JP2022067072A - Culture vessel - Google Patents

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

The present invention relates to a culture vessel.

Cultured cells are widely used in basic research for elucidating life phenomena and drug discovery research. In particular, the three-dimensional cell mass (spheroid) in which the cells obtained by the three-dimensional culture are aggregated has a three-dimensional structure similar to that in the living body, so that the cells obtained by the two-dimensional culture are more in vivo than the cells obtained by the two-dimensional culture. It is expected to have similar biological activity. As the three-dimensional culture, for example, a culture container (microprocessed container) in which a large number of fine wells (microwells) having a pore diameter of 100 to 1,000 μm were formed on the bottom surface of each well of the microplate, the bottom surface of the dish, or the like was used. A culture method is known (Patent Document 1). When cells are sown in a microfabrication container, the cells associate in each microwell to form a cell mass.

When measuring biological activities such as cell viability and the action of enzymes in cells, the amount of light emitted or fluorescence is measured by a plate reader or cell imager using a reagent that emits light in response to cells or enzymes or emits fluorescence. Measure the amount and measure its localization. The method of optically measuring a cell mass cultured in a microfabrication container as it is in a microwell has an advantage that a plurality of cell masses can be measured at one time.

Japanese Patent No. 6400575

However, in the conventional microprocessed container as in Patent Document 1, when the cell mass and the reaction solution in the fine well are optically measured, a secondary and unnecessary image is easily reflected, so that the amount of light emission and the amount of fluorescence are emitted. The measurement accuracy of the optics is low, and it is difficult to analyze the localization.

It is an object of the present invention to provide a culture vessel capable of efficiently measuring a plurality of cell masses and reducing secondary image reflection in the optical measurement of cell masses formed in each fine well. And.

The present invention has the following aspects.
[1] A culture vessel having a plurality of fine wells.
For the opening shape of the fine well, the radii of the concentric two circles obtained by the minimum region center method of roundness measurement specified in JIS B0621 are R ₁ , r ₁ (however, R ₁ > r ₁ ), respectively. A culture vessel containing a non-circular fine well having r ₁ / R ₁ of 0.75 to 0.95.
[2] The minimum of the non-circular shape of the fine well with respect to the plan view shape at the position where the ratio of the distance in the depth direction from the bottom of the fine well to the depth of the fine well is 0.5. When the radii of the concentric two circles obtained by the region center method are R ₂ and r ₂ (where R ₂ > r ₂ ), respectively, is r ₂ / R ₂ 0.80 to 0.95? ,
Alternatively, the radius of the concentric two circles obtained by the minimum region center method is R3 _, respectively, for the plan view shape at the position where the distance in the depth direction from the bottom of the non-perfect circular shape is 100 microns. The culture vessel of [1], wherein r ₃ / R ₃ is 0.80 to 0.95 when r ₃ (where R ₃ > r ₃ ).
[3] The non-circular shape of the opening of the fine well is not line-symmetrical with respect to a straight line passing through the center of the concentric two circles obtained by the minimum region center method, [1] or [2]. Culture container.
[4] Any of [1] to [3], wherein the opening shape of the fine well having an irregular circular shape is not point-symmetrical with respect to the center of the concentric two circles obtained by the minimum region center method. Culture container.

According to the present invention, it is possible to provide a culture vessel capable of efficiently measuring a plurality of cell masses and reducing secondary image reflection in the optical measurement of the cell mass formed in each fine well.

It is sectional drawing which showed the culture container of an embodiment. FIG. 3 is an enlarged plan view showing a part of fine wells on the bottom surface of the culture vessel of FIG. 1. FIG. 2 is a cross-sectional view taken along the line AA of the fine well shown in FIG. It is an optical measurement result of the cell mass in a fine well in an Example and a comparative example.

The meanings and definitions of the terms used herein are as follows.
The numerical range represented by "-" means a numerical range in which the numerical values before and after "-" are the lower limit value and the upper limit value.
The "fine well" is composed of a bottom portion, a side wall portion that rises at an inclination angle (angle with respect to the horizontal direction) larger than the bottom portion, and an upper portion that extends at an inclination angle smaller than the side wall portion. Each of the bottom portion, the side wall portion, and the upper portion includes an embodiment in which the inclination angle of the surface is constant, an embodiment in which the surface has a constant radius of curvature, and an embodiment in which the inclination angle and the radius of curvature gradually change in the cross section in the depth direction.
The "open end of the fine well" is the surface of the fine well on the first reference plane, with the horizontal surface whose reference height is the point where the side wall of the fine well changes to the upper part. It is a boundary line that goes around the entire circumference. The "point that changes from the side wall to the upper part" is the angle of inclination of the surface from the side surface to the upper part (when the horizontal direction is 0 °) in the cross section in the depth direction passing through the deepest part of the fine well. It is assumed that the point at which the inclination) changes from 65 ° or more to 65 ° or less (the inclination angle of the tangent to the surface is 65 °, and if the inclination angle changes suddenly, the tangent of 65 ° cannot always be drawn). If the point of change from the side wall to the upper part is not a constant height in the circumferential direction of the opening of the fine well, the lowest point among the points of change from the side wall of the fine well to the upper part is taken as the reference height. .. As the surface forming the fine well, the bottom surface of the container can be exemplified in the culture container in the form of a flask, a dish, etc., and the bottom surface of the well can be exemplified in the culture container in the form of a microplate.
The "opening shape of the fine well" is a plan view shape of the opening of the fine well on the first reference plane.
The "diameter of the opening of the fine well" is the diameter of the opening on the first reference plane of the fine well, and if the opening shape is not a perfect circle, it is the diameter of the circumscribed circle with respect to the opening shape.
The "opening area of the fine well" is the area of the opening of the fine well on the first reference plane.
The "depth of the fine well" is the distance between the deepest part of the fine well and the first reference plane.

Hereinafter, an example of an embodiment of the culture vessel of the present invention will be shown and described with reference to the drawings. It should be noted that the dimensions and the like of the figures exemplified in the following description are examples, and the present invention is not necessarily limited to them, and the present invention can be appropriately modified without changing the gist thereof. ..

As shown in FIGS. 1 to 3, the culture vessel 1 of the present embodiment includes a disk-shaped bottom wall portion 10 and a peripheral wall portion 12 that rises vertically from the peripheral edge of the bottom wall portion 10. The culture vessel 1 has a plurality of fine wells 20 on the bottom surface 10a on the upper side of the bottom wall portion 10. That is, the culture container 1 is a microfabricated container having a plurality of microwells 20.

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, the radii of concentric circles obtained by the minimum region center method of roundness measurement specified in JIS B0621 are R ₁ , r ₁ (however, R ₁ > r ₁ ), respectively. ), R ₁ / R ₁ is 0.75 to 0.95.

The method of obtaining r ₁ and R ₁ under condition 1 will be described more specifically. Using the minimum region center method (MZC), two circles having the smallest radius difference are obtained as two concentric circles sandwiching the opening shape of the fine well 20 on the first reference plane k (FIG. 3). Then, the radius of the circle having the larger radius among the two concentric circles is R ₁ , and the radius of the circle having the smaller radius is r ₁ .

In the non-circular fine well 20 satisfying the condition 1, when the cell mass formed in the fine well 20 is optically measured, the light emitted from the cell mass is reflected on the surface of the fine well 20 and is reflected. Strengthening due to the interference of reflected light is reduced. This reduces secondary (unwanted) light reflections in the optical measurement of cell mass. Therefore, the measurement accuracy of the amount of light emission and the amount of fluorescence is improved, and the analysis of localization becomes easy.

r ₁ / R ₁ is 0.75 to 0.95, preferably 0.83 to 0.92, and more preferably 0.85 to 0.90. When r ₁ / R ₁ is not more than the upper limit of the above range, the reflection of secondary light due to the intensification due to reflection and interference on the surface of the fine well in the optical measurement is reduced. When r ₁ / R ₁ is equal to or higher than the lower limit of the above range, a sufficient space is secured in the fine well 20, so that a cell mass having a sufficient size can be efficiently formed.

R ₁ is preferably 120 to 1000 μm, more preferably 200 to 600 μm, and even more preferably 260 to 450 μm. When R ₁ is equal to or higher than the lower limit of the above range, it is easy to grow a cell mass of sufficient size. When R ₁ is not more than the upper limit of the above range, the cell mass in the fine well tends to stay constant.

r ₁ is preferably 85 to 850 μm, more preferably 110 to 605 μm, and even more preferably 230 to 380 μm. When r ₁ is not more than the lower limit of the above range, it is easy to grow a cell mass of sufficient size. When r ₁ is not more than the upper limit of the above range, the cell mass in the fine well tends to stay constant.

The plurality of fine wells included in the culture vessel 1 may be non-circular fine wells 20 that all satisfy condition 1, some of which are non-circular fine wells 20 that satisfy condition 1, and the rest are non-circular fine wells 20. The fine well 20 may not satisfy the condition 1. The ratio of the number of fine wells 20 satisfying the condition 1 to the total number of fine wells 20 possessed by the culture vessel 1 is preferably 80 to 100%, more preferably 85 to 100%, further preferably 90 to 100%, and 100%. Especially preferable.

The minimum region center method for roundness measurement specified in JIS B0621 for the plan view shape at the position where the ratio of the distance in the depth direction from the bottom of the fine well 20 to the depth of the fine well 20 is 0.5. Let the radii of the _two concentric circles obtained in ( _R2 > r2 be _R2 > r2, _respectively ). Further, for the plan view shape at the position where the distance from the bottom of the fine well 20 in the depth direction is 100 microns, the radii of the concentric two circles obtained by the minimum region center method are R ₃ , r ₃ (however, respectively). R ₃ > r ₃ ). It is preferable that the non-circular fine well 20 satisfying the condition 1 further satisfies the following condition 2.
Condition 2: r ₂ / R ₂ is 0.80 to 0.95, or r ₃ / R ₃ is 0.80 to 0.95.

The method of obtaining r ₂ and R ₂ under the condition 2 will be described more specifically. As shown in FIG. 3, when the depth of the fine well 20 is H, the distance h from the bottom (deepest part) of the fine well 20 in the depth direction is h = 0.5 × H. The plane parallel to the 1 reference plane k is defined as the second reference plane m. For a closed line shape that orbits the surface of the fine well 20 over the entire circumference on the second reference surface m, the minimum region center method is used, and the radius is set as two concentric circles sandwiching the line shape on the second reference surface m. Find the two circles with the smallest difference. Then, the radius of the circle having the larger radius among the two concentric circles is R ₂ , and the radius of the circle having the smaller radius is r ₂ .
Instead of the _second reference plane _, r3 and R3 have a plane parallel to the first reference plane k at a position where the distance from the bottom (deepest part) of the fine well 20 in the depth direction is 100 microns. ₃ Obtained by the same method as r2 and _R2 except that the reference plane is used.

When the non-circular fine well 20 further satisfies the condition 2 in addition to the condition 1, the light emitted from the cell mass is reflected on the surface of the fine well 20 and the reflected light is reflected in the optical measurement of the cell mass. The intensification due to the interference of the light is further reduced, and the reflection of secondary light is further suppressed.

r2 / _R2 is 0.80 to 0.95, preferably 0.83 to 0.92, and _more preferably 0.85 to 0.90. When r ₂ / R ₂ is not more than the upper limit of the above range, the reflection of secondary light due to the intensification due to reflection or interference on the surface of the fine well in the optical measurement is further reduced. When r ₂ / R ₂ is equal to or greater than the lower limit of the above range, sufficient space is likely to be secured in the fine well 20, so that a cell mass having a sufficient size can be efficiently formed.

_R2 is preferably 100 to 800 μm, more preferably 150 to 500 μm, and even more preferably 180 to 400 μm. When R ₂ is equal to or higher than the lower limit of the above range, it is easy to grow a cell mass of sufficient size. When R ₂ is not more than the upper limit of the above range, the cell mass in the fine well tends to stay constant.

r2 is preferably 50 to 800 μm, _more preferably 100 to 460 μm, and even more preferably 120 to 300 μm. When r ₂ is not more than the lower limit of the above range, it is easy to grow a cell mass of sufficient size. When r ₂ is not more than the upper limit of the above range, the cell mass in the fine well tends to stay constant.

It is preferable that a plurality of fine wells 20 having a uniform size are formed on the bottom surface 10a of the culture vessel from the viewpoint that a cell mass having a uniform size can be easily obtained.
The average diameter D of the openings of the fine wells 20 is preferably 100 to 2000 μm, more preferably 200 to 1400 μm, and even more preferably 400 to 800 μm. When the average diameter D of the openings of the fine wells 20 is not less than the lower limit of the above range, it is easy to prevent the cell mass from becoming too small. When the average diameter D of the openings of the fine wells 20 is not more than the upper limit of the above range, the gap between the fine wells and the fine wells can be reduced. As a result, the number of cell masses formed increases. The average diameter D of the openings of the fine wells is a value obtained by measuring and averaging the diameters of two arbitrary fine wells. The measurement of the diameter of the opening of the fine well can be performed by three-dimensional observation with a laser microscope.

The opening area of the fine well 20 is preferably 6600 to 270000 μm ² , more preferably 100,000 to 960000 μm ² , and even more preferably 200,000 to 500,000 μm ² . When the opening area of the fine well 20 is equal to or greater than the lower limit of the above range, it is easy to grow a cell mass having a sufficient size. When the opening area of the fine well 20 is not more than the upper limit of the above range, the cell mass in the fine well tends to stay constant.

The average depth H (FIG. 3) of the fine well 20 is preferably 120 to 800 μm, more preferably 150 to 650 μm, and even more preferably 180 to 500 μm. When the average depth H of the fine well 20 is at least the lower limit of the above range, the cell mass in the fine well tends to stay constant. When the average depth H of the fine well 20 is not more than the upper limit of the above range, the bubbles can be easily removed when a solution such as a medium is added. The average depth H of the fine wells is a value obtained by measuring and averaging the depths of the fine wells (distance between the first reference plane and the deepest part of the fine wells) for any two fine wells. The depth of the fine well can be measured by three-dimensional observation with a laser microscope or by dividing the bottom wall of the container in the height direction so as to pass through the deepest part of the fine well and observing the cross section with a microscope.

The arrangement pattern of the fine wells 20 is not particularly limited, and may be formed in a regular pattern, irregularly formed, or a mixture of regular portions and irregular portions. As a regular arrangement pattern, for example, a pattern in which fine wells are arranged at each vertex of a square arranged without gaps, a pattern in which fine wells are arranged at each vertex of a regular hexagon arranged without gaps and a fine well in the center, and a staggered pattern are used. It can be exemplified.

The opening shape of the fine well 20 is not particularly limited, and examples thereof include a circular shape, an elliptical shape, a polygonal shape, a donut shape, and an irregular shape. The opening shape of the fine well 20 satisfying the condition 1 is preferably a shape that is not axisymmetric with respect to a straight line passing through the center of the concentric two circles obtained by the minimum region center method in a plan view. Further, it is also preferable that the opening shape of the fine well 20 satisfying the condition 1 is not point-symmetrical with respect to the center of the concentric two circles obtained by the minimum region center method in a plan view. If the opening shape of the fine well 20 satisfying the condition 1 is an irregular shape having no symmetry factor as described above, the surface of the fine well 20 is less likely to be strengthened by light reflection or interference. Secondary light reflections in targeted measurements are further reduced.

The number of fine wells 20 contained in the culture vessel 1 is preferably 10 pieces / cm ² or more, more preferably 10 to 10000 pieces / cm ² , still more preferably 15 to 5000 pieces / cm ² , and 20 to 1000 per unit area. Pieces / cm ² are particularly preferred.

Examples of the material of the culture container 1 include synthetic resin and glass.
Synthetic resins include polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, polyethylene terephthalate (PET), polyvinyl chloride, high density polyethylene, polyether sulfan, PET copolymer, and Permanox (Thermofisher Scientific trademark). ), Cycloolefin polymer resin, Cytop (AGC trademark), acrylic resin, polycarbonate resin, silicone resin, and examples thereof. Of these, polystyrene resin or silicone resin is preferable from the viewpoint of ease of molding and microfabrication. The synthetic resin constituting the culture container 1 may be one kind or two or more kinds.

Examples of the glass include quartz glass, borosilicate glass, phosphoric acid glass, and chemically strengthened glass. The glass constituting the microplate 1 may be one kind or two or more kinds.

The peripheral wall portion 12 of the culture vessel 1 may be transparent or opaque. When the peripheral wall portion 12 is opaque, the color tone is more preferably black or white, which makes it difficult for light to pass through. By making the peripheral wall portion 12 opaque, it is possible to reduce the difficulty of observing due to the reflection of light when observing with a microscope. The method for making the peripheral wall portion 12 opaque is not particularly limited, and for example, a method of adding fine particles, a method of adding a coloring agent such as a pigment, or the like can be used. In the case of black, carbon or the like can be used, and in the case of white, titanium oxide or the like can be used.

The method for producing the culture container 1 is not particularly limited, and can be molded by, for example, an injection molding method. As a method for forming the fine well 20, for example, laser irradiation can be exemplified. When the bottom surface 10a of the resin culture container 1 is irradiated with laser light, the synthetic resin constituting the bottom surface 10a is dissolved and vaporized to form a fine well 20 having a very smooth surface. A bank portion may be formed by raising the melted synthetic resin around the opening of the fine well 20.

The laser light source is not particularly limited, and a CO ₂ laser can be exemplified. The arrangement and size of the fine wells 20 can be adjusted by adjusting irradiation conditions such as the irradiation position and output of the laser beam. By performing laser irradiation without changing the laser output and the laser irradiation time, the size of each fine well 20 can be made uniform.

On the surface of the fine well 20, a low adhesion coat film that suppresses cell adhesion or an easy adhesion coat film that adheres cells may be formed. As the easy-adhesion coating film, for example, collagen or gelatin is used. The formation of the low adhesive coat film facilitates the removal of cell clumps from the fine wells 20.

The low adhesion coat film can be formed, for example, by applying a cell adhesion inhibitor. Examples of the cell adhesion inhibitor include phospholipid polymers (2-methacryloyloxyethyl phosphorylcholine and the like), polyhydroxyethyl methacrylates, fluorine-containing compounds, and polyethylene glycol. As the cell adhesion inhibitor, one type may be used alone, or two or more types may be used in combination.

If at least the bottom wall portion 10 of the culture vessel 1 is molded with a resin having a cell adhesion inhibitory effect such as a silicone resin or a synthetic resin containing the cell adhesion inhibitor, a low adhesion coat film will not be formed. However, it is possible to prevent cells from adhering to the surface of the fine well.

Hereinafter, the action and effect of the culture vessel 1 will be described. The closer the opening shape of the fine well is to a perfect circle, the more the light emission and fluorescence from the cell mass are reflected on the surface of the fine well 20 and interfere with each other to strengthen each other, so that a secondary and unnecessary image is captured. Easy to get in. On the other hand, since the culture vessel 1 has a non-circular fine well 20 whose opening shape satisfies the condition 1, reflection of light on the surface of the fine well 20 and strengthening due to interference are suppressed, and secondary. The reflection of a clear image is reduced. In addition, since a uniform cell mass of sufficient size can be efficiently formed in the fine well, it is possible to measure a plurality of cell masses at one time.

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 in the shape of a flask, or may be a microplate in which a plurality of fine wells are formed on the bottom surfaces of the plurality of wells. In addition, it is possible to appropriately replace the components in the embodiment with well-known components without departing from the spirit of the present invention, and the above-mentioned modifications may be appropriately combined.

Hereinafter, the present invention will be specifically described with reference to Examples, but the present invention is not limited to the following description.

[Example 1]
Using polystyrene resin, the culture container of the microplate illustrated in FIG. 1 was molded by injection molding. Next, a CO ₂ laser was irradiated using a laser irradiation device to form a plurality of non-circular fine wells. The culture surface was then coated with a low cell adhesion coat. The formed fine wells had r ₁ / R ₁ of 0.77, r ₂ / R ₂ of 0.85, an average diameter D of 570 μm, an opening area of 255000 μm ² , and an average depth of 190 μm.

[Comparative Example 1]
A culture vessel was prepared in the same manner as in Example 1 except that fine wells having a perfect circular opening were formed. The formed fine wells had r ₁ / R ₁ of 1, r ₂ / R ₂ of 1, an average diameter D of 560 μm, an opening area of 246000 μm ² , and an average depth H of 360 μm.

[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 planting was 9,600, and DMEM (Dalbeco) was used. Modified-Eagle's medium (made by Thermo Fisher Scientific) containing 10% KSR (KnockOut Serum Replacement, manufactured by Thermo Fisher Scientific) and 0.1% CellTracker ^TM Orange (manufactured by Thermo Fisher Scientific) at 37 ° C., 5 The cells were cultured for 2 days under the condition of% CO ₂ atmosphere. After that, the cell mass in the fine well was optically measured (excitation light 540 nm, fluorescence emission 580 nm) using a fluorescence microscope IX73 (manufactured by Olympus Corporation), 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)
◯: Fluorescence reflection cannot be seen anywhere in the fine well.
X: Fluorescence reflection can be seen in the entire opening or fine well.

FIG. 4A shows the result of Example 1, and FIG. 4B shows the result of Comparative Example 1. The cells fluoresce and appear glowing white. In Example 1, only the cell mass emits fluorescence, and the reflection of fluorescence from the wall surface of the fine well or the like is not observed, so that it can be confirmed that there is no reflection of the secondary image. However, in Comparative Example 1, not only the cell mass but also the wall surface of the well glows white, and the fluorescence emitted from the cell mass is reflected by the wall surface of the fine well, and the reflection of the secondary image can be confirmed. From the above, it can be said that the configuration of Comparative Example 1 is not preferable in terms of observability, and Example 1 is superior in terms of observability.

1 ... culture vessel, 10 ... bottom wall, 10a ... bottom, 12 ... peripheral wall, 20 ... fine well.

Claims (4)

A culture vessel with multiple microwells,
For the opening shape of the fine well, the radii of the concentric two circles obtained by the minimum region center method of roundness measurement specified in JIS B0621 are R ₁ , r ₁ (however, R ₁ > r ₁ ), respectively. A culture vessel containing a non-circular fine well having r ₁ / R ₁ of 0.75 to 0.95. The minimum region center method with respect to the plan view shape of the non-circular shape of the fine well at a position where the ratio of the distance in the depth direction from the bottom of the fine well to the depth of the fine well is 0.5. When the radii of the concentric two circles obtained in (1) are R ₂ and r ₂ (where R ₂ > r ₂ ), respectively, is r ₂ / R ₂ 0.80 to 0.95?
Alternatively, the radius of the concentric two circles obtained by the minimum region center method is R3 _, respectively, for the plan view shape at the position where the distance in the depth direction from the bottom of the non-perfect circular shape is 100 microns. The culture vessel according to claim 1, wherein r ₃ / R ₃ is 0.80 to 0.95 when r ₃ (where R ₃ > r ₃ ). According to claim 1 or 2, the opening shape of the fine well having a non-circular shape is not line-symmetrical with respect to a straight line passing through the center of the concentric two circles obtained by the minimum region center method in a plan view. The culture vessel described. Any one of claims 1 to 3, wherein the opening shape of the fine well having a non-circular shape is not point-symmetrical with respect to the center of the concentric two circles obtained by the minimum region center method in a plan view. The culture vessel described in the section.

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