JP5731421B2 - Cell culture devices - Google Patents
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- JP5731421B2 JP5731421B2 JP2012030204A JP2012030204A JP5731421B2 JP 5731421 B2 JP5731421 B2 JP 5731421B2 JP 2012030204 A JP2012030204 A JP 2012030204A JP 2012030204 A JP2012030204 A JP 2012030204A JP 5731421 B2 JP5731421 B2 JP 5731421B2
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- 238000004113 cell culture Methods 0.000 title claims description 55
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 57
- 239000000463 material Substances 0.000 claims description 57
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 57
- 229920001343 polytetrafluoroethylene Polymers 0.000 claims description 25
- 239000004810 polytetrafluoroethylene Substances 0.000 claims description 25
- 229920002379 silicone rubber Polymers 0.000 claims description 5
- 239000004945 silicone rubber Substances 0.000 claims description 5
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims 5
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims 5
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims 5
- 210000004027 cell Anatomy 0.000 description 14
- 210000002889 endothelial cell Anatomy 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 8
- 238000007789 sealing Methods 0.000 description 8
- 210000003494 hepatocyte Anatomy 0.000 description 7
- 230000005499 meniscus Effects 0.000 description 7
- 230000015572 biosynthetic process Effects 0.000 description 6
- 230000003908 liver function Effects 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 4
- 238000000034 method Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 210000000013 bile duct Anatomy 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000005228 liver tissue Anatomy 0.000 description 2
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- 108010042086 Collagen Type IV Proteins 0.000 description 1
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- 208000009331 Experimental Sarcoma Diseases 0.000 description 1
- 102000007547 Laminin Human genes 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
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- 210000002469 basement membrane Anatomy 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/12—Well or multiwell plates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
- C12M25/14—Scaffolds; Matrices
Description
本発明は、細胞の足場となるゲルが収容される培養室を備えた細胞培養デバイスに関する。 The present invention relates to a cell culture device including a culture chamber in which a gel serving as a cell scaffold is accommodated.
細胞培養は、一般的にシャーレ等の容器に細胞及び液体状の培地を収容した状態で行なわれる。しかし、近年、半導体製造分野での微細加工技術の進歩に伴って、医療やバイオテクノロジーの研究分野でも微細加工技術によって製造されたマイクロデバイスの応用が進められている。そして、マイクロデバイスを用いた細胞培養が行われるようになっている（例えば、特許文献１を参照。）。 Cell culture is generally performed in a state where cells and a liquid medium are contained in a container such as a petri dish. However, in recent years, with the progress of microfabrication technology in the semiconductor manufacturing field, the application of microdevices manufactured by microfabrication technology has been promoted in the medical and biotechnology research fields. And cell culture using a micro device is performed (for example, refer to patent documents 1).
細胞培養用のマイクロデバイス（細胞培養デバイス）は、平板状基材の内部に培養室と微小流路が形成されて成るものである。その培養室に細胞及び培地が収容されて細胞培養が行なわれる。 A microdevice for cell culture (cell culture device) is formed by forming a culture chamber and a microchannel inside a flat substrate. Cells and medium are accommodated in the culture chamber, and cell culture is performed.
ところで、近年、人工臓器の開発に期待が寄せられている。生体の化学工場にたとえられる肝臓は、判明しているだけで５００種類以上の代謝反応を行なっているとされる。このような肝臓の機能を人工的な装置で全て補うことは中長期的にみても極めて困難であると考えられている。そこで、生体の肝細胞を利用することが提案されている。このような技術は肝細胞と人工装置の組み合わせであることからハイブリッド型の人工肝臓と呼ばれる。 By the way, in recent years, there are expectations for the development of artificial organs. It is said that the liver, which is compared to a biological chemical factory, is carrying out more than 500 kinds of metabolic reactions only by being known. It is considered that it is extremely difficult to supplement all the functions of the liver with an artificial device in the medium to long term. Thus, it has been proposed to use living hepatocytes. Such a technique is called a hybrid type artificial liver because it is a combination of hepatocytes and an artificial device.
従来の人工肝臓システムでは、ブタなどから取り出した肝組織を用いて肝機能を補助することを目的としている。
肝臓は生体内では肝実細胞の他に、類洞内皮細胞などが規則正しく配列され、細胞間のシグナル伝達や体液の循環が正常な機能を維持するために重要な役割を果たしていると考えられる。特に、肝実質細胞と類洞内皮細胞は並行して並んでおり、それらの細胞列間は直接接触していない。それらの間にはディッセ腔と呼ばれる細胞外マトリックスがある。
A conventional artificial liver system aims to assist liver function using liver tissue taken out from pigs and the like.
In the living body, in addition to liver hepatocytes, sinusoidal endothelial cells and the like are regularly arranged in the living body, and it is considered that the signal transmission between the cells and the circulation of body fluids play an important role in maintaining normal functions. In particular, hepatic parenchymal cells and sinusoidal endothelial cells are juxtaposed in parallel, and the cell rows are not in direct contact. Between them is an extracellular matrix called the Diesse space.
それぞれの細胞列の外側は、肝実質細胞側は胆管、類洞内皮細胞側は血管となっている。肝機能を有する人工肝システムを確立するためには、これらの細胞種の規則正しい配置と細胞外マトリックスの構築が重要である。薬物や栄養物は血管側から供給され、胆管側に排出される。培養した細胞を用いた人工肝システムヘの取り組みもなされているが、安定したシステムは構築できていない。 Outside each cell line, hepatocytes are bile ducts and sinusoidal endothelial cells are blood vessels. In order to establish an artificial liver system with liver function, regular arrangement of these cell types and construction of an extracellular matrix are important. Drugs and nutrients are supplied from the blood vessel side and discharged to the bile duct side. Although efforts have been made to artificial liver systems using cultured cells, a stable system has not been established.
また、肝細胞の培養そのものが難しく、その上、生体内の肝臓に近い組織を形成することはさらに困難である。このような理由から、長期間にわたって安定な肝機能を維持できる人エシステムは構築できていない。 In addition, it is difficult to culture hepatocytes, and it is further difficult to form a tissue close to the liver in the living body. For these reasons, human systems that can maintain stable liver function over a long period of time have not been established.
この問題の対策として、本願発明者らは、ＥＨＳ−ｇｅｌ（Engelbreth-Holm-Swarm sarcoma-derived matrix）上でネットワーク構造を構築した類洞内皮細胞に肝実質細胞が組み込まれていくことを見いだし（非特許文献１）、生体内に近い構造の肝組織デバイスを提案している。 As a countermeasure against this problem, the present inventors have found that hepatocytes are incorporated into sinusoidal endothelial cells in which a network structure is constructed on EHS-gel (Engelbreth-Holm-Swarm sarcoma-derived matrix) ( Non-Patent Document 1), a liver tissue device having a structure close to that of a living body is proposed.
しかしながら、細胞培養デバイスの培養室にＥＨＳ−ｇｅｌを塗布すると、ゲルが培養室の内壁側面に引っ張られて凹型のメニスカスが形成され、ゲル表面の平坦な部分が中央部に限られてしまうという問題があった。内皮細胞がネットワーク構造を形成するのはゲルが平坦な部分のみである。したがって、上記問題は培養室の小容量化や肝機能の十分な発現を妨げる。 However, when EHS-gel is applied to the culture chamber of the cell culture device, the gel is pulled to the inner wall side surface of the culture chamber to form a concave meniscus, and the flat portion of the gel surface is limited to the central portion. was there. Endothelial cells form a network structure only on the flat part of the gel. Therefore, the above problem hinders the reduction of the culture chamber capacity and sufficient expression of liver function.
本発明は、細胞の足場材となるゲルが収容される培養室を備えた細胞培養デバイスにおいて、ゲル表面の平坦な部分の面積を大きくすることを目的とする。 An object of the present invention is to increase the area of a flat portion of a gel surface in a cell culture device including a culture chamber in which a gel serving as a cell scaffold is accommodated.
本発明にかかる細胞培養デバイスは、細胞の足場材となるゲルが収容される培養室を備えた細胞培養デバイスであって、上記培養室の内壁側面はゲルに濡れない素材で形成された部分とゲルに濡れる素材で形成された部分とを備え、上記ゲルに濡れない素材は、上記内壁側面において上記培養室の底面から所定の間隔をもって、かつ上方から見て環状に配置されており、上記ゲルに濡れない素材と上記底面との間の上記内壁側面は上記ゲルに濡れる素材によって形成されていることを特徴とする細胞培養デバイスである。
ここで、「ゲルに濡れない素材」とは固体状になる前の流動性をもつ状態のゲルとの接触角が９０度以上の素材である。「ゲルに濡れる素材」とは固体状になる前の流動性をもつ状態のゲルとの接触角が９０度未満の素材である。
The cell culture device according to the present invention is a cell culture device including a culture chamber in which a gel serving as a cell scaffold is accommodated, and the inner wall side surface of the culture chamber is formed of a material that does not wet the gel; A portion formed of a material that wets the gel, and the material that does not wet the gel is arranged on the inner wall side surface at a predetermined interval from the bottom surface of the culture chamber and in an annular shape when viewed from above, the gel The cell culture device is characterized in that the inner wall side surface between the material that does not get wet and the bottom surface is formed of the material that gets wet with the gel.
Here, the “material that does not wet the gel” is a material having a contact angle of 90 degrees or more with the gel in a fluid state before becoming a solid. The “material that wets the gel” is a material having a contact angle of less than 90 degrees with the gel in a fluid state before becoming a solid.
本発明の細胞培養デバイスでは、培養室の内壁側面はゲルに濡れない素材で形成された部分とゲルに濡れる素材で形成された部分とを備えている。ゲルに濡れない素材は、培養室の底面から所定の間隔をもって、かつ上方から見て環状に配置されている。さらに、ゲルに濡れない素材と培養室の底面との間の培養室の内壁側面はゲルに濡れる素材によって形成されている。固体化する前の流動性をもつ状態のゲルが培養室内に注入される際、ゲルの表面が濡れない素材で形成された部分とゲルに濡れる素材で形成された部分の境界の高さになるように、所定量のゲルが培養室内に注入される。これにより、従来の細胞培養デバイスを用いた場合に比べて、ゲル表面に形成されるメニスカスは小さくなる又は形成されない。 In the cell culture device of the present invention, the inner wall side surface of the culture chamber includes a portion formed of a material that does not wet the gel and a portion formed of a material that wets the gel. The material that does not wet the gel is arranged in a ring shape with a predetermined interval from the bottom surface of the culture chamber as viewed from above. Furthermore, the inner wall side surface of the culture chamber between the material that does not wet the gel and the bottom surface of the culture chamber is formed of the material that wets the gel. When a gel with fluidity before solidification is injected into the culture chamber, the surface of the gel becomes the height of the boundary between the part formed with a material that does not wet and the part formed with the material that wets the gel Thus, a predetermined amount of gel is injected into the culture chamber. Thereby, compared with the case where the conventional cell culture device is used, the meniscus formed in the gel surface becomes small or is not formed.
本発明の細胞培養デバイスにおいて、上記ゲルに濡れない素材の例としてＰＴＦＥ（polytetrafluoroethylene）又はフッ素樹脂が挙げられる。ただし、本発明の細胞培養デバイスにおいて、ゲルに濡れない素材はこれらに限定されない。 In the cell culture device of the present invention, examples of the material that does not wet the gel include PTFE (polytetrafluoroethylene) or fluororesin. However, in the cell culture device of the present invention, the material that does not wet the gel is not limited thereto.
また、上記ゲルに濡れない素材は多孔質のものである例を挙げることができる。ただし、本発明の細胞培養デバイスにおいて、ゲルに濡れない素材は多孔質のものに限定されない。 An example in which the material that does not wet the gel is porous. However, in the cell culture device of the present invention, the material that does not wet the gel is not limited to a porous material.
また、上記ゲルに濡れる素材の例としてＰＤＭＳ（polydimethylsiloxane）又はシリコーンゴムが挙げられる。ただし、本発明の細胞培養デバイスにおいて、ゲルに濡れる素材はこれらに限定されない。 Examples of the material that gets wet with the gel include PDMS (polydimethylsiloxane) or silicone rubber. However, in the cell culture device of the present invention, the material wetted by the gel is not limited to these.
本発明の細胞培養デバイスにおいて、上記培養室は、下層側ＰＤＭＳシート、ＰＴＦＥシート、上層側ＰＤＭＳシートが下層側から順に積層された積層体に対して積層方向に形成された貫通孔と、上記下層側ＰＤＭＳシートの上記ＰＴＦＥシートとは反対側の面に配置された底板によって形成された空間を備えており、上記培養室内壁に露出した上記ＰＴＦＥシートの部分によって上記ゲルに濡れない素材で形成された部分が構成され、上記培養室内壁に露出した上記下層側ＰＤＭＳシートの部分によって上記ゲルに濡れる素材で形成された部分が構成されている例を挙げることができる。ただし、本発明の細胞培養デバイスにおいて、培養室の構成はこれに限定されない。 In the cell culture device of the present invention, the culture chamber includes a through hole formed in a stacking direction with respect to a stacked body in which a lower layer side PDMS sheet, a PTFE sheet, and an upper layer side PDMS sheet are stacked in order from the lower layer side, and the lower layer side The side PDMS sheet has a space formed by a bottom plate disposed on the surface opposite to the PTFE sheet, and is formed of a material that does not wet the gel by the portion of the PTFE sheet exposed on the culture chamber wall. An example in which a portion formed of a material that gets wet with the gel is formed by the portion of the lower layer side PDMS sheet exposed on the culture chamber wall. However, in the cell culture device of the present invention, the configuration of the culture chamber is not limited to this.
本発明の細胞培養デバイスは、培養室の内壁側面に関し、ゲルに濡れない素材で形成された部分とゲルに濡れる素材で形成された部分とを備えている。ゲルに濡れない素材は、培養室の内壁側面において培養室の底面から所定の間隔をもって、かつ上方から見て環状に配置されている。ゲルに濡れない素材と培養室の底面との間の培養室の内壁側面はゲルに濡れる素材によって形成されている。本発明の細胞培養デバイスにおいて、ゲルの表面が濡れない素材で形成された部分とゲルに濡れる素材で形成された部分の境界の高さになるように、所定量のゲルが培養室内に注入されことにより、従来の細胞培養デバイスを用いた場合に比べて、ゲル表面に形成されるメニスカスは小さくなる又は形成されない。これにより、培養室内に塗布されたゲルの表面の平坦な部分の面積は、従来の細胞培養デバイスを用いた場合に比べて大きくなる。 The cell culture device of the present invention relates to the inner wall side surface of the culture chamber, and includes a portion formed of a material that does not wet the gel and a portion formed of a material that wets the gel. The material that does not wet the gel is arranged on the inner wall side surface of the culture chamber at a predetermined interval from the bottom surface of the culture chamber and in an annular shape when viewed from above. The inner wall side surface of the culture chamber between the material that does not wet the gel and the bottom surface of the culture chamber is formed of the material that wets the gel. In the cell culture device of the present invention, a predetermined amount of gel is injected into the culture chamber so that the surface of the gel is at the height of the boundary between the portion formed of the material that does not wet and the portion formed of the material that wets the gel. Thereby, compared with the case where the conventional cell culture device is used, the meniscus formed in the gel surface becomes small or is not formed. Thereby, the area of the flat part of the surface of the gel apply | coated in the culture chamber becomes large compared with the case where the conventional cell culture device is used.
図１は本発明の細胞培養デバイスの一実施例の断面図である。図２は同実施例の平面図である。図１の断面は図２のＡ−Ａ位置に対応している。図３は同実施例を構成する各部材を分解して示した平面図である。 FIG. 1 is a cross-sectional view of one embodiment of the cell culture device of the present invention. FIG. 2 is a plan view of the embodiment. The cross section in FIG. 1 corresponds to the position AA in FIG. FIG. 3 is an exploded plan view showing each member constituting the embodiment.
細胞培養デバイス１は、大きく分けて本体３とカバー部材５から成る。本体３は、底板７、下層側ＰＤＭＳシート９、ＰＴＦＥシート１１、上層側ＰＤＭＳシート１３、流路形成用ＰＤＭＳシート１５及び封止用ＰＤＭＳ１７を備えている。 The cell culture device 1 is roughly composed of a main body 3 and a cover member 5. The main body 3 includes a bottom plate 7, a lower layer side PDMS sheet 9, a PTFE sheet 11, an upper layer side PDMS sheet 13, a flow path forming PDMS sheet 15, and a sealing PDMS 17.
底板７は例えばガラス基板で形成されている。底板７の寸法は例えば長さが２０ｍｍ（ミリメートル）、幅が２０ｍｍ、厚みが１ｍｍである。底板７は例えば樹脂基板や細胞培養用ディッシュなど、ガラスとは異なる素材で形成されたものであってもよい。底板７は、後述するゲルに濡れる素材で形成されていることが好ましい。ただし、底板７の素材はゲルに濡れない素材であってもよい。 The bottom plate 7 is formed of, for example, a glass substrate. The dimensions of the bottom plate 7 are, for example, a length of 20 mm (millimeters), a width of 20 mm, and a thickness of 1 mm. The bottom plate 7 may be made of a material different from glass, such as a resin substrate or a cell culture dish. The bottom plate 7 is preferably formed of a material that gets wet with the gel described later. However, the material of the bottom plate 7 may be a material that does not wet the gel.
下層側ＰＤＭＳシート９、上層側ＰＤＭＳシート１３及び流路形成用ＰＤＭＳシート１５はＰＤＭＳ（東レ・ダウコーニング株式会社製、ＳＩＬＰＯＴ１８４）で形成されている。ＰＤＭＳシート９，１３，１５の寸法は例えば長さが１８ｍｍ、幅が１８ｍｍ、厚みが０.５ｍｍである。 The lower layer side PDMS sheet 9, the upper layer side PDMS sheet 13, and the flow path forming PDMS sheet 15 are formed of PDMS (SILPOT 184, manufactured by Toray Dow Corning Co., Ltd.). The dimensions of the PDMS sheets 9, 13, and 15 are, for example, 18 mm in length, 18 mm in width, and 0.5 mm in thickness.
ＰＴＦＥシート１１はＰＴＦＥ（日本ゴア株式会社製、ゴア（登録商標）ハイパーシート（登録商標）ガスケット）で形成されている。ＰＴＦＥシート１１の寸法は例えば長さが１８ｍｍ、幅が１８ｍｍ、厚みが約０.５ｍｍである。 The PTFE sheet 11 is formed of PTFE (manufactured by Nippon Gore Co., Ltd., Gore (registered trademark) Hypersheet (registered trademark) gasket). The dimensions of the PTFE sheet 11 are, for example, a length of 18 mm, a width of 18 mm, and a thickness of about 0.5 mm.
カバー部材５は例えばシリコーンゴムで形成されている。カバー部材５の寸法は例えば長さが２０ｍｍ、幅が２０ｍｍ、厚みが３ｍｍである。 The cover member 5 is made of, for example, silicone rubber. The cover member 5 has, for example, a length of 20 mm, a width of 20 mm, and a thickness of 3 mm.
下層側ＰＤＭＳシート９、ＰＴＦＥシート１１、上層側ＰＤＭＳシート１３及び流路形成用ＰＤＭＳシート１５は、それらのシート９，１１，１３，１５の周囲に形成された封止用ＰＤＭＳ１７によって一体化されている。ＰＤＭＳ１７を用いて封止した後の平面寸法は例えば長さが２０ｍｍ、幅が２０ｍｍである。 The lower layer side PDMS sheet 9, the PTFE sheet 11, the upper layer side PDMS sheet 13 and the flow path forming PDMS sheet 15 are integrated by a sealing PDMS 17 formed around the sheets 9, 11, 13, 15. Yes. The planar dimensions after sealing with PDMS 17 are, for example, 20 mm in length and 20 mm in width.
シート９，１１，１３，１５の中央に、それぞれ直径６ｍｍの貫通孔９ａ，１１ａ，１３ａ，１５ａが形成されている。貫通孔９ａ，１１ａ，１３ａ，１５ａ及び底板７によって培養室３ａが形成されている。なお、培養室３ａの内壁に露出したＰＴＦＥシート１１の部分によって、本発明の細胞培養デバイスにおける「ゲルに濡れない素材で形成された部分」が構成されている。また、培養室３ａの内壁に露出した下層側ＰＤＭＳシート９の部分によって、本発明の細胞培養デバイスにおける「ゲルに濡れる素材で形成された部分」が構成されている。 Through holes 9a, 11a, 13a, and 15a each having a diameter of 6 mm are formed at the centers of the sheets 9, 11, 13, and 15, respectively. The culture chamber 3a is formed by the through holes 9a, 11a, 13a, 15a and the bottom plate 7. The portion of the PTFE sheet 11 exposed on the inner wall of the culture chamber 3a constitutes a “portion formed of a material that does not wet the gel” in the cell culture device of the present invention. In addition, the portion of the lower layer PDMS sheet 9 exposed on the inner wall of the culture chamber 3a constitutes the “portion formed of the material that wets the gel” in the cell culture device of the present invention.
流路形成用ＰＤＭＳシート１５の隅には直径１.５ｍｍの貫通孔１５ｂが形成されている。流路形成用ＰＤＭＳシート１５の下面（上層側ＰＤＭＳシート１３側の面）に深さ０.１ｍｍの凹部１５ｃが形成されている。凹部１５ｃは貫通孔１５ａの周縁部に設けられた幅１ｍｍのドーナツ状の溝と、その溝と貫通孔１５ｂを連結する幅２ｍｍの溝とで形成されている。 A through-hole 15b having a diameter of 1.5 mm is formed in the corner of the PDMS sheet 15 for flow path formation. A recess 15c having a depth of 0.1 mm is formed on the lower surface (surface on the upper layer side PDMS sheet 13 side) of the PDMS sheet 15 for flow path formation. The recess 15c is formed by a doughnut-shaped groove having a width of 1 mm provided at the peripheral edge of the through hole 15a, and a groove having a width of 2 mm connecting the groove and the through hole 15b.
カバー部材５に、貫通孔１５ｂに対応する位置に直径１.５ｍｍの貫通孔５ｂが形成されている。カバー部材５には、貫通孔５ｂが設けられた隅と対向する隅に直径１.５ｍｍの貫通孔５ａも形成されている。カバー部材５の下面（本体３側の面）に深さ０.１ｍｍの凹部５ｃが形成されている。凹部５ｃはカバー部材５の中央部に設けられた直径６ｍｍの凹部と、その凹部と貫通孔５ａを連結する幅２ｍｍの溝とで形成されている。 A through hole 5b having a diameter of 1.5 mm is formed in the cover member 5 at a position corresponding to the through hole 15b. The cover member 5 is also formed with a through hole 5a having a diameter of 1.5 mm at a corner facing the corner where the through hole 5b is provided. A recess 5c having a depth of 0.1 mm is formed on the lower surface of the cover member 5 (the surface on the main body 3 side). The recess 5c is formed by a recess having a diameter of 6 mm provided in the center of the cover member 5 and a groove having a width of 2 mm connecting the recess and the through hole 5a.
本体３の製造工程の一例について説明する。
貫通孔１５ａ，１５ｂが形成されておらず、凹部１５ｃが形成されている流路形成用ＰＤＭＳシート１５に対して貫通孔１５ｂが形成される。貫通孔１５ｂが形成された流路形成用ＰＤＭＳシート１５と、貫通孔１３ａが形成されていない上層側ＰＤＭＳシート１３とが貼り合わせられる。その際、シート１３，１５の接合面にはプラズマ処理が施される。貼り合わせられたシート１３，１５の中央部に貫通孔１３ａ，１５ａが形成される。
An example of the manufacturing process of the main body 3 will be described.
The through holes 15a and 15b are not formed, and the through holes 15b are formed on the flow path forming PDMS sheet 15 in which the recesses 15c are formed. The flow path forming PDMS sheet 15 in which the through holes 15b are formed and the upper layer side PDMS sheet 13 in which the through holes 13a are not formed are bonded together. At that time, the plasma treatment is performed on the joint surfaces of the sheets 13 and 15. Through holes 13a and 15a are formed at the center of the bonded sheets 13 and 15.
貫通孔９ａが形成されていない下層側ＰＤＭＳシート９の上に、貫通孔１１ａが形成されていないＰＴＦＥシート１１が配置される。このときのＰＴＦＥシート１１の厚みは０.５ｍｍである。ＰＴＦＥシート１１の上に、シート１３，１５の積層体が配置される。 A PTFE sheet 11 having no through hole 11a is disposed on the lower PDMS sheet 9 having no through hole 9a. The thickness of the PTFE sheet 11 at this time is 0.5 mm. On the PTFE sheet 11, a laminate of sheets 13 and 15 is disposed.
シート９，１１，１３，１５の積層体の周囲が封止用ＰＤＭＳ１７によって封止される。これにより、シート９，１１，１３，１５が一体化される。このとき、流路形成用ＰＤＭＳシート１５が下層側ＰＤＭＳシート９側に加圧された状態で封止処理が行なわれることにより、多孔質のＰＴＦＥシート１１の厚みが減少した状態で、一体化されたシート９，１１，１３，１５及び封止用ＰＤＭＳ１７が形成される。すなわち、流路形成用ＰＤＭＳシート１５への加圧の程度を調節することにより、シート９，１１，１３，１５の積層体の厚みを調節できる。なお、封止処理の際に、下層側ＰＤＭＳシート９が流路形成用ＰＤＭＳシート１５側に加圧されることによっても同じ作用及び効果が得られる。 The periphery of the laminate of the sheets 9, 11, 13, and 15 is sealed with the sealing PDMS 17. Thereby, the sheets 9, 11, 13, and 15 are integrated. At this time, the sealing process is performed in a state where the flow path forming PDMS sheet 15 is pressed to the lower PDMS sheet 9 side, so that the porous PTFE sheet 11 is integrated with a reduced thickness. Sheets 9, 11, 13, and 15 and sealing PDMS 17 are formed. That is, by adjusting the degree of pressure applied to the flow path forming PDMS sheet 15, the thickness of the laminate of the sheets 9, 11, 13, 15 can be adjusted. In addition, the same operation and effect can be obtained by pressing the lower layer side PDMS sheet 9 toward the flow path forming PDMS sheet 15 side during the sealing process.
一体化されたシート９，１１，１３，１５及び封止用ＰＤＭＳ１７のシート９，１１に対して、貫通孔１３ａ，１５ａの位置に合わせて貫通孔９ａ，１１ａが形成される。下層側ＰＤＭＳシート９の下面（ＰＴＦＥシート１１とは反対側の面）に底板７が貼り合わされる。これにより、貫通孔９ａ，１１ａ，１３ａ，１５ａ及び底板７で構成された培養室３ａが形成される。 Through holes 9a and 11a are formed in the integrated sheets 9, 11, 13, and 15 and the sheets 9 and 11 of the sealing PDMS 17 in accordance with the positions of the through holes 13a and 15a. The bottom plate 7 is bonded to the lower surface of the lower layer side PDMS sheet 9 (surface opposite to the PTFE sheet 11). Thereby, the culture chamber 3a comprised by the through-holes 9a, 11a, 13a, 15a and the bottom plate 7 is formed.
このようにして形成された本体３の上面（流路用ＰＤＭＳシート１５の上層側ＰＤＭＳシート１３とは反対側の面）にカバー部材５が貼り付けられることにより、細胞培養デバイス１が形成される。本体３とカバー部材５は自己吸着によってある程度の液密を保てる。本体３とカバー部材５を固定具により固定すれば、より安定したデバイスを形成できる。 The cell culture device 1 is formed by affixing the cover member 5 to the upper surface of the main body 3 thus formed (the surface opposite to the upper PDMS sheet 13 of the flow path PDMS sheet 15). . The main body 3 and the cover member 5 can maintain a certain degree of liquid tightness by self-adsorption. If the main body 3 and the cover member 5 are fixed by a fixture, a more stable device can be formed.
細胞培養デバイス１が使用されるときの動作について説明する。
本体３及びカバー部材５をオートクレーブやアルコール等によって滅菌処理する。カバー部材５を外した状態で培養室３ａの底面に細胞の足場材となるゲル１９を塗布する。ゲル１９としては、例えばＥＨＳ−ｇｅｌを好適に用いることができる。ＥＨＳ−ｇｅｌは、ＥＨＳマウス肉腫細胞から単離した基底膜調製物であり、ラミニン、ＩＶ型コラーゲン及びプロテオグリカンを豊富に含んでいる。このＥＨＳ−ｇｅｌは低温で液状化し、常温で固体状となる。
An operation when the cell culture device 1 is used will be described.
The main body 3 and the cover member 5 are sterilized with an autoclave or alcohol. With the cover member 5 removed, a gel 19 serving as a cell scaffold is applied to the bottom surface of the culture chamber 3a. As the gel 19, for example, EHS-gel can be suitably used. EHS-gel is a basement membrane preparation isolated from EHS mouse sarcoma cells and is rich in laminin, type IV collagen and proteoglycans. This EHS-gel liquefies at a low temperature and becomes solid at a normal temperature.
本体３が冷却した板の上に置かれる。冷却されたＥＨＳ−ｇｅｌがマイクロピペットで１４μＬ（マイクロリットル）によって培養室３ａに流し込まれる。培養室３ａに収容されたＥＨＳ−ｇｅｌは、冷えて粘性の低いうちに培養室３内に均等になるように拡げられる。ここで、底板７はゲル１９（ＥＨＳ−ｇｅｌ）に濡れる素材で形成されていることが好ましい。底板７がゲル１９に濡れない素材で形成されている場合、ゲル１９と底板７との間に気泡が形成されることが懸念されるからである。 The body 3 is placed on a cooled plate. The cooled EHS-gel is poured into the culture chamber 3a by 14 μL (microliter) with a micropipette. The EHS-gel accommodated in the culture chamber 3a is spread evenly in the culture chamber 3 while it is cooled and has low viscosity. Here, the bottom plate 7 is preferably formed of a material that gets wet with the gel 19 (EHS-gel). This is because when the bottom plate 7 is formed of a material that does not wet the gel 19, it is feared that bubbles are formed between the gel 19 and the bottom plate 7.
培養室３ａに収容されたゲル１９の厚みは均等になれば約０.５ｍｍである。培養室３ａの内壁側面において、最下層の下層側ＰＤＭＳシート９の厚みは０.５ｍｍである。下層側ＰＤＭＳシート９の上層にはＰＴＦＥシート１１が配置されている。ゲル１９と下層側ＰＤＭＳシート９の接触角は９０度未満である。ゲル１９とＰＴＦＥシート１１の接触角は９０以上である。すなわち、培養室３ａに収容されたゲル１９において、ＰＴＦＥシート１１で形成された培養室３ａの内壁側面の部分にはゲル１９が濡れないので、メニスカスが形成されない。これにより、培養室３ａに収容されたゲル１９の表面はほぼ平坦になる。
本体３がインキュベーターの中に移動される。培養室３ａに収容されたゲル１９は固体化する。
If the thickness of the gel 19 accommodated in the culture chamber 3a becomes equal, it is about 0.5 mm. On the inner wall side surface of the culture chamber 3a, the lowermost layer PDMS sheet 9 is 0.5 mm in thickness. A PTFE sheet 11 is disposed on the upper layer of the lower PDMS sheet 9. The contact angle between the gel 19 and the lower layer side PDMS sheet 9 is less than 90 degrees. The contact angle between the gel 19 and the PTFE sheet 11 is 90 or more. That is, in the gel 19 accommodated in the culture chamber 3a, the gel 19 does not get wet on the inner wall side surface portion of the culture chamber 3a formed of the PTFE sheet 11, so that no meniscus is formed. Thereby, the surface of the gel 19 accommodated in the culture chamber 3a becomes substantially flat.
The main body 3 is moved into the incubator. The gel 19 accommodated in the culture chamber 3a is solidified.
図４は、本実施例の細胞培養デバイスに塗布されたゲルの写真である。図５は、従来技術の細胞培養デバイスに塗布されたゲルの写真である。図４及び図５において、白い部分がゲル表面の平坦な部分を示す。従来技術としては培養室の内壁側面がＰＤＭＳのみで形成されたものを用いた。 FIG. 4 is a photograph of the gel applied to the cell culture device of this example. FIG. 5 is a photograph of a gel applied to a prior art cell culture device. In FIG.4 and FIG.5, a white part shows the flat part of the gel surface. As the prior art, the one in which the inner wall side surface of the culture chamber was formed only by PDMS was used.
本実施例（図４）と従来技術（図５）を比較すると、本実施例のほうが従来技術に比べてゲル表面の平坦な部分の面積が大きいことがわかる。従来技術の細胞培養デバイスでは、培養室内に収容されたゲル１９に凹状のメニスカスが形成されているからである。 When this example (FIG. 4) is compared with the prior art (FIG. 5), it can be seen that the area of the flat portion of the gel surface is larger in this example than in the prior art. This is because in the conventional cell culture device, a concave meniscus is formed in the gel 19 accommodated in the culture chamber.
細胞培養デバイス１によって細胞培養を行なう際には、ゲル１９が塗布された培養室３ａに、カバー部材５を外した状態で培地及び細胞を収容し、その後、カバー部材５を本体３に取り付ける。このとき、カバー部材５を構成するシリコーンゴムの自己吸着性によりカバー部材５が本体３に密着する。なお、培養室３ａに収容される細胞は組織片の形で培養室３ａに収容してもよいし、個々の細胞に分離させた状態で収容してもよい。 When cell culture is performed by the cell culture device 1, the culture medium and cells are accommodated in the culture chamber 3 a coated with the gel 19 with the cover member 5 removed, and then the cover member 5 is attached to the main body 3. At this time, the cover member 5 comes into close contact with the main body 3 due to the self-adsorption property of the silicone rubber constituting the cover member 5. Note that the cells accommodated in the culture chamber 3a may be accommodated in the culture chamber 3a in the form of tissue pieces, or may be accommodated in a state of being separated into individual cells.
図１中の矢印を参照して細胞培養デバイス１に供給される培地の流れを説明する。
貫通孔５ｂに培地が供給される。供給された培地は貫通孔１５ｂ及び凹部１５ｃを介して培養室３ａ内に導入される。培養室３ａへの培地の導入に伴い、培養室３ａ内の培地の一部は、凹部５ｃ及び貫通孔５ａを介して細胞培養デバイス１の外部へ排出される。
The flow of the medium supplied to the cell culture device 1 will be described with reference to the arrows in FIG.
A culture medium is supplied to the through-hole 5b. The supplied medium is introduced into the culture chamber 3a through the through hole 15b and the recess 15c. With the introduction of the culture medium into the culture chamber 3a, a part of the culture medium in the culture chamber 3a is discharged outside the cell culture device 1 through the recess 5c and the through hole 5a.
図６は、本実施例の細胞培養デバイスで培養された内皮細胞のネットワーク形成状態を示す写真である。図７は、従来の細胞培養デバイスで培養された内皮細胞のネットワーク形成状態を示す写真である。 FIG. 6 is a photograph showing the network formation state of the endothelial cells cultured with the cell culture device of this example. FIG. 7 is a photograph showing a network formation state of endothelial cells cultured with a conventional cell culture device.
従来技術（図７）では、培養室内に収容されたゲル１９に凹状のメニスカスが形成されているために、ゲル１９表面に平坦な部分が少なく、均一な内皮細胞２１のネットワークが形成されていない。
本実施例（図６）では、培養室内に収容されたゲル１９のメニスカスが小さいので、従来技術に比べてゲル１９表面の平坦な部分が大きく、比較的均一な内皮細胞２１のネットワークが形成されている。
In the prior art (FIG. 7), since a concave meniscus is formed in the gel 19 accommodated in the culture chamber, there are few flat portions on the surface of the gel 19 and a uniform network of endothelial cells 21 is not formed. .
In this example (FIG. 6), the meniscus of the gel 19 accommodated in the culture chamber is small, so that the flat portion of the surface of the gel 19 is larger than in the prior art, and a relatively uniform network of endothelial cells 21 is formed. ing.
以上の実施例は本発明の一例であり、本発明は上記実施例に限定されるものではなく、本発明の範囲内で種々の変更が可能である。例えば、上記細胞培養デバイスを構成する各部材の素材や寸法、配置などは、上記実施例に限らず、種々の変更が可能である。 The above embodiment is an example of the present invention, and the present invention is not limited to the above embodiment, and various modifications can be made within the scope of the present invention. For example, the material, dimensions, arrangement, and the like of each member constituting the cell culture device are not limited to the above-described embodiments, and various changes can be made.
例えば、ＰＤＭＳに替えてシリコーンゴムが用いられてもよい。また、ＰＴＦＥに替えて他のフッ素樹脂が用いられてもよい。 For example, silicone rubber may be used instead of PDMS. In addition, other fluororesins may be used instead of PTFE.
また、上記実施例では、培養室３ａの内壁側面は４層のシート９，１１，１３，１５によって形成されているが、本発明はこれに限定されない。本発明の細胞培養デバイスにおいて、培養室の内壁側面は、ゲルに濡れない素材で形成された部分とゲルに濡れる素材で形成された部分とを備え、ゲルに濡れない素材は、培養室の内壁側面において培養室の底面から所定の間隔をもって、かつ上方から見て環状に配置されており、ゲルに濡れない素材と培養室の底面との間の内壁側面はゲルに濡れる素材によって形成されている構成であれば、どのような構成であってもよい。 Moreover, in the said Example, although the inner wall side surface of the culture chamber 3a is formed with the sheet | seat 9, 11, 13, 15 of 4 layers, this invention is not limited to this. In the cell culture device of the present invention, the inner wall side surface of the culture chamber includes a portion formed of a material that does not wet the gel and a portion formed of a material that wets the gel, and the material that does not wet the gel is the inner wall of the culture chamber. The side wall is arranged in a ring shape with a predetermined interval from the bottom surface of the culture chamber as viewed from above, and the inner wall side surface between the material that does not wet the gel and the bottom surface of the culture chamber is formed of the material that wets the gel. Any configuration is possible as long as it is configured.
また、上記実施例において、流路（溝５ｃ，１５ｃ）の構成は種々の変更が可能である。例えば、カバー部材５に形成された溝５ｃは、流路用ＰＤＭＳシート１５の上面に形成されていてもよいし、流路用ＰＤＭＳシート１５の下面又は上層側ＰＤＭＳシート１３の上面に形成されていてもよい。また、流路用ＰＤＭＳシート１５に形成された溝１５ｃは上層側ＰＤＭＳシート１３の上面に形成されていてもよい。 Moreover, in the said Example, various changes are possible for the structure of a flow path (grooves 5c and 15c). For example, the groove 5 c formed in the cover member 5 may be formed on the upper surface of the flow path PDMS sheet 15, or formed on the lower surface of the flow path PDMS sheet 15 or the upper surface side PDMS sheet 13. May be. Further, the groove 15 c formed in the flow path PDMS sheet 15 may be formed on the upper surface of the upper PDMS sheet 13.
また、カバー部材５とＰＤＭＳシート１５の間で貫通孔１５ａに対応する部分にフィルターを配置するようにしてもよい。これにより、ゲル１９による流路の詰まりを軽減することができる。 Further, a filter may be arranged between the cover member 5 and the PDMS sheet 15 in a portion corresponding to the through hole 15a. Thereby, the clogging of the flow path by the gel 19 can be reduced.
１ 細胞培養デバイス
３ａ 培養室
７ 底板
９ 下層側ＰＤＭＳシート（ゲルに濡れる素材）
１１ ＰＴＦＥシート（ゲルに濡れない素材）
１３ 上層側ＰＤＭＳシート
１９ ゲル
1 Cell culture device 3a Culture chamber 7 Bottom plate 9 Lower layer side PDMS sheet (material that gets wet with gel)
11 PTFE sheet (material that does not wet the gel)
13 Upper layer side PDMS sheet 19 Gel
Claims (5)
前記培養室の内壁側面はゲルに濡れない素材で形成された部分とゲルに濡れる素材で形成された部分とを備え、
前記ゲルに濡れない素材は、前記内壁側面において前記培養室の底面から所定の間隔をもって、かつ上方から見て環状に配置されており、
前記ゲルに濡れない素材と前記底面との間の前記内壁側面は前記ゲルに濡れる素材によって形成されていることを特徴とする細胞培養デバイス。 In a cell culture device having a culture chamber in which a gel serving as a cell scaffold is accommodated,
The inner wall side surface of the culture chamber comprises a portion formed of a material that does not wet the gel and a portion formed of a material that wets the gel,
The material that does not wet the gel is arranged in a ring shape at a predetermined interval from the bottom surface of the culture chamber on the inner wall side surface, as viewed from above,
The cell culture device, wherein the side surface of the inner wall between the material that does not wet the gel and the bottom surface is formed of the material that wets the gel.
前記培養室内壁に露出した前記ＰＴＦＥシートの部分によって前記ゲルに濡れない素材で形成された部分が構成され、
前記培養室内壁に露出した前記下層側ＰＤＭＳシートの部分によって前記ゲルに濡れる素材で形成された部分が構成されている請求項１に記載の細胞培養デバイス。 The culture chamber includes at least a lower layer side PDMS sheet, a PTFE sheet, a through hole formed in the stacking direction with respect to a stacked body in which the upper layer side PDMS sheet is stacked in that order from the lower layer side, and the PTFE of the lower layer side PDMS sheet. It has a space formed by a bottom plate placed on the surface opposite to the sheet,
A portion formed of a material not wetted by the gel is constituted by a portion of the PTFE sheet exposed on the culture chamber wall;
The cell culture device according to claim 1, wherein a portion formed of a material that gets wet with the gel is constituted by a portion of the lower layer PDMS sheet exposed to the culture chamber wall.
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