c) 該封止装置を嵌通して該回転式円筒型培養槽
の外部から培養液面の内部に気体を導入すべく
設置された気体供給管および該気体供給管に連
設され、該円筒型培養槽の底部に設けられた気
体噴出用多孔中空体、
(d) 該封止装置を嵌通して該回転式円筒型培養槽
の内部の培養液面上の空間部から外部に気体を
排出すべく設置された気体排出管、
(e) 該封止装置を嵌通して該回転式円筒型培養槽
内の底部に設けられた供給排出管、および
(f) 該円筒型培養槽を該中心軸のまわりに回転さ
せるための回転装置、
を備えることを特徴とする回転式円筒型培養装置
である。 To summarize the invention, the invention provides: (a) a circular or cylindrical shape disposed in the center of at least one end of the invention; a rotary cylindrical culture tank having a mold opening; (b) a sealing device that is in sliding contact with the opening and fixedly installed on the central axis of the rotary cylindrical culture tank to prevent rotation; c) A gas supply pipe installed to introduce gas from the outside of the rotary cylindrical culture tank to the inside of the culture liquid surface by fitting the sealing device, and a gas supply pipe connected to the gas supply pipe, and the cylindrical type (d) a porous hollow body for blowing out gas provided at the bottom of the culture tank; (e) a supply and exhaust pipe fitted through the sealing device and provided at the bottom of the rotary cylindrical culture tank; This is a rotary cylindrical culture device characterized by comprising: a rotation device for rotating around the cylindrical culture device.
本発明の回転式円筒型培養装置を図１ないし図
２に示した。 A rotary cylindrical culture apparatus of the present invention is shown in FIGS. 1 and 2.
図１に、本発明の回転式円筒型培養装置の回転
式円筒型培養槽１の概略図を示し、図２にはロー
ラー型回転架台に載せた回転式円筒型培養槽の概
略図を示す。 FIG. 1 shows a schematic diagram of a rotary cylindrical culture tank 1 of the rotary cylindrical culture apparatus of the present invention, and FIG. 2 shows a schematic diagram of the rotary cylindrical culture tank mounted on a roller-type rotating stand.
図１から明らかなように、回転式円筒型培養装
置の円筒型培養槽１はその中心軸２のまわりに回
転できるように設置されており、その際円筒型培
養槽１はその中心軸２が水平であるように設置さ
れていることが好ましいが、さらにその中心軸２
が水平軸から45゜以内の傾斜角を有するように設
置されていても差し支えない。中心軸２の水平軸
からの傾斜角が45゜を越えると、増殖した細胞等
の沈降を防止するためには高速で回転させること
が必要になり、その結果細胞等が壊われ易くなつ
たり、増殖を円滑に行わせることが困難になるよ
うににる。該円筒型培養槽１の側面部の少なくと
も一端の中心部には、封止装置４を摺接設置する
ための円形または円筒型の開口部３が設けられて
いる。 As is clear from FIG. 1, the cylindrical culture tank 1 of the rotary cylindrical culture device is installed so that it can rotate around its central axis 2; It is preferable that it is installed horizontally, but furthermore, its central axis 2
may be installed with an angle of inclination of up to 45° from the horizontal axis. If the angle of inclination of the central axis 2 from the horizontal axis exceeds 45°, it will be necessary to rotate at high speed in order to prevent the multiplied cells from settling, and as a result, the cells may be easily broken. It becomes difficult for the cells to proliferate smoothly. At the center of at least one end of the side surface of the cylindrical culture tank 1, a circular or cylindrical opening 3 is provided in which a sealing device 4 is slidably installed.
本発明の回転式円筒型培養装置の該円筒型培養
槽１の前記開口部３には、該円筒型培養槽１の中
心軸上に該開口部に摺接するように封止装置が設
置される。その際、この封止装置は該円筒型培養
槽１と共に回転しないように固定されて設置され
る。該封止装置としては、培養槽１からの培養液
の漏れを防止することができしかも雑菌の侵入を
防止することができ、かつ培養槽の回転が円滑に
実施できるものならばどのようなタイプのもので
も差し支えないが、具体的には、Ｏリングシー
ル、グランドシール、メカニカルシールなどを採
用することができ、封止装置４の外周に、開口部
３の内周に摺接するシール部材５が装着された封
止装置を採用することができる。 A sealing device is installed in the opening 3 of the cylindrical culture tank 1 of the rotary cylindrical culture device of the present invention so as to be in sliding contact with the opening on the central axis of the cylindrical culture tank 1. . At this time, this sealing device is fixedly installed together with the cylindrical culture tank 1 so as not to rotate. Any type of sealing device may be used as long as it can prevent the leakage of the culture solution from the culture tank 1, prevent the invasion of bacteria, and allow smooth rotation of the culture tank. Specifically, an O-ring seal, a gland seal, a mechanical seal, etc. may be used, and a sealing member 5 that slides on the outer periphery of the sealing device 4 and the inner periphery of the opening 3 may be used. A fitted sealing device may be employed.
本発明の回転式円筒型培養装置において、該封
止装置４を嵌通して該封止装置４の外部から円筒
型培養液面の内部に気体供給管６、気体排出管８
および供給排出管９の少なくとも３本の管が嵌通
挿着される。これらのうちで気体供給管６は該封
止装置４を嵌通して該回転式円筒型培養槽１の外
部から培養液面の内部に気体を導入すべく挿着さ
れており、さらにその先端は該円筒型培養槽１の
内底部に設けられた気体噴出用多孔中空体７に連
設されている。この気体噴出用多孔中空体７は培
養液中に分子状酸素含有気体を気泡状態で可能な
かぎり均一に分散供給できる構造を有している多
孔中空体が好適であり、具体的には多孔質材料に
よりできた多孔管、直径２mm以下の孔を複数個備
えた多孔管等を例示することができる。また、気
体排出管８は、該封止装置４を嵌通して該回転式
円筒型培養槽１の内部の培養液面上の空間部から
外部に気体を排出すべく挿着されており、培養槽
の内部の先端は培養液面上の空間部に位置してい
る。さらに、供給排出管９は、該円筒型培養槽１
の外部から該封止装置を嵌通してその先端は該円
筒型培養槽１内の底部に達するように設置されて
いる。該供給排出管９は、該封止装置４の外部か
ら円筒型培養槽１の内部に植物の細胞等や栄養源
を含んだ培地を供給したり、円筒型培養槽１の内
部から生成物を含む培養液を外部に排出させるた
めに使用される。 In the rotary cylindrical culture device of the present invention, a gas supply pipe 6 and a gas discharge pipe 8 are inserted into the cylindrical culture liquid surface from the outside of the sealing device 4 through the sealing device 4.
At least three pipes, ie, supply and discharge pipes 9, are fitted and inserted. Among these, the gas supply pipe 6 is inserted through the sealing device 4 to introduce gas from the outside of the rotary cylindrical culture tank 1 to the inside of the culture solution surface, and the tip thereof is It is connected to a porous hollow body 7 for blowing out gas provided at the inner bottom of the cylindrical culture tank 1 . This porous hollow body 7 for blowing out gas is preferably a porous hollow body that has a structure that can disperse and supply molecular oxygen-containing gas in the form of bubbles as uniformly as possible into the culture solution. Examples include a perforated pipe made of a material, a perforated pipe provided with a plurality of holes having a diameter of 2 mm or less, and the like. Further, the gas discharge pipe 8 is inserted through the sealing device 4 to discharge gas to the outside from the space above the culture solution level inside the rotary cylindrical culture tank 1. The tip inside the tank is located in the space above the culture solution surface. Furthermore, the supply and discharge pipe 9 is connected to the cylindrical culture tank 1.
The sealing device is inserted from the outside of the cylindrical culture tank 1 so that its tip reaches the bottom of the cylindrical culture tank 1. The supply/discharge pipe 9 is used to supply a culture medium containing plant cells and other nutrients from the outside of the sealing device 4 to the inside of the cylindrical culture tank 1, and to supply products from the inside of the cylindrical culture tank 1. It is used to drain the culture solution to the outside.
本発明の回転式円筒型培養装置において、該円
筒型培養槽１の内部には、前記気体供給管６、気
体排出管８および供給排出管９の他に、必要に応
じて、該円筒型培養槽１の中の培養液の温度を制
御するために該封止装置４を嵌通して該円筒型培
養槽１の培養液内部に熱交換器１０および温度検
知器１３を挿着させておくこともできる。 In the rotary cylindrical culture apparatus of the present invention, the cylindrical culture tank 1 includes, in addition to the gas supply pipe 6, gas discharge pipe 8, and supply and discharge pipe 9, as necessary. In order to control the temperature of the culture solution in the tank 1, a heat exchanger 10 and a temperature sensor 13 are inserted into the culture solution of the cylindrical culture tank 1 by fitting the sealing device 4 therethrough. You can also do it.
本発明の回転式円筒型培養装置において、該気
体供給管６、該気体排出管８、該供給排出管９お
よび必要に応じて設置された熱交換器１０ならび
に温度検知器１３のすべての装置を、該円筒型培
養槽１の一側端壁の中心部の開口部３に摺接した
封止装置に嵌通して挿着させることができるし、
これらのうちの一部を該円筒型培養槽１の一側端
壁の開口部３に摺接した封止装置４に嵌通して挿
着させかつ残りの付属装置を他側端壁の他の他端
の開口部に同様に摺接した封止装置に嵌通して挿
着させていても差し支えない。また、該円筒型培
養槽１の槽長が長いかまたは内容積が著しく大き
い場合には、該円筒型培養槽１の両側端の各々の
開口部に摺接設置された封止装置にそれぞれ該気
体供給管、該気体排出管、該体給排出管、ならび
に必要に応じて熱交換器、温度検知器が嵌通して
挿着されていても差し支えない。 In the rotary cylindrical culture apparatus of the present invention, all the devices including the gas supply pipe 6, the gas discharge pipe 8, the supply and discharge pipe 9, and the heat exchanger 10 and temperature sensor 13 installed as necessary. , the cylindrical culture tank 1 can be inserted by fitting into a sealing device that is in sliding contact with the opening 3 in the center of one end wall of the cylindrical culture tank 1;
A part of these is fitted and inserted into the sealing device 4 that is in sliding contact with the opening 3 of the end wall on one side of the cylindrical culture tank 1, and the remaining accessory devices are inserted into the opening 3 of the end wall of the other side. There is no problem even if it is fitted and inserted into a sealing device that is in sliding contact with the opening at the other end. In addition, if the length of the cylindrical culture tank 1 is long or the internal volume is extremely large, the sealing devices installed in sliding contact with the openings at both ends of the cylindrical culture tank 1 may be used. The gas supply pipe, the gas discharge pipe, the body supply and discharge pipe, and if necessary, a heat exchanger and a temperature sensor may be fitted and inserted therethrough.
本発明の回転式円筒型培養装置において、該気
体供給管６の内部には円筒型培養槽内の培養液が
逆流しないように外側の端部から空気などの酸素
含有気体が圧入され、該気体排出管８の一方の端
部は該円筒型培養槽内部の培養液面の上部の空間
部に設置されており、他の端部は培養槽の外部に
設置されている。また、該供給排出管９の一方の
端部は該円筒型培養槽１の内部の培養液の底部に
設置されており、他の端部は培養槽の外部に設置
されており、原料の植物の細胞等や培養液を槽内
に供給する際は圧入あるいはポンプ等により供給
してもよいし、培養液を排出する際は重力差によ
り排出する方法あるいはポンプ等により排出する
方法を採用することができる。また、該円筒培養
槽１の内部に設置された熱交換器１０の熱媒体の
供給管１１および熱媒体の排出管１２はそれぞれ
該封止装置４を嵌通して挿着され、それぞれの端
部は培養槽の外部に設置され、熱媒体はポンプ等
により循環される。該円筒型培養槽１内部の培養
液の温度は所望の温度に保たれる。 In the rotary cylindrical culture apparatus of the present invention, an oxygen-containing gas such as air is pressurized into the gas supply pipe 6 from the outer end so that the culture solution in the cylindrical culture tank does not flow back. One end of the discharge pipe 8 is installed in the space above the culture solution level inside the cylindrical culture tank, and the other end is installed outside the culture tank. Also, one end of the supply/discharge pipe 9 is installed at the bottom of the culture solution inside the cylindrical culture tank 1, and the other end is installed outside the culture tank, and is connected to the raw material plant. When supplying cells, etc. and culture solution into the tank, they may be supplied by press-fitting or using a pump, etc., and when discharging the culture solution, a method of discharging by gravity difference or a method of discharging with a pump, etc. may be adopted. Can be done. Further, the heat medium supply pipe 11 and the heat medium discharge pipe 12 of the heat exchanger 10 installed inside the cylindrical culture tank 1 are respectively inserted through the sealing device 4, and the respective ends is installed outside the culture tank, and the heat medium is circulated by a pump or the like. The temperature of the culture solution inside the cylindrical culture tank 1 is maintained at a desired temperature.
本発明の回転式円筒型培養装置には、該円筒型
培養槽１をその中心軸のまわりに回転させるため
の回転装置が設置されている。回転装置として
は、該円筒型培養槽１の中心軸２と平行方行に並
べられかつ固定された複数個の回転ローラーを有
する架台上に該円筒型培養槽１を置き、ローラー
を回転させることにより該円筒型培養槽１を回転
させる装置；該培養槽１の２個以上の複数個所の
円周上に歯車を設置した該円筒型培養槽１を該歯
車と噛み合うように設置された複数個の歯車を有
する架台上に置き、歯車を回転させることにより
該筒型培養槽１を回転させる装置などを例示する
ことができる。図２には、回転装置として２本の
固定された回転ローラー１４および１４′を有す
る回転ローラー架台１６上に該円筒型培養槽１を
設置した回転式円筒型培養装置を示す。 The rotary cylindrical culture device of the present invention is equipped with a rotation device for rotating the cylindrical culture tank 1 around its central axis. As a rotating device, the cylindrical culture tank 1 is placed on a pedestal having a plurality of fixed rotating rollers arranged parallel to the central axis 2 of the cylindrical culture tank 1, and the rollers are rotated. A device for rotating the cylindrical culture tank 1; a device in which gears are installed on the circumference of two or more locations of the culture tank 1; a plurality of gears are installed so as to mesh with the gears; An example of this device is a device that rotates the cylindrical culture tank 1 by placing it on a pedestal having a gear and rotating the gear. FIG. 2 shows a rotary cylindrical culture device in which the cylindrical culture tank 1 is installed on a rotating roller mount 16 having two fixed rotating rollers 14 and 14' as a rotating device.
次に、本発明の円筒型培養装置を使用した植物
の細胞等の培養方法について簡単に説明する。 Next, a method for culturing plant cells using the cylindrical culture device of the present invention will be briefly described.
該円筒型培養槽１には培養液が供給排出管９か
ら導入され、その量は気体排出管８の先端が培養
液中に没しない範囲において任意に設定できる
が、該円筒型培養槽１の内容積の通常90vol％以
下、好ましくは30ないし80vol％の範囲である。
また、該円筒型培養槽１には植物の細胞等が供給
排出管９から供給され、その際の供給方法として
培養液と共に供給することもできるし、空気など
の気体と共に送入することもできる。 A culture solution is introduced into the cylindrical culture tank 1 from the supply/discharge pipe 9, and the amount can be set arbitrarily within the range that the tip of the gas discharge pipe 8 is not submerged in the culture solution. It is usually 90 vol% or less of the internal volume, preferably in the range of 30 to 80 vol%.
Further, plant cells and the like are supplied to the cylindrical culture tank 1 from the supply/discharge pipe 9, and in this case, the supply method can be such that they are supplied together with the culture solution, or they can also be supplied together with a gas such as air. .
該円筒型培養槽１への培養液の供給は回分法、
間欠法または連続法のいずれの方法によつても実
施することができる。 The culture solution is supplied to the cylindrical culture tank 1 by a batch method,
It can be carried out by either an intermittent method or a continuous method.
該円筒培養槽１からの培養液の排出は、供給排
出管９を通して行われ、排出方法としては回分
法、間欠法または連続法のいずれの方法を採用す
ることもできる。 The culture solution is discharged from the cylindrical culture tank 1 through the supply/discharge pipe 9, and any of the batch method, intermittent method, or continuous method can be adopted as the discharge method.
該円筒型培養槽１には、その中の培養液中に気
体供給管６およびそれに連設された気体噴出用多
孔中空体７を通して酸素含有気体が気泡として供
給される。酸素含有気体の供給量は植物の細胞等
の種類、その濃度および培養液量によつても異な
るが、いずれの培養の場合にも酸素移動容量係数
を必要な値にまで高めることができる。 Oxygen-containing gas is supplied to the cylindrical culture tank 1 as bubbles into the culture solution therein through a gas supply pipe 6 and a porous hollow body 7 for blowing out gas connected thereto. Although the amount of oxygen-containing gas supplied varies depending on the type of plant cells, their concentration, and the amount of culture solution, the oxygen transfer capacity coefficient can be increased to the required value in any culture.
該円筒型培養槽１の培養液の温度は植物の細胞
等の培養に好適な温度に維持される。そのための
方法としては、本発明の回転式円筒型培養装置全
体を一定温度に制御した部屋に設置する方法、本
発明の回転式円筒型培養装置全体を一定温度に制
御した浴槽に設置する方法、図１に示したように
円筒型培養槽１内に設置した熱交換器１０に熱媒
体を通じることにより制御する方法等を採用する
ことができる。これらの培養液の温度を制御する
方法のうちでは、第三番目の該円筒型培養槽１内
に設置した熱交換器を使用する方法が培養液の温
度を正確に制御することができ、他の方法にくら
べて温度制御のための装置が小型であるので好適
である。 The temperature of the culture solution in the cylindrical culture tank 1 is maintained at a temperature suitable for culturing plant cells and the like. Methods for this include a method in which the entire rotary cylindrical culture device of the present invention is installed in a room controlled at a constant temperature, a method in which the entire rotary cylindrical culture device of the present invention is installed in a bathtub controlled at a constant temperature, As shown in FIG. 1, a method of controlling by passing a heat medium through a heat exchanger 10 installed in a cylindrical culture tank 1 can be adopted. Among these methods of controlling the temperature of the culture solution, the third method of using a heat exchanger installed in the cylindrical culture tank 1 is able to accurately control the temperature of the culture solution; This method is preferable because the device for temperature control is smaller than that of the above method.
該円筒型培養槽１を前記回転装置によつて回転
させることにより培養増殖が実施される。該円筒
型培養槽１の回転速度は培養する植物の細胞等の
性質によつても異なるが、通常25回／分以下、好
ましくは１ないし20回／分の範囲であり、また該
円筒型培養槽１の周速度は通常２ｍ／分以上、好
ましくは４ないし60ｍ／分の範囲である。本発明
の回転式円筒型培養装置を使用すると、100以
上の工業的規模の大型の培養槽の場合でも前記回
転速度および周速度とすることにより、細胞等の
沈降や堆積を防止することができ、しかも従来の
撹拌型培養槽にくらべて細胞等に過度の衝撃を与
えないので好適である。 Culture propagation is carried out by rotating the cylindrical culture tank 1 using the rotation device. The rotation speed of the cylindrical culture tank 1 varies depending on the properties of the cells of the plant to be cultured, but is usually 25 times/min or less, preferably in the range of 1 to 20 times/min. The circumferential speed of tank 1 is usually 2 m/min or more, preferably in the range of 4 to 60 m/min. When the rotary cylindrical culture device of the present invention is used, sedimentation and accumulation of cells, etc. can be prevented by setting the rotational speed and circumferential speed as described above even in the case of a large industrial scale culture tank of 100 or more. Moreover, it is preferable because it does not give an excessive shock to the cells etc. compared to the conventional stirring type culture tank.
従来の撹拌羽根を備えた培養装置を用いた培養
では培養細胞等の壁への付着が著しいため、培養
の途中あるいは終了後に付着物を除去することが
必要であり、そのためこれらの方法では長期間の
培養を継続することができない。しかし、本発明
の回転式円筒型培養装置では培養中に壁への細胞
等の付着はほとんど起こらないために、長期間の
培養を継続することができるという特徴もある。 When culturing using conventional culture equipment equipped with stirring blades, cultured cells adhere to the walls significantly, so it is necessary to remove the adhered substances during or after culturing. cannot continue culturing. However, in the rotary cylindrical culture apparatus of the present invention, adhesion of cells, etc. to the walls hardly occurs during culture, so that culture can be continued for a long period of time.
本発明の回転式円筒型培養装置を使用した植物
の組織培養の例を次に具体的に示す。 A specific example of plant tissue culture using the rotary cylindrical culture device of the present invention will be shown below.
実施例 １
図１および図２に示した形状で内容積200の
回転式円筒型培養装置にCa（NO3）2・4H2O、
KNO3、KCl、NaH2PO4、MgSO4、H3BO4、
ZnSO4・7H2O、NaFeEDTA・3H2O、CuSO4・
5H2O、Na2SO4、インドール酢酸、シヨ糖、水
を含む培地160を培地供給口９を通して入れ、
円筒型培養槽１を回転しながら熱交換器１０にス
チームを通すことによつて槽内全体を滅菌した。Example 1 Ca(NO 3 ) 2 ·4H 2 O,
KNO3 , KCl , NaH2PO4 , MgSO4 , H3BO4 ,
ZnSO4・7H2O , NaFeEDTA・3H2O , CuSO4・
A medium 160 containing 5H 2 O, Na 2 SO 4 , indole acetic acid, sucrose, and water is introduced through the medium supply port 9,
The entire interior of the cylindrical culture tank 1 was sterilized by passing steam through the heat exchanger 10 while rotating the tank.
熱交換器に冷却水を通して培養液の温度を25℃
にした後、培地供給口９を通して無菌的にムラサ
キのカルスを湿潤重量で4.8Kg供給し、空気供給
口６を通して無菌空気を毎分32の速度で供給
し、槽を3rpmの速度で回転させて培養を14日間
行つた。この時KLa（酸素移動容量係数）の値は
9.9で、通気しない場合のKLaは0.002であつた。
また槽の周速度は5.5ｍ／分であつた。培養終了
後培養液を分析した結果、乾燥細胞収量は1.3Kg、
細胞中のシコニン系色素の含有率は13重量％であ
つた。培養終了後の槽内には何等付着物はほとん
どなく、簡単な洗浄作業の後、次の培養を行うこ
とが出来た。 Cooling water is passed through the heat exchanger to bring the temperature of the culture solution to 25℃.
After that, 4.8 kg of wet weight of purple callus was aseptically supplied through the medium supply port 9, sterile air was supplied through the air supply port 6 at a rate of 32 per minute, and the tank was rotated at a speed of 3 rpm. Culture was carried out for 14 days. At this time, the value of KLa (oxygen transfer capacity coefficient) is
9.9, and KLa without ventilation was 0.002.
The peripheral speed of the tank was 5.5 m/min. As a result of analyzing the culture solution after culturing, the dry cell yield was 1.3Kg.
The content of shikonin pigment in the cells was 13% by weight. After the culture was completed, there was almost no deposit inside the tank, and after a simple cleaning operation, the next culture could be carried out.
比較例 １
本発明によらない通常のたて型通気撹拌槽（内
容積200）で変形パドル撹拌翼を20rpmで回転
させて実施例１と同じ培地を用いて培養を行つた
ところ細胞収量は1.0Kg、色素の含有率は13重量
％であつた。また培養槽の内壁および撹拌器のシ
ヤフトには多量のカルスを主成分とする付着物が
認められ、次の培養を行うためには専用の器具で
付着物を削り落してから洗浄することが必要であ
つた。Comparative Example 1 When culture was carried out using the same medium as in Example 1 with a modified paddle stirring blade rotated at 20 rpm in a normal vertical aeration stirring tank (inner volume 200) not according to the present invention, the cell yield was 1.0. kg, and the pigment content was 13% by weight. In addition, a large amount of deposits mainly composed of callus were found on the inner walls of the culture tank and the shaft of the stirrer, and in order to perform the next culture, it is necessary to scrape off the deposits with a special tool and then wash them. It was hot.
図１は、本発明の回転式円筒型培養装置の円筒
型培養槽１の概略図を示し、図２には該回転型培
養槽１をローラー型架台上に設置した回転式円筒
型培養装置の概略図を示す。
FIG. 1 shows a schematic diagram of a cylindrical culture tank 1 of a rotary cylindrical culture apparatus of the present invention, and FIG. A schematic diagram is shown.
Claims (1)
角を有する中心軸のまわりに回転できるように
設置され、かつ少なくとも一端の中心部に円形
または円筒型の開口部を有する回転式円筒培養
槽、 (b) 該開口部に摺接し、かつ回転しないように該
回転式円筒型培養槽の該中心軸上に固定設置さ
れた封止装置、 (c) 該封止装置を嵌通して該回転式円筒型培養槽
の外部から培養液面の内部に気体を導入すべく
設置された気体供給管および該気体供給管に連
設され、該円筒型培養槽の底部に設けられた気
体噴出用多孔中空体、 (d) 該封止装置を嵌通し、該回転式円筒型培養槽
の内部の培養液面上の空間部から外部に気体を
排出すべく設置された気体排出管、 (e) 該封止装置を嵌通して該回転式円筒型培養槽
内の底部に設けられた供給排出管、および (f) 該円筒型培養槽を該中心軸のまわりに回転さ
せるための回転装置、 を備えることを特徴とする回転式円筒型培養装
置。[Scope of Claims] 1 (a) A circular or cylindrical opening in the center of at least one end, installed so as to be rotatable about a horizontal axis or a central axis having an angle of inclination within 45° from the horizontal axis; (b) a sealing device that is in sliding contact with the opening and fixedly installed on the central axis of the rotary cylindrical culture tank so as not to rotate; (c) the sealing device; A gas supply pipe installed to introduce gas from the outside of the rotary cylindrical culture tank to the inside of the culture solution surface by fitting the device, and a gas supply pipe connected to the gas supply pipe and installed at the bottom of the cylindrical culture tank. (d) a porous hollow body provided for blowing out gas; (d) a gas installed to fit through the sealing device and discharge gas to the outside from a space above the culture liquid level inside the rotary cylindrical culture tank; (e) a supply and exhaust pipe fitted through the sealing device and provided at the bottom of the rotary cylindrical culture tank; and (f) for rotating the cylindrical culture tank about the central axis. A rotary cylindrical culture device characterized by comprising: a rotation device for.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1484382A JPS58134989A (en) | 1982-02-03 | 1982-02-03 | Cylindrical rotary cultivation apparatus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1484382A JPS58134989A (en) | 1982-02-03 | 1982-02-03 | Cylindrical rotary cultivation apparatus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS58134989A JPS58134989A (en) | 1983-08-11 |
| JPS6328588B2 true JPS6328588B2 (en) | 1988-06-09 |
Family
ID=11872313
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1484382A Granted JPS58134989A (en) | 1982-02-03 | 1982-02-03 | Cylindrical rotary cultivation apparatus |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS58134989A (en) |
Families Citing this family (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0646956B2 (en) * | 1984-01-27 | 1994-06-22 | フ−パ−・トレ−デイング・カンパニ−・エヌ・ベ− | Efficient in vitro production of serum-free and mitogen-free interkin-2. |
| JPS60232088A (en) * | 1984-05-04 | 1985-11-18 | Japan Synthetic Rubber Co Ltd | Method for cultivating cell and apparatus therefor |
| JPH0640831B2 (en) * | 1985-01-31 | 1994-06-01 | 日本合成ゴム株式会社 | Method for producing culture medium containing physiologically active substance secreted from cells |
| JPH0644860B2 (en) * | 1985-07-03 | 1994-06-15 | 日本合成ゴム株式会社 | Incubator |
| JPS6279772A (en) * | 1985-10-04 | 1987-04-13 | Iwashiya Seibutsu Kagaku:Kk | Method for stirring culture solution and device therefor |
| JPH02142461A (en) * | 1988-11-24 | 1990-05-31 | Sakai Eng Kk | Rotary biochemical reactor |
| JPH02265468A (en) * | 1989-04-06 | 1990-10-30 | Shibata Hario Glass Kk | Rotating bottle type culture device |
| JPH0779677B2 (en) * | 1991-10-08 | 1995-08-30 | 株式会社荏原製作所 | Incubator |
| US5443985A (en) * | 1993-07-22 | 1995-08-22 | Alberta Research Council | Cell culture bioreactor |
| JP5899349B1 (en) * | 2015-03-25 | 2016-04-06 | 株式会社神鋼環境ソリューション | Paramylon manufacturing method |
-
1982
- 1982-02-03 JP JP1484382A patent/JPS58134989A/en active Granted
Also Published As
| Publication number | Publication date |
|---|---|
| JPS58134989A (en) | 1983-08-11 |
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