JP2015073520A - Cell cultivation container - Google Patents
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- JP2015073520A JP2015073520A JP2013213988A JP2013213988A JP2015073520A JP 2015073520 A JP2015073520 A JP 2015073520A JP 2013213988 A JP2013213988 A JP 2013213988A JP 2013213988 A JP2013213988 A JP 2013213988A JP 2015073520 A JP2015073520 A JP 2015073520A
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- 238000004113 cell culture Methods 0.000 title claims abstract description 77
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 239000001963 growth medium Substances 0.000 claims abstract description 4
- 239000000463 material Substances 0.000 claims description 21
- 239000002184 metal Substances 0.000 claims description 9
- 229910052751 metal Inorganic materials 0.000 claims description 9
- 239000000835 fiber Substances 0.000 claims description 7
- 229920005989 resin Polymers 0.000 claims description 7
- 239000011347 resin Substances 0.000 claims description 7
- 229920003002 synthetic resin Polymers 0.000 claims description 7
- 239000000057 synthetic resin Substances 0.000 claims description 7
- 238000004891 communication Methods 0.000 claims description 3
- 239000011521 glass Substances 0.000 claims description 3
- 229920002678 cellulose Polymers 0.000 claims description 2
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- 239000011148 porous material Substances 0.000 claims description 2
- 239000002759 woven fabric Substances 0.000 claims description 2
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- 229920002223 polystyrene Polymers 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 1
- 244000025254 Cannabis sativa Species 0.000 description 1
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 description 1
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- 150000002739 metals Chemical class 0.000 description 1
- 239000002557 mineral fiber Substances 0.000 description 1
- 230000002572 peristaltic effect Effects 0.000 description 1
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- 229920002338 polyhydroxyethylmethacrylate Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 229910052895 riebeckite Inorganic materials 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
Description
本発明は、細胞や組織片などの被培養物を培養してスフェロイド（細胞凝集塊）を作製する細胞培養容器に関する。 The present invention relates to a cell culture vessel for producing a spheroid (cell aggregate) by culturing a culture object such as a cell or a tissue piece.
近年、細胞を二次元的に培養する単層培養に代わって、細胞を培養して三次元的に凝集させるスフェロイド培養が注目されている。スフェロイド培養は、単層培養に比べて、生体内の細胞に近い状態を構築することができるため、細胞が生体内で有する特異的な機能を引き出すことができる。 In recent years, spheroid culture in which cells are cultured and aggregated three-dimensionally is attracting attention instead of monolayer culture in which cells are cultured two-dimensionally. Since spheroid culture can construct a state closer to cells in a living body than monolayer culture, it can draw out a specific function that cells have in vivo.
スフェロイド培養を行う従来の培養基材として、例えば、特許文献１に記載される容器がある。この容器は、底面に互いに間隔を空けて複数の窪み部が形成されている。 As a conventional culture substrate for spheroid culture, for example, there is a container described in Patent Document 1. In this container, a plurality of depressions are formed on the bottom surface at intervals.
スフェロイド培養は、スフェロイド前駆体としての細胞が撹拌された培養液を容器内に流し込み、窪み部内で細胞を培養する。そうすると、窪み部内の細胞は、窪み部の形状・大きさに対応して培養されて、三次元的に凝集して、スフェロイドを形成する。 In spheroid culture, a culture solution in which cells as spheroid precursors are agitated is poured into a container, and the cells are cultured in the depression. If it does so, the cell in a hollow part will be culture | cultivated according to the shape and magnitude | size of a hollow part, and it will aggregate three-dimensionally, and will form a spheroid.
また、特許文献１の容器と比較し、所望の大きさのスフェロイドを均一に形成でき、培養を効率的に行える培養容器が提案されている（特許文献２参照）。
この容器は、被培養物が培養される隔室を形成する窪み部が基材表面に複数形成されて、互いに近接する前記窪み部の間の基材表面が非平坦面であることを特徴としている。そのため、この容器は窪み部の間に平坦面がなく、窪み部の大きさに対応したスフェロイドの他に二次元的に培養されたり、窪みの大きさの影響を受けないランダムな大きさのスフェロイドが形成されることがない。
Moreover, compared with the container of patent document 1, the culture container which can form spheroid of a desired magnitude | size uniformly and can culture | cultivate efficiently is proposed (refer patent document 2).
The container is characterized in that a plurality of depressions forming a compartment in which a culture object is cultured are formed on the substrate surface, and the substrate surface between the depressions adjacent to each other is a non-flat surface. Yes. Therefore, this container does not have a flat surface between the depressions, and in addition to the spheroids corresponding to the size of the depressions, it is cultured two-dimensionally or is not affected by the size of the depressions. Is not formed.
ところが、上述した特許文献２の容器を用いてスフェロイド培養を行う際、容器の移動等で容器が大きく搖動すると、培養した細胞が当初居た窪み部から飛び出し隣接する他の窪み部に移動するという現象が確認された。
特許文献２の容器は、ひとつの窪み部に対して１つのスフェロイドを形成することで、所望の均一な大きさのスフェロイドを得るものである。しかしながら、前述のとおり容器の搖動によって培養途中の細胞が隣接する他の窪み部に移動すると、特定の窪み部には複数の細胞が存在することになる。そのため、形成されるスフェロイドの大きさがランダムとなるおそれがある。ランダムな大きさのスフェロイドは、窪みの大きさに対応した均一なスフェロイドと比較して、生理機能が異なったり、幹細胞培養の場合には、分化のステージが異なったりなど、培養容器内の細胞群の均一性が低くなり、実験データ評価等に支障をきたす。
However, when the spheroid culture is performed using the container of Patent Document 2 described above, if the container is shaken greatly by moving the container or the like, the cultured cells jump out of the originally existing recess and move to another adjacent recess. The phenomenon was confirmed.
The container of patent document 2 obtains the spheroid of a desired uniform magnitude | size by forming one spheroid with respect to one hollow part. However, as described above, when cells in the middle of culture are moved to another adjacent dent by the peristaltic movement of the container, a plurality of cells are present in the specific dent. Therefore, the size of the formed spheroids may be random. Randomly sized spheroids have different physiological functions compared to uniform spheroids corresponding to the size of the depression, and in the case of stem cell culture, the differentiation stage is different. The homogeneity of the data becomes low, which hinders the evaluation of experimental data.
そこで、本発明は、上記の課題を解決するためになされたものであり、容器が大きく搖動した際に生じる細胞の隣接する窪み部への移動を抑制することで、所望の大きさのスフェロイドを均一に形成でき、培養を効率的に行うことが可能な細胞培養容器の提供を目的とする。 Therefore, the present invention has been made to solve the above-described problem, and suppresses the movement of cells to the adjacent depressions that occur when the container is swung greatly, so that a spheroid having a desired size can be obtained. An object of the present invention is to provide a cell culture vessel that can be uniformly formed and that can be cultured efficiently.
上記の目的を達成するために、本発明に係る細胞培養容器は、被培養物が培養される隔室を形成する窪み部が複数形成された培養面と、前記培養面を底面に備える容器本体と、複数の窪み部の上部に載置され前記窪み部の開口を塞ぐ透液性蓋体とを備え、前記培養面は、互いに近接する前記窪み部の間の頂部が非平坦面からなり、前記透液性蓋体は、前記容器本体内の培養液中に浸漬された状態で前記窪み部の間の頂部との距離が、前記窪み部内で培養された前記被培養物の外径寸法よりも小さくなるよう配置されていることを特徴とする。 In order to achieve the above object, a cell culture container according to the present invention includes a culture surface in which a plurality of depressions forming a compartment in which a culture object is cultured is formed, and a container body having the culture surface on the bottom surface And a permeable lid that is placed on top of the plurality of depressions and closes the openings of the depressions, and the culture surface has a non-planar surface between the depressions adjacent to each other, The liquid permeable lid is immersed in the culture medium in the container main body, and the distance from the top between the recesses is larger than the outer diameter of the culture object cultured in the recesses. Is also arranged to be smaller.
本発明によれば、所望の大きさのスフェロイドを均一に形成し、細胞の培養を効率的に行うことができる。 According to the present invention, spheroids having a desired size can be uniformly formed, and cells can be cultured efficiently.
［第１の実施形態］
本発明の第１の実施形態に係る細胞培養容器について、図１ないし図３を用いて説明する。図１は、細胞培養容器の縦断面図である。図２は、細胞培養容器の平面図である。図３は、図１に記載の細胞培養容器の窪み部付近の部分拡大図である。
[First Embodiment]
A cell culture container according to a first embodiment of the present invention will be described with reference to FIGS. FIG. 1 is a longitudinal sectional view of a cell culture container. FIG. 2 is a plan view of the cell culture container. FIG. 3 is a partially enlarged view of the vicinity of the recess of the cell culture container shown in FIG.
本実施形態に係る細胞培養容器１０は、細胞を培養して三次元的に凝集させてなるスフェロイドを作製する細胞培養容器である。 The cell culture container 10 according to the present embodiment is a cell culture container for producing spheroids obtained by culturing cells and aggregating them three-dimensionally.
まず、本実施形態に係る細胞培養容器１００の構成について説明する。
細胞培養容器１００は、図１および図２に示したように、容器本体１０、透液性蓋体２０、保持具３０とを備えている。この実施形態において、容器本体１０の内側の底板部１１の上面が培養面１５に相当する部分となる。培養面１５は、例えばポリスチレンなどの合成樹脂材から構成されている。本実施形態では、細胞培養容器１００は、合成樹脂材料を用いた射出成形により得られる。なお、本実施形態では、透液性蓋体２０は、透液性シート２１からなる。
First, the configuration of the cell culture container 100 according to the present embodiment will be described.
As shown in FIGS. 1 and 2, the cell culture container 100 includes a container body 10, a liquid-permeable lid 20, and a holder 30. In this embodiment, the upper surface of the bottom plate portion 11 inside the container body 10 is a portion corresponding to the culture surface 15. The culture surface 15 is made of a synthetic resin material such as polystyrene. In this embodiment, the cell culture container 100 is obtained by injection molding using a synthetic resin material. In the present embodiment, the liquid-permeable lid 20 includes a liquid-permeable sheet 21.
容器本体１０は、円板状の底板部１１、および、環状の側壁部１２を有している。側壁部１２は、底板部１１の外周縁から起立している。本実施形態では、底板部１１の直径は、８５ｍｍ、底板部１１の厚さは、１ｍｍに設計されている。また、側壁部１２の高さは、２０ｍｍに設計されている。 The container body 10 includes a disk-shaped bottom plate portion 11 and an annular side wall portion 12. The side wall portion 12 stands from the outer peripheral edge of the bottom plate portion 11. In this embodiment, the diameter of the bottom plate portion 11 is designed to be 85 mm, and the thickness of the bottom plate portion 11 is designed to be 1 mm. Further, the height of the side wall portion 12 is designed to be 20 mm.
底板部１１上面の培養面１５（すなわち、被培養物５０が培養される隔室が形成される領域）には、複数の窪み部１７が形成されている。窪み部１７の内面は、滑らかな凹面になっている。窪み部１７は、被培養物が培養される隔室（ウェル：ｗｅｌｌ）を形成する。本実施形態では、直径８５ｍｍの円形の培養面１５に１４２００個程度（約２５０個／ｃｍ２）の窪み部１７が形成されている。１つの窪み部１７には、所望の大きさの均一な１つの被培養物５０（スフェロイド）が最終的に培養される。 A plurality of depressions 17 are formed on the culture surface 15 on the upper surface of the bottom plate portion 11 (that is, a region where a compartment in which the culture object 50 is cultured is formed). The inner surface of the recess 17 is a smooth concave surface. The hollow part 17 forms a compartment (well) in which the culture object is cultured. In this embodiment, about 14200 (about 250 / cm 2 ) depressions 17 are formed on a circular culture surface 15 having a diameter of 85 mm. One cultivated object 50 (spheroid) having a desired size and uniform size is finally cultured in one depression 17.
透液性蓋体２０は、複数の窪み部１７の開口を一括して塞ぐことで、移動等により容器本体１０が大きく搖動する際、窪み部１７にて培養されている被培養物５０が、隣接する他の窪み部１７に移動することを抑制するものである。そのため、透液性蓋体２０は、容器本体１０内に満たされている培養液１６が透過でき、かつ窪み部１７に存在する被培養物５０（培養中の細胞）が通過しない程度の大きさの透孔を有している。また、透液性蓋体２０は、容器本体１０内の培養液１６に浸漬され、培養面１５の互いに近接する窪み部１７の間の頂部の上方に載置される。 The permeable lid 20 collectively closes the openings of the plurality of depressions 17 so that when the container main body 10 is greatly swung by movement or the like, the culture object 50 cultured in the depressions 17 is It suppresses moving to the other adjacent dent part 17. Therefore, the liquid-permeable lid 20 has a size that allows the culture solution 16 filled in the container body 10 to pass therethrough and does not allow the culture object 50 (cells being cultured) present in the recess 17 to pass. Has through holes. In addition, the liquid-permeable lid 20 is immersed in the culture solution 16 in the container body 10 and placed on the top of the culture surface 15 between the adjacent recesses 17.
透液性蓋体２０は、金属、樹脂、セルロース、ガラス、天然繊維からなる群より選択される１種以上の材質からなる。また、透液性蓋体２０は、織物、不織繊維、スポンジ状物、多孔質材、板厚方向に連通孔が形成された板状物からなる群より選択される１種以上の形態からなる。なお、天然繊維とは、植物繊維（綿、麻等）、動物繊維（羊毛、絹、カシミヤ等）、鉱物繊維（石綿等）を包含するものある。
透液性蓋体２０は、例えばステンレス等の金属製メッシュより構成されている。金属製メッシュは、材質、メッシュ数、目開き、線径、開口率、織方（例えば、平織や綾織）などのバリエーションが多く、被培養物の大きさに応じた適宜のものを選択して用いることができる。また、金属製メッシュは、樹脂製メッシュと比較して自重によって撓みにくいため、使用時に枠体を用いずに平坦な状態を維持することができる。
また、透液性蓋体２０は、例えば金属、樹脂、ガラス等からなり板厚方向に連通孔が形成された板状物であってもよい。板状物は、前述の金属製メッシュと同様に自重によって撓みにくいため、使用時に枠体を用いずに平坦な状態を維持することができる。
The liquid-permeable lid 20 is made of one or more materials selected from the group consisting of metals, resins, cellulose, glass, and natural fibers. Further, the liquid-permeable lid 20 is made of one or more types selected from the group consisting of woven fabrics, non-woven fibers, sponge-like materials, porous materials, and plate-like materials having communication holes formed in the plate thickness direction. Become. Natural fibers include plant fibers (cotton, hemp, etc.), animal fibers (wool, silk, cashmere, etc.), and mineral fibers (asbestos, etc.).
The liquid-permeable lid 20 is made of a metal mesh such as stainless steel, for example. There are many variations of metal mesh, such as material, number of meshes, mesh size, wire diameter, opening ratio, weaving method (for example, plain weave or twill weave), and select the appropriate one according to the size of the culture object. Can be used. Further, since the metal mesh is less likely to be bent by its own weight compared to the resin mesh, it can maintain a flat state without using a frame during use.
Further, the liquid-permeable lid 20 may be a plate-like object made of, for example, metal, resin, glass, or the like and having communication holes formed in the plate thickness direction. Since the plate-like object is unlikely to be bent by its own weight like the above-described metal mesh, it can maintain a flat state without using a frame during use.
保持具３０は、透液性蓋体２０を容器本体１０の窪み部１７側（すなわち、培養面１５側）に付勢した状態で保持するためのものである。保持具３０は、一端が透液性蓋体２０を窪み部側に付勢するように押え、他端が容器本体１０の側壁部１２に固定される。一端は、透液性蓋体２０と接着されていても、接着されていなくてもよい。また、他端は、クリップのように側壁部１２を挟み込むようになっていてもよいし、その他の手段で固定されてもよい。保持具３０は、樹脂成型品であってもよいし、金属線の成形品であってもよく、その他前述の機能を備えればどのような素材や形態であってもよい。また、保持具３０は、単一または複数のいずれであってもよい。 The holder 30 is for holding the liquid-permeable lid 20 in a state of being biased toward the recessed portion 17 side (that is, the culture surface 15 side) of the container body 10. One end of the holder 30 is pressed so as to urge the liquid-permeable lid 20 toward the recessed portion, and the other end is fixed to the side wall portion 12 of the container body 10. One end may be bonded to the liquid-permeable lid 20 or may not be bonded. Further, the other end may be configured to sandwich the side wall portion 12 like a clip, or may be fixed by other means. The holder 30 may be a resin molded product, a metal wire molded product, or any other material or form as long as it has the aforementioned functions. Moreover, the holder 30 may be either single or plural.
蓋４０（図示しない）は、容器本体１０に上方の開口部に対応した形状に形成されている。蓋４０は、細胞の培養環境を維持するために、細胞の培養中は容器本体１０に被せられて使用される。 The lid 40 (not shown) is formed in the container body 10 in a shape corresponding to the upper opening. The lid 40 is used by being put on the container body 10 during the cell culture in order to maintain the cell culture environment.
次いで、図３を用いて、容器本体１０と透液性蓋体２０との位置関係について説明する。前述のとおり、透液性蓋体２０は、複数の窪み部１７の開口を一括して塞ぐことで、窪み部１７にて培養されている被培養物５０が、隣接する他の窪み部１９に移動することを抑制するものである。そのため、窪み部の間の頂部１８と前記透液性蓋体２０との距離をａ、窪み部内で培養された被培養物の外径寸法をｂとした場合、距離ａが寸法ｂよりも小さい。これにより、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から飛び出しそうになったとしても、透液性蓋体２０が隣接する他の窪み部１９への移動を阻止する。なお、被培養物の外径寸法ｂとは、個々の被培養物５０の外径寸法の最大値を平均した値である。容器本体１０中の被培養物５０は個数が膨大であるため、外径寸法ｂを求める際は、被培養物５０が均一に培養されていることを前提に、適宜の数量の外径寸法を測定し、そのデータを用いて外径寸法ｂを求めてもよい。 Next, the positional relationship between the container body 10 and the liquid-permeable lid 20 will be described with reference to FIG. As described above, the liquid-permeable lid 20 collectively closes the openings of the plurality of depressions 17 so that the culture object 50 cultured in the depressions 17 is placed in the other depressions 19 adjacent thereto. It suppresses moving. Therefore, when the distance between the top 18 between the depressions and the liquid-permeable lid 20 is a, and the outer diameter dimension of the culture object cultured in the depressions is b, the distance a is smaller than the dimension b. . Thereby, even if the container main body 10 swings greatly and the culture object 50 in the dent part 17 is likely to jump out of the opening of the dent part 17, the other dent part 19 to which the liquid-permeable lid 20 is adjacent is disposed. Stop moving to. In addition, the outer diameter dimension b of the culture object is a value obtained by averaging the maximum values of the outer diameter dimensions of the individual culture objects 50. Since the number of the culture objects 50 in the container body 10 is enormous, when determining the outer diameter dimension b, an appropriate number of outer diameter dimensions are set on the assumption that the culture object 50 is uniformly cultured. The outer diameter dimension b may be obtained by measuring and using the data.
容器本体１０と透液性蓋体２０との位置関係における「距離ａが寸法ｂよりも小さい」は、対応する頂部１８と透液性蓋体２０との間で全体の４０％以上の割合で満たしていれば、被培養物５０が窪み部１７からの飛び出し隣接する他の窪み部１９に移動するのを抑制できる。前記関係を満たしている割合は、好ましくは５０％以上であり、より好ましくは６０％以上である。他方、前記関係を満たしている割合が９５％を超えると、各部材に非常に高い平坦性が求められ、製造コストが高くなる。前記関係を満たしている割合は、好ましくは９０％以下であり、さらに好ましくは８５％以下である。 The “distance a is smaller than the dimension b” in the positional relationship between the container body 10 and the liquid-permeable lid 20 is a ratio of 40% or more of the total between the corresponding top portion 18 and the liquid-permeable lid 20. If satisfy | filling, it can suppress that the to-be-cultured material 50 jumps out from the hollow part 17, and moves to the other hollow part 19 adjacent. The ratio satisfying the relationship is preferably 50% or more, and more preferably 60% or more. On the other hand, if the ratio satisfying the relationship exceeds 95%, very high flatness is required for each member, and the manufacturing cost increases. The ratio satisfying the relationship is preferably 90% or less, and more preferably 85% or less.
本実施形態では、窪み部１７は、容器本体１０の培養面１５に対してレーザ光を照射することにより形成される。レーザ照射は、底板部１１の上面に対して、レーザ光を深さ方向に照射して行う。 In the present embodiment, the recess 17 is formed by irradiating the culture surface 15 of the container body 10 with laser light. Laser irradiation is performed by irradiating the upper surface of the bottom plate portion 11 with laser light in the depth direction.
まず、底板部１１の平面方向をｘ−ｙ軸とした場合、レーザ照射装置の照射部をｘ軸の正方向に走査させつつ、一定の間隔（例えば８００μｍ）ごとにレーザ光を照射して、ｘ軸方向に並んだ複数の窪み部１７を形成する。続けて、照射部をｙ軸方向に一定の距離（例えば４００μｍ）だけ走査させた後、照射部をｘ軸の負方向に走査させつつ、一定の間隔（例えば８００μｍ）ごとにレーザ光を照射して、ｘ軸方向に並んだ複数の窪み部１７を形成する。同様に、照射部をｙ軸方向に一定の距離（例えば４００μｍ）だけ走査させる。これを繰り返して、底板部１１の上面に規則的に配列された複数の窪み部１７を形成する。 First, when the plane direction of the bottom plate portion 11 is the xy axis, the laser beam is irradiated at regular intervals (for example, 800 μm) while scanning the irradiation unit of the laser irradiation device in the positive direction of the x axis, A plurality of depressions 17 arranged in the x-axis direction are formed. Subsequently, after the irradiation unit is scanned by a certain distance (for example, 400 μm) in the y-axis direction, the laser beam is irradiated at regular intervals (for example, 800 μm) while the irradiation unit is scanned in the negative direction of the x-axis. Thus, a plurality of depressions 17 arranged in the x-axis direction are formed. Similarly, the irradiation unit is scanned by a certain distance (for example, 400 μm) in the y-axis direction. By repeating this, a plurality of depressions 17 regularly arranged on the upper surface of the bottom plate part 11 are formed.
窪み部１７は、培養面１５の単位面積あたり、１０個／ｃｍ２〜１００００個／ｃｍ２、形成するのが好ましい。さらに好ましくは、２０個／ｃｍ２〜８０００個／ｃｍ２、さらに好ましくは、２０個／ｃｍ２〜３０００個／ｃｍ２である
上記した数値範囲を示す「〜」とは、特段の定めがない限り、その前後に記載された数値を下限値及び上限値として含む意味で使用され、以下本明細書において「〜」は、同様の意味をもって使用される。
The depressions 17 are preferably formed at 10 pieces / cm 2 to 10,000 pieces / cm 2 per unit area of the culture surface 15. More preferably, it is 20 pieces / cm 2 to 8000 pieces / cm 2 , more preferably 20 pieces / cm 2 to 3000 pieces / cm 2. As long as it is used, the numerical values described before and after it are used as the lower limit value and the upper limit value. In the following description, “˜” is used with the same meaning.
本実施形態では、レーザ光源には、ＣＯ２レーザを用い、レーザ光は、出力１０Ｗ、照射速度６１００ｍｍ／ｍｉｎでパルス照射される。照射スポットの形状は、円形であり、その直径は、約４００μｍである。スフェロイドは、小さすぎると所望の生理機能が生じず、また、大きすぎるとスフェロイドの中心部が壊死してしまう。これを考慮すると、照射スポットの直径は、２０μｍ〜１５００μｍが適当である。 In the present embodiment, a CO 2 laser is used as the laser light source, and the laser beam is pulse-irradiated at an output of 10 W and an irradiation speed of 6100 mm / min. The shape of the irradiation spot is circular and its diameter is about 400 μm. If the spheroid is too small, a desired physiological function does not occur, and if it is too large, the center part of the spheroid is necrotized. Considering this, the appropriate diameter of the irradiation spot is 20 μm to 1500 μm.
培養面１５（底板部１１の上面）にレーザ光が照射されると、底板部１１を構成する合成樹脂材が溶解して、窪み部１７が形成される。さらに、窪み部１７の開口周辺には、溶解した合成樹脂材が盛り上がって、土手部が形成されてもよい。互いに隣り合った２個の窪み部１７は、１個または複数個の土手部を介して形成されており、互いに近接する窪み部１７間の頂部１８には、平坦面が残らない。すなわち、互いに近接する窪み部１７間の培養面が非平坦面になっている。 When the laser beam is applied to the culture surface 15 (the upper surface of the bottom plate portion 11), the synthetic resin material constituting the bottom plate portion 11 is dissolved, and the recess portion 17 is formed. Further, a melted synthetic resin material may be formed around the opening of the recess portion 17 to form a bank portion. The two depressions 17 adjacent to each other are formed via one or a plurality of bank parts, and a flat surface does not remain at the top 18 between the depressions 17 adjacent to each other. That is, the culture surface between the depressions 17 adjacent to each other is a non-flat surface.
レーザ光の照射位置や出力量などの照射条件を調節することにより、近接する窪み部１７間の距離、窪み部１７の径・深さ、土手部の幅・高さなどを調節できる。本実施形態では、互いに近接する窪み部１７間の培養基材表面に平坦面が残らないように、すなわち、互いに近接する窪み部１７間の頂部１８が非平坦面になるように、レーザ光の照射条件が設定されて、レーザ照射が行われる。 By adjusting the irradiation conditions such as the irradiation position and output amount of the laser beam, the distance between the adjacent depressions 17, the diameter / depth of the depressions 17, the width / height of the bank, etc. can be adjusted. In the present embodiment, the laser light is emitted so that no flat surface remains on the surface of the culture substrate between the recesses 17 adjacent to each other, that is, the top 18 between the recesses 17 adjacent to each other is a non-flat surface. Irradiation conditions are set, and laser irradiation is performed.
なお、窪み部１７の深さ（すなわち、レーザ照射前の底板部１１）の上面を基準とした深さ）ｄは、１０μｍ〜１５００μｍに設計されることが望ましく、本実施形態では、２００μｍ±２０μｍに設計されている。なお、底板部１１の厚さは、深さｄに応じて貫通しないよう適宜設計される。また、略楕円形の窪み部１７の開口の長径（レーザ照射前の底板部１４の上面での長径）Ｄは、１０μｍ〜１５００μｍに設計されることが望ましく、本実施形態では、５００μｍ±２０μｍに設計されている。さらに、土手部の高さ（すなわち、図４、５に示したように、レーザ照射前の底板部１４の上面を基準とした高さ）ｈは、１０μｍ〜５０μｍに設計されることが望ましく、本実施形態では、２５μｍ±５μｍに設計されている。 In addition, it is desirable that the depth d (that is, the depth based on the upper surface of the bottom plate portion 11 before laser irradiation) d is designed to be 10 μm to 1500 μm, and in this embodiment, 200 μm ± 20 μm. Designed to. In addition, the thickness of the bottom plate part 11 is appropriately designed so as not to penetrate depending on the depth d. Further, the major axis (major axis on the upper surface of the bottom plate part 14 before laser irradiation) D of the substantially elliptical hollow part 17 is desirably designed to be 10 μm to 1500 μm. In this embodiment, the major axis is 500 μm ± 20 μm. Designed. Furthermore, the height of the bank portion (that is, the height based on the upper surface of the bottom plate portion 14 before laser irradiation as shown in FIGS. 4 and 5) h is preferably designed to be 10 μm to 50 μm, In this embodiment, it is designed to be 25 μm ± 5 μm.
底板部１１の上面、すなわち培養面は、細胞接着抑制剤（図示省略）により被膜されていることが好ましい。細胞接着抑制剤は、細胞が底板部１１の上面、特に、窪み部１７の内面に接着するのを抑制する役割を果たす。細胞接着抑制剤としては、例えば、リン脂質ポリマー、ポリヒドロキシエチルメタアクリレート、あるいは、ポリエチレングリコールなどが用いられる。 The upper surface of the bottom plate portion 11, that is, the culture surface is preferably coated with a cell adhesion inhibitor (not shown). The cell adhesion inhibitor plays a role of inhibiting the cells from adhering to the upper surface of the bottom plate portion 11, particularly the inner surface of the recess portion 17. As the cell adhesion inhibitor, for example, phospholipid polymer, polyhydroxyethyl methacrylate, polyethylene glycol or the like is used.
次に、本実施形態に係る細胞培養容器１００を用いた被培養物５０の培養方法について説明する。 Next, a method for culturing the culture object 50 using the cell culture container 100 according to the present embodiment will be described.
被培養物であるスフェロイド前駆体としての細胞を培養液１６に入れて、撹拌する。撹拌後、培養液１６を液面が窪み部１７の頂部１８より上となるように容器本体１０内に流し入れる。そうすると、培養液１６中の細胞は、沈殿して、窪み部１７内に収まる。 Cells to be cultured as spheroid precursors are placed in the culture medium 16 and stirred. After stirring, the culture solution 16 is poured into the container body 10 so that the liquid level is above the top 18 of the recess 17. Then, the cells in the culture solution 16 are settled and fit in the recess 17.
その後、容器本体１０に蓋４０を被せて、数日〜数十日間放置する。窪み部１７内の細胞は、培養され、増殖する。このとき、窪み部１７の内面が細胞接着抑制剤により被膜されているため、細胞は、窪み部１７の形状・大きさに対応して、三次元的に凝集する。こうして、スフェロイドが得られる。 Thereafter, the container body 10 is covered with a lid 40 and left for several days to several tens of days. The cells in the depression 17 are cultured and proliferated. At this time, since the inner surface of the depression 17 is coated with the cell adhesion inhibitor, the cells aggregate three-dimensionally according to the shape and size of the depression 17. Thus, a spheroid is obtained.
本実施形態に係る細胞培養容器では、隣接する窪み部１７間の頂部１８は、非平坦面になっている。そのため、単層培養されたり、不均一なスフェロイドが形成されにくく、均一なスフェロイドが形成される確率が高く、スフェロイド培養を効率良く行うことができる。 In the cell culture container according to the present embodiment, the top portion 18 between the adjacent recess portions 17 is a non-flat surface. Therefore, it is difficult to form a monolayer culture or heterogeneous spheroids, the probability that uniform spheroids are formed is high, and spheroid culture can be performed efficiently.
スフェロイドの用途や培養する細胞の種類などに応じて、作製すべきスフェロイドの大きさはそれぞれ異なる。そのため、スフェロイドの作製にあたっては、所望するスフェロイドの大きさに対応した窪み部１７を備えた培養基材１を用意する必要がある。ここで、本実施形態では、レーザ照射により窪み部１７を形成する。したがって、照射位置や出力量などの照射条件を調節することによって、培養面１５に任意の大きさの窪み部１７を容易に形成することができる。 Depending on the use of the spheroid and the type of cells to be cultured, the size of the spheroid to be produced varies. Therefore, when producing a spheroid, it is necessary to prepare the culture base material 1 provided with the hollow part 17 corresponding to the magnitude | size of the desired spheroid. Here, in this embodiment, the hollow part 17 is formed by laser irradiation. Therefore, by adjusting the irradiation conditions such as the irradiation position and the output amount, it is possible to easily form the recessed portion 17 having an arbitrary size on the culture surface 15.
また、容器本体１０として、ポリスチレンなどの透明な合成樹脂材を使用し、この合成樹脂材に対して、レーザ光照射加工により窪み部を形成した場合、窪み部１７の断面形状は上開きとなり、かつ、レーザ光の熱により窪み部１７の内面が滑らかになって、透過光が乱反射するのを低減することができ、窪み部１７内で培養されるスフェロイドの顕微鏡による観察を、容易に行うことができる。 Further, when a transparent synthetic resin material such as polystyrene is used as the container body 10 and a hollow portion is formed by laser light irradiation processing on this synthetic resin material, the cross-sectional shape of the hollow portion 17 becomes an open top, In addition, the inner surface of the recess portion 17 is smoothed by the heat of the laser beam, and it is possible to reduce the irregular reflection of the transmitted light, and the observation of the spheroid cultured in the recess portion 17 with a microscope can be easily performed. Can do.
次に、本実施形態に係る細胞培養容器１００における透液性蓋体２０の使用方法について説明する。
細胞培養容器１００内の被培養物５０の観察や培養液１６の交換の際に、細胞培養容器１００を移動することがある。その際、細胞培養容器１００を移動する前に、蓋４０を細胞培養容器１００から取り外し、事前に滅菌処理された透液性蓋体２０を静かに培養液１６に浸漬する。それにより、透液性蓋体２０は、窪み部１７の頂部１８の上方に載置される。
次いで、事前に滅菌処理された保持具３０を用いて、透液性蓋体２０を窪み部１７の方向に付勢し、その状態を維持するように容器本体の側壁部１２に他端を挟み込むようにして固定する。
Next, the usage method of the liquid-permeable cover body 20 in the cell culture container 100 which concerns on this embodiment is demonstrated.
When observing the culture object 50 in the cell culture container 100 or exchanging the culture solution 16, the cell culture container 100 may be moved. In that case, before moving the cell culture container 100, the lid | cover 40 is removed from the cell culture container 100, and the liquid-permeable cover body 20 sterilized previously is gently immersed in the culture solution 16. FIG. Thereby, the liquid permeable lid 20 is placed above the top 18 of the recess 17.
Next, using the holder 30 that has been sterilized in advance, the liquid-permeable lid 20 is urged in the direction of the recess 17 and the other end is sandwiched between the side walls 12 of the container body so as to maintain the state. To fix.
これにより、窪み部１７の頂部１８と透液性蓋体２０との距離を、窪み部１７内の被培養物５０の外径寸法よりも小さくする。
このようにした上で容器本体１０を移動することで、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から隣接する他の窪み部１９に移動することを抑制できる。
As a result, the distance between the top 18 of the recess 17 and the liquid-permeable lid 20 is made smaller than the outer diameter of the culture object 50 in the recess 17.
By moving the container main body 10 after doing in this way, the container main body 10 swings greatly, and the to-be-cultured object 50 in the hollow part 17 moves to the other hollow part 19 adjacent from the opening of the hollow part 17. This can be suppressed.
［第２の実施形態］
本発明の第２の実施形態に係る細胞培養容器２００について、図４および図５を用いて説明する。図４は、細胞培養容器２００の縦断面図である。図５は、細胞培養容器２００の平面図である。なお、本実施形態は、第１の実施形態の変形例であって、第１の実施形態と同一部分または類似部分には、同一符号を付して、重複説明を省略する。
[Second Embodiment]
A cell culture container 200 according to a second embodiment of the present invention will be described with reference to FIGS. 4 and 5. FIG. 4 is a longitudinal sectional view of the cell culture container 200. FIG. 5 is a plan view of the cell culture container 200. In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.
第１の実施形態では、透液性蓋体２０は、透液性シート２１からなる。一方、本実施形態では、透液性蓋体２０は、透液性シート２１および枠体２２からなる。枠体２２は、円環状の部材であり、透液性シート２１の外周を保持するものである。枠体２２を用いることで、自重により撓みやすいもの、例えば樹脂製フィルタや紙製フィルタなどの安価なものを透液性シート２１として使用することができる。これは、枠体２２によって透液性シート２１の外周を保持するため、透液性シート２１が柔軟であっても使用時に撓むことなく平坦性がよい状態で用いることができるためである。
枠体２２は、保持具３０に固定されていても、固定されていなくてもよい。また、枠体２２は保持具３０と一体成形されていてもよい。
枠体２２による透液性シート２１の保持方法は、接着剤を用いた接着、熱融着等の適宜方法を用いることができる。
In the first embodiment, the liquid-permeable lid 20 includes a liquid-permeable sheet 21. On the other hand, in the present embodiment, the liquid-permeable lid 20 includes a liquid-permeable sheet 21 and a frame body 22. The frame 22 is an annular member and holds the outer periphery of the liquid-permeable sheet 21. By using the frame body 22, an inexpensive material such as a resin filter or a paper filter that is easily bent due to its own weight can be used as the liquid-permeable sheet 21. This is because the outer periphery of the liquid-permeable sheet 21 is held by the frame body 22, so that even if the liquid-permeable sheet 21 is flexible, it can be used in a state of good flatness without being bent during use.
The frame body 22 may or may not be fixed to the holder 30. Further, the frame body 22 may be integrally formed with the holder 30.
As a method for holding the liquid-permeable sheet 21 by the frame body 22, an appropriate method such as adhesion using an adhesive or heat fusion can be used.
本実施形態に係る細胞培養容器２００における透液性蓋体２０の使用方法については、第１の実施形態と同様である。
細胞培養容器２００を用いることで、容器本体１０を移動する際に、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から隣接する他の窪み部１７に移動することを抑制できる。
About the usage method of the liquid-permeable cover body 20 in the cell culture container 200 which concerns on this embodiment, it is the same as that of 1st Embodiment.
By using the cell culture container 200, when moving the container body 10, the container body 10 swings greatly, and the culture object 50 in the recess part 17 is adjacent to the other recess part 17 from the opening of the recess part 17. It can suppress moving to.
［第３の実施形態］
本発明の第３の実施形態に係る細胞培養容器３００について、図６および図７を用いて説明する。図６は、細胞培養容器３００の縦断面図である。図７は、細胞培養容器３００の平面図である。なお、本実施形態は、第１の実施形態の変形例であって、第１の実施形態と同一部分または類似部分には、同一符号を付して、重複説明を省略する。
[Third Embodiment]
A cell culture container 300 according to a third embodiment of the present invention will be described with reference to FIGS. 6 and 7. FIG. 6 is a longitudinal sectional view of the cell culture container 300. FIG. 7 is a plan view of the cell culture container 300. In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.
第１の実施形態では、透液性蓋体２０は、保持具３０を用いて容器本体１０の窪み部１７側に付勢された状態で保持される。一方、本実施形態では、保持具３０を用いず、透液性蓋体２０は、その外周の少なくとも一部が容器本体１０の内側面に圧接されることで、容器本体１０の窪み部１７の頂部１８と透液性蓋体２０との距離を一定以下とした状態で保持される。
透液性蓋体２０は、第１の実施形態に記載されたものと同様のものを用いることができる。そして、外周の少なくとも一部を曲げ、曲げた先端部が容器本体１０の内側面に圧接される。なお、透液性蓋体２０の外周の曲げる程度は、容器本体１０の内径寸法等によって、適宜調整する。また、なお、透液性蓋体２０は、外周の一部のみが曲げられていてもよいし、全周が曲げられていてもよい。
In the first embodiment, the liquid-permeable lid 20 is held in a state where it is urged toward the recessed portion 17 side of the container body 10 using the holder 30. On the other hand, in this embodiment, the holder 30 is not used, and the liquid-permeable lid 20 has at least a part of the outer periphery thereof pressed against the inner side surface of the container body 10, thereby The distance between the top 18 and the liquid-permeable lid 20 is maintained in a state where the distance is constant or less.
The liquid-permeable lid 20 can be the same as that described in the first embodiment. Then, at least a part of the outer periphery is bent, and the bent tip is pressed against the inner surface of the container body 10. The degree of bending of the outer periphery of the liquid-permeable lid 20 is appropriately adjusted according to the inner diameter dimension of the container body 10 and the like. In addition, as for the liquid-permeable cover body 20, only a part of outer periphery may be bent, and the perimeter may be bent.
次に、本実施形態に係る細胞培養容器３００における透液性蓋体２０の使用方法について説明する。
細胞培養容器３００内の被培養物５０の観察や培養液１６の交換の際に、細胞培養容器３００を移動することがある。その際、細胞培養容器３００を移動する前に、蓋４０を細胞培養容器３００から取り外す。
そして、透液性蓋体２０を培養液１６に浸漬する。その際、透液性蓋体２０の外周先端部を容器本体１０の内側面に圧接した状態で、容器本体１０の下方に押し下げる。
このようにすることで、容器本体１０の窪み部１７の頂部１８と透液性蓋体２０との距離を一定以下とした状態で、透液性蓋体２０は保持される。
Next, the usage method of the liquid-permeable cover body 20 in the cell culture container 300 which concerns on this embodiment is demonstrated.
When observing the culture object 50 in the cell culture container 300 or exchanging the culture solution 16, the cell culture container 300 may be moved. At that time, the lid 40 is removed from the cell culture container 300 before the cell culture container 300 is moved.
Then, the permeable lid 20 is immersed in the culture solution 16. At this time, the outer peripheral tip of the liquid-permeable lid 20 is pressed down to the lower side of the container body 10 while being pressed against the inner surface of the container body 10.
By doing in this way, the liquid-permeable cover body 20 is hold | maintained in the state which made the distance of the top part 18 of the hollow part 17 of the container main body 10 and the liquid-permeable cover body 20 or less constant.
これにより、窪み部１７の頂部１８と透液性蓋体２０との距離を、窪み部１７内の被培養物５０の外径寸法よりも小さくする。
このようにした上で容器本体１０を移動することで、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から隣接する他の窪み部１９に移動することを抑制できる。
As a result, the distance between the top 18 of the recess 17 and the liquid-permeable lid 20 is made smaller than the outer diameter of the culture object 50 in the recess 17.
By moving the container main body 10 after doing in this way, the container main body 10 swings greatly, and the to-be-cultured object 50 in the hollow part 17 moves to the other hollow part 19 adjacent from the opening of the hollow part 17. This can be suppressed.
［第４の実施形態］
本発明の第４の実施形態に係る細胞培養容器４００について、図８および図９を用いて説明する。図８は、細胞培養容器４００の縦断面図である。図９は、細胞培養容器４００の平面図である。なお、本実施形態は、第３の実施形態の変形例であって、第３の実施形態と同一部分または類似部分には、同一符号を付して、重複説明を省略する。
[Fourth Embodiment]
A cell culture container 400 according to a fourth embodiment of the present invention will be described with reference to FIGS. 8 and 9. FIG. 8 is a longitudinal sectional view of the cell culture container 400. FIG. 9 is a plan view of the cell culture container 400. In addition, this embodiment is a modification of 3rd Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 3rd Embodiment, and duplication description is abbreviate | omitted.
第３の実施形態では、透液性蓋体２０は、透液性シート２１からなる。一方、本実施形態では、透液性蓋体２０は、透液性シート２１および枠体２２からなる。枠体２２は、円環状の部材であり、透液性シート２１の外周を保持するものである。枠体２２を用いることで、自重により撓みやすいもの、例えば樹脂製フィルタや紙製フィルタなどの安価なものを透液性シート２１として使用することができる。これは、枠体２２によって透液性シート２１の外周を保持するため、透液性シート２１が柔軟であっても使用時に撓むことなく平坦性がよい状態で用いることができるためである。 In the third embodiment, the liquid-permeable lid 20 includes a liquid-permeable sheet 21. On the other hand, in the present embodiment, the liquid-permeable lid 20 includes a liquid-permeable sheet 21 and a frame body 22. The frame 22 is an annular member and holds the outer periphery of the liquid-permeable sheet 21. By using the frame body 22, an inexpensive material such as a resin filter or a paper filter that is easily bent due to its own weight can be used as the liquid-permeable sheet 21. This is because the outer periphery of the liquid-permeable sheet 21 is held by the frame body 22, so that even if the liquid-permeable sheet 21 is flexible, it can be used in a state of good flatness without being bent during use.
また、本実施形態では、第３の実施形態と同様に、保持具３０を用いず、透液性蓋体２０は、その外周の少なくとも一部が容器本体１０の内側面に圧接されることで、容器本体１０の窪み部１７の頂部１８と透液性蓋体２０との距離を一定以下とした状態で保持される。
透液性蓋体２０は、第２の実施形態と同様に透液性シート２１と枠体２２によって構成される。ただし、本実施形態では、枠体２２もよって透液性蓋体２０を容器本体１０の内側面に圧接して保持するため、枠体２２は外周の少なくとも一部が曲げられているか、曲った状態で成形されることが好ましい。なお、枠体２２の外周の曲げる程度は、容器本体１０の内径寸法によって、適宜調整する。また、枠体２２に硬質ゴム等の弾性体を用いれば、枠体２２の外周を曲げることなく、透液性蓋体２０を容器本体１０の内側面に圧接することができる。
Further, in the present embodiment, as in the third embodiment, the holder 30 is not used, and the liquid-permeable lid 20 is in pressure contact with the inner surface of the container body 10 at least at a part of the outer periphery thereof. The distance between the top portion 18 of the hollow portion 17 of the container body 10 and the liquid-permeable lid 20 is held in a state where the distance is constant or less.
The liquid-permeable cover 20 is comprised by the liquid-permeable sheet 21 and the frame 22 similarly to 2nd Embodiment. However, in this embodiment, since the frame body 22 also holds the liquid-permeable lid 20 in pressure contact with the inner surface of the container body 10, the frame body 22 is bent or bent at least partly on the outer periphery. It is preferable to be molded in a state. The degree of bending of the outer periphery of the frame body 22 is appropriately adjusted according to the inner diameter dimension of the container body 10. If an elastic body such as hard rubber is used for the frame body 22, the liquid-permeable lid 20 can be pressed against the inner surface of the container body 10 without bending the outer periphery of the frame body 22.
本実施形態に係る細胞培養容器４００における透液性蓋体２０の使用方法については、第３の実施形態と同様である。
細胞培養容器４００を用いることで、容器本体１０を移動する際に、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から隣接する他の窪み部１９に移動することを抑制できる。
About the usage method of the liquid-permeable cover body 20 in the cell culture container 400 which concerns on this embodiment, it is the same as that of 3rd Embodiment.
By using the cell culture container 400, when moving the container body 10, the container body 10 swings greatly, and the object to be cultured 50 in the recess part 17 is adjacent to the other recess part 19 from the opening of the recess part 17. It can suppress moving to.
［第５の実施形態］
本発明の第５の実施形態に係る細胞培養容器５００について、図１０および図１１を用いて説明する。図１０は、細胞培養容器５００の縦断面図である。図１１は、細胞培養容器５００の切断平面図（図１０に記載のｘ−ｘラインで切断した際の平面図）である。なお、本実施形態は、第１の実施形態の変形例であって、第１の実施形態と同一部分または類似部分には、同一符号を付して、重複説明を省略する。
[Fifth Embodiment]
A cell culture container 500 according to a fifth embodiment of the present invention will be described with reference to FIGS. 10 and 11. FIG. 10 is a longitudinal sectional view of the cell culture container 500. FIG. 11 is a cut plan view of the cell culture container 500 (plan view when cut along the xx line shown in FIG. 10). In addition, this embodiment is a modification of 1st Embodiment, Comprising: The same code | symbol is attached | subjected to the same part or similar part as 1st Embodiment, and duplication description is abbreviate | omitted.
第１の実施形態では、透液性蓋体２０は、保持具３０を用いて容器本体１０の窪み部１７側に付勢された状態で保持される。一方、本実施形態では、保持具３０の代わりに、蓋４０の裏面に設けた押さえ具４５により、透液性蓋体２０を容器本体１０の窪み部１７側に付勢された状態で保持する。
蓋４０の裏面に設けた押さえ具４５は、透液性蓋体２０を上方から押さえることができれば、どのような形状であってもよい。例えば、押さえ具４５は、透液性蓋体２０の外周と寸法が略同一の筒状体や透液性蓋体２０の外周に沿うように配置した複数の棒状体や板状体であってもよい。
In the first embodiment, the liquid-permeable lid 20 is held in a state where it is urged toward the recessed portion 17 side of the container body 10 using the holder 30. On the other hand, in this embodiment, instead of the holder 30, the liquid-permeable lid 20 is held in a state of being urged toward the recessed portion 17 side of the container body 10 by a presser 45 provided on the back surface of the lid 40. .
The presser 45 provided on the back surface of the lid 40 may have any shape as long as the liquid-permeable lid 20 can be pressed from above. For example, the pressing tool 45 is a plurality of rod-like bodies or plate-like bodies arranged along the outer circumference of the cylindrical body or the liquid-permeable lid body 20 whose dimensions are substantially the same as the outer circumference of the liquid-permeable lid body 20. Also good.
本実施形態に係る細胞培養容器５００における透液性蓋体２０の使用方法については、透液性蓋体２０を保持具３０に替えて押さえ具４５によって保持することを除いて、第１の実施形態と同様である。
細胞培養容器５００を用いることで、容器本体１０を移動する際に、容器本体１０が大きく搖動して、窪み部１７内の被培養物５０が窪み部１７の開口から隣接する他の窪み部１９に移動することを抑制できる。
About the usage method of the liquid-permeable cover body 20 in the cell culture container 500 which concerns on this embodiment, it replaces the liquid-permeable cover body 20 with the holder 30, and hold | maintains with the pressing tool 45 1st implementation. It is the same as the form.
By using the cell culture container 500, when moving the container body 10, the container body 10 swings greatly, and the culture object 50 in the recess part 17 is adjacent to the other recess part 19 from the opening of the recess part 17. It can suppress moving to.
［他の実施形態］
上記の各実施形態に係る細胞培養容器は、代表的な例示であって、本発明はこれらに限定されるものではない。例えば、透液性蓋体２０を金属製メッシュのような培養液１６に浸漬した際に浮遊しない素材、すなわち透液性蓋体２０の自重により培養面１５上に載置されるもので構成してもよい。これにより、透液性蓋体２０を容器本体１０の内側面へ圧接したり保持具３０を用いる等の透液性蓋体２０の保持手段を用いることなく、透液性蓋体２０を適切な位置に配置することができる。
また、上記の各実施形態に係る細胞培養容器の使用方法の説明において、透液性蓋体２０は細胞培養容器の移動の前に容器本体１０に載置するとしたが、常時培養液１６中に浸漬した状態としてもよい。この場合、透液性蓋体２０がメッシュ状であれば、透明な蓋体４０を付けた状態であっても窪み部１７内で培養されるスフェロイドの顕微鏡による観察が可能である。
[Other Embodiments]
The cell culture container according to each of the above embodiments is a representative example, and the present invention is not limited to these. For example, the liquid-permeable lid 20 is made of a material that does not float when immersed in the culture solution 16 such as a metal mesh, that is, a material that is placed on the culture surface 15 by its own weight. May be. Accordingly, the liquid-permeable lid 20 can be appropriately attached without using the holding means of the liquid-permeable lid 20 such as pressing the liquid-permeable lid 20 against the inner surface of the container body 10 or using the holder 30. Can be placed in position.
In the description of the method for using the cell culture container according to each of the embodiments described above, the liquid-permeable lid 20 is placed on the container body 10 before the cell culture container is moved. It is good also as an immersed state. In this case, if the liquid-permeable lid 20 is in a mesh shape, it is possible to observe the spheroids cultured in the recess 17 with a microscope even when the transparent lid 40 is attached.
本発明の細胞培養容器は、所望の大きさのスフェロイドを均一に形成でき、培養を効率的に行うことが可能である。 The cell culture container of the present invention can uniformly form spheroids of a desired size, and can be cultured efficiently.
１０…容器本体、１１…容器本体の底板部、１２…容器本体の側壁部、１５…培養面、１６…培養液、１７，１９…窪み部、１８…頂部、２０…透液性蓋体、２１…透液性シート、２２…枠体、３０…保持具、４０…蓋、４５…押さえ具、５０…被培養物、１００，２００，３００，４００，５００…細胞培養容器。
DESCRIPTION OF SYMBOLS 10 ... Container main body, 11 ... Bottom plate part of container main body, 12 ... Side wall part of container main body, 15 ... Culture surface, 16 ... Culture solution, 17, 19 ... Depression part, 18 ... Top part, 20 ... Liquid-permeable lid, DESCRIPTION OF SYMBOLS 21 ... Liquid-permeable sheet, 22 ... Frame body, 30 ... Holder, 40 ... Lid, 45 ... Holding tool, 50 ... Culture object, 100, 200, 300, 400, 500 ... Cell culture container.
Claims (13)
前記培養面を底面に備える容器本体と、
複数の窪み部の上部に載置され前記窪み部の開口を塞ぐ透液性蓋体とを備え、
前記培養面は、互いに近接する前記窪み部の間の頂部が非平坦面からなり、
前記透液性蓋体は、前記容器本体内の培養液中に浸漬された状態で前記窪み部の間の頂部との距離が、前記窪み部内で培養された前記被培養物の外径寸法よりも小さくなるよう配置されていることを特徴とする細胞培養容器。 A culture surface on which a plurality of depressions forming a compartment in which the culture object is cultured are formed;
A container body provided with the culture surface on the bottom surface;
A liquid-permeable lid that is placed on top of the plurality of depressions and closes the opening of the depressions;
The culture surface comprises a non-flat surface at the top between the recesses adjacent to each other,
The liquid permeable lid is immersed in the culture medium in the container main body, and the distance from the top between the recesses is larger than the outer diameter of the culture object cultured in the recesses. A cell culture vessel characterized by being arranged to be smaller.
The cell culture container according to any one of claims 1 to 12, wherein a material of the container body is made of a synthetic resin.
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