JP2018085978A - Culture vessel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a culture vessel that is capable of arranging substance to be cultured such as cells in a uniformly dispersed manner for a plurality of wells.SOLUTION: A culture vessel of the present invention comprises a plurality of wells and partition wall parts. The partition wall part has a shape in which the center is recessed in a height direction, and partitions the wells.SELECTED DRAWING: Figure 3A

Description

  The present invention relates to a culture container for culturing a culture object such as a cell.

  For example, spheroid culture in which cells derived from humans or animals are three-dimensionally aggregated while being artificially cultured in a culture vessel or the like is known. Since spheroid culture can construct a state close to a three-dimensional cell structure in which cells interact with each other in a living body, a more specific function can be derived compared to monolayer culture or the like.

  Here, as a cell culture vessel, a culture vessel or the like in which a depression having a cell low adhesion surface is provided on the lowermost surface of each well serving as a cell accommodating portion has been proposed (see Patent Document 1). There is also known a culture vessel or the like that includes a plurality of wells and a bank portion interposed between adjacent wells, and the well and the bank portion are configured by a continuous curved surface (see Patent Document 2).

JP 2009-50194 A International Publication No. 2012/036011

  By the way, in such a culture container, in order to obtain spheroids (cell aggregates) of uniform size, a plurality of cells to be cultured are uniformly dispersed in each well in the culture container. Arrangement is important and improvements are needed in this regard.

  The present invention has been made to solve the above-described problem, and an object of the present invention is to provide a culture container capable of uniformly dispersing and arranging a culture object such as a cell in a plurality of wells. .

  The culture container of the present invention includes a plurality of wells and a partition wall. The partition wall has a shape in which the center is recessed in the height direction and partitions the wells.

  ADVANTAGE OF THE INVENTION According to this invention, the culture container which can disperse | distribute to-be-cultured objects, such as a cell, uniformly with respect to each well can be provided.

The top view which shows roughly the culture container which concerns on the 1st Embodiment of this invention. FIG. 2 is a vertical sectional view schematically showing the structure of the culture vessel of FIG. 1. The perspective view which expands and shows the structure of the culture base material with which the culture container of FIG. 1 is provided. 3A is a cross-sectional view of the culture substrate shown in FIG. BB sectional drawing of the culture base material shown to FIG. 3A. The perspective view which expands and shows the structure of the culture base material which arranged the partition part in the honeycomb form. CC sectional drawing of the culture | cultivation base material shown to FIG. 4A. Sectional drawing which shows the glass substrate used as the base material of the culture base material of FIG. 3A. Sectional drawing which shows the state which formed the seed hole in the glass substrate of FIG. 5A. Sectional drawing which shows the state which etched the glass substrate which provided the seed hole of FIG. 5B, and formed the well. The flowchart which shows the manufacturing method of the culture base material of FIG. 3A. The top view which shows roughly the culture container which concerns on the 2nd Embodiment of this invention. FIG. 8 is a vertical sectional view schematically showing the structure of the culture vessel of FIG. 7.

Hereinafter, embodiments of the present invention will be described with reference to the drawings.
<First Embodiment>
As shown in FIGS. 1 and 2, the culture container 10 of the present embodiment spheroids (cell aggregates) obtained by three-dimensionally aggregating cells in the course of culturing while culturing cells to be cultured. It is a bottomed cylindrical cell culture container for obtaining. In this culture vessel 10, cells and a culture solution (medium) are accommodated. Note that the shape of the culture vessel 10 is not limited to a bottomed cylindrical shape, and for example, a plate shape or a flask shape may be employed.

  As shown in FIGS. 1 to 3A, a culture vessel 10 includes a resin-made container main body (dish) 16 including a disc-shaped bottom portion 12, a peripheral wall portion 11, and a ring-shaped leg portion 12b, and a plurality of wells ( well) 21 made of glass having a culture surface 15a on which a well 21 is formed. The culture substrate 15 may be configured as a single substrate (single substrate) having a predetermined number of wells, or a plurality of the above-mentioned single substrates having a predetermined number of wells are unitized. It is also possible to apply things. Moreover, such a culture substrate 15 is formed in a rectangular shape. On the other hand, a rectangular through hole 12 a having a slightly smaller size than the culture substrate 15 is formed in the bottom 12 of the container body 16.

  The culture substrate 15 has a culture surface 15a facing the through hole 12a (exposed from the through hole 12a), and the peripheral portion 15b of the culture substrate 15 on the culture surface 15a side is on the bottom surface of the bottom 12 It is attached (joined) to the outer edge portion of the through hole 12a. The peripheral part 15b of the culture base material 15 is configured as a flat part in which the well 21 is not formed, and adhesion with the bottom part 12 of the container body 16 to be joined is enhanced.

  The peripheral wall portion 11 is configured in a shape rising from the peripheral edge portion of the bottom portion 12 (a state in which the peripheral edge portion is erected). In addition, the bottom part 12, the surrounding wall part 11, and the leg part 12b with which the container main body 16 is provided may be comprised by integral components, and each may be comprised by an individual component. The culture vessel 10 can also include a lid for covering the opening at the upper end of the vessel body 16. In addition, as shown in FIG. 2, by using the ring-shaped legs 12 b that protrude toward the bottom of the culture substrate 15 for alignment, it is possible to store a plurality of culture vessels 10 in a stacked state. It becomes possible.

  Here, the structure of the culture substrate 15 will be described in detail. As shown in FIGS. 3A to 3C, the plurality of wells 21 on the culture surface 15a of the culture substrate 15 are compartments (indentations) for accommodating and culturing cells to obtain spheroids. In addition, a partition wall 22 that partitions adjacent wells 21 is provided on the culture surface 15a. The partition wall portion 22 has a shape in which the center (center portion) is recessed in the height direction.

  More specifically, as shown in FIG. 3B, the partition wall 22 is recessed in an arc shape when viewed from the thickness direction of the partition wall 22 (when the AA cross section cut along the ridge line of the partition wall 22 is viewed). It is out. Further, as shown in FIGS. 3B and 3C, the distal end portion of the partition wall portion 22 is configured to have a sharp shape (in a protruding shape). Further, such partition walls 22 are arranged in a lattice pattern as shown in FIG. 3A. Here, when observing the cells cultured in the well 21 with a microscope, the ridgelines of the partition walls 22 arranged in a lattice form shown in FIG. 3A are used as reference for the X-axis and Y-axis in the scanning direction of the microscope. It is also possible to use it. Moreover, since the material of the culture substrate 15 including the well 21 and the partition wall 22 is made of glass, the light transmittance is improved, and the visibility with a microscope can be improved.

  In addition, the ridgeline of the partition part 22 arranged in the grid | lattice form and any side part of the rectangular culture base material 15 main body have a parallel positional relationship. Further, as shown in FIGS. 3A to 3C, the partition wall portions 22 arranged in a lattice shape are configured so that the height of the intersection portion 22a is the highest, while the central portion between the intersection portions 22a. Is configured to be the lowest.

  In the culture container 10 of the present embodiment, a plurality of cells (cell suspension) to be cultured are put into the culture container 10 and then the main body of the culture container 10 is swung so that a concave wall in the center is formed. The cells move relatively easily between the plurality of wells 21 via the portion 22. Thereby, a plurality of cells can be uniformly distributed and arranged in each well 21 in the culture vessel 10. As a result, spheroids (cell aggregates) having a uniform size can be obtained in each well 21. 3A to 3C show an example in which the partition walls 22 are arranged in a grid pattern, but the partition walls 22 may be arranged in a honeycomb pattern as shown in FIGS. 4A and 4B.

Here, it is preferable that h / d, which is the relationship between the depth h of the arcuately recessed portion in the partition wall 22 and the depth d of the well 21, is 0.1 or more and 0.6 or less, and h / d Is more preferably 0.2 or more and 0.5 or less, and h / d is more preferably 0.3 or more and 0.4 or less.
If h / d is 0.1 or more, the cells are smoothly moved between the wells 21, and if it is 0.6 or less, the spheroid cells are not easily moved between the wells 21 during spheroid culture. This is because spheroid cells are stabilized.
Further, as shown in FIGS. 3C and 4B, the depth h of the recess in the partition wall 22 is preferably 10 μm or more and 600 μm or less, more preferably h is 20 μm or more and 500 μm or less, and h is 30 μm or more and 300 μm or less. More preferably. The depth h is appropriately selected depending on the size of the single cell, but if the depth h is 10 μm or more, the single cell can easily get over, and if the depth h is 600 μm or less, the spheroid cell moves between the wells 21 during spheroid culture. This is because spheroid cells are not easily generated and spheroid cells are stabilized.
The depth h of the portion recessed in the above-mentioned arc shape is substantially the same depth for each well when a plurality of wells are formed at substantially equal intervals. On the other hand, when the interval is not equal, the depth h may be different for each well. At this time, it is preferable that h / d satisfies 0.1 or more and 0.6 or less in all the partition walls.

  Further, as described above, the distal end portion of the partition wall portion 22 is configured in a sharp shape (in a protruding shape) as shown in FIGS. 3C and 4B. That is, since there is no flat surface at the boundary portion between the adjacent wells 21, it is possible to prevent the cells from staying at the boundary portion of the well 21 (the seeded cells surely fall into the well 21). It becomes possible to suppress the cells from becoming spheroids. Here, the meaning that a cell does not become a spheroid means that it is a monolayer culture or a single cell suspension culture, a non-spherical layered culture, a culture in which cells are adhered to the culture surface, or a single cell that is not taken up by a spheroid Including things.

Moreover, it is preferable to form the wells 21 per unit area of the culture surface 15a at 10 pieces / cm ^{2 to} 20000 pieces / cm ² . More preferably, 15 / cm ² to 10000 pieces / cm ^2, more preferably, is 20 / cm ² ~ 5000 pieces / cm ^2. In the present embodiment, “˜” indicating a numerical range is used in the sense of including the numerical values described before and after the numerical value as a lower limit value and an upper limit value.

  Further, the surface of each of the plurality of wells 21 and the partition wall 22 (culture surface 15a) is subjected to a surface treatment for suppressing cell adhesion. Specifically, a film (coat layer) using a cell adhesion inhibitor (protein low adhesive) is formed on the surfaces of the well 21 and the partition wall 22. Examples of the cell adhesion inhibitor include phospholipid polymers (such as 2-methacryloyloxyethyl phosphorylcholine), polyhydroxyethyl methacrylate, fluorine-containing compounds, and polyethylene glycol. Since such a coating is formed on the culture surface 15a including the well 21 and the partition wall 22, the cells do not adhere to the culture surface, so that the cells aggregate to form spheroids, The spheroid can be easily taken out from the well 21.

  Next, the manufacturing method of the culture base material 15 with which the culture container 10 is provided is demonstrated mainly based on sectional drawing shown to FIG. 5A-FIG. 5C, and the flowchart shown in FIG. As shown in FIGS. 5A and 6, first, a rectangular glass substrate (for example, 75 mm long × 25 mm wide [or 6 inch square] × 0.5 mm thick non-alkali glass substrate serving as a base material of the culture substrate 15) ) 15c is prepared (S [Step] 1).

Next, a PET (polyethylene terephthalate) film is attached to the upper and lower surfaces (one and the other main surfaces) of the glass substrate 15c (S2). Subsequently, using a CO ₂ laser, as shown in FIGS. 5B and 6, a plurality of seed holes 15 d serving as a base of the well are formed at a predetermined pitch (for example, on the culture surface formation side (upper surface side) of the glass substrate 15 c (for example, Drilling is performed at a pitch of 800 μm (S3). After the formation of the seed hole 15d, the PET film is peeled from the glass substrate 15c (S4), and annealing is performed under the condition that the temperature environment of 710 ° C. is continued for 1 hour (S5).

  Next, after a protective film is attached to the non-culture surface side (lower surface side) of the glass substrate 15c (S6), shower etching is performed by pouring hydrofluoric acid (S7). Thereby, as shown to FIG. 5C, the several well 21 and the partition part 22 are formed on the culture surface 15a. Since the etching in this case is isotropic etching, the depth of the well 21 becomes the depth of the seed hole 15d, and the size of the well 21 in the width direction depends on the etching time. In the shower etching, generation of a residue (an unnecessary product due to a reaction between aluminum and hydrofluoric acid) is suppressed. Moreover, it may replace with such shower etching and may apply the general etching which immerses the whole glass substrate 15c in etching liquid. Examples of the glass that can be used here include soda lime glass, borosilicate glass, quartz glass, and glass for chemical strengthening.

  Thereafter, for example, when a glass substrate for taking a plurality of culture substrates is applied, the glass substrate is cut into substrate units (units of the culture substrate 15) (S8), and further, inspection and cleaning are performed. Perform (S9). Next, after coating the cell surface non-adhesive coating material obtained by hydrolyzing the silane coupling agent on the culture surface 15a at room temperature, the cell non-adhesive coat layer is formed by, for example, heat drying for 2 hours in a 120 ° C temperature environment. It forms on the culture surface 15a (S10).

  Subsequently, since the glass material is discolored in the sterilization treatment with γ-rays, EOG sterilization (60 ° C.) is performed on the glass substrate on which the cell non-adhesive coat is formed without using this. Sterilization using ethylene oxide gas) or autoclave sterilization (sterilization for 20 minutes in 121 ° C. saturated steam) (S11). The culture substrate 15 can be obtained through such sterilization treatment (S12).

  Next, a method for forming a spheroid using the culture vessel 10 according to the present embodiment will be described. First, a plurality of cells (cell suspension) to be cultured are put into the culture container 10 and then the main body of the culture container 10 is swung so that the plurality of cells are separated from the central recessed wall portion 22. Thus, the cells are moved between the wells 21, whereby a plurality of cells are uniformly distributed and arranged in the wells 21 in the culture vessel 10.

  Thereafter, the culture vessel 10 to which the culture medium (medium) has been added is cultured or incubated for several hours to several days, for example, in a culture apparatus maintained in a 5% carbon dioxide gas atmosphere at 37 ° C. under saturated steam. Since the cells in the well 21 are coated with a cell adhesion inhibitor on the inner surface of the well 21, the cells adhere to each other without adhering to the well 21 or the partition wall 22, and spheroids (cell aggregation) Lump) is formed. At this time, the cells aggregate three-dimensionally according to the shape and size of the well 21. Thereafter, by further culturing, the cells constituting the spheroids proliferate and differentiate to display any physiological activity.

  As described above, according to the culture container 10 of the present embodiment, the cells to be cultured can be uniformly distributed and arranged in each well. This makes it possible to obtain a spheroid having a uniform size.

<Second Embodiment>
Next, a second embodiment will be described based on FIGS. In FIGS. 7 and 8, the same components as those in the first embodiment shown in FIGS. 1 and 2 are given the same reference numerals and redundant description is omitted.

  As shown in FIGS. 7 and 8, the culture container 30 according to the second embodiment is replaced with the container body 16 and the rectangular culture substrate 15 provided in the culture container 10 of the first embodiment. A container body 36 and a disk-shaped glass culture substrate 35 are provided. A through hole is not formed in the bottom portion 32 of the container body 36 of the culture container 30, and the disc-shaped culture substrate 35 is formed on the top surface of the bottom portion 32 of the container body 36 as shown in FIGS. 7 and 8. It is joined to. In addition, the culture substrate 35 is provided with a plurality of wells 21 and partition walls 22 having the structure shown in FIG. 3A on the culture surface 35a, similarly to the culture substrate 15.

  Here, unlike the culture vessel 10 in which the culture surface 15a side (flat peripheral edge 15b of the culture substrate 15) is joined to the container body 16, the culture vessel 30 is provided on the back side (non-culture surface) of the culture surface 35a. Side) is joined to the container body 36. For this reason, the well 21 is also formed in the peripheral portion 35 b of the culture substrate 35. By effectively using such a space, cells can be cultured efficiently.

  In addition, it can replace with forming a well in the peripheral part 35b of the culture base material 35, and the peripheral part 35b of the culture base material 35 can also be comprised as a flat part. Numbers (addresses) or marks indicating identification numbers for specifying individual wells may be written on the flat peripheral edge 35b.

  According to the culture container 30 according to the second embodiment, similarly to the culture container 10 of the first embodiment, cells to be cultured can be uniformly distributed and arranged in each well. In addition, it is possible to simplify the structure of the main body of the culture container and improve the efficiency when culturing cells.

  Although the present invention has been specifically described above with reference to the embodiment, the present invention is not limited to the embodiment as it is, and various modifications can be made without departing from the scope of the invention at the implementation stage. For example, some constituent elements may be deleted from all the constituent elements shown in the embodiment, or a plurality of constituent elements disclosed in the above embodiments may be combined as appropriate. For example, instead of the glass culture substrates 15 and 35, resin culture substrates may be applied. In the case of a resin culture substrate, the well seed hole can be formed by die molding, reheat press processing, or the like.

  DESCRIPTION OF SYMBOLS 10,30 ... Culture container, 15, 35 ... Culture substrate, 15a, 35a ... Culture surface of culture substrate, 15b, 35b ... Periphery of culture substrate, 15c ... Glass substrate, 15d ... Seed hole, 16, 36 ... container body, 21 ... well, 22 ... partition wall, 22a ... intersection part of partition wall.

Claims (10)

Multiple wells,
A partition wall having a shape in which the center is recessed in the height direction and partitioning the wells;
A culture vessel comprising: The partition wall is recessed in an arc shape,
The culture container according to claim 1. The tip of the partition wall is configured with a pointed shape,
The culture container according to claim 1 or 2. The partition walls are arranged in a lattice shape or a honeycomb shape,
The culture container according to any one of claims 1 to 3. The partition wall has the highest intersection part height,
The culture container according to claim 4. H / d, which is the relationship between the depth h of the arc-shaped portion of the partition wall and the depth d of the well, is 0.1 or more and 0.6 or less.
The culture container according to any one of claims 2 to 5. The depth h of the recess in the partition wall is 10 μm or more and 600 μm or less.
The culture container according to any one of claims 1 to 6. The material constituting the plurality of wells and the partition wall is glass.
The culture container according to any one of claims 1 to 7. The plurality of wells and the partition wall are provided on a predetermined base material,
A container body to which the substrate is attached;
The culture container according to any one of claims 1 to 8. Each surface of the plurality of wells and the partition wall is subjected to a surface treatment for suppressing cell adhesion,
The culture container according to any one of claims 1 to 9.

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