Culture container

Abstract

To provide a culture vessel whereby: in optical measurement of cell clusters each formed by fine wells, the cell clusters can be efficiently measured, and reflections of secondary images are reduced.SOLUTION: A culture vessel 1 includes fine wells 20, the fine wells 20 each having an out-of-round shape with r1 / R1 of 0.75-0.95 where R1 and r1 (R1>r1) are, respectively, the radii of concentric two circles, determined by the minimum zone center method of roundness measurement defined by JIS B0621 with respect to the opening shapes of the fine wells 20.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] This invention relates to a culture vessel. [Background technology]

[0002] Cultured cells are widely used in basic research to elucidate life phenomena and in drug discovery research. In particular, three-dimensional cell aggregates (spheroids) obtained by three-dimensional culture have a three-dimensional structure similar to that in vivo, and are therefore expected to have biological activity closer to that in vivo than cells obtained by two-dimensional culture. As a three-dimensional culture method, for example, a culture vessel (microfabricated vessel) is known in which many microwells with a pore size of 100 to 1,000 μm are formed on the bottom surface of each well of a microplate or the bottom surface of a dish (Patent Document 1). When cells are seeded in a microfabricated vessel, the cells associate in each microwell and cell aggregates are formed.

[0003] When measuring biological activity such as cell viability or the activity of intracellular enzymes, reagents that react with cells or enzymes to emit light or fluorescence are used, and the amount of light emitted or fluorescence is measured using a plate reader or cell imager, or their localization is measured. A method of optically measuring cell aggregates cultured in microfabricated containers directly within microwells has the advantage of being able to measure multiple cell aggregates at once. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Patent No. 6400575 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] However, with conventional microfabricated containers such as those described in Patent Document 1, when optically measuring cell aggregates or reaction solutions within microwells, secondary and unwanted images are easily captured, resulting in low measurement accuracy for luminescence and fluorescence, and making localization analysis difficult.

[0006] The present invention aims to provide a culture vessel that enables efficient measurement of multiple cell aggregates formed in each microwell, while reducing the inclusion of secondary images. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] A culture vessel having multiple microwells, A culture vessel containing non-circular microwells in which, with respect to the opening shape of the microwells, the radii of two concentric circles determined by the minimum area center method for roundness measurement specified in JIS B0621 are R1 and r1, respectively (where R1 > r1), and r1 / R1 is between 0.75 and 0.95. [2] For the plan view shape of the non-circular microwell at a position where the ratio of the distance from the bottom of the microwell to the depth of the microwell is 0.5, when the radii of the two concentric circles obtained by the minimum area center method are R2 and r2 respectively (where R2 > r2), is r2 / R2 between 0.80 and 0.95? Alternatively, the culture vessel of [1] wherein, with respect to the plan view shape at a position where the distance in the depth direction from the bottom of the non-circular microwell is 100 microns, the radii of the two concentric circles determined by the minimum area center method are R3 and r3, respectively (where R3 > r3), and r3 / R3 is 0.80 to 0.95. [3] The culture vessel according to [1] or [2], wherein the opening shape of the non-circular microwell is not symmetrical with respect to a straight line passing through the centers of two concentric circles determined by the minimum region center method. [4] A culture vessel according to any of [1] to [3], wherein the opening shape of the non-circular microwell is not point-symmetric with respect to the center of the two concentric circles determined by the minimum region center method. [Effects of the Invention]

[0008] According to the present invention, a culture vessel is provided that allows for efficient measurement of multiple cell aggregates formed in each microwell, while reducing the appearance of secondary images. [Brief explanation of the drawing]

[0009] [Figure 1] This is a cross-sectional view showing a culture vessel according to the embodiment. [Figure 2] This is a plan view showing a magnified view of some of the microwells on the bottom surface of the culture vessel in Figure 1. [Figure 3] Figure 2 is a cross-sectional view of the microwell (AA). [Figure 4] These are the optical measurement results of cell aggregates in microwells in the examples and comparative examples. [Modes for carrying out the invention]

[0010] The meanings and definitions of terms used in this specification are as follows: A numerical range represented by "~" means a range of numbers whose lower and upper limits are the numbers before and after the "~". A "microwell" consists of a bottom, a side wall rising at a larger angle of inclination (angle relative to the horizontal) than the bottom, and an upper part that widens at a smaller angle of inclination than the side wall. The bottom, side wall, and upper part each include a configuration in which the angle of inclination of the surface is constant in the depth direction cross-section, a configuration in which the surface has a constant radius of curvature, and a configuration in which the angle of inclination and radius of curvature change gradually. The "opening end of a microwell" is defined as a boundary line that encircles the entire surface of the microwell on a horizontal plane, with the first reference plane being the first reference plane, where the height of the horizontal plane is defined as the point where the side wall of the microwell changes upward. The "point where the side wall changes upward" is defined as the point in the depth direction cross-section passing through the deepest part of the microwell where the surface inclination angle (inclination with the horizontal direction as 0°) changes from 65° or more to 65° or less from the side to the top (the point where the inclination angle of the tangent to the surface is 65°; however, if the inclination angle changes abruptly, a tangent of 65° cannot necessarily be drawn). If the point where the side wall changes upward is not at a constant height in the circumferential direction of the opening of the microwell, the lowest point among the points where the side wall of the microwell changes upward is defined as the reference height. Examples of surfaces forming a microwell include the bottom surface of the container in the case of culture vessels such as flasks and dishes, and the bottom surface of the well in the case of culture vessels in the form of microplates. The "opening shape of the microwell" refers to the plan view shape of the opening at the first reference plane of the microwell. "The diameter of the microwell opening" refers to the diameter of the opening on the first reference plane of the microwell; if the opening shape is not a perfect circle, it refers to the diameter of the circumscribed circle for that opening shape. "Microwell opening area" refers to the area of ​​the opening of the microwell on the first reference plane. "Microwell depth" is the distance between the deepest part of the microwell and the first reference plane.

[0011] The following describes an example of an embodiment of the culture vessel of the present invention, with reference to the drawings. Note that the dimensions and other specifications shown in the following description are examples only, and the present invention is not necessarily limited to them. It can be implemented with appropriate modifications without altering its essence.

[0012] As shown in Figures 1-3, the culture vessel 1 of this embodiment comprises a disc-shaped bottom wall portion 10 and a peripheral wall portion 12 that rises vertically from the periphery of the bottom wall portion 10. The culture vessel 1 has a plurality of microwells 20 on the upper bottom surface 10a of the bottom wall portion 10. In other words, the culture vessel 1 is a microfabricated container having a plurality of microwells 20.

[0013] The culture vessel 1 includes a non-circular fine well 20 that satisfies the following condition 1. Condition 1: For the opening shape of the fine well 20, when the radii of two concentric circles obtained by the minimum region center method for circularity measurement defined in JIS B0621 are R1 and r1 (where R1 > r1), r1 / R1 is 0.75 to 0.95.

[0014] The method for obtaining r1 and R1 in Condition 1 will be described more specifically. Using the minimum region center method (MZC), on the first reference plane k (Figure 3), two concentric circles that sandwich the opening shape of the fine well 20 are obtained, and the two circles with the smallest radius difference are determined. Then, the radius of the larger circle of the two concentric circles is set as R1, and the radius of the smaller circle is set as r1.

[0015] In the non-circular fine well 20 that satisfies Condition 1, when optically measuring the cell mass formed in the fine well 20, the reflection of the light emitted from the cell mass on the surface of the fine well 20 and the enhancement due to the interference of the reflected light are reduced. As a result, in the optical measurement of the cell mass, the secondary (unnecessary) light reflection is reduced. Therefore, the measurement accuracy of the light emission amount and fluorescence amount is increased, and the analysis of localization is also facilitated.

[0016] r1 / R1 is 0.75 to 0.95, preferably 0.83 to 0.92, and more preferably 0.85 to 0.90. If r1 / R1 is below the upper limit value of the above range, the secondary light reflection caused by the reflection and enhancement due to interference on the fine well surface in optical measurement is reduced. If r1 / R1 is above the lower limit value of the above range, a sufficient space is ensured within the fine well 20, so that a cell mass of sufficient size can be efficiently formed.

[0017] R1 is preferably 120 to 1000 μm, more preferably 200 to 600 μm, and even more preferably 260 to 450 μm. If R1 is above the lower limit value of the above range, it is easy to grow a cell mass of sufficient size. If R1 is below the upper limit value of the above range, the cell mass in the fine well is likely to remain constant.

[0018] r1 is preferably 85 to 850 μm, more preferably 110 to 605 μm, and even more preferably 230 to 380 μm. If r1 is above the lower limit of the above range, it is easier to grow cell aggregates of sufficient size. If r1 is below the upper limit of the above range, the cell aggregates in the microwells tend to remain constant.

[0019] The multiple microwells in the culture vessel 1 may all be non-circular microwells 20 that satisfy condition 1, or some may be non-circular microwells 20 that satisfy condition 1 and the remainder may be microwells 20 that do not satisfy condition 1. The ratio of the number of microwells 20 that satisfy condition 1 to the total number of microwells 20 in the culture vessel 1 is preferably 80-100%, more preferably 85-100%, even more preferably 90-100%, and particularly preferably 100%.

[0020] For a plan view shape at a position where the ratio of the distance from the bottom of the microwell 20 in the depth direction to the depth of the microwell 20 is 0.5, the radii of two concentric circles determined by the minimum area center method for roundness measurement specified in JIS B0621 are R2 and r2, respectively (where R2 > r2). Also, for a plan view shape at a position where the distance from the bottom of the microwell 20 in the depth direction is 100 microns, the radii of two concentric circles determined by the same minimum area center method are R3 and r3, respectively (where R3 > r3). ​​A non-circular microwell 20 that satisfies condition 1 is preferably further satisfied with condition 2 below. Condition 2: r2 / R2 is between 0.80 and 0.95, or r3 / R3 is between 0.80 and 0.95.

[0021] Let's explain in more detail how to find r2 and R2 in condition 2. As shown in Figure 3, when the depth of the microwell 20 is H, the second reference plane m is the plane parallel to the first reference plane k at the position where the distance h from the bottom (deepest part) of the microwell 20 in the depth direction is h = 0.5 × H. For a closed linear shape that encircles the entire surface of the microwell 20 on the second reference plane m, the minimum region center method is used to find two concentric circles on the second reference plane m that have the smallest difference in radius. Then, of these two concentric circles, the radius of the circle with the larger radius is taken as R2, and the radius of the circle with the smaller radius is taken as r2. r3 and R3 are determined in the same way as r2 and R2, except that instead of a second reference plane, a third reference plane is defined as a plane parallel to the first reference plane k at a position where the distance from the bottom (deepest part) of the microwell 20 in the depth direction is 100 microns.

[0022] The non-circular microwells 20 satisfy condition 2 in addition to condition 1, which further reduces the reflection of light emitted from the cell aggregate at the surface of the microwells 20 and the constructive interference of reflected light during optical measurements of the cell aggregate, thereby further suppressing secondary light reflection.

[0023] The r2 / R2 ratio is between 0.80 and 0.95, preferably between 0.83 and 0.92, and more preferably between 0.85 and 0.90. If r2 / R2 is below the upper limit of the above range, secondary light reflections caused by constructive interference due to reflection and interference on the surface of the microwells are further reduced during optical measurements. If r2 / R2 is above the lower limit of the above range, sufficient space is easily secured within the microwells 20, allowing for the efficient formation of cell aggregates of sufficient size.

[0024] R2 is preferably 100 to 800 μm, more preferably 150 to 500 μm, and even more preferably 180 to 400 μm. If R2 is above the lower limit of the above range, it is easier to grow cell aggregates of sufficient size. If R2 is below the upper limit of the above range, the cell aggregates in the microwells tend to remain constant.

[0025] r2 is preferably 50 to 800 μm, more preferably 100 to 460 μm, and even more preferably 120 to 300 μm. If r2 is above the lower limit of the above range, it is easier to grow cell aggregates of sufficient size. If r2 is below the upper limit of the above range, the cell aggregates in the microwells tend to remain constant.

[0026] It is preferable that the bottom surface 10a of the culture vessel has multiple microwells 20 of uniform size, as this makes it easier to obtain cell aggregates of uniform size. The average diameter D of the openings of the microwells 20 is preferably 100 to 2000 μm, more preferably 200 to 1400 μm, and even more preferably 400 to 800 μm. If the average diameter D of the openings of the microwells 20 is above the lower limit of the above range, it is easier to prevent the size of the cell aggregates from becoming too small. If the average diameter D of the openings of the microwells 20 is below the upper limit of the above range, the gaps between the microwells can be reduced. As a result, the number of cell aggregates formed increases. The average diameter D of the openings of the microwells is the average value obtained by measuring the diameters of any two microwells. The diameter of the openings of the microwells can be measured by three-dimensional observation using a laser microscope.

[0027] The opening area of ​​the microwell 20 is 6600 to 2700000 μm². 2 Preferably, 100,000 to 960,000 μm 2 More preferably, 200,000 to 500,000 μm 2 This is even more preferable. If the opening area of ​​the microwells 20 is greater than or equal to the lower limit of the range, it is easier to grow cell aggregates of sufficient size. If the opening area of ​​the microwells 20 is less than or equal to the upper limit of the range, the cell aggregates in the microwells tend to remain constant.

[0028] The average depth H of the microwells 20 (Figure 3) is preferably 120 to 800 μm, more preferably 150 to 650 μm, and even more preferably 180 to 500 μm. If the average depth H of the microwells 20 is above the lower limit of the above range, the cell aggregates in the microwells tend to remain constant. If the average depth H of the microwells 20 is below the upper limit of the above range, bubbles escape well when a solution such as a culture medium is added. The average depth H of the microwells is the average value obtained by measuring the depth of any two microwells (distance between the first reference plane and the deepest part of the microwell). The depth of the microwells can be measured by three-dimensional observation with a laser microscope, or by cutting the bottom wall of the container in the height direction so as to pass through the deepest part of the microwell, and observing the cross-section with a microscope.

[0029] The arrangement pattern of the microwells 20 is not particularly limited and may be formed in a regular pattern, an irregular pattern, or a mixture of regular and irregular parts. Examples of regular arrangement patterns include a pattern in which microwells are placed at each vertex of a square arranged without gaps, a pattern in which microwells are placed at each vertex and the center of a regular hexagon arranged without gaps, and a staggered pattern.

[0030] The aperture shape of the microwell 20 is not particularly limited, and examples include circular, elliptical, polygonal, donut-shaped, and irregular shapes. For the aperture shape of the microwell 20 satisfying condition 1, it is preferable that, in a plan view, it is not line-symmetric with respect to a line passing through the centers of two concentric circles determined by the minimum region center method. Furthermore, for the aperture shape of the microwell 20 satisfying condition 1, it is also preferable that, in a plan view, it is not point-symmetric with respect to the centers of two concentric circles determined by the minimum region center method. If the aperture shape of the microwell 20 satisfying condition 1 is an irregular shape without symmetry factors, constructive interference due to light reflection and interference on the surface of the microwell 20 becomes even less likely, further reducing secondary light reflection in optical measurements.

[0031] The number of microwells 20 in culture vessel 1 is 10 per unit area / cm². 2The above is preferable, with 10 to 10,000 pieces / cm². 2 More preferably, 15 to 5000 pieces / cm 2 More preferably, 20 to 1000 pieces / cm 2 That is particularly preferable.

[0032] Examples of materials for the culture vessel 1 include synthetic resin or glass. Examples of synthetic resins include polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, polyethylene terephthalate (PET), polyvinyl chloride, high-density polyethylene, polyethersulfan, PET copolymer, Permanox (Thermo Fisher Scientific trademark), cycloolefin polymer resin, Cytop (AGC trademark), acrylic resin, polycarbonate resin, and silicone resin. Among these, polystyrene resin or silicone resin is preferred in terms of ease of molding and microfabrication. The synthetic resin constituting the culture vessel 1 may be one type or two or more types.

[0033] Examples of glass include quartz glass, borosilicate glass, phosphate glass, and chemically strengthened glass. The glass constituting microplate 1 may consist of one type or two or more types.

[0034] The peripheral wall 12 of the culture vessel 1 may be transparent or opaque. If the peripheral wall 12 is opaque, a color that does not easily transmit light, such as black or white, is more preferable. By making the peripheral wall 12 opaque, the difficulty in observation due to light reflection during microscopic observation can be reduced. There are no particular limitations on the method of making the peripheral wall 12 opaque; for example, methods such as adding fine particles or adding colorants such as pigments can be used. For black, carbon can be used, and for white, titanium dioxide can be used.

[0035] The method for manufacturing the culture vessel 1 is not particularly limited, and for example, it can be molded by injection molding. As a method for forming the microwells 20, laser irradiation can be exemplified. When laser light is irradiated onto the bottom surface 10a of the resin culture vessel 1, the synthetic resin constituting the bottom surface 10a dissolves and vaporizes, forming microwells 20 with a very smooth surface. Around the opening of the microwells 20, the dissolved synthetic resin may rise up, forming a ridge.

[0036] The laser light source is not particularly limited, but a CO2 laser can be used as an example. The arrangement and size of the microwells 20 can be adjusted by adjusting the irradiation conditions such as the irradiation position and output of the laser beam. By performing laser irradiation without changing the laser output and laser irradiation time, the size of each microwell 20 can be made uniform.

[0037] The surface of the microwells 20 may be coated with a low-adhesion coating that inhibits cell adhesion or with an easy-adhesion coating that promotes cell adhesion. For example, collagen or gelatin can be used as the easy-adhesion coating. The formation of a low-adhesion coating makes it easier to remove cell aggregates from the microwells 20.

[0038] A low-adhesion coating film can be formed, for example, by applying a cell adhesion inhibitor. Examples of cell adhesion inhibitors include phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, and polyethylene glycol. One cell adhesion inhibitor may be used alone, or two or more may be used in combination.

[0039] Furthermore, if at least the bottom wall portion 10 of the culture vessel 1 is molded with a resin that has a cell adhesion inhibitory effect, such as silicone resin, or a synthetic resin containing the aforementioned cell adhesion inhibitor, it is possible to suppress cell adhesion to the surface of the microwells without forming a low-adhesion coating film.

[0040] The following describes the effects of culture vessel 1. The closer the opening shape of the microwells is to a perfect circle, the more the light emission and fluorescence from the cell aggregate are reflected and interfered with by the surface of the microwells 20 during optical measurements, making it easier for secondary and unwanted images to be captured. In contrast, culture vessel 1 has microwells 20 with an opening shape that is not perfectly circular, satisfying condition 1. Therefore, the reinforcement due to light reflection and interference on the surface of the microwells 20 is suppressed, and the capture of secondary images is reduced. In addition, since uniform cell aggregates of sufficient size can be efficiently formed within the microwells, it becomes possible to measure multiple cell aggregates at once.

[0041] It should be noted that the culture vessel of the present invention is not limited to the culture vessel 1 described above. For example, the culture vessel of the present invention may be flask-shaped, or it may be a microplate in which multiple microwells are formed on the bottom surface of multiple wells. Furthermore, it is possible to replace the components in the above embodiments with well-known components as appropriate, without departing from the spirit of the present invention, and the above modifications may be combined as appropriate. [Examples]

[0042] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following description.

[0043] [Example 1] A microplate culture vessel, as illustrated in Figure 1, was formed by injection molding using polystyrene resin. Next, a CO2 laser was irradiated using a laser irradiation device to create multiple non-circular microwells. Then, a low-cell adhesion coating was applied to the culture surface. The formed microwells had an r1 / R1 ratio of 0.77, an r2 / R2 ratio of 0.85, an average diameter D of 570 μm, and an opening area of ​​255,000 μm². 2 The average depth H was 190 μm.

[0044] [Comparative Example 1] A culture vessel was prepared in the same manner as in Example 1, except that a fine well with a circular opening shape was formed. For the formed fine well, r1 / R1 was 1, r2 / R2 was 1, the average diameter D was 560 μm, the opening area was 246000 μm 2 , and the average depth H was 360 μm.

[0045] [Optical measurement of cell mass] Using the microplates obtained in each example, Panc-1 cells (human pancreatic adenocarcinoma cells, obtained from ATCC) were seeded in each well so that the number of cells per seeding was 9,600, and 10% KSR (KnockOut Serum Replacement, manufactured by Thermo Fisher Scientific), 0.1% CellTracker TM Orange (manufactured by Thermo Fisher Scientific) was cultured in a medium containing Orange at 37 °C in a 5% CO2 atmosphere for 2 days. Then, optical measurement (excitation light 540 nm, fluorescence emission 580 nm) of the cell mass in the fine well was performed using a fluorescence microscope IX73 (manufactured by Olympus), and the reflection of the secondary image was evaluated according to the following criteria. The results are shown in FIG. 4 and Table 1. (Evaluation criteria) ○: No fluorescence reflection can be seen anywhere in the fine well. ×: Fluorescence reflection can be seen at the opening or throughout the fine well.

[0046]

Table 1

[0047] Figure 4(a) shows the results of Example 1, and Figure 4(b) shows the results of Comparative Example 1. The cells fluoresce and are displayed as white light. In Example 1, only the cell aggregates fluoresce, and no reflection of fluorescence from the microwell walls is observed, confirming that there is no secondary image reflection. However, in Comparative Example 1, not only the cell aggregates but also the well walls glow white, and the fluorescence emitted from the cell aggregates is reflected from the microwell walls, confirming the reflection of secondary images. Therefore, the observationability of the configuration in Comparative Example 1 is undesirable, and Example 1 is superior in terms of observationability. [Explanation of Symbols]

[0048] 1...Culture vessel, 10...Bottom wall, 10a...Bottom surface, 12...Surface wall, 20...Microwell.

Claims

1. A culture vessel having multiple microwells, For the opening shape of the microwell, the radii of the two concentric circles obtained by the minimum area center method of roundness measurement specified in JIS B0621 are R. １ ,r １ (However, R １ >r １ When this is the case, r １ / R １ A culture vessel comprising non-circular microwells having a diameter of 0.75 to 0.95, wherein the opening shape of the microwells is, in plan view, not symmetrical with respect to a straight line passing through the centers of two concentric circles determined by the minimum region center method, or not symmetrical with respect to the straight line and not point-symmetrical with respect to the center.

2. For the planar shape of the position where the ratio of the depth-direction distance from the bottom of the micro well to the depth of the micro well in the non-circular micro well is 0.5, the radii of the concentric two circles obtained by the minimum region center method are respectively R ２ , r ２ (where R ２ > r ２ ).), when r ２ / R ２ is 0.80 to 0.95, or Alternatively, for a plan view shape at a position where the distance in the depth direction from the bottom of the non-circular microwell is 100 microns, the radii of the two concentric circles obtained by the minimum region center method are R. ３ ,r ３ (However, R ３ >r ３ When this is the case, r ３ / R ３ A culture vessel according to claim 1, wherein the ratio is 0.80 to 0.

95.

3. The culture vessel according to claim 1 or 2, characterized in that the microwells are arranged without gaps on the bottom surface of the culture vessel.

Images