Method for producing fixed products of cultured cells

Abstract

[Problem] To provide a method with which it becomes possible to appropriately handle a plurality of fixed products of cultured cells. [Solution] A method for producing fixed products of cultured cells, which is a method for producing a plurality of cultured cell specimens using a specimen production apparatus 1 which includes an upper wall 9 and a body part 7 that has a plurality of holes 5 corresponding to the shapes of a plurality of multi-well-shaped tools 3. The method includes: a cultured cell obtaining step in which cultured cells are obtained by culturing cells of interest in the plurality of holes 5; a first fixed product obtaining step in which first fixed products, in which the cultured cells are fixed in the plurality of holes 5, are obtained by introducing a first coagulant into the upper wall 9; and a second fixed product obtaining step in which, after removing the specimen production apparatus 1 from the first fixed products 11 obtained in the first fixed product obtaining step, second fixed products 13 each containing the first fixed product 11 are obtained by fixing a part from which the specimen production apparatus 1 has been removed with a second coagulant.

Description

Method for producing a cultured cell fixture

[0001] This invention relates to a method for producing a cultured cell fixture and the like.

[0002] Japanese Patent No. 6626884 describes a method for in vitro production of a cell layer and a device used for the production method, which enable a cell layer such as an organoid to be arranged, cultured, and examined by a predetermined method. Japanese Patent No. 6698710 describes a multi-well-shaped device. This multi-well-shaped device is a device for cell culture developed from a microtiter plate. This multi-well-shaped device has a container and a plurality of culture dishes arranged in the container. Japanese Patent No. 7009714 describes a cell culture container. Each culture part of the multi-well-shaped device functions as a cell culture container. Thus, devices and methods for culturing organoids and cells are known.

[0003] On the other hand, three-dimensional cell culture models such as spheroids and organoids are minute. Therefore, it is not easy to handle those specimens. Also, it is desired to examine changes in cells under a plurality of conditions for three-dimensional cell culture models created under different conditions.

[0004] Japanese Patent No. 6626884, Japanese Patent No. 6698710, Japanese Patent No. 7009714

[0005] An object of this invention is to provide a method capable of appropriately handling a plurality of cultured cell fixtures and a device used for such a method. Examples of the cultured cell fixture are three-dimensional cell culture models such as spheroids and organoids.

[0006] The above problem is basically based on the finding that by using a container or jig that further combines compartmentalized containers capable of culturing cultured cell fixtures together, the cultured cell fixtures produced under different conditions can be subjected to specimen processing under the same conditions.

[0007] The first invention relates to a specimen preparation device. This specimen preparation device 1 is a device for culturing multiple cell-fixed specimens, such as spheroids, under different conditions. This specimen preparation device 1 has a main body 7 and an upper wall 9. The main body 7 has a plurality of holes 5. Each of the plurality of holes 5 corresponds to the shape of a multi-well instrument 3. Therefore, each hole 5 can accommodate the corresponding multi-well instrument 3. The upper wall 9 is a wall that surrounds the entire set of multiple multi-well instruments when multiple multi-well instruments 3 are placed in the plurality of holes 5.

[0008] The second invention relates to a method for producing multiple cultured cell specimens using a specimen preparation device 1. This method includes a cultured cell acquisition step, a first fixative acquisition step, and a second fixative acquisition step. The cultured cell acquisition step is a step for obtaining cultured cells by culturing target cells in a plurality of wells 5 (preferably multi-well type instruments 3 installed in each of the plurality of wells 5). The first fixative acquisition step is a step for obtaining a first fixative 11 in which cultured cells in the plurality of multi-well type instruments 3 are fixed by introducing a first coagulant into the upper wall 9. The second fixative acquisition step is a step for obtaining a second fixative 13 including the first fixative 11 by removing the specimen preparation device 1 from the first fixative 11 obtained in the first fixative acquisition step, and then fixing the part from which the specimen preparation device 1 was removed with a second coagulant.

[0009] This invention provides a method for appropriately handling multiple cultured cell fixatives, and an apparatus used in such a method.

[0010] Figure 1 is a conceptual diagram illustrating a specimen preparation device. Figure 1(a) shows an example of the configuration of the specimen preparation device, and Figure 1(b) shows the specimen preparation device with a multi-well instrument installed. Figure 2 is a conceptual diagram illustrating an example of a specimen preparation device. Figures 2(a) to (c) show examples of specimen preparation devices having multiple cylindrical holes, and Figures 2(d) to (f) show examples of specimen preparation devices having multiple rectangular holes. Figure 3 is a conceptual diagram illustrating a multi-well instrument. Figure 3(a) is a photograph instead of a drawing showing an example of a multi-well instrument (CuPS). Figure 3(b) is a photograph instead of a drawing showing another example of a multi-well instrument (CuPS). Figure 3(c) is a conceptual diagram showing a view of the multi-well instrument from above. Figure 3(d) is a conceptual diagram illustrating the notches on the outer circumference of the multi-well instrument. Figure 4 is a conceptual diagram illustrating a substance injection device. Figure 4(a) shows a perspective view of the substance injection device. Figure 4(b) shows a rear view of the substance injection device. Figure 4(c) shows the substance injection device set in the specimen preparation device. Figure 5 is a conceptual diagram for explaining the substance volume adjustment device. Figure 6 is a conceptual diagram for explaining the adapter. Figure 6(a) shows an external view of the adapter. Figure 6(b) shows an external view of the adapter connected to the substance injection device containing cultured cell fixation. Figure 6(c) shows a view from below of the adapter connected to the substance injection device containing cultured cell fixation. Figure 6(d) shows the adapter connected to the substance injection device placed in a container. Figure 7 is a conceptual diagram for explaining the containment container. Figure 8 is a conceptual diagram for explaining the method of producing cultured cell fixation. Figure 9 is a diagram for explaining the concept of the method of producing cultured cell fixation. Figure 10 is a conceptual diagram for explaining the fitting rod (jig). Figure 10(a) is a diagram for explaining the external appearance of the fitting rod. Figure 10(b) is a diagram for explaining the cross-section of the fitting rod. Figure 10(c) is a diagram illustrating the relationship between the fitting rod and the multi-well instrument. Figure 11 is a conceptual diagram illustrating the first fixative acquisition process. Figure 11(a) shows the state before gel injection. Figure 11(b) shows the state after the gel has been injected into the specimen preparation device via the substance injection device.Figure 12 is a conceptual diagram showing the removal of the specimen preparation device from the first fixed object obtained in the first fixed object acquisition process. Figure 12(a) shows the first fixed object (specimen preparation device 1 with the gelled substance injection device set in place). Figure 12(b) shows the removal of the specimen preparation device from the substance injection device containing the first fixed object. Figure 12(c) shows the specimen preparation device 1 after it has been removed from the substance injection device containing the first fixed object. Figure 13 is a diagram illustrating an example of the second fixed object acquisition process. Figure 13(a) shows the process of placing paraffin in the containment container. Figure 13(b) shows the substance injection device set in the containment container. Figure 13(c) shows the removal of the substance injection device from the containment container. Figure 13(d) shows the substance injection device after it has been removed from the containment container. Figure 13(e) shows a cross-sectional view of the substance injection device. Figure 14 is a photograph replacing a drawing of the jig actually created in the embodiment. Figure 15 is a photograph replacing a drawing of the first fixing object in the embodiment. Figure 16 is a photograph replacing a drawing of the adapter coupled with the first fixing object installed in a container containing a chemical solution. Figure 17 is a photograph replacing a drawing of the obtained second fixing object. Figure 18 is a photograph replacing a drawing of the obtained specimen.

[0011] Figure 1 is a conceptual diagram illustrating a specimen preparation device. Figure 1(a) shows an example of the configuration of the specimen preparation device, and Figure 1(b) shows the specimen preparation device with a multi-well instrument installed. This specimen preparation device 1 is a device for culturing multiple cell-fixed specimens, such as spheroids, under different conditions. As shown in Figure 1, this specimen preparation device 1 has a main body 7 and an upper wall 9. This specimen preparation device 1 functions as a device for processing multiple compartmentalized containers together. Because of this configuration, specimens from different experimental conditions can be processed under the same conditions using this specimen preparation device.

[0012] The main body 7 has a plurality of holes 5. The main body 7 is usually made of a hard material such as resin or metal. Each of the multiple holes 5 may be a recess formed in the main body 7. The shape of the holes 5 corresponds to the shape of the multi-well instrument 3. Therefore, each hole 5 can accommodate the corresponding multi-well instrument 3. In other words, each hole 5 functions as a multi-well instrument housing for accommodating the multi-well instrument 3. When the main body 7 has a plurality of holes 5, the shapes of each hole 5 may be the same or different. It is preferable that each hole 5 is separated so as not to interfere with each other. In the example in Figure 1, a housing 2 is formed surrounding the upper wall 9. Since the holes 5 correspond to the shape of the multi-well instrument 3 (multi-well instruments 3 can be accommodated or set in the holes 5), not only can cells be cultured using the holes 5 as well, but cells can also be cultured using the multi-well instrument 3.

[0013] In the example shown in Figure 1, a connecting element (projection) 4 for connecting with a substance injection device 21, which will be described later, is formed. In the example shown in Figure 1, two projections 4 are provided around the multiple holes 5 at symmetrical positions. The connecting element 4 only needs to correspond to the connecting element 25 of the substance injection device 21, and if the connecting element 25 of the substance injection device 21 is a projection, the connecting element 4 in the main body 7 may be a corresponding recess. The connecting element (projection) 4 may be, for example, a projection (cylinder, elliptical cylinder, or prism) or a recess (cylindrical recess, elliptical recess, or prism-shaped recess). If there are multiple connecting elements 4, they may be only projections or only recesses, or they may be both projections and recesses.

[0014] In the example shown in Figure 1, there is an adapter coupling groove 6 having a shape corresponding to the adapter coupling column 45, which will be described later. In the example shown in Figure 1, the adapter coupling groove 6 is a groove located on the outside of the opposing upper wall 9 (or opposing wall of the housing 2). This adapter coupling groove 6 is continuous from the bottom edge to the top edge on the outside of the upper wall 9 (or opposing wall of the housing 2).

[0015] In the example shown in Figure 1, there is a removal groove 8 for removing the substance injection device, which is set in the sample preparation device 1, from the sample preparation device. The removal groove 8 is preferably located on the upper wall 9 (or the opposing wall of the housing 2) where the adapter coupling groove 6 is not formed. The removal groove 8 is preferably located on the upper edge of the upper wall 9 (or housing 2). The removal groove 8 is preferably semicircular or arched.

[0016] The multi-well instrument 3 has multiple wells in a single instrument. Cells can be cultured in each well of the multi-well instrument 3. The upper wall 9 is a wall that surrounds the entire set of multi-well instruments when multiple multi-well instruments 3 are placed in multiple holes 5. The upper wall 9 may also be located in the main body 7 of the specimen preparation device 1 and surround it.

[0017] In the example shown in Figure 1, the inner surfaces of the opposing upper walls 9 form a recessed portion 10. In other words, the upper walls 9 are located on the main body 7 of the specimen preparation device 1 and surround the main body 7, thereby forming the recessed portion 10. It is preferable that the substance injection device 21 has a shape corresponding to this recessed portion 10. In this case, it is possible to prevent the substance injection device 21 from shifting after it has been set in the specimen preparation device 1.

[0018] Figure 2 is a conceptual diagram illustrating an example of a specimen preparation apparatus. Figures 2(a) to (c) show examples of specimen preparation apparatuses having multiple cylindrical holes, and Figures 2(d) to (f) show examples of specimen preparation apparatuses having multiple rectangular holes. In the example in Figure 2(a), for example, experiments can be conducted under different conditions for each multi-well instrument 3 installed in each hole.

[0019] Figure 3 is a conceptual diagram illustrating a multi-well apparatus. The multi-well apparatus 3 is a container partitioned by multiple wells (culture grooves). Figure 3(a) is a photograph instead of a drawing showing an example of a multi-well apparatus (CuPS). Figure 3(b) is a photograph instead of a drawing showing another example of a multi-well apparatus (CuPS). Figure 3(c) is a conceptual diagram showing a view of the multi-well apparatus from above. Figure 3(d) is a conceptual diagram illustrating the notches present on the outer circumference of the multi-well apparatus. As shown in Figure 3, the multi-well apparatus 3 has multiple culture sections. An example of the multi-well apparatus 3 is described in Japanese Patent Publication No. 6698710. The multi-well apparatus 3 is suitable for culturing biological materials such as cells. Each culture section 3 is typically a well (a depression, a recess).

[0020] The multi-well apparatus 3 has multiple culture sections in its central region. The multi-well apparatus 3 has a shape in which the central region is surrounded by an outer wall. Preferably, the outer wall of the multi-well apparatus 3 extends to a position higher than the culture sections. As shown in Figure 3, it is preferable that the outer wall of the multi-well apparatus 3 has multiple notches. As shown in Figure 3, the outer wall of the multi-well apparatus 3 may have an outer layer (the region in Figure 3 where multiple notches exist) and an inner layer inside the outer layer. In the example in Figure 3, there are no notches in the inner layer, so the state in which the outer wall surrounds the culture sections can be maintained. In the example in Figure 3, four notches are formed in each multi-well apparatus 3. The number of notches may be one, two, three or more, or there may be no notches. As shown in Figure 3(d), in this example, the notches are formed in an inverted L shape. In this example, each notch is composed of a first notch that extends vertically downward and a second notch that extends laterally from the first notch. The width of the first notch is preferably 1.2 to 20 times the width of the second notch, but may also be 1.5 to 10 times, or 2 to 7 times. Having such a width makes it easier to rotate after inserting the hook portion 19 into the notch 17. The length of the second notch is preferably, for example, 0.5 mm to 1 cm, but may also be 0.5 mm to 5 mm.

[0021] Figure 4 is a conceptual diagram illustrating the substance injection device. Figure 4(a) shows a perspective view of the substance injection device. Figure 4(b) shows a view of the substance injection device from the rear. Figure 4(c) shows the substance injection device set in the specimen preparation device. The substance injection device 21 is an element for injecting a substance into multiple holes 5 of the specimen preparation device 1. As shown in Figure 4, the substance injection device 21 is (partially or entirely) housed within the upper wall 9 of the specimen preparation device 1 and has a substance injection section 23 that has a space connected to the holes 5 of the specimen preparation device 1. In the example of Figure 4(b), the substance injection section 23 has multiple holes corresponding to the multiple holes 5 of the specimen preparation device 1. Therefore, the substance is injected into the multiple holes 5 of the specimen preparation device 1 through the holes of the substance injection section 23. Since multi-well type instruments 3 are set in the multiple holes 5, the substance is injected into the culture grooves of the multi-well type instruments 3.

[0022] As shown in Figure 4, the substance injector 21 may have a coupling element 25 for coupling with the specimen preparation device 1 at a position corresponding to the coupling element 4 of the specimen preparation device 1. The substance injector 21 should have the coupling element 25 at a position where, for example, when the substance injector 21 is set in the specimen preparation device 1, the coupling element 4 of the specimen preparation device 1 and the coupling element 25 of the substance injector 21 will fit together. As explained above, the coupling element 25 of the substance injector 21 only needs to correspond to the coupling element 4 of the specimen preparation device 1, and if the coupling element 25 of the substance injector 21 is a projection, the coupling element 4 in the main body 7 may be a corresponding recess.

[0023] As shown in Figure 4, the substance injector 21 may have a main body 27 and a lower part 29 provided at the bottom of the main body 27. For example, a hole in the lower part 29 of the substance injector 21 communicates with a plurality of holes 5 of the specimen preparation device 1. When the substance injector 21 is set in the specimen preparation device 1, it is preferable that the lower part 29 is housed within the upper wall 9 of the specimen preparation device 1. When the substance injector 21 is set in the specimen preparation device 1, it is preferable that a part of the bottom of the main body 27 is above the removal groove 8 of the specimen preparation device 1. When the substance injector 21 is set in the specimen preparation device 1, a part of the bottom of the main body 27 is above the removal groove 8 of the specimen preparation device 1, making it easier to remove the substance injector 21 from the specimen preparation device 1. In the example in Figure 4, an injection port for injecting a substance is formed on the upper surface of the main body 27.

[0024] As shown in Figure 4, it is preferable that the outer shape of the lower part 29 or the main body 27 of the substance injector 21 has a shape corresponding to the recess 10 of the specimen preparation device 1. In this case, it is possible to prevent them from shifting after the substance injector 21 has been set in the specimen preparation device 1. Also, as shown in Figure 4, the outer shape of the lower part 29 or the main body 27 of the substance injector 21 has an adapter coupling groove 26 which has a shape corresponding to the coupling column 45 of the adapter, which will be described later. In the example in Figure 4, the adapter coupling groove 26 is a groove located on the outside of the opposing walls of the lower part 29 and the opposing walls of the main body 27. This adapter coupling groove 26 is located on the outside of the lower part 29 (or the opposing walls of the housing 2) from the bottom edge to the top edge.

[0025] Figure 5 is a conceptual diagram illustrating a substance quantity adjustment device. The substance quantity adjustment device (gel reduction unit) 31 is an element for adjusting the amount of substance (e.g., gel) present in the substance injection device 21 or the specimen preparation device 1 by inserting it into the substance injection section 23 of the substance injection device 21. The substance quantity adjustment device 31 has a shape that corresponds to part or all of the shape of the substance injection section 23 of the substance injection device 21. In the example shown in Figure 5, the substance quantity adjustment device 31 has a plurality of legs 33. Each leg 33 corresponds to a hole 5 in the specimen preparation device 1. In the example shown in Figure 5, the tip 35 of the leg 33 of the substance quantity adjustment device 31 can reach the hole 5 of the specimen preparation device 1 or approach the vicinity of the hole 5 of the specimen preparation device 1. In particular, the substance quantity adjustment device 31 is an optional element.

[0026] Figure 6 is a conceptual diagram illustrating the adapter. Figure 6(a) shows an external view of the adapter. Figure 6(b) shows an external view of the adapter coupled to a substance injection device containing cultured cells. Figure 6(c) shows a view from below of the adapter coupled to the substance injection device containing cultured cells. Figure 6(d) shows the adapter coupled to the substance injection device placed in a container. The adapter 41 (adapter for post-gelation treatment) is coupled to the substance injection device 21 (substance injection device 21 after the specimen preparation device 1 has been removed) and is a device for treating the cultured cells supported on the substance injection device 21 with a chemical substance. With the substance injection device 21 set in the adapter 41, the adapter 41 is placed in a container. The container contains a solution containing a chemical substance. Then, the cultured cells contained in the adapter 41 can be treated with a chemical substance. The adapter 41 has a frame 43 and connecting columns 45 located on the sides of the frame 43. The connecting column 45 also serves as a handle. The adapter 41 is constructed from a frame 43, preferably with an opening on its bottom. In the example shown in Figure 6, a notch 47 is present in the center of the side of the frame 43. When the adapter 41 is placed in the container, the solution inside the container moves into the adapter 41 through the notch 47 and comes into contact with the cultured cell fixation material. An example of a container is the containment container 51, which will be described later.

[0027] Figure 7 is a conceptual diagram illustrating the containment container. The containment container 51 is a container shaped to connect with the substance injection device 21. The containment container 51 contains, for example, a coagulant (second coagulant). In this state, it connects with the substance injection device 21. In the example of Figure 7, there is also a removal groove 53 on the upper edge of the frame of the containment container 51 for removing the substance injection device 21 from the containment container 51.

[0028] Next, a method for producing cultured cell fixatives will be described. Figure 8 is a conceptual diagram illustrating the method for producing cultured cell fixatives. In Figure 8, S represents a step. As shown in Figure 8, the method for producing cultured cell fixatives includes a cultured cell acquisition step, a first fixative acquisition step, and a second fixative acquisition step. Examples of cultured cell fixatives are three-dimensional cell culture models such as spheroids and organoids. Hereinafter, cultured cell fixatives will be described mainly in terms of spheroids, but this invention is not limited to spheroids. A cultured cell fixative refers to an aggregate of cells formed by the adhesion of cultured cells to each other without adhering to a culture vessel. In the example shown in Figure 8, the steps include a cultured cell acquisition step (S101), a packing step (S102), a first fixative acquisition step (S103), an action step (S104), a second fixative acquisition step (S105), a section preparation step (S106), and a post-processing step (S107) in this order. The method for producing fixed cultured cell specimens involves using a gel or other material (coagulant) to simultaneously hold multiple specimens while maintaining a flat plane, and then treating them with chemical substances. The specimens are then held in place on the same plane using a substance capable of holding specimens, such as a gel or curable resin, while the specimen preparation process is carried out.

[0029] Figure 9 is a diagram illustrating the concept of a method for producing a cultured cell fixation. In other words, the method for producing a cultured cell fixation involves solidifying spheroids with a gel or similar material. When the specimen preparation device 1 is removed, the gel with the spheroids integrated into it can be separated from the substance injection device 21. With the spheroids in this state, the adapter 41 is attached and placed in a container containing a drug solution (chemical substance). This allows the spheroids to be uniformly treated with the drug solution. During these steps, the binding column 45 of the adapter 41 can be grasped and used as a handle to facilitate the work. After removing the adapter 41, the gel fixation 11 containing the spheroids is placed in a containment container 51 loaded with paraffin (coagulant), and the paraffin is solidified. After the paraffin has solidified, when the containment container 51 is removed, a fixation 13 is obtained in which the spheroids are contained in a gel having the shape of wells (culture grooves), and the gel is surrounded by paraffin. In this fixation 13, the spheroids are located in positions that reflect the positions of the wells. Therefore, by excising the layer containing the spheroid, a section (specimen) containing the spheroid can be obtained. The second coagulant is not limited to paraffin.

[0030] A spheroid refers to a state in which cells form simple clusters (aggregates) through mutual adhesion, constructing a three-dimensional structure. The term spheroid, which refers to the three-dimensional culture of cell populations, was introduced in 1971. As the name suggests ("sphere" + "oid"), spheroids are usually spherical in shape. Cells normally adhere to a container via a scaffold, but if there is no scaffold or the scaffold is weak, they will form a three-dimensional structure simply by adhering to each other. Generally, methods that prevent the formation of a scaffold are used to create spheroids by utilizing this characteristic. Known methods for creating spheroids include, for example, methods using non-adhesive containers, methods using structures created with engineering-based microfabrication technology, rotational culture methods where the culture vessel is rotated, and hanging drop methods where three-dimensional structures are built in a droplet.

[0031] Similar to spheroids are "organoids." The term "organoid" is derived from "organ" + "oid," meaning something resembling an organ. Generally, organoids are considered to be tissues obtained by culturing stem cells in three dimensions. Recently, however, the term organoid is also used to refer to cell groups created three-dimensionally by mixing different types of differentiated cells derived from stem cells, or cells that were already differentiated. Therefore, regardless of whether the original cells were all stem cells, it is more appropriate to describe organoids in general terms as "tissues artificially created using cell technology to resemble organs." Biologically, organoids can be described as "organ-like aggregates formed autonomously by artificially mimicking the biological processes in development using means such as drugs or constructing an environment, from stem cells or progenitor cells that contribute to organ formation." Research to date has shown that organoids can reproduce the structure and physiological functions of human organs in detail, and they are being used in disease elucidation and drug development. Research on organoids is progressing due to the trend towards shortening clinical trial periods and replacing animal experiments. Spheroid culture and organoid culture are performed because it has been shown that enabling cells to communicate (interact) directly or indirectly with each other allows them to exhibit functions that are not observed in individual cells.

[0032] Regarding the need to prepare specimens of minute samples, as mentioned above, spheroid and organoid research has revealed cellular functions that could not be observed in conventional two-dimensional planar culture, and techniques for culturing cells in a spherical shape are attracting attention. Although specimen preparation is considered for the analysis of spheroids and organoids, the size of spheroids and organoids is small, about 0.05 mm to 2 mm, and generally the specimens are difficult to handle. When cultured cell fixatives are manufactured based on general preparation methods, the positions of the generated cultured cell fixatives are not consistent, making it difficult to remove multiple specimens simultaneously. In particular, it is common practice to prepare sections in order to analyze the prepared cultured cell fixatives. To prepare sections, it is desirable to cut multiple specimens within the same plane. However, normally, the positions of the generated cultured cell fixatives are not consistent, so it is desirable to have the positions of the cultured cell fixatives approximately on the same plane. As will be described later, by using the method for producing cultured cell fixatives of this invention, the positions of cultured cell fixatives present in multiple wells can be made approximately on the same plane.

[0033] Regarding the need to prepare specimens from multiple samples, when investigating cellular changes in spheroids or organoids under different conditions such as drug treatment or exposure to different environments, it is crucial to be able to simultaneously evaluate multiple conditions. Scientifically, all conditions other than those being investigated must be identical. To ensure identical sample processing, it is desirable to be able to handle multiple samples simultaneously. As described later, by using the method for producing cultured cell fixatives of this invention, it is possible to simultaneously evaluate multiple conditions regarding cellular changes under different conditions such as drug treatment or exposure to different environments.

[0034] Cell Culture Acquisition Step (S101) The cell culture acquisition step is a step of culturing target cells in a plurality of wells 5 (preferably a plurality of multi-well type instruments 3 installed in each of the plurality of wells 5) to obtain cultured cells. In this step, for example, as shown in Figure 1(b), a plurality of multi-well type instruments 3 are installed in the specimen preparation device 1. However, cells may be cultured directly in each of the plurality of wells 5 without installing the multi-well type instruments 3. When using the multi-well type instruments 3, it is preferable to use a jig (fitting rod). The fitting rod is a jig (fitting rod) that makes it easier to install and remove the multi-well type instruments from the specimen preparation device 1. Using the jig, the multi-well type instruments 3 are installed in each of the plurality of wells 5 of the specimen preparation device 1. Culture medium is injected into each well of the multi-well type instrument 3, and the sample to be converted into cells is injected. Then, culture is carried out so that the target cell fixative such as spheroids can be obtained. Methods for obtaining cell fixatives such as spheroids are well known.

[0035] Figure 10 is a conceptual diagram illustrating a fitting rod (jig). Figure 10(a) is a diagram illustrating the external appearance of the fitting rod. Figure 10(b) is a diagram illustrating a cross-section of the fitting rod. Figure 10(c) is a diagram illustrating the relationship between the fitting rod and the multiwell-type instrument 3. The fitting rod is a jig used to remove the multiwell-type instrument 3 from the hole 5. The first embodiment of the fitting rod has a fitting rod body and an alignment rod that penetrates the inside of the fitting rod body so as to be movable in the axial direction of the fitting rod. The second embodiment of the fitting rod has the outer wall of the multiwell-type instrument 3 provided with a plurality of notches, and the end of the fitting rod on the multiwell-type instrument 3 side has a shape (connecting part, hook part) that fits into the plurality of notches. The third embodiment of the fitting rod has the fitting rod body having a lower part of the body on the multiwell-type instrument 3 side, an upper part of the body located on the opposite side of the lower part of the body, and a ring-shaped part located between the lower parts of the body. Furthermore, the width WR of the ring-shaped portion is wider than the width WB of the lower part of the main body, and the length LD of the lower part of the main body is shorter than the length LU of the upper part of the main body. The first to third embodiments may each be any combination. Preferred examples of fitting rods will be described below.

[0036] As shown in Figure 10(c), it is preferable that the end of the alignment rod on the multiwell-type device 3 side is wider than the portion housed inside the fitting rod body. It is also preferable that the end of the alignment rod opposite to the multiwell-type device 3 side is wider than the portion housed inside the fitting rod body. Furthermore, it is preferable that the width of these ends is wider than the width of the hole portion (width of the cavity portion) around the alignment rod inside the fitting rod body. Having such a shape prevents the alignment rod from coming out when it moves inside the fitting rod body. The end of the fitting rod body on the multiwell-type device 3 side may have a shape that fits inside the outer wall of the multiwell-type device 3.

[0037] The end of the fitting rod body on the multiwell-type device 3 side preferably has a hook portion (connecting portion with the multiwell-type device 3) that fits into a plurality of notches. It is preferable that each hook portion has a shape that fits into the corresponding notch. As shown in Figure 10, in this example, the hook portion is formed in an inverted L shape. In this example, each hook portion consists of a body portion that extends vertically downward and a tip portion that extends laterally from the body portion. The width of the body portion is preferably 1.2 to 20 times the width of the tip portion, and may be 1.5 to 10 times, or 2 to 7 times. Having such a width makes it easier to rotate after inserting the hook portion 19 into the notch 17. The length of the tip portion is preferably, for example, 0.5 mm to 1 cm, and may be 0.5 mm to 5 mm.

[0038] Furthermore, it is preferable that the length of the alignment rod, including both ends, is longer than the length of the fitting rod body. Because the alignment rod is longer than the fitting rod body, pressing the outer end allows force to be applied to the inner end, making it easier to remove the fitting rod from the multi-well instrument 3. It is preferable that the alignment rod is 2 mm or longer than the fitting rod body, but it may also be 5 mm or longer, 1 cm or longer, or 1.5 cm or longer (10 cm or less).

[0039] The size of the fitting rod can be adjusted as appropriate to match the size of the corresponding multi-well instrument. The fitting rod body can be molded from, for example, metal, stainless steel, plastic, or ceramic. The alignment rod can be molded from, for example, metal, stainless steel, plastic, elastomer, or ceramic. The end of the alignment rod is preferably made of plastic or elastomer. If the length (axial direction) of the fitting rod body is LB, an example of LB is 2 cm to 30 cm, but it may also be 3 cm to 20 cm, 4 cm to 15 cm, or 5 cm to 15 cm. If the length of the lower part of the body is LD, it may be 0.1 LB ≤ LD ≤ 0.4 LB, 0.2 LB ≤ LD ≤ 0.4 LB, or 0.25 LB ≤ LD ≤ 0.35 LB. In other words, the ring-shaped portion is preferably located on the side of the fitting rod body that is on the multi-well instrument 3 side. If LR is the length of the ring-shaped portion (the axial length of the fitting rod body, which corresponds to the thickness of the ring-shaped portion), then it may be 0.01LB ≤ LR ≤ 0.1LB, 0.02LB ≤ LR ≤ 0.09LB, or 0.025LR ≤ LR ≤ 0.08LB. The length LU of the upper part of the body can be appropriately derived from the above. If WB is the width of the fitting rod body (if the fitting rod body is polygonal, the diameter of the circumscribed circle of the polygon, and so on), then WB should be adjusted to match the size of the multi-well instrument 3. An example of WB is 0.5 cm or more and 4 cm or less, but it may also be 1 cm or more and 2.5 cm or 1 cm or more and 2 cm or less. If WA is the width of the end of the alignment rod, then 0.1WB ≤ WA ≤ 0.9WB, 0.2WB ≤ WA ≤ 0.8WB, or 0.3WB ≤ WA ≤ 0.7WB. If WR is the width of the ring-shaped portion, then 1.01WB ≤ WR ≤ 1.5WB, 1.02WB ≤ WR ≤ 1.3WB, or 1.05WB ≤ WR ≤ 1.2WB.

[0040] Filling process (S102) The filling process is a process in which a filler is filled into multiple notches present in the outer walls of multiple multi-well type instruments 3, before the first fixed object acquisition process. This process is optional. In particular, if a multi-well type instrument 3 is not used, the filling process is unnecessary. In particular, if there are no notches in the multi-well type instrument 3, this process is unnecessary. If there are notches in the multi-well type instrument 3, this process is included so that the solidifying agent does not enter the notches. The filler may be a solidifying agent (e.g., gel) or a resin.

[0041] First Fixed Material Acquisition Step (S103) The first fixed material acquisition step is a step of introducing a first coagulant into the upper wall 9 to obtain a first fixed material 11 in which cultured cells in multiple holes 5 (multiple multiwell-type instruments 3) are fixed. Figure 11 is a conceptual diagram for explaining the first fixed material acquisition step. Figure 11(a) shows the state before the gel is injected (the state in which the substance injection device is attached to the specimen preparation device). Figure 11(b) shows the state after the gel has been injected into the specimen preparation device via the substance injection device. As shown in Figure 11(a), the substance injection device 21 is attached to the specimen preparation device 1. The substance injection device 21 can be used to inject a coagulant such as a gel into multiple holes 5 or into each of the multiple multiwells (culture grooves) present in each of the multiwell-type instruments 3. The following explanation will focus on gels, but the first coagulant is not limited to gels and only needs to be able to fix cultured cells. Coagulants are well known, and examples of coagulants are gels and paraffins. With the substance injection device 21 attached to the specimen preparation device 1, the coagulant is injected. As shown in Figure 11(b), a gel or similar substance will be present at the injection port of the substance injection device. If the amount of gel or other coagulant is too large, the amount of gel or other substance can be adjusted by inserting the substance volume adjustment device (gel reduction unit) shown in Figure 5 into the injection port of the substance injection device. After injecting the coagulant into each well, the coagulant is allowed to solidify. In this way, the gel or other coagulant and the specimen (cultured cells) can be integrated. An example of the first coagulant is a gel. The first fixed object 11 is the portion transferred with gel, and may include the gel in the well, the cultured cells in the well, and the gel at the top of the well.

[0042] Figure 12 is a conceptual diagram showing how the specimen preparation device is removed from the first fixed object obtained in the first fixed object acquisition process. Figure 12(a) shows the first fixed object (the specimen preparation device 1 with the gelled substance injection device 21 set in place). Figure 12(b) shows how the specimen preparation device 1 is removed from the substance injection device 21 containing the first fixed object 11. Figure 12(c) shows the specimen preparation device 1 after it has been removed from the substance injection device 21 containing the first fixed object 11. As shown in Figure 12(a), after gelation, the substance injection device 21 and the specimen preparation device 1 are joined together to form the first fixed object 11. After the gel has solidified, they are separated. As shown in Figure 12(b), the removal groove 8 is located on the upper edge of the upper wall 9 (or housing 2) of the specimen preparation device 1. A removal tool (e.g., a flathead screwdriver) is inserted into this gap. The removal tool is then inserted under the bottom of the main body 27 of the substance injection device 21. In this state, the removal tool is rotated. The torque then removes the specimen preparation device 1 from the substance injection device 21 containing the first fixed object 11. If the removal groove 8 is semicircular or arched, such removal becomes easier. In this way, as shown in Figure 12(c), the specimen preparation device 1 is removed from the first fixed object 11 (and the substance injection device 21 containing it). Then, for example, a specimen can be produced in which spheroids are embedded in a gel and positioned on the same plane. In the example shown in Figure 12, cultured cells such as spheroids are embedded in a coagulant and are positioned approximately on the same plane. In the example shown in Figure 12, the first fixed object 11 is exposed after the specimen preparation device 1 has been removed. As will be described later, various treatments may be performed on this exposed first fixed object 11.

[0043] Action Step (S104) The action step is a step for causing the cultured cells contained in the first fixture 11 to act on a chemical substance by treating the first fixture 11 with the chemical substance after the first fixture acquisition step. For example, the substance injector 21 containing the first fixture 11 is attached to the adapter 41. Then, the adapter 41 to which the substance injector 21 is attached is immersed in a solution carrying a solution containing the chemical substance. Then, as shown in FIG. 6(d), the solution moves into the adapter 41 through the notch 47 of the adapter 41 and contacts the first fixture 11. In this way, the first fixture 11 can be treated with the chemical substance. At this time, if a set of these tools is used, the first fixture 11 (for example, spheroids fixed to a gel) can be appropriately treated with a small amount of the chemical substance.

[0044] Second Fixture Acquisition Step (S105) The second fixture acquisition step is a step of obtaining a second fixture 13 including the first fixture 11 by fixing the portion from which the specimen preparation device 1 has been removed with a second coagulant after removing the specimen preparation device 1 from the first fixture 11 obtained in the first fixture acquisition step. This step may be performed after the action step or before the action step.

[0045] Figure 13 is a diagram illustrating an example of the second fixed object acquisition process. Figure 13(a) shows the process of placing paraffin in the containment container. Figure 13(b) shows the substance injection device 21 set in the containment container 51. Figure 13(c) shows the process of removing the substance injection device 21 from the containment container 51. Figure 13(d) shows the state after the substance injection device 21 has been removed from the containment container 51. Figure 13(e) shows a cross-sectional view of the substance injection device 21. If the second fixed object acquisition process is performed after the operation process, the adapter 41 is moved from the container. As the adapter 41 moves, the substance injection device 21 (and the first fixed object 11) also move from the container. Meanwhile, as shown in Figure 13(a), a coagulant such as paraffin is placed in the containment container. In that state, as shown in Figure 13(b), the substance injection device 21 is set in the containment container 51. The containment container 51 is a container having a shape that connects to the substance injection device 21. In this state, the paraffin is solidified. As shown in Figure 13(c), the substance injection device 21 is removed from the containment container 51 using a removal jig. As shown in Figure 13(d), the paraffin solidifies in a state that reflects the shape of the bottom surface of the containment container 51. In this way, a second fixation object 13 containing the first fixation object 11 can be obtained. In this case, as shown in Figure 13(e), a second fixation object 13 is obtained in which the paraffin surrounds the first fixation object 11 while maintaining the shape of the first fixation object 11 (shape of the culture groove).

[0046] The process may further include a step after the second fixative acquisition step in which the second fixative is treated with a chemical substance to allow the cultured cells contained in the second fixative to interact with the chemical substance. The second action step may be performed after sections have been obtained in the section preparation step described later.

[0047] Section Preparation Step (S106) The section preparation step is a step of preparing sections using the second solid 13. The second solid 13 is, for example, the first solid 11 surrounded by paraffin while maintaining the shape of the first solid 11. Therefore, in the second solid 13, specimens such as spheroids are maintained on the same plane. The second solid 13 is cut to a thickness including this same plane. Then, specimens cultured and experimented under a plurality of conditions will be included in one section. Since this includes a plurality of cultures cultured and experimented under the same conditions, it can be accurately evaluated.

[0048] Post-treatment Step (S107) The post-treatment step is a step of performing treatment on the obtained sections. The post-treatment step may include the above-described action steps. Also, the obtained sections may be observed. Further, the obtained sections may be made into specimens. These steps are well-known.

[0049] Note that, after the second solid acquisition step, an action step may be performed. In this case, the action step is a step of causing the cultured cells contained in the second solid to act on a chemical substance by treating the second solid with the chemical substance after the second solid acquisition step. A first action step may be performed before the second solid acquisition step, and a second action step may be performed after the second solid acquisition step.

[0050] Each jig made of resin was created using a 3D printer. Each jig can also be manufactured using a mold. FIG. 14 is a photograph replacing a drawing showing the jig actually created in the example. A multi-well-shaped instrument 3 was installed in the specimen preparation device 1. In that state, spheroids were cultured. After culturing the spheroids, the substance injector 21 was set in the specimen preparation device 1. Gel was injected into the specimen preparation device 1 through the substance injector 21. The injected gel also reached the culture grooves of the multi-well-shaped instrument 3. In that state, the specimen preparation device 1 was left standing still to solidify the gel. In this way, solidified spheroids were obtained. Then, the specimen preparation device 1 was separated from the substance injector 21.

[0051] Figure 15 is a photograph that replaces the drawing showing the first fixed object in the embodiment. In the example shown in Figure 15, the image is inverted vertically for clarity. As shown in Figure 15, the first fixed object 11 contained a gel that reflected the shape of the culture groove containing the spheroid. The first fixed object 11 was connected to the adapter 41. The adapter 41 connected to the first fixed object 11 was placed in a container containing the drug solution.

[0052] Figure 16 is a photograph, replacing the diagram, showing the adapter coupled to the first fixed object placed in a container containing the chemical solution. In this example, the chemical solution was present at a higher level than the spheroid, and the chemical solution acted with the spheroid via the gel. Paraffin was poured into the containment container 51. Then, the first fixed object 11, after the chemical solution had acted on it, was placed in the containment container 51. After waiting for the paraffin to solidify, the second fixed object 13 was obtained. The containment container 51 and the second fixed object 13 were separated.

[0053] Figure 17 is a photograph, replacing the drawing, showing the second fixed object obtained. The second fixed object obtained in this way was a solid object containing spheroids on a certain plane. Therefore, by excising the layer containing the spheroids, a section (specimen) containing spheroids could be obtained.

[0054] Figure 18 is a photograph of the obtained specimen, which replaces the diagram. As shown in Figure 18, this method allowed for the appropriate acquisition and evaluation of spheroids.

[0055] This invention can be used in fields such as physicochemical instruments for cell culture and cell culture.

[0056] 1 Specimen preparation device 3 Multiwell instrument 5 Hole section 7 Main body 9 Upper wall 11 First fixing object 13 Second fixing object

Claims

1. A method for producing multiple cultured cell specimens using a specimen preparation device (1), comprising: a main body (7) having a plurality of holes (5) corresponding to the shapes of a plurality of multi-well instruments (3); and an upper wall (9) surrounding the entire perimeter of the plurality of multi-well instruments (3) when the plurality of multi-well instruments (3) are placed in the plurality of holes (5), the method comprising: a cultured cell acquisition step of culturing target cells in the plurality of holes (5) to obtain cultured cells; a first fixation acquisition step of introducing a first coagulant into the upper wall (9) to obtain a first fixation in which the cultured cells in the plurality of holes (5) are fixed; and a second fixation acquisition step of removing the specimen preparation device (1) from the first fixation (11) obtained in the first fixation acquisition step, and then fixing the portion from which the specimen preparation device (1) was removed with a second coagulant to obtain a second fixation (13) including the first fixation (11), A method for producing fixed cultured cell products.

2. A method for producing a cultured cell fixative according to claim 1, wherein the cultured cells are spheroids.

3. A method for producing a cultured cell fixation product according to claim 1, further comprising a filler filling step, which is a step of filling a plurality of notches present on the outer wall of the plurality of multi-well type instruments (3) with fillers, before the first fixation product acquisition step.

4. A method for producing a cultured cell fixative according to claim 2, further comprising a step of treating the first fixative (11) with a chemical substance after the first fixative acquisition step, thereby causing the cultured cells contained in the first fixative (11) to react with the chemical substance.

5. A method for producing a cultured cell fixative according to claim 2, further comprising a step of treating the second fixative with a chemical substance before the second fixative acquisition step, thereby causing the cultured cells contained in the second fixative to react with the chemical substance.

6. A method for producing a cultured cell fixative according to any one of claims 1 to 5, further comprising a sectioning step of preparing sections using the second solid material.

7. A specimen preparation device (1) comprising: a main body (7) having multiple holes (5) corresponding to the shapes of multiple multi-well instruments (3); and an upper wall (9) that surrounds the entire perimeter of the multiple multi-well instruments when the multiple multi-well instruments (3) are placed in the multiple holes (5).

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