JP5578779B2 - Spheroid culture method and spheroid culture vessel - Google Patents

Description

  The present invention relates to a method for culturing spheroids using adherent cells, and a culture vessel suitable for spheroid culture.

  In the cell culture technique, attention is focused on spheroid culture capable of constructing a three-dimensional tissue equivalent to that in a living body. Spheroids are also called cell clusters or cell aggregates, and are mainly formed from adherent cells such as hepatocytes and cancer cells. In spheroid culture, cells aggregate in three dimensions to form a lump, so that the specific functions of the cells can be maintained for a long period of time compared to a monolayer culture method in which cells aggregate in two dimensions. Because of these characteristics, spheroid culture is expected to be used in the fields of drug toxicity and pharmacological activity evaluation simulators, hybrid artificial organs, bioreactors, and the like.

  In spheroid culture, it is desired to control the number of cells that gather. Since spheroids do not have a luminal structure corresponding to the capillaries of living bodies, differences occur in the supply of oxygen and nutrients and the removal of waste products between cells in the central part and cells on the outer surface. For example, when the spheroids become large, cells in the central part can become cell-dead due to oxygen deficiency or nutrient deficiency. Such a difference between the state of the cell in the central portion and the state of the cell on the outer surface can be a difference in, for example, evaluating the toxicity and pharmacological activity of the drug. If the state of the cell in the center of the spheroid and the state of the cell on the outer surface can be made equivalent, in the evaluation of the toxicity and pharmacological activity of the drug, not only the life and death of the spheroid but also the uniqueness of the cell in the spheroid Functional improvement or reduction, histological examination of a biologically similar three-dimensional structure formed by spheroids, and the like can be realized. Evaluation of drug toxicity and pharmacological activity by such spheroids can replace part of animal experiments.

  Conventionally, roller bottle culture, spinner flask culture, hanging drop culture, and the like are known as spheroid culture. A 96-well plate for spheroid culture is commercially available.

  Patent Document 1 discloses a cell culture chip provided with cell culture cells that are regularly arranged in an array, honeycomb, or the like on a substrate surface to aggregate and hold seed cells. The cell culture cell is filled with a culture solution in which seed cells are dispersed, and the seed cells are implanted in the cell culture cell. Further, while the cell culture chip is continuously or intermittently rotated horizontally or reciprocally swung, Spheroids can be formed by agglutination culture of seed cells in a cell culture cell.

  Patent Document 2 discloses a cell tissue provided with a tissue body forming region including a first region exhibiting cell adhesion and a second region surrounding the first region and exhibiting lower cell adhesion than the first region. A body microdevice is disclosed. The cells seeded in the first region and the second region of the cell tissue microdevice form an intercellular bond with each other, and the cells are detached from the second region while maintaining the intercellular bond. A cell tissue in which cells are three-dimensionally connected can be formed.

JP-A-2005-27598 JP 2006-122012 A

  For example, in the spheroid culture using the above-described 96-well plate, it is necessary to inject the suspension of adherent cells into the 96-wells, and the operation is complicated, and the size of the spheroid in each hole is controlled. There is a problem that it is difficult to do.

  If the cell culture chip described in Patent Document 1 is used, seed cells implanted in each cell culture cell can be cultured into spheroids of uniform size. However, according to Patent Document 1, in order to form seed cells into spheroids in a cell culture cell, the cell culture chip needs to be horizontally rotated or reciprocally rocked continuously or intermittently. If the cell culture chip is rotated horizontally, the seed cells in the cell culture cell may jump out of the cell due to centrifugal force, etc., or may enter another cell. Spheroids may not be formed. In order to prevent such problems, the rotational speed and duration such as horizontal rotation must be set appropriately after considering the affinity between the seed cell and the cell culture cell. There is a problem that an experiment for setting conditions is required, and quality control in commercial production becomes complicated.

  In the cell tissue microdevice described in Patent Document 2, as a tissue body forming region, a first region showing cell adhesiveness and a second region surrounding the first region and showing lower cell adhesiveness than the first region are shown. A region needs to be formed. These regions can be formed by surface processing of the substrate, etc., but the cell adhesion of the second region such that cells attached to the second region are detached from the second region while maintaining cell-cell bonding with the cells attached to the first region. It is difficult to set gender. In addition, it is difficult to maintain the cell adhesiveness of such a second region constant in a plurality of tissue body formation regions. Therefore, commercial mass production of cell tissue microdevices is difficult, and even if spheroids are formed in the tissue formation region, it is difficult to uniformly control the size of the spheroids.

  The present invention has been made in view of these circumstances, and an object thereof is to provide means suitable for culturing a large amount of spheroids of a uniform size by a simple operation.

  (1) The spheroid culturing method according to the present invention is a method for suspending adherent cells in a culture vessel having a plurality of non-cell-adhesive depressions having a slope that continues from the cell-non-adhesive bottom surface to the lowest position in the direction of gravity. A first step of injecting a suspension, and a second step of allowing the culture vessel into which the suspension has been injected to stand to form spheroids in the recessed portion of the culture vessel.

  The culture container has a dish shape such as a petri dish, for example, and can hold a liquid such as a cell suspension or a culture medium. The bottom surface of this culture container is non-cell-adhesive. Cell non-adhesive refers to surface performance to which adherent cells cannot adhere. Here, the fact that the adherent cells cannot adhere does not mean that the adherent cells do not adhere at all, but the adherent cells do not inhibit the formation of the spheroid targeted by the present invention. The main point is that it should not adhere. Such cell non-adhesion may be defined, for example, by the water contact angle. The water contact angle is preferably 30 degrees or less, and more preferably 10 degrees or less.

  The cell non-adhesiveness described above can be realized, for example, when the cell culture container is formed as a molded article of a highly hydrophilic substance such as polyethylene glycol, polyhydroxyethyl methacrylate, ethylene vinyl alcohol copolymer, or the bottom surface of the cell culture container. However, it can be realized by coating with a substance capable of making the surface performance hydrophilic, such as a surfactant or phospholipid, or by imparting hydrophilicity by surface treatment such as plasma treatment.

  A plurality of recesses are provided on the bottom surface of the culture vessel. The recessed portion is a bottomed hole that is recessed from the bottom surface in the direction of gravity. The surface of the recessed portion is also non-cell-adhesive like the bottom surface. Each recess has a slope that continues to the lowest position. For example, such a recess is formed by laminating a thin film on a flat base material in which the bottom surface and the recess are integrally formed of plastic or the like, the recess is cut on the bottom surface, or a plurality of holes are formed. The thin film may be formed by being deformed so as to sink into the hole.

  In the first step, a suspension of adherent cells is injected into the culture vessel. Examples of adherent cells include cancer cells such as human osteosarcoma cells and hepatocytes. The adherent cells are uniformly diffused into the liquid medium to prepare a suspension. As the medium, a known medium suitable for culturing adherent cells is used. The number of adherent cells is appropriately set in consideration of the capacity of the culture vessel, the size of the recess, the size of the target spheroid, etc., for example, about tens of thousands to several million per mL Adjusted in range. This suspension is developed only once for the culture vessel. That is, an amount of the suspension in which the entire bottom surface of the culture vessel is immersed is injected into the culture vessel.

  In the second step, the culture container into which the suspension has been injected is allowed to stand. At this time, the culture vessel may be maintained in a desired temperature range or humidity range by an incubator or the like. The standing time is appropriately set in consideration of the type of adherent cells, the target spheroid size, and the like, and is, for example, about several hours to several days. By this standing, adherent cells in the suspension settle by gravity and reach the bottom surface or the recessed portion of the culture vessel.

  Adherent cells that have reached the recessed portion of the culture vessel settle along the slope and reach the lowest position of the recessed portion. Since each surface of the recessed portion is non-cell-adhesive, adherent cells settle along the slope by gravity without attaching to the wall surface in the recessed portion. As a result, the adherent cells that have reached the recess are gathered to the lowest position, and the adherent cells are bonded to each other at the lowest position to form a spheroid. In a stationary culture vessel, the number of adherent cells reaching the recess is proportional to the opening area of the recess, so that by providing a plurality of uniform recesses, the size of each of the recesses is equal. Each spheroid is formed.

  (2) The culture container is provided with an inlet for allowing the culture medium to flow into the culture container and an outlet for allowing the culture medium to flow out of the culture container. And a third step of injecting a new medium into the culture container through the inlet and discharging the medium in the culture container through the outlet to the culture container placed. Also good.

  The culture medium inlet and outlet are provided in the dish-shaped culture container as described above. The medium can be flowed into the culture vessel from the outside through the inlet. The medium can be discharged from the inside of the culture vessel to the outside through the outlet.

  In the third step, after the culture vessel is allowed to stand in the second step described above and the adherent cells in the suspension reach the recess, a new medium flows from the outside into the culture vessel through the inlet. And the culture medium in the culture vessel flows out to the outside through the outlet. Thereby, when the adherent cells form spheroids in the recessed portion, the medium in the culture vessel is exchanged.

  (3) The present invention is a spheroid culture vessel comprising a non-cell-adhesive bottom surface and a wall provided surrounding the bottom surface, and a plurality of non-cell-adhesive surfaces formed on the bottom surface. A recessed part is provided, and each said recessed part may be grasped | ascertained as a spheroid culture container which has a slope which continues to the lowest position in a gravitational direction.

  (4) Moreover, each said recessed part may be regularly arrange | positioned at the said bottom face. As a result, the adherent cells that settle in the suspension and reach the recesses are even for the recesses.

  (5) Examples of the inclined surface include hemispherical or conical shapes arranged around the lowermost position.

  According to the present invention, when the suspension of adherent cells is developed once in the culture container and the culture container is left still, the adherent cells in the suspension settle by gravity and are recessed in the culture container. In the recessed part, adherent cells settle along the slope and gather to the lowest position, and adherent cells bind to each other at the lowest position to form spheroids. Thereby, spheroids of a uniform size are cultured in large quantities by a simple operation.

  Hereinafter, preferred embodiments of the present invention will be described. In addition, this embodiment is only one embodiment of this invention, and it cannot be overemphasized that an embodiment can be changed in the range which does not change the summary of this invention.

[Explanation of drawings]
FIG. 1 is a front view of a spheroid culture vessel 10 according to the first embodiment of the present invention. The right side view, left side view, and rear view of the spheroid culture vessel 10 are the same as the front view, and are omitted here. FIG. 2 is a plan view of the spheroid culture vessel 10. FIG. 3 is a bottom view of the spheroid culture vessel 10. In FIG. 3, if the spheroid culture vessel 10 is made of a transparent material, the recessed portion 20 appears through the bottom 12 as in FIG. 4 is a cross-sectional view showing a cross section taken along the line IV-IV in FIG. FIG. 5 is an enlarged plan view showing a region V in FIG. FIG. 6 is an enlarged cross-sectional view showing a region VI in FIG. FIG. 7 is a cross-sectional view showing the first step of the spheroid culture method using the spheroid culture vessel 10. FIG. 8 is an enlarged cross-sectional view showing the second step of the spheroid culture method using the spheroid culture vessel 10. FIG. 9 is a cross-sectional view showing a third step of the spheroid culture method according to the second embodiment. FIG. 10 is a plan view showing the spheroid culture vessel 10 according to the third embodiment. 11 is a cross-sectional view showing a cross section taken along the line XI-XI in FIG. FIG. 12 is an enlarged plan view showing a region XII in FIG. FIG. 13 is an enlarged cross-sectional view showing a region XIII in FIG. FIG. 14 is a cross-sectional view showing a spheroid culture vessel 10 according to the fourth embodiment. FIG. 15 is an enlarged cross-sectional view showing a region XV in FIG. FIG. 16 is a cross-sectional view showing a spheroid culture vessel 10 according to the fifth embodiment. FIG. 17 is an enlarged cross-sectional view showing a region XVII in FIG. FIG. 18 is a schematic diagram showing a method for manufacturing the spheroid culture vessel 10. 19 and 20 show the observation results of the first example. FIG. 21 is a cross-sectional view showing the spheroid culture vessel 10 according to the second embodiment. FIG. 22 shows the observation results of the second example. The scale bars shown in FIGS. 19, 20, and 22 are all 100 μm.

[First Embodiment]
The first embodiment of the present invention will be described below.

[Spheroid culture vessel 10]
As shown in FIGS. 1 to 4, the spheroid culture vessel 10 has a dish shape such as a petri dish, the side wall 11 has a cylindrical shape and surrounds a disk-shaped bottom 12, and faces upward in a substantially vertical direction from the bottom 12. Is standing. A liquid such as a cell suspension or a medium is held in the internal space of the spheroid culture vessel 10 surrounded by the side wall 11 and the bottom 12.

  The inner surface 13 that is the inner side of the container in the side wall 11 and the bottom surface 14 that is the inner side of the container in the bottom 12 are non-cell-adhesive. This cell non-adhesiveness is imparted by coating the inner surface 13 and the bottom surface 14 made of hydrophobic plastic with an amphiphilic polymer. Of both the amphiphilic polymers, the hydrophobic portion is adsorbed on the inner surface 12 and the bottom surface 14, and the hydrophilic portion is exposed to the inside of the container. Thereby, a hydrophilic coating is applied to the inner surface 13 and the bottom surface 14, and adherent cells do not adhere to the inner surface 13 and the bottom surface 14.

[Concave part 20]
As shown in FIGS. 2 and 4, the bottom surface 14 is provided with a plurality of recessed portions 20 that are recessed in the direction of gravity (downward in FIG. 4). The recessed portion 20 is a bottomed hole having a hemispherical shape as will be described later. As shown in FIG. 2, each recess 20 has a circular boundary 21 with the bottom surface 14 in plan view. The plurality of recessed portions 20 are regularly and densely arranged in the entire area of the bottom surface 14.

  As shown in FIG. 5, the distance L between the center O of each recessed portion 20 and the center O of the adjacent recessed portion 20 is constant in each recessed portion 20 in plan view. Further, the distance L is not more than three times the distance R (corresponding to the radius of the circular boundary 21 in plan view) from the center O of each recessed portion 20 to the boundary 21 with the bottom surface 14 in plan view. That is, each recessed part 20 is disposed closer to the adjacent recessed part 20 than the distance R.

  Each recessed part 20 is depressed in a hemispherical shape from the bottom surface 14, and as shown in FIG. 6, each recessed part 20 has a semicircular outline in a longitudinal sectional view. The lowest position 22 in the semicircular contour is the deepest position in each recess 20. The depth to the lowest position 22 in each recess 20 is equal to the distance R described above.

  In each recessed portion 20, a slope 23 inclined in a spherical shape is formed from the boundary 21 to the lowest position 22. The slope 23 is a smooth surface that continues from the boundary 21 to the lowest position 22, and has a point-symmetric shape from the lowest position 22 to the boundary 21. The lowermost position 22 and the slope 23 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.

[Spheroid culture method]
Below, the spheroid culture | cultivation method using the spheroid culture container 10 mentioned above is demonstrated.

The spheroid culture method in this embodiment is roughly divided into the following two steps.
(1) A first step of injecting a suspension of adherent cells into the spheroid culture vessel 10.
(2) A second step in which the spheroid culture vessel 10 into which the suspension has been injected is allowed to stand and spheroids are formed in each recess 20.

  As shown in FIG. 7, in the first step, a suspension 30 of adherent cells is injected into the spheroid culture vessel 10. As the adherent cells, for example, cancer cells such as human osteosarcoma cells or hepatocytes are used depending on the purpose. The adherent cells are uniformly diffused into the liquid medium, and the suspension 30 is adjusted. The number of adherent cells is adjusted, for example, in the range of about tens of thousands to several million per 1 mL of the medium. The suspension 30 is developed only once with respect to the spheroid culture vessel 10.

  In the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. At this time, the spheroid culture vessel 10 is maintained in a desired temperature range or humidity range by an incubator or the like. The standing time is, for example, about 1 day to several days.

  As shown in FIG. 8 (A), in the spheroid culture container 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle down due to gravity, and reach the bottom surface 14 or the recessed portion 20 of the spheroid culture container 10. To reach. In FIG. 8, the downward direction is the direction of gravity. As described above, since the plurality of recessed portions 20 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 20. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 21 of each recess 20 in a plan view (corresponding to the vertical projection area of the recess 20). Since they are the same, the number of adherent cells reaching each recess 20 is equal.

  Adherent cells 31 that have reached the respective recessed portions 20 settle down along the slopes 23 of the respective recessed portions 20 and move toward the lowest position 22 due to gravity. Since the lowest position 22 and the slope 23 of each recess 20 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 23 in each recess 20 but settle along the slope 23 by gravity. As a result, as shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 20 are gathered to the lowest position 22, and the adherent cells 31 are bonded to each other at the lowest position 22, and the spheroid 32. Is formed. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 20, spheroids 32 having an equal size are formed in each recess 20.

[Effects of First Embodiment]
As described above, when the suspension 30 of the adherent cells 31 is developed once in the spheroid culture vessel 10 and the spheroid culture vessel 10 is allowed to stand, the adherent cells 31 in the suspension 30 are separated by gravity. It sinks and reaches each concave part 20, and in each concave part 20, the adherent cells 31 settle along the slope 23 and gather to the lowest position 22. Are combined to form a spheroid 32. As a result, a large amount of spheroids 32 having a uniform size is cultured by a simple operation.

[Second Embodiment]
The second embodiment of the present invention will be described below. In the second embodiment, an inlet 15 and an outlet 16 for medium exchange are provided in the spheroid culture container 10, and medium exchange (third step) in the spheroid culture container 10 is performed in the second step described above. In other respects, the spheroid culture vessel 10 is used to perform spheroid culture except that it is different from the first embodiment. In the following, detailed description will be made only for points different from the first embodiment, and detailed description of points similar to those of the first embodiment will be omitted. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.

  As shown in FIG. 9, the spheroid culture vessel 10 according to the second embodiment is provided with a hole penetrating the side wall 11 in the thickness direction (horizontal direction), and an inlet 15 and an outlet 16 are formed. Yes. The inflow port 15 and the outflow port 16 form a flow path that passes through the side wall 11 and communicates with the inside and outside of the spheroid culture vessel 10. Although only a pair of inlets 15 and outlets 16 are shown in the figure, a plurality of inlets 15 and outlets 16 may be provided for one spheroid culture vessel 10. In addition, the inflow port 15 and the outflow port 16 are preferably provided at symmetrical positions with respect to the center of the spheroid culture vessel 10.

  Although not shown in FIG. 9, a tube or the like is connected to the inflow port 15 on the outside of the side wall 11 to form a flow path through which a new medium flows into the spheroid culture vessel 10 through the inflow port 15. A tube or the like is connected to the outflow port 16 on the outside of the side wall 11 to form a flow path through which the medium in the spheroid culture vessel 10 flows out through the outflow port 16. Moreover, the pump for making a culture medium flow to the inflow port 15 side or the outflow port 16 side may be provided as needed.

  In the second embodiment, in the second step in the first embodiment, a new medium is injected into the spheroid culture container 10 through the inlet 15 and the medium in the spheroid culture container 10 is discharged through the outlet 16. Steps are performed.

  In the third step, the spheroid culture vessel 10 is allowed to stand in the second step described above, and the adherent cells 31 in the suspension 30 reach the respective recessed portions 20 to form spheroids. A new medium is introduced into the spheroid culture vessel 10 from the outside through 15, and the medium in the spheroid culture vessel 10 is flowed out through the outlet 16. Thereby, when the adherent cell 31 forms the spheroid 32 in the recessed part 20, the medium exchange in the spheroid culture vessel 10 is performed.

  In the medium exchange described above, a medium flow occurs in the spheroid culture vessel 10, but the spheroid 32 flows out together with the medium by controlling the medium flow to such an extent that the spheroid 32 does not jump out of the recessed portion 20. Is prevented.

  By the medium exchange described above, it is possible to form a large spheroid 32 that requires a relatively long culture period, or to store the formed spheroid 32 for a long period of time.

[Third Embodiment]
The third embodiment of the present invention will be described below. The third embodiment is the same as the first embodiment except that the shape of the recessed portion 40 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 40 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 40 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.

[Recess 40]
As shown in FIGS. 10 and 11, the bottom surface 14 is provided with a plurality of recessed portions 40 that are recessed in the direction of gravity (downward in FIG. 11). The recessed portion 40 is a bottomed hole having a generally inverted conical shape as will be described later. As shown in FIGS. 10 and 12, each recess 40 has a circular boundary 41 with the bottom surface 14 in plan view. The plurality of recessed portions 40 are regularly and densely arranged in the entire area of the bottom surface 14.

  As shown in FIG. 12, the distance L between the center O of each recessed portion 40 and the center O of the adjacent recessed portion 40 is constant in each recessed portion 40 in plan view. Further, the distance L is not more than three times the distance R from the center O of each recessed portion 40 to the boundary 41 with the bottom surface 14 in plan view (the plan view corresponds to the radius of the circular boundary 41). That is, each recess 40 is arranged closer to the adjacent recess 40 than the distance R.

  Each recessed portion 40 is recessed in a generally inverted conical shape from the bottom surface 14, and as shown in FIG. 13, each recessed portion 40 has a trapezoidal outline whose bottom side is short in a longitudinal sectional view. The lowest position 42 in the trapezoidal contour is the deepest position in each recessed portion 40. The depth to the lowest position 42 in each recess 40 is equal to the distance R described above.

  In each recessed portion 40, the boundary 41 to the lowest position 42 is a slope 43 that is inclined linearly in a longitudinal sectional view. The lowermost position 42 is circular in plan view, and the slope 43 is a smooth surface that continues from the boundary 41 to the lowermost position 42 as a curved surface that surrounds the lowermost position 42. It has a point-symmetric shape up to 41. The lowest position 42 and the slope 43 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.

  Also in this embodiment, the same spheroid culture method as in the first embodiment described above is performed. In the first step, the adherent cell suspension 30 is developed only once with respect to the spheroid culture vessel 10, and in the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. .

  As shown in FIG. 8 (A), in the spheroid culture vessel 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle by gravity, and the bottom surface 14 or the recessed portion of the spheroid culture vessel 10. Reach 40. As described above, since the plurality of recessed portions 40 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 40. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 41 of each recessed portion 40 in a plan view (corresponding to the vertical projection area of the recessed portion 40). Since they are the same, the number of adherent cells reaching each recess 40 is equal.

  As shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 40 settle down along the slopes 43 of the respective recessed portions 40 by gravity and travel toward the lowest position 42. Since the lowest position 42 and the slope 43 of each recess 40 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 43 in each recess 40, but settle along the slope 43 by gravity. As a result, the adherent cells 31 that have reached the respective recesses 40 are gathered to the lowest position 42, and the adherent cells 31 are bonded to each other at the lowest position 42, thereby forming a spheroid 32. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 40, spheroids 32 having an equal size are formed in each recess 40.

  As described above, also in the recessed portion 40 according to the third embodiment, the same effect as the first embodiment is exhibited. Moreover, also in 3rd Embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture container 10 similarly to 2nd Embodiment, and the culture medium of the spheroid culture container 10 is carried out like the 3rd step mentioned above. Exchanges may be made.

[Fourth Embodiment]
Below, 4th Embodiment of this invention is described. The fourth embodiment is the same as the first embodiment except that the shape of the recessed portion 50 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 20 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 50 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.

[Recess 50]
As shown in FIGS. 14 and 15, the bottom surface 14 is provided with a plurality of recessed portions 50 that are recessed in the direction of gravity (downward in FIG. 14). The recessed portion 50 is a bottomed hole having an inverted conical shape as will be described later. Similarly to the recessed portion 20 shown in FIG. 2, each recessed portion 50 has a circular boundary 51 with the bottom surface 14 in plan view. The plurality of recessed portions 50 are regularly and densely arranged in the entire area of the bottom surface 14.

  The distance L between the center O of each recessed portion 50 and the center O of the adjacent recessed portion 50 is the distance R from the center O of each recessed portion 50 to the boundary 51 with the bottom surface 14 (the recessed portion having a circular shape in plan view). The arrangement of the recessed portions 50 on the bottom surface 14 is the same as that of the first embodiment, such as being equal to or less than 3 times the radius of the portion 50. Therefore, detailed description thereof is omitted here.

  Each recessed portion 50 is recessed in an inverted conical shape from the bottom surface 14, and as shown in FIG. 15, each recessed portion 50 has an inverted triangular outline in a longitudinal sectional view. The lowest position 52 in the inverted triangular contour is the deepest position in each recessed portion 50. The depth to the lowest position 52 in each recess 50 is equal to the distance R described above.

  In each recessed part 50, the boundary 51 to the lowest position 52 is a slope 53 that is inclined linearly in a longitudinal sectional view. The slope 53 is a smooth surface that continues from the boundary 51 to the lowest position 52 as a curved surface surrounding the lowest position 52, and has a point-symmetric shape from the lowest position 52 to the boundary 51. The lowest position 52 and the slope 53 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.

  Also in this embodiment, the same spheroid culture method as in the first embodiment described above is performed. In the first step, the adherent cell suspension 30 is developed only once with respect to the spheroid culture vessel 10, and in the second step, the spheroid culture vessel 10 into which the suspension 30 has been injected is allowed to stand. .

  As shown in FIG. 8 (A), in the spheroid culture vessel 10 that has been allowed to stand, the adherent cells 31 in the suspension 30 settle by gravity, and the bottom surface 14 or the recessed portion of the spheroid culture vessel 10. Reach 50. As described above, since the plurality of recessed portions 50 are densely arranged in the entire area of the bottom surface 14, most of the adherent cells 31 reach the recessed portion 50. In addition, the adherent cells 31 are uniformly dispersed in the suspension 30 and the area surrounded by the boundary 51 of each recessed portion 50 in a plan view (corresponding to the vertical projection area of the recessed portion 50). Since they are the same, the number of adherent cells reaching each recess 50 is equal.

  As shown in FIG. 8B, the adherent cells 31 that have reached the respective recessed portions 50 settle down along the slopes 53 of the respective recessed portions 50 and move toward the lowest position 52 due to gravity. Since the lowest position 52 and the slope 53 of each recess 50 are non-cell-adhesive, the adherent cells 31 do not adhere to the slope 53 in each recess 50, but settle along the slope 53 by gravity. As a result, the adherent cells 31 that have reached the respective recessed portions 50 are gathered to the lowermost position 52, and the adherent cells 31 are bonded to each other at the lowermost position 52, whereby the spheroid 32 is formed. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach each recess 50, spheroids 32 having an equal size are formed in each recess 50.

  As described above, also in the recessed portion 50 according to the fourth embodiment, the same effect as the first embodiment is exhibited. Moreover, also in 4th Embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture container 10 similarly to 2nd Embodiment, and the culture medium of the spheroid culture container 10 is carried out like the 3rd step mentioned above. Exchanges may be made.

[Fifth Embodiment]
Hereinafter, a fifth embodiment of the present invention will be described. The fifth embodiment is the same as the first embodiment except that the shape of the recessed portion 60 is different from the recessed portion 20 of the first embodiment. Moreover, even if the recessed part 20 differs, the spheroid culture | cultivation method is performed similarly to 1st Embodiment and 2nd Embodiment. Below, detailed description is given only about the recessed part 60 different from 1st Embodiment, and detailed description is abbreviate | omitted about the point similar to 1st Embodiment. In addition, in each figure, the member etc. which were attached with the same referential mark as 1st Embodiment are the same as that of 1st Embodiment.

[Recess 60]
As shown in FIGS. 16 and 17, the recessed portion 60 is different from the recessed portion 20 of the first embodiment in that the depth to the lowest position 62 is larger than the distance R. Since the arrangement of the plurality of recessed portions 60 provided on the bottom surface 14 is the same as that of the recessed portion 20 of the first embodiment, detailed description thereof is omitted here.

  Each recessed portion 60 is recessed in a substantially hemispherical shape from the bottom surface 14, and as shown in FIG. 17, each recessed portion 60 has a dome-shaped outline in a longitudinal sectional view. The lowest position 62 in the dome-shaped contour is the deepest position in each recess 60. The depth to the lowest position 62 in each recessed portion 60 is larger than the circular radius formed by the boundary 61 between the bottom surface 14 and the recessed portion 60, that is, the distance R.

  In each recessed portion 60, a vertical wall 63 is formed from the boundary 21 in the direction of gravity (downward in FIG. 17), and from the vertical wall 63 to the lowest position 62, a slope 64 inclined in a spherical shape is formed. Yes. The vertical wall 63 and the inclined surface 64 are smooth surfaces that continue from the boundary 61 to the lowest position 62, and have a point-symmetric shape from the lowest position 62 to the boundary 61. The lowest position 62, the vertical wall 63, and the inclined surface 64 are also non-cell-adhesive with the same coating as the inner surface 13 and the bottom surface 14 described above.

  Also in this embodiment, the same spheroid culturing method as that in the first embodiment described above is performed, so that the adherent cells 31 that have reached the respective recessed portions 60 settle down along the slope 64 due to gravity and reach the bottom. At the lowest position 62, the adherent cells 31 are joined together to form a spheroid 32. In the stationary spheroid culture vessel 10, when an equal number of adherent cells reach the respective recessed portions 60, spheroids 32 having an equal size are formed in each recessed portion 60.

  As described above, also in the recessed portion 60 according to the fifth embodiment, the same effect as that of the first embodiment is exhibited. Also in the fifth embodiment, the inlet 15 and the outlet 16 are provided in the spheroid culture vessel 10 as in the second embodiment, and the medium of the spheroid culture vessel 10 is provided as in the third step described above. Exchanges may be made. By increasing the depth of the recessed portion 60, the spheroid 32 is prevented from flowing out of the recessed portion 60 in the medium exchange.

[Method for Producing Spheroid Culture Container 10]
Below, an example of the manufacturing method of the spheroid culture container 10 mentioned above is shown.

  As shown in FIG. 18A, a formwork 80 is made from acrylic or the like. The mold 80 has an internal space 81 processed into a shape corresponding to the inner surface of the spheroid culture vessel 10. The interior space 81 of the mold 80 is filled with a polydimethylsiloxane solution (hereinafter also referred to as “PDMS solution”) and solidified.

  A mold 82 is formed in the mold 80 by solidification of the PDMS solution. As shown in FIG. 18B, the mold 82 is removed from the formwork 80. Then, the surface of the mold 82 is plasma treated.

  As shown in FIG. 18C, the mold 82 is placed in the center of the petri dish-shaped container 83, and the PDMS solution 84 is poured into the container 83 until the mold 82 is immersed. Then, the PDMS solution 84 is solidified in the container 83.

  As the PDMS solution 84 is solidified, the spheroid culture vessel 10 is formed in the vessel 83. As shown in FIG. 18D, the spheroid culture vessel 10 is taken out from the vessel 83, and the mold 82 is further removed. Thereby, mass production of the spheroid container 10 becomes possible.

  In the following, examples of the present invention are described.

(First embodiment)
[Spheroid culture vessel]
The spheroid culture container 10 according to the first embodiment described above was produced. In detail, the spheroid culture container 10 in which the recessed part 20 was formed was shape | molded using polyethylene, and the spheroid culture container 10 was immersed in 70% ethanol for 1 hour, and sterilized. After the sterilized spheroid culture vessel 10 was dried in a clean bench, 2 mL of 4% pluronic F-127 aqueous solution was injected and left overnight in a CO ₂ incubator maintained at 37 ° C. Thereafter, the spheroid culture vessel 10 was washed twice with a phosphate buffer.

[Suspension of adherent cells]
Using human osteosarcoma cells MG63 as adherent cells, the suspension was adjusted to 1 million cells / 2 mL using αMEM medium containing 10% FBS.

[Spheroid culture]
2 mL of a suspension of human osteosarcoma cells MG63 was injected into the spheroid culture vessel 10 and cultured in an incubator (37 ° C., 5% CO ₂ ) for 3 days. After the culture, the spheroids formed in the recess 20 of the spheroid culture vessel 10 were observed using an optical microscope (Leica DM14000). The observation results ((A) 50 times, (B) 100 times) in the recessed portion 20 of the spheroid culture vessel 10 are shown in FIG. 19, and the observation results of the spheroids collected from the spheroid culture vessel 10 ((A) 50 times, (B) 100 times) is shown in FIG.

  As shown in FIG. 19, it was observed that one spheroid was formed for one recess 20. Further, as shown in FIG. 20, it was observed that a large amount of spheroids of almost uniform size were formed. The average diameter (N = 35) of the collected spheroids was 231.3 ± 7.9 μm.

(Second embodiment)
[Spheroid culture vessel]
The spheroid culture container 10 according to the first embodiment described above was produced. Specifically, as shown in FIG. 21, a polydimethylsiloxane thin film 72 was laminated on an acrylic plate 70 in which a plurality of circular holes 71 were formed. The thin film 72 was sandwiched between an acrylic plate 70 and a cylindrical culture tank 73 made of polydimethylsiloxane so that the cell suspension was held in a space surrounded by the thin film 72 and the culture tank 73. By decompressing the inside of each circular hole 71 through the flow path 74 formed in the acrylic plate 70, the thin film 72 was sucked into the circular hole 71 and deformed, and the deformed thin film 72 was used as the recessed portion 20. The surfaces of the thin film 72 and the culture tank 73 were coated with a 4% pluronic F-127 aqueous solution in the same manner as in the first embodiment described above.

[Suspension of adherent cells]
Human osteosarcoma cells MG63 and human liver cancer-derived cells HepG2 are used as adherent cells, respectively, and 4 million cells, 2 million cells, and 1 million cells per spheroid culture vessel 10 using αMEM medium containing 10% FBS. The suspensions were adjusted so that there were 1, 500,000, 250,000, and 125,000, respectively.

[Spheroid culture]
Spheroid culture was performed in the same manner as in the first example, and the spheroids formed in the recessed portion 20 of the spheroid culture vessel 10 were observed using an optical microscope (200 times). The result of human osteosarcoma cell MG63 in each cell number is shown in FIG. 22 (A), and the result of human liver cancer-derived cell HepG2 in each cell number is shown in FIG. 22 (B).

  As shown in FIG. 22, it was observed that spheroids having a size corresponding to the number of human osteosarcoma cells MG63 or human hepatoma-derived cells HepG2 in the suspension were formed in the respective recessed portions 20.

FIG. 1 is a front view of a spheroid culture vessel 10 according to the first embodiment of the present invention. FIG. 2 is a plan view of the spheroid culture vessel 10. FIG. 3 is a bottom view of the spheroid culture vessel 10. 4 is a cross-sectional view showing a cross section taken along the line IV-IV in FIG. FIG. 5 is an enlarged plan view showing a region V in FIG. FIG. 6 is an enlarged cross-sectional view showing a region VI in FIG. FIG. 7 is a cross-sectional view showing the first step of the spheroid culture method using the spheroid culture vessel 10. FIG. 8 is an enlarged cross-sectional view showing the second step of the spheroid culture method using the spheroid culture vessel 10. FIG. 9 is a cross-sectional view showing a third step of the spheroid culture method according to the second embodiment. FIG. 10 is a plan view showing the spheroid culture vessel 10 according to the third embodiment. 11 is a cross-sectional view showing a cross section taken along the line XI-XI in FIG. FIG. 12 is an enlarged plan view showing a region XII in FIG. FIG. 13 is an enlarged cross-sectional view showing a region XIII in FIG. FIG. 14 is a cross-sectional view showing a spheroid culture vessel 10 according to the fourth embodiment. FIG. 15 is an enlarged cross-sectional view showing a region XV in FIG. FIG. 16 is a cross-sectional view showing a spheroid culture vessel 10 according to the fifth embodiment. FIG. 17 is an enlarged cross-sectional view showing a region XVII in FIG. FIG. 18 is a schematic diagram showing a method for manufacturing the spheroid culture vessel 10. FIG. 19 shows the observation results of the first example. FIG. 20 shows the observation results of the first example. FIG. 21 is a cross-sectional view showing the spheroid culture vessel 10 according to the second embodiment. FIG. 22 shows the observation results of the second example.

Explanation of symbols

10 ... Culture container 11 ... Side wall (wall)
14 ... Bottom 15 ... Inlet 16 ... Outlet 20, 40, 50, 60 ... Depressed part 22, 42, 52, 62 ... Lowermost position 23, 43, 53, 64 ..Slope 30 ... Suspension 31 ... Adherent cells 32 ... Spheroid

Claims (1)

A plurality of recessed portions of the non-cell-adhesive to have a slope hemispherical or conical shape continues from the cell non-adhesive bottom surface to the surrounding lowermost position in the gravity direction, the thickness direction side walls rising from the bottom upward A first step of injecting a suspension of adherent cells into a culture vessel having an inlet and an outlet penetrating to
A second step of allowing the culture vessel infused with the suspension to stand to form spheroids in the recessed portion of the culture vessel;
A new culture medium is injected into the culture container through the inflow port and the culture medium in the culture container is discharged through the outflow port to the culture container in which the suspension is injected and left still. A spheroid culture method comprising 3 steps .