JP2008054603A - Cell culture apparatus - Google Patents

Cell culture apparatus Download PDF

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JP2008054603A
JP2008054603A JP2006236817A JP2006236817A JP2008054603A JP 2008054603 A JP2008054603 A JP 2008054603A JP 2006236817 A JP2006236817 A JP 2006236817A JP 2006236817 A JP2006236817 A JP 2006236817A JP 2008054603 A JP2008054603 A JP 2008054603A
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Mikio Segawa
幹雄 世川
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Shibuya Corp
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Shibuya Kogyo Co Ltd
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell culture apparatus which can well be carried and facilitates works for connecting culture containers and liquid supply containers to a liquid circuit. <P>SOLUTION: This cell culture apparatus 1 has a liquid supply circuit 3 for connecting culture containers 13, 14 to liquid supply containers 21 to 26. The liquid supply circuit 3 is formed by integrating the first branched passage 32 and the second branched passage 33 comprising a plurality of liquid delivery tubes with two sheet-like members 35. An opening portion 35A for a liquid supply pump 36 as a pump means and opening portions 35B, 35C for pinch valves PV11, PV12 as valve means are formed at prescribed positions of the sheet-like members 35. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は再生医療等で実施される細胞培養のための操作を自動化した細胞培養装置に関する。   The present invention relates to a cell culture apparatus in which operations for cell culture performed in regenerative medicine or the like are automated.

従来、細胞を培養するための培地の交換などの手作業を省略できるようにした細胞培養装置が提案されている（例えば特許文献１、特許文献２）。
こうした従来の細胞培養装置は、細胞を培養させる培養容器と、少なくとも培地と薬剤をそれぞれ収容した複数の給液容器と、上記培養容器と上記給液容器とを接続して該給液容器から培養容器に対して培地および薬剤を流入可能とする液回路とを備えて、給液容器から培養容器への送液を自働制御するようにしている。
特開２００１−２７５６５９号公報 特開２００４−８９０９５号公報 2. Description of the Related Art Conventionally, a cell culture device has been proposed in which manual work such as replacement of a medium for culturing cells can be omitted (for example, Patent Document 1 and Patent Document 2).
Such a conventional cell culture device is a culture vessel for culturing cells, a plurality of liquid supply containers each containing at least a medium and a drug, the culture container and the liquid supply container connected to each other, and culturing from the liquid supply container A liquid circuit that allows the culture medium and the drug to flow into the container is provided to automatically control the liquid feeding from the liquid supply container to the culture container.
JP 2001-275659 A JP 2004-89095 A

上記従来の細胞培養装置における液回路は複数のチューブを備えており、これら複数のチューブの一端を培養容器と接続し、他端を給液容器と接続するようにしている。
これら培養容器、給液容器、チューブは、実施する培養毎に新しいものを使用するため、そのつど細胞培養装置に決められた液回路を形成するよう接続する必要があり、作業が煩雑であり、接続ミスが生じる虞があった。 The liquid circuit in the conventional cell culture apparatus includes a plurality of tubes, and one end of each of the plurality of tubes is connected to the culture container, and the other end is connected to the liquid supply container.
Since these culture containers, liquid supply containers, and tubes use new ones for each culture to be performed, it is necessary to connect to form a liquid circuit determined for each cell culture device, and the work is complicated. There was a risk of connection errors.

上述した事情に鑑み、請求項１に記載した本発明は、培地や薬液を収容した複数の給液容器と、細胞を培養するための複数の培養容器と、これらを接続する送液用のチューブを備え、上記培養容器に対し上記チューブを介して送液するようにした細胞培養装置において、
上記チューブを介して送液するポンプ手段と、上記チューブを外部から閉塞させるバルブ手段を備えるとともに、上記チューブは、上記複数の給液容器に接続できるよう分岐された第１分岐通路と、上記複数の培養容器に接続できるよう分岐された第２分岐通路とを備えており、これら分岐通路が所定位置に位置するように上記チューブをシート状部材に配置させて固定し、該シート状部材には、固定されたチューブが上記ポンプ手段および上記バルブ手段と対応する位置に、これらポンプ手段およびバルブ手段を挿入可能な開口部が形成されていることを特徴とするものである。 In view of the circumstances described above, the present invention described in claim 1 includes a plurality of liquid supply containers containing a culture medium and a chemical solution, a plurality of culture containers for culturing cells, and a liquid-feeding tube connecting them. In a cell culture apparatus that is configured to send liquid via the tube to the culture container,
Pump means for feeding liquid through the tube, valve means for closing the tube from the outside, the tube branching so as to be connected to the plurality of liquid supply containers, and the plurality A second branch passage branched so as to be connected to the culture vessel, and the tube is arranged and fixed on the sheet-like member so that these branch passages are located at predetermined positions. The fixed tube has an opening into which the pump means and the valve means can be inserted at a position corresponding to the pump means and the valve means.

このような構成によれば、上記チューブはシート状部材に固定されて液回路は全体としてシート状となるので、可搬性が良好になるとともに、培養容器および給液容器への液回路の接続作業も容易になる。   According to such a configuration, since the tube is fixed to the sheet-like member and the liquid circuit becomes a sheet as a whole, the portability is improved, and the connection operation of the liquid circuit to the culture container and the liquid supply container Will also be easier.

以下図示実施例について本発明を説明すると、図１において１は細胞を継代培養する細胞培養装置であり、その本体１Ａの上面を示している。
この細胞培養装置１は、細胞を培養する培養部２と、給液回路３を介して上記培養部２へ培地やその他の液体を供給する給液部４と、上記培養部２で使用した液体を排液回路５を介して回収する排液部６と、滅菌ガス供給装置７および気体供給装置８と接続させた循環回路１１と、ポンプ手段として給液部４に備えた給液ポンプ３６、排液部６に備えた排液ポンプ４６、および、バルブ手段として所定位置に配置した多数のピンチバルブＰＶの作動を制御する制御装置１２とを備えており、これらがそれぞれ本体１Ａ上面の同一平面上に配置されている。 Hereinafter, the present invention will be described with reference to the illustrated embodiment. In FIG. 1, reference numeral 1 denotes a cell culture apparatus for subculturing cells, and shows the upper surface of the main body 1A.
The cell culture apparatus 1 includes a culture unit 2 for culturing cells, a liquid supply unit 4 for supplying a culture medium and other liquids to the culture unit 2 via a liquid supply circuit 3, and a liquid used in the culture unit 2. A liquid draining part 6 for collecting the liquid through a draining circuit 5, a circulation circuit 11 connected to the sterilizing gas supply device 7 and the gas supply device 8, a liquid supply pump 36 provided in the liquid supply unit 4 as pump means, A drainage pump 46 provided in the drainage unit 6 and a control device 12 for controlling the operation of a number of pinch valves PV arranged at predetermined positions as valve means are provided, each of which is on the same plane on the upper surface of the main body 1A. Is placed on top.

培養部２には、１個の一次培養容器１３と、この一次培養容器１３と同じ容器を２個並列に並べた二次培養容器１４ａ、１４ｂとが備えられ、これら一次培養容器１３と二次培養容器１４ａ、１４ｂを載置させそれらを約３７℃に加温および保温するプレートヒータからなる加温体１５が設けられている。
一次培養容器１３はほぼ直方体に形成され、その長手方向の一端側を供給口１３Ａとしてあり、他端側を排出口１３Ｂとしている。また、これと同様に各二次培養容器１４ａ、１４ｂも長手方向の一端側を供給口１４Ａとしてあり、他端側を排出口１４Ｂとしている。
そして、上記各培養容器１３、１４は並列に配置され、各供給口１３Ａ、１４Ａに給液回路３を接続し、各排出口１３Ｂ、１４Ｂに排液回路５を接続している。 The culture unit 2 includes one primary culture container 13 and secondary culture containers 14a and 14b in which two same containers as the primary culture container 13 are arranged in parallel. There is provided a heating body 15 composed of a plate heater on which the culture vessels 14a and 14b are placed and heated and maintained at about 37 ° C.
The primary culture vessel 13 is formed in a substantially rectangular parallelepiped shape, and one end side in the longitudinal direction is a supply port 13A, and the other end side is a discharge port 13B. Similarly, each of the secondary culture vessels 14a and 14b also has one end side in the longitudinal direction as a supply port 14A and the other end side as a discharge port 14B.
The culture vessels 13 and 14 are arranged in parallel, the liquid supply circuit 3 is connected to the supply ports 13A and 14A, and the drainage circuit 5 is connected to the discharge ports 13B and 14B.

給液部４には、給液容器として、接着系細胞を収容した第１容器２１と、加湿用の純水を収容した第２容器２２と、細胞の剥離剤としてトリプシンを収容した第３容器２３と、培地を収容した第４容器２４と、血清を収容した第５容器２５と、さらに培養容器の洗浄に用いるＰＢＳ（燐酸緩衝生理食塩水）を収容した第６容器２６とが備えられている。
これら各給液容器２１〜２６には、それぞれ送液用のシリコーンチューブからなる給排部２１Ａ〜２６Ａを設けてあり、各給排部２１Ａ〜２６Ａを介して給液回路３と接続するようになっている。なお、各給液容器２１〜２６の容量は各々の必要量に応じて設定されている。
さらに、各給液容器２１〜２６は、図２に示すように偏平に形成されており、各給排部２１Ａ〜２６Ａを平行に揃えた状態で、各給液容器２１〜２６を２枚の薄い矩形状のシリコーン製のシート状部材２７ａ、２７ｂで挟み込み、これらシート状部材２７ａ、２７ｂ同士を接着して固定し、一枚の偏平なシート状体２７を構成している。これにより、各給液容器２１〜２６は、給排部２１Ａ〜２６Ａの先端をシート状体２７の一辺から突出させた状態で一体化されている。 The liquid supply unit 4 includes a first container 21 containing adhesive cells as a liquid supply container, a second container 22 containing pure water for humidification, and a third container containing trypsin as a cell peeling agent. 23, a fourth container 24 containing a culture medium, a fifth container 25 containing serum, and a sixth container 26 containing PBS (phosphate buffered saline) used for washing the culture container. Yes.
Each of these liquid supply containers 21 to 26 is provided with supply / discharge portions 21A to 26A each made of a silicone tube for liquid supply, and is connected to the liquid supply circuit 3 via each of the supply / discharge portions 21A to 26A. It has become. In addition, the capacity | capacitance of each liquid supply containers 21-26 is set according to each required amount.
Furthermore, each liquid supply container 21-26 is formed flat as shown in FIG. 2, and each liquid supply container 21-26 is made into two sheets in the state which aligned each supply-and-discharge part 21A-26A in parallel. The sheet-like member 27a, 27b made of thin rectangular silicone is sandwiched between the sheet-like members 27a, 27b. The sheet-like members 27a, 27b are bonded and fixed to form a flat sheet-like body 27. Thereby, each liquid supply container 21-26 is integrated in the state which made the front-end | tip of supply / discharge part 21A-26A protrude from the one side of the sheet-like body 27. As shown in FIG.

各給液容器２１〜２６を一体化させたシート状体２７は、細胞培養装置１の本体１Ａの側部寄りの所定位置に設けられたペルチェ素子による冷却体２８上に載置されるようになっており、この冷却体２８によって各給液容器２１〜２６の内容物が常に約４℃に保冷されるようになっている。
そして、上記冷却体２８の隣接位置にシート状体２７から突出した各給排部２１Ａ〜２６Ａに対応させてピンチバルブＰＶ１〜ＰＶ６が設けられており、それらピンチバルブＰＶ１〜ＰＶ６が備えるピンチャーＰＶＡ、ＰＶＢの間に、給排部２１Ａ〜２６Ａを位置させるようになっている。
各ピンチバルブＰＶ１〜ＰＶ６は、制御装置１２によって各々が独立して作動を制御されるようになっており、制御装置１２がピンチャーＰＶＡ、ＰＶＢを開放させた状態では給排部２１Ａ〜２６Ａは流通可能となり、他方、閉鎖させた状態では給排部２１Ａ〜２６Ａは外部から閉塞されて、内部の流通が阻止されるようになっている。なお、本実施例において、全てのピンチバルブＰＶの動作、構成は共通している。 The sheet-like body 27 in which the liquid supply containers 21 to 26 are integrated is placed on the cooling body 28 by a Peltier element provided at a predetermined position near the side of the main body 1A of the cell culture device 1. The cooling body 28 keeps the contents of the liquid supply containers 21 to 26 at about 4 ° C. at all times.
And pinch valves PV1 to PV6 are provided corresponding to the respective supply / discharge portions 21A to 26A protruding from the sheet-like body 27 at positions adjacent to the cooling body 28, and the pincher PVA included in these pinch valves PV1 to PV6, The supply / discharge portions 21A to 26A are positioned between the PVBs.
The operation of each pinch valve PV1 to PV6 is controlled independently by the control device 12, and when the control device 12 opens the pinchers PVA and PVB, the supply / discharge sections 21A to 26A are in circulation. On the other hand, in the closed state, the supply / exhaust portions 21A to 26A are closed from the outside, and the internal circulation is prevented. In this embodiment, all the pinch valves PV have the same operation and configuration.

給液回路３はシリコーンチューブを連結させて構成されており、その中心として給液ポンプ３６に装着される主液通路３１を備え、この主液通路３１には上流側となる一端側で分岐させる第１分岐通路３２と、下流側となる他端側で分岐させる第２分岐通路３３とが接続されている。
第１分岐通路３２は、各給液容器２１〜２６の給排部２１Ａ〜２６Ａに対応させて６本に分岐させた分岐接続部３２Ａ〜３２Ｆを備えており、他方、第２分岐通路３３は、一次培養容器１３の供給口１３Ａと２つの二次培養容器１４ａ、１４ｂの各供給口１４Ａに対応させて３本に分岐させた分岐接続部３３Ａ〜３３Ｃを備えている。
第１分岐通路３２の分岐接続部３２Ａ〜３２Ｆは、３２Ａから順番に給液容器２１〜２６の各給排部２１Ａ〜２６Ａと着脱可能に接続されるようになっており、第２分岐通路３３の分岐接続部３３Ａ〜３３Ｃは、分岐接続部３３Ａを一次培養容器１３の供給口１３Ａに固着させ、３３Ｂを二次培養容器１４Ａの供給口１４Ａに、３３Ｃを二次培養容器１４ｂの供給口１４Ａにそれぞれ固着して接続させている。また、第１分岐通路３２には分岐接続部３２Ａと直交するように分岐接続部３２Ｇを設けてあり、該分岐接続部３２Ｇと滅菌ガス供給装置７および気体供給装置８とが接続された循環回路１１の下流側端部１１Ａと着脱可能に接続するようになっており、この分峡接続部３２Ｇに対応させてピンチバルブＰＶ７が設けられている。 The liquid supply circuit 3 is configured by connecting a silicone tube, and has a main liquid passage 31 attached to the liquid supply pump 36 at the center thereof, and the main liquid passage 31 is branched at one end side which is an upstream side. The first branch passage 32 is connected to the second branch passage 33 that branches on the other end side that is the downstream side.
The first branch passage 32 includes branch connection portions 32A to 32F branched into six corresponding to the supply / discharge portions 21A to 26A of the respective liquid supply containers 21 to 26, while the second branch passage 33 has In addition, branch connection portions 33A to 33C branched into three corresponding to the supply port 13A of the primary culture container 13 and the supply ports 14A of the two secondary culture containers 14a and 14b are provided.
The branch connection portions 32A to 32F of the first branch passage 32 are detachably connected to the supply / discharge portions 21A to 26A of the liquid supply containers 21 to 26 in order from 32A. The branch connection portions 33A to 33C fix the branch connection portion 33A to the supply port 13A of the primary culture vessel 13, 33B to the supply port 14A of the secondary culture vessel 14A, and 33C to the supply port 14A of the secondary culture vessel 14b. Are fixedly connected to each other. The first branch passage 32 is provided with a branch connection portion 32G orthogonal to the branch connection portion 32A, and the circulation circuit in which the branch connection portion 32G is connected to the sterilizing gas supply device 7 and the gas supply device 8. 11 is detachably connected to the downstream end portion 11A, and a pinch valve PV7 is provided corresponding to the branch connection portion 32G.

培養部２と給液部４の間には、一次培養容器１３および二次培養容器１４ａ、１４ｂに接続された分岐接続部３３Ａ〜３３Ｃの各位置に対応させてピンチバルブＰＶ８〜ＰＶ１０を設けており、制御装置１２の指令によりこれらを開閉作動させることで、分岐接続部３３Ａ〜３３Ｃの内部の流通を開放、遮断するようになっている。
また、給液部４には給液回路３の主液通路３１に対応させて給液ポンプ３６が設けられており、主液通路３１のチューブを湾曲させて該給液ポンプ３６のガイド部３６Ａに装着し、ローター３６Ｂを回転（図１の場合は時計回転）させることで、主液通路３１を構成するチューブが外部から扱かれて該チューブを介して給液容器２１〜２６側から培養容器１３、１４ａ、１４ｂの方向へ送液できるようになっている。よって、制御装置１２の指令によってピンチバルブＰＶ１〜ＰＶ６のいずれかと、ピンチバルブＰＶ８〜ＰＶ１０のいずれかを開放させた状態で、給液ポンプ３６を作動させることで、給液容器２１〜２６の必要な液体を培養容器１３、１４ａ、１４ｂのいずれかに供給することができるようになっている。
給液回路３にはさらに、第２分岐通路３３の分岐接続部３３Ａと主液通路３１の給液ポンプ３６の上流側とを直接的に接続する戻し通路３４を備えている。また、該戻し通路３４に対応させてピンチバルブＰＶ１１が設けられるとともに、分岐接続部３３Ａと分岐接続部３３Ｂの間で第２分岐通路３３を閉塞可能なピンチバルブＰＶ１２が設けられている。該戻し通路３４は一次培養容器１３から二次培養容器１４ａ、１４ｂへ送液を行う場合に使用し、ピンチバルブＰＶ８、ＰＶ９、ＰＶ１１を開放し、ＰＶ１０、ＰＶ１２を閉鎖した状態で給液ポンプ３６を回転させることで、戻し通路３４を流通させて一次培養容器１３から二次培養容器１４ａへの送液が可能となり、この状態からＰＶ９を閉鎖させてＰＶ１０を開放することで一次培養容器１３から二次培養容器１４bへの送液が可能となる。 Between the culture part 2 and the liquid supply part 4, pinch valves PV8 to PV10 are provided corresponding to the respective positions of the branch connection parts 33A to 33C connected to the primary culture container 13 and the secondary culture containers 14a and 14b. In addition, by opening and closing them according to a command from the control device 12, the flow in the branch connection portions 33A to 33C is opened and shut off.
The liquid supply unit 4 is provided with a liquid supply pump 36 corresponding to the main liquid passage 31 of the liquid supply circuit 3. The tube of the main liquid passage 31 is curved to guide the 36 A of the liquid supply pump 36. The tube constituting the main liquid passage 31 is handled from the outside by rotating the rotor 36B (clockwise in the case of FIG. 1), and the culture vessel is supplied from the side of the liquid supply containers 21 to 26 through the tube. The liquid can be fed in the direction of 13, 14a, 14b. Therefore, it is necessary to supply the liquid supply containers 21 to 26 by operating the liquid supply pump 36 in a state where any one of the pinch valves PV1 to PV6 and any one of the pinch valves PV8 to PV10 is opened by a command of the control device 12. Liquid can be supplied to any one of the culture vessels 13, 14a, 14b.
The liquid supply circuit 3 further includes a return passage 34 that directly connects the branch connection portion 33A of the second branch passage 33 and the upstream side of the liquid supply pump 36 of the main liquid passage 31. Further, a pinch valve PV11 is provided corresponding to the return passage 34, and a pinch valve PV12 capable of closing the second branch passage 33 between the branch connection portion 33A and the branch connection portion 33B is provided. The return passage 34 is used when liquid is fed from the primary culture container 13 to the secondary culture containers 14a and 14b. The liquid supply pump 36 is opened with the pinch valves PV8, PV9 and PV11 open and the PV10 and PV12 closed. By rotating the return passage 34, the liquid can be fed from the primary culture vessel 13 to the secondary culture vessel 14a. From this state, the PV 9 is closed and the PV 10 is opened to release the PV 10 from the primary culture vessel 13. The liquid can be sent to the secondary culture container 14b.

この様な給液回路３もまた、図２に示すように、複数のシリコーンチューブを連結させて、第１分岐通路３２、主液通路３１、第２分岐通路３３、戻し通路３４を構成し、これを２枚の薄い矩形状のシリコーン製のシート状部材３５ａ、３５bで上下から挟み込み、これらシート状部材３５ａ、３５ｂ同士を接着して、必要な配置に位置決めした状態にてチューブを固定し、１枚の偏平なシート状体３５を構成して一体化させている。この様なシー卜状体３５においては、その一辺には第１分岐通路３２の分岐接続部３２Ａ〜３２Ｆが、給液容器２１〜２６の各給排部２１Ａ〜２６Ａと着脱可能となるように開口され、これと対向する辺からは分岐接続部３３Ａ〜３３Ｃが突出されて、培養容器１３、１４の供給口１３Ａ、１４Ａと固着されている。また、分岐接続部３２Ａ〜３２Ｆが配置された上記一辺と、分岐接続部３２Ａ側で交差する辺には分岐接続部３２Ｇが突出されている。
また、該シート状体３５においては、給液ポンプ３６の位置に対応する部分を矩形状にくり抜き、該給液ポンプ３６を挿入可能な開口部３５Ａとしてあり、シート状体３５を細胞培養装置１の本体１Ａ上に載置させた際に、給液ポンプ３６が開口部３５Ａ内に位置するようになっている。開口部３５Ａには主液通路３１を構成するチューブが給液ポンプ３６の装着に必要な長さだけ引き出されて露出されている。さらに、ピンチバルブＰＶ１１、ＰＶ１２の位置に対応する部分も矩形状にくり抜いて、これらピンチバルブＰＶ１１、ＰＶ１２を挿入可能な開口部３５Ｂ、３５Ｃとしてあり、シート状体３５を細胞培養装置１の本体１Ａ上に載置させた際に、開口部３５ＢにはピンチバルブＰＶ１１が位置し、開口部３５ＣにはピンチバルブＰＶ１２が位置するようになっている。開口部３５Ｂには戻り通路３４のチューブが露出され、開口部３５Ｃには分岐接続部３３Ａと分岐接続部３３Ｂの間となる第２分岐通路３３のチューブが露出されている。 Such a liquid supply circuit 3 also includes a first branch passage 32, a main liquid passage 31, a second branch passage 33, and a return passage 34 by connecting a plurality of silicone tubes, as shown in FIG. This is sandwiched from above and below by two thin rectangular silicone sheet-like members 35a, 35b, these sheet-like members 35a, 35b are bonded together, and the tube is fixed in a state where it is positioned as required, One flat sheet 35 is constructed and integrated. In such a sheath-like body 35, branch connection portions 32 </ b> A to 32 </ b> F of the first branch passage 32 can be attached to and detached from the supply / discharge portions 21 </ b> A to 26 </ b> A of the liquid supply containers 21 to 26 on one side thereof. The branch connection portions 33A to 33C protrude from the side opposite to the opening and are fixed to the supply ports 13A and 14A of the culture vessels 13 and 14, respectively. Further, a branch connection portion 32G protrudes from the side where the branch connection portions 32A to 32F are arranged and the side intersecting on the branch connection portion 32A side.
In addition, in the sheet-like body 35, a portion corresponding to the position of the liquid supply pump 36 is cut out in a rectangular shape to form an opening 35A into which the liquid supply pump 36 can be inserted. The liquid supply pump 36 is positioned in the opening 35A when placed on the main body 1A. A tube constituting the main liquid passage 31 is drawn out through the opening 35A by a length necessary for mounting the liquid supply pump 36 and exposed. Furthermore, the portions corresponding to the positions of the pinch valves PV11 and PV12 are also cut out in a rectangular shape to form openings 35B and 35C into which the pinch valves PV11 and PV12 can be inserted. The sheet-like body 35 is used as the main body 1A of the cell culture device 1. When placed on top, the pinch valve PV11 is positioned in the opening 35B, and the pinch valve PV12 is positioned in the opening 35C. The tube of the return passage 34 is exposed at the opening 35B, and the tube of the second branch passage 33 between the branch connection portion 33A and the branch connection portion 33B is exposed at the opening 35C.

排液部６を構成する排液回路５も、上記給液回路３と同様にシリコーンチューブを連結させて構成されており、その中心として排液ポンプ４６に装着される主排液通路４３と、この主排液通路４３を上流側で分岐させる排液分岐通路４４とを備えている。排液分岐通路４４は３本の分岐接続部４４Ａ〜４４Ｃに分岐させてあり、分岐接続部４４Ａを一次培養容器１３の排液口１３Ｂに固着させ、４４Ｂを二次培養容器１４ａの排液ロ１４Ｂに、４４Ｃを二次培養容器１４ｂの排液口１４Ｂにそれぞれ固着させて接続している。また、排液分岐通路４４には分岐接続部４４Ａと直交するように分岐接続部４４Ｄを設けてあり、該分岐接続部４４Ｄと滅菌ガス供給装置７および気体供給装置８と接続された循環回路１１の上流側端部１１Ｂと着脱可能に接続するようになっている。   The drainage circuit 5 constituting the drainage unit 6 is also configured by connecting a silicone tube in the same manner as the liquid supply circuit 3, and a main drainage passage 43 attached to the drainage pump 46 as the center thereof, A drainage branch passage 44 that branches the main drainage passage 43 on the upstream side is provided. The drainage branch passage 44 is branched into three branch connection portions 44A to 44C. The branch connection portion 44A is fixed to the drainage port 13B of the primary culture vessel 13, and 44B is drained from the secondary culture vessel 14a. 44B is fixedly connected to the drainage port 14B of the secondary culture vessel 14b. Further, the drainage branch passage 44 is provided with a branch connection portion 44D so as to be orthogonal to the branch connection portion 44A, and the circulation circuit 11 connected to the branch connection portion 44D and the sterilizing gas supply device 7 and the gas supply device 8. It connects with the upstream edge part 11B of this machine so that attachment or detachment is possible.

培養部２と排液部６の間には、一次培養容器１３および二次培養容器１４ａ、１４ｂに接続された分岐接続部４４Ａ〜４４Ｃの各位置に対応させてピンチバルブＰＶ１３〜ＰＶ１５を設けており、制御装置１２の指令によりこれらを開閉作動させることで、分岐接続部４４Ａ〜４４Ｃの内部の流通を開放、遮断するようになっている。また、分岐接続部４４Ｄの位置に対応させてピンチバルブＰＶ１６を設けている。
排液部６には排液回路５の主排液通路４３に対応させて排液ポンプ４６が設けられており、主排液通路４３のチューブを湾曲させて該排液ポンプ４６のガイド部４６Ａに装着し、ローター４６Ｂを回転（図１の場合は時計回転）させることで、主排液通路４３を構成するチューブが外部から扱かれて、該チューブを介して両培養容器１３、１４ａ、１４ｂ側から、主排液通路４３の下流側端部４３Ｂに接続した排液容器６Ａの方向へ送液できるようになっている。さらに、排液部６には排液ポンプ４６の手前に位置させてピンチバルブＰＶ１７を設けており、該ピンチバルブＰＶ１７により主排液通路４３を排液ポンプ４６の上流側で開閉するようになっている。
これにより、制御装置１２の指令によってピンチバルブＰＶ１３〜ＰＶ１５のいずれかと、ピンチバルブＰＶ１７を開放させた状態で、排液ポンプ４６を作動させることで、培養容器１３、１４ａ、１４ｂのいずれかから排液容器６Ａに排液できるようになっている。 Between the culture part 2 and the drainage part 6, pinch valves PV13 to PV15 are provided corresponding to the positions of the branch connection parts 44A to 44C connected to the primary culture container 13 and the secondary culture containers 14a and 14b. In addition, by opening and closing them according to a command from the control device 12, the flow in the branch connection portions 44A to 44C is opened and shut off. A pinch valve PV16 is provided in correspondence with the position of the branch connection portion 44D.
The drainage unit 6 is provided with a drainage pump 46 corresponding to the main drainage passage 43 of the drainage circuit 5. The tube of the main drainage passage 43 is bent to guide the guide unit 46 </ b> A of the drainage pump 46. And the tubes constituting the main drainage passage 43 are handled from the outside by rotating the rotor 46B (clockwise in the case of FIG. 1), and both culture vessels 13, 14a, 14b are passed through the tubes. From the side, liquid can be fed in the direction of the drainage container 6 </ b> A connected to the downstream end 43 </ b> B of the main drainage passage 43. Further, the drainage section 6 is provided with a pinch valve PV17 positioned in front of the drainage pump 46, and the main drainage passage 43 is opened and closed upstream of the drainage pump 46 by the pinch valve PV17. ing.
As a result, the drainage pump 46 is operated in a state where any one of the pinch valves PV13 to PV15 and the pinch valve PV17 are opened according to a command from the control device 12, thereby draining from any of the culture vessels 13, 14a, 14b. The liquid can be drained into the liquid container 6A.

この様な排液回路５についても給液回路３と同様に、複数のシリコーンチューブを連結させて、排液分岐通路４４、主排液通路４３を構成し、これを２枚の薄い矩形状のシリコーン製のシー卜状部材４５ａ、４５ｂで上下から挟み込み、これらシート状部材４５ａ、４５ｂ同士を接着して、必要な配置に位置決めした状態にてチューブを固定し、１枚の扁平なシート状体４５を構成して一体化させている。この様なシート状体４５においては、その一辺には排液分岐通路４４の分岐接続部４４Ａ〜４４Ｃが突出されて培養容器１３、１４ａ、１４ｂの排液口１３Ｂ、１４Ｂと固着され、これと対向する辺には主排液通路４３の下流側端部４３Ｂが突出されている。さらに、分岐接続部４４Ａ〜４４Ｃが突出された一辺と交差する分岐接続部４４Ａ側の辺には、分岐接続部４４Ｄが突出されている。   Similarly to the liquid supply circuit 3, such a drain circuit 5 is configured by connecting a plurality of silicone tubes to form a drain branch passage 44 and a main drain passage 43, which are formed into two thin rectangular shapes. One flat sheet-like body that is sandwiched from above and below by silicone sheath-like members 45a and 45b, and that the sheet-like members 45a and 45b are bonded to each other, and the tube is fixed in a required arrangement. 45 is configured and integrated. In such a sheet-like body 45, branch connection portions 44A to 44C of the drainage branch passage 44 protrude on one side and are fixed to the drainage ports 13B and 14B of the culture vessels 13, 14a and 14b. A downstream end 43 </ b> B of the main drainage passage 43 protrudes from the opposite side. Further, a branch connection portion 44D protrudes from the side on the branch connection portion 44A side that intersects one side from which the branch connection portions 44A to 44C protrude.

また、該シート状体４５においては、排液ポンプ４６の位置およびこの上流側に隣接するピンチバルブＰＶ１７の位置に対応する部分を、両者を網羅するように矩形を合わせて多角形状にくり抜き、これら排液ポンプ４６、ピンチバルブＰＶ１７を挿入可能な開口部４５Ａとしてあり、シート状体４５を細胞培養装置１の本体１Ａ上に載置させた際に、排液ポンプ４６およびピンチバルブＰＶ１７が開口部４５Ａ内に位置するようになっている。開口部４５Ａには主排液通路４３を構成するチューブが、ピンチバルブＰＶ１７のピンチャーＰＶＡ、ＰＶＢの間に位置され、さらに排液ポンプ４６の装着に必要な長さだけ引き出されて露出されている。   Further, in the sheet-like body 45, a portion corresponding to the position of the drainage pump 46 and the position of the pinch valve PV17 adjacent to the upstream side is cut out into a polygonal shape by combining rectangles so as to cover both, The drainage pump 46 and the pinch valve PV17 serve as an opening 45A into which the drainage pump 46 and the pinch valve PV17 are opened when the sheet 45 is placed on the main body 1A of the cell culture device 1. It is located within 45A. A tube constituting the main drainage passage 43 is positioned between the pinchers PVA and PVB of the pinch valve PV17 in the opening 45A, and is further drawn out and exposed by a length necessary for mounting the drainage pump 46. .

循環回路１１は、細胞培養装置１の本体上１Ａに設けられた滅菌ガス供給装置７および気体供給装置８と接続されて、細胞培養装置１に設けられて、その上流側端部１１Ｂは排液回路５と接続され、下流側端部１１Ａは給液回路３と接続されるようになっている。
循環回路１１は上流側端部１１Ｂの下流で第１通路４１と第２通路４２に分岐され、第１通路４１は滅菌ガス供給装置７と接続され、第２通路４２は気体供給装置８に接続されている。また．第１通路４１と第２通路４２は、滅菌ガス供給装置７および気体供給装置８の下流側で合流されて下流側端部１１Ａに至っている。
滅菌ガス供給装置７の上流側には、第１通路４１に対応させてピンチバルブＰＶ１８が、下流側にはピンチバルブＰＶ１９がそれぞれ設けられ、気体供給装置８の上流側には、第２通路４２に対応させてピンチバルブＰＶ２０が、下流側にはピンチバルブＰＶ２１がそれぞれ設けられている。
滅菌ガス供給装直７は過酸化水素蒸気等の滅菌ガスを発生させ、循環回路１１から給液回路３を介して培養容器１３、１４ａ、１４ｂへ供給し、排液回路５から回収するようになっている。また、気体供給装置８は第２容器２２の純水を供給されて蒸気を発生させ、これをボンベ等から供給された二酸化炭素とともに循環回路１１から給液回路３を介して培養容器１３、１４ａ、１４ｂへ供給し、排液回路５から回収してこれを循環させるようになっている。このような気体供給装置８では、その内部で回路内の圧力を検出して蒸気供給を制御するとともに、二酸化炭素濃度を検出して二酸化炭素の供給を制御することで、培養容器１３、１４ａ、１４ｂ内を湿度１００％に維持するとともに二酸化炭素濃度を所定濃度に保つようにしている。 The circulation circuit 11 is connected to the sterilization gas supply device 7 and the gas supply device 8 provided on the main body 1A of the cell culture device 1, and is provided in the cell culture device 1, and its upstream end 11B is drained. Connected to the circuit 5, the downstream end 11 </ b> A is connected to the liquid supply circuit 3.
The circulation circuit 11 is branched into a first passage 41 and a second passage 42 downstream of the upstream end portion 11B, the first passage 41 is connected to the sterilizing gas supply device 7, and the second passage 42 is connected to the gas supply device 8. Has been. Also. The first passage 41 and the second passage 42 are joined downstream of the sterilizing gas supply device 7 and the gas supply device 8 to reach the downstream end portion 11A.
A pinch valve PV18 is provided on the upstream side of the sterilizing gas supply device 7 in correspondence with the first passage 41, a pinch valve PV19 is provided on the downstream side, and a second passage 42 is provided on the upstream side of the gas supply device 8. A pinch valve PV20 and a pinch valve PV21 are provided on the downstream side.
The sterilization gas supply device 7 generates a sterilization gas such as hydrogen peroxide vapor and supplies the sterilization gas from the circulation circuit 11 to the culture vessels 13, 14 a and 14 b via the liquid supply circuit 3 and collects it from the drainage circuit 5. It has become. Further, the gas supply device 8 is supplied with pure water from the second container 22 to generate steam, and this is combined with carbon dioxide supplied from a cylinder or the like to the culture containers 13 and 14a from the circulation circuit 11 through the liquid supply circuit 3. , 14b, recovered from the drain circuit 5 and circulated. In such a gas supply device 8, the pressure in the circuit is detected inside to control the vapor supply, and the carbon dioxide concentration is detected to control the supply of carbon dioxide, whereby the culture vessels 13, 14 a, The inside of 14b is maintained at 100% humidity, and the carbon dioxide concentration is maintained at a predetermined concentration.

以上のように構成した細胞培養装置１によって細胞を培養する場合の作業手順と作動を説明する。
上記細胞培養装置１による細胞の培養作業の開始前の状態では、各給液容器２１〜２６、給液回路３、一次培養容器１３，二次培養容器１４ａ、１４ｂおよび排液回路５は、細胞培養装置１の本体１Ａに取付けられておらず、各給液容器２１〜２６はシート状体２７として一体化された状態で、予め滅菌されて包装されている。また、シート状体３５として一体化された給液回路３、および、シート状体４５として一体化された排液回路５は、それぞれ培養容器１３、１４ａ、１４ｂに接続された状態にて予め滅菌されて、シート状体３５,４５をそれぞれ培養容器１３，１４に重ねるように折り畳んだ状態で包装されている。
先ず、作業者はクリーンベンチ等の無菌空間内で、第１給液容器２１〜第６給液容器２６にそれぞれ収容すべき液体を各給排部２１Ａ〜２６Ａを介して注入し、各給排部２１Ａ〜２６Ａを図示しないピンチコックで挟んで密封する。
また、培養容器１３、１４ａ、１４ｂを培養部２の加温体１５上に載置するとともに、シート状体３５からなる給液回路３を給液部４の所定位置に装着し、シート状体４５からなる排液回路５を排液部６の所定位置に装着する。 The operation procedure and operation in the case of culturing cells with the cell culture device 1 configured as described above will be described.
In the state before the start of the cell culture operation by the cell culture apparatus 1, each of the liquid supply containers 21 to 26, the liquid supply circuit 3, the primary culture container 13, the secondary culture containers 14a and 14b, and the drain circuit 5 The liquid supply containers 21 to 26 are not attached to the main body 1 </ b> A of the culture apparatus 1, and are sterilized and packaged in advance in an integrated state as a sheet-like body 27. Further, the liquid supply circuit 3 integrated as the sheet-like body 35 and the drain circuit 5 integrated as the sheet-like body 45 are sterilized in advance in a state of being connected to the culture vessels 13, 14a, 14b, respectively. Thus, the sheet-like bodies 35 and 45 are packaged in a folded state so as to overlap the culture vessels 13 and 14, respectively.
First, an operator injects the liquids to be stored in the first liquid supply container 21 to the sixth liquid supply container 26 through the supply / discharge sections 21A to 26A in an aseptic space such as a clean bench. The parts 21A to 26A are sealed with a pinch cock (not shown).
In addition, the culture vessels 13, 14 a, and 14 b are placed on the warming body 15 of the culture unit 2, and the liquid supply circuit 3 including the sheet-like body 35 is mounted at a predetermined position of the liquid supply unit 4, A drainage circuit 5 consisting of 45 is mounted at a predetermined position of the drainage section 6.

給液回路３の装着にあたっては、第２分岐通路３３の各分岐接続部３３Ａ〜３３Ｃをそれぞれに対応する開放状態のピンチバルブＰＶ８〜ＰＶ１０のピンチャーＰＶＡ、ＰＶＢ間に位置させる。また、シート状体３５の開口部３５Ｂに露出する戻し通路３４のチューブをピンチバルブＰＶ１１のピンチャーＰＶＡ、ＰＶＢ間に位置させるとともに、開口部３５Ｃに露出する第２分岐通路３３のチューブをピンチバルブＰＶ１２のピンチャーＰＶＡ、ＰＶＢ間に位置させる。
さらに開口部３５Ａに露出する主液通路３１のチューブを、湾曲させてポンプ３６のガイド部３６Ａに装着させる。また、第１分岐通路３２の分岐接続部３２Ｇを循環回路１１の下流側端部１１Ａに接続するとともに、ピンチバルブＰＶ７のピンチャーＰＶＡ、ＰＶＢの間に位置させる。
一方、排液回路５の装着にあたっては、排液分岐通路４４の各分岐接続部４４Ａ〜４４Ｃをそれぞれに対応する開放状態のピンチバルブＰＶ１３〜ＰＶ１５のピンチャーＰＶＡ、ＰＶＢ間に位置させ、さらに、開口部４５Ａに露出する主排液通路４３のチューブをピンチバルブＰＶ１７のピンチャーＰＶＡ，ＰＶＢ間に位置させるとともに、湾曲させて排液ポンプ４６のガイド部４６Ａに装着させる。
また、主排液通路４３の下流側端部４３Ｂを排液容器６Ａに接続するとともに、分岐接続部４４Ｄを循環回路１１の上流側端部１１Ｂに接続するとともに、ピンチバルブＰＶ１６のピンチャーＰＶＡ、ＰＶＢ間に位置させる。 In mounting the liquid supply circuit 3, the branch connection portions 33A to 33C of the second branch passage 33 are positioned between the pinchers PVA and PVB of the corresponding pinch valves PV8 to PV10 in the open state. Further, the tube of the return passage 34 exposed at the opening 35B of the sheet-like body 35 is positioned between the pinchers PVA and PVB of the pinch valve PV11, and the tube of the second branch passage 33 exposed at the opening 35C is pinched by the pinch valve PV12. Between the pinchers PVA and PVB.
Further, the tube of the main liquid passage 31 exposed in the opening 35A is bent and attached to the guide portion 36A of the pump 36. Further, the branch connection portion 32G of the first branch passage 32 is connected to the downstream end portion 11A of the circulation circuit 11, and is positioned between the pinchers PVA and PVB of the pinch valve PV7.
On the other hand, when the drainage circuit 5 is mounted, the branch connection portions 44A to 44C of the drainage branch passage 44 are positioned between the corresponding pinchers PVA and PVB of the open pinch valves PV13 to PV15, and the openings The tube of the main drainage passage 43 exposed to the part 45A is positioned between the pinchers PVA and PVB of the pinch valve PV17, and is bent and attached to the guide part 46A of the drainage pump 46.
Further, the downstream end portion 43B of the main drainage passage 43 is connected to the drainage container 6A, the branch connection portion 44D is connected to the upstream end portion 11B of the circulation circuit 11, and the pinchers PVA and PVB of the pinch valve PV16 are connected. Position between.

また、シート状体２７からなる各容器２１〜２６については、給液部４の冷却体２８上に載置させ、かつすでに装着されている給液回路３の各分岐接続部３２Ａ〜３２Ｆと対向する各給排部２１Ａ〜２６Ａを各々接続するとともに、ピンチコックで挟んだ位置よりも先で各ピンチバルブＰＶ１〜ＰＶ６のピンチャーＰＶＡ、ＰＶＢ間に位置させてから、制御装置１２を介した手動操作によりピンチバルブＰＶ１〜ＰＶ６を一斉に閉塞状態とし、その後で各給排部２１Ａ〜２６Ａからピンチコックを取り外す。   In addition, the containers 21 to 26 made of the sheet-like body 27 are placed on the cooling body 28 of the liquid supply unit 4 and face the branch connection portions 32A to 32F of the liquid supply circuit 3 that is already mounted. Each of the supply / discharge portions 21A to 26A to be connected to each other and positioned between the pinchers PVA and PVB of the pinch valves PV1 to PV6 before the position sandwiched by the pinch cocks, and then manually operated via the control device 12 To simultaneously close the pinch valves PV1 to PV6, and then remove the pinch cocks from the supply / discharge portions 21A to 26A.

以上のようにして、各給液容器２１〜２６、給液回路３、培養容器１３、１４ａ、１４ｂ、排液回路５の細胞培養装置１本体１Ａへの装着が完了したら、培養作業を開始する前に、今回接続したチューブの接続箇所および気体供給装置８の滅菌を行う。
すなわち、制御装置１２は、ピンチバルブＰＶ１〜ＰＶ６は閉鎖したまま、ピンチバルブＰＶ１９、ＰＶ７、ＰＶ１２、ＰＶ８、ＰＶ１３、ＰＶ１６、ＰＶ１８を開放させ、他のピンチバルブＰＶは閉鎖した状態にて、滅菌ガス供給装置７および給液ポンプ３６を作動させる。これにより、滅菌ガスが給液回路３から一次培養容器１３を通過し、排液回路５を経て循環回路１１へ流通する。これにより、今回の接続箇所に滅菌ガスが行き渡り滅菌される。制御装置１２は所定時間の間、滅菌ガスを循環させると、滅菌ガスに替えて無菌エアを循環させてエアレーションにより滅菌ガス成分を除去する。
接続箇所の滅菌が終了したら、ピンチバルブＰＶ７、ＰＶ１２、ＰＶ８、ＰＶ１３、ＰＶ１６を閉じてＰＶ２１、ＰＶ２０を開き、気体供給装置８を対象に滅菌とエアレーションを行う。 As described above, when the liquid supply containers 21 to 26, the liquid supply circuit 3, the culture containers 13, 14a and 14b, and the drainage circuit 5 are attached to the cell culture apparatus 1 main body 1A, the culture work is started. Before, the connection part of the tube connected this time and the gas supply apparatus 8 are sterilized.
That is, the control device 12 opens the pinch valves PV19, PV7, PV12, PV8, PV13, PV16, and PV18 while the pinch valves PV1 to PV6 are closed, while the other pinch valves PV are closed, and the sterilizing gas The supply device 7 and the liquid supply pump 36 are operated. As a result, the sterilizing gas passes through the primary culture container 13 from the liquid supply circuit 3 and flows to the circulation circuit 11 through the drain circuit 5. As a result, the sterilized gas is spread over the current connection location and sterilized. When the sterilization gas is circulated for a predetermined time, the control device 12 circulates aseptic air instead of the sterilization gas and removes the sterilization gas component by aeration.
When the sterilization of the connection portion is completed, the pinch valves PV7, PV12, PV8, PV13, and PV16 are closed, PV21 and PV20 are opened, and the gas supply device 8 is sterilized and aerated.

このようにして、滅菌操作が終了すると、制御装置１２はピンチバルブＰＶ２、ＰＶ１２、ＰＶ８、ＰＶ１３、ＰＶ１６、ＰＶ２０を開放させ、他のピンチバルブＰＶは閉鎖させた状態で給液ポンプ３６を作動させて、第２容器２２内の純水を気体供給装置８に内蔵したバッファタンクに送液する。
気体供給装置８に純水が供給されたら、一次培養のための細胞の播種を行う。すなわち、制御装置１２はピンチバルブＰＶ２を閉じてピンチバルブＰＶ１を開き、また、ピンチバルブＰＶ１６、ＰＶ２０を閉じてＰＶ１７を開いて給液ポンプ３６および排液ポンプ４６を作動させる。これにより、第１容器２１内の細胞が一次培養容器１３に供給される。これに引き続き、ピンチバルブＰＶ１を閉じてピンチバルブＰＶ５を開いて、一次培養容器１３に所定量の血清を供給し、さらにピンチバルブＰＶ５を閉じてピンチバルブＰＶ４を開いて所定量の培地を供給する。なお、一次培養容器１３への供給の間、排液ポンプ４６を作動させることで、容器やチューブ内に収容されている気体を、ベントフィルタを介して外気に開放されている排液容器６Ａに送気している。 Thus, when the sterilization operation is completed, the control device 12 opens the pinch valves PV2, PV12, PV8, PV13, PV16, PV20 and operates the liquid supply pump 36 with the other pinch valves PV closed. Then, the pure water in the second container 22 is sent to a buffer tank built in the gas supply device 8.
When pure water is supplied to the gas supply device 8, seeding of cells for primary culture is performed. That is, the control device 12 closes the pinch valve PV2 and opens the pinch valve PV1, and closes the pinch valves PV16 and PV20 and opens PV17 to operate the liquid supply pump 36 and the drainage pump 46. Thereby, the cells in the first container 21 are supplied to the primary culture container 13. Subsequently, the pinch valve PV1 is closed and the pinch valve PV5 is opened to supply a predetermined amount of serum to the primary culture vessel 13, and the pinch valve PV5 is closed and the pinch valve PV4 is opened to supply a predetermined amount of medium. . In addition, by operating the drainage pump 46 during the supply to the primary culture container 13, the gas stored in the container or the tube is transferred to the drainage container 6A that is opened to the outside air through the vent filter. I'm airing.

このように、細胞、血清、培地が一次培養容器１３に供給されたら、制御装置１２は、ピンチバルブＰＶ２１、ＰＶ７、ＰＶ１２、ＰＶ８、ＰＶ１３、ＰＶ１６、ＰＶ２０を開放させ、その他のピンチバルブＰＶは閉鎖させ、給液ポンプ３６は作動させた状態として、循環回路１１の第２通路４２、給液回路３、一次培養容器１３、排液回路５から再び第２通路４２という循環経路を形成し、気体供給装置８から蒸気と二酸化炭素を供給して循環させるようになっている。このようにして一次培養容器１３内で所定時間にわたって一次培養が行われ、この培養中においては、加温体１５によって一次培養容器１３は３７℃に保温されており、また、上記循環経路は閉回路として形成されているため、気体供給装置８で蒸気を発生させることで、一次培養容器１３内の湿度をほぼ１００％に保つことができる。また、二酸化炭素を循環させながら、気体供給装置８で濃度を測定し消費分を補うため、一次培養容器１３内を必要な二酸化炭素濃度に保つことができる。また、気体供給装置８で圧力を測定し、圧が高まり過ぎた場合には、ピンチバルブＰＶ１７を開放し、排液ポンプ４６を作動させて圧を大気圧に下げるようにする。   As described above, when cells, serum, and culture medium are supplied to the primary culture container 13, the control device 12 opens the pinch valves PV21, PV7, PV12, PV8, PV13, PV16, PV20, and closes the other pinch valves PV. Then, the supply pump 36 is operated, and a circulation path called the second passage 42 is formed again from the second passage 42 of the circulation circuit 11, the supply circuit 3, the primary culture vessel 13, and the drain circuit 5, Steam and carbon dioxide are supplied from the supply device 8 and circulated. Thus, the primary culture is performed in the primary culture container 13 for a predetermined time. During the culture, the primary culture container 13 is kept at 37 ° C. by the warming body 15 and the circulation path is closed. Since it is formed as a circuit, the humidity in the primary culture vessel 13 can be maintained at almost 100% by generating steam with the gas supply device 8. Moreover, since the density | concentration is measured with the gas supply apparatus 8 and a consumption part is supplemented, circulating the carbon dioxide, the inside of the primary culture container 13 can be maintained at a required carbon dioxide density | concentration. Further, when the pressure is measured by the gas supply device 8 and the pressure is excessively increased, the pinch valve PV17 is opened, and the drainage pump 46 is operated to reduce the pressure to the atmospheric pressure.

一次培養容器１３で培養を開始してから所定時間が経過したら、制御装置１２は排液部６のピンチバルブＰＶ１３、ＰＶ１７を開放するとともに、ピンチバルブＰＶ１６、ＰＶ２０は閉じて排液ポンプ４６を所定時間作動させ、一次培養容器１３の培地を排液容器６へ排出させる。なお、この際には細胞は一次培養容器１３の底部に接着しており流出することはない。
この後、制御装置１２は、給液ポンプ３６は作動させたまま、ピンチバルブＰＶ２１、ＰＶ７は閉鎖してピンチバルブＰＶ５を開放し、一次培養容器１３に給液容器２５から血清を所定量供給するとともに、引き続いてピンチバルブＰＶ５を閉じてＰＶ４を開き、給液容器２４から培地を所定量供給する。これにより、培地交換作業が完了し、再び気体供給装置８から蒸気と二酸化炭素を供給させる循環経路を形成して、一次培養が継続される。 When a predetermined time elapses after the culture is started in the primary culture vessel 13, the control device 12 opens the pinch valves PV13 and PV17 of the drainage unit 6, closes the pinch valves PV16 and PV20, and sets the drainage pump 46 to the predetermined level. It is operated for a time, and the medium in the primary culture container 13 is discharged to the drainage container 6. At this time, the cells adhere to the bottom of the primary culture vessel 13 and do not flow out.
After that, the control device 12 closes the pinch valves PV21 and PV7 and opens the pinch valve PV5 while operating the liquid supply pump 36, and supplies a predetermined amount of serum from the liquid supply container 25 to the primary culture container 13. At the same time, the pinch valve PV5 is closed and the PV4 is opened, and a predetermined amount of medium is supplied from the liquid supply container 24. As a result, the medium exchange operation is completed, a circulation path for supplying steam and carbon dioxide from the gas supply device 8 is formed again, and the primary culture is continued.

必要な回数の培地交換を行いながら、培養開始から所定時間が経過して培養が充分に進行したら、一次培養容器１３から二次培養容器１４ａ、１４ｂへ細胞を移す継代作業に入る。すなわち、制御装置１２は気体供給装置８からの供給状態のまま、ピンチバルブＰＶ１３、ＰＶ１７を開放するとともにピンチバルブＰＶ１６、ＰＶ２０を閉鎖し、排液ポンプ４６を所定時間作動させて、一次培養容器１３から培地を排液容器６Ａへ排出し、さらに、ピンチバルブＰＶ６を開放し、給液ポンプ３６は作動させてピンチバルブＰＶ２１、ＰＶ７を閉鎖し、給液容器２６からＰＢＳを一次培養容器１３に供給する。供給後、再びピンチバルブＰＶ２１、ＰＶ７を開いてＰＶ６を閉じ、一次培養容器１３からＰＢＳを排液させて容器内を洗い流す。
その後、ピンチバルブＰＶ２１、ＰＶ１７を閉鎖し、ＰＶ３を開放させて、給液容器２３から一次培養容器１３にトリプシンを供給し、細胞を一次培養容器１３の底部から剥離させる。なお、この際に、容器に振動を与えたり、傾斜させる機構を備えても良い。
この後、制御装置１２はピンチバルブＰＶ３を閉鎖するとともにＰＶ４を開放させて、給液ポンプ３６は作動させた状態にて、一次培養容器１３に培地を所定量供給してから、ピンチバルブＰＶ８，ＰＶ１１、ＰＶ９を開き、ＰＶ１２を閉鎖状態として、戻し通路３４を使用して一次培養容器１３から二次培養容器１４ａへ細胞を送り、さらに所定時間経過後、ピンチバルブＰＶ９を閉じてＰＶ１０を開き、二次培養容器１４ｂへ細胞を送る。なお、この間、ピンチバルブＰＶ１７は閉鎖状態として排液ポンプ４６を停止させ、ピンチバルブＰＶ１３とＰＶ１４を開放して二次培養容器１４ａから一次培養容器１３へ、また、ＰＶ１３とＰＶ１５を開放して二次培養容器１４ｂから一次培養容器１３へ気体が流通できるようにしている。 When the culture is sufficiently advanced after a predetermined time has passed from the start of the culture while performing the necessary number of medium exchanges, a subculture operation for transferring the cells from the primary culture vessel 13 to the secondary culture vessels 14a and 14b is started. That is, the control device 12 opens the pinch valves PV13 and PV17 and closes the pinch valves PV16 and PV20 while the supply state from the gas supply device 8 is maintained, and operates the drainage pump 46 for a predetermined period of time. The medium is discharged to the drainage container 6A, the pinch valve PV6 is opened, the feed pump 36 is operated to close the pinch valves PV21 and PV7, and PBS is supplied from the feed container 26 to the primary culture container 13. To do. After the supply, the pinch valves PV21 and PV7 are opened again to close the PV6, and the PBS is drained from the primary culture vessel 13 to wash out the inside of the vessel.
Thereafter, the pinch valves PV21 and PV17 are closed, PV3 is opened, trypsin is supplied from the liquid supply container 23 to the primary culture container 13, and the cells are detached from the bottom of the primary culture container 13. At this time, a mechanism for applying vibration or tilting the container may be provided.
Thereafter, the control device 12 closes the pinch valve PV3 and opens the PV4, and with the supply pump 36 operating, supplies a predetermined amount of medium to the primary culture vessel 13, and then the pinch valves PV8, PV11, PV9 are opened, PV12 is closed, cells are sent from the primary culture vessel 13 to the secondary culture vessel 14a using the return passage 34, and after a predetermined time, the pinch valve PV9 is closed and PV10 is opened, Cells are sent to the secondary culture container 14b. During this time, the pinch valve PV17 is closed and the drainage pump 46 is stopped, the pinch valves PV13 and PV14 are opened, the secondary culture vessel 14a is transferred to the primary culture vessel 13, and the PV13 and PV15 are opened. Gas is allowed to flow from the primary culture vessel 14b to the primary culture vessel 13.

これらに引き続き、ピンチバルブＰＶ５、ＰＶ９を開いて給液ポンプ３６を作動させた状態で、ＰＶ１３、ＰＶ１５は閉じてＰＶ１４、ＰＶ１７を開き排液ポンプ４６を作動させて二次培養容器１４ａに血清を供給してトリプシンの作用を失活させ、その後、ピンチバルブＰＶ５を閉じてＰＶ４を開き、培地を供給する。これに引き続き、ピンチバルブＰＶ９、ＰＶ１４を閉鎖状態とし、ＰＶ１０、ＰＶ１５を開放状態として、二次培養容器１４ｂを対象に血清と培地の供給を行う。これにより、継代作業が完了し、二次培養容器１４ａ、１４ｂにおいて二次培養が始まる。
その後は、一次培養の場合と同様にして、両二次培養容器１４ａ、１４ｂ毎に培地交換作業を行いながら、所定時間にわたって培養を行い、充分な培養が確認されたら培養作業を終了する。
なお、この二次培養の際には、二次培養容器１４ａ、１４ｂに対して、それぞれ所定時間ずつ交互に蒸気と二酸化炭素を循環させて供給しており、二次培養容器１４ａの場合はピンチバルブＰＶ２１、ＰＶ７、ＰＶ９、ＰＶ１４、ＰＶ１６、ＰＶ２０を開放状態とし、他のピンチバルブＰＶは閉鎖状態として循環経路を形成し、二次培養容器１４ｂへ切り替える場合は、ＰＶ９、ＰＶ１４を閉じてＰＶ１０、ＰＶ１５を開くようにする。いずれの場合も給液ポンプ３６は作動状態としている。 Subsequently, with the pinch valves PV5 and PV9 opened and the feed pump 36 operated, the PV13 and PV15 closed, the PV14 and PV17 opened and the drainage pump 46 operated to supply serum to the secondary culture vessel 14a. Then, trypsin is deactivated, and then the pinch valve PV5 is closed and PV4 is opened to supply the medium. Following this, the pinch valves PV9 and PV14 are closed, PV10 and PV15 are opened, and serum and medium are supplied to the secondary culture vessel 14b. Thereby, the subculture work is completed, and the secondary culture is started in the secondary culture containers 14a and 14b.
Thereafter, in the same manner as in the case of the primary culture, the culture is performed for a predetermined time while performing the medium exchange operation for each of the secondary culture containers 14a and 14b, and the culture operation is terminated when sufficient culture is confirmed.
In this secondary culture, steam and carbon dioxide are alternately circulated and supplied to the secondary culture vessels 14a and 14b for a predetermined time, respectively. In the case of the secondary culture vessel 14a, a pinch is used. When the valves PV21, PV7, PV9, PV14, PV16, PV20 are opened, and the other pinch valves PV are closed to form a circulation path and are switched to the secondary culture vessel 14b, PV9, PV14 are closed and PV10, Open PV15. In any case, the liquid supply pump 36 is in an operating state.

上述した本実施例によれば、制御装置１２によって各ピンチバルブＰＶ、給液ポンプ３６、排液ポンプ４６を作動させることにより、自動的に細胞培養を実施することができる。また給液回路３、排液回路５は、全体としてシート状となっており、各液回路を構成するチューブは位置決めされているので、可搬性が良好となり、また、接続作業も容易で、接続ミスを防止することができる。   According to the present embodiment described above, cell culture can be automatically performed by operating each pinch valve PV, the liquid supply pump 36, and the drainage pump 46 by the control device 12. In addition, the liquid supply circuit 3 and the drain circuit 5 are in the form of a sheet as a whole, and the tubes constituting each liquid circuit are positioned, so that the portability is good and the connection work is also easy. Mistakes can be prevented.

なお、上記実施例においては、給液回路３、培養容器１３、１４および排液回路５を予め接続して一体化しているが、各々を分割し、または、給液容器２１〜２６を一体化したシート状体２７を加えて一体化するようにしても良い。   In addition, in the said Example, although the liquid supply circuit 3, the culture vessels 13 and 14 and the drainage circuit 5 are connected and integrated previously, each is divided | segmented or the liquid supply containers 21-26 are integrated. The sheet-like body 27 may be added and integrated.

本発明の一実施例を示す細胞培養装置１の平面図。The top view of the cell culture apparatus 1 which shows one Example of this invention. シート状体２７、シート状体３５を示す斜視図。The perspective view which shows the sheet-like body 27 and the sheet-like body 35. FIG.

符号の説明Explanation of symbols

１…細胞培養装置 ３…給液回路
１３…一次培養容器 １４…二次培養容器
２１…第１容器（給液容器） ２２…第２容器（給液容器）
２３…第３容器（給液容器） ２４…第４容器（給液容器）
２５…第５容器（給液容器） ２６…第６容器（給液容器）
３１…主液通路 ３２…第１分岐通路
３３…第２分岐通路 ３４…戻し通路
３５…シート状体 ３５ａ、３５ｂ…シート状部材
３５Ａ…開口部 ３５Ｂ…開口部
３５Ｃ…開口部 ３６…給液ポンプ（ポンプ手段）
ＰＶ１〜２１…ピンチバルブ（バルブ手段） DESCRIPTION OF SYMBOLS 1 ... Cell culture apparatus 3 ... Liquid supply circuit 13 ... Primary culture container 14 ... Secondary culture container 21 ... 1st container (liquid supply container) 22 ... 2nd container (liquid supply container)
23 ... 3rd container (liquid supply container) 24 ... 4th container (liquid supply container)
25 ... 5th container (liquid supply container) 26 ... 6th container (liquid supply container)
31 ... Main liquid passage 32 ... First branch passage 33 ... Second branch passage 34 ... Return passage 35 ... Sheet-like body 35a, 35b ... Sheet-like member 35A ... Opening portion 35B ... Opening portion 35C ... Opening portion 36 ... Liquid supply pump (Pump means)
PV1-21 ... Pinch valve (valve means)

Claims (3)

培地や薬液を収容した複数の給液容器と、細胞を培養するための複数の培養容器と、これらを接続する送液用のチューブを備え、上記培養容器に対し上記チューブを介して送液するようにした細胞培養装置において、
上記チューブを介して送液するポンプ手段と、上記チューブを外部から閉塞させるバルブ手段を備えるとともに、上記チューブは、上記複数の給液容器に接続できるよう分岐された第１分岐通路と、上記複数の培養容器に接続できるよう分岐された第２分岐通路とを備えており、これら分岐通路が所定位置に位置するように上記チューブをシート状部材に配置させて固定し、該シート状部材には、固定されたチューブが上記ポンプ手段および上記バルブ手段と対応する位置に、これらポンプ手段およびバルブ手段を挿入可能な開口部が形成されていることを特徴とする細胞培養装置。 Provided with a plurality of liquid supply containers containing a culture medium and a chemical solution, a plurality of culture containers for culturing cells, and a liquid feeding tube for connecting them, and the liquid is fed to the culture container via the tube In such a cell culture apparatus,
Pump means for feeding liquid through the tube, valve means for closing the tube from the outside, the tube branching so as to be connected to the plurality of liquid supply containers, and the plurality A second branch passage branched so as to be connected to the culture vessel, and the tube is arranged and fixed on the sheet-like member so that these branch passages are located at predetermined positions. A cell culture apparatus, wherein an opening into which the pump means and the valve means can be inserted is formed at a position where the fixed tube corresponds to the pump means and the valve means. 上記培養容器は一次培養容器と二次培養容器からなり、上記チューブは、一端側に上記第１分岐通路を接続し、他端側に第２分岐通路を接続した主液通路を備えるとともに、該主液通路を上記ポンプ手段に対応させて配置し、さらに、上記第２分岐通路と上記主液通路のポンプ手段の上流側とを接続する戻し通路を備え、該戻し通路を使用して一次培養容器から二次培養容器へ送液することを特徴とする請求項１に記載の細胞培養装置。   The culture vessel includes a primary culture vessel and a secondary culture vessel, and the tube includes a main liquid passage having the first branch passage connected to one end side and the second branch passage connected to the other end side, The main liquid passage is arranged corresponding to the pump means, and further includes a return passage connecting the second branch passage and the upstream side of the pump means of the main liquid passage, and primary culture is performed using the return passage. The cell culture apparatus according to claim 1, wherein the liquid is fed from the container to the secondary culture container. 該細胞培養装置は、上記給液容器から上記培養容器へ送液するための給液回路と、上記培養容器から排液するための排液回路と、上記給液回路と上記排液回路とを接続する循環回路を備え．該循環回路に気体供給装置を接続して、上記循環回路、上記給液回路、上記培養容器および上記排液回路を介して気体を循環させて、上記培養容器に気体を供給するようにしたことを特徴とする請求項２または３に記載の細胞培養装置。   The cell culture apparatus includes: a liquid supply circuit for supplying liquid from the liquid supply container to the culture container; a drain circuit for discharging liquid from the culture container; the liquid supply circuit and the drain circuit. It has a circulating circuit to connect. A gas supply device is connected to the circulation circuit, and the gas is circulated through the circulation circuit, the liquid supply circuit, the culture vessel, and the drainage circuit to supply gas to the culture vessel. The cell culture device according to claim 2 or 3, wherein
JP2006236817A 2006-08-31 2006-08-31 Cell culture apparatus Withdrawn JP2008054603A (en)

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JP2014166144A (en) * 2013-02-28 2014-09-11 Hitachi Ltd Cell culture device
WO2015111347A1 (en) * 2014-01-27 2015-07-30 株式会社 日立ハイテクノロジーズ Flow-path control method, and cell culture device
JP2017012051A (en) * 2015-06-30 2017-01-19 東洋製罐グループホールディングス株式会社 Kit for cell culture, sampling method of content liquid of kit for cell culture, and sampling auxiliary tool
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JPWO2016013070A1 (en) * 2014-07-23 2017-04-27 株式会社日立製作所 Liquid feeding device and cell culture device
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014166144A (en) * 2013-02-28 2014-09-11 Hitachi Ltd Cell culture device
WO2015111347A1 (en) * 2014-01-27 2015-07-30 株式会社 日立ハイテクノロジーズ Flow-path control method, and cell culture device
JPWO2015111347A1 (en) * 2014-01-27 2017-03-23 株式会社日立ハイテクノロジーズ Flow path control method and cell culture apparatus
US10208277B2 (en) 2014-01-27 2019-02-19 Hitachi High-Technologies Corporation Flow-path control method, and cell culture device
JPWO2016013070A1 (en) * 2014-07-23 2017-04-27 株式会社日立製作所 Liquid feeding device and cell culture device
JP2017012051A (en) * 2015-06-30 2017-01-19 東洋製罐グループホールディングス株式会社 Kit for cell culture, sampling method of content liquid of kit for cell culture, and sampling auxiliary tool
JP2017023088A (en) * 2015-07-24 2017-02-02 Ckd株式会社 Culture unit and culture apparatus comprising the same
WO2020256014A1 (en) * 2019-06-20 2020-12-24 ヤマト科学株式会社 Rearing device
JP2021000006A (en) * 2019-06-20 2021-01-07 ヤマト科学株式会社 Raising apparatus
WO2022091648A1 (en) * 2020-10-29 2022-05-05 株式会社アステック Cell culturing device and cell culturing method
JP7072310B1 (en) * 2020-10-29 2022-05-20 株式会社アステック Cell culture device and cell culture method

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