WO2018123663A1 - Cell culture substrate and method for producing same - Google Patents

Abstract

Provided is a cell culture substrate having multiple microwells formed on one culturing surface thereof, whereby it becomes possible to prevent the migration of spheroids from the microwells to form spheroids having uniform sizes without the need to form the microwells too deeply. A culture substrate having a culturing surface on which cells are to be cultured, said culture substrate being characterized in that the culturing surface has multiple microwells formed thereon, each of the microwells has a first well having an inclined surface and a second well formed in the first well, a protrusion is formed on the boundary between the first well and the second well, and the culturing surface is a poorly cell-adhesive surface.

Description

Cell culture substrate and method for producing the same

The present invention relates to a cell culture substrate for culturing a culture object such as a cell to obtain a spheroid (cell aggregate) and a method for producing the same.

Spheroid culture in which cells derived from humans or animals are artificially cultured in a culture vessel or the like and aggregated three-dimensionally is well known. In spheroid culture, the cell population forms a three-dimensional structure, and the cells interact with each other, so it is thought that it can be cultured or maintained in a state that is closer to the three-dimensional structure in vivo. It is known to show superior properties compared to planar adhesion culture. Actually, spheroid culture is often used for anticancer drug screening using cancer cells and proliferation and differentiation of pluripotent stem cells.

In addition, in the cell culture container provided with a plurality of microwells for collecting cells by gravity at the bottom of the recess, a recess for containing cells and culture solution is provided at the bottom of the container body. There is also known one in which the side surface of the concave portion is formed by an inclined surface so that the opening area is expanded as it approaches the open end (see Patent Document 1).

In addition, a cell culture container is also known in which spheroids and cells are difficult to move to other wells during medium replacement by providing an inclination in a plurality of microwells provided on the culture surface and increasing the depth thereof ( Patent Document 2).

Japanese Patent Laying-Open No. 2015-029431 International Publication No. 2014/196204

However, in the case of applying a cell culture container such as Patent Document 1 in which a large number of wells capable of culturing in large quantities are applied at the same time, the inside of the culture container is moved when the container is moved or when suction or addition is performed by medium exchange. There is a risk that the culture medium of the above will flow greatly. As a result, cells and formed spheroids may jump out of the well and move into another well. As a result, the formation efficiency of spheroids is reduced, and it is difficult to obtain spheroids of uniform size. Problems such as becoming.

In Patent Document 2, in order to solve the movement of spheroids from within the well, an inclination (1 to 20 °) is formed, or the depth of the well is made deeper than before (Examples and comparisons in Patent Document 2) See example). However, when using a petri dish-shaped culture container that has been used conventionally, the well is formed deeply with respect to the thickness of the bottom, so that the strength of the bottom is reduced, and there is a risk of breakage, cracking, etc. is there.

Therefore, in the present invention, in a culture substrate in which a plurality of microwells are formed on one culture surface, the migration of spheroids from the microwells is suppressed without increasing the depth of the microwells, and the size is uniform. A cell culture substrate capable of forming a spheroid and a method for producing the same are provided.

The present invention is a culture substrate having a culture surface for culturing cells, wherein the culture surface has a plurality of microwells, and the microwell has a first well having an inclined surface and the first well. A second well existing in the well, and a protrusion at a boundary between the first well and the second well, and the culture surface is a low cell adhesion surface It is characterized by being.

The culture surface preferably has an inclined wall on its outer peripheral surface.
The first well is preferably arranged densely on the culture surface.
The diameter of the opening of the second well is preferably 0.05 mm to 3 mm.
Preferably, the first well has an inclined surface inside, and the inclination angle of the inclined surface is more than 0 ° and less than 90 °.

The depth of the second well is preferably 0.1 to 2 times the diameter of the opening.
The diameter of the opening of the first well is preferably 1.5 to 5 times the diameter of the opening of the second well.
The depth of the first well is preferably 0.1 to 3 times the diameter of the opening.
The combined depth of the first well and the second well is preferably 0.1 to 0.9 times the thickness of the culture surface.

The method for producing a culture substrate of the present invention is a method for producing a culture substrate having a culture surface for culturing cells, the first well having an inclined surface, and being present in the first well. A plurality of microwells are formed on the culture surface so as to have a protrusion at the boundary between the first well and the second well.
In the method for producing a culture substrate, it is preferable to form the second well after forming the first well.
In the method for producing a culture substrate, the first well is preferably formed simultaneously with the culture substrate.
In the method for producing a culture substrate, it is preferable that after forming the first well, a second well is formed in the first well by a laser.
In the method for producing a culture substrate, the protrusions are preferably formed by a laser.
In the method for producing a culture substrate, it is preferable that a low cell adhesion treatment is performed on the culture surface after the formation of the microwell.

According to the present invention, even if a plurality of microwells for forming spheroids are formed on the culture surface of a conventional culture substrate, the movement of cells and spheroids between wells due to the flow of the culture medium (culture solution) is reduced. In addition, it is possible to provide a cell culture substrate capable of culturing a large amount of spheroids having a uniform size.

An example of the top view of the culture substrate of the present invention is shown. An example of the AA ′ cross-sectional view of the culture substrate of the present invention is shown. An example of the top view of the microwell in the culture base material of this invention is shown. An example of the sectional view of the microwell in the culture substrate of the present invention is shown. An example of the enlarged view of the microprotrusion in the microwell in the culture base material of this invention is shown. An example of the variation of the opening shape of the 1st well in the culture base material of this invention is shown. An example of sectional drawing of the 1st well (before 2nd well formation) in the culture substrate of the present invention is shown. An example of the variation of the opening shape of the 2nd well in the culture base material of this invention is shown. An example of a variation of the BB ′ cross-sectional view of the second well in the culture substrate of the present invention is shown.

Hereinafter, embodiments will be described with reference to the drawings. In addition, this invention is not restricted to embodiment described below, It can change suitably.

The culture substrate 1 of the present invention has a culture surface on which at least one surface of the substrate can obtain spheroids in which cells are three-dimensionally aggregated during the culture process, and the culture surface has a plurality of microwells. . Examples of the shape of the culture substrate 1 include petri dishes, flasks, multilayer flasks, microplates, sheets, inserts, and the like, but the shape is not particularly limited. After forming a microwell on a sheet-like member, it may be used by placing it on a container such as a petri dish. In the present specification and drawings, an example in which a microwell 3 is provided on a culture surface 2 of a petri dish will be described.

As shown in FIG. 1, one culture substrate 1 of the embodiment of the present invention mainly includes a bottom portion and a peripheral wall portion. The bottom is configured in a disc shape and has a culture surface 2 with a plurality of microwells 3. The culture surface 2 is formed at the bottom.

The peripheral wall rises from the peripheral edge of the bottom. The shape of the peripheral wall portion is a state in which the peripheral edge portion is erected. The disk-shaped bottom part is formed with a diameter of, for example, 85 mm and a thickness of, for example, 1 mm. The height of the peripheral wall portion is, for example, 20 mm with respect to the bottom portion. In addition, the bottom part and the surrounding wall part are comprised by the integral component. Further, the cell culture substrate 1 may include a lid for covering the opening.

The culture surface 2 in the culture substrate 1 of the present invention is preferably a surface to which cells do not adhere or are difficult to adhere (hereinafter referred to as cell low adhesion surface) in order to form spheroids. In order to form a low cell adhesion surface when adhered to the culture surface 2, the culture surface 2 is treated with low cell adhesion (hereinafter referred to as cell low adhesion treatment), or the container itself is formed of a low cell adhesion resin. May be. Examples of the low cell adhesion treatment include coating with a phospholipid polymer (polymer such as 2-methacryloyloxyethyl phosphorylcholine), poly (hydroxyethyl methacrylate), a fluorine-containing compound, or polyethylene glycol, plasma treatment, Surface treatment such as corona discharge and UV ozone treatment may be performed. Examples of the low cell adhesion resin include silicone resin and resin mixed with a cell low adhesion coating component. Note that the low cell adhesion treatment is not limited to the culture surface 2, and the same treatment may be applied to the peripheral wall portion of the culture surface and the like.

As shown in FIGS. 1 and 2, the culture substrate 1 of the present invention includes a plurality of microwells 3 on a culture surface 2. Spheroids are cultured in each microwell 3. It is preferable that the microwells 3 are densely arranged on the culture surface 2 and that there is no flat surface between the microwells 3 (a non-flat surface). For example, by setting a non-flat surface between the microwells 3, the seeded cells always fall into the microwell 3, so that the cells can be prevented from staying outside the microwell 3. Thereby, it becomes possible to suppress that a cell does not become a spheroid. Here, “non-flat” in the present specification refers to not being horizontal with respect to the bottom surface of the culture surface 2 (the bottom surface of the culture substrate). “Dense” means that the microwells 3 are formed densely and there is no flat surface between the wells. In addition, “cells do not become spheroids” means that the cultured cells are spheroid-like in monolayer culture, single cell suspension culture, non-spherical layered culture, or adhesion culture in which cells adhere to the culture surface 2 and are cultured. It means not to be.

As shown in FIG. 1, the microwell 3 includes a first well 31 and a second well 32. The first well 31 and the second well 32 may be formed integrally, or the second well 32 may be formed after the first well 31 is formed. In this specification, the case where the second well 32 is formed after the first well 31 is formed will be described.
In FIG. 3, the first well 31 is indicated by the opening diameter 311 and the depth 312, the second well 32 is indicated by the opening diameter 321 and the depth 322, and the microwell 3 is indicated by the depth 30.

The second well 32 is formed at the bottom of the first well 31. The second well 32 is formed by opening the bottom of the first well 31 and has the bottom of the opening (hereinafter referred to as the second opening). The second opening refers to the point where the tilt angle of the first well has changed. As the opening shape of the second well 32, as in the example shown in FIG. 8, there are a plurality of openings (circle (a), polygons (b) (c) (e) (f), donut shape (d)). It can be suitably selected from the shape (g) and the like. The shape of the second well 32 can be appropriately selected from a conical shape, a prismatic shape, a U-bottom shape, a V-bottom shape, and the like. In particular, if the shape does not have a flat portion, for example, a U-bottom shape. It is preferable because cells that have fallen into the well are easily collected and a uniform sphere is easily formed. An example of the cross-sectional shape of the second well is shown in FIG.

The diameter 321 of the second opening is preferably 0.05 mm to 3 mm. If the diameter 321 of the second opening is less than 0.05 mm, it may be difficult to take out the spheroids. Moreover, when the diameter 321 of the second opening is more than 3 mm, the spheroids may be likely to jump out of the well. The diameter 321 of the second opening is more preferably 0.1 mm to 2.5 mm, and still more preferably 0.15 mm to 2.0 mm.

The depth 322 of the second well is preferably 0.1 to 2 times the diameter 321 of the second opening. If the depth 322 of the second well is less than 0.1 times the diameter of the second opening, there is a problem that spheroids are easily spilled. In addition, if the depth 322 of the second well is more than twice the diameter 321 of the second opening, it becomes difficult to take out spheroids and the bottom surface strength is lowered. The depth 322 of the second well is more preferably 0.2 to 1.8 times the diameter 321 of the second well, and still more preferably 0.3 to 1.7 times. Note that the depth 322 of the second well indicates the value of the longest portion of the height from the second opening to the bottom.

The first well 31 has an opening (hereinafter referred to as a first opening) and an inclined surface toward the bottom. Moreover, the shape seen from the upper surface of the 1st opening part can be suitably selected from a honeycomb type, a lattice type | mold, a circle | round | yen, etc. as shown in FIG. For example, the honeycomb type is preferable because the distance between adjacent wells becomes equal, so that the same amount of medium can easily enter each well and spheroids can be formed under more uniform conditions. The diameter 311 of the first opening is preferably 1.5 to 5 times the diameter 321 of the second well. If the ratio is less than 1.5 times, it becomes difficult to form the protrusions 5 and the ratio of the second wells 32 is increased, so that the size of the spheroids formed in one microwell 3 is increased. If the medium consumption increases, the medium replacement frequency increases, and there is a possibility that troubles may occur during the culture, or nutrients may not be sufficiently distributed to the cells. If the ratio is more than 5 times, the ratio of the second well 32 becomes small, and the size of the spheroid formed in one microwell 3 becomes small, so that the culture efficiency may be lowered. The diameter 311 of the first opening is more preferably 1.8 to 4.5 times the diameter 321 of the second opening, and even more preferably 2 to 3.5 times.
The depth 312 of the first well is preferably 0.1 to 3 times the diameter 311 of the first opening. If it is less than 0.1 times, depending on the viscosity of the medium, the surface tension of the medium may be lost and the slope may be insufficient. If it exceeds 3 times, the second well cannot be deepened depending on the thickness 21 of the culture surface. The depth 312 of the first well is more preferably 0.13 to 2.5 times the diameter 311 of the first opening, and even more preferably 0.15 to 2 times. The diameter 311 and depth 312 of the first opening can be appropriately adjusted according to the size of the cells to be cultured and the desired spheroid.

Further, the inclined surface of the first well preferably has an inclination angle 313 (θ) of more than 0 ° and less than 90 °. When the inclination angle θ is 0 ° (flat surface), depending on the viscosity of the medium, the medium does not flow to the second well due to the influence of the surface tension, and the spheroid size tends to vary. In addition, when the inclination angle 313 (θ) is 90 ° or more, the spheroids are likely to be difficult to take out because the shape tends to be deep. The inclination angle 313 (θ) is more preferably 5 ° to 80 °, and still more preferably 8 ° to 70 °. Since the optimum angle varies depending on the viscosity of the medium, it can be adjusted as appropriate within the above range.

The first well 31 and the second well 32 are connected to each other. The combined depth 30 of the first well and the second well (that is, the depth of the microwell 3) is 0.1 to 0.9 times the thickness 21 of the culture surface of the culture substrate 1. preferable. If the ratio is less than 0.1 times, a well having a sufficient depth cannot be formed. If the ratio exceeds 0.9 times, the strength of the culture surface 2 of the culture substrate 1 may decrease. It is preferably 0.3 to 0.8 times, and more preferably 0.4 to 0.7 times.

It is preferable to form a protrusion 5 as shown in FIG. 3 and FIG. 4 between the first well 31 and the second well 32 (bonded portion, hereinafter referred to as a boundary portion). The protrusion 5 is for suppressing the cells that have fallen into the second well 32 and the spheroids formed in the second well 32 from moving outside the second well 32. Due to the presence of the protrusions 5, when the culture substrate 1 is tilted at the time of medium exchange or the like, spheroids and cells are blocked by the protrusions 5 and can be retained in the second well 32. It is preferable that the protrusion 5 has a shape protruding at the boundary portion into the second opening. The protrusion 5 protrudes into the second opening as long as the cells fall into the second well 32 through the second opening and the spheroids formed in the second well 43 can be taken out. May be. For example, when the diameter 321 of the opening of the second well is about 250 μm and the desired spheroid diameter is about 150 μm, the protrusion 5 is preferably about 2 μm to 5 μm. The shape of the protrusion 5 is not particularly limited, but the point where the cell or spheroid contacts has a sharp corner so as not to damage the cell or spheroid or leave the cell on the protrusion 5. It is preferable that it is a non-flat surface which does not have.

Microwell 3 in culture substrate 1 of the present invention, per unit area of the culture surface 2, 10 pieces ^/ cm 2 ~ 10000 pieces / cm ^2, is preferably formed. When the number of microwells 3 is less than 10 / cm ² , the number of microwells 3 decreases, and the number of spheroids that can be formed at one time decreases, so that the culture efficiency may decrease. When microwell 3 is 10,000 / cm ^2, greater than that greater number of microwells 3, since the increase in the number of spheroids formed at one time, medium exchange frequency is increased by a medium consumption increases There is a risk that labor may occur during the culture, or the cells may not be sufficiently fed with nutrients. More preferably, it is 15 pieces / cm ² to 5000 pieces / cm ² , and further preferably 20 pieces / cm ² to 1000 pieces / cm ² . The number of microwells 3 can be appropriately adjusted depending on the size of the culture substrate 1 to be used and the size and number of desired spheroids.

The microwell 3 preferably has a uniform well size in order to make the size of the obtained spheroid uniform. Since the size of the spheroid depends on the size of the well, if the size of the well is different, the size of the formed spheroid is not uniform, which is not preferable.

In the culture substrate 1 of the present invention, although not essential, the following configurations may be selected and combined.

Depending on the shape of the culture substrate 1, for example, in the case of a petri dish, there may be a surface that is not wide enough to form a microwell 3 having the same size as the other microwells 3 in the vicinity of the peripheral wall. In this case, if the surface on which the microwell 3 cannot be formed is left as a flat surface, the cells may fall, and the cells may not become spheroids. Therefore, when the surface where the microwell 3 cannot be formed remains, it is preferable to make the surface non-flat. For example, the inclined wall 4 is formed as shown in FIG. 2 (a), the peripheral wall is thickened as shown in FIG. 2 (b), a part forming a non-flat surface is placed, and a partition is attached. Good. Further, the above-mentioned correspondence is not limited to use when the microwell 3 having a uniform size cannot be formed. For example, when the microwell 3 is formed only on a part of the culture surface 2 and the number of microwells 3 is specified. It is also possible to use it when desired.

In order to further suppress the movement of spheroids from inside the microwell 3, a partition may be provided. The partition may be joined to the culture surface 2, may be placed, or may be one that does not contact the culture surface 2 (for example, a drop lid shape). The material of the partition is not particularly limited, but may be the same material as the culture substrate 1 or may be a different material, or may be made of a membrane-like material that passes only the medium without passing through the cells.

The center portion of the first well 31 and the center portion of the second well 32 may or may not match. By making them coincide, the length of the inclined surface of the first well 31 becomes uniform, so that the cells easily fall uniformly. Moreover, by not matching, the well opening has a tilted shape, and the cells can be prevented from popping out when the container is tilted.

Subsequently, a method for producing the culture substrate 1 of the present invention will be described. In addition, a manufacturing method can be suitably changed with the raw material of a base material to be used, a desired shape, a magnitude | size, etc.

The culture substrate 1 main body (the state before formation of the microwell) may be manufactured by a method suitable for the material and size of the substrate. The material of the substrate can be appropriately selected from resin, glass, metal, or a combination thereof. In the case of resin, for example, in addition to acrylic resin, polystyrene resin, polyester resin, polycarbonate resin, polypropylene resin, etc., a mixture of the above-mentioned substances with low cell adhesion, or a colorant (for example, titanium oxide, carbon, etc.) A mixture or the like can be used. The culture substrate can be suitably selected from, for example, injection molding, press molding, vacuum molding, blow molding and the like.

In the microwell 3, the first well 31 and the second well 32 may be formed at a time, and the second well 32 may be formed after the first well 31 is formed. Examples of the method for forming the microwell include a mold (molded together when the base body is manufactured), laser (CO ₂ laser, YAG laser, excimer laser, etc.), nanoimprint, press and the like. When the second well 32 is formed after the first well 31 is formed, the first well 31 is formed in advance with a mold, and then the second well 32 is formed with a laser. It becomes easy to standardize. In addition, when the second well is formed on the resin base material with a CO ₂ laser, the resin is dissolved and vaporized, so that the surface becomes a smooth surface, so that it becomes easy to form a beautiful spherical spheroid. In addition, since the smooth protrusion 5 is formed at the boundary portion, the spheroid is hardly damaged and a preferable form is obtained.

When the second well 32 is formed with a CO ₂ laser after the first well 31 is formed, the intensity of the laser light is preferably 5 to 500 W. The higher the strength, the deeper the second well 32 and the wider the diameter 321 of the second opening. That is, the higher the strength, the larger spheroids can be formed. The irradiation spot is preferably circular, but the shape is not particularly limited as long as a well capable of aggregating cells can be formed. The diameter of the irradiation spot is suitably 20 μm to 1500 μm. The laser irradiation position can also be adjusted as appropriate according to the desired well shape. Further, the strength may be appropriately adjusted depending on the material of the culture substrate 1. For example, 5-30 W is preferable for polystyrene, and 80-200 W is preferable for glass.

When the culture surface 2 of the culture substrate 1 is not a low cell adhesion surface (not molded from a low cell adhesion material), it is necessary to separately perform a cell low adhesion treatment. As a low adhesion treatment method, the culture surface 2 is immersed in a liquid containing a low adhesion substance and fixed to the culture surface 2, or the low adhesion substance is mixed with a UV curable resin and fixed by UV irradiation. And a method of attaching a sheet containing a substance that has low adhesion. Even when the culture substrate 1 is formed of a material having low cell adhesion, the low adhesion treatment may be performed together.
The entire contents of the specification, claims, drawings and abstract of Japanese Patent Application No. 2016-256320 filed on Dec. 28, 2016 are cited here as disclosure of the specification of the present invention. Incorporated.

1: culture substrate, 2: culture surface, 21: thickness of culture surface, 3: microwell, 30: depth of whole microwell, 31: first well, 311: diameter of first well, 312: Depth of first well, 313: inclination angle of first well, 32: second well, 321: diameter of second well, 322: depth of second well, 4: inclined wall, 5 : Projection

Claims (15)

1. A culture substrate having a culture surface for culturing cells,
   The culture surface has a plurality of microwells;
   The microwell has a first well having an inclined surface and a second well existing in the first well, and a boundary between the first well and the second well Has a protrusion on the part,
   The culture substrate, wherein the culture surface is a low cell adhesion surface.
2. The culture substrate according to claim 1, wherein the culture surface has an inclined wall on an outer peripheral surface thereof.
3. The culture substrate according to claim 1 or 2, wherein the first wells are densely arranged on the culture surface.
4. The culture substrate according to any one of claims 1 to 3, wherein the diameter of the opening of the second well is 0.05 mm to 3 mm.
5. The culture substrate according to any one of claims 1 to 4, wherein the first well has an inclined surface therein, and the inclined angle of the inclined surface is more than 0 ° and less than 90 °.
6. The culture substrate according to any one of claims 1 to 5, wherein the depth of the second well is 0.1 to 2 times the diameter of the opening.
7. The culture substrate according to any one of claims 1 to 6, wherein the diameter of the opening of the first well is 1.5 to 5 times the diameter of the opening of the second well.
8. The culture substrate according to any one of claims 1 to 7, wherein the depth of the first well is 0.1 to 3 times the diameter of the opening.
9. The culture substrate according to any one of claims 1 to 8, wherein the combined depth of the first well and the second well is 0.1 to 0.9 times the thickness of the culture surface. .
10. A method for producing a culture substrate having a culture surface for culturing cells,
    A first well having an inclined surface; and a second well existing in the first well; and a protrusion at a boundary between the first well and the second well. And forming a plurality of microwells on the culture surface.
11. The method for producing a culture substrate according to claim 10, wherein the second well is formed after forming the first well.
12. The method for producing a culture substrate according to claim 10 or 11, wherein the first well is formed simultaneously with the culture substrate.
13. The method for producing a culture substrate according to any one of claims 10 to 12, wherein after the formation of the first well, a second well is formed in the first well by a laser.
14. The method for producing a culture substrate according to any one of claims 10 to 13, wherein the protrusion is formed by a laser.
15. The method for producing a culture substrate according to any one of claims 10 to 14, wherein a low cell adhesion treatment is performed on the culture surface after forming the microwell.

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