JP2010233456A - Cell assembly-forming tool, cell assembly-culturing tool, cell assembly-transferring kit and method for culturing cell assembly - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cell assembly-forming tool enabling the floating cell assemblies to be precisely arranged on a cell-bondable surface. <P>SOLUTION: The cell assembly-forming tool (1) includes a first base material (10) having through-holes (13) formed therein, a second base material (20) separably stuck to the surface (12) of one side of the first base material (10) and covering the one side of the through-holes (13), and cell-non-bondable wells (30) formed by covering the one side of the through-holes (13) with the second base material (20). <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a cell assembly forming device, a cell assembly culture device, a cell assembly transfer kit, and a cell assembly culturing method, and in particular, a device for precisely placing a floating cell assembly on a cell adhesive surface. And a method.

  Attempts have been made to induce differentiation of undifferentiated stem cells such as ES cells (Embryonic Stem Cells) and iPS cells (Induced Pluripotent Stem Cells) into various functional cells such as hepatocytes, cardiomyocytes, and neurons. . As a differentiation induction method, first, in a culture solution, a cell aggregate called an embryoid body is formed in a floating state from a stem cell, and then the embryoid body is adhered to a cell adhesive surface. A method of adding a differentiation-inducing factor to an embryoid body is used (for example, Non-Patent Document 1 and Non-Patent Document 2).

  In such differentiation induction methods, conventionally, floating embryoid bodies are adhered to the cell-adhesive surface using a hanging drop method or a floating embryoid body formed in a non-cell-adhesive culture vessel is collected. Then, the culture solution in which the embryoid body is dispersed is put into a cell-adhesive culture dish using an instrument such as a pipette.

Exp. Physiol. 85, 645-651, 2000 Mol. Med. 6, 88-95, 2000

  However, in the above prior art, a large number of floating embryoid bodies are collectively transferred to the cell-adhesive surface. For example, there is a problem that a plurality of floating embryoid bodies adhere to each other in a culture solution and fuse. There was a problem that a plurality of embryoid bodies adhered on the cell adhesive surface in a non-uniform distribution.

  The present invention has been made in view of the above problems, and is a cell assembly forming device, a cell assembly culture device, and a cell assembly transcription capable of precisely arranging a floating cell assembly on a cell adhesive surface. It is an object of the present invention to provide a kit and a method for culturing cell aggregates.

  A cell assembly forming device according to an embodiment of the present invention for solving the above-described problem is detachably bonded to a first base material in which a through hole is formed and a surface on one side of the first base material. A second base material covering the one side of the through hole, and a cell non-adhesive well formed by covering the one side of the through hole with the second base material. To do. ADVANTAGE OF THE INVENTION According to this invention, the cell aggregate formation instrument which can arrange | position the floating cell aggregate precisely on a cell adhesive surface can be provided.

The area of the bottom surface of the well may be in the range of 1 × 10 ² to 2 × 10 ⁶ μm ² . In this way, a cell aggregate having an appropriate size can be efficiently formed. The well may be formed in a tapered shape in which the area of the opening on the other surface of the first base material is smaller than the area of the bottom surface. In this way, the position where the cell aggregate is transferred can be controlled more precisely. Further, the cell assembly forming device is a cell assembly forming device used in combination with a culture device having a cell adhesive surface, and the other surface of the first substrate is used as the cell adhesive surface. It is good also as having a positioning structure which contacts the said culture device in the state contact | abutted and fixes a mutual relative position. In this way, the position where the cell aggregate is transferred can be controlled more precisely.

  A cell assembly transcription kit according to an embodiment of the present invention for solving the above-described problems is any one of the above-described cell assembly formation device and the surface of the other side of the first base material of the cell assembly formation device. And a culture device having a cell-adhesive surface that abuts. ADVANTAGE OF THE INVENTION According to this invention, the cell aggregate transcription | transfer kit which can arrange | position the floating cell aggregate precisely on a cell adhesive surface can be provided.

  In addition, at least one of the cell assembly forming device and the culture device is in contact with the other in a state where the surface of the other side of the first base material is in contact with the cell adhesive surface, and is relative to each other. It is good also as having a positioning structure which fixes various positions. In this way, the position where the cell aggregate is transferred can be controlled more precisely.

  A cell assembly culture device according to an embodiment of the present invention for solving the above-described problem is a cell adhesive surface that abuts the surface on the other side of the first base material of any one of the above-described cell assembly formation devices. And a positioning structure for fixing the relative positions of the first and second base members in contact with the cell assembly-forming device in a state where the other surface of the first base member is in contact with the cell adhesive surface. It is characterized by that. ADVANTAGE OF THE INVENTION According to this invention, the cell aggregate culture instrument which can arrange | position the floating cell aggregate precisely on a cell adhesive surface can be provided.

  The cell assembly culturing method according to an embodiment of the present invention for solving the above-described problem is an assembly formation step of forming a suspended cell assembly in the well of any one of the above-described cell assembly formation devices. And the other surface of the first substrate of the cell assembly forming device is brought into contact with a predetermined cell adhesive surface, and the cell assembly in the well is allowed to settle on the cell adhesive surface. And a transfer step of peeling the second base material from the first base material, and an assembly culture step of culturing the cell aggregate adhered to the cell adhesive surface. ADVANTAGE OF THE INVENTION According to this invention, the culture method of the cell aggregate which can arrange | position a floating cell aggregate on a cell adhesive surface precisely can be provided.

  According to the present invention, there are provided a cell assembly forming device, a cell assembly culture device, a cell assembly transcription kit, and a cell assembly culture method capable of precisely placing floating cell assemblies on a cell adhesive surface. can do.

It is a perspective view which shows the 1st base material and the 2nd base material which comprise an example of the cell assembly formation instrument which concerns on one Embodiment of this invention. It is sectional drawing of the 1st base material and the 2nd base material which were cut | disconnected by the surface which passes the II-II line shown in FIG. It is a perspective view which shows an example of the cell assembly formation instrument which concerns on one Embodiment of this invention. It is sectional drawing of the cell assembly formation instrument cut | disconnected by the surface which passes along the IV-IV line shown in FIG. It is the perspective view which looked at the cell assembly formation instrument shown in FIG. 3 from the 2nd base material side. It is a perspective view which shows a mode that the 2nd base material of the cell assembly formation apparatus shown in FIG. 5 is peeled from a 1st base material. It is sectional drawing which shows the 1st base material which comprises the other example of the cell assembly formation instrument which concerns on one Embodiment of this invention. It is sectional drawing which shows the other example of the cell assembly formation instrument which concerns on one Embodiment of this invention. It is explanatory drawing which shows the main processes contained in an example of the cultivation method of the cell aggregate | assembly which concerns on one Embodiment of this invention. It is a perspective view which shows a mode that the cell assembly formation instrument which concerns on one Embodiment of this invention is immersed in a culture solution within a culture container, and is culture | cultivated. It is sectional drawing of the cell assembly formation apparatus and culture container cut | disconnected by the surface which passes along the XI-XI line shown in FIG. It is sectional drawing of the cell assembly formation apparatus and culture container shown in FIG. It is a perspective view which shows an example of the cell assembly formation instrument which has the positioning structure which concerns on one Embodiment of this invention. It is a perspective view which shows an example of the cell aggregate formation instrument shown in FIG. 13, and the cell aggregate culture instrument which has a positioning structure. It is a perspective view which shows a mode that the cell aggregate formation instrument shown in FIG. 14 was made to contact | abut to a cell aggregate culture instrument. It is a perspective view which shows a mode that a cell aggregate is cultured with the cell aggregate culture instrument shown in FIG. It is a perspective view which shows an example of the cell assembly culture instrument which has the positioning structure which concerns on one Embodiment of this invention. It is a perspective view which shows a mode that the cell aggregate formation instrument was made to contact | abut to the cell aggregate culture instrument shown in FIG. It is explanatory drawing which shows an example of the microscope picture image | photographed in the Example which performed formation and transcription | transfer of the cell aggregate using the cell aggregate formation instrument and mouse | mouth ES cell which concern on one Embodiment of this invention. It is explanatory drawing which shows an example of the microscope picture image | photographed in the Example which performed formation and transcription | transfer of the cell aggregate using the cell aggregate formation instrument which concerns on one Embodiment of this invention, and a primary rat hepatocyte. It is explanatory drawing which shows an example of the microscope picture image | photographed in the Example which formed and transferred the cell aggregate using the cell aggregate formation instrument and HepG2 cell which concern on one Embodiment of this invention. It is explanatory drawing which shows the other example of the microscope picture image | photographed in the Example which formed and transferred the cell aggregate using the cell aggregate formation instrument and HepG2 cell which concern on one Embodiment of this invention.

  Hereinafter, a cell assembly formation device, a cell assembly culture device, a cell assembly transfer kit, and a cell assembly culture method according to an embodiment of the present invention will be described with reference to the drawings.

  FIG. 1 is a perspective view showing a first base material 10 and a second base material 20 constituting an example of a cell assembly forming device (hereinafter referred to as “forming device 1”) according to the present embodiment. FIG. 2 is a cross-sectional view of the first base material 10 and the second base material 20 cut along a plane passing through the line II-II shown in FIG. FIG. 3 is a perspective view of the forming instrument 1 having the first base material 10 and the second base material 20 shown in FIGS. 1 and 2. 4 is a cross-sectional view of the forming instrument 1 cut along a plane passing through the line IV-IV shown in FIG. FIG. 5 is a perspective view of the forming instrument 1 shown in FIG. 3 as viewed from the second base material 20 side. FIG. 6 is a perspective view showing a state where the second base material 20 of the forming instrument 1 shown in FIG.

  As shown in FIGS. 1 to 6, the forming instrument 1 has a first base material 10 and a second base material 20. In the present embodiment, each of the first base material 10 and the second base material 20 is a flat plate-shaped body having a predetermined thickness. That is, the first base material 10 has a flat upper surface 11 and a lower surface 12, and the second base material 20 also has a flat upper surface 21 and a lower surface 22. In addition, the shape of the 1st base material 10 and the 2nd base material 20 is not restricted to a tetragon | quadrangle, For example, it can be set as another polygon, circular, an ellipse, and other shapes.

  The material constituting the first base material 10 and the second base material 20 is not particularly limited. For example, polystyrene, polyethylene, polypropylene, polycarbonate, polyamide, polyacetal, polyester (polyethylene terephthalate, etc.), polyurethane, polysulfone, polyacrylate, Synthetic resins such as polymethacrylate (polymethyl methacrylate (PMMA), etc.), polyvinyl, etc., silicon-based resins such as PDMS (Poly-Dimethylsiloxane), synthetic rubbers such as EPDM (Ethylene Propylene Diene Monomer), natural rubber, glass, ceramics, stainless steel One type or two or more types selected from the group consisting of metal materials such as steel can be used. The material constituting the first substrate 10 and the material constituting the second substrate 20 may be the same or different.

  A through hole 13 is formed in the first base material 10. The through hole 13 is formed so as to penetrate from the upper surface 11 to the lower surface 12 of the first base material 10. In the present embodiment, a plurality of through holes 13 are formed. The plurality of through holes 13 are regularly arranged at regular intervals.

  For forming the through hole 13, any processing method selected according to the purpose can be used. That is, for example, the through-hole 13 can be formed in the previously formed first base material 10 by drilling using a machining center or the like, optical fine processing using a laser, etching, embossing, or the like. Further, for example, the through hole 13 can be formed in the first base material 10 simultaneously with the molding of the first base material 10 by injection molding, press molding, stereolithography, or the like.

  The second substrate 20 is detachably bonded to the lower surface 12 of the first substrate 10. Here, “peelable adhesion” is a strength that does not allow the second base material 20 to peel from the first base material 10 while the cell assembly is formed using the forming tool 1 as described later. As shown in FIG. 6, the second base material 20 can be peeled off from the first base material 10 according to a tool such as tweezers P or an operator's hand. It is.

  Such “peelable adhesion” can be performed by any technical method such as selection of materials constituting the first base material 10 and the second base material 20 and use of an adhesive exhibiting an appropriate strength of adhesive force. This can be achieved by adopting means.

  That is, for example, a portion of the lower surface 12 of the first base material 10 that is bonded to the peripheral portion of the opening 13b below the through hole 13 or the upper surface 21 of the second base material 20 is bonded to the peripheral surface. It can be formed of an adhesive material that can be in close contact only by being pressed.

  In this case, for example, the lower surface 12 of the first base material 10 and the upper surface 21 of the second base material 20 are combined, and the first base material 10 and the second base material 20 are simply pressed and used. The second base material 20 can be detachably bonded to the first base material 10 without doing so.

  Such an adhesive material can be appropriately determined depending on the combination with the material constituting the mating surface. Specifically, for example, among the above-mentioned materials, a silicon-based resin such as PDMS, a synthetic rubber such as EPDM, and natural rubber can be used. Among these, a silicon-based resin can be preferably used, and PDMS is particularly used. It can be preferably used.

  In addition, the whole 1st base material 10, the whole 2nd base material 20, the whole lower surface 12 of the said 1st base material 10, or the whole upper surface 21 of the said 2nd base material 20 is formed with an adhesive material. You can also. Specifically, for example, the entire first base material 10 can be formed of synthetic resin, silicon-based resin, or glass, and the entire second base material 20 can be formed of silicon-based resin.

  Moreover, the 2nd base material 20 can also be adhere | attached on the 1st base material 10 so that peeling is possible, without using an adhesive agent by thermocompression bonding. That is, for example, a combination in which the peripheral portion of the opening 13b of the through hole 13 in the lower surface 12 of the first base material 10 and the portion bonded to the peripheral portion in the upper surface 21 of the second base material 20 can be thermocompression bonded. The thermoplastic resin can be used. Specifically, for example, the first base material 10 and the second base material 20 are formed of PMMA, and the lower surface 12 of the first base material 10 and the upper surface 21 of the second base material 20 are in the vicinity of the softening point of PMMA. The second base material 20 is bonded to the first base material 10 by being pressure-bonded in a heated state at a temperature of (for example, a temperature in the range of 100 to 130 ° C.) and held for a predetermined time (for example, 1-2 hours). It can be made to adhere peelably.

  Further, for example, the second base material 20 can be detachably bonded to the lower surface 12 of the first base material 10 using a predetermined adhesive. As such an adhesive, it is possible to appropriately select and use an adhesive that exhibits an adhesive strength with an appropriate strength capable of realizing the above-described peelable adhesion. Specifically, for example, a known adhesive containing a silicone resin such as PDMS, a dental adhesive resin, or a UV (ultraviolet) curable resin as an active ingredient can be used.

  As shown in FIG. 6, the second base material 20 preferably has flexibility so that it can be gradually peeled off from the end when it is peeled off from the first base material 10. In this embodiment, the 2nd base material 20 is formed in the flexible sheet form. Therefore, for example, as shown in FIG. 6, by capturing the end portion 20a of the second base material 20 with an instrument such as tweezers P or the hand of the operator, the second base material 20 is gradually removed from the end portion 20a. Can be peeled off.

  As a material constituting such a flexible sheet-like second base material 20, for example, a silicon-based resin such as PDMS, a flexible synthetic resin, synthetic rubber, or natural rubber can be used. Moreover, the thickness of the 2nd base material 20 can be made into the range of 5-5000 micrometers, for example, and it is preferable to set it as the range of 50-1000 micrometers.

  The second base material 20 is preferably excellent in gas permeability. That is, the 2nd base material 20 can be comprised with the material excellent in gas permeability, such as silicon resin (for example, PDMS), for example. In this case, when the thickness of the second substrate 20 is in the above range, the gas permeability of the second substrate can be made particularly excellent. Moreover, the 2nd base material 20 can also be used as the base material by which the gas permeability was improved by forming the micropore which does not allow the cell to be used to pass through with a predetermined aperture ratio, for example. When the second base material 20 is excellent in gas permeability, the cells held in the through holes 13 of the first base material 10 are surely supplied with sufficient oxygen to maintain their survival. Can be supplied.

  When the second base material 20 is bonded to the first base material 10 without using an adhesive, as shown in FIG. 6, an instrument such as tweezers P is connected to the end 20 a of the second base material 20 and the By inserting it between the first substrate 10, the end 20 a can be easily captured. Even if the second base material 20 is bonded to the first base material 10 via an adhesive, a part of the edge of the second base material 20 (for example, the corner shown in FIG. 6). The part 20a) or the entire part (entire circumference) is left as a non-adhesive part (that is, a part or the whole of the edge part is not adhered to the first substrate 10), thereby the non-adhesive part. Can be easily captured and the second substrate 20 can be peeled off as shown in FIG.

  Further, as described later, from the viewpoint of convenience when observing cells or cell aggregates cultured using the forming tool 1 by optical means such as a microscope, the first base material 10 and the second base The material constituting the material 20 is preferably a translucent material, particularly preferably a transparent material. That is, for example, the entire first base material 10 is formed of a transparent synthetic resin (for example, PMMA), a silicon-based resin (for example, PDMS) or glass, and the entire second base material 20 is formed of a transparent silicon-based resin. (For example, PDMS) is preferable.

  Such a 2nd base material 20 is adhere | attached on the lower surface 12 of the said 1st base material 10 so that the downward side of the through-hole 13 of the 1st base material 10 may be covered. As a result, the forming instrument 1 has the cell non-adhesive well 30 formed by covering the lower side of the through hole 13 of the first base material 10 with the second base material 20. That is, in the present embodiment, the forming instrument 1 has a plurality of wells 30 as shown in FIGS. 3 and 4. The plurality of wells 30 are regularly arranged at regular intervals.

  The well 30 is formed as a hole with a bottom that opens in the upper surface 11. That is, the bottom surface 30 b (see FIG. 4) of the well 30 is a part of the upper surface 21 of the second base material 20 exposed in the through hole 13. Further, the inner side surface 30c of the well 30 is an inner surface 13c of the through hole 13 (see FIG. 2).

  The bottom surface 30b and the inner side surface 30c are non-cell-adhesive. Here, the cell adhesive surface and the cell non-adhesive surface will be described. For example, when a cell settles on the surface in a solution used for culture, the cell adhesive surface changes its shape from a spherical shape and adheres in a relatively flat shape. It is a surface that can

  On the other hand, a cell non-adhesive surface means that, for example, when a cell settles on the surface in a solution used for culture, the cell hardly changes its shape from a spherical shape and does not adhere at all. Or a surface that adheres very weakly. Cells on the non-cell-adhesive surface cannot be adhered to the surface at all, so they remain in a spherical shape in the solution, or are extremely weakly adhered to the surface, so they can be easily removed from the surface by the flow of the culture solution. Detach into

  The cell non-adhesive surface in the forming instrument 1 is, for example, physically or chemically fixed to the surface of the material constituting the first base material 10 or the material constituting the second base material 20. It can be set as the surface formed by this. The cell non-adhesive substance is not particularly limited as long as it is a substance that does not bind to cell surface molecules such as proteins and sugar chains present in the cell membrane of the cell to be used.

  That is, for example, polymers exhibiting extremely high hydrophilicity in solution (polyethylene glycol and derivatives thereof), MPC (2-methacryloyloxyethyl phosphorylcholine), poly-HEMA (polyhydroxyethyl methacrylate), SPC (segmented polyurethane) Or a protein (albumin or the like) obtained from a living body can be appropriately selected and used depending on the cell type.

  These non-cell-adhesive substances are, for example, a method of drying an aqueous solution containing them on the bottom surface 30b and the inner side surface 30c of the well 30, a functional group of the substance in the aqueous solution, and the bottom surface 30b and the inner side surface 30c. A method in which a chemical reaction (for example, a condensation reaction between functional groups such as a carboxyl group or an amino group) is caused to form a covalent bond with a bonded functional group, or the substance has in the aqueous solution. It can be immobilized on the bottom surface 30b and the inner side surface 30c by a method of bonding a thiol group and a metal (platinum, gold, etc.) thin film previously formed on the bottom surface 30b and the inner side surface 30c.

  Such a process of imparting cell non-adhesiveness to the bottom surface 30b and the inner side surface 30c of the well 30 may be performed on the forming instrument 1 assembled by bonding the second substrate 20 to the first substrate 10. Or it can be applied to each of the first base material 10 and the second base material 20 prior to the assembly of the forming tool 1. For example, by performing a cell non-adhesive treatment after the assembly of the forming tool 1, a portion of the first base material 10 and the second base material 20 that is exposed in the forming tool 1 (for example, the bottom surface 30b of the well 30). In addition, cell non-adhesiveness can be selectively and efficiently imparted to the inner surface 30c and the upper surface 11) of the first base material 10.

The area of the bottom surface 30b of the well 30 (that is, the area of the opening 13b of the through hole 13 in the lower surface 12 of the first base material 10) can be set in a range of 1 × 10 ² to 2 × 10 ⁶ μm ² , for example. , Preferably it can be set as the range of 7 * 10 < ³ > -8 * 10 < ⁵ > micrometer < ² >.

  Moreover, when the bottom surface 30b of the well 30 is circular like this embodiment, the diameter can be made into the range of 10-1500 micrometers, for example, Preferably it can be set as the range of 100-1000 micrometers. . The shape of the bottom surface 30b of the well 30 is not limited to a circle, and may be a polygon or an ellipse, for example.

  When the area of the bottom surface 30b of the well 30 is smaller than the lower limit of the above range, it is not easy to reliably hold the cells in the well 30. In addition, when the area of the bottom surface 30b of the well 30 is larger than the upper limit of the above range, the number of cells seeded in the well 30 increases, so that the cell aggregates formed in the well 30 are increased. The size may be too large. If the size of the cell aggregate becomes too large, the cells located inside the cell aggregate may not receive sufficient nutrients and oxygen from the culture solution outside the cell aggregate and may die. .

  Further, when the number of cells seeded in the well 30 increases, a plurality of cell aggregates may be formed in one well 30. A plurality of cell aggregates usually become heterogeneous populations having different sizes, and are fused together in a floating state to form a giant cell aggregate having an irregular shape.

  On the other hand, when the area of the bottom surface 30b of the well 30 is within the above range, a cell aggregate having a uniform size within a predetermined range (for example, a spherical cell aggregate having a diameter within the predetermined range) in the well 30. Can be reliably formed one by one. In this case, the forming tool 1 can have a large number of minute wells 30. For this reason, even when rare cells are used, a large number of cell aggregates having a uniform size can be efficiently formed.

  The area of the opening 30a in the upper surface 11 of the well 30 (that is, the area of the opening 13a of the through hole 13 in the upper surface 11 of the first base material 10) is also preferably set in the same range as the bottom surface 30b.

  Further, the area of the opening 30a of the well 30 can be approximately the same as the area of the bottom surface 30b, but can be smaller than the area of the bottom surface 30b, for example.

  FIG.7 and FIG.8 is sectional drawing which shows the 1st base material 10 and the forming instrument 1 in this case, respectively. In the example shown in FIGS. 7 and 8, the well 30 (through hole 13) has an opening 30 a (through hole 13) in the upper surface 11 of the first base material 10 rather than the area of the bottom surface 30 b (opening 13 b of the through hole 13). The opening 13a) is formed in a tapered shape with a small area.

  Further, in this example, the opening 30a of the well 30 is formed to be a concentric circle having a diameter smaller than that of the bottom surface 30b. That is, the opening 30a is formed at a position corresponding to the central portion of the bottom surface 30b. When the well 30 is formed in such a tapered shape, as will be described later, the transfer of the cell aggregate formed in the well 30 can be performed very precisely.

  The depth of the well 30 (that is, the length from the upper surface 11 to the lower surface 12, the thickness of the first base material 10) is preferably in the range of 2.5 to 3000 μm, for example, in the range of 50 to 2000 μm. More preferably.

  When the depth of the well 30 is smaller than the lower limit of the above range, the cells or cell aggregates held in the well 30 may be detached from the well 30 during the culture operation. Further, when the depth of the well 30 is larger than the upper limit of the above range, oxygen and nutrients are sufficiently supplied from the culture solution outside the well 30 to the cells and cell aggregates held in the well 30. It may not be possible to supply. On the other hand, when the depth of the well 30 is within the above range, cells and cell aggregates can be stably cultured in a good state in the well 30.

  Depth ratio (aspect ratio) of the well 30 to the representative length of the bottom surface 30b of the well 30 (when the bottom surface 30b is circular or polygonal, the diameter of the circle or the diagonal length of the polygon, respectively) Is preferably in the range of 0.5 to 2.0, for example. Specifically, for example, when the diameter of the circular bottom surface 30b is a 10 μm circle that is the lower limit of the above range, the depth of the well 30 is preferably 5 μm to 20 μm. For example, when the diameter of the circular bottom surface 30b is 1500 μm which is the upper limit of the above range, the depth of the well 30 is preferably 750 μm to 3000 μm.

  When the aspect ratio is smaller than the lower limit of the above range, the cells or cell aggregates held in the well 30 may be detached from the well 30 during the culture operation. When the aspect ratio is larger than the upper limit of the above range, oxygen or nutrients may not be sufficiently supplied from the culture solution outside the well 30 to the cells or cell aggregates held in the well 30. is there. On the other hand, when the aspect ratio of the well 30 is within the above range, cells and cell aggregates can be stably cultured in a good state in the well 30.

  In the present embodiment, the area of the upper surface 21 of the second base material 20 is smaller than the area of the lower surface 12 of the first base material 10. That is, as shown in FIG. 5, in the forming instrument 1, a part of the lower surface 12 of the first base material 10 is not covered with the second base material 20. Therefore, for example, as shown in FIG. 6, in the operation of peeling the second base material 20 from the first base material 10, a portion of the first base material 10 that is not covered with the second base material 20 On the other hand, it is possible to reliably and efficiently peel the second base material 20 by pinching and peeling the end portion 20a of the second base material 20 with an instrument such as tweezers P or the operator's hand. it can. In addition, the method of pressing down the first base material 10 is not particularly limited. For example, a weight having a predetermined shape is placed only on a portion of the lower surface 12 of the first base material 10 that is not covered with the second base material 20. It is possible to adopt a method or a method of pressing with an instrument such as tweezers P or an operator's hand.

  Next, a method for culturing a cell aggregate according to this embodiment (hereinafter referred to as “main culturing method”) will be described. FIG. 9 is an explanatory diagram showing main steps included in an example of the main culture method. FIG. 10 is a perspective view showing a state in which the forming instrument 1 is immersed in a culture solution in a predetermined culture vessel 40 and culture is performed. 11A to 11E are cross-sectional views of the forming instrument 1 and the culture vessel 40 cut along a plane passing through the line XI-XI shown in FIG.

  As shown in FIG. 9, the main culture method includes an assembly formation step S100, a transfer step S200, and an assembly culture step S300. In the assembly forming step S100, a floating cell assembly is formed in the well 30 of the forming instrument 1.

  That is, first, cells are seeded in the well 30 of the forming instrument 1 and cultured. Here, the cells are not particularly limited as long as they can form a cell aggregate by forming a bond with each other. That is, any type of cells according to the purpose can be appropriately selected and used regardless of the type of animal, organ, or tissue from which it is derived.

  Specifically, for example, any organ or tissue (liver, pancreas, heart, cartilage, bone, fat, kidney, nerve, skin, bone marrow of a human or non-human animal (monkey, pig, dog, rat, mouse, etc.) Primary cells derived from embryos, etc.), established cell lines, or cells obtained by genetic manipulation of these cells.

  More specifically, for example, ES cells, iPS cells, neural stem cells, mesenchymal stem cells, tissue stem cells (somatic stem cells), hematopoietic stem cells, cancer stem cells, and other undifferentiated stem cells or progenitor cells can be used. In addition, for example, cells derived from digestive organs such as liver cells and pancreatic cells, cells derived from circulatory organs such as kidney cells, nervous cells and cardiomyocytes, connective tissues such as fat cells and skin dermis Differentiated fibroblasts, epithelial cells derived from epithelial tissues such as skin epidermis, bone cells, cartilage cells, cells derived from eye tissues such as retina, vascular cells, blood cells, germ cells, etc. Cells can also be used. Also, cancerous cells can be used. As such a cell, one type of cell can be used alone, or two or more types of cells can be mixed and used at an arbitrary ratio.

The number of cells to be seeded per well 30 can be appropriately determined according to conditions such as the size of the well 30, the size of individual cells, and the size of the cell aggregate to be formed. That is, the density of cells to be seeded in the well 30 is preferably in the range of, for example, 2 to 1.0 × 10 ⁵ cells / mm ² per unit area of the bottom surface 30 b of the well 30. More preferably, the range is 0.0 × 10 ⁴ / mm ² . The number of cells seeded in the well 30 is preferably in the range of 2 to 1.5 × 10 ⁵ per well 30, for example, and preferably in the range of 10 to 5.0 × 10 ^4. More preferably.

  This is because the number of cells necessary to form a cell aggregate per well 30 needs to be seeded. If the number of seeded cells is too large, the cell aggregate formed from the cells is huge. This is because the cells inside it may become necrotic due to lack of nutrients and oxygen.

  Since the volume of the well 30 of the forming tool 1 is small, it is preferable to perform culture by immersing the entire forming tool 1 in the culture solution in the assembly forming step S100. That is, for example, as shown in FIG. 10, the forming tool 1 is placed on the bottom surface 41 of a predetermined culture container 40, and the whole forming tool 1 is immersed in the culture medium M to culture cells. . As such a culture container 40, for example, a culture dish made of a transparent synthetic resin, which is widely used for cell culture, can be used.

  As the culture solution M, any aqueous solution containing necessary salts and nutrient components at an appropriate concentration can be used so that the survival state and function of the cells to be used can be maintained. Specifically, for example, a culture solution in which an antibiotic or serum is added to a basal medium such as DMEM (Dulbecco's Modified Eagle's Medium), a buffer solution such as phosphate buffer (PBS), so-called physiological saline, or the like. Can be used.

  FIG. 11A is a cross-sectional view of the forming instrument 1 and the culture container 40 showing how individual cells C in a state dispersed in the well 30 are cultured. In cell culture, as shown in FIGS. 10 and 11A, the forming tool 1 is arranged such that the second base material 20 is on the lower side. Therefore, when the forming tool 1 is left still, the individual cells C seeded in the well 30 settle on the bottom surface 30b of the well 30, as shown in FIG. 11A. As described above, since the bottom surface 30b and the inner side surface 30c of the well 30 are non-cell-adhesive, the cells C are not substantially adhered to the bottom surface 30b and the inner side surface 30c, and are cultured in a floating state.

  In the assembly forming step S100, the cells C are cultured in the well 30 for a predetermined period, so that the cells C are assembled three-dimensionally to form a cell assembly. FIG. 11B is a cross-sectional view of the forming instrument 1 and the culture vessel 40 showing how the cell aggregate T is formed in the well 30.

  Since the bottom surface 30b and the inner side surface 30c of the well 30 are non-cell-adhesive and the cells C can form a bond with each other, the cells C form a bond with each other and gradually gather as the culture time elapses. Finally, as shown in FIG. 11B, a cell aggregate T including a plurality of cells C aggregated three-dimensionally is formed. In addition, in this cell aggregate T, it can be said that the more advanced structure | tissue is formed because the individual cell C aggregates three-dimensionally. Therefore, the cell aggregate T can also be called a cell tissue body.

  Although the shape of the cell aggregate T is not particularly limited, for example, it can be spherical as shown in FIG. 11B. In addition, since the well 30 is non-cell-adhesive, the cell aggregate T is held in a suspended state in the culture medium M in the well 30.

  As shown in FIG. 11A, such a cell aggregate T can be formed by so-called stationary culture in which the cells C are cultured in a state where they are settled on the bottom surface 30b of the well 30. Further, by tilting the forming instrument 1 to the extent that cells do not spill out of the well 30, and collecting and holding the cells in a part of the well 30 (for example, the edge portion of the bottom surface 30b) by the action of gravity, It is also possible to promote the formation of the cell tissue body T in a stationary state. The cell aggregate T can also be formed by culturing the forming device 1 while turning it at a constant angular velocity so as to draw an arc when viewed from above (so-called turning culture). In any case, the plurality of cells C in contact with each other in the well 30 form a bond with each other and form a cell aggregate within a predetermined culture period.

  The size of the cell aggregate T formed in the aggregate formation step S100 is not particularly limited as long as the survival and function of the cells constituting the cell aggregate T can be maintained at a predetermined level. That is, when the cell aggregate T is a spherical body, the diameter can be, for example, in the range of 30 to 1000 μm, and preferably in the range of 50 to 700 μm. In the assembly forming step S <b> 100, cell assemblies T having such a size range are formed one by one in each well 30. Note that the size of the cell aggregate T can be adjusted by the number of cells seeded in the well 30 as described above, for example.

  In the subsequent transfer step S200, the cell aggregate T is transferred from the forming device 1 to the cell adhesive surface. That is, first, the upper surface 11 of the first base material 10 of the forming instrument 1 is brought into contact with a predetermined cell adhesive surface.

  The cell adhesive surface for transferring the cell aggregate T is not particularly limited as long as it is a surface to which the cells C constituting the cell aggregate T can adhere. That is, the cell adhesive surface can be, for example, a surface of a material itself such as a synthetic resin or a surface formed by physically or chemically fixing a cell adhesive substance on the surface of the material. . The cell adhesion substance is not particularly limited as long as it is a substance capable of binding to a specific cell surface molecule such as a protein (eg, integrin or sugar chain receptor) existing in the cell membrane of the cell to be used. be able to.

  That is, for example, proteins obtained from living bodies (collagen, fibronectin, laminin, etc.), specific amino acid sequences showing cell adhesion (arginine, glycine, aspartic acid (so-called RGD) sequences, etc.), specific sugar chain sequences, etc. A synthesized cell-adhesive substance having a (galactose side chain or the like) can be appropriately selected and used depending on the cell type.

  These cell-adhesive substances are, for example, a method of drying an aqueous solution containing them on a predetermined material surface, and between a functional group of the substance in the aqueous solution and a functional group bonded to the material surface. A method in which a chemical reaction (for example, a condensation reaction between a carboxyl group, an amino group, etc.) is caused to form a covalent bond, or a thiol group possessed by the substance in the aqueous solution and a metal previously formed on the material surface (Platinum, gold, etc.) It can be immobilized on the surface of the material by a method of bonding with a thin film. As such a cell adhesive surface, for example, the bottom surface of a culture vessel such as a culture dish or the surface of a predetermined culture substrate can be used.

  FIG. 11C is a cross-sectional view showing a state in which the upper surface 11 of the first base material 10 of the forming instrument 1 is brought into contact with the cell adhesive surface 51 that is the bottom surface of the culture vessel 50. The culture vessel 50 is, for example, a transparent synthetic resin culture dish similar to the culture vessel 40 shown in FIGS. 10, 11A and 11B, and a cell adhesive substance such as collagen is fixed to the bottom surface thereof. Can be used.

  As shown in FIG. 11C, in the transfer step S200, the forming tool 1 is turned upside down, and the forming tool 1 is attached to the cell with the upper surface 11 on which the opening 30a of the well 30 is formed facing downward. Placed on the conductive surface 51. Then, the cell aggregate T in the well 30 of the forming device 1 is settled on the cell adhesive surface 51 by the action of gravity. When the second base material 20 is excellent in gas permeability as described above, oxygen is sufficiently supplied from the culture medium M outside the well 30 to the cell aggregate T in the well 30. Can be supplied to.

  Further, in the transfer step S200, the second substrate 20 is peeled from the first substrate 10 that is in contact with the cell adhesive surface 51. This peeling can be performed, for example, in the manner shown in FIG. 6 when the second substrate 20 is formed in a flexible sheet shape. That is, first, the end 20a of the second base material 20 is peeled off from the lower surface 12 of the first base material 10 and pinched by an instrument such as tweezers P or the operator's hand.

  Next, the second base material 20 is gradually peeled off from the first base material 10 from the end 20a. Finally, the entire second base material 20 is removed from the first base material 10. In this manner, the second base material 20 can be peeled off slowly and slowly without detaching the cell aggregate T from the well 30.

  FIG. 11D is a cross-sectional view of the first base material 10 and the culture vessel 50 from which the second base material 20 has been peeled off. By peeling and removing the second base material 20 constituting the bottom surface 30b of the well 30 as described above, the opening 13b on the lower surface 12 side of the through hole 13 of the first base material 10 is again formed as shown in FIG. 11D. Opened.

  As a result, the cell aggregate T held in the through hole 13 can receive a sufficient supply of oxygen and nutrients from the culture medium M through the opening 13b. Therefore, the cell aggregate T can be sufficiently adhered to the cell adhesive surface 51 while maintaining the survival and function of the cell aggregate T held in the through hole 13.

  The timing at which the second substrate 20 is peeled in the transfer step S200 is after the cell aggregate T has settled on the cell adhesive surface 51, and the survival rate of the cells contained in the cell aggregate T is equal to or higher than a predetermined level. It is not particularly limited as long as it is within a period that can be maintained. This timing varies depending on conditions such as the state of the cell aggregate T, the temperature of the culture medium M, and the concentration of oxygen and nutrients contained in the culture medium M. For example, the cell aggregate T is on the cell adhesive surface 51. It can be in the range of 1 to 180 minutes after settling.

  In the subsequent assembly culture step S300, the cell assembly T adhered to the cell adhesive surface 51 is cultured. That is, for example, as shown in FIG. 11D, the cell aggregate T can be cultured as it is in the through hole 13 of the first base material 10 placed on the cell adhesive surface 51.

  Alternatively, the cell aggregate T can be cultured on the cell adhesive surface 51 from which the first base material 10 has been removed. That is, in this case, after peeling the second base material 20 from the first base material 10 in the transfer step S200, the first base material 10 is further detached from the cell adhesive surface 51 to culture the cell aggregate T. To do. FIG. 11E is a cross-sectional view of the culture vessel 50 showing a state in which the cell aggregate T is cultured in a state where the first base material 10 is removed from the cell adhesive surface 51.

  The timing at which the first substrate 10 is removed from the cell adhesive surface 51 is not particularly limited as long as the cell aggregate T adheres to the cell adhesive surface 51 to such an extent that the cell aggregate T does not float in the culture medium M. That is, for example, after the cell aggregate T adheres to the cell adhesive surface 51 in the through hole 13 of the first base material 10, the first base material 10 is kept in contact with the cell adhesive surface 51 and is predetermined. The first substrate 10 can be removed from the cell-adhesive surface 51, and the culture can be continued.

  Thus, in the assembly culture step S300, the cell assembly T is cultured in a state of being adhered to the position corresponding to the well 30 in which the cell assembly T is accommodated in the cell adhesive surface 51. That is, in the present embodiment, the plurality of cell aggregates T formed by the plurality of wells 30 regularly arranged are regularly arranged at positions corresponding to the plurality of wells 30 on the cell adhesive surface 51. Cultured in a state of being placed in

  Here, when the well 30 of the forming instrument 1 is formed in a tapered shape as shown in FIGS. 7 and 8, the position where the cell aggregate T is adhered to the cell adhesive surface 51 is more precisely defined. Can be controlled.

  FIG. 12A is a cross-sectional view of the forming tool 1 and the culture vessel 40 when the forming tool 1 shown in FIG. 8 is used in the assembly forming step S100. FIG. 12B is a cross-sectional view of the forming tool 1 and the culture vessel 50 when the forming tool 1 shown in FIG. 8 is used in the transfer step S200. 12A and 12B correspond to FIGS. 11B and 11C, respectively.

  In this case, in the culturing step S100, as shown in FIG. 12A, the cell aggregate T is formed in the well 30 of the forming instrument 1 as described above. Then, in the subsequent transfer step S200, as shown in FIG. 12B, the upper and lower sides of the forming tool 1 are turned over to bring the upper surface 11 of the first base material 10 into contact with the cell adhesive surface 51 of the culture vessel 50.

  Here, as described above, the opening 30a in the upper surface 11 of the well 30 has a smaller area than the bottom surface 30b and is concentrically formed at a position corresponding to the central portion of the bottom surface 30b. The cell aggregate T settles in the well 30 from the bottom surface 30b side to the opening 30a side by the action of gravity while being guided by the tapered inner side surface 30c.

  Therefore, as shown in FIG. 12B, the cell aggregate T settles and adheres to a position (in the opening 30a) corresponding to the central portion of the bottom surface 30b of the well 30 in the cell adhesive surface 51. As described above, by using the tapered well 30, even when the size of the cell aggregate T is smaller than the bottom surface 30 b of the well 30, the cell aggregate T is removed from the cell adhesive surface 51. Thus, it is possible to accurately transfer to a position in the range of the narrower opening 30a.

  Thus, the forming device 1 is used in combination with a culture device having a cell adhesive surface. Therefore, the cell assembly transfer kit (hereinafter referred to as “transfer kit”) according to the present embodiment makes the above-described forming tool 1 and the upper surface 11 of the first base material 10 of the forming tool 1 contact each other. A culture device having a cell adhesive surface (for example, the culture vessel 50 described above).

  In addition, the cell assembly culture device (hereinafter referred to as “culture device”) according to the present embodiment is a device used in combination with the forming device 1, and is a cell for attaching and culturing the cell assembly T. Has an adhesive surface. The culture instrument is not particularly limited as long as it is a structure having a cell-adhesive surface with which the upper surface 11 of the forming instrument 1 can be brought into contact. That is, this culture instrument can be, for example, a culture vessel 50 having a cell-adhesive bottom surface as described above, and a flat culture substrate similar to the first substrate 10 as described later. It can also be.

  Further, at least one of the forming device 1 and the culture device is brought into contact with the other in a state where the upper surface 11 of the first base material 10 is in contact with the cell adhesive surface of the culture device, and the relative position of each other is fixed. It is good also as having the positioning structure to do. That is, the forming device 1 and the culture device can have a positioning structure formed at a corresponding position in a shape in which one is fitted or locked to the other, for example.

  An example of such a case is shown in FIGS. FIG. 13 is a perspective view showing an example of the forming instrument 1 having a positioning structure. FIG. 14 is a perspective view showing an example of the forming tool 1 shown in FIG. 13 and a culture tool 60 having a positioning structure corresponding to the forming tool 1. FIG. 15 is a perspective view showing a state in which the forming tool 1 shown in FIG. FIG. 16 is a perspective view showing a state in which the cell aggregate T formed by the forming instrument 1 is cultured on the cell adhesive surface 61 of the culture instrument 60. Note that the transcription kit can include, for example, a forming device 1 and a culture device 60 as shown in FIGS. 14 and 15.

  In this example, as shown in FIG. 13, the forming instrument 1 has a plurality of convex portions 14 projecting at a predetermined height on the upper surface 11 of the first base material 10. On the other hand, as shown in FIG. 14, the culture instrument 60 has a plurality of recesses with a predetermined depth to be fitted to the projections 14 at positions corresponding to the projections 14 of the forming instrument 1 on the upper surface 61. 62.

  Then, in the transfer step S200, the corresponding convex portions 14 and concave portions 62 shown in FIG. 14 are fitted, and the upper surface 11 of the forming device 1 is brought into contact with the upper surface 61 of the culture device 60 as shown in FIG. Make contact. As a result, the first base material 10 and the second base material 20 can be simply and reliably fixed in a predetermined positional relationship.

  That is, the well 30 of the forming instrument 1 can be disposed at a predetermined position on the cell adhesive surface 61. Therefore, as shown in FIG. 16, the cell aggregate T can be transferred to a predetermined position on the cell adhesive surface 61. As described above, by using the forming tool 1 and the culture tool 60 having the positioning structure, the cell aggregate T can be accurately and reliably disposed at a desired position on the cell adhesive surface 61.

  In addition, when using the culture instrument 60 as shown in FIGS. 14-16, the said culture instrument 60 and the formation instrument 1 are contained in the culture solution M in a predetermined culture container like the culture container 40 shown in FIG. It is preferable to transfer or culture the cell aggregate T in a state immersed in the cell.

  As shown in FIG. 13, when the upper surface 11 or other part of the forming instrument 1 has a convex part, the convex part can also be used as a handle. That is, for example, the operator can easily perform operations such as movement of the forming tool 1 by pinching the convex portion with a tool such as tweezers. In addition, it is good also as the formation instrument 1 having a recessed part and having the convex part which the culture instrument 60 fits into the said recessed part.

  In addition, the culture instrument 60 may be formed with a lattice-like pattern that divides an area corresponding to each of the plurality of wells 30 of the forming instrument 1 (that is, an area where the cell aggregate T is to be transferred). That is, in the example shown in FIGS. 14 to 16, a lattice-like pattern 63 including a plurality of vertical lines 63 a and a plurality of horizontal lines 63 b orthogonal to each other is formed on the culture instrument 60. The two vertical lines 63a and the two horizontal lines 63b that intersect with each other form a grid 63c that is a rectangular region. The position of the grid 63c corresponds to the position of the well 30 of the forming tool 1.

  Therefore, as shown in FIG. 15, each well 30 of the forming instrument 1 is in contact with the cell adhesive surface 61 in the state where the forming instrument 1 is in contact with the cell adhesive surface 61. It is arranged within the range. Then, as shown in FIG. 16, each cell aggregate T is transferred within the range of the grid 63 c of each lattice on the cell adhesive surface 61. As a result, the cell aggregates T can be cultured one by one within the range of the mesh 63c of each lattice.

  Such a lattice-like pattern 63 is convenient for identifying each cell aggregate T adhered to the cell adhesive surface 61 when observing using an optical observation device such as a phase contrast microscope. is there. In addition, the lattice-like pattern 63 can be formed as a pattern drawn (printed) on the cell adhesive surface 61 or the surface on the back side thereof, or as a groove formed on the cell adhesive surface 61, for example. Can be formed.

  In addition, one of the forming device 1 and the culture device 60 may have a positioning structure formed so as to contact the outer peripheral portion of the other and fix the relative positional relationship with each other. 17 and 18 show an example of this case. FIG. 17 is a perspective view showing an example of the culture instrument 60 having a plurality of convex portions 64 that abut on the outer peripheral portion of the first base material 10. 18 is a perspective view showing a state in which the forming instrument 1 shown in FIG. 5 is brought into contact with the cell adhesive surface 61 of the culture instrument 60 shown in FIG.

  As shown in FIG. 17, the plurality of convex portions 64 of the culture instrument 60 are formed in a shape and a position that contact the side surface 15 (see FIG. 18) of the first base material 10 of the forming instrument 1. Specifically, each convex portion 64 is formed in a frame shape that abuts so as to surround a bent corner portion of the side surface 15 of the first base material 10.

  Accordingly, as shown in FIG. 18, the first base material 10 and the second base material 20 are determined in advance by bringing the plurality of convex portions 64 and the side surface 15 of the first base material 10 into contact with each other. Therefore, it can be simply and reliably fixed by the positional relationship.

  According to the cell assembly formation device, the cell assembly culture device, the cell assembly transcription kit, and the cell assembly culture method described above, for example, it is possible to easily and reliably cultivate rare cells using rare reagents. It can be applied to various industrial uses such as drug discovery, regenerative medicine, basic research using cultured cells.

  Specifically, for example, a plurality of floating embryoid bodies formed using the forming device 1 are arranged on the cell adhesive surfaces 51 and 61 of the culture devices 50 and 60 in a predetermined regular arrangement. Micropatterning can be performed precisely.

  In addition, by making the intervals between the embryoid bodies on the cell adhesive surfaces 51 and 61 regularly constant, the state of the cells constituting the embryoid bodies can be maintained uniformly. Furthermore, since the distance between adjacent embryoid bodies can be maintained at a predetermined value, it is possible to construct a culture system in which adverse effects due to mutual interference between embryoid bodies after adhesion are reduced. In addition, important basic research can be carried out efficiently and reliably, such as cell differentiation induction experiments and drug response tests in a state where the gene expression and cell morphology of the target embryoid body are uniform. .

  Next, specific examples according to the present embodiment will be described.

  First, 143 circular through holes 13 having a diameter of 800 μm were formed on the first base material 10 made of a flat plate made of PMMA (24 mm × 24 mm, thickness 800 μm) by a drilling process using a machining center. The through holes 13 were regularly arranged so that the distance between the centers thereof was 880 μm.

  On the other hand, the 2nd base material 20 which consists of a sheet | seat made from PDMS (13 mm x 13 mm, thickness 400 micrometers) was prepared. And the 2nd base material 20 is aligned so that the opening 13b below the through-hole 13 of the 1st base material 10 may be covered, and the said 2nd base material 20 is said 1st base material, without using an adhesive agent. 10 was pressed against the lower surface 12. Thus, as shown in FIGS. 3 to 5, the second base material 20 was detachably bonded to the lower surface 12 of the first base material 10.

  Next, a sputtering process using a sputtering apparatus was performed from the upper surface 11 side of the first base material 10. By this sputtering treatment, a metal thin film capable of binding thiol groups was formed on the upper surface 11 of the first base material 10, the bottom surface 30b of the well 30, and the inner side surface 30c.

Then, an ethanol solution containing a cell non-adhesive synthetic polymer (chemical formula: CH ₃ (CH ₂ CH ₂ ) _n SH) having a molecular weight of 30,000 and a thiol group is injected into the well 30 at a concentration of 5 mM. At the same time, it was applied to the upper surface 11 of the first substrate 10.

  Thereafter, the first base material 10 and the second base material 20 are allowed to stand for a predetermined time in a nitrogen atmosphere, so that the cell non-adhesive polymer is allowed to adhere to the upper surface 11 of the first base material 10, the bottom surface 30b of the well 30, and the inner surface. Immobilized on the side surface 30c. As a result, the upper surface 11 of the first substrate 10, the bottom surface 30b of the well 30 (the portion exposed in the through hole 13 in the upper surface 21 of the second substrate 20), and the inner surface 30c became a cell non-adhesive surface. .

  In this way, the forming tool 1 having the transparent first base material 10 made of PMMA, the transparent second base material 20 made of PDMS, and 143 wells 30 having a diameter of the bottom surface 30b of 800 μm was manufactured.

In each well 30 of the forming device 1, 5 × 10 ² mouse ES cells (Dainippon Sumitomo Pharma Co., Ltd.) were seeded. As a culture solution, a DMEM medium containing 15% fetal bovine serum, 1% nucleoside, 1% non-essential amino acid, 1% 2-mercaptoethanol, 1% glutamine was used.

  Then, by culturing the mouse ES cells for 3 days, one embryoid body, which is a spherical cell aggregate containing the mouse ES cells assembled three-dimensionally, was formed in each well 30. FIG. 19A is an example of a photograph of the embryoid body T in a floating state held in the well 30 of the forming device 1 on the third day of culture taken under a phase contrast microscope. The formed embryoid body T had a diameter in the range of 155 to 195 μm (172 ± 8 μm: arithmetic mean ± standard deviation).

  Next, a commercially available culture dish (diameter 35 mm, AGC Techno Glass Co., Ltd.) was prepared as the culture container 50. The bottom surface of the culture dish coated with collagen was used as the cell adhesive surface 51.

  Then, the upper surface 11 of the forming device 1 was brought into contact with the cell adhesive surface 51 of the culture vessel 50, and the embryoid body T in the well 30 of the forming device 1 was allowed to settle on the cell adhesive surface 51. FIG. 19B is an example of a photograph immediately after the upper surface 11 of the forming instrument 1 is brought into contact with the cell adhesive surface 51.

  Contact of the forming tool 1 with the cell adhesive surface 51 was performed as follows. That is, first, the cell culture surface 51 was wetted with the culture solution by putting the culture solution into the culture vessel 50 and then discarding the culture solution. Then, the upper surface 11 of the forming device 1 inclined so as not to spill the embryoid body T from the well 30 was combined with the cell adhesive surface 51 wetted with the culture solution. Thus, the upper surface 11 of the forming device 1 was brought into contact with the cell adhesive surface 51 while the embryoid body T was held in the well 30.

  Further, a metal ring-shaped weight was placed on the forming device 1 in a state where the forming device 1 was in contact with the cell adhesive surface 51. By this weight, four corner portions not covered with the second base material 20 of the first base material 10 of the forming tool 1 were pressed. Thereby, the upper surface 11 of the forming instrument 1 and the cell adhesive surface 51 were brought into close contact with each other.

  When 10 minutes passed after this contact, as shown in FIG. 6, the second base material 20 was gradually peeled from the end portion 20 a using tweezers P and removed from the first base material 10. FIG. 19C is an example of a photograph immediately after the second substrate 20 is peeled from the first substrate 10.

  Thereafter, the embryoid body T in each well 30 was adhered to the cell adhesive surface 51 by maintaining the culture vessel 50 at 37 ° C. for 60 minutes. Then, after the embryoid body T was adhered, the first substrate 10 was removed from the cell adhesive surface 51. FIG. 19D is an example of a photograph immediately after removing the first substrate 10 from the cell adhesive surface 51.

  As a result, as shown in FIG. 10D, a plurality of embryoid bodies T having a uniform size regularly arranged at predetermined intervals remained on the cell adhesive surface 51. FIG. 19E is an example of a photograph of an embryoid body T that has been cultured for 1 hour in a state in which the first substrate 10 is removed from the cell adhesive surface 51 and adhered to the cell adhesive surface 51. FIG. 19F is an example of a photograph of an embryoid body T cultured for 3 hours in a state where the first substrate 10 is removed from the cell adhesive surface 51 and adhered to the cell adhesive surface 51.

  The embryoid body T could be further cultured in a state where it was adhered to the cell adhesive surface 51. As the culture time passed, the cells constituting the embryoid body T adhered to the cell adhesive surface 51 and migrated and spread.

  A forming instrument 1 was produced in the same manner as in Example 1 described above. That is, first, 143 circular through holes 13 having a diameter of 800 μm were formed on the first base material 10 made of a flat plate made of PMMA (24 mm × 24 mm, thickness 800 μm) by a drilling process using a machining center. The through holes 13 were regularly arranged so that the distance between the centers thereof was 880 μm.

  On the other hand, the 2nd base material 20 which consists of a sheet | seat made from PDMS (13 mm x 13 mm, thickness 400 micrometers) was prepared. And the 2nd base material 20 was adhere | attached on the lower surface 12 of the 1st base material 10 so that peeling was possible, without using an adhesive agent. Further, in the same manner as in Example 1 described above, the cell non-adhesive substance was fixed to give the well 30 cell non-adhesiveness. In this way, the forming instrument 1 having the transparent first base material 10 made of PMMA, the transparent second base material 20 made of PDMS, and 143 wells 30 having a diameter of the bottom surface 30b of 800 μm was manufactured.

1.4 × 10 ³ primary rat hepatocytes were seeded in each well 30 of the forming device 1. As a culture solution, 60 mg / L proline, 50 μg / L EGF, 10 mg / L insulin, 7.5 mg / L hydrocortisone, 0.1 μM copper sulfate pentahydrate, 3 μg / L selenate, Add 50 pM zinc sulfate heptahydrate, 50 μg / L linoleic acid, 58.8 mg / L penicillin, 100 mg / L streptomycin, 1.05 g / L sodium bicarbonate, 1.19 g / L HEPES DMEM serum-free medium was used. Primary rat hepatocytes were prepared from rat liver by a known collagenase perfusion method.

  Then, the primary rat hepatocytes are swirl cultured (40 rpm) for 7 days, thereby forming one spheroid, which is a spherical cell aggregate containing the three-dimensionally assembled primary rat hepatocytes, in each well 30. I let you. FIG. 20A is an example of a photograph in which a floating spheroid T held in the well 30 of the forming instrument 1 on the seventh day of culture is photographed under a phase contrast microscope. The diameter of the formed spheroid T was in the range of 235 to 285 μm (259 ± 10 μm: arithmetic mean ± standard deviation).

  Next, as the culture vessel 50, a commercially available culture dish having a bottom surface that is the cell adhesive surface 51 was prepared as in Example 1 described above. Then, the upper surface 11 of the forming instrument 1 was brought into contact with the cell adhesive surface 51 of the culture vessel 50, and the spheroid T in the well 30 of the forming instrument 1 was allowed to settle on the cell adhesive surface 51. FIG. 20B is an example of a photograph immediately after the upper surface 11 of the forming tool 1 is brought into contact with the cell adhesive surface 51.

  When 10 minutes passed after the contact, the second base material 20 was gradually peeled off from the end using tweezers P and removed from the first base material 10. FIG. 20C is an example of a photograph immediately after the second substrate 20 is peeled from the first substrate 10.

  Thereafter, the spheroid T was adhered to the cell adhesive surface 51 in each well 30 by maintaining the culture vessel 50 at 37 ° C. for 1 day. Then, after the spheroid T was adhered, the first substrate 10 was removed from the cell adhesive surface 51. FIG. 20D is an example of a photograph immediately after the first substrate 10 is removed from the cell adhesive surface 51.

  As a result, a plurality of spheroids T of uniform size regularly arranged at predetermined intervals remained on the cell adhesive surface 51. FIG. 20E is an example of a photograph of spheroid T cultured for one day with the first substrate 10 removed from the cell adhesive surface 51 and then adhered to the cell adhesive surface 51. FIG. 20F is an example of a photograph of spheroid T cultured for 2 days with the first substrate 10 removed from the cell adhesive surface 51 and then adhered to the cell adhesive surface 51.

  The spheroid T was able to be further cultured in a state of being adhered to the cell adhesive surface 51. As the culture time passed, the cells constituting the spheroid T adhered to the cell adhesive surface 51 and migrated and spread.

  A forming instrument 1 was produced in the same manner as in Example 1 described above. That is, first, 270 circular through holes 13 having a diameter of 600 μm were formed on the first base material 10 made of a flat plate made of PMMA (24 mm × 24 mm, thickness 600 μm) by a drilling process using a machining center. The through holes 13 were regularly arranged such that the distance between the centers thereof was 660 μm.

  On the other hand, the 2nd base material 20 which consists of a sheet | seat made from PDMS (13 mm x 13 mm, thickness 400 micrometers) was prepared. And the 2nd base material 20 was adhere | attached on the lower surface 12 of the 1st base material 10 so that peeling was possible, without using an adhesive agent. Further, in the same manner as in Example 1 described above, the cell non-adhesive substance was fixed to give the well 30 cell non-adhesiveness. In this way, the forming tool 1 having the transparent first base material 10 made of PMMA, the transparent second base material 20 made of PDMS, and 270 wells 30 having a diameter of the bottom surface 30b of 600 μm was manufactured.

In each well 30 of the forming instrument 1, 1.0 × 10 ³ HepG2 cells (RIKEN / Bioresource Center) were seeded. The culture medium used was Williams medium E medium supplemented with 58.8 mg / L penicillin, 100 mg / L streptomycin, 2.2 g / L sodium bicarbonate and 10% fetal bovine serum.

  Then, by culturing the HepG2 cells for 5 days, one spheroid, which is a spherical cell aggregate containing the HepG2 cells assembled three-dimensionally, was formed in each well 30. FIG. 21A is an example of a photograph in which a floating spheroid T held in the well 30 of the forming instrument 1 on the seventh day of culture is photographed under a phase contrast microscope. The diameter of the formed spheroid T was in the range of 240 to 280 μm (259 ± 8 μm: arithmetic mean ± standard deviation).

  Next, a commercial culture dish having a bottom surface, which is a cell adhesive surface 51 coated with collagen, was prepared as the culture vessel 50 in the same manner as in Example 1 described above. Then, the upper surface 11 of the forming device 1 was brought into contact with the cell adhesive surface 51 of the culture device 50, and the spheroid T in the well 30 of the forming device 1 was settled on the cell adhesive surface 51. FIG. 21B is an example of a photograph immediately after the upper surface 11 of the forming instrument 1 is brought into contact with the cell adhesive surface 51.

  When 10 minutes passed after the contact, the second base material 20 was gradually peeled off from the end using tweezers P and removed from the first base material 10. FIG. 21C is an example of a photograph immediately after the second substrate 20 is peeled from the first substrate 10.

  Thereafter, the spheroid T was adhered to the cell adhesive surface 51 in each well 30 by maintaining the culture vessel 50 at 37 ° C. for 1 day. Then, after the spheroid T was adhered, the first substrate 10 was removed from the cell adhesive surface 51. FIG. 21D is an example of a photograph immediately after the first substrate 10 is removed from the cell adhesive surface 51.

  As a result, a plurality of spheroids T of uniform size regularly arranged at predetermined intervals remained on the cell adhesive surface 51. FIG. 21E is an example of a photograph of spheroid T cultured for 2 days with the first substrate 10 removed from the cell adhesive surface 51 and then adhered to the cell adhesive surface 51. FIG. 21F is an example of a photograph of spheroid T cultured for 4 days with the first substrate 10 removed from the cell adhesive surface 51 and then adhered to the cell adhesive surface 51.

  Spheroid T was able to be further cultured in a state where it adhered to the cell adhesive surface 51. As the culture time passed, the cells constituting the spheroid T adhered to the cell adhesive surface 51 and migrated and spread.

  A forming instrument 1 was produced in the same manner as in Example 1 described above. That is, first, 672 circular through holes 13 having a diameter of 300 μm were formed on the first base material 10 made of a flat plate made of PMMA (24 mm × 24 mm, thickness 400 μm) by a drilling process using a machining center. The through holes 13 were regularly arranged so that the distance between the centers thereof was 400 μm.

  On the other hand, the 2nd base material 20 which consists of a sheet | seat made from PDMS (13 mm x 13 mm, thickness 400 micrometers) was prepared. And the 2nd base material 20 was adhere | attached on the lower surface 12 of the 1st base material 10 so that peeling was possible, without using an adhesive agent. Further, in the same manner as in Example 1 described above, the cell non-adhesive substance was fixed to give the well 30 cell non-adhesiveness. In this way, the forming instrument 1 having the transparent first base material 10 made of PMMA, the transparent second base material 20 made of PDMS, and 672 wells 30 having a diameter of the bottom surface 30b of 300 μm was manufactured.

In each well of the forming instrument 1, 5.6 × 10 ² HepG2 cells (RIKEN Bioresource Center) were seeded. The culture medium used was Williams medium E medium supplemented with 58.8 mg / L penicillin, 100 mg / L streptomycin, 2.2 g / L sodium bicarbonate and 10% fetal bovine serum.

  Then, by culturing the HepG2 cells for 7 days, one spheroid, which is a spherical cell aggregate including the HepG2 cells assembled three-dimensionally, was formed in each well 30. FIG. 22A is an example of a photograph taken under a phase contrast microscope of a floating spheroid T held in the well 30 of the forming instrument 1 on the seventh day of culture. The diameter of the formed spheroid T was in the range of 195 to 235 μm (208 ± 8 μm: arithmetic mean ± standard deviation).

  Next, as the culture vessel 50, a commercially available culture dish having a bottom surface that is the cell adhesive surface 51 was prepared as in Example 1 described above. Then, the upper surface 11 of the forming instrument 1 was brought into contact with the cell adhesive surface 51 of the culture vessel 50, and the spheroid T in the well 30 of the forming instrument 1 was allowed to settle on the cell adhesive surface 51. FIG. 22B is an example of a photograph immediately after the upper surface 11 of the forming instrument 1 is brought into contact with the cell adhesive surface 51.

  When 10 minutes passed after the contact, the second base material 20 was gradually peeled off from the end using tweezers P and removed from the first base material 10. FIG. 22C is an example of a photograph immediately after the second substrate 20 is peeled from the first substrate 10.

  Thereafter, the spheroid T was adhered to the cell adhesive surface 51 in each well 30 by maintaining the culture vessel 50 at 37 ° C. In Example 4, the spheroid T was cultured in the through hole 13 of the first base material 10 while the first base material 10 was in contact with the cell adhesive surface 51. FIG. 22D is an example of a photograph after the spheroid T is transferred to the cell adhesive surface 51 and then cultured in the through hole 13 of the first substrate 10 for 3 days. FIG. 22E is an example of a photograph after the spheroid T is transferred to the cell adhesive surface 51 and cultured in the through hole 13 of the first substrate 10 for 10 days. As the culture time passed, the cells constituting the spheroid T adhered to the cell adhesive surface 51 and migrated and spread.

  DESCRIPTION OF SYMBOLS 1 Cell assembly formation instrument, 10 1st base material, 11 upper surface, 12 lower surface, 13 through-hole, 13a opening, 13b opening, 13c inner side surface, 14 convex part, 15 side surface, 20 2nd base material, 21 upper surface, 22 Lower surface, 30 well, 30a opening, 30b bottom surface, 30c inner surface, 40 culture container, 41 bottom surface, 50 culture container, 51 cell adhesive surface, 60 cell assembly culture device, 61 cell adhesive surface, 62 recess, 63 lattice Pattern, 63a vertical line, 63b horizontal line, 63c lattice, 64 convex part, C cell, M culture solution, P tweezers, T cell aggregate (embryoid body, spheroid).

Claims (8)

A first substrate having a through hole formed thereon;
A second base material that is releasably adhered to the surface of one side of the first base material and covers the one side of the through hole;
A non-cell-adhesive well formed by covering the one side of the through hole with the second substrate;
A device for forming cell aggregates, comprising: The area of the bottom surface of the well is in the range of 1 × 10 ² to 2 × 10 ⁶ μm ^2. The cell aggregate formation device according to claim 1, wherein 3. The cell assembly according to claim 1, wherein the well is formed in a tapered shape in which an area of an opening on the other surface of the first base material is smaller than an area of a bottom surface thereof. Body shaping equipment. A cell assembly forming device used in combination with a culture device having a cell adhesive surface,
It has a positioning structure which contacts the said culture instrument in the state which made the other side surface of said 1st base material contact | abut to the said cell-adhesive surface, and fixes each other's relative position. The cell aggregate formation device described in any one of 1 to 3. The cell assembly formation device according to any one of claims 1 to 4,
A culture instrument having a cell-adhesive surface that abuts the surface of the other side of the first substrate of the cell assembly-forming instrument;
A cell assembly transcription kit comprising: At least one of the cell assembly-forming instrument and the culture instrument is in contact with the other in a state where the surface on the other side of the first substrate is in contact with the cell-adhesive surface, and relative positions to each other. The cell assembly transcription kit according to claim 5, wherein the kit has a positioning structure for fixing the cell assembly. A cell-adhesive surface that abuts the surface on the other side of the first base material of the cell assembly forming device according to any one of claims 1 to 4;
A positioning structure for fixing the relative position of each other in contact with the cell assembly forming device in a state where the other surface of the first base material is in contact with the cell adhesive surface;
A cell assembly culture device characterized by comprising: An assembly formation step of forming a suspended cell assembly in the well of the cell assembly formation device according to any one of claims 1 to 4,
The other side surface of the first base material of the cell assembly forming device is brought into contact with a predetermined cell adhesive surface, and the cell aggregate in the well is allowed to settle on the cell adhesive surface. A transfer step of peeling the second substrate from the first substrate;
An assembly culture step of culturing the cell assembly adhered to the cell adhesive surface;
A method for culturing a cell aggregate, comprising: