JP6501255B2 - Culture apparatus and cell culture method using the culture apparatus - Google Patents
Culture apparatus and cell culture method using the culture apparatus Download PDFInfo
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Description
本発明は、培養液を含む筒状体の下で細胞を培養する、静水圧を細胞に付与するための培養装置であって、筒状体中に培養液を入れることで細胞に微細な圧力（０．００５−０．１気圧程度）を付加できる培養装置及び該培養装置を用いた細胞培養方法に関する。 The present invention is a culture apparatus for culturing cells under a cylinder containing a culture solution, and applying a hydrostatic pressure to the cells, wherein a minute pressure is applied to the cells by placing the culture solution in the cylinder. The present invention relates to a culture apparatus capable of adding (about 0.005 to 0.1 atm) and a cell culture method using the culture apparatus.
従来から、細胞に圧力を付加しながら培養しようとする試みが数多くなされている。
例えば、特許文献１では圧力制御もなし得る細胞培養器および培養細胞処理システムが提案されている。
しかし、特許文献１の培養装置は耐圧容器と、該耐圧容器に雰囲気ガスを供給するガス供給装置と、該雰囲気ガスの圧力を制御するガス圧力制御装置とを備えてなる複雑な構造を有する大がかりな培養装置であるため、取り扱いが煩雑であり、そのうえ高価で手に入れづらいという問題点を有する。また、Ｏ２ガスを供給するＯ２ガス供給機構と、ＣＯ２ガスを供給するＣＯ２ガス供給機構と、Ｎ２ガスを供給するＮ２ガス供給機構とを備えてなるのが好ましいとされており、雰囲気ガスを供給するにも別途構成が必要になり一層取扱いが煩雑になり、高いコストがかかるという問題点を有する。
Conventionally, many attempts have been made to culture while applying pressure to cells.
For example, Patent Document 1 proposes a cell incubator and a cultured cell processing system that can also perform pressure control.
However, the culture apparatus of Patent Document 1 has a complicated structure including a pressure resistant container, a gas supply device for supplying the atmosphere gas to the pressure resistant container, and a gas pressure control device for controlling the pressure of the atmosphere gas. It is a complicated culture apparatus, so it is cumbersome to handle and is expensive and difficult to obtain. Also, it is the O 2 gas supply mechanism for supplying an O 2 gas, and CO 2 gas supply mechanism for supplying CO 2 gas, as made by a N 2 gas supply mechanism for supplying the N 2 gas is preferable In order to supply the atmosphere gas, a separate configuration is required, which makes the handling more complicated and increases the cost.
特許文献２では、高圧下でも培養液を培養槽に的確に供給でき、しかも培養液の成分測定をも可能にし得る高圧培養装置、及びこれを用いた深水生物の育成方法が提案されている。
この装置は小型化が容易な簡易な構成を有することを特徴としていると記載されているが、各種センサや制御手段を備えており、特許文献１と同様に複雑な構造を有する培養装置であると言え、取扱いが煩雑であり、そのうえ高価で手に入れづらいという問題点を有する。
特許文献２記載の培養装置は１−２００ＭＰａ（メガパスカル）程度の加圧を付与することを想定した装置であり、例えばヘクトパスカル（メガパスカルの１／１０４）のような非常に弱い圧力を付加することができないという問題点を有する。
この培養装置は培養槽を使用して細胞を培養するため、少量の細胞を培養するには適さないという問題点を有する。
Patent Document 2 proposes a high-pressure culture apparatus capable of accurately supplying a culture solution to a culture tank even under high pressure and also capable of measuring components of the culture solution, and a method for cultivating deep-water organisms using the same.
Although this device is described as having a simple configuration that is easy to miniaturize, it is a culture device having various sensors and control means, and having a complicated structure as in Patent Document 1 However, the handling is complicated, and it is expensive and difficult to obtain.
The culture apparatus described in Patent Document 2 is an apparatus assuming application of pressure of about 1 to 200 MPa (megapascals), and for example, a very weak pressure such as hectopascal (1/10 4 of megapascals) is added. It has the problem that it can not do.
This culture device has the problem that it is not suitable for culturing a small amount of cells, since the cells are cultured using a culture tank.
本発明は、上記した従来技術の問題点を解決すべくなされたものであって、非常に弱い圧力を付加することができる安価な培養装置を提供するものである。 The present invention has been made to solve the problems of the prior art described above, and provides an inexpensive culture apparatus capable of applying very weak pressure.
請求項１に係る発明は、有孔プレート材からなる基盤と、該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、該培養容器の上に密閉手段を介して設けられ、一方の面に突出部が設けられ、他方の面に着脱自在に液体培地を含む筒状体が立設され、該筒状体を支持するための支持プレートとを備え、該支持プレートと突出部を貫通する孔である第二貫通孔が穿設され、該培養容器が該基盤と該支持プレートの間に該突出部の先端と該密閉手段を介して挟持され、該第一貫通孔と該第二貫通孔が同軸状に位置づけられて固定され、前記培養容器の底面を液体培地に浸漬させるために前記基盤の下に受け皿が配され、前記培養容器の底面が半透膜であり、前記筒状体は、液体培地を加えることで細胞に０．０３〜０．１気圧の圧力を付与し、前記密閉手段は、前記筒状体につながる前記第二貫通孔から液体培地が漏出することを防ぐことを特徴とする、静水圧を細胞に付与するための培養装置に関する。
The invention according to claim 1 comprises a substrate comprising a perforated plate material, a culture vessel comprising cells and liquid medium disposed so as to cover the first through holes of the substrate, and a sealing means on the culture vessel. And a support plate for supporting the cylindrical body, the cylindrical body including the liquid culture medium being erected and provided on one side thereof and the projection on the other side. A second through-hole, which is a hole penetrating through the support plate and the protrusion, is bored, and the culture vessel is sandwiched between the base and the support plate via the sealing means with the tip of the protrusion, One through hole and the second through hole are coaxially positioned and fixed, and a pan is disposed under the base to immerse the bottom of the culture vessel in a liquid medium, and the bottom of the culture vessel is semi-permeable Makudea is, the tubular body is 0.03 to 0.1 air to the cells by addition of liquid medium The pressure of applying the sealing means may prevent the liquid medium from leaking out of the second through-hole leading to the tubular body, relates to a culture apparatus for applying hydrostatic pressure to the cells.
請求項２に係る発明は、有孔プレート材からなる基盤と、該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、該培養容器の底面上にその一端が配され、液体培地を含む筒状体と、該筒状体を支持するための支持手段と、該筒状体と該培養容器の底面の間に挟持される密閉手段と、前記培養容器の底面を液体培地に浸漬させるために該基盤の下に配される受け皿とを備え、前記培養容器の底面が半透膜であり、前記筒状体は、液体培地を加えることで細胞に０．０３〜０．１気圧の圧力を付与し、前記密閉手段は、前記筒状体につながる第二貫通孔から液体培地が漏出することを防ぐことを特徴とする、静水圧を細胞に付与するための培養装置に関する。
The invention according to claim 2 comprises a substrate comprising a perforated plate material, a culture vessel comprising cells and liquid medium disposed so as to cover the first through holes of the substrate, and one end thereof on the bottom surface of the culture vessel A cylindrical body containing a liquid culture medium, a support means for supporting the cylindrical body, a sealing means held between the cylindrical body and the bottom surface of the culture vessel, and the culture vessel And a receptacle disposed under the base for immersing the bottom surface in the liquid medium, wherein the bottom surface of the culture vessel is a semipermeable membrane, and the tubular body is used to add 0. 1 to the cells by adding the liquid medium. applying pressure of 03 to 0.1 atm, the sealing means may prevent the liquid medium from leaking out of the second through hole that connected to the tubular body, imparts hydrostatic pressure to the cells The present invention relates to a culture apparatus for
請求項４に係る発明は、前記培養容器の底面と前記第一貫通孔及び少なくともその周辺部の間に多孔質シートが配されることを特徴とする、請求項１乃至３のいずれかに記載の培養装置に関する。
Invention is characterized in that a porous sheet is disposed between the bottom surface and the first through-hole and at least a peripheral portion of the culture vessel, according to any one of claims 1 to 3 according to claim 4 Culture apparatus of
請求項５に係る発明は、前記多孔質シートにおける孔の直径が０．０２−０．５ｍｍの範囲内であり、１ｃｍ２当たり１００−３０００個の孔を有することを特徴とする、請求項４記載の培養装置に関する。
The invention according to claim 5, wherein the diameter of the pores in the porous sheet is in the range of 0.02-0.5Mm, characterized by having a 1 cm 2 per 100-3000 amino holes, claim 4 It relates to the described culture apparatus.
請求項６に係る発明は、前記半透膜における孔の直径が０．７−２．０μｍの範囲内である場合１ｃｍ２当たり５．０×１０５−５．０×１０７個の孔を有し、前記半透膜における孔の直径が０．１−０．７μｍの範囲内である場合１ｃｍ２当たり５．０×１０７−１．０×１０９個の孔を有することを特徴とする、請求項５記載の培養装置に関する。
The invention according to claim 6 provides 5.0 × 10 5 -5.0 × 10 7 holes per 1 cm 2 when the diameter of the holes in the semipermeable membrane is in the range of 0.7-2.0 μm. Characterized in that it has 5.0 × 10 7 -1.0 × 10 9 holes per 1 cm 2 when the diameter of the holes in the semipermeable membrane is in the range of 0.1-0.7 μm. The culture apparatus according to claim 5, wherein
請求項７に係る発明は、前記密閉手段がＯ−リングであることを特徴とする、請求項１乃至６のいずれかに記載の培養装置に関する。
The invention according to claim 7 relates to the culture apparatus according to any one of claims 1 to 6 , wherein the sealing means is an O-ring.
請求項８に係る発明は、前記受け皿および／または、前記筒状体が蓋を有することを特徴とする、請求項１乃至７のいずれかに記載の培養装置に関する。
The invention according to claim 8 relates to the culture apparatus according to any one of claims 1 to 7 , characterized in that the saucer and / or the cylindrical body has a lid.
請求項３に係る発明は、前記筒状体は、少なくとも５０ｃｍの高さを有することを特徴とする、請求項１又は２記載の培養装置に関する。
The invention according to claim 3 relates to the culture apparatus according to claim 1 or 2 , wherein the cylindrical body has a height of at least 50 cm .
請求項９に係る発明は、前記基盤の側面に第一貫通孔と交わる第三貫通孔を有することを特徴とする、請求項１乃至８のいずれかに記載の培養装置に関する。 The invention according to claim 9 relates to the culture apparatus according to any one of claims 1 to 8, characterized in that the side surface of the base has a third through hole intersecting with the first through hole.
請求項１０に係る発明は、請求項１乃至９のいずれかに記載の培養装置を用いて細胞を培養する方法に関する。 The invention according to claim 10 relates to a method for culturing cells using the culture apparatus according to any one of claims 1 to 9.
請求項１１に係る発明は、前記細胞が、呼吸器系上皮細胞、消化器系粘膜上皮細胞、消化器系腺上皮細胞、表皮細胞、神経細胞、上衣細胞、骨・軟骨細胞、滑膜細胞、及び間葉系細胞からなる群から選択されることを特徴とする、請求項１０記載の方法に関する。 In the invention according to claim 11, the cells may be respiratory epithelial cells, digestive mucosal epithelial cells, digestive glandular epithelial cells, epidermal cells, neurons, ependymal cells, bones / chondrocytes, synovial cells, The method according to claim 10, wherein the method is selected from the group consisting of mesenchymal cells and mesenchymal cells.
請求項１に係る発明によれば、有孔プレート材からなる基盤と、該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、該培養容器の上に密閉手段を介して設けられ、一方の面に突出部が設けられ、他方の面に着脱自在に液体培地を含む筒状体が立設され、該筒状体を支持するための支持プレートとを備え、該支持プレートと突出部を貫通する孔である第二貫通孔が穿設され、該培養容器が該基盤と該支持プレートの間に該突出部の先端と該密閉手段を介して挟持され、該第一貫通孔と該第二貫通孔が同軸状に位置づけられて固定されていることで、培養容器内の細胞の上に筒状体が位置するため、筒状体に液体培地を加えることで細胞に圧力を付加することができる。このため、例えば培養容器の底面から３０ｃｍの高さにまで液体培地を加えた場合、０．０３気圧程度の微細な圧力を細胞に付加することができる。ひいては、このような微細な圧力が発症の原因とされる頭蓋内圧亢進による脳浮腫や緑内障等に関連する脳神経細胞・上衣細胞、網膜神経節細胞をこの培養装置を用いて培養することで、上記疾患の病態の分子生物学的メカニズムの研究を詳細に行うことができる。また、支持プレートで筒状体を支持することで筒状体は安定した状態で立設することができる。さらに、密閉手段を有することで筒状体につながる第二貫通孔から液体培地が漏出するのを防ぐことができる。
上記培養装置は従来の培養装置と比較して非常に簡易な構造であるため、培養装置ごとオートクレーブやアルコール殺菌などの滅菌処理をすることができ、且つ安価で提供することができる。
前記培養容器の底面を液体培地に浸漬させるために前記基盤の下に受け皿を配し、前記培養容器の底面が半透膜であることで、受け皿に含まれる液体培地から培養容器に含まれる液体培地へＯ２及びＣＯ２が供給でき、温度やｐＨの変化も少なくなるため、数日間の培養が可能となる。
さらに、従来の培養装置と比べて小型であるため、少量の細胞の培養に適している。
According to the first aspect of the present invention, there is provided a base comprising a perforated plate material, a culture vessel containing cells and liquid medium disposed so as to cover the first through holes of the base, and a culture vessel sealed on the culture vessel. A cylindrical body including a liquid culture medium is provided so as to be detachably mounted on the other surface, and a support plate for supporting the cylindrical body. A second through hole, which is a hole passing through the support plate and the protrusion, is formed, and the culture vessel is sandwiched between the base and the support plate via the sealing means with the tip of the protrusion; Since the tubular body is positioned on the cells in the culture vessel by coaxially positioning and fixing the first through hole and the second through hole, the liquid medium is added to the tubular body. Pressure can be applied to the cells. For this reason, for example, when the liquid medium is added to a height of 30 cm from the bottom of the culture vessel, a minute pressure of about 0.03 atm can be applied to the cells. As a result, the above culture apparatus is used to culture the above-mentioned culture devices of brain nerve cells, ependymal cells and retinal ganglion cells associated with brain edema and glaucoma due to increased intracranial pressure caused by the occurrence of such minute pressure. Detailed research on molecular biological mechanisms of disease states can be conducted. In addition, the cylindrical body can be erected in a stable state by supporting the cylindrical body with the support plate. Furthermore, by having the sealing means, it is possible to prevent the liquid culture medium from leaking out through the second through hole connected to the cylindrical body.
Since the above-mentioned culture apparatus has a very simple structure as compared with the conventional culture apparatus, the whole culture apparatus can be subjected to sterilization processing such as autoclave and alcohol sterilization and can be provided at low cost.
In order to immerse the bottom surface of the culture vessel in a liquid medium, a saucer is disposed under the base, and the bottom surface of the culture vessel is a semipermeable membrane, so that the liquid medium contained in the culture vessel from the liquid medium contained in the saucer Since O 2 and CO 2 can be supplied to the medium, and changes in temperature and pH are also reduced, culture for several days becomes possible.
Furthermore, because they are smaller than conventional culture devices, they are suitable for culture of small amounts of cells.
請求項２に係る発明によれば、有孔プレート材からなる基盤と、該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、該培養容器の底面上にその一端が配され、液体培地を含む筒状体と、該筒状体を支持するための支持手段と、該筒状体と該培養容器の底面の間に挟持される密閉手段とを備えていることで、請求項１記載の培養装置とは異なる構成を有するものの、請求項１記載の培養装置と同様に培養容器内の細胞の上に筒状体が位置するため、筒状体に液体培地を加えることで細胞に圧力を付加することができる。このため例えば培養容器の底面から３０ｃｍの高さにまで液体培地を加えた場合、０．０３気圧程度の微細な圧力を細胞に付加することができる。また、密閉手段を有することで筒状体につながる第二貫通孔から液体培地が漏出するのを防ぐことができる。
上記培養装置は従来の培養装置と比較して非常に簡易な構造であるため、培養装置ごとオートクレーブやアルコール殺菌などの滅菌処理をすることができ、且つ安価で提供することができる。
前記培養容器の底面を液体培地に浸漬させるために前記基盤の下に受け皿を配し、前記培養容器の底面が半透膜であることで、受け皿に含まれる液体培地から培養容器に含まれる液体培地へＯ２及びＣＯ２が供給でき、温度やｐＨの変化も少なくなるため、数日間の培養が可能となる。
さらに、従来の培養装置と比べて小型であるため、少量の細胞の培養に適している。
According to the second aspect of the present invention, there is provided a base comprising a perforated plate material, a culture vessel containing cells and liquid medium disposed so as to cover the first through holes of the base, and a bottom surface of the culture vessel. A tubular body including a liquid medium, a support means for supporting the tubular body, and a sealing means held between the tubular body and the bottom surface of the culture vessel; In the same manner as in the culture apparatus according to claim 1, the cylindrical body is positioned above the cells in the culture vessel, so that the liquid in the cylindrical body is different. Pressure can be applied to the cells by adding media. Therefore, for example, when the liquid medium is added to a height of 30 cm from the bottom of the culture vessel, a minute pressure of about 0.03 atm can be applied to the cells. Further, by having the sealing means, it is possible to prevent the liquid culture medium from leaking out from the second through hole connected to the cylindrical body.
Since the above-mentioned culture apparatus has a very simple structure as compared with the conventional culture apparatus, the whole culture apparatus can be subjected to sterilization processing such as autoclave and alcohol sterilization and can be provided at low cost.
In order to immerse the bottom surface of the culture vessel in a liquid medium, a saucer is disposed under the base, and the bottom surface of the culture vessel is a semipermeable membrane, so that the liquid medium contained in the culture vessel from the liquid medium contained in the saucer Since O 2 and CO 2 can be supplied to the medium, and changes in temperature and pH are also reduced, culture for several days becomes possible.
Furthermore, because they are smaller than conventional culture devices, they are suitable for culture of small amounts of cells.
請求項４に係る発明によれば、多孔質シートを培養容器の底面と第一貫通孔及びその周辺部の間に配することで、培養容器内のガス分圧平衡を保ちつつ培養容器の底面が筒状体に含まれる液体培地による圧力で下方に湾曲するのを防ぐことができる。
According to the invention of claim 4 , by disposing the porous sheet between the bottom surface of the culture container and the first through hole and the periphery thereof, the gas partial pressure balance in the culture container is maintained while the bottom surface of the culture container is maintained. Can be prevented from bending downward by the pressure of the liquid medium contained in the tubular body.
請求項５に係る発明によれば、多孔質シートの孔の直径が０．０２−０．５ｍｍの範囲内であり、１ｃｍ２当たり１００−３０００個の孔を有することで、培養容器内のガス分圧平衡を保ちつつ培養容器の底面が筒状体に含まれる液体培地による圧力で下方に湾曲するのをより効果的に防ぐことができる。さらに、筒状体に含まれる液体培地の液面が低下するのを防ぐことができるため、長期間（数日間）にわたる培養を可能にする。
According to the invention of claim 5 , the diameter of the pores of the porous sheet is in the range of 0.02 to 0.5 mm, and the gas in the culture vessel is provided by having 100 to 3000 pores per 1 cm 2. It is possible to more effectively prevent the bottom of the culture vessel from being bent downward by the pressure of the liquid medium contained in the tubular body while maintaining partial pressure equilibrium. Furthermore, since the liquid level of the liquid medium contained in the tubular body can be prevented from lowering, culture over a long period (several days) is enabled.
請求項６に係る発明によれば、半透膜の孔の直径が０．７−２．０μｍの範囲内である場合１ｃｍ２当たり５．０×１０５−５．０×１０７個の孔を有し、前記半透膜における孔の直径が０．１−０．７μｍの範囲内である場合１ｃｍ２当たり５．０×１０７−１．０×１０９個の孔を有することで、多孔質シートと組み合わせた際に、ガス分圧平衡を維持（培養中の酸素分圧を維持）しつつ、受け皿に含まれる液体培地から培養容器に含まれる液体培地へＯ２及びＣＯ２が供給でき、数日間の培養に適した培養装置とすることができる。
According to the invention of claim 6, when the diameter of the pores of the semipermeable membrane is in the range of 0.7-2.0 μm, 5.0 × 10 5 -5.0 × 10 7 pores per 1 cm 2 And having a diameter of 5.0 × 10 7 -1.0 × 10 9 holes per 1 cm 2 when the diameter of the holes in the semipermeable membrane is in the range of 0.1-0.7 μm, When combined with the porous sheet, O 2 and CO 2 are supplied from the liquid culture medium contained in the receiver to the liquid culture medium contained in the culture vessel while maintaining gas partial pressure equilibrium (maintaining oxygen partial pressure during culture) And a culture apparatus suitable for several days of culture.
請求項７に係る発明によれば、密閉手段をＯ−リングとすることで筒状体又は第二貫通孔に含まれる液体培地が外部に漏れるのを効果的に防ぐことができる。接着剤等の液体培地に溶解するような化学物質を使用しないため、培養している細胞に悪影響を与えることなく、筒状体に含まれる液体培地の漏出を防ぐことができる。
According to the seventh aspect of the present invention, by making the sealing means an O-ring, the liquid culture medium contained in the cylindrical body or the second through hole can be effectively prevented from leaking to the outside. Since no chemical substance that dissolves in a liquid medium such as an adhesive is used, leakage of the liquid medium contained in the tubular body can be prevented without adversely affecting the cells in culture.
請求項８係る発明によれば、受け皿が蓋を有することで受け皿に含まれる液体培地の蒸発を防ぐことができる。
According to the eighth aspect of the present invention, evaporation of the liquid culture medium contained in the saucer can be prevented by having the lid with the saucer.
請求項８に係る発明によれば、筒状体が蓋を有することで筒状体に含まれる液体培地の蒸発を防ぐことができる。 According to the invention which concerns on Claim 8, evaporation of the liquid culture medium contained in a cylindrical body can be prevented because a cylindrical body has a lid | cover.
請求項９に係る発明によれば、基盤の側面に第一貫通孔と交わる第三貫通孔を有することで第一貫通孔内に空気が入ってしまっても第三貫通孔から液体培地を注入することで空気を抜くことができる。 According to the invention of claim 9, the side surface of the base has the third through hole which intersects the first through hole, and even if air enters the first through hole, the liquid medium is injected from the third through hole. You can purge the air by doing this.
請求項１０に係る発明によれば、請求項１乃至９のいずれかに記載の培養装置を用いて細胞を培養することで、ヒトの種々の疾患（緑内障、頭蓋内圧亢進による脳浮腫等）の原因となる０．０３気圧程度の圧力を付加しながら細胞を培養することができるため、軽微な圧力が如何にして種々の細胞の変性・アポトーシスを惹起し、その結果として病変や病態が形成されるのかについて、その分子生物学的なメカニズムを解明するための培養環境を提供することができる。 According to the invention as set forth in claim 10, by culturing cells using the culture device according to any one of claims 1 to 9, various human diseases (glaucoma, brain edema due to increased intracranial pressure, etc.) Since cells can be cultured while applying the pressure of about 0.03 atmosphere, which is the cause, slight pressure causes degeneration and apoptosis of various cells, resulting in formation of lesions and pathological conditions. It is possible to provide a culture environment for elucidating its molecular biological mechanism.
請求項１１に係る発明によれば、細胞が呼吸器系上皮細胞、消化器系粘膜上皮細胞、消化器系腺上皮細胞、表皮細胞、神経細胞、上衣細胞、骨・軟骨細胞、滑膜細胞、及び間葉系細胞からなる群から選択されることで、軽微な圧力が原因である種々の疾患の中でも例えば気道内圧上昇に伴う肺傷害、腸内圧亢進に伴う粘膜傷害、緑内障、及び頭蓋内圧亢進による脳浮腫に関係する細胞が軽微な圧力を付加された場合その分子生物学的なメカニズムの解明につながる。また、表皮細胞や骨細胞等は日常的に軽微な圧力を受けているため、その圧力がこれらの細胞にどのような影響を与えているかを分子生物学的に解析するのに役立つ。 According to the invention of claim 11, the cells are respiratory system epithelial cells, digestive system mucosal epithelial cells, digestive system glandular epithelial cells, epidermal cells, neural cells, ependymal cells, bone / chondrocytes, synovial cells, Among various diseases caused by slight pressure, it is selected from the group consisting of and mesenchymal cells, for example, lung injury associated with an increase in airway pressure, mucosal injury associated with increased intestinal pressure, glaucoma, and intracranial pressure increase If cells involved in brain edema are exposed to slight pressure, it leads to elucidation of the molecular biological mechanism. In addition, since epidermal cells and bone cells and the like are routinely subjected to slight pressure, they are useful for molecular biological analysis of how the pressure affects these cells.
以下、本発明に係る培養装置及び該培養装置を使用した細胞培養方法の好適な実施形態について、図面を参照しながら詳細に説明するが、本発明の技術的範囲がこれらの記載によって制限されるべきものではない。 Hereinafter, preferred embodiments of a culture apparatus according to the present invention and a cell culture method using the culture apparatus will be described in detail with reference to the drawings, but the technical scope of the present invention is limited by these descriptions. It should not be.
＜実施形態１＞
以下、本発明に係る培養装置の実施形態１について、図面を参照しながら詳細に説明する。
図１及び２は、本発明の実施形態１に係る培養装置を示す全体構成図及び縦断面図である。
本実施形態（実施形態１）に係る培養装置（１）は、孔が穿設された有孔のプレート材（以下、単に有孔プレート材という）からなる基盤（２）と、基盤（２）の第一貫通孔（３）を覆うように配された細胞と液体培地を含む培養容器（４）と、培養容器（４）の上に密閉手段（５）を介して設けられ、一方の面に突出部（６）が設けられ、他方の面に着脱自在に液体培地を含む筒状体（７）が立設され、筒状体（７）を支持するための支持プレート（８）とを備え、支持プレート（８）と突出部（６）を貫通する孔である第二貫通孔（９）が穿設され、培養容器（４）が基盤（２）と支持プレート（８）の間に突出部（６）の先端と密閉手段（５）を介して挟持され、第一貫通孔（３）と第二貫通孔（９）が同軸状に位置づけられて固定される。本発明に係る培養装置（１）をこのような構成にすることで、培養容器（４）内の細胞の上に筒状体（７）が位置するため、筒状体（７）に液体培地を加えることで細胞に圧力を付加することができる。例えば培養容器（４）の底面から３０ｃｍの高さにまで液体培地を加えた場合、０．０３気圧程度の微細な圧力を細胞に付加することができる。支持プレート（８）で筒状体（７）を支持することで筒状体（７）は安定した状態で立設できる。密閉手段（５）を有することで筒状体（７）につながる第二貫通孔（９）から液体培地が漏出するのを防ぐことができる。
本実施形態の培養装置は従来の培養装置と比較して非常に簡易な構造であるため、培養装置ごとオートクレーブやアルコール殺菌などの滅菌処理をすることができ、且つ安価で提供することができる。
First Embodiment
Hereinafter, Embodiment 1 of the culture apparatus according to the present invention will be described in detail with reference to the drawings.
1 and 2 are an overall configuration view and a longitudinal sectional view showing a culture apparatus according to Embodiment 1 of the present invention.
A culture apparatus (1) according to the present embodiment (embodiment 1) includes a base (2) made of a perforated plate material (hereinafter, simply referred to as a perforated plate material) in which holes are bored, and a base (2) Provided on the culture vessel (4) containing cells and liquid medium arranged to cover the first through hole (3) and the culture vessel (4) via the sealing means (5), A cylindrical body (7) having a liquid medium detachably mounted on the other surface, and a support plate (8) for supporting the cylindrical body (7). And a second through hole (9), which is a hole penetrating through the support plate (8) and the projection (6), is provided, and the culture vessel (4) is between the base (2) and the support plate (8) The first through hole (3) and the second through hole (9) are coaxially positioned and fixed while being held between the tip of the protrusion (6) and the sealing means (5). That. By forming the culture apparatus (1) according to the present invention in such a configuration, the tubular body (7) is positioned above the cells in the culture vessel (4), and thus the liquid medium is used in the tubular body (7). The pressure can be applied to the cells by adding. For example, when the liquid medium is added to a height of 30 cm from the bottom of the culture vessel (4), a minute pressure of about 0.03 atm can be applied to the cells. By supporting the cylindrical body (7) with the support plate (8), the cylindrical body (7) can be erected in a stable state. By having the sealing means (5), it is possible to prevent the liquid culture medium from leaking out through the second through hole (9) connected to the cylindrical body (7).
The culture apparatus of the present embodiment has a very simple structure as compared with the conventional culture apparatus, and therefore, the whole culture apparatus can be subjected to sterilization processing such as autoclave and alcohol sterilization, and can be provided at low cost.
本発明における基盤（２）、筒状体（７）及び支持プレート（８）の素材は特に限定されないが、オートクレーブや紫外線滅菌、次亜塩素酸消毒等の滅菌処理が施されることを考慮し、ポリエチレン、ポリスチレン、ポリアクリレート、ポリメタクリレート、ポリ塩化ビニル、ポリカーボネート等の熱可塑性樹脂の他、フェノール樹脂などの熱硬化性樹脂を使用したもの、アクリル製や耐熱ガラス製のもの等耐熱性、耐薬品性に優れた素材を使用することが好ましい。 The material of the base (2), the cylindrical body (7) and the support plate (8) in the present invention is not particularly limited, but in consideration of sterilization such as autoclave or ultraviolet sterilization or hypochlorous acid sterilization being applied. Other than thermoplastic resins such as polyethylene, polystyrene, polyacrylate, polymethacrylate, polyvinyl chloride and polycarbonate, those using thermosetting resin such as phenol resin, those made of acrylic or heat-resistant glass, etc. It is preferable to use a material having excellent chemical properties.
基盤（２）の側面には、第一貫通孔（３）と交わる第三貫通孔（図示せず）を有することが望ましい。第三貫通孔を有することで第一貫通孔（３）内に空気が入ってしまっても第三貫通孔から液体培地を注入することで空気を抜くことができる。
第三貫通孔の数及び孔径は特に限定されない。
It is desirable that the side surface of the base (2) has a third through hole (not shown) intersecting with the first through hole (3). Even if air enters the first through hole (3) by having the third through hole, air can be removed by injecting the liquid medium from the third through hole.
The number and the hole diameter of the third through holes are not particularly limited.
筒状体（７）を支持プレート（８）に固定するための方法は特に限定されないが、筒状体（７）に含まれる液体培地が漏出するのを防ぐために、筒状体（７）と支持プレート（８）の接続部にねじ切り加工等互いに摺合せが良く気密性を高めるような加工を施すことが望ましい。
筒状体（７）及び支持プレート（８）は別体であって上記のように接続されているものであっても、一体のものであっても良い。
Although the method for fixing the cylindrical body (7) to the support plate (8) is not particularly limited, in order to prevent the liquid medium contained in the cylindrical body (7) from leaking, the cylindrical body (7) and It is desirable that the connection portion of the support plate (8) be processed such as thread cutting so as to have good mutual contact and improve air tightness.
The tubular body (7) and the support plate (8) may be separate bodies and connected as described above, or may be integral.
筒状体（７）は液体培地の蒸発を防ぐための蓋を有するものであっても、図１及び２に示す如く蓋を有さないものであっても良い。 The tubular body (7) may have a lid for preventing evaporation of the liquid culture medium, or may not have a lid as shown in FIGS.
支持プレート（８）を安定させるために、支持プレート（８）と基盤（２）を互いに固定する固定手段を有することが好ましい。固定手段としては特に限定されないが、図２に示す如くネジ（１０）等が挙げられる。
固定手段の個数は特に限定されないが、支持プレート（８）全体を安定させるために３つ以上であることが好ましい。
In order to stabilize the support plate (8) it is preferred to have fixing means for fixing the support plate (8) and the base (2) to each other. The fixing means is not particularly limited, but as shown in FIG. 2, a screw (10) and the like can be mentioned.
The number of fixing means is not particularly limited, but is preferably three or more in order to stabilize the entire support plate (8).
密閉手段（５）は、筒状体（７）と培養容器（４）の底面の間の気密性を高め、半透膜である培養容器（４）の底面を傷つけたり変形させたりするものでないことが好ましい。例としてシリコン製のＯ−リング等が挙げられる。また、密閉手段（５）として接着剤等化学的に筒状体（７）と培養容器（４）の底面を接着させるものであっても良いが、液体培地の化学的性質や細胞の状態に影響を与えないようなものでなければならない。 The sealing means (5) enhances the airtightness between the cylindrical body (7) and the bottom surface of the culture vessel (4), and does not damage or deform the bottom surface of the culture vessel (4) which is a semipermeable membrane. Is preferred. An example is an O-ring made of silicon or the like. Alternatively, the sealing means (5) may be such that an adhesive or the like chemically adheres the cylindrical body (7) to the bottom of the culture vessel (4), but depending on the chemical properties of the liquid medium and the state of cells. It must be such as not to affect.
培養容器（４）の底面を液体培地に浸漬させるために基盤（２）の下に受け皿（１１）を配し、培養容器（４）の底面が半透膜であることで、受け皿（１１）に含まれる液体培地から培養容器（４）に含まれる液体培地へＯ２及びＣＯ２が供給でき、温度やｐＨの変化も少なくなるため、数日間の培養が可能となる。 In order to immerse the bottom of the culture vessel (4) in the liquid medium, the receiving tray (11) is disposed under the base (2), and the bottom of the culture vessel (4) is a semipermeable membrane. Since O 2 and CO 2 can be supplied from the liquid medium contained in to the liquid medium contained in the culture vessel (4), changes in temperature and pH are also reduced, and culture for several days becomes possible.
受け皿（１１）はシャーレのように比較的浅いものであっても、ビーカーのように比較的深いものであっても良く、蓋を有するものであっても図１及び２に示す如く蓋がないものであっても良い。素材は液体培地を入れるのに好適なものであれば特に限定されず、入手しやすいことからプラスチック製のものやガラス製のものが挙げられる。 The saucer (11) may be relatively shallow like a petri dish or relatively deep like a beaker, and even if it has a lid, it has no lid as shown in FIGS. 1 and 2. It may be something. The material is not particularly limited as long as it is suitable for containing a liquid culture medium, and may be made of plastic or glass because it is easy to obtain.
受け皿（１１）から培養容器（４）中へ液体培地を流入させるために、培養容器（４）の底面は半透膜であることが好ましい。培養する細胞として動物細胞（長径１０−３０μｍ程度）が挙げられることから、動物細胞は通過できないが液体培地は通過でき、且つｐＨの変化やＯ２及びＣＯ２の分圧に変化を与えないような孔の直径（０．７−２．０μｍ）及び密度（１ｃｍ２当たり５．０×１０５−５．０×１０７個）、乃至は孔の直径（０．１−０．７μｍ）及び密度（１ｃｍ２当たり０．５−５．０×１０8個）であることが望ましい。孔の直径及び密度が上記範囲より小さい半透膜だと受け皿（１１）から培養容器（４）へ液体培地が供給されにくくなるため好ましくなく、孔の直径及び密度が上記範囲より大きい半透膜だと筒状体（７）に含まれる液体培地の液面が短時間で低下してしまい、圧力を一定に保つことができないため好ましくない。 The bottom surface of the culture vessel (4) is preferably a semipermeable membrane in order to allow the liquid medium to flow from the receiving tray (11) into the culture vessel (4). Since cells to be cultured include animal cells (about 10-30 μm in major diameter), animal cells can not pass but liquid media can pass, and changes in pH and partial pressures of O 2 and CO 2 are not changed. Pore diameter (0.7-2.0 μm) and density (5.0 × 10 5 -5.0 × 10 7 / cm 2 ), or pore diameter (0.1-0.7 μm) and It is desirable to have a density (0.5-5.0 × 10 8 per 1 cm 2 ). A semipermeable membrane having a pore diameter and density smaller than the above range is not preferable because the liquid medium is difficult to be supplied from the receiving tray (11) to the culture vessel (4), and a semipermeable membrane having a pore diameter and density larger than the above range If this is the case, the liquid level of the liquid culture medium contained in the cylindrical body (7) drops in a short time, and the pressure can not be kept constant, which is not preferable.
第一貫通孔（３）を介して受け皿（１１）から培養容器（４）へ液体培地が供給されやすくするために、受け皿（１１）を底上げするための底上げシート（１２）を有することが好ましい。底上げシート（１２）の素材は、液体培地の化学的な性質に影響を与えない素材であれば特に限定されず、シリコン製のもの等が挙げられる。 It is preferable to have a bottom raising sheet (12) for raising the pan (11) so that the liquid medium can be easily supplied from the pan (11) to the culture vessel (4) through the first through hole (3) . The material of the bottom-raising sheet (12) is not particularly limited as long as it does not affect the chemical properties of the liquid medium, and examples thereof include those made of silicon.
本発明に係る培養装置（１）は、多孔質シート（１３）を培養容器（４）の底面と第一貫通孔（３）及びその周辺部の間に配することが好ましい。多孔質シート（１３）を使用することで培養容器（４）内のガス分圧平衡を保ちつつ培養容器（４）の底面が筒状体（７）に含まれる液体培地による圧力で下方に湾曲するのを防ぐことができる。
多孔質シート（１３）の孔の直径は０．５−１．０ｍｍであり、密度は１ｃｍ２当たり１００−５００個であることが望ましい。孔の直径は０．５ｍｍより小さいと受け皿（１１）から培養容器（４）の間で液体培地が効率的に交換されないため、ｐＨ、Ｏ２分圧、ＣＯ２分圧等が変化してしまい好ましくない。１．０ｍｍより大きいと筒状体（７）内の液体培地の液面が短時間で下がってしまうため圧力を一定に保つことができず好ましくない。また、例えば金網のような孔径の大きなものを多孔質シート（１３）として用いると同様に筒状体（７）内の液体培地の液面が短時間で下がってしまうため圧力を一定に保つことができず好ましくない。
In the culture apparatus (1) according to the present invention, it is preferable to dispose the porous sheet (13) between the bottom surface of the culture container (4) and the first through hole (3) and the periphery thereof. By using the porous sheet (13), the bottom of the culture vessel (4) is curved downward by the pressure of the liquid medium contained in the cylindrical body (7) while maintaining the gas partial pressure equilibrium in the culture vessel (4) You can prevent it.
The diameter of the pores of the porous sheet (13) is preferably 0.5 to 1.0 mm, and the density is preferably 100 to 500 per 1 cm 2 . If the diameter of the hole is smaller than 0.5 mm, the liquid medium is not efficiently exchanged between the receiving plate (11) and the culture vessel (4), so pH, O 2 partial pressure, CO 2 partial pressure, etc. change. Not desirable. If it is larger than 1.0 mm, the liquid level of the liquid culture medium in the cylindrical body (7) falls in a short time, and the pressure can not be kept constant, which is not preferable. Also, for example, when using a metal mesh having a large pore diameter as the porous sheet (13), the liquid level of the liquid culture medium in the cylindrical body (7) is lowered in a short time, and the pressure is kept constant. Unable to
多孔質シート（１３）を使用する際、半透膜の孔の直径が０．７−１．５μｍの範囲内である場合１ｃｍ２当たり０．５−５．０×１０６個の孔を有し、前記半透膜における孔の直径が０．１−０．７μｍの範囲内である場合１ｃｍ２当たり５．０×１０７−１．０×１０９個の孔を有することが好ましい。この範囲外、例えば孔の直径が０．４μｍで密度が１ｃｍ２当たり１．６×１０６個の半透膜を使用すると、筒状体（７）内のガス分圧平衡を維持（培養中の酸素分圧を維持）することができないため好ましくない（下記実施例１参照）。 When using a porous sheet (13), when the diameter of the pores of the semipermeable membrane is in the range of 0.7-1.5 μm, the membrane has 0.5-5.0 × 10 6 pores per 1 cm 2 It is preferable to have 5.0 × 10 7 -1.0 × 10 9 pores per 1 cm 2 when the diameter of the pores in the semipermeable membrane is in the range of 0.1-0.7 μm. Outside this range, for example, when the diameter of the pore is 0.4 μm and the density is 1.6 × 10 6 semipermeable membranes per 1 cm 2 , gas partial pressure equilibrium in the cylindrical body (7) is maintained (during culture) Oxygen partial pressure can not be maintained), which is not preferable (see Example 1 below).
＜実施形態２＞
図３は、本発明の実施形態２に係る培養装置を示す縦断面図である。
上記実施形態の他にも、図３に示すような培養装置が実施形態２として挙げられる。
実施形態１と実施形態２の相違点は、第一実施形態においては支持プレート（８）があり、支持プレート（８）の一方の面に突出部（６）が設けられ、他方の面に着脱自在に筒状体（７）が立設されることで筒状体（７）を支持プレート（８）によって支えているのに対し、第二実施形態においては支持プレート（８）を有さず、筒状体（７）が密閉手段（５）を介して培養容器（４）の底面に直接接続される点である。
支持プレート（８）がない代わりに、図３に図示の如く支持手段（１４）を使用して筒状体（７）を支えても、支持手段（１４）がなくても良い。
支持手段（１４）は、筒状体（７）を支持できるものであれば特に限定されないが、図３に図示の如く筒状体（７）を直接挟持する支持体（１５）と、支持体（１５）と基盤（２）とをつなぐ枠体（１６）からなるものであってもよい。
Second Embodiment
FIG. 3 is a longitudinal sectional view showing a culture apparatus according to Embodiment 2 of the present invention.
In addition to the above embodiment, a culture apparatus as shown in FIG. 3 can be mentioned as a second embodiment.
The difference between the first embodiment and the second embodiment is that in the first embodiment, there is a support plate (8), a protrusion (6) is provided on one surface of the support plate (8), and the other surface is detachable. While the cylindrical body (7) is supported by the support plate (8) by the cylindrical body (7) being set up freely, the second embodiment does not have the support plate (8). The point is that the cylindrical body (7) is directly connected to the bottom of the culture vessel (4) through the sealing means (5).
Instead of the absence of the support plate (8), the support means (14) may be used to support the tubular body (7) as shown in FIG. 3 or without the support means (14).
The support means (14) is not particularly limited as long as it can support the cylindrical body (7), but as shown in FIG. 3, a support (15) for directly holding the cylindrical body (7), and a support It may consist of a frame (16) which connects (15) and a base (2).
＜実施形態３：実施形態１又は２の培養装置を用いた細胞を培養する方法＞
軽微な圧力を付加しつつ細胞を培養する方法において、上記実施形態１及び２で挙げられている培養装置を使用することができる。
培養条件は培養する細胞の種類等によって決定されるべきであるので特に限定されないが、動物細胞を用いて数日間培養する場合であれば、培養液のｐＨの安定性を保つためにＣＯ２インキュベーター内で培養することが好ましい。培養する温度も培養細胞の種類等に応じて適宜変更し得るが、室温よりも高い温度で培養する場合、培養液の蒸発を防ぐために受け皿（１１）や筒状体（７）は蓋を有するものを使用するのが好ましい。
培養する細胞として、例えばマウス等の実験動物やヒト由来の動物細胞が挙げられる。より具体的には、ヒトあるいはその他の実験動物由来の呼吸器系上皮細胞、消化器系粘膜上皮細胞、消化器系腺上皮細胞、表皮細胞、神経細胞、上衣細胞、骨・軟骨細胞（前駆細胞を含む）、滑膜細胞、及び間葉系細胞（線維芽細胞など）が挙げられる。これらを本発明の培養装置で培養することで、軽微な圧力（０．００５−０．１気圧程度）を細胞に付加することによる細胞への影響を研究することができ、この軽微な圧力が原因となる疾患、例えば気道内圧上昇に伴う肺傷害、緑内障、頭蓋内圧亢進による脳浮腫等の疾患の分子生物学的なメカニズムの解明につながる。また、表皮細胞や骨細胞等は日常的に軽微な圧力を受けているため、その圧力がこれらの細胞にどのような影響を与えているかを分子生物学的に解析するのに役立つ。
Embodiment 3: Method for Cultivating Cells Using the Culture Device of Embodiment 1 or 2>
In the method of culturing cells while applying a slight pressure, the culture apparatus described in Embodiments 1 and 2 can be used.
The culture conditions are not particularly limited because they should be determined depending on the type of cells to be cultured, etc. However, in the case of culturing for several days using animal cells, a CO 2 incubator is used to maintain pH stability of the culture solution. It is preferable to culture it internally. The temperature to be cultured can also be changed as appropriate depending on the type of cultured cells, etc., but when culturing at a temperature higher than room temperature, the saucer (11) and the tubular body (7) have a lid to prevent evaporation of the culture solution. It is preferred to use one.
Examples of cells to be cultured include experimental animals such as mice and animal cells of human origin. More specifically, respiratory system epithelial cells derived from human or other experimental animals, digestive system mucosal epithelial cells, digestive system glandular epithelial cells, epidermal cells, neurons, ependymal cells, bone and chondrocytes (progenitor cells And synovial cells, and mesenchymal cells (such as fibroblasts). By culturing them in the culture apparatus of the present invention, it is possible to study the influence on the cells by applying a slight pressure (about 0.005 to 0.1 atm) to the cells, and this slight pressure is It leads to elucidation of the molecular biological mechanism of diseases causing diseases such as lung injury associated with increased airway pressure, glaucoma, brain edema caused by increased intracranial pressure and the like. In addition, since epidermal cells and bone cells and the like are routinely subjected to slight pressure, they are useful for molecular biological analysis of how the pressure affects these cells.
＜実施例１：本発明における半透膜の選択＞
本発明における培養容器の底面に使用する半透膜として適切なものを選択するため、半透膜の孔が小さなもの（孔の直径：０．４μｍ）を用いて培養した場合と、半透膜の孔が大きなもの（孔の直径：１．０μｍ）を用いて培養した場合を比較した。比較実験の詳細を以下に示す。
<Example 1: Selection of semipermeable membrane in the present invention>
In order to select an appropriate semipermeable membrane to be used on the bottom of the culture vessel in the present invention, the semipermeable membrane is cultured using one having a small pore of the semipermeable membrane (diameter of the pore: 0.4 μm) The case where culture was carried out using a large pore (pore diameter: 1.0 μm) was compared. The details of the comparative experiment are shown below.
約１．０×１０４個のＲＬＥ−６ＴＮ肺上皮細胞を、２ｍｌのRPMI-1640培養液（10%牛血清含有）に懸濁し、０．４μｍ及び１．０μｍにおいて共に孔の密度は１ｃｍ２当たり約１．６×１０６個であるBD Biosciences社製の半透膜付きBD Falcon Cell Culture Insert（６ウェル用）内に播種した後、２日間ＣＯ２インキュベーター（エスペック社製）中において３７℃で前培養した。その後、Cell Culture Insertを、多孔質シート（孔の直径が約０．１５ｍｍ、孔の密度が１ｃｍ２当たり約２００個である、厚さ約１ｍｍのシリコンゴムシート）が配された本発明の培養装置に移し、受け皿（１０ｃｍシャーレ）に６０ｍｌのRPMI-1640培養液を加え、液面高が５０ｃｍになるように底面が円形であって直径が１．３ｃｍである筒状体にRPMI-1640培養液を加えた。また、受け皿と筒状体は蓋をして用いた。
上記のように準備した培養装置において、ＲＬＥ−６ＴＮ肺上皮細胞を３日間ＣＯ２インキュベーター中において３７℃で培養し、その間のｐＨ、ＣＯ２分圧、Ｏ２分圧及び細胞の状態の違いを観察した。観察結果を以下表１及び表２（ｐＨ、ＣＯ２分圧、Ｏ２分圧の変化）及び図４（細胞の状態の違い）に示す。以下表１及び表２に記載されている数値は、３回の上記比較実験で得られた結果の平均及びその標準偏差を示したものである。
About 1.0 × 10 4 RLE-6TN lung epithelial cells are suspended in 2 ml of RPMI-1640 culture medium (containing 10% bovine serum), and the density of pores is 1 cm 2 at 0.4 μm and 1.0 μm. The cells were seeded in BD Biosciences with semipermeable membrane BD Falcon Cell Culture Insert (for 6 wells) each having about 1.6 × 10 6 cells, and then at 37 ° C. in a CO 2 incubator (manufactured by Espec) for 2 days. Pre-cultured. After that, the culture of the present invention in which the cell culture insert is arranged with a porous sheet (a silicon rubber sheet having a diameter of about 0.15 mm and a density of the holes about 200 per 1 cm 2 ) Transfer to a device, add 60 ml of RPMI-1640 culture solution to a receiving dish (10 cm petri dish), and make a cylinder with a circular bottom and a diameter of 1.3 cm so that the liquid level is 50 cm. RPMI-1640 culture The solution was added. Moreover, the saucer and the cylindrical body were used with a lid.
In the culture apparatus prepared as described above, RLE-6TN lung epithelial cells are cultured at 37 ° C. in a CO 2 incubator for 3 days, and the difference between pH, CO 2 partial pressure, O 2 partial pressure, and cell condition among them is I observed it. The observation results are shown in Table 1 and Table 2 (pH, change in partial pressure of CO 2 , change in partial pressure of O 2 ) and FIG. 4 (difference in cell condition). The numerical values described in Tables 1 and 2 below show the average of the results obtained in the three comparison experiments and the standard deviation thereof.
上記表１及び２に示すように、孔の直径が１．０μｍの半透膜を使用した場合、受け皿内のｐＨ、ＣＯ２分圧、Ｏ２分圧と比較して筒状体内のｐＨ、ＣＯ２分圧、Ｏ２分圧は大きな変化がなかった。
しかし、孔の直径が０．４μｍの半透膜を使用した場合、受け皿内のｐＨ、ＣＯ２分圧、Ｏ２分圧と比較して筒状体内のｐＨ、ＣＯ２分圧、Ｏ２分圧の変化は大きかった。より具体的には、筒状体内において日を追うごとにｐＨは低下し、ＣＯ２分圧は上昇し、Ｏ２分圧は低下していた。
上記の結果から、孔の直径が小さい半透膜、つまり透過性の低い半透膜を使用すると筒状体内のｐＨ、ＣＯ２分圧、Ｏ２分圧が大きく変化してしまい、数日間の培養には適さないことが理解できる。
As shown in Tables 1 and 2 above, when a semipermeable membrane having a pore diameter of 1.0 μm is used, the pH in the receiver, the pH in the tubular body in comparison with the CO 2 partial pressure, and the O 2 partial pressure, The CO 2 partial pressure and the O 2 partial pressure did not change significantly.
However, if the diameter of the holes was used 0.4μm semipermeable membranes, pH of the saucer, CO 2 partial pressure, O 2 partial pressure and the tubular body of pH compared, CO 2 partial pressure, O 2 minutes The change in pressure was large. More specifically, the pH decreased with the passage of time in the cylinder, the partial pressure of CO 2 increased, and the partial pressure of O 2 decreased.
From the above results, it can be seen that the use of a semipermeable membrane having a small pore diameter, that is, a semipermeable membrane having low permeability, causes a large change in pH, CO 2 partial pressure and O 2 partial pressure in the cylindrical body, It can be understood that it is not suitable for culture.
図４は、（Ａ）孔の直径が０．４μｍの半透膜を使用した場合と、（Ｂ）孔の直径が１．０μｍの半透膜を使用した場合との、細胞の状態の違いを示す図である。
図４（Ａ）において多数見られる白色の粒子は細胞の死骸である。
図４が示すように、孔の直径が小さい半透膜（０．４μｍ）で３日間培養した場合と孔の直径が大きい半透膜（１．０μｍ）で３日間培養した場合を比較すると、細胞の生存率が、孔の直径が小さい半透膜を使用した場合において著しく低いことがわかる。この細胞の生存率の差の一因として、表１及び表２に示された筒状体内のｐＨ、ＣＯ２分圧、Ｏ２分圧の変化が挙げられる。
したがって、孔の密度が同じ場合、細胞の生存率からも孔の直径が大きい半透膜、つまり透過性の高い半透膜を使用する方が、本発明の培養装置における数日間の培養には適しているといえる。
FIG. 4 shows the difference in the state of cells between (A) using a semipermeable membrane having a diameter of 0.4 μm and (B) using a semipermeable membrane having a diameter of 1.0 μm. FIG.
White particles seen in large numbers in FIG. 4 (A) are dead cells.
As FIG. 4 shows, when culture | cultivating the case where it is 3 days with a semipermeable membrane (0.4 micrometer) with a small diameter of a hole for 3 days, and the case where it culture | cultivates with a semipermeable membrane (1.0 micrometer) with a large diameter of a hole for 3 days, It can be seen that cell viability is significantly lower when using semipermeable membranes with small pore diameters. As a cause of this difference in cell viability, changes in pH, CO 2 partial pressure, and O 2 partial pressure in the cylinder shown in Tables 1 and 2 can be mentioned.
Therefore, if the pore density is the same, using a semipermeable membrane having a large pore diameter, that is, a highly permeable semipermeable membrane, is more preferable for culture in the culture apparatus of the present invention for several days. It can be said that it is suitable.
＜実施例２：圧力を付加した培養による培養細胞への影響＞
上記実施例１で数日間の培養に適していることが示された透過性の高い半透膜（本実施例においては孔の直径１．０μｍ、孔の密度１ｃｍ２当たり１．６×１０６個）を使用して、筒状体内に含まれる液体培地の液面高が低い（１０ｃｍ）場合と高い（４０ｃｍ）場合で、細胞の状態の違いを観察した。
本実施例は、培養細胞としてＮＣＩ−Ｈ４４１肺胞上皮細胞を使用し、半透膜を孔の直径１．０μｍ、孔の密度１ｃｍ２当たり１．６×１０６個のものに統一し、筒状体内に含まれる液体培地の液面高を１０ｃｍ又は４０ｃｍとしたことを除いて、上記実施例１と同様の培養条件で実施した。結果を図５に示す。
<Example 2: Effect on culture cells by pressure-added culture>
A highly permeable semipermeable membrane which has been shown to be suitable for culture for several days in Example 1 above (in this example, the pore diameter is 1.0 μm, and the density of pores is 1.6 × 10 6 / cm 2) The difference in the state of cells was observed when the liquid surface height of the liquid medium contained in the tubular body was low (10 cm) and high (40 cm), using the
In this example, NCI-H441 alveolar epithelial cells are used as cultured cells, and the semipermeable membrane is standardized to a pore diameter of 1.0 μm and a pore density of 1.6 × 10 6 cells per 1 cm 2. The culture was carried out under the same culture conditions as in Example 1 except that the liquid surface height of the liquid culture medium contained in the matrix was 10 cm or 40 cm. The results are shown in FIG.
図５は、（Ａ）１０ｃｍ液面高、及び（Ｂ）４０ｃｍ液面高で３日間培養した後の肺胞上皮細胞の様子を示す図である。
図５（Ａ）と比較して（Ｂ）は細胞数が顕著に少ないことが見てとれる。
図５に示すように、１０ｃｍ液面高で培養した場合と比較して４０ｃｍ液面高で培養した場合は顕著に細胞の増殖が抑制されているのがわかる。
FIG. 5 is a diagram showing the appearance of alveolar epithelial cells after culturing for 3 days at (A) 10 cm liquid level and (B) 40 cm liquid level.
It can be seen that the number of cells is significantly smaller in (B) than in FIG. 5 (A).
As shown in FIG. 5, it can be seen that the cell growth is significantly suppressed when culturing at a 40 cm liquid surface height as compared to when culturing at a 10 cm liquid surface height.
一般的に０．０３気圧程度の圧力を付加した細胞は何らかの傷害を受ける可能性が推測されており、本実施例により、本発明の培養装置を用いることによって微細な圧力を付加しながら細胞を培養することができることが示された。
よって、本発明である培養装置は、細胞に微細な圧力（０．００５−０．１気圧程度）を付加しながら培養できるため、例えば気道内圧上昇に伴う肺傷害、緑内障、頭蓋内圧亢進による脳浮腫等の疾患の原因となる肺胞上皮細胞、消化管せん粘膜上皮細胞、網膜神経節細胞、脳神経細胞及び上衣細胞に対する微細な圧力の影響を調べ、これらの疾患の分子生物学的なメカニズムを解明するために好適に利用される。また、表皮細胞や骨細胞等は日常的に軽微な圧力を受けているため、その圧力がこれらの細胞にどのような影響を与えているかを分子生物学的に解析するために好適に利用される。
Generally, it is assumed that cells to which a pressure of about 0.03 atm is applied may be damaged in any way, and according to this embodiment, cells are exposed while applying minute pressure by using the culture apparatus of the present invention. It has been shown that it can be cultured.
Therefore, since the culture apparatus of the present invention can culture cells while applying a minute pressure (about 0.005 to 0.1 atm) to cells, for example, lung injury associated with an increase in airway pressure, glaucoma, brain due to increased intracranial pressure To investigate the effect of minute pressure on alveolar epithelial cells, gastrointestinal arachnoid epithelial cells, retinal ganglion cells, cranial nerve cells and ependymal cells that cause diseases such as edema, It is suitably used for clarification. In addition, since epidermal cells, bone cells, etc. are routinely subjected to slight pressure, they are suitably used for molecular biological analysis of how the pressure affects these cells. Ru.
１ 培養装置
２ 基盤
３ 第一貫通孔
４ 培養容器
５ 密閉手段
６ 突出部
７ 筒状体
８ 支持プレート
９ 第二貫通孔
１０ 固定手段
１１ 受け皿
１２ 底上げシート
１３ 多孔質シート
１４ 支持手段
１５ 支持体
１６ 枠体
Reference Signs List 1 culture apparatus 2 base 3 first through hole 4 culture vessel 5 sealing means 6 projecting portion 7 cylindrical body 8 support plate 9 second through hole 10 fixing means 11 saucer 12 bottom raising sheet 13 porous sheet 14 support means 15 support 16 Frame
Claims (11)
該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、
該培養容器の上に密閉手段を介して設けられ、一方の面に突出部が設けられ、他方の面に着脱自在に液体培地を含む筒状体が立設され、該筒状体を支持するための支持プレートとを備え、
該支持プレートと突出部を貫通する孔である第二貫通孔が穿設され、該培養容器が該基盤と該支持プレートの間に該突出部の先端と該密閉手段を介して挟持され、該第一貫通孔と該第二貫通孔が同軸状に位置づけられて固定され、
前記培養容器の底面を液体培地に浸漬させるために前記基盤の下に受け皿が配され、
前記培養容器の底面が半透膜であり、
前記筒状体は、液体培地を加えることで細胞に０．０３〜０．１気圧の圧力を付与し、
前記密閉手段は、前記筒状体につながる前記第二貫通孔から液体培地が漏出することを防ぐことを特徴とする、静水圧を細胞に付与するための培養装置。 A base made of perforated plate material,
A culture vessel containing cells and liquid medium arranged to cover the first through hole of the base;
A cylindrical body including a liquid culture medium is provided upright on the culture vessel via a sealing means, a protrusion is provided on one side, and the other side is detachably mounted, and the cylindrical body is supported. With a support plate for
A second through hole, which is a hole passing through the support plate and the protrusion, is bored, and the culture vessel is sandwiched between the base and the support plate via the sealing means with the tip of the protrusion. The first through hole and the second through hole are coaxially positioned and fixed;
A pan is placed under the base to immerse the bottom of the culture vessel in liquid medium,
The bottom of the culture vessel is a semipermeable membrane,
The cylinder applies a pressure of 0.03 to 0.1 atm to the cells by adding a liquid medium,
The culture apparatus for applying hydrostatic pressure to cells, wherein the sealing means prevents the liquid culture medium from leaking through the second through hole connected to the cylindrical body.
該基盤の第一貫通孔を覆うように配された細胞と液体培地を含む培養容器と、
該培養容器の底面上にその一端が配され、液体培地を含む筒状体と、
該筒状体を支持するための支持手段と、
該筒状体と該培養容器の底面の間に挟持される密閉手段と、
前記培養容器の底面を液体培地に浸漬させるために該基盤の下に配される受け皿とを備え、
前記培養容器の底面が半透膜であり、
前記筒状体は、液体培地を加えることで細胞に０．０３〜０．１気圧の圧力を付与し、
前記密閉手段は、前記筒状体につながる第二貫通孔から液体培地が漏出することを防ぐことを特徴とする、静水圧を細胞に付与するための培養装置。 A base made of perforated plate material,
A culture vessel containing cells and liquid medium arranged to cover the first through hole of the base;
A cylindrical body having one end disposed on the bottom surface of the culture vessel and containing a liquid medium;
Support means for supporting the tubular body;
Sealing means sandwiched between the cylindrical body and the bottom surface of the culture vessel;
And a receptacle disposed under the base for immersing the bottom surface of the culture vessel in the liquid medium.
The bottom of the culture vessel is a semipermeable membrane,
The cylinder applies a pressure of 0.03 to 0.1 atm to the cells by adding a liquid medium,
The sealing means may prevent the liquid medium from the second through hole that connected to the tubular body to leak, the culture device for applying hydrostatic pressure to the cells.
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