WO2019131673A1

WO2019131673A1 - Cell culture vessel

Info

Publication number: WO2019131673A1
Application number: PCT/JP2018/047648
Authority: WO
Inventors: 晃輔堀; 紀之河原
Original assignee: 株式会社幹細胞＆デバイス研究所
Priority date: 2017-12-26
Filing date: 2018-12-25
Publication date: 2019-07-04
Also published as: JPWO2019131673A1; JP7278523B2

Abstract

[Problem] To provide a cell culture vessel having a pre-prepared cell retaining region. [Solution] A protruding section 105 of each well 100 in a multiwell plate W1 is formed as an island shape protruding from a bottom surface section 101 toward the inner space S100 side. A cell retaining space 107 is formed as a hollow space in the form of a hollow cylinder from the top surface P105u of the protruding section 105 to the bottom surface section 101. In other words, the cell retaining space 107 is formed as a recess from the top surface P105u of the protruding section 105 to the bottom surface section 101. Thus, in the multiwell plate W1, the cell retaining space 107 is formed in a predetermined position in each well 100. As a result, it is possible to fix the site where cultured cells are retained when cells are cultured in the wells 100. In addition, since a cell retaining space 107 with a predetermined capacity is provided, it is possible to avoid differences in the amount of cultured cells in the wells 100.

Description

Cell culture vessel

The present invention relates to a cell culture vessel, and in particular to one having a cell holding area in advance.

A conventional cell culture vessel will be described using a plastic vessel 10. As shown in FIG. 7, the plastic container 10 is a container for culturing living cells such as living cells and tissues, and is a container for imaging (or observing) the cultured living cells with a phase contrast microscope. is there. Further, the plastic container 10 is a so-called multi-well plate, and has a plurality of circularly opened wells 11. The well 11 is a recess for containing living cells to be imaged after culture and culture and a culture solution for culturing the living cells. Although FIG. 7 shows a 96-well plate having 96 wells 11 in 8 rows and 12 columns as an example, the plastic container 10 may have at least two wells 11. The number of is arbitrary. In addition to the 96-well plate, the wells 11 are often 6, 24, or 384 multi-well plates.

Suitable materials for the plastic container 10 are, for example, polyethylene terephthalate (PET), polystyrene (PS), polycarbonate (PC), TAC (triacetyl cellulose), polyimide (PI), nylon (Ny), low density polyethylene (LDPE) And medium density polyethylene (MDPE), vinyl chloride, vinylidene chloride, polyphenylene sulfide, polyether sulfone, polyethylene naphthalate, polypropylene, acrylic materials such as urethane acrylate, cellulose, glass and the like. Further, resins such as biodegradable polymers such as polylactic acid, polyglycolic acid, polycaprolactone or copolymers thereof can be used. Among these, polyethylene terephthalate, polystyrene and polycarbonate can be preferably used, and polystyrene can be particularly preferably used. It is because cytotoxicity is low. In addition, the surface of the plastic container 10 may be subjected to any surface treatment (for example, irradiation with plasma, corona, microwave, electron beam, ultraviolet light, etc.).

As shown in FIG. 8, the well 11 is a non-through hole and is opened on the surface of the plastic container 10. The living cells 13 to be cultured and the culture solution 12 (for example, serum solution) for culturing the living cells 13 are injected into the wells 11 from the opening. When the plastic container 10 is used, since the well 11 is open, the culture solution 12 injected into the well 11 is always in the vicinity of the opening of the well 11 during the culture of the living cells 13 and during imaging with a phase contrast microscope. Contact the air. For this reason, the cultured living cells 13 are imaged (observed) by a phase contrast microscope while being alive.

In the plastic container 10, the well 11 has an opening diameter R of 0.5 mm or more and less than 2 cm, and a depth D of 2 mm or more and less than 2 cm. The diameter R of the opening of the well 11 is preferably 0.5 mm or more and less than 2 cm because it is preferable to obtain data in which the number of living cells 13 cultured in the same environment is uniformly converged. . The diameter R of the opening of the well 11 is more preferably 1 mm or more. The present invention is particularly useful because when the diameter R of the opening of the well 11 is less than 2 cm, the meniscus of the liquid surface 12a of the culture solution 12 becomes remarkable, and the diameter R of the opening of the well 11 is 1 cm or less It is particularly suitable in the case. For example, in the case of a 96-well plate having 96 wells 11, the diameter of the opening of the well 11 is 6 mm, for example, in the case of a 384-well plate having 384 wells 11, the diameter of the opening of the well 11 is 3 mm is there.

The depth D of the well 11 is the height from the bottom surface 11b of the well 11 to the opening (surface of the plastic container 10), and the depth D of the well 11 is 2 mm or more. This is for securing a sufficient medium for culturing 13 and for securing a sufficient amount of culture solution 12 for culturing living cells 13. The depth D of the well 11 is formed to be less than 2 cm in order to prevent the decrease in the amount of light of the peripheral vision due to vignetting when observed with a transmission type microscope. If the depth D of the well 11 is in the range of 3 mm or more and 1 cm or less, it is particularly suitable for culture and imaging of the living cell 13. Moreover, it is preferable that the quantity of the culture solution 12 put into the well 11 is 1/2 or less of the depth D of the well 11 (as mentioned above, refer patent document 1).

JP, 2016-67322, A

The aforementioned plastic container 10 has the following points to be improved. In the plastic container 10, the living cells 13 are cultured at the bottom of the well 11. The bottom of the well 11 has a relatively large area in view of the cells to be cultured, and it is unknown at which position of the bottom the cell is to be cultured and to what extent. Therefore, even if the conditions for cell culture, the number of cells, the amount of medium, the temperature, and the like are the same, cells are not uniformly cultured at the bottom of the well 11. For this reason, also in the well 11, there is a point to be improved that the result may be different depending on the position of the cell target to be experimented.

The present inventors have found that, as a result of intensive studies aimed at overcoming the problems of the prior art described above, it is possible to provide a cell culture vessel having a cell holding region in advance, and to complete the present invention. It reached.
That is, the object of the present invention is achieved by the following invention.
(1)
A cell culture vessel for culturing cells in a culture medium stationed in a cell culture space formed by a bottom surface and a side, comprising:
A protrusion protruding from the bottom surface to the cell culture space;
A cell holding space formed concavely from the top surface to the bottom surface in the protrusion;
A cell culture vessel having
(2)
In the cell culture vessel according to (1),
The cell culture vessel is
Being a well placed in a multiwell plate,
A cell culture vessel characterized by
(3)
In the cell culture vessel according to (1) or (2),
The body is
It is formed by the upper surface and the inner surface of at least a hollow column shape,
The cell holding space is
Being a hollow space of the hollow column shape;
A cell culture vessel characterized by
(4)
In any of the cell culture vessels according to (1) to (3),
The cell holding space is
It has been subjected to a hydrophilic treatment to facilitate retention of the cells,
A cell culture vessel characterized by
(5)
In the cell culture vessel according to (4),
The cell holding space is
A cell culture vessel having a contact angle of less than 60 ° at least in a region forming the bottom of the cell holding space.
(6)
In any of the cell culture vessels according to (1) to (5),
The cell holding space is
Having the cells to be cultured,
A cell culture vessel characterized by
(7)
In any of the cell culture vessels according to (1) to (6),
The cell culture container which has a contact angle of 60 degrees or more other than the surface which forms the said cell holding | maintenance space.
(8)
In any of the cell culture vessels according to (1) to (7),
A cell culture vessel, wherein a volume of the cell holding space is 30 μL to 0.5 μL.
(9)
In any of the cell culture vessels according to (1) to (8),
A cell culture vessel, wherein a region forming the bottom of the cell holding space is formed of a material having a total light transmittance of 80% or more.
(10)
In the cell culture vessel according to (9),
The cell holding space is
The area forming the bottom is polystyrene (PS), glass, polycarbonate (PC), cycloolefin polymer (COP), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), and A cell culture vessel formed of one or more materials selected from the group consisting of acrylic resin (PMMA).

The means for solving the problems in the present invention and the effects of the invention will be shown below.

The cell culture vessel according to the present invention is a cell culture vessel used for culturing cells in a culture medium stationed in a cell culture space formed by a bottom surface and a side surface, and protrudes from the bottom surface to the cell culture space And the cell holding space formed in a concave shape from the top surface toward the bottom surface.

Thus, the cell holding space is formed at a predetermined position with respect to the cell culture vessel, so that the cells are cultured in the cell culture vessel, and the place where the cultured cells are held can be fixed.

In addition, since the cell holding space having a predetermined capacity is arranged with respect to the cell culture vessel, it is possible to prevent a difference in the amount of cells to be cultured among the cell culture vessels. Thereby, when performing the comparison experiment and the comparison test on cells cultured in each cell culture vessel, the conditions before the experiment in each cell culture vessel can be made the same.

Furthermore, since cells can be cultured in the cell holding space, desired cells can be cultured in three dimensions.

In the cell culture vessel according to the present invention, the cell culture vessel is a well disposed in a multiwell plate.

Thereby, in the multi-well plate, the cell holding space is formed at a predetermined position for each well, so that the cells are cultured in each well, and the place where the cultured cells are held is fixed.

The cell culture vessel according to the present invention is characterized in that the main body is formed by at least an upper surface and an inner side surface of a hollow column shape, and the cell holding space is a hollow space of the hollow column shape.

Thereby, the cell holding area forming device for cell culture containers can be easily generated.

In the cell culture vessel according to the present invention, the cell holding space is characterized in that a surface coating treatment is performed to facilitate holding of the cells.

This allows cells to be easily seeded and held in the cell holding space.

In the cell culture vessel according to the present invention, the cell holding space has the cells to be cultured.

This can provide a cell culture vessel in which cells are seeded at a fixed position in advance.

FIG. 1 is a view showing a multi-well plate W1 which is an example of a cell culture vessel according to the present invention, in which A shows a plan view, B shows a front view, and C shows an X1-X1 cross section of A. It is a figure which shows the well 100 of multi-well plate W1. FIG. 1 is a view showing a multi-well plate W2 which is an example of a cell culture vessel according to the present invention, wherein A shows a plan view, B shows a front view, and C shows an X3-X3 cross section of A. FIG. It is a figure which shows the well 200 of multi-well plate W2. FIG. 1 is a view showing a multi-well plate W3 which is an example of a cell culture vessel according to the present invention, wherein A shows a plan view, B shows a front view, and C shows an X5-X5 cross section of A. FIG. It is a figure which shows the well 300 of multi-well plate W3. It is a figure which shows the conventional cell culture container. It is a figure which shows the conventional cell culture container. It is a figure which shows the shape of cell holding | maintenance space.

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

The cell culture vessel according to the present invention will be described using a multiwell plate W1 having 96 wells as one embodiment. The multiwell plate W1 is a device for culturing cells in a culture medium and performing predetermined experiments and tests on the cultured cells.

First Configuration The configuration of the multi-well plate W1 will be described with reference to FIG. Here, FIG. 1A shows a plan view of the multiwell plate W1, FIG. 1B shows a front view of the multiwell plate W1, and FIG. 1C shows an X1-X1 cross section of FIG. 1A.

As shown in FIG. 1A, the multiwell plate W1 has 96 wells 100. Each well 100 is arranged in a multiwell plate W1 in a matrix of 8 × 12. As shown in FIG. 1B, the well 100 has a bottom surface portion 101 and a side surface portion 103. The bottom surface portion 101 and the side surface portion 103 form a cylindrical internal space S100. A medium or the like for cell culture is stored in the internal space S100.

Also, as shown in FIG. 1C, the well 100 has a protrusion 105 and a cell holding space 107. In FIG. 2, the enlarged view of the circle part A of FIG. 1C is shown. The protrusion 105 is formed in a hollow cylindrical shape (see FIG. 1A). The projecting portion 105 is formed such that the center of the hollow cylindrical shape coincides with the center of the bottom surface portion 101. Further, the protruding portion 105 is formed in an island shape so as to protrude from the bottom surface portion 101 toward the internal space S100 side. The height of the protrusion 105, that is, the height from the top surface P 105 u to the bottom portion 101 is lower than the general medium storage height for cell culture in the inner space S 100. Thus, the cell holding space 107 can be disposed in the medium to culture the cells in the medium.

As shown in FIG. 2, the cell holding space 107 is formed as a hollow cylindrical hollow space from the top surface P 105 u of the protrusion 105 to the bottom surface 101. That is, the cell holding space 107 is formed in a concave shape from the top surface P 105 u of the protrusion 105 toward the bottom surface 101. Thus, in the multiwell plate W1, the cell holding space 107 is formed at a predetermined position with respect to each well 100. For this reason, cells are cultured in the well 100, and the place where the cultured cells are held is fixed.

In addition, since the cell holding space 107 having a predetermined capacity is disposed with respect to the wells 100, it is possible to prevent differences in the amount of cells to be cultured among the wells 100. As a result, when performing comparison experiments and comparison tests on cells cultured in each well 100, the conditions before the experiment in each well 100 can be made the same. Similarly, the conditions before the experiment can be made the same between the multiwell plates W1.

Furthermore, since cells can be cultured in the cell holding space 107, desired cells can be cultured in three dimensions.

Cell Culture Method in Second Multi-Well Plate W1 The cell culture method in the multi-well plate W1 will be described by taking a case where cardiomyocytes are cultured as an example. The user applies a cell retention area generation process to the cell retention space 107 to facilitate retention of the seeded cardiomyocytes. The cell holding area generation process has a hydrophilic process, a washing process, and a surface coating process.

The user first applies hydrophilic treatment to the cell holding space 107. The hydrophilic treatment is performed, for example, by discharging a cow's serum sucked into a pipette into the cell holding space 107, retaining the cow's serum in the cell holding space 107 for a predetermined time, and then suctioning it with an aspirator.

Next, the user performs a washing process on the cell holding space 107 subjected to the hydrophilic process. The washing treatment is performed, for example, by aspirating pure water using a pipette, discharging it into the cell holding space 107, and aspirating using an aspirator.

Furthermore, the user applies a surface coating treatment to the washed cell holding space 107. In the surface coating process, for example, a commercially available surface coating material, such as Geltrex (trademark), is aspirated with a pipette, discharged into the cell holding space 107, and dried for a predetermined time. Drying is carried out at a humidity of 60% or more for 30 minutes to 2 hours.

Then, the user disseminates cardiomyocytes in the cell holding space 107 using a cell suspension containing cardiomyocytes. Thereby, the cell suspension containing cardiomyocytes is accurately placed at a predetermined position of the well 100, specifically, the position where the cell holding space 107 is formed, that is, the cardiomyocytes are seeded and held can do. The user similarly disseminates cardiomyocytes for the required wells 100.

The cell culture vessel according to the present invention will be described using a multiwell plate W2 having 96 wells as one embodiment. In the multi-well plate W1 described above, each well 100 has the projecting portion 105 projecting like an island. On the other hand, the multi-well plate W2 has a projecting portion 205 (described later) having a shape different from that of the above-mentioned projecting portion 105. In the following, the same components as those of the first embodiment are denoted by the same reference numerals, and the detailed description is omitted.

First Configuration The configuration of the multi-well plate W2 will be described with reference to FIG. Here, FIG. 3A shows a plan view of the multiwell plate W2, FIG. 3B shows a front view of the multiwell plate W2, and FIG. 3C shows an X3-X3 cross section of FIG. 3A.

As shown in FIG. 3A, the multiwell plate W2 has 96 wells 200. Each well 200 is arranged in a multiwell plate W2 in a matrix of 8 × 12. As shown in FIG. 3B, the well 200 has a bottom surface portion 101 and a side surface portion 103.

Further, as shown in FIG. 3C, the well 200 has a protrusion 205 and a cell holding space 107. FIG. 4 shows an enlarged view of the circle portion P3 of FIG. 3C. The protrusion 205 is formed in a hollow cylindrical shape (see FIG. 3A). The projecting portion 205 is arranged such that the center of the hollow cylindrical shape coincides with the center of the bottom portion 101. In addition, the projecting portion 205 is formed to project from the bottom surface portion 101 toward the internal space S100 (see FIG. 3). The projecting portion 205 is formed such that the outer surface P 205 s of the projecting portion 205 and the side surface portion 103 are integrated, and the lower surface P 205 b of the projecting portion 205 and the bottom surface portion 101 are integrated. The height of the projecting portion 205, that is, the height from the top surface P205u to the bottom portion 101 is lower than the general medium storage height for cell culture in the internal space S100. Thereby, the cell holding space 107 is disposed in the medium, and the cells can be cultured in the medium.

As shown in FIG. 4, the cell holding space 107 is formed as a hollow cylindrical hollow space from the bottom surface portion 101 to the top surface P 205 u of the projecting portion 205. That is, the cell holding space 107 is formed in a concave shape from the top surface P 205 u of the protrusion 205 toward the bottom surface 101. Thus, in the multiwell plate W2, the cell holding space 107 is formed at a predetermined position with respect to each well 200. For this reason, the place where a cell is hold | maintained in the well 200 is fixed.

In addition, since the cell holding space 107 having a predetermined capacity is arranged with respect to the wells 200, it is possible to prevent differences in the amount of cells to be cultured between the wells 200. Thus, when performing comparison experiments symmetrically on cells cultured in each well 200, the conditions before the experiment in each well 200 can be made the same. Similarly, the conditions before the experiment can be made the same between the multiwell plates W2.

The cell culture vessel according to the present invention will be described using a multiwell plate W3 having 96 wells as one embodiment. In the multi-well plate W2 described above, the lower surface P205b of the protruding portion 205 and the bottom surface portion 101 are integrated such that the outer surface P205s of the protruding portion 205 and the side surface portion 103 are integrated. It had the protrusion part 205 formed in this way. On the other hand, the multi-well plate W3 has a protrusion 305 (described later) having a shape different from that of the protrusion 205 described above. In the following, the same components as those in the first embodiment and the second embodiment are denoted by the same reference numerals, and detailed description will be omitted.

First Configuration The configuration of the multi-well plate W3 will be described with reference to FIG. Here, FIG. 5A shows a plan view of the multiwell plate W3, FIG. 5B shows a front view of the multiwell plate W3, and FIG. 5C shows an X5-X5 cross section of FIG. 5A.

As shown in FIG. 5A, the multiwell plate W3 has 96 wells 300. Each well 300 is arranged in a multiwell plate W3 in a matrix of 8 × 12. As shown in FIG. 5B, the well 300 has a bottom surface portion 301 and a side surface portion 303. The bottom surface portion 301 is formed as a surface that closes one end of the lower portion of the inner side surface P 305 i (described later) of the projecting portion 305.

In addition, as shown in FIG. 5C, the well 300 has a protrusion 305 and a cell holding space 107. FIG. 6 shows an enlarged view of the circle portion P5 of FIG. 5C. The protrusion 305 is formed of a hollow cylindrical upper surface P 305 u and an inner side surface P 305 i (see FIG. 5A). The protruding portion 305 is arranged such that the center of the partial shape of the hollow cylindrical shape formed by the top surface P 305 u and the inner side surface P 305 i coincides with the center of the bottom surface portion 301. The protrusion 305 is formed to protrude from the bottom surface 301 toward the internal space S100. The height of the protrusion 305, that is, the height from the top surface P305u to the bottom portion 301, is lower than the general medium storage height for cell culture in the internal space S100. Thus, the cell holding space 107 can be disposed in the medium to culture the cells in the medium.

As shown in FIG. 6, the cell holding space 107 is formed as a hollow cylindrical hollow space from the bottom surface portion 301 to the upper surface P 305 u of the projecting portion 305. That is, the cell holding space 107 is formed in a concave shape from the top surface P 305 u of the protrusion 305 toward the bottom surface 301. Thus, in the multiwell plate W3, the cell holding space 107 is formed at a predetermined position with respect to each well 300. For this reason, the place where a cell is hold | maintained in the well 300 is fixed.

In addition, since the cell holding space 107 having a predetermined capacity is arranged with respect to the wells 300, it is possible to prevent differences in the amount of cells to be cultured among the wells 300. As a result, when performing comparison experiments on cells cultured in each well 300 symmetrically, the conditions before the experiment in each well 300 can be made the same. Similarly, the conditions before the experiment can be made the same between the multiwell plates W3.

Other Embodiments
(1) Shape of Protrusions: In the above-described first embodiment, the protrusion 105 has a hollow cylindrical shape, but is not limited to the illustrated one as long as it protrudes from the bottom surface 101 toward the internal space S100. . For example, it may be a hollow prism having a prismatic shape. In addition, the cylindrical shape may be removed from the prismatic shape. The same applies to Example 2 and Example 3.

(2) Cell Holding Space 107: In Example 1 described above, the well 100 has one cell holding space 107, but may have a plurality of cell holding spaces. The same applies to Example 2 and Example 3.

(3) Cell to be Measured: In Example 1 described above, cardiomyocytes were used as cells cultured in the medium using the well 100, but the present invention is not limited to the illustrated one. For example, it may be a nerve cell. In addition, it may be neural cells derived from pluripotent stem cells. Examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells) and iPS cells. The same applies to Example 2 and Example 3.

(4) Multi-Well Plate: In Example 1 described above, the multi-well plate W1 having the wells 100 arranged in a matrix of 8 × 12 was used, but if it has a plurality of wells, It is not limited to the illustrated one. For example, it may have wells arranged in a 3 × 4, 4 × 6 matrix. The same applies to Example 2 and Example 3.

(5) Hydrophilic treatment: In the above-mentioned Example 1, although the hydrophilic treatment was performed using bovine serum, it is not limited to the exemplified one as long as at least the cell holding space 107 can be made hydrophilic. For example, physical treatment using plasma treatment, corona discharge treatment, microwave, electron beam or ultraviolet radiation, collagen, fibronectin, vitronectin, laminin, matrigel, gelatin, geltrex, synthetic amino acid chain, poly-L-ornithine, A protein-amino acid-matrix coating treatment using albumin or the like, or a compound treatment using an oxidizing agent, a hydrophilic substance, a hydrogel, a vitrifying agent or the like may be used. The same applies to Example 2 and Example 3.

(6) Washing treatment: In the above-mentioned Example 1, although the washing treatment was performed using pure water, it is not limited to the illustrated one as long as at least the cell holding space 107 can be washed. In addition, if it is not necessary, in the cell holding area generation process, the washing process may not be performed. The same applies to Example 2 and Example 3.

(7) Surface coating treatment: In the above-mentioned Example 1, although the surface coating treatment was performed using geltorex, at least the cell holding space 107 is limited to the exemplified one as long as it can cover the surface. I will not. For example, protein-amino acid-matrix coating treatment using collagen, fibronectin, vitronectin, laminin, matrigel, gelatin, geltrex, synthetic amino acid chain, poly-L-ornithine, albumin or the like may be used. In addition, if it is not necessary, it is not necessary to perform the surface coating process in the cell holding area generation process. The same applies to Example 2 and Example 3.

(8) Contact Angle: The cell suspension stored in the cell holding space 107 does not flow out to the area other than the cell holding space 107 with respect to the faces other than the face forming the cell holding space 107 in Example 1 described above. It is desirable to have a contact angle. Specifically, it is desirable that the surfaces other than the surface forming the cell holding space 107 have a contact angle greater than 60 °. The same applies to the other embodiments.

(9) Size of cell holding space: In Example 1 described above, the size of the cell holding space 107 is the same as the size of the cell discharged once from the pipette used when storing the cell suspension in the cell holding space 107. The size of the suspension may be similar to that of the suspension. Specifically, the volume of the cell holding space 107 is preferably about 30 μL to 0.5 μL.

(10) Member for Forming Cell Holding Space: In Example 1 described above, a region forming the bottom of the cell holding space 107 in the bottom portion 101, that is, a hollow cylinder of at least the protrusion 105 in the bottom portion 101. It is desirable to form the area corresponding to the part by a transparent material. Thus, the operation can be carried out while confirming the state of cell culture in the cell holding space 107 from the outside, particularly from the bottom side. The same applies to the other embodiments. Examples of the transparent material include polystyrene (PS), glass, polycarbonate (PC), cycloolefin polymer (COP), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), acrylic Although resin (PMMA) etc. are mentioned, it does not limit to these.

In the well 200 (see FIG. 4) in the second embodiment, the bottom portion 101 is formed of a transparent material, and the well 200 is formed by bonding with the composite of the side surface portion 103 and the projecting portion 205. May be Alternatively, the well 200 may be formed by forming the bottom surface portion 101 and the projecting portion 205 with a transparent material and bonding the side surface portion 103 whose both ends are open. Similarly, in the well 300 (see FIG. 6) in the third embodiment, the bottom surface portion 301 is formed of a transparent material, and a composite of a hollow cylindrical upper surface P 305 u, an inner side surface P 305 i, and a side surface portion 301 and both ends The well 300 may be formed by bonding with the open side surface portion 103. Alternatively, the well 300 may be formed by forming the bottom surface portion 301 and the projecting portion 305 with a transparent material and bonding the side surface portion 103 whose both ends are open. In addition, it is made to use an adhesive material with low cytotoxicity, such as a silicon adhesive material, for joining of both members.

In addition, it is desirable that the region forming the bottom of the cell holding space in each example have a contact angle of less than 60 ° in order to facilitate cell holding.

(11) Material of Well: In Example 1 described above, the well 100 may be formed of polystyrene, silicon, glass, polycarbonate or the like.

(12) Shape of Cell Holding Space: In Example 1 described above, the protrusion 105 forming the cell holding space 107 is a hollow cylindrical shape, and the cell holding space 107 is a cylindrical shape, but the cell suspension is The cell holding space 107 is not limited to the illustrated one as long as the cell holding space 107 can hold the For example, as shown in FIG. 9, a protrusion 505 having a shape obtained by removing a truncated cone shape from a cylindrical shape may be used. In this case, the side surface P505 can be tapered downward, so that the cell suspension can be stably stored in the inverted truncated conical cell holding space 507. The same applies to the other embodiments.

The cell culture vessel according to the present invention can be used, for example, in a multiwell plate.

W1 multi-well plate 100 well 101 bottom portion 103 side portion 105 protrusion P 105 u top surface 107 cell holding space S 100 internal space W 2 multi-well plate 200 well 205 protrusion P 205 s outer surface P 205 b lower surface P 205 u upper surface W 3 multi-well plate 300 well 301 lower surface portion 303 side surface 305 protrusion P305i inner side P305u upper surface

Claims

A cell culture vessel for culturing cells in a culture medium stationed in a cell culture space formed by a bottom surface and a side, comprising:
A protrusion protruding from the bottom surface to the cell culture space;
A cell holding space formed concavely from the top surface to the bottom surface in the protrusion;
A cell culture vessel having
In the cell culture vessel according to claim 1,
The cell culture vessel is
Being a well placed in a multiwell plate,
A cell culture vessel characterized by
In the cell culture vessel according to claim 1 or 2,
The body is
It is formed by the upper surface and the inner surface of at least a hollow column shape,
The cell holding space is
Being a hollow space of the hollow column shape;
A cell culture vessel characterized by
The cell culture vessel according to any one of claims 1 to 3,
The cell holding space is
It has been subjected to a hydrophilic treatment to facilitate retention of the cells,
A cell culture vessel characterized by
In the cell culture vessel according to claim 4,
The cell holding space is
A cell culture vessel having a contact angle of less than 60 ° at least in a region forming the bottom of the cell holding space.
The cell culture vessel according to any one of claims 1 to 5, wherein
The cell holding space is
Having the cells to be cultured,
A cell culture vessel characterized by
In the cell culture vessel according to any one of claims 1 to 6,
The cell culture container which has a contact angle of 60 degrees or more other than the surface which forms the said cell holding | maintenance space.
The cell culture vessel according to any one of claims 1 to 7, wherein
A cell culture vessel, wherein a volume of the cell holding space is 30 μL to 0.5 μL.
The cell culture vessel according to any one of claims 1 to 8, wherein
A cell culture vessel, wherein a region forming the bottom of the cell holding space is formed of a material having a total light transmittance of 80% or more.
In the cell culture vessel according to claim 9,
The cell holding space is
The area forming the bottom is polystyrene (PS), glass, polycarbonate (PC), cycloolefin polymer (COP), cyclic olefin copolymer (COC), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), and A cell culture vessel formed of one or more materials selected from the group consisting of acrylic resin (PMMA).