JP2015216880A

JP2015216880A - Method and device for supplying carriers and cell culture system

Info

Publication number: JP2015216880A
Application number: JP2014103298A
Authority: JP
Inventors: 知弥小島; Tomoya Kojima; 浩介石井; Kosuke Ishii; 佐藤　健治; Kenji Sato; 健治佐藤
Original assignee: IHI Corp
Current assignee: IHI Corp
Priority date: 2014-05-19
Filing date: 2014-05-19
Publication date: 2015-12-07
Anticipated expiration: 2034-05-19
Also published as: JP6349949B2

Abstract

PROBLEM TO BE SOLVED: To provide a method and device for supplying carriers, which can supply carriers to a culture tank uniformly and a cell culture system.SOLUTION: The method for supplying carriers comprises the steps of: supplying carriers suspended in cell adhesion substance solution to a carrier supply vessel 8; discharging the cell adhesion substance solution after the carriers are precipitated; supplying a medium in the culture tank to the carrier supply vessel; suspending the carriers in the medium by shaking; and introducing the medium where the carriers are suspended to the culture tank while keeping shaking.

Description

本発明は、培地中に懸濁させた担体を培養槽に均等に供給する担体供給方法及び担体供給装置及び細胞培養システムに関するものである。 The present invention relates to a carrier supply method, a carrier supply apparatus, and a cell culture system that uniformly supply a carrier suspended in a culture medium to a culture tank.

動物細胞、植物細胞等の細胞には、それぞれ培養の際に増殖用の足場を必要とする付着性の細胞と、増殖用の足場を必要としない浮遊性の細胞とがあり、培養した細胞は有用物質の生産等に広く使用されている。 Animal cells, plant cells, and other cells each have an adherent cell that requires a scaffold for growth and a floating cell that does not require a scaffold for growth. Widely used in the production of useful substances.

Ｖｅｒｏ細胞やＥＳ細胞、ｉＰＳ細胞等の付着性の動物細胞の培養方法の１つとして、浮遊培養法がある。浮遊培養法は、マイクロキャリア等の微小な担体に細胞を付着させ、ボトル等の容器に導入された培地中に担体を浮遊させ培養を行う。その際に担体に付着した細胞に撹拌等で生じる剪断応力を許容剪断応力（細胞が破損しない強さの剪断応力）以下で与えることで、効率よく細胞の培養を行なうものである。 One method for culturing adherent animal cells such as Vero cells, ES cells, and iPS cells is a suspension culture method. In the suspension culture method, cells are attached to a microcarrier such as a microcarrier, and the carrier is suspended in a medium introduced into a container such as a bottle and cultured. In this case, cells are efficiently cultured by applying a shear stress generated by stirring or the like to the cells attached to the carrier at or below an allowable shear stress (a shear stress that does not damage the cells).

担体を用いた浮遊培養を行なう為の担体の供給方法として、担体を手動で供給する場合と、担体を自動で供給する場合の２つの方法がある。 There are two methods of supplying a carrier for carrying out suspension culture using a carrier: a case where the carrier is manually supplied and a case where the carrier is automatically supplied.

担体を手動で供給する場合には、予めＰＢＳ（ＰｈｏｓｐｈａｔｅＢｕｆｆｅｒｅｄＳａｌｉｎｅ：リン酸緩衝生理食塩水）等の緩衝液に懸濁させておいた担体をピペットで吸取り、担体を細胞や培地と共に培養槽に供給する。その後、撹拌等により細胞の付着した担体を培地中に浮遊させることで、細胞の培養を行う。 When manually supplying the carrier, the carrier suspended in a buffer solution such as PBS (Phosphate Buffered Saline) in advance is sucked with a pipette, and the carrier is put into the culture tank together with the cells and the medium. Supply. Thereafter, the cells are cultured by suspending the carrier to which the cells are attached by stirring or the like in the medium.

又、担体を自動で供給するものとして、特許文献１に示されるものがあり、第２供給槽に予め貯溜されたマイクロディスク浮遊液を、第２供給ポンプにより第２供給管を介してチャンバ内に供給する構成が開示されている。 In addition, as an apparatus for automatically supplying a carrier, there is one disclosed in Patent Document 1, in which a microdisk floating liquid stored in advance in a second supply tank is supplied into a chamber through a second supply pipe by a second supply pump. A configuration for supplying to is disclosed.

然し乍ら、担体を手動で供給する場合、担体を懸濁させたＰＢＳ等からの回収に作業者の技術が必要であり、作業も繁雑である。又、ピペット等のツールが汚染されていた場合には、結果の信頼性、再現性を失う虞れがある。 However, when the carrier is manually supplied, the operator's technique is required for recovery from the PBS or the like in which the carrier is suspended, and the work is complicated. Further, when a tool such as a pipette is contaminated, there is a risk that the reliability and reproducibility of the result may be lost.

又、特許文献１の場合、マイクロディスクをＰＢＳ溶液等に懸濁させた後、静置するとマイクロディスクが沈殿することから、単に第２供給ポンプによりマイクロディスク浮遊液を移送しようとすると、マイクロディスクを均一に供給することができない。マイクロディスクを均一に供給できないことで、細胞の付着していないマイクロディスクができる、或は細胞が付着しすぎて細胞が増殖する余地がないマイクロディスクができる等の問題が発生する。 In the case of Patent Document 1, since the microdisk is precipitated when suspended after being suspended in a PBS solution or the like, the microdisk floating liquid is simply transferred by the second supply pump. Cannot be supplied uniformly. The inability to supply the microdisk uniformly causes problems such as the formation of a microdisk with no cells attached, or the formation of a microdisk with too much cell attachment and no room for cell proliferation.

尚、特許文献２には、マイクロキャリアで満たされたスラリー作製槽に培地貯液槽から培地を供給し、撹拌装置により前記スラリー作製槽に設けられた撹拌翼を回転させることで、マイクロキャリアスラリーを作製する構成が開示されている。 In Patent Document 2, a medium is supplied from a medium storage tank to a slurry preparation tank filled with microcarriers, and a stirring blade provided in the slurry preparation tank is rotated by a stirrer so that a microcarrier slurry is obtained. A configuration for producing the above is disclosed.

特開２００５−２３７２７４号公報JP 2005-237274 A 特開平７−１３５９６８号公報JP 7-135968 A

本発明は斯かる実情に鑑み、担体を培養槽に均一に供給可能とした担体供給方法及び担体供給装置及び細胞培養システムを提供するものである。 In view of such circumstances, the present invention provides a carrier supply method, a carrier supply device, and a cell culture system that can uniformly supply a carrier to a culture tank.

本発明は、担体供給容器に細胞接着物質溶液に懸濁された担体を供給する工程と、該担体が沈降した後に前記細胞接着物質溶液を排出する工程と、培養槽内の培地を前記担体供給容器に供給する工程と、振盪で前記培地中に前記担体を懸濁させる工程と、振盪を維持した状態で前記担体を懸濁させた前記培地を前記培養槽に導入する工程とを有する担体供給方法に係るものである。 The present invention includes a step of supplying a carrier suspended in a cell adhesion substance solution to a carrier supply container, a step of discharging the cell adhesion substance solution after the carrier has settled, and supplying a medium in a culture tank to the carrier A carrier supply comprising: supplying the container; suspending the carrier in the medium by shaking; and introducing the medium in which the carrier is suspended in a state where shaking is maintained into the culture tank. It concerns the method.

又本発明は、前記細胞接着物質溶液を排出する工程と、前記培養槽内の前記培地を前記担体供給容器に供給する工程との間に、該担体供給容器に薬液を供給する工程と、振盪で前記担体供給容器を撹拌して前記薬液で前記担体を洗浄する工程と、該担体を沈降させる工程と、前記薬液を排出する工程とを有する担体供給方法に係るものである。 Further, the present invention provides a step of supplying a chemical solution to the carrier supply container between the step of discharging the cell adhesion substance solution and the step of supplying the medium in the culture tank to the carrier supply container. The carrier supply method includes a step of stirring the carrier supply container and washing the carrier with the chemical solution, a step of precipitating the carrier, and a step of discharging the chemical solution.

又本発明は、担体供給容器と、該担体供給容器に担体を供給する担体供給手段と、前記担体供給容器を振盪させ前記担体を浮遊させて培地中に懸濁させる振盪手段と、前記担体供給容器内の液体を排出する排出手段と、前記担体供給容器に細胞を培養する培養槽から培地を供給すると共に、前記振盪手段により懸濁された前記培地を培養槽に導入する培地給排手段とを具備する担体供給装置に係るものである。 The present invention also provides a carrier supply container, carrier supply means for supplying a carrier to the carrier supply container, shaking means for shaking the carrier supply container to suspend the carrier and suspending it in a medium, and carrier supply Discharge means for discharging the liquid in the container; medium supply / discharge means for supplying the medium from a culture tank for culturing cells to the carrier supply container and introducing the culture medium suspended by the shaking means into the culture tank; The present invention relates to a carrier supply device comprising:

又本発明は、前記担体供給手段と前記培地給排手段は、それぞれ担体供給管と培地給排管とを有し、前記担体供給管の下流端部は前記担体供給容器の内部に延出し、前記担体供給管及び前記培地給排管が前記下流端部の上流で接続された担体供給装置に係るものである。 In the present invention, the carrier supply means and the medium supply / discharge means each have a carrier supply pipe and a medium supply / discharge pipe, and the downstream end of the carrier supply pipe extends into the carrier supply container, The carrier supply pipe and the culture medium supply / discharge pipe are related to a carrier supply apparatus connected upstream of the downstream end portion.

又本発明は、前記担体供給容器に薬液を供給する薬液供給手段を更に具備し、該薬液供給手段は薬液供給管を有し、該薬液供給管は前記下流端部よりも上流で前記担体供給管に接続された担体供給装置に係るものである。 The present invention further includes a chemical solution supply means for supplying a chemical solution to the carrier supply container, the chemical solution supply means having a chemical solution supply pipe, and the chemical solution supply pipe supplies the carrier upstream of the downstream end. The present invention relates to a carrier supply device connected to a pipe.

又本発明は、前記担体供給容器は逆円錐状又は逆角錐状であり、前記担体供給容器の頂角は、４５°〜１２０°に含まれる担体供給装置に係るものである。 According to the present invention, the carrier supply container has an inverted conical shape or an inverted pyramid shape, and the apex angle of the carrier supply container relates to a carrier supply device included in 45 ° to 120 °.

更に又本発明は、前記担体供給装置を有する細胞導入装置と、細胞培養装置と、細胞剥離装置と、細胞回収装置とを具備し、前記細胞導入装置が前記細胞培養装置に担体と細胞と培地とを導入し、前記細胞培養装置が前記担体を前記培地に浮遊させて細胞を前記担体上で培養し、前記細胞剥離装置が前記細胞培養装置から供給された前記培地に含まれる前記担体から細胞を剥離させ、前記細胞回収装置が前記細胞剥離装置に剥離させた細胞を回収する細胞培養システムに係るものである。 Furthermore, the present invention includes a cell introduction device having the carrier supply device, a cell culture device, a cell peeling device, and a cell recovery device, and the cell introduction device includes a carrier, a cell, and a medium in the cell culture device. And the cell culture device suspends the carrier in the medium and cultures the cells on the carrier, and the cell peeling device supplies cells from the carrier contained in the medium supplied from the cell culture device. And the cell collection device collects the cells separated by the cell separation device.

本発明によれば、担体供給容器に細胞接着物質溶液に懸濁された担体を供給する工程と、該担体が沈降した後に前記細胞接着物質溶液を排出する工程と、培養槽内の培地を前記担体供給容器に供給する工程と、振盪で前記培地中に前記担体を懸濁させる工程と、振盪を維持した状態で前記担体を懸濁させた前記培地を前記培養槽に導入する工程とを有するので、該培養槽で特に幹細胞を培養する際に前記担体上に細胞の塊が形成されることがなく、未分化状態が維持されて該担体上に均一に細胞を培養させることができる。 According to the present invention, a step of supplying a carrier suspended in a cell adhesion substance solution to a carrier supply container, a step of discharging the cell adhesion substance solution after the carrier has settled, and a medium in a culture tank include the medium Supplying the carrier supply container, suspending the carrier in the culture medium by shaking, and introducing the culture medium in which the carrier is suspended in the culture tank while shaking is maintained. Therefore, when stem cells are cultured in the culture tank, cell clumps are not formed on the carrier, and the undifferentiated state can be maintained and the cells can be uniformly cultured on the carrier.

又本発明によれば、担体供給容器と、該担体供給容器に担体を供給する担体供給手段と、前記担体供給容器を振盪させ前記担体を浮遊させて培地中に懸濁させる振盪手段と、前記担体供給容器内の液体を排出する排出手段と、前記担体供給容器に細胞を培養する培養槽から培地を供給すると共に、前記振盪手段により懸濁された前記培地を培養槽に導入する培地給排手段とを具備するので、前記振盪手段により前記担体供給容器を振盪させることで、前記担体供給容器の下端まで満遍なく撹拌することができ、前記担体を前記培地中に均一に懸濁させ、前記担体を前記培養槽に均一に供給することができる。 According to the invention, a carrier supply container, carrier supply means for supplying a carrier to the carrier supply container, shaking means for suspending the carrier in a medium by shaking the carrier supply container, A discharge means for discharging the liquid in the carrier supply container, and a medium supply / discharge for supplying the medium from the culture tank for culturing the cells to the carrier supply container and introducing the culture medium suspended by the shaking means into the culture tank. The carrier supply container is shaken by the shaking means so that the lower end of the carrier supply container can be uniformly stirred, the carrier is uniformly suspended in the medium, and the carrier Can be uniformly supplied to the culture tank.

更に又本発明によれば、前記担体供給装置を有する細胞導入装置と、細胞培養装置と、細胞剥離装置と、細胞回収装置とを具備し、前記細胞導入装置が前記細胞培養装置に担体と細胞と培地とを導入し、前記細胞培養装置が前記担体を前記培地に浮遊させて細胞を前記担体上で培養し、前記細胞剥離装置が前記細胞培養装置から供給された前記培地に含まれる前記担体から細胞を剥離させ、前記細胞回収装置が前記細胞剥離装置に剥離させた細胞を回収するので、前記担体を前記細胞培養装置に自動で均一に供給でき、細胞と前記担体との比が容易に調整され、自動培養の効率を向上させることができるという優れた効果を発揮する。 Furthermore, according to the present invention, the apparatus includes a cell introduction device having the carrier supply device, a cell culture device, a cell detachment device, and a cell recovery device, and the cell introduction device includes a carrier and a cell in the cell culture device. And the medium, the cell culture device suspends the carrier in the medium and cultures the cells on the carrier, and the cell peeling device is contained in the medium supplied from the cell culture device. And the cell collection device collects the cells detached by the cell separation device, so that the carrier can be automatically and uniformly supplied to the cell culture device, and the ratio of cells to the carrier can be easily achieved. It is adjusted and exhibits an excellent effect that the efficiency of automatic culture can be improved.

本発明の実施例に係る細胞培養システムを示す概略構成図である。It is a schematic block diagram which shows the cell culture system which concerns on the Example of this invention. 本発明の第１の実施例に係る担体供給容器及び周辺部を示す概略立断面図であり、（Ａ）は逆円錐部の頂角が大きい場合を示し、（Ｂ）は該逆円錐部の頂角が小さい場合を示している。BRIEF DESCRIPTION OF THE DRAWINGS It is a general | schematic sectional drawing which shows the support | carrier supply container and peripheral part which concern on 1st Example of this invention, (A) shows the case where the apex angle of an inverted cone part is large, (B) shows this inverted cone part. The case where the apex angle is small is shown. 本発明の第１の実施例に係る担体供給方法を説明するフローチャートである。It is a flowchart explaining the carrier supply method which concerns on 1st Example of this invention. 本発明の第２の実施例に係る担体供給容器及び周辺部を示す概略立断面図であり、（Ａ）は逆円錐部の頂角が大きい場合を示し、（Ｂ）は該逆円錐部の頂角が小さい場合を示している。It is a schematic elevation sectional view showing a carrier supply container and a peripheral part according to a second embodiment of the present invention, (A) shows a case where the apex angle of the inverted conical part is large, and (B) shows the inverted conical part. The case where the apex angle is small is shown.

以下、図面を参照しつつ本発明の実施例を説明する。 Embodiments of the present invention will be described below with reference to the drawings.

先ず、図１に於いて、本発明の実施例に係る細胞培養システムについて説明する。 First, referring to FIG. 1, a cell culture system according to an embodiment of the present invention will be described.

細胞培養システム１は、主に細胞導入装置２、細胞培養装置３、細胞剥離装置４、細胞回収装置５によって構成され、細胞の導入から培養、剥離、回収迄の一連の処理を行う。尚、図１中、実線の矢印は各構成同士が常時接続されている状態を示し、破線の矢印は各構成同士が必要に応じてその都度接続される状態を示している。 The cell culture system 1 is mainly composed of a cell introduction device 2, a cell culture device 3, a cell detachment device 4, and a cell recovery device 5, and performs a series of processes from cell introduction to culture, detachment, and recovery. In FIG. 1, solid arrows indicate a state in which the respective components are always connected, and broken arrows indicate a state in which the respective components are connected as necessary.

前記細胞導入装置２は、主に担体供給部６、薬液タンク７、担体供給容器８、細胞供給部９、冷蔵庫１１等により冷蔵保存された培地タンク１２から構成されている。 The cell introduction apparatus 2 is mainly composed of a medium supply tank 6, a chemical solution tank 7, a carrier supply container 8, a cell supply part 9, a refrigerator 11 and the like, which are stored in a refrigerator.

前記担体供給部６には、細胞接着物質の溶液中に懸濁された担体１３が貯溜され、前記薬液タンク７には、所定の薬液が貯溜され、前記培地タンク１２には培地１４が貯溜されている。前記細胞導入装置２は、前記細胞培養装置３の培養槽１５に、前記担体１３、細胞、前記培地１４をそれぞれ導入する様になっている。尚、前記薬液タンク７に貯溜される薬液としては、ＰＢＳ、ＨＢＳ、ＴＢＳ、ＢＢＳ等が用いられる。 A carrier 13 suspended in a cell adhesion substance solution is stored in the carrier supply unit 6, a predetermined chemical solution is stored in the chemical solution tank 7, and a medium 14 is stored in the medium tank 12. ing. The cell introduction device 2 introduces the carrier 13, the cells, and the medium 14 into the culture tank 15 of the cell culture device 3. As the chemical solution stored in the chemical solution tank 7, PBS, HBS, TBS, BBS or the like is used.

前記細胞培養装置３は、主に内部を恒温状態に維持する恒温槽１６と、該恒温槽１６内に設けられた前記培養槽１５と、培地循環ポンプ１７と、廃液タンク１８とから構成されている。前記細胞培養装置３は、前記細胞導入装置２から導入された前記担体１３を前記培地１４中に浮遊させ、前記培地循環ポンプ１７により前記培地１４を循環させることで、細胞に許容剪断応力以下の剪断応力を与え、前記担体１３上で細胞を培養する。 The cell culture device 3 is mainly composed of a thermostat 16 for maintaining the inside at a constant temperature, the culture tank 15 provided in the thermostat 16, a medium circulation pump 17, and a waste liquid tank 18. Yes. The cell culture device 3 suspends the carrier 13 introduced from the cell introduction device 2 in the medium 14 and circulates the medium 14 by the medium circulation pump 17, so that the cell has an allowable shear stress or less. Shear stress is applied and the cells are cultured on the carrier 13.

前記細胞剥離装置４は、主に剥離槽１９、緩衝液や剥離液等の各種薬液が貯溜された薬液タンク２１から構成され、該薬液タンク２１から供給された薬液と、剪断応力付与手段（図示せず）により与えられる剪断応力により、前記剥離槽１９内で前記担体１３から細胞を剥離させる。更に、前記細胞回収装置５は、前記細胞剥離装置４により剥離された細胞のみを回収する様になっている。 The cell peeling device 4 is mainly composed of a peeling tank 19, a chemical tank 21 in which various chemical solutions such as a buffer solution and a peeling solution are stored, a chemical solution supplied from the chemical solution tank 21, and a shear stress applying means (see FIG. The cells are peeled from the carrier 13 in the peeling tank 19 by a shearing stress applied by an unillustrated). Further, the cell collection device 5 collects only the cells detached by the cell separation device 4.

前記細胞導入装置２について更に詳しく説明する。 The cell introduction device 2 will be described in more detail.

前記担体供給部６には、前記担体１３上で細胞、特に多能性幹細胞を未分化状態を維持したまま培養する為の細胞接着物質溶液、例えばカドヘリン、マトリゲル、ラミニン等の溶液が貯溜されると共に、該溶液中に１８５μｍ程度の球状、或はディスク状のマイクロキャリアである前記担体１３が懸濁されている。 The carrier supply unit 6 stores a cell adhesive substance solution for culturing cells, particularly pluripotent stem cells while maintaining an undifferentiated state on the carrier 13, such as a solution of cadherin, matrigel, laminin or the like. At the same time, the carrier 13 which is a spherical or disk-shaped microcarrier of about 185 μm is suspended in the solution.

又、前記薬液タンク７には、前記担体１３を洗浄する為の緩衝液、例えばＰＢＳ（リン酸緩衝生理食塩水）が貯溜されている。 The chemical solution tank 7 stores a buffer solution for washing the carrier 13, for example, PBS (phosphate buffered saline).

前記担体供給部６には担体供給管２２の上流端が接続され、該担体供給管２２の下流端が前記担体供給容器８に接続されている。前記担体供給部６は、前記担体供給管２２を介して前記担体供給容器８に接続され、前記担体供給管２２には、上流側から順に担体供給バルブ２３、ポンプ２４が設けられている。尚、前記担体供給部６、前記担体供給管２２、前記担体供給バルブ２３、前記ポンプ２４により担体供給手段が構成される。 An upstream end of a carrier supply pipe 22 is connected to the carrier supply unit 6, and a downstream end of the carrier supply pipe 22 is connected to the carrier supply container 8. The carrier supply unit 6 is connected to the carrier supply container 8 via the carrier supply pipe 22, and the carrier supply pipe 22 is provided with a carrier supply valve 23 and a pump 24 in order from the upstream side. The carrier supply unit 6, the carrier supply pipe 22, the carrier supply valve 23, and the pump 24 constitute a carrier supply unit.

又、前記薬液タンク７には薬液供給管２５の上流端が接続され、該薬液供給管２５の下流端が前記担体供給管２２の前記担体供給バルブ２３と前記ポンプ２４との間に接続されている。前記薬液タンク７は、前記薬液供給管２５、前記担体供給管２２を介して前記担体供給容器８に接続され、前記薬液供給管２５には薬液供給バルブ２６が設けられている。尚、前記薬液タンク７、前記薬液供給管２５、前記担体供給管２２、前記薬液供給バルブ２６、前記ポンプ２４により薬液供給手段が構成される。 The chemical liquid tank 7 is connected to an upstream end of a chemical liquid supply pipe 25, and a downstream end of the chemical liquid supply pipe 25 is connected between the carrier supply valve 23 of the carrier supply pipe 22 and the pump 24. Yes. The chemical liquid tank 7 is connected to the carrier supply container 8 via the chemical liquid supply pipe 25 and the carrier supply pipe 22, and a chemical liquid supply valve 26 is provided in the chemical liquid supply pipe 25. The chemical liquid tank 7, the chemical liquid supply pipe 25, the carrier supply pipe 22, the chemical liquid supply valve 26, and the pump 24 constitute chemical liquid supply means.

前記担体供給バルブ２３と前記薬液供給バルブ２６のいずれか一方を開放し、いずれか他方を閉塞した状態で、前記ポンプ２４を駆動することで、前記担体供給管２２より細胞接着物質溶液に懸濁された前記担体１３と薬液のいずれか一方を選択的に前記担体供給容器８に供給できる様になっている。 By suspending either one of the carrier supply valve 23 or the chemical solution supply valve 26 and closing the other, the pump 24 is driven to be suspended in the cell adhesive substance solution from the carrier supply tube 22. One of the carrier 13 and the chemical solution thus prepared can be selectively supplied to the carrier supply container 8.

又、前記担体供給容器８は、排液管２７を介して前記廃液タンク１８に接続され、前記排液管２７に排液ポンプ２８が設けられている。該排液ポンプ２８を駆動させることで、前記排液管２７を介して前記担体供給容器８内の液体を前記廃液タンク１８に排出可能となっている。尚、前記排液管２７、前記排液ポンプ２８により排出手段が構成される。 The carrier supply container 8 is connected to the waste liquid tank 18 via a drain pipe 27, and a drain pump 28 is provided in the drain pipe 27. By driving the drainage pump 28, the liquid in the carrier supply container 8 can be discharged to the waste liquid tank 18 through the drainage pipe 27. The drainage pipe 27 and the drainage pump 28 constitute a draining means.

又、前記担体供給容器８は、前記担体供給管２２の中途部に接続された培地給排管２９を介して前記培養槽１５に接続されている。又、前記培地給排管２９には正転及び逆転が可能な培地給排ポンプ３１が設けられている。該培地給排ポンプ３１を駆動させることで、前記担体供給容器８中の担体懸濁液（後述）を前記培養槽１５に供給できると共に、該培養槽１５内の前記培地１４を前記担体供給容器８内に導入できる様になっている。尚、前記培地給排管２９、前記担体供給管２２、前記培地給排ポンプ３１により培地給排手段が構成される。 The carrier supply container 8 is connected to the culture tank 15 through a medium supply / discharge tube 29 connected to the middle of the carrier supply tube 22. The medium supply / discharge pipe 29 is provided with a medium supply / discharge pump 31 capable of normal rotation and reverse rotation. By driving the medium supply / discharge pump 31, a carrier suspension (described later) in the carrier supply container 8 can be supplied to the culture tank 15, and the medium 14 in the culture tank 15 is supplied to the carrier supply container. 8 can be introduced. The medium supply / discharge pipe 29, the carrier supply pipe 22, and the medium supply / discharge pump 31 constitute medium supply / discharge means.

前記培地タンク１２は培地供給管３２を介して前記培養槽１５に接続され、前記培地供給管３２には培地供給ポンプ３３が設けられている。該培地供給ポンプ３３を駆動させることで、前記培養槽１５内に培地を供給できる。更に、前記細胞供給部９は前記培地給排管２９の中途部に接続された細胞供給管３４を介して前記培養槽１５に接続され、前記細胞供給管３４には細胞供給ポンプ３５が設けられ、該細胞供給ポンプ３５を駆動させることで、前記培養槽１５内に細胞を供給できる。 The medium tank 12 is connected to the culture tank 15 via a medium supply pipe 32, and a medium supply pump 33 is provided in the medium supply pipe 32. The medium can be supplied into the culture tank 15 by driving the medium supply pump 33. Further, the cell supply unit 9 is connected to the culture tank 15 via a cell supply tube 34 connected to the middle part of the medium supply / exhaust tube 29, and a cell supply pump 35 is provided in the cell supply tube 34. The cells can be supplied into the culture tank 15 by driving the cell supply pump 35.

次に、図２（Ａ）（Ｂ）に於いて、前記担体供給容器８及びその周辺部の詳細について説明する。 Next, with reference to FIGS. 2A and 2B, details of the carrier supply container 8 and its peripheral portion will be described.

該担体供給容器８は、水平断面積が上方に向って漸次増大する逆円錐部３６と、該逆円錐部３６の上端に連続する円筒部３７とから構成されている。尚、前記逆円錐部３６の頂角は所定の角度αを有しており、例えば頂角αは４５°〜１２０°の範囲で任意の角度が選択される。尚、４５°は前記担体供給容器８を振盪させた場合に該担体供給容器８内の液体を撹拌可能な下限値であり、１２０°は前記逆円錐部３６に堆積した前記担体１３を前記逆円錐部３６の頂部に収集し、効率的に回収可能な角度の上限値となっている。又、本実施例に於いては、図２（Ａ）に示される様に頂角αを９０°とした場合と、図２（Ｂ）に示される様に頂角αを６０°とした場合について説明する。 The carrier supply container 8 includes an inverted conical portion 36 whose horizontal cross-sectional area gradually increases upward, and a cylindrical portion 37 continuous with the upper end of the inverse conical portion 36. The apex angle of the inverted conical portion 36 has a predetermined angle α. For example, the apex angle α is selected in the range of 45 ° to 120 °. 45 ° is a lower limit value at which the liquid in the carrier supply container 8 can be stirred when the carrier supply container 8 is shaken, and 120 ° is the reverse of the carrier 13 deposited on the inverted conical portion 36. The upper limit value of the angle that can be collected and efficiently collected at the top of the conical portion 36 is set. In this embodiment, when the apex angle α is 90 ° as shown in FIG. 2 (A) and when the apex angle α is 60 ° as shown in FIG. 2 (B). Will be described.

前記担体供給容器８の下部には、該担体供給容器８を振盪可能に保持する振盪手段である振盪機構３８が設けられている。該振盪機構３８は、前記担体供給容器８を水平方向に大きく往復動させるもの、該担体供給容器８を小刻みに振動させるもの、該担体供給容器８を大きくゆっくりと円運動させるもの等が適宜選択される。又、往復動、振動、円運動の複合動作を行わせてもよい。 At the lower part of the carrier supply container 8, a shaking mechanism 38, which is a shaking means for holding the carrier supply container 8 so as to be shaken, is provided. The shaking mechanism 38 is appropriately selected from those that reciprocate the carrier supply container 8 in the horizontal direction, those that vibrate the carrier supply container 8 in small increments, and those that cause the carrier supply container 8 to move in a large and slow circle. Is done. Further, a combined operation of reciprocation, vibration, and circular motion may be performed.

前記担体供給管２２の下流端部は、鉛直方向に屈曲されて屈曲部２２ａが形成される。該屈曲部２２ａは、前記担体供給容器８の軸心に沿って上方から該担体供給容器８に挿入され、前記屈曲部２２ａは前記逆円錐部３６の底部迄延出している。前記屈曲部２２ａの下端は前記逆円錐部３６の頂部に位置し、前記屈曲部２２ａの下端と前記逆円錐部３６の頂点とは供給に支障がない様接近していることが好ましい。尚、前記屈曲部２２ａの内径は、前記担体１３が詰まらない大きさ、例えば２ｍｍ〜３ｍｍ程度となっている。 The downstream end portion of the carrier supply pipe 22 is bent in the vertical direction to form a bent portion 22a. The bent portion 22 a is inserted into the carrier supply container 8 from above along the axis of the carrier supply container 8, and the bent portion 22 a extends to the bottom of the inverted conical portion 36. The lower end of the bent portion 22a is preferably located at the top of the inverted conical portion 36, and the lower end of the bent portion 22a and the apex of the inverted conical portion 36 are preferably close to each other so as not to interfere with the supply. The inner diameter of the bent portion 22a is such that the carrier 13 is not clogged, for example, about 2 mm to 3 mm.

又、前記屈曲部２２ａの上流端に前記培地給排管２９が接続され、前記担体供給管２２、前記屈曲部２２ａ、前記培地給排管２９はＴ字状の配管を構成している。更に、前記担体供給管２２の前記屈曲部２２ａの上流側に前記薬液供給管２５が接続されている。 The medium supply / discharge pipe 29 is connected to the upstream end of the bent part 22a, and the carrier supply pipe 22, the bent part 22a, and the medium supply / discharge pipe 29 constitute a T-shaped pipe. Further, the chemical solution supply pipe 25 is connected to the upstream side of the bent portion 22 a of the carrier supply pipe 22.

前記逆円錐部３６の周面の所要位置には、前記排液管２７が接続されている。尚、該排液管２７が接続される高さは、前記担体供給管２２により供給された前記担体１３が沈降し、堆積した堆積層３９よりも上方となっており、前記担体供給容器８より液体を排出する際に、液体と共に前記担体１３が排出されない様になっている。 The drainage pipe 27 is connected to a required position on the peripheral surface of the inverted conical portion 36. The drainage pipe 27 is connected to a height higher than the deposited layer 39 where the carrier 13 supplied by the carrier supply pipe 22 settles and deposits, and from the carrier supply container 8. When the liquid is discharged, the carrier 13 is not discharged together with the liquid.

尚、前記担体供給容器８、前記担体供給手段、前記薬液供給手段、前記培地給排手段、前記排出手段、前記振盪機構３８等により担体供給装置が構成される。 The carrier supply device is constituted by the carrier supply container 8, the carrier supply means, the chemical solution supply means, the medium supply / discharge means, the discharge means, the shaking mechanism 38, and the like.

次に、図３のフローチャートを用い、前記担体供給容器８から前記培養槽１５に前記担体１３を供給する場合について説明する。 Next, the case where the carrier 13 is supplied from the carrier supply container 8 to the culture tank 15 will be described with reference to the flowchart of FIG.

ＳＴＥＰ：０１先ず、前記担体供給バルブ２３を開放し、前記薬液供給バルブ２６を閉塞した状態で、前記ポンプ２４を駆動させる。該ポンプ２４の駆動により、前記担体供給部６から細胞接着物質、例えばカドヘリン溶液と前記担体１３との懸濁液が屈曲部２２ａより前記担体供給容器８の頂部近傍に供給される。 STEP: 01 First, the carrier 24 is opened while the chemical solution supply valve 26 is closed, and the pump 24 is driven. By driving the pump 24, a cell adhesion substance, for example, a suspension of the cadherin solution and the carrier 13, is supplied from the carrier supply unit 6 to the vicinity of the top of the carrier supply container 8 from the bent portion 22a.

ＳＴＥＰ：０２前記担体１３の供給後、前記担体供給容器８を所定時間静置し、前記担体１３を沈降させ、前記逆円錐部３６の頂部に前記堆積層３９を形成させる。前記担体１３の沈降速度は、数ｃｍ／ｍｉｎ程度であり、前記担体供給容器８の静置時間は前記カドヘリン溶液の液位や前記逆円錐部３６の頂角α等により決定される。 (Step 02) After supplying the carrier 13, the carrier supply container 8 is allowed to stand for a predetermined time, the carrier 13 is allowed to settle, and the deposited layer 39 is formed on the top of the inverted conical portion 36. The settling speed of the carrier 13 is about several cm / min, and the standing time of the carrier supply container 8 is determined by the liquid level of the cadherin solution, the apex angle α of the inverted conical portion 36, and the like.

尚、図２（Ａ）に示される様に、頂角αが大きい場合、或は前記担体１３がディスク状等容易に移動しない形状の場合、前記逆円錐部３６の斜面に沈降した前記担体１３が前記逆円錐部３６の斜面に留まり易く、下方に移動し難い。この場合、前記振盪機構３８を駆動させ、前記担体供給容器８に小刻みな振動を与えるか、或はゆっくりと円運動させ、前記担体１３の移動を促進してもよい。 As shown in FIG. 2A, when the apex angle α is large, or when the carrier 13 has a shape such as a disk that does not move easily, the carrier 13 that sinks on the inclined surface of the inverted conical portion 36. However, it is easy to stay on the slope of the inverted conical portion 36 and hardly move downward. In this case, the movement of the carrier 13 may be promoted by driving the shaking mechanism 38 and applying a slight vibration to the carrier supply container 8 or slowly moving it.

又、図２（Ｂ）に示される様に、頂角αが小さい場合、前記逆円錐部３６の斜面に沈降した前記担体１３は、斜面に沿って降下するので、該担体１３の形状に拘わらず容易に前記逆円錐部３６の下方に移動し、前記振盪機構３８による前記担体供給容器８への振盪動作は省略することもできる。 In addition, as shown in FIG. 2B, when the apex angle α is small, the carrier 13 that has settled on the slope of the inverted conical portion 36 descends along the slope, and therefore, regardless of the shape of the carrier 13. It can be easily moved below the inverted conical portion 36, and the shaking operation to the carrier supply container 8 by the shaking mechanism 38 can be omitted.

ＳＴＥＰ：０３前記逆円錐部３６の頂部に前記堆積層３９が形成されると、次に前記排液ポンプ２８を駆動させ、前記担体供給容器８中のカドヘリン溶液を前記廃液タンク１８に排出する。この時、前記排液管２７が前記堆積層３９よりも上方に接続されているので、カドヘリン溶液と共に前記担体１３が排出されるのを防止できる。 (Step 03) When the deposited layer 39 is formed on the top of the inverted conical portion 36, the drainage pump 28 is driven to discharge the cadherin solution in the carrier supply container 8 to the waste liquid tank 18. At this time, since the drainage pipe 27 is connected above the deposition layer 39, it is possible to prevent the carrier 13 from being discharged together with the cadherin solution.

ＳＴＥＰ：０４カドヘリン溶液の排出後、前記薬液供給バルブ２６を開放し、前記担体供給バルブ２３を閉塞した状態で、前記ポンプ２４を駆動させる。該ポンプ２４の駆動により、前記薬液タンク７から、例えばＰＢＳが前記屈曲部２２ａより前記担体供給容器８の底部に供給される。 (Step 04) After the cadherin solution is discharged, the pump 24 is driven with the chemical solution supply valve 26 opened and the carrier supply valve 23 closed. By driving the pump 24, for example, PBS is supplied from the bent portion 22 a to the bottom of the carrier supply container 8 from the chemical solution tank 7.

ＳＴＥＰ：０５ＰＢＳの供給後、前記振盪機構３８を駆動させ、前記担体供給容器８を大きく往復動させることで、該担体供給容器８の内部が撹拌され、前記担体１３の表面に付着したカドヘリン溶液がＰＢＳにより洗浄される。 STEP: 05 After supplying PBS, the shaking mechanism 38 is driven to reciprocate the carrier supply container 8 so that the inside of the carrier supply container 8 is stirred, and the cadherin solution attached to the surface of the carrier 13 Is washed with PBS.

この時、図２（Ａ）に示される様に、頂角αが大きい場合、ＰＢＳの液位が低くなるので、液体は水平方向に移動し易く、小さな振動、或は小さな振幅で前記担体供給容器８全体を撹拌することができ、該担体供給容器８に与える往復動の振動数、振幅等を小さくすることができる。 At this time, as shown in FIG. 2A, when the apex angle α is large, the liquid level of the PBS is low, so that the liquid easily moves in the horizontal direction, and the carrier is supplied with small vibration or small amplitude. The entire container 8 can be agitated, and the frequency and amplitude of the reciprocating motion applied to the carrier supply container 8 can be reduced.

ＳＴＥＰ：０６前記担体供給容器８の撹拌後、該担体供給容器８を所定時間静置し、前記担体１３を沈降させて前記逆円錐部３６の頂部に前記堆積層３９を形成させる。 (Step 06) After the carrier supply container 8 is agitated, the carrier supply container 8 is allowed to stand for a predetermined time, the carrier 13 is allowed to settle, and the deposited layer 39 is formed on the top of the inverted conical portion 36.

この時、頂角αが大きい、或は前記担体１３の形状がディスク状等である場合、沈降した該担体１３が前記逆円錐部３６の斜面に沿って移動し難くなる。従って、前記振盪機構３８を駆動させ、前記担体供給容器８に小刻みな振動を与えるか、或はゆっくりと円運動させることで、前記逆円錐部３６の斜面に沈降した前記担体１３を移動させ、前記逆円錐部３６の頂部に前記堆積層３９を形成させることができる。 At this time, when the apex angle α is large or the shape of the carrier 13 is a disk shape or the like, the sedimented carrier 13 is difficult to move along the slope of the inverted conical portion 36. Accordingly, by driving the shaking mechanism 38 and applying a slight vibration to the carrier supply container 8 or slowly moving it circularly, the carrier 13 that has settled on the inclined surface of the inverted conical portion 36 is moved, The deposited layer 39 can be formed on the top of the inverted conical portion 36.

又、頂角αが小さい、或は前記担体１３の形状が球状である場合、前記逆円錐部３６の斜面に沈降した前記担体１３は、容易に前記逆円錐部３６の斜面に沿って頂部に移動するので、前記振盪機構３８による前記担体供給容器８への振盪動作は省略してもよい。 In addition, when the apex angle α is small or the shape of the carrier 13 is spherical, the carrier 13 that has settled on the slope of the inverted conical portion 36 can easily reach the top along the slope of the inverted cone portion 36. Since it moves, the shaking operation to the carrier supply container 8 by the shaking mechanism 38 may be omitted.

ＳＴＥＰ：０７前記担体１３が沈降し、前記逆円錐部３６の頂部に前記堆積層３９が形成されると、次に前記排液ポンプ２８を駆動させ、前記担体供給容器８中のＰＢＳを前記廃液タンク１８に排出する。 (Step 07) When the carrier 13 is settled and the deposited layer 39 is formed on the top of the inverted conical portion 36, the drainage pump 28 is driven next, and the PBS in the carrier supply container 8 is replaced with the waste liquid. Discharge into the tank 18.

ＳＴＥＰ：０８ＰＢＳの排出後、前記培地給排ポンプ３１を逆転駆動させ、前記培地給排管２９、前記屈曲部２２ａを介して前記培養槽１５中の前記培地１４を前記担体供給容器８内に供給する。 (Step 08) After discharging the PBS, the medium supply / discharge pump 31 is driven in reverse, and the medium 14 in the culture tank 15 is put into the carrier supply container 8 via the medium supply / discharge pipe 29 and the bent portion 22a. Supply.

ＳＴＥＰ：０９前記培地１４の供給後、前記振盪機構３８を駆動させ、前記担体供給容器８を往復動させて該担体供給容器８内を撹拌する。該担体供給容器８内が撹拌されることで、前記担体１３が前記培地１４中に浮遊し、懸濁されて担体懸濁液が生成される。 (Step 09) After supplying the culture medium 14, the shaking mechanism 38 is driven to reciprocate the carrier supply container 8 to stir the inside of the carrier supply container 8. By stirring the inside of the carrier supply container 8, the carrier 13 floats in the medium 14 and is suspended to produce a carrier suspension.

尚、前記振盪機構３８による振盪動作に加え、前記培地給排ポンプ３１に正転駆動及び逆転駆動を繰返し行わせ、前記担体供給容器８に対して前記培地１４を出し入れすることで、該培地１４を撹拌し、前記担体１３を浮遊させ、懸濁させてもよい。この場合、前記培地給排ポンプ３１及び前記振盪機構３８が振盪手段を構成する。 In addition to the shaking operation by the shaking mechanism 38, the medium supply / discharge pump 31 is repeatedly driven to rotate forward and reversely, and the medium 14 is taken in and out of the carrier supply container 8. The carrier 13 may be suspended and suspended. In this case, the culture medium supply / discharge pump 31 and the shaking mechanism 38 constitute shaking means.

ＳＴＥＰ：１０最後に、前記振盪機構３８による振盪動作を維持した状態で、前記培地給排ポンプ３１を正転駆動させ、前記屈曲部２２ａ、前記培地給排管２９を介して前記担体供給容器８中の前記担体懸濁液を前記培養槽１５内に供給することで、前記担体供給容器８から前記培養槽１５へ前記担体１３が均一に懸濁された状態で自動で供給することができる。 STEP: 10 Finally, in a state where the shaking operation by the shaking mechanism 38 is maintained, the medium supply / discharge pump 31 is driven to rotate forward, and the carrier supply container 8 is passed through the bent portion 22a and the medium supply / discharge pipe 29. By supplying the carrier suspension in the culture tank 15, the carrier 13 can be automatically supplied from the carrier supply container 8 to the culture tank 15 in a state of being uniformly suspended.

上述の様に、第１の実施例では、前記担体供給容器８の下部が、水平断面積が上方に向って漸次増大する前記逆円錐部３６となっているので、カドヘリン溶液やＰＢＳ中で沈降した前記担体１３を斜面に沿って降下させ、該担体１３を１箇所（頂部）に集めることができるので、該担体１３の回収が容易となる。 As described above, in the first embodiment, the lower portion of the carrier supply container 8 is the inverted conical portion 36 whose horizontal cross-sectional area gradually increases upward, so that it settles in a cadherin solution or PBS. Since the carrier 13 can be lowered along the slope and the carrier 13 can be collected at one place (top), the carrier 13 can be easily recovered.

又、前記担体供給容器８の下部が前記逆円錐部３６となっているので、前記振盪機構３８により前記担体供給容器８を振盪させることで、前記逆円錐部３６の頂部迄満遍なく撹拌することができ、前記担体１３を前記培地１４中に容易且つ均一に懸濁させることができる。従って、前記担体１３を前記培養槽１５に自動で均一の状態で供給することができるので、細胞と前記担体１３との比を容易に調整でき、自動培養の効率を向上させることができる。 Further, since the lower part of the carrier supply container 8 is the inverted conical part 36, the carrier supply container 8 is shaken by the shaking mechanism 38, so that the top of the reverse cone part 36 can be uniformly stirred. The carrier 13 can be easily and uniformly suspended in the medium 14. Therefore, since the carrier 13 can be automatically and uniformly supplied to the culture tank 15, the ratio of cells to the carrier 13 can be easily adjusted, and the efficiency of automatic culture can be improved.

又、前記担体供給容器８の下部が前記逆円錐部３６となっているので、円柱状の場合と比べて前記担体供給容器８の容積を減少させることができ、前記排液管２７を介して前記担体供給容器８中の液体を排出する際の、残留液量を低減させることができる。 Further, since the lower part of the carrier supply container 8 is the inverted conical part 36, the volume of the carrier supply container 8 can be reduced as compared with the case of a columnar shape, via the drainage pipe 27. The amount of residual liquid when the liquid in the carrier supply container 8 is discharged can be reduced.

又、前記担体供給管２２の前記屈曲部２２ａが、上方から前記逆円錐部３６の頂部迄延出しているので、前記屈曲部２２ａからの液体の給排動作によっても前記担体１３を懸濁させることができ、より容易に該担体１３を前記培地１４中に懸濁させることができる。 Further, since the bent portion 22a of the carrier supply pipe 22 extends from above to the top of the inverted conical portion 36, the carrier 13 is suspended by the liquid supply / discharge operation from the bent portion 22a. The carrier 13 can be suspended in the medium 14 more easily.

又、前記屈曲部２２ａの上流端で前記担体供給管２２と前記培地給排管２９が接続され、前記屈曲部２２ａよりも上流側で前記担体供給管２２と前記薬液供給管２５とが接続されているので、前記担体１３及びＰＢＳの供給、及び前記培地１４の給排が全て前記屈曲部２２ａを介して行われることとなり、配管の本数が低減され、前記担体供給装置の小型化を図ることができる。 Further, the carrier supply pipe 22 and the culture medium supply / discharge pipe 29 are connected at the upstream end of the bent part 22a, and the carrier supply pipe 22 and the chemical solution supply pipe 25 are connected upstream of the bent part 22a. Therefore, the supply of the carrier 13 and PBS and the supply / discharge of the culture medium 14 are all performed through the bent portion 22a, the number of pipes is reduced, and the carrier supply device can be downsized. Can do.

前記担体１３は、カドヘリン等の細胞接着物質溶液に懸濁された状態で前記担体供給部６に保管され、細胞接着物質溶液と共に前記担体供給容器８に供給される様になっているので、細胞を培養する際に前記担体１３上に細胞が固まった状態で培養されることがなく、未分化状態が維持されて該担体１３上に均一に細胞を培養させることができる。 The carrier 13 is stored in the carrier supply unit 6 in a state suspended in a cell adhesion substance solution such as cadherin, and is supplied to the carrier supply container 8 together with the cell adhesion substance solution. When the cells are cultured, the cells are not cultured on the carrier 13 in a solid state, and the cells are uniformly cultured on the carrier 13 while maintaining an undifferentiated state.

更に、図２（Ａ）に示される様に、頂角αが大きい場合、前記逆円錐部３６の水平断面積が大きくなり、カドヘリン溶液やＰＢＳ等前記担体供給容器８中に供給される液体の液位が低くなるので、前記担体１３の沈降に要する時間を短縮することができ、前記培養槽１５に対する前記担体１３の供給処理の時間を短縮することができる。 Further, as shown in FIG. 2A, when the apex angle α is large, the horizontal cross-sectional area of the inverted conical portion 36 becomes large, and the liquid supplied into the carrier supply container 8 such as cadherin solution or PBS Since the liquid level is lowered, the time required for the carrier 13 to settle can be shortened, and the time for supplying the carrier 13 to the culture tank 15 can be shortened.

又、頂角αが大きい場合、前記担体供給容器８中に供給される液体の液位当たりの容積が大きくなるので、懸濁可能な前記担体１３の量を増大させることができる。更に、頂角αが大きいと、液体が水平方向に移動し易いので、振盪効果が大きい。 Further, when the apex angle α is large, the volume per liquid level of the liquid supplied into the carrier supply container 8 becomes large, so that the amount of the carrier 13 that can be suspended can be increased. Furthermore, when the apex angle α is large, the liquid is easy to move in the horizontal direction, so that the shaking effect is large.

又、図２（Ｂ）に示される様に、頂角αが小さい場合、前記逆円錐部３６の斜面に沈降した前記担体１３は、形状に拘わらず容易に前記逆円錐部３６の頂部に移動するので、該逆円錐部３６の斜面に沈降した前記担体１３を下方に移動させる為の振盪動作を省略することができる。 As shown in FIG. 2B, when the apex angle α is small, the carrier 13 that has settled on the slope of the inverted conical portion 36 easily moves to the apex of the inverted conical portion 36 regardless of the shape. Therefore, the shaking operation for moving the carrier 13 that has settled on the inclined surface of the inverted conical portion 36 downward can be omitted.

又、頂角αが小さい場合、前記逆円錐部３６の水平断面積が小さくなり、容積が小さくなるので、排液の際に前記担体供給容器８内に残留する液体の液量をより低減させることができる。 In addition, when the apex angle α is small, the horizontal cross-sectional area of the inverted conical portion 36 is reduced and the volume is reduced, so that the amount of liquid remaining in the carrier supply container 8 during drainage is further reduced. be able to.

従って、使用する前記担体１３の形状、供給する該担体１３の量等、種々の条件に応じて最適な頂角αを有する前記担体供給容器８を用いることで、効率よく、且つ均一に懸濁された前記担体１３を前記培養槽１５内に自動で供給することができる。 Therefore, the carrier supply container 8 having the optimum apex angle α according to various conditions such as the shape of the carrier 13 to be used and the amount of the carrier 13 to be supplied can be efficiently and uniformly suspended. The carried carrier 13 can be automatically supplied into the culture tank 15.

尚、第１の実施例では、前記担体１３の表面に付着したカドヘリン溶液をＰＢＳで洗浄した後、前記培養槽１５に供給しているが、前記担体１３として洗浄が不要な担体を用いることもできる。この場合、表面にカドヘリン溶液が付着した担体を前記培養槽１５に供給することとなるので、図３中のＳＴＥＰ：０４〜ＳＴＥＰ：０７の行程を省略することができる。 In the first embodiment, the cadherin solution adhering to the surface of the carrier 13 is washed with PBS and then supplied to the culture tank 15. However, a carrier that does not require washing may be used as the carrier 13. it can. In this case, since the carrier with the cadherin solution attached to the surface is supplied to the culture tank 15, the steps from STEP: 04 to STEP: 07 in FIG. 3 can be omitted.

次に、図４（Ａ）（Ｂ）に於いて、本発明の第２の実施例について説明する。尚、図４（Ａ）（Ｂ）中、図２（Ａ）（Ｂ）中と同等のものには同符号を付し、その説明を省略する。又、第２の実施例に於いても、逆円錐部３６の頂角は４５°〜１２０°の範囲で任意の角度が選択される様になっており、図４（Ａ）は頂角αを９０°とした担体供給容器８を示し、図４（Ｂ）は頂角αを６０°とした該担体供給容器８を示している。 Next, a second embodiment of the present invention will be described with reference to FIGS. In FIGS. 4A and 4B, the same components as those in FIGS. 2A and 2B are denoted by the same reference numerals, and the description thereof is omitted. Also in the second embodiment, the apex angle of the inverted conical portion 36 is selected in the range of 45 ° to 120 °, and FIG. 4 (A) shows the apex angle α. FIG. 4B shows the carrier supply container 8 with the apex angle α being 60 °.

第２の実施例では、担体供給管２２が前記担体供給容器８の下方に配設され、下流端部が上方に屈曲して屈曲部２２ｂを形成しており、該屈曲部２２ｂは前記逆円錐部３６の頂点に開口している。尚、前記屈曲部２２ｂの径は、例えば２ｍｍ〜３ｍｍ程度であり、担体１３が詰まらない大きさとなっている。 In the second embodiment, the carrier supply pipe 22 is disposed below the carrier supply container 8, and the downstream end is bent upward to form a bent portion 22b. The bent portion 22b is the inverted cone. An opening is made at the apex of the portion 36. The diameter of the bent portion 22b is, for example, about 2 mm to 3 mm, and is a size that does not clog the carrier 13.

第２の実施例に於いても、カドヘリン溶液排出後の前記担体１３をＰＢＳで洗浄し、ＰＢＳを排出した後に培地１４（図１参照）を前記担体供給容器８内に供給し、振盪機構３８を駆動させて前記担体供給容器８を撹拌することで、前記担体１３を前記培地１４中に浮遊させ、均一に懸濁させた担体懸濁液を生成することができる。 Also in the second embodiment, the carrier 13 after the cadherin solution is discharged is washed with PBS, and after the PBS is discharged, the culture medium 14 (see FIG. 1) is supplied into the carrier supply container 8, and the shaking mechanism 38 By driving the carrier supply container 8, the carrier 13 can be suspended in the medium 14 and a carrier suspension in which the carrier 13 is uniformly suspended can be generated.

更に、前記振盪機構３８による振盪動作を継続した状態で、培地給排ポンプ３１（図１参照）を駆動させることで、前記担体１３を均一に懸濁させた前記担体懸濁液を自動で培養槽１５（図１参照）に供給することができる。 Further, by driving the culture medium supply / discharge pump 31 (see FIG. 1) while the shaking operation by the shaking mechanism 38 is continued, the carrier suspension in which the carrier 13 is uniformly suspended is automatically cultured. It can supply to the tank 15 (refer FIG. 1).

１細胞培養システム２細胞導入装置
３細胞培養装置４細胞剥離装置
５細胞回収装置６担体供給部
７薬液タンク８担体供給容器
１３担体１４培地
１５培養槽２２担体供給管
２４ポンプ２６薬液供給管
２７排液管２８排液ポンプ
２９培地給排管３６逆円錐部
３７円筒部３８振盪機構
３９堆積層 DESCRIPTION OF SYMBOLS 1 Cell culture system 2 Cell introduction apparatus 3 Cell culture apparatus 4 Cell peeling apparatus 5 Cell collection apparatus 6 Carrier supply part 7 Chemical liquid tank 8 Carrier supply container 13 Carrier 14 Medium 15 Culture tank 22 Carrier supply pipe 24 Pump 26 Chemical liquid supply pipe 27 Exhaust Liquid pipe 28 Drain pump 29 Medium supply / drain pipe 36 Reverse cone part 37 Cylindrical part 38 Shaking mechanism 39 Deposition layer

Claims

担体供給容器に細胞接着物質溶液に懸濁された担体を供給する工程と、該担体が沈降した後に前記細胞接着物質溶液を排出する工程と、培養槽内の培地を前記担体供給容器に供給する工程と、振盪で前記培地中に前記担体を懸濁させる工程と、振盪を維持した状態で前記担体を懸濁させた前記培地を前記培養槽に導入する工程とを有することを特徴とする担体供給方法。 Supplying the carrier suspended in the cell adhesion substance solution to the carrier supply container; discharging the cell adhesion substance solution after the carrier settles; and supplying the culture medium in the culture tank to the carrier supply container. And a step of suspending the carrier in the medium by shaking, and a step of introducing the medium in which the carrier is suspended in a state in which the carrier is suspended into the culture vessel. Supply method.

前記細胞接着物質溶液を排出する工程と、前記培養槽内の前記培地を前記担体供給容器に供給する工程との間に、該担体供給容器に薬液を供給する工程と、振盪で前記担体供給容器を撹拌して前記薬液で前記担体を洗浄する工程と、該担体を沈降させる工程と、前記薬液を排出する工程とを有する請求項１の担体供給方法。 Between the step of discharging the cell adhesion substance solution and the step of supplying the medium in the culture tank to the carrier supply container, the step of supplying a chemical to the carrier supply container, and the carrier supply container by shaking The carrier supply method according to claim 1, comprising: a step of washing the carrier with the chemical solution, a step of precipitating the carrier, and a step of discharging the chemical solution.

担体供給容器と、該担体供給容器に担体を供給する担体供給手段と、前記担体供給容器を振盪させ前記担体を浮遊させて培地中に懸濁させる振盪手段と、前記担体供給容器内の液体を排出する排出手段と、前記担体供給容器に細胞を培養する培養槽から培地を供給すると共に、前記振盪手段により懸濁された前記培地を培養槽に導入する培地給排手段とを具備することを特徴とする担体供給装置。 A carrier supply container, a carrier supply means for supplying a carrier to the carrier supply container, a shaking means for shaking the carrier supply container to suspend the carrier and suspending it in a medium, and a liquid in the carrier supply container. A discharge means for discharging, and a medium supply / discharge means for supplying the medium from the culture tank for culturing the cells to the carrier supply container and for introducing the medium suspended by the shaking means into the culture tank. A carrier supply device.

前記担体供給手段と前記培地給排手段は、それぞれ担体供給管と培地給排管とを有し、前記担体供給管の下流端部は前記担体供給容器の内部に延出し、前記担体供給管及び前記培地給排管が前記下流端部の上流で接続された請求項３の担体供給装置。 The carrier supply means and the medium supply / discharge means each have a carrier supply pipe and a medium supply / discharge pipe, and a downstream end of the carrier supply pipe extends into the carrier supply container, and the carrier supply pipe and The carrier supply apparatus according to claim 3, wherein the culture medium supply / discharge pipe is connected upstream of the downstream end portion.

前記担体供給容器に薬液を供給する薬液供給手段を更に具備し、該薬液供給手段は薬液供給管を有し、該薬液供給管は前記下流端部よりも上流で前記担体供給管に接続された請求項３又は請求項４の担体供給装置。 The apparatus further comprises a chemical supply means for supplying the chemical supply to the carrier supply container, the chemical supply means having a chemical supply pipe, and the chemical supply pipe is connected to the carrier supply pipe upstream from the downstream end. The carrier supply device according to claim 3 or 4.

前記担体供給容器は逆円錐状又は逆角錐状であり、前記担体供給容器の頂角は、４５°〜１２０°に含まれる請求項３〜請求項５のいずれか１項に記載の担体供給装置。 The carrier supply device according to any one of claims 3 to 5, wherein the carrier supply container has an inverted conical shape or an inverted pyramid shape, and an apex angle of the carrier supply container is included in 45 ° to 120 °. .

請求項３〜請求項６のいずれか１項に記載の担体供給装置を用いた細胞培養システムであって、前記担体供給装置を有する細胞導入装置と、細胞培養装置と、細胞剥離装置と、細胞回収装置とを具備し、前記細胞導入装置が前記細胞培養装置に担体と細胞と培地とを導入し、前記細胞培養装置が前記担体を前記培地に浮遊させて細胞を前記担体上で培養し、前記細胞剥離装置が前記細胞培養装置から供給された前記培地に含まれる前記担体から細胞を剥離させ、前記細胞回収装置が前記細胞剥離装置に剥離させた細胞を回収することを特徴とする細胞培養システム。 A cell culture system using the carrier supply device according to any one of claims 3 to 6, wherein the cell introduction device, the cell culture device, the cell peeling device, and the cell having the carrier supply device A recovery device, wherein the cell introduction device introduces a carrier, cells, and a medium into the cell culture device, the cell culture device suspends the carrier in the medium, and cultures the cells on the carrier, The cell culturing apparatus wherein the cell detaching apparatus detaches cells from the carrier contained in the medium supplied from the cell culturing apparatus, and the cell recovery apparatus recovers the cells detached by the cell detaching apparatus. system.