JP4820979B2 - Cell culture equipment - Google Patents

Cell culture equipment Download PDF

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JP4820979B2
JP4820979B2 JP2004297582A JP2004297582A JP4820979B2 JP 4820979 B2 JP4820979 B2 JP 4820979B2 JP 2004297582 A JP2004297582 A JP 2004297582A JP 2004297582 A JP2004297582 A JP 2004297582A JP 4820979 B2 JP4820979 B2 JP 4820979B2
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泰二郎 末田
政則 宮崎
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Hiroshima University NUC
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    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/40—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pressure

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Description

本発明は、細胞培養装置に係り、特に拍動を与えながら細胞培養を行う細胞培養装置に関する。   The present invention relates to a cell culture device, and more particularly to a cell culture device that performs cell culture while applying pulsation.

心臓冠動脈や末梢動脈の閉塞疾患に対するバイパス手術において、患者の内皮細胞により被覆された血栓が形成されにくい合成繊維製の人工血管を使用する外科治療が注目されている。そのような人工血管を多くの人が利用できるようにするために、血管を構成する種々の細胞を効率的に培養することができるような細胞培養装置が求められている。   In bypass surgery for obstructive diseases of the cardiac coronary artery and peripheral artery, surgical treatment using an artificial blood vessel made of a synthetic fiber in which a thrombus covered with a patient's endothelial cells is hard to be formed has attracted attention. In order to make such an artificial blood vessel available to many people, a cell culture device capable of efficiently culturing various cells constituting the blood vessel is required.

細胞培養を効率的に行うには、血液の流れや血圧などの生体環境を模した条件下で細胞培養を行うことが好ましく、そのため拍動を与えながら細胞培養を行うことができる細胞培養装置が種々提案されている。   In order to efficiently perform cell culture, it is preferable to perform cell culture under conditions simulating a biological environment such as blood flow and blood pressure. Therefore, a cell culture apparatus capable of performing cell culture while applying pulsation is provided. Various proposals have been made.

例えば、特許文献１には、動物の心臓血管領域を形成しうる細胞または組織を収容する培養槽と、ポンプから機械弁を介して緩衝槽へ培養液が供給されるのに連動して、前記緩衝槽から前記培養槽に培養液を供給するための緩衝手段と、前記培養槽から排出される培養液を導管を通して貯蔵槽に流入させて、該培養液を、大気圧下で貯蔵槽に一時的に貯蔵するための回収貯蔵手段と、前記ポンプにより、前記貯蔵槽から機械弁を介してポンプに培養液を吸引させるとともに、該吸引に連動して前記緩衝槽に培養液を加圧して間欠的に供給するための加圧手段とを備える細胞培養装置が提案されている。   For example, Patent Document 1 discloses that a culture tank containing cells or tissues capable of forming a cardiovascular region of an animal and a culture solution supplied from a pump to a buffer tank via a mechanical valve, Buffer means for supplying the culture medium from the buffer tank to the culture tank, and the culture liquid discharged from the culture tank are allowed to flow into the storage tank through a conduit, and the culture liquid is temporarily stored in the storage tank under atmospheric pressure. The collection and storage means for storing the liquid and the pump causes the pump to suck the culture medium from the storage tank via the mechanical valve, and pressurizes the culture medium to the buffer tank in conjunction with the suction to intermittently And cell pressurizers for supplying the cells are proposed.

また、特許文献２には、生物の細胞を培養する細胞培養装置に於いて、循環する培養液の流量を制御する流量制御手段と、上記培養液の流量を測定する流量測定手段と、上記培養液の圧力を制御する圧力制御手段と、上記培養液の圧力を測定する圧力測定手段と、制御目標となる流量と圧力の波形情報が供給される制御手段とを具備し、上記圧力制御手段と上記流量制御手段との相互の干渉を相殺する様に制御目標に到達させる様に上記制御手段を介して制御して成る細胞培養装置が提案されている。   Patent Document 2 discloses a flow rate control means for controlling the flow rate of a circulating culture solution, a flow rate measurement means for measuring the flow rate of the culture solution, and the culture medium in a cell culture apparatus for culturing living cells. Pressure control means for controlling the pressure of the liquid, pressure measuring means for measuring the pressure of the culture liquid, and control means for supplying flow rate and pressure waveform information as a control target, the pressure control means, There has been proposed a cell culture device which is controlled via the control means so as to reach a control target so as to cancel the mutual interference with the flow rate control means.

特開2004-49185号公報Japanese Patent Laid-Open No. 2004-49185 特開平9-313166号公報JP-A-9-313166

しかし、特許文献１に提案された細胞培養装置は、バルーンの膨張・収縮により拍動を与えるバルーンポンプ等のポンプを使用するものであるから、ポンプの送り出し側及び戻り側に開閉弁を設けなければならず、また、緩衝槽等のサージ圧吸収手段を設けなければならない。さらに、バルーンポンプ制御のために高価・大容積の制御装置を設けなければならない。   However, since the cell culture device proposed in Patent Document 1 uses a pump such as a balloon pump that gives pulsation by inflation / deflation of the balloon, it is necessary to provide opening / closing valves on the delivery side and return side of the pump. In addition, surge pressure absorbing means such as a buffer tank must be provided. Furthermore, an expensive and large-volume control device must be provided for controlling the balloon pump.

特許文献２に提案された細胞培養装置は、培養液の拍動流と拍動圧を制御するために高価・複雑な装置を設けなければならない。このように従来の細胞培養装置は、構造が複雑で高価な装置になり易いという問題があった。また、操作及び維持管理が難しいという問題があった。   The cell culture device proposed in Patent Document 2 must be provided with an expensive and complicated device in order to control the pulsating flow and pulsating pressure of the culture solution. Thus, the conventional cell culture apparatus has a problem that the structure is complicated and the apparatus is likely to be expensive. In addition, there is a problem that operation and maintenance are difficult.

本発明は、かかる従来の細胞培養装置の問題点に鑑み、拍動を与えながら細胞培養を行うことができ、簡単でコンパクトな構造をなし操作や維持管理が容易な細胞培養装置を提供することを目的とする。   In view of the problems of the conventional cell culture apparatus, the present invention provides a cell culture apparatus that can perform cell culture while applying pulsation, has a simple and compact structure, and is easy to operate and maintain. With the goal.

本発明に係る細胞培養装置は、細胞の培養を行う培養室、培養液の一時貯留を行う培養液槽、培養液中の酸素及び二酸化炭素分圧の調整を行うガス交換手段、培養液の加温手段及び前記培養室に培養液を供給する培養液供給手段が導管により連結されてなり、前記培養液供給手段は、ポンプと、該ポンプの最大吐出量が制御できるとともに吐出量が周期的に変化するように制御することができるポンプ制御装置と、前記導管内を流れる培養液の流量を制御することができる流量調整弁と、を備えてなる。   The cell culture device according to the present invention includes a culture chamber for culturing cells, a culture solution tank for temporarily storing the culture solution, a gas exchange means for adjusting oxygen and carbon dioxide partial pressure in the culture solution, and adding the culture solution. The culture medium supply means for supplying the culture medium to the temperature means and the culture chamber is connected by a conduit. The culture medium supply means can control the pump, the maximum discharge amount of the pump, and the discharge amount periodically. The pump control apparatus which can be controlled to change, and the flow regulating valve which can control the flow volume of the culture solution which flows in the said conduit | pipe are provided.

上記発明において、ポンプは遠心ポンプを用いるのが好ましく、ポンプ制御装置はファンクションゼネレータを用いるのが好ましい。また、ガス交換手段は、酸素及び二酸化炭素分圧が所定値に保持されるガス室及び該ガス室内に収容されたシリコンチューブからなるものが好ましい。   In the above invention, the pump is preferably a centrifugal pump, and the pump control device is preferably a function generator. Further, the gas exchange means is preferably composed of a gas chamber in which oxygen and carbon dioxide partial pressures are maintained at predetermined values and a silicon tube accommodated in the gas chamber.

本発明に係る細胞培養装置は、拍動を与えながら細胞培養を行うことができ、簡単でコンパクトな構造をなし操作が容易である。また、簡単な構造であるから維持管理に特別の工数を要しない。   The cell culture device according to the present invention can perform cell culture while applying pulsation, has a simple and compact structure, and is easy to operate. In addition, since the structure is simple, no special man-hours are required for maintenance.

本発明に係る細胞培養装置の実施形態を図に基づいて説明する。図１は、本細胞培養装置の一実施例のレイアウトを説明する模式図である。本細胞培養装置は、図１に示すように、培養室10、培養液槽15、ガス交換手段20、加温手段30及び培養液供給手段40を有し、それらが導管60で連結されて閉回路を構成してなる。   An embodiment of a cell culture device according to the present invention will be described with reference to the drawings. FIG. 1 is a schematic diagram for explaining the layout of one embodiment of the present cell culture apparatus. As shown in FIG. 1, this cell culture apparatus has a culture chamber 10, a culture solution tank 15, a gas exchange means 20, a heating means 30, and a culture solution supply means 40, which are connected by a conduit 60 and closed. A circuit is constructed.

本細胞培養装置は、培養液供給手段40により所要の拍動流及び拍動圧が与えられる構成になっている。すなわち、培養液供給手段40はポンプ41、ポンプ制御装置43及び流量調整弁45を備え、ポンプ制御装置43によりポンプ41の最大吐出量を制御するとともに吐出量を周期的に変化させ、さらに、流量調整弁45により導管60内を流れる培養液の流量を制御することにより、所要の拍動流及び拍動圧を得ることができる。   The present cell culture device is configured such that a required pulsatile flow and pulsatile pressure are applied by the culture solution supply means 40. That is, the culture solution supply means 40 includes a pump 41, a pump control device 43, and a flow rate adjustment valve 45. The pump control device 43 controls the maximum discharge amount of the pump 41 and periodically changes the discharge amount. The required pulsatile flow and pulsation pressure can be obtained by controlling the flow rate of the culture fluid flowing in the conduit 60 by the regulating valve 45.

ポンプ41は、安定した流量を供給することができ信頼性の高い定常流型のポンプを用いるのがよい。例えば遠心ポンプは、弁が不要で血栓発生の可能性が少なく、比較的吐出量が多い割に小型で信頼性が高いので好適である。   The pump 41 is preferably a steady flow pump that can supply a stable flow rate and has high reliability. For example, a centrifugal pump is suitable because it does not require a valve, has a low possibility of thrombus generation, and is relatively small and highly reliable for a relatively large discharge amount.

ポンプ制御装置43は、上述のようにポンプの最大吐出量が制御できるとともに吐出量が周期的に変化するように制御できるものを用いる。そのようなポンプ制御装置43として、ファンクションゼネレータを使用するのがよい。これによれば、例えば、任意の振幅及び周波数の正弦波状の電圧をポンプ41に印加することができ、適当な振幅V及び周波数fを選択することにより、ポンプの吐出量を周期的に変動させるとともにその吐出量の最大値及び周波数を容易に制御することができる。   As the pump control device 43, a device that can control the maximum discharge amount of the pump as described above and can control the discharge amount to periodically change is used. As such a pump control device 43, a function generator is preferably used. According to this, for example, a sinusoidal voltage having an arbitrary amplitude and frequency can be applied to the pump 41, and by appropriately selecting the amplitude V and the frequency f, the pump discharge amount is periodically changed. At the same time, the maximum value and frequency of the discharge amount can be easily controlled.

流量調整弁45は、導管60のいずれかの部分に設置し、導管60内を流れる培養液の流量を容易かつ微細に調整することができるものがよい。例えばフローコントロールピンチバルブを使用することができる。フローコントロールピンチバルブは簡単な構造で容易かつ微細な流量調整ができるので好適である。   The flow rate adjusting valve 45 is preferably installed in any part of the conduit 60 and can easily and finely adjust the flow rate of the culture solution flowing in the conduit 60. For example, a flow control pinch valve can be used. The flow control pinch valve is suitable because it can easily and finely adjust the flow rate with a simple structure.

本細胞培養装置における培養室10は、公知のものを使用することができる。培養室10の内部を拍動する培養液が円滑に流れ、その培養液が培養室10の内部に設置した人工血管に適切に供給されるような培養室であればよい。なお、培養室はその内部を容易に観察することができるような、例えば、透明なアクリル製とするのがよい。   A well-known thing can be used for the culture room 10 in this cell culture apparatus. Any culture chamber may be used as long as the culture medium that pulsates inside the culture chamber 10 flows smoothly and is appropriately supplied to the artificial blood vessel installed inside the culture chamber 10. The culture chamber is preferably made of, for example, transparent acrylic so that the inside can be easily observed.

培養液槽15は、培養液中に含有される空気の排出（空気抜き）を行うため、培養液を一時貯留するためのものである。従って、培養液槽15は、密封可能で貯留培養液16の表面上に空間部17を有し、貯留培養液16に含まれる空気が十分に抜ける程度の大きさを有するものであればよい。なお、培養液槽15には、本細胞培養装置を組み立てた当初に該装置内に残留する空気を抜くため、フィルター18及び開閉弁19を設けるのがよい。また、培養液槽15は、培養液の温度を所定温度に維持するために、例えば所定温度に保持されたウォーターバスに浸漬させておくのがよい。   The culture solution tank 15 is for temporarily storing the culture solution in order to discharge (bleed out) air contained in the culture solution. Therefore, the culture medium tank 15 is not limited as long as it can be sealed, has the space 17 on the surface of the stored culture medium 16, and has a size that allows air contained in the stored culture medium 16 to escape sufficiently. The culture medium tank 15 is preferably provided with a filter 18 and an on-off valve 19 in order to remove air remaining in the apparatus when the present cell culture apparatus is assembled. The culture medium tank 15 is preferably immersed in a water bath maintained at a predetermined temperature, for example, in order to maintain the temperature of the culture liquid at a predetermined temperature.

ガス交換手段20は、公知のものを使用することができる。しかしながら、ガス交換手段20は、図１に示すように、導管60に連結されたシリコンチューブ22を酸素及び二酸化炭素分圧が所定値に保持されたガス室21、例えば公知のCO2インキュベータ内に収容し、シリコンチューブ22を介してガス交換をさせる構造のものがよい。このガス交換手段20に用いるシリコンチューブ22は、実験用に使用される公知のもので足りるが、ガス交換を十分に行うためには、ガス室21内を通すシリコンチューブ22の長さを1m以上、好ましくは1.5〜2mとするのがよい。 As the gas exchange means 20, a known one can be used. However, as shown in FIG. 1, the gas exchange means 20 has a silicon tube 22 connected to a conduit 60 in a gas chamber 21 in which oxygen and carbon dioxide partial pressures are maintained at predetermined values, for example, a known CO 2 incubator. A structure that accommodates gas and exchanges gas through the silicon tube 22 is preferable. The silicon tube 22 used for the gas exchange means 20 may be a known one used for experiments, but in order to sufficiently perform gas exchange, the length of the silicon tube 22 passing through the gas chamber 21 is 1 m or more. Preferably, the thickness is 1.5 to 2 m.

係るガス交換手段20により、従来のような細胞培養装置全体をCO2インキュベータ内に収容した構造のものと異なり、細胞培養装置を構成する培養室10及び培養液槽20の配置や大きさの自由度が増し、コンパクトな細胞培養装置を構成することができる。例えば、培養室10内の培養細胞の状態をリアルタイムに顕微鏡で観察することができる培養室10をCO2インキュベータの外に設けた細胞培養装置を容易に構成することができる。また、多数の培養室10を並列させてなる細胞培養装置を容易に構成することがでる。さらに、本細胞培養装置の組み立ては、先ず、CO2インキュベータのない無菌室で各構成部品と導管60からなる閉回路を構成し、次に、シリコンチューブ22をCO2インキュベータのサンプルポートからその内部に入れ込んで装置全体を組み立てることができるので、容易に本装置を組み立てることができる。また、一つの細胞培養装置でCO2インキュベータを使用中であっても、新たな細胞培養装置を容易に組み立てることができる。高価な中空糸人工肺を用いたガス交換装置を用いなくても、安価にガス交換手段20を構成することができるという利点もある。 Unlike the conventional structure in which the entire cell culture apparatus is accommodated in the CO 2 incubator, the gas exchange means 20 can freely arrange and size the culture chamber 10 and the culture liquid tank 20 constituting the cell culture apparatus. The degree is increased and a compact cell culture apparatus can be constructed. For example, a cell culture apparatus in which the culture chamber 10 that can observe the state of the cultured cells in the culture chamber 10 with a microscope in real time is provided outside the CO 2 incubator can be easily configured. In addition, it is possible to easily configure a cell culture apparatus in which a large number of culture chambers 10 are arranged in parallel. Furthermore, in assembling the cell culture apparatus, first, a closed circuit consisting of each component and the conduit 60 is constructed in a sterile room without a CO 2 incubator, and then the silicon tube 22 is connected from the sample port of the CO 2 incubator to its inside. It is possible to assemble the entire apparatus by inserting it into the apparatus, so that the apparatus can be easily assembled. Further, even when a CO 2 incubator is being used with one cell culture apparatus, a new cell culture apparatus can be easily assembled. There is also an advantage that the gas exchange means 20 can be constructed at low cost without using a gas exchange device using an expensive hollow fiber artificial lung.

加温手段30は、培養液の温度を所定温度に加温できるものであればよい。例えば、図１に示すように培養液が流れる螺旋状の塩化ビニールで構成された熱交換器32を恒温槽31内に設けた構造のものを使用することができる。恒温槽31には、所定温度に保持されたウォーターバスを使用することができる。   The heating means 30 only needs to be capable of heating the temperature of the culture solution to a predetermined temperature. For example, as shown in FIG. 1, the thing of the structure which provided the heat exchanger 32 comprised with the helical vinyl chloride through which a culture solution flows in the thermostat 31 can be used. As the thermostat 31, a water bath maintained at a predetermined temperature can be used.

加温手段30は、それ自体が独立した図１に示すような構造のものでなくてもよい。例えば、培養液槽15内の培養液を所定温度に加温することができる構造のものでもよい。しかしながら図１のような螺旋状の塩化ビニールで構成された熱交換器32を恒温槽31内に設けた構造のものは、培養液の加温を迅速に行うことができ、装置全体がコンパクトになり使用する培養液の量を少なくすることができるので好ましい。   The heating means 30 may not have a structure as shown in FIG. For example, a structure in which the culture solution in the culture solution tank 15 can be heated to a predetermined temperature may be used. However, the structure with the heat exchanger 32 made of spiral vinyl chloride as shown in FIG. 1 provided in the thermostatic chamber 31 can quickly heat the culture solution, and the entire apparatus is compact. It is preferable because the amount of the culture solution to be used can be reduced.

以上本発明に係る細胞培養装置は、簡単かつコンパクトな構造で培養液に所要の拍動流及び拍動圧を与えることができる。また、簡単な構造であるから容易に操作、調整することができ、維持管理にも特別の工数を要しない。さらに、各機能部分を導管60で連結して閉回路を構成しているので、各構成部品の滅菌、補修、交換等を安価かつ容易に行うことができる。なお、本細胞培養装置には、図１に示すように、培養液の流量及び圧力を調整するための流量計51、圧測定器53、温度管理のための温度計55及び培養液の交換を行うための開閉弁47を設けるのがよい。   As described above, the cell culture device according to the present invention can give a required pulsatile flow and pulsation pressure to a culture solution with a simple and compact structure. In addition, since it has a simple structure, it can be easily operated and adjusted, and no special man-hours are required for maintenance. Furthermore, since each functional part is connected by a conduit 60 to form a closed circuit, each component can be sterilized, repaired, replaced, etc. at low cost and easily. In the present cell culture apparatus, as shown in FIG. 1, a flow meter 51 for adjusting the flow rate and pressure of the culture solution, a pressure measuring device 53, a thermometer 55 for temperature management, and replacement of the culture solution are provided. An on-off valve 47 is preferably provided.

図１に示した構造の細胞培養装置を組み立て、培養液に拍動を与えた場合の圧力及び流量測定試験を行った。各構成部品は、それぞれエチレンオキサイドガスで滅菌した後、無菌室内で組み立てて細胞培養装置を構成した。培養室10はアクリル樹脂製の内容積が3.6ml（長さ×幅×高さが10×0.6×0.6cm）、培養液槽15は円筒型アクリル樹脂製の内容積が140ml（半径×高さが2.5×７cm）のものを用いた。ガス交換手段20はCO2インキュベータ（三洋電機バイオメディカ株式会社製MCO-17AIC型）内に長さ1.8m、外径8mm、内径6mmの実験用シリコンチューブを収容することによって構成し、CO2インキュベータ内は空気95vol%、二酸化炭素5vol%、湿度100%の環境に保った。加温手段30は塩化ビニール製コイル状チューブを37℃に保持したウォーターバス（アズワン株式会社製サーマルロボ）に浸漬することによって構成した。培養液供給手段40は、遠心ポンプ（株式会社イワキ製マグネットポンプMD-15R）、ファンクションゼネレータ（菊水電子工業株式会社製FGE3250）及びフローコントロールピンチバルブから構成した。さらに圧測定器（日本光電工業株式会社製LIFEKIT POLYGRAPH SYSTEM）、流量計（日本光電工業株式会社製ELECTROMAGNETIC BLOOD FLOWMETER）、温度計（アズワン株式会社製デジタル温度計）を用いた。導管60には外径8mm、内径6mmの塩化ビニールチューブを用いた。培養液は、Medium 199（GIBCO Invitrogen社製）、10%胎児ウシ血清（Hyclone Laboratories 社製）を用いた。 A cell culture apparatus having the structure shown in FIG. 1 was assembled, and a pressure and flow rate measurement test was performed when pulsation was given to the culture solution. Each component was sterilized with ethylene oxide gas and assembled in a sterile room to constitute a cell culture device. The culture chamber 10 has an acrylic resin internal volume of 3.6 ml (length x width x height 10 x 0.6 x 0.6 cm), and the culture bath 15 has a cylindrical acrylic resin internal volume of 140 ml (radius x height) Of 2.5 × 7 cm). Gas exchange means 20 is constituted by accommodating CO 2 incubator (Sanyo Biomedical Corp. MCO-17AIC type) length within 1.8 m, an outer diameter of 8 mm, a silicon tube for an inner diameter of 6mm experiments, CO 2 incubator The inside was maintained in an environment of 95 vol% air, 5 vol% carbon dioxide, and 100% humidity. The heating means 30 was configured by immersing a coiled tube made of vinyl chloride in a water bath (Astro Corporation's thermal robot) maintained at 37 ° C. The culture solution supply means 40 was composed of a centrifugal pump (Magnet pump MD-15R manufactured by Iwaki Co., Ltd.), a function generator (FGE3250 manufactured by Kikusui Electronics Co., Ltd.), and a flow control pinch valve. Furthermore, a pressure measuring instrument (LIFEKIT POLYGRAPH SYSTEM manufactured by Nihon Kohden Kogyo Co., Ltd.), a flow meter (ELECTROMAGNETIC BLOOD FLOWMETER manufactured by Nihon Koden Kogyo Co., Ltd.), and a thermometer (digital thermometer manufactured by As One Co., Ltd.) were used. For the conduit 60, a vinyl chloride tube having an outer diameter of 8 mm and an inner diameter of 6 mm was used. Medium 199 (GIBCO Invitrogen) and 10% fetal bovine serum (Hyclone Laboratories) were used as the culture solution.

図２は、遠心ポンプに正弦波状の電圧を印加し周波数1Hzの拍動が得られるように調整した場合の回路内の圧力及び流量の変化状態を示す。図２（a）は、収縮期血圧／拡張期血圧が120／70mmHg、流量が平均100mL/minに調整されている状態を示す。図２（b）は収縮期血圧／拡張期血圧が120／70mmHg、流量が平均10mL/minに調整されている状態を示し、図２（c）は収縮期血圧／拡張期血圧が40／20mmHg、流量が平均100mL/minに調整されている状態を示す。   FIG. 2 shows changes in pressure and flow rate in the circuit when a sinusoidal voltage is applied to the centrifugal pump and adjustment is made so that a pulsation with a frequency of 1 Hz is obtained. FIG. 2A shows a state in which the systolic blood pressure / diastolic blood pressure is adjusted to 120/70 mmHg and the flow rate is adjusted to an average of 100 mL / min. Fig. 2 (b) shows the systolic blood pressure / diastolic blood pressure being 120/70 mmHg and the flow rate adjusted to an average of 10 mL / min, and Fig. 2 (c) is the systolic blood pressure / diastolic blood pressure being 40/20 mmHg. The flow rate is adjusted to an average of 100 mL / min.

図３は、３日間にわたる培養液の酸素分圧と二酸化炭素分圧の変化の状態（図３（a））、pHの変化の状態（図３（b））、温度の変化の状態（図３（c））を示す。図３より、培養液は、酸素分圧が約160mmHg、二酸化炭素分圧が35〜45mmHg、pHが7．35〜7．34、温度が37℃に維持されていることが分かる。これらの数値は生体の平均的な値であり、一般に培養液が３日間ごとに交換されることを考慮すると、本細胞培養装置によれば生体を模した環境下で細胞培養を行うことができることが分かる。   FIG. 3 shows changes in oxygen partial pressure and carbon dioxide partial pressure of the culture solution over three days (FIG. 3 (a)), changes in pH (FIG. 3 (b)), and changes in temperature (FIG. 3). 3 (c)). From FIG. 3, it can be seen that the culture liquid is maintained at an oxygen partial pressure of about 160 mmHg, a carbon dioxide partial pressure of 35 to 45 mmHg, a pH of 7.35 to 7.34, and a temperature of 37 ° C. These numerical values are average values of the living body, and generally considering that the culture medium is changed every 3 days, the cell culturing apparatus can perform cell culture in an environment simulating the living body. I understand.

本発明に係る細胞培養装置のレイアウトを説明する模式図である。It is a schematic diagram explaining the layout of the cell culture apparatus which concerns on this invention. 拍動を与えた培養液の圧力と流量の変化の状態を示すグラフである。It is a graph which shows the state of the change of the pressure and flow volume of the culture solution which gave the pulsation. ３日間にわたる培養液の酸素分圧と二酸化炭素分圧の変化の状態、pHの変化の状態、温度の変化の状態を示すグラフである。It is a graph which shows the state of the change of the oxygen partial pressure of a culture solution over 3 days, the carbon dioxide partial pressure, the state of change of pH, and the state of change of temperature.

符号の説明Explanation of symbols

10 培養室
15 培養液槽
16 貯留培養液
17 空間部
18 フィルター
19 開閉弁
20 ガス交換手段
21 ガス室
22 シリコンチューブ
30 加温手段
31 恒温槽
32 熱交換器
40 培養液供給手段
41 ポンプ
43 ポンプ制御装置
45 流量調整弁
51 流量計
53 圧測定器
55 温度計
60 導管
10 Incubation room
15 Culture tank
16 Reservoir culture
17 Space
18 Filter
19 On-off valve
20 Gas exchange means
21 Gas chamber
22 Silicon tube
30 Heating means
31 Thermostatic bath
32 heat exchanger
40 Medium supply means
41 pump
43 Pump controller
45 Flow control valve
51 Flow meter
53 Pressure measuring instrument
55 Thermometer
60 conduit

Claims (2)

細胞の培養を行う培養室、培養液の一時貯留を行う培養液槽、培養液中の酸素及び二酸化炭素分圧の調整を行うガス交換手段、培養液の加温手段並びに前記培養室に培養液を供給する培養液供給手段が導管により連結されてなり、
前記ガス交換手段は、酸素及び二酸化炭素分圧が所定値に保持されたガス室と、そのガス室内に収容され前記導管に連通したシリコンチューブとを有し、そのシリコンチューブを介して前記培養室及び培養液槽に供給される培養液中の酸素及び二酸化炭素分圧を調整することができ、
前記培養液供給手段は、ポンプと、該ポンプの最大吐出量が制御できるとともに吐出量が周期的に変化するように制御することができるファンクションゼネレータと、前記導管内を流れる培養液の流量を制御することができる流量調整弁と、を備えてなる細胞培養装置。 Culture chamber for culturing the cells, the culture solution tank for temporarily storing the culture solution, a gas exchange means to adjust the oxygen and carbon dioxide partial pressure in the culture solution, culture medium heating means and said culture chamber of the culture solution The culture medium supply means for supplying
The gas exchange means includes a gas chamber in which oxygen and carbon dioxide partial pressures are maintained at predetermined values, and a silicon tube accommodated in the gas chamber and communicated with the conduit, and the culture chamber is interposed via the silicon tube. And the oxygen and carbon dioxide partial pressure in the culture solution supplied to the culture solution tank can be adjusted,
The culture solution supply means controls a pump, a function generator capable of controlling the maximum discharge amount of the pump and controlling the discharge amount periodically, and a flow rate of the culture solution flowing in the conduit A cell culture device comprising: a flow control valve capable of performing the operation. ポンプは、遠心ポンプであることを特徴とする請求項１に記載の細胞培養装置。   The cell culture device according to claim 1, wherein the pump is a centrifugal pump.
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