Method of Using a Cell Culture Chip

Abstract

To provide a method of using a cell culture chip which enables a liquid retained in an opening to be sucked by a simple operation procedure while leaving a liquid in a channel.SOLUTION: A cell culture chip includes: a bottom portion; a base portion formed on an upper surface of the bottom portion; a first well provided by opening the base portion in a first direction extending from a portion of a main surface of the base portion toward the bottom portion; a second well provided by opening the base portion in the first direction at a position separated from the first well in a second direction parallel to the main surface; and a tubular chamber that provides communication between the first and second wells in the second direction. This method includes the steps of: (a) charging culture media into the first well and the chamber; and (b) sucking the culture media in the first well with a predetermined suction force to expose the bottom face of the first well without causing the culture media in the chamber to flow out into the first well.SELECTED DRAWING: Figure 5C

Description

Technical Field

[0001] The present invention relates to a method of using a cell culture chip.

Background Art

[0002] Cells exist in the "extracellular microenvironment" in a living body / tissue, which is composed of (i) soluble factors such as growth factors, vitamins, and gas molecules, (ii) insoluble factors such as extracellular matrix proteins, hardness, and pressure, and (iii) cell-cell interactions. The functions of cells are controlled while these factors are complexly and strictly regulated. That is, in order to freely control the functions of target cells such as human pluripotent stem cells (human ES / iPS cells), which are expected to be used in regenerative medicine, cell transplantation therapy, drug development, etc., it is essential to freely control this extracellular microenvironment.

[0003] Conventionally, the culture and experiments of cells including human ES / iPS cells have been carried out in a two-dimensional environment using culture dishes and plates. However, cells are originally placed in a three-dimensional environment, and it is considered that their original functions cannot be expressed in a two-dimensional environment. In tissue engineering using human ES / iPS cells, it is also very important to prepare a three-dimensional environment.

[0004] In addition, the size of the extracellular microenvironment is also a very important factor. Cells are controlled in a microenvironment on the micrometer (μm) scale in the living body. However, with conventional culture methods, it has been difficult to control factors in such a microspace. Furthermore, it has been almost impossible to comprehensively analyze these factors. Due to such circumstances, a technique for creating a three-dimensional cell culture environment, which has been difficult with conventional methods, has been desired.

[0005] As a means for realizing such a three-dimensional cell culture environment, conventionally, a microchannel chip disclosed in Patent Document 1 below has been proposed.

[0006] FIG. 13 is a perspective view schematically showing the structure of a microchannel chip disclosed in Patent Document 1 below. The microchannel chip 100 includes a base material 101 and a resin film 102. On the main surface side of the base material 101, openings are formed at two locations, and these openings constitute an inlet 111 and an outlet 112. Further, a groove 110 for a flow path is formed so as to communicate with these openings (111, 112). The groove 110 is covered with the resin film 102 and constitutes a tubular flow path. FIG. 14 is a cross-sectional view schematically showing the structure of the microchannel chip 100.

[0007] At the time of inspection, a target liquid sample is introduced from the inlet 111 in the direction of d111. This liquid sample flows through the groove 110 toward the outlet 112. A part of the groove 110 also serves as a detection unit. For example, a substance that emits fluorescence in response to a detection target substance such as a specific protein is immobilized in the middle of the groove 110. By observing this part (detection unit) using a fluorescence microscope, it is determined whether or not a specific detection target substance is contained in the liquid sample.

Prior Art Document

Patent Document

[0008]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0009] The microchannel chip is used for culturing cells in a culture space under a predetermined environment and observing the state of these cells. When culturing, a liquid culture medium is used as the culture medium.

[0010] When culturing cells under specific environmental conditions over a certain period of time (number of days), it may be necessary to replace the culture medium (culture solution) for the purpose of maintaining the culture environment, supplying nutrients to the cells, removing waste products, etc. For example, when culturing cells using the microchannel chip 100 shown in FIG. 13, when it becomes necessary to replace the culture solution, the culture solution is removed from the inlet 111 or the outlet 112, and at the same time, a new culture solution is replenished (added). Specifically, a vacuum operation is performed from the inlet 111 or the outlet 112 using a pipette with its interior depressurized, thereby removing the culture solution. Hereinafter, the case of performing the operation of extracting the culture solution using a pipette from the inlet 111 side will be described, but the case of extracting from the outlet 112 side is the same.

[0011] When performing the suction operation of the culture solution with a pipette with the tip of the pipette positioned on the inlet 111 side, if the suction force is not precisely adjusted, not only the culture solution in the inlet 111 but also the culture solution in the groove 110 will be sucked. This point will be described in detail with reference to FIG. 15.

[0012] FIG. 15 is a drawing schematically showing the change in the remaining amount of the culture solution when sucking the culture solution from the conventional microchannel chip 100 described above with reference to FIGS. 13 and 14. In FIG. 15, the change (decrease) in the remaining amount of the culture solution is schematically shown in the order of (a), (b), (c), and (d) as time passes. Also, in FIG. 15, hatching is applied to the region where the culture solution 130 exists.

[0013] When sucking the culture solution from the conventional microchannel chip 100, with the tip of the pipette 120 inserted into the inlet 111, the culture solution 130 is sucked in the d120 direction. More specifically, while pressing the tip of the pipette 120 against the bottom surface or the side wall near the bottom surface of the inlet 111, the culture solution 130 is sucked. As the suction of the culture solution 130 progresses, the liquid level of the culture solution 130 gradually decreases (see FIGS. 15(a) and (b)).

[0014] When the suction of the culture solution 130 further progresses, as shown in FIG. 15(c), the bottom surface of the inlet 111 is exposed. At this point, as shown in FIG. 15(c), a small amount of the culture solution 130 remains in the corner portion of the inlet 111.

[0015] When the suction of the culture solution 130 is further advanced from the state of FIG. 15(c), as shown in FIG. 15(d), while leaving the culture solution 130 in the corner portion of the inlet 111 as it is, the culture solution 130 that was present in the groove 110 constituting the flow path is guided (in the d121 direction) to the inlet 111 side along the inner wall of the groove 110, and then is sucked by the pipette 120. As a result, as shown in FIG. 15(d), a part of the culture solution 130 in the groove 110 is sucked by the pipette 120.

[0016] As described above, the suction of the culture solution 130 is performed, for example, to exchange the culture solution 130. That is, after the culture solution 130 stored on the inlet 111 side is removed as shown in FIG. 15(d), for example, a new culture solution (herein described as "culture solution 130a" for convenience) is introduced from the inlet 111 side. This new culture solution 130a flows into the groove 110 through the inlet 111. As a result, the old culture solution 130 that was already present in the groove 110 is pushed out to the outlet 112 side. Thereby, the culture solution 130 in the groove 110 is exchanged for the new culture solution 130a.

[0017] However, as shown in FIG. 15(d), when the new culture solution 130a is introduced, if a part of the culture solution 130 in the groove 110 has been sucked, bubbles may remain in the boundary region between the new culture solution 130a and the existing culture solution 130 in the groove 110. When the new culture solution 130a is gradually introduced in a state where these bubbles are present, the bubbles are moved so as to be pushed out of the groove 110 by the culture solution 130a. At this time, in the groove 110, more specifically, the cells cultured in a state of adhering to the bottom surface of the groove 110 may be peeled off by the force generated at the interface of the bubbles and may be washed away together with the culture solution (130 / 130a).

[0018] Even when the cells can be retained in the groove 110 during the replacement operation of the culture medium, the replacement operation of the culture medium may be completed with air bubbles remaining in the groove 110. In this case, since the volume occupied by the air bubbles and the surface of the culture chamber cannot be used for culturing, they will decrease disorderly from the original total number of cells and total amount of the culture medium, and accurate cell tests cannot be performed. In addition, the flow and diffusion of the original culture medium designed in the channel shape may be inhibited, and there is a risk that cells cannot be cultured under the intended environment. For example, there is a risk that the concentration of the culture medium components transported to the cells is disturbed, the shear stress applied to the cells changes, and waste products and residues generated from the cells remain.

[0019] For example, bioactive substances (e.g., cytokines, hormones, lipids, extracellular matrix, microRNAs, exosomes, nutrients, or drugs that exhibit endocrine effects, etc.) are released from the cells. When this bioactive substance collides with the wall surface covering the groove 110 and is returned to the cell side, it may act on this cell. However, if air bubbles are formed in the groove 110, the flow of this bioactive substance may be obstructed, which may affect the culture state of the cells.

[0020] Due to the above circumstances, when replacing the culture medium 130, it is necessary to adjust the suction force while avoiding sucking the culture medium 130 in the groove 110. However, in the conventional microchannel chip 100, the openings (inlet 111 / outlet 112) are generally configured in a common cylindrical shape (circular cylindrical shape), and no consideration is given to adjusting the suction force of the culture medium 130. Therefore, when performing the suction operation of the culture medium 130, a precise adjustment is required, that is, the suction operation is interrupted immediately before the suction of the culture medium 130 existing in the groove 110 is started while removing the culture medium 130 stored in the openings (inlet 111 / outlet 112).

[0021] Therefore, when performing the suction operation, there is a problem that strict adjustment skills are required for the operator and the reproducibility is poor. In addition, the existence of such circumstances also becomes an obstacle to automating the suction operation.

[0022] In view of the above problems, an object of the present invention is to provide a method for using a cell culture chip that enables suction of the liquid stored in the opening while leaving the liquid in the flow path by a simple operation procedure.

Means for Solving the Problems

[0023] The cell culture chip according to the present invention a bottom, a substrate portion formed on the upper surface of the bottom, a first well formed by opening the substrate portion in a first direction from a part of the main surface, which is the surface of the substrate portion opposite to the bottom, toward the bottom; a second well formed by opening the substrate portion in the first direction at a position spaced from the first well in a second direction parallel to the main surface; a tubular chamber that connects the first well and the second well in the second direction by a region sandwiched between the bottom and the substrate portion; The first well is characterized by having a shape in which the capillary force of the first well is lower than the capillary force of the chamber.

[0024] The present invention is a method for using the cell culture chip, comprising: a step (a) of filling a culture solution into the first well and the chamber; and a step (b) of exposing the bottom surface of the first well without allowing the culture solution in the chamber to flow into the first well by sucking the culture solution in the first well with a predetermined suction force.

[0025] In this specification, the term "capillary force of the well" refers to the capillary force generated at the corner portion where the inner surface (inner wall) of the well and the bottom surface (inner bottom wall) of the well intersect.

[0026] As described above with reference to FIG. 15, when the suction operation by the pipette 120 is continued with the conventional microchannel chip 100 filled with the culture solution 130, although a part of the culture solution 130 remains in the opening (inlet 111), the suction of the culture solution 130 stored in the channel (groove 110) is started. This is considered to be because, as shown in FIG. 15(c), the capillary force of the liquid pool portion of the culture solution 130 formed at the corner portion of the inlet 111 is higher than the capillary force of the groove 110.

[0027] Both the inner wall of the groove 110 and the bottom surface of the opening (inlet 111) are highly hydrophilic, and their surfaces are in a state of being thinly wetted. Therefore, when the suction operation is continued from the state of FIG. 15(c), the culture solution 130 stored in the groove 110, whose capillary force is lower than that of the corner portion of the inlet 111, moves along the inner wall of the groove 110 and the bottom surface of the inlet 111 toward the corner portion side of the inlet 111 with a high capillary force. As a result, as shown in FIG. 15(d), almost none of the culture solution 130 stored in the corner portion of the inlet 111 is sucked, and it is considered that the suction of the culture solution 130 in the groove 110 continues.

[0028] On the other hand, according to the cell culture chip of the present invention, the first well has a shape in which the capillary force of the first well is lower than the capillary force of the chamber. As a result, when the suction is started from the first well side, the suction of the liquid (culture solution) stored in the chamber is not started until the suction of the liquid stored in the first well is substantially completed. Therefore, the operator only needs to perform the suction of the liquid with a suction force sufficient to suck the liquid stored in the first well and stop the suction operation when the suction of the first well is completed, and there is no need to strictly adjust the suction force and suction time during the suction operation.

[0029] As a result, compared with the operation of replacing the culture solution for the conventional chip, the operation of the operator is simplified and no specialized skills are required, so the workability is improved. In addition, it is only necessary to suck with a suction force sufficient to suck the liquid stored in the first well, and strict adjustment is not required, so the suction operation can be automated.

[0030] The step (b) may be performed until the culture solution stored in the first well is completely sucked.

[0031] The usage method may also include a step (c) of filling the first well with a new culture solution after the step (b).

[0032] In the cell culture chip, the bottom portion and the base portion may be configured by being integrally formed of the same material, or may be configured of different materials.

[0033] The chamber can be a chamber (culture chamber) that constitutes a culture space. Further, the chamber may be configured to include the culture chamber and a communication channel that communicates the culture chamber and the first well in a second direction. In the latter case, the capillary force of the first well can be set to be lower than the capillary force of the communication channel that constitutes the chamber.

[0034] In the cell culture chip, the first well may have a reduced diameter region in which the opening diameter continuously decreases without increasing as it approaches the bottom at a position closer to the bottom than the main surface of the base portion.

[0035] According to the above configuration, the first well has a shape in which the opening diameter at a position close to the chamber is smaller than the opening diameter at a position far from the chamber. According to such a shape, the capillary force at the position of the bottom surface of the first well can be reduced.

[0036] The first well has an inner wall formed of the base portion, The inner wall may include a curved surface or a plane non-parallel to the main surface in the reduced-diameter region of the first well.

[0037] According to such a configuration, in the vicinity of the bottom surface of the first well, an inclined surface is formed at the corner portion, and the corner angle of the corner portion between this inclined surface and the bottom surface of the first well becomes an obtuse angle. As a result, the capillary force at the corner portion of the first well is reduced.

[0038] The cell culture chip has a communication well formed continuously with the first well in the first direction, and communicating the reduced-diameter region of the first well and the chamber in the first direction. The communication well may have the bottom as the bottom surface and an opening diameter smaller than that of the reduced-diameter region of the first well.

[0039] As described above, since the first well has the reduced-diameter region, it has a shape in which the opening diameter becomes smaller as it approaches the bottom at a position close to the bottom. Therefore, at the position where the opening diameter is the smallest, the wall thickness of the base portion becomes small, and there may be difficulties during molding.

[0040] On the other hand, by providing the communication well as in the above configuration, the wall thickness of the base portion located around the communication well is ensured, so that the molding is facilitated. Further, since the opening diameter of this communication well is smaller than that of the reduced-diameter region of the first well, it does not prevent only the liquid stored in the first well from being sucked during liquid suction. That is, even if a part of the liquid remains in the communication well, the liquid stored in the first well can be substantially completely sucked. In other words, even in a configuration provided with a communication well, it is possible to substantially completely suck the liquid stored in the first well without sucking the liquid stored in the chamber.

[0041] In the cell culture chip, the bottom surface of the first well may be composed of the bottom portion.

[0042] Also, in the cell culture chip, the second well may have a shape in which the capillary force of the second well is lower than the capillary force of the chamber. In this case, liquid can be sucked while leaving the liquid in the chamber from either the first well or the second well.

[0043] The second well may have a reduced diameter region that continuously decreases without increasing the opening diameter as it approaches the bottom at a position closer to the bottom than the main surface of the base portion.

[0044] Moreover, the cell culture chip according to the present invention a bottom portion, a base portion formed on the upper surface of the bottom portion, a first well formed by opening the base portion in a first direction from a part of the main surface, which is the surface opposite to the bottom of the base portion, toward the bottom portion, a second well formed by opening the base portion in the first direction at a position spaced apart from the first well in a second direction parallel to the main surface, a tubular chamber that connects the first well and the second well in the second direction by a region sandwiched between the bottom portion and the base portion, The first well is characterized by having a reduced diameter region that continuously decreases without increasing the opening diameter at a position closer to the bottom than the main surface of the base portion.

Advantages of the Invention

[0045] According to the cell culture chip of the present invention, it is possible to suck the liquid stored in the opening while leaving the liquid in the flow path by a simple operation procedure.

Brief Description of the Drawings

[0046]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5A

Figure 5B

Figure 5C

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12A

Figure 12B

Figure 12C

Figure 12D

Figure 13

Figure 14

Figure 15

Embodiments for Carrying Out the Invention

[0047] Embodiments of the cell culture chip according to the present invention will be described with reference to the drawings. It should be noted that the following drawings are merely schematically illustrated. That is, the dimensional ratio on the drawing does not necessarily match the actual dimensional ratio, and the dimensional ratios are not necessarily the same among the drawings.

[0048] FIG. 1 is a plan view schematically showing the structure of an embodiment of the cell culture chip. FIG. 2 is a schematic cross-sectional view when the cell culture chip 1 shown in FIG. 1 is cut along the line A1-A1 in FIG. 1. Hereinafter, FIG. 1 will be described as corresponding to the XZ plan view when the cell culture chip 1 is viewed from the Y direction, and FIG. 2 will be described as corresponding to the XY plan view when the cell culture chip 1 cut in the XY plane is viewed from the Z direction.

[0049] The cell culture chip 1 includes a bottom portion 3 and a base portion 5. The base portion 5 includes a first well 10 and a second well 20 that are opened in the Y direction toward the bottom portion 3 from a part of the surface (main surface 5a) on the side opposite to the bottom portion 3. That is, the first well 10 has an opening surface 10a on the main surface 5a side of the base portion 5 and is opened in the Y direction toward the bottom portion 3. Similarly, the second well 20 has an opening surface 20a on the main surface 5a side of the base portion 5 and is opened in the Y direction toward the bottom portion 3. That is, the Y direction corresponds to the "first direction".

[0050] The opening surface 10a of the first well 10 and the opening surface 20a of the second well 20 are arranged at positions spaced apart in a direction parallel to the main surface 5a of the base portion 5. Here, it will be described assuming that both are arranged at positions spaced apart from each other in the X direction. In this case, the X direction corresponds to the "second direction". Note that the opening surface 10a of the first well 10 and the opening surface 20a of the second well 20 may be spaced apart in the Z direction, or may be spaced apart in both the X direction and the Z direction. The "second direction" corresponds to the direction from the opening surface 10a of the first well 10 toward the opening surface 20a of the second well 20.

[0051] The base portion 5 has a tubular recess at a position on the bottom 3 side, and a chamber 7 is formed by the region sandwiched between this recess and the bottom 3. In the present embodiment, the chamber 7 constitutes a space for culturing cells.

[0052] In the present embodiment, one end of the chamber 7 is connected to the first well 10 via the communication well 19, and the other end is connected to the second well 20 via the communication well 29. Note that the communication well 19 is connected to the first well 10 in the Y direction, and the bottom 3 constitutes the bottom surface of the communication well 19. Similarly, the communication well 29 is connected to the second well 20 in the Y direction, and the bottom 3 constitutes the bottom surface of the communication well 29.

[0053] In the present embodiment, the first well 10 and the second well 20 have a region where the opening diameter decreases without increasing as approaching the bottom 3 at a position closer to the bottom 3 than the main surface 5a. This point will be described with reference to FIG. 3. FIG. 3 is an enlarged view of the vicinity of the first well 10 in FIG. 2.

[0054] As shown in Fig. 3, the first well 10 is located closer to the bottom 3 than the main surface 5a of the base portion 5. As it approaches the bottom 3, that is, as it progresses in the +Y direction, the opening diameter 10b decreases without increasing. This region is hereinafter referred to as the "diameter-reducing region 11". In the present embodiment, the first well 10 has a substantially uniform opening diameter 10b at a position closer to the main surface 5a of the base portion 5 than the diameter-reducing region 11. That is, the inner wall 10c of the first well 10 forms an inclined surface within the diameter-reducing region 11.

[0055] Fig. 4 is a drawing schematically showing a state in which the cell culture chip 1 of the present embodiment is filled with the culture solution 30. For convenience of illustration, in Fig. 4, the region where the culture solution 30 exists is hatched, and the bottom 3 and the base portion 5 are not hatched. In the following drawings, when the region where the culture solution 30 exists is to be clearly shown, it will be illustrated in the same manner.

[0056] When filling the cell culture chip 1 with the culture solution 30, the culture solution 30 is injected from the side of the first well 10 or the second well 20. For example, when the culture solution 30 is injected from the side of the first well 10, the culture solution 30 flows into the second well 20 side through the communication well 19 and the chamber 7. When a predetermined amount or more of the culture solution 30 is injected into the cell culture chip 1, the chamber 7 located between the first well 10 and the second well 20 is filled with the culture solution 30. Thereby, cells can be cultured in the chamber 7.

[0057] As shown in Fig. 4, the mode of extracting the culture solution 30 using the pipette 31 from the state where the cell culture chip 1 is filled with the culture solution 30 will be described. Hereinafter, the case where the culture solution 30 is extracted by sucking the culture solution 30 using the pipette 31 from the side of the first well 10 will be described.

[0058] Figs. 5A to 5C are drawings schematically showing a state in which the culture solution 30 is being sucked using the pipette 31. The mode in which the suction of the culture solution 30 progresses in the order of Figs. 5A, 5B, and 5C is shown.

[0059] When the tip of the pipette 31 is inserted into the first well 10 and the suction operation is started, the liquid level of the culture solution 30 gradually begins to drop (see Fig. 5A), and eventually drops into the reduced-diameter region 11 (see Fig. 5B). Thereafter, when the suction operation is further continued, the liquid level of the culture solution 30 drops into the communication well 19 (see Fig. 5C). Note that when the suction operation is continued at a suction force that is greater than the suction force required to completely suck the culture solution 30 stored in the first well 10 and within a range of a predetermined suction force or less, the liquid level of the culture solution 30 further drops, and eventually the bottom 3 is exposed (see Fig. 5C). At this time, a liquid pool of the culture solution 30 is formed at the corner portion in the communication well 19 (reference numeral 30a). However, even if the suction operation is further continued, the suction of the culture solution 30 does not progress.

[0060] That is, according to the configuration of the present embodiment, at the moment immediately after the suction of the culture solution 30 stored in the first well 10 is completed, the culture solution 30 does not flow from the chamber 7 toward the first well 10 side. And even if the suction operation from the pipette 31 is continued at a constant suction force, the suction of the culture solution 30 does not progress.

[0061] Regarding the reason for such a phenomenon, the present inventor considers as follows.

[0062] Fig. 6 is a drawing schematically showing a state in which the culture solution 130 filled in the conventional microchannel chip 100 is being sucked, and is substantially the same drawing as Fig. 15(c).

[0063] When the angle of the corner portion of the inlet 111 (hereinafter, described as "opening 111" here) is φ and the distance between both ends of the portion where the meniscus of the culture solution 130 (130a) stored in the corner portion contacts the corner portion is D1, the capillary force P1 generated in the meniscus of the culture solution 130a is represented by the following formula (1). In the following formula (1), γ represents the surface tension and θ represents the contact angle of the culture solution 130 (130a). P1≒2·γcos(θ+φ / 2) / (D1 / 2) ····(1)

[0064] On the other hand, when the inner diameter of the groove 110 is D2, the capillary force P2 generated in the meniscus of the culture solution 130 (130b) stored in the groove 110 is similarly expressed by the following formula using γ and θ. However, in the following formula (2), since the groove 110 has inner wall surfaces facing each other in parallel and the angle between both ends of the portion where the meniscus of the culture solution 130b is in contact is 0°, the φ component is calculated as 0. P2≒2·γcos(θ) / (D2 / 2) ····(2)

[0065] For example, when the contact angle θ of the culture solution 130 is 20° and the inner diameter D2 of the groove 110 is 400 μm, when the distance D1 between both ends of the portion where the meniscus of the culture solution 130a is in contact with the corner portion of the opening 111 is approximately 180 μm, P1 = P2 is satisfied. In other words, when the distance D1 < 180 μm, the capillary force P1 generated in the meniscus of the culture solution 130a is greater than the capillary force P2 generated in the meniscus of the culture solution 130b stored in the groove 110, and P1 > P2.

[0066] This means that in the conventional microchannel chip 100 shown in FIG. 6, when trying to suck the culture solution 130a remaining in the corner portion of the opening 111 in order to take out the culture solution 130a from the opening 111, the culture solution 130a is not sucked, but the culture solution 130b in the groove 110 is sucked instead. This state is consistent with the fact described above with reference to FIG. 15(d).

[0067] Based on this fact, in the structure shown in FIG. 3, by making the capillary force of the meniscus of the culture solution 30 stored in the corner portion of the first well 10 smaller than the capillary force of the meniscus of the culture solution 30 in the chamber 7, even if the culture solution 30 in the first well 10 is sucked with a suction force sufficient to suck all of it, it can be seen that the outflow of the culture solution 30 from the chamber 7 side can be prevented.

[0068] Here, as described above with reference to FIG. 3, the cell culture chip 1 of the present embodiment includes a reduced-diameter region 11 on the bottom 3 side of the first well 10, where the opening diameter 10b decreases as it progresses in the +Y direction. The corner angle φ of the corner portion in the reduced-diameter region 11 becomes an obtuse angle as shown in FIG. 5B and is larger than the opening 111 of the conventional microchannel chip shown in FIG. 6. As a result, the value of cos(θ + φ / 2) in the above formula (1) decreases. Therefore, in the state of FIG. 5B, the value of the capillary force P1 generated in the meniscus of the culture solution 30 stored in the corner portion in the reduced-diameter region 11 is lower than the value of the capillary force P1 generated in the meniscus of the culture solution 130 (130a) stored in the corner portion of the opening 111 in the conventional structure shown in FIG. 6.

[0069] As a result, the capillary force P1 generated in the meniscus of the culture solution 30 stored in the corner portion in the reduced-diameter region 11 becomes smaller than the capillary force P2 of the meniscus of the culture solution 30 in the chamber 7. Thus, even if the suction of the culture solution 30 with the pipette 31 is continued from the state of FIG. 5B, the suction of the culture solution 30 can be continued without storing the culture solution 30 in the corner portion of the first well 10, and the state shown in FIG. 5C can be reached.

[0070] Incidentally, the cell culture chip 1 of the present embodiment includes a communication well 19, and the corner angle of the corner portion of the communication well 19 may be approximately 90°, for example, similar to the opening 111 of the conventional microchannel chip 100. In this case, when the suction operation is continued from the state of FIG. 5B, the culture solution 30 (30a) is stored in the corner portion of the communication well 19. However, in the state before the state of FIG. 5C, since at least the culture solution 30 in the first well 10 has been completely removed, it is possible to end the suction operation at this point and introduce a new replacement culture solution from the first well 10 side.

[0071] When the contact angle θ of the culture solution 30 and the height of the chamber 7 are fixed, the preferable minimum value of the length D (the length of the chamfered part) of the inclined surface when the inner wall 10c in the reduced-diameter region 11 of the first well 10 is viewed from the Z direction is as shown in Table 1 below. By making the inner wall 10c in the reduced-diameter region 11 have an inclined surface larger than the value described in the table, the capillary force at the corner part in the first well 10 can be made lower than the capillary force of the chamber 7.

[0072]

Table 1

[0073] Specific dimensional examples of the cell culture chip 1 are as follows. · The height (the length in the Y direction) of the base part 5 is 1 mm or more and 20 mm or less, and is 3 mm as an example. · The height (the length in the Y direction) of the bottom part 3 is 0.1 mm or more and 5 mm or less, and is 1 mm as an example. · The height (the length in the Y direction) of the chamber 7 is 200 μm or more and 2000 μm or less, and is 400 μm as an example.

[0074] · The opening diameter 10b on the opening surface 10a side of the first well 10 is 0.5 mm or more and 40 mm or less, and is 2 mm as an example. · Among the first wells 10, the minimum value of the opening diameter 10b in the reduced-diameter region 11 is 0.5 mm or more and 40 mm or less, and is 1.75 mm as an example. Also, the length of the inclined surface when the inner wall 10c in this reduced-diameter region 11 is viewed from the Z direction is 20 μm or more and 2000 μm or less, and is 180 μm as an example. · The opening diameter of the communication well 19 is 0.2 mm or more and 39 mm or less, and is 1.75 mm as an example. Also, the height (the length in the Y direction) of the communication well 19 is 0.2 mm or more and 3 mm or less, and is 0.6 mm as an example.

[0075] · The separation distance between the central axis of the first well 10 and the central axis of the second well 20 is 2 mm or more and 40 mm or less, and is 9 mm as an example. · The opening diameter on the opening surface 20a side of the second well 20 is 0.5 mm or more and 40 mm or less, and is 1 mm as an example. · Among the second well 20, the minimum value of the opening diameter within the diameter-reduced region is 0.5 mm or more and 40 mm or less, and is 0.75 mm as an example. Also, the length of the inclined surface when the inner wall 10c within this diameter-reduced region 11 is viewed from the Z direction is 20 μm or more and 2000 μm or less, and is 180 μm as an example. · The opening diameter of the communication well 29 is 0.2 mm or more and 39 mm or less, and is 0.7 mm as an example. Also, the height (length in the Y direction) of the communication well 19 is 0.2 mm or more and 2000 mm or less, and is 0.6 mm as an example.

[0076] (Modification example) The cell culture chip 1 of the present embodiment can be variously modified. This will be described below.

[0077] 〈1〉 The cell culture chip 1 of the present embodiment described above has a structure having a diameter-reduced region similar to the diameter-reduced region 11 of the first well 10 also on the second well 20 side. Thereby, even when the culture solution 30 is sucked by the pipette 31 from the second well 20 side, for the same reason, it is possible to remove the culture solution in the second well 20 while retaining the culture solution 30 in the chamber 7. However, the present invention does not exclude a structure having a diameter-reduced region only on one well (first well 10 / second well 20) side.

[0078] <2>Figure 3 shows a case where the inner wall 10c of the first well 10 in the reduced-diameter region 11 is a flat surface. However, from the perspective of reducing the capillary force at the corner portion of the first well 10, it is not necessarily required that the inner wall 10c be a flat surface, and it may be configured as a curved surface. FIG. 7 is a drawing schematically showing the structure in the vicinity of the first well 10 when the inner wall 10c of the first well 10 in the reduced-diameter region 11 is configured as a curved surface, following FIG. 3.

[0079] <3>Figures 2 and 3 show a case where the first well 10 has a mode in which the opening diameter 10b decreases symmetrically with respect to the central axis in the reduced-diameter region 11. However, since the central axis in the reduced-diameter region 11 is different from the central axis on the main surface 5a side of the base portion 5 rather than the reduced-diameter region 11, the first well 10 may have an eccentric shape. FIGS. 8 and 9 are diagrams showing the cell culture chip 1 having such a structure, following FIGS. 1 and 2, respectively. In this aspect, the inner wall in the reduced-diameter region 11 may be formed as a curved surface as shown in FIG. 7, or the reduced-diameter region 11 may exist only in the first well 10 and may not have a reduced-diameter region on the second well 20 side.

[0080] <4>As shown in FIG. 10, the cell culture chip 1 may not be provided with the communication well (19 / 29). Even in such a configuration, since the first well 10 is provided with a reduced-diameter region, for the reasons described above, the culture solution 30 in the first well 10 can be removed without substantially sucking the culture solution 30 from the chamber 7 side.

[0081] However, as shown in FIG. 2, since the cell culture chip 1 is provided with the communication well (19 / 29), a thickness (wall thickness) corresponding to the height of the communication well (19 / 29) is ensured in the base portion 5 located at this position. Therefore, from the perspective of enabling stable molding during the manufacture of the cell culture chip 1, it is preferable to provide the communication well (19 / 29).

[0082] <5>As shown in FIG. 11, the chamber 7 provided in the cell culture chip 1 may be configured to include a culture chamber 7a that substantially constitutes a space for culturing cells, and a communication flow path (7b / 7c) that communicates the culture chamber 7a with each well (10 / 20) and has a smaller opening diameter than the culture chamber 7a. In this case, it may be assumed that the reduced-diameter region 11 is formed so that the capillary force of the first well 10 is smaller than the capillary force of the communication flow path 7b.

[0083] In the structure of the cell culture chip 1 shown in FIGS. 1 and 2, as described above, the chamber 7 constitutes a space for culturing cells. That is, the chamber 7 corresponds to the culture chamber.

[0084] [Alternative Embodiment] Hereinafter, an alternative embodiment will be described.

[0085] <1>In the above embodiment, the bottom portion 3 and the base portion 5 provided in the cell culture chip 1 have been described as being made of separate members, but the bottom portion 3 and the base portion 5 may be constituted by integrally molding the same member.

[0086] <2>The opening diameter 10b of the first well 10 of the cell culture chip 1 described with reference to FIG. 3 is substantially uniform at a position closer to the main surface 5a of the base portion 5 than the reduced-diameter region 11. However, the first well 10 may have a structure in which the opening diameter 10b decreases over the entire length from the opening surface 10a toward the bottom portion 3 side and has a reduced-diameter region 11.

[0087] <3>According to the cell culture chip 1 of each of the above embodiments, it has been described that it is possible to extract the culture solution 30 filled inside from the first well 10 side or the second well 20 side while retaining the culture solution 30 in the chamber 7. However, the content to be extracted from the cell culture chip 1 while being retained in the chamber 7 is not limited to the culture solution 30 and may be any liquid.

[0088] 〈4〉In the above embodiment, the case where the pipette 31 is used when sucking the culture solution 30 from the cell culture chip 1 has been exemplified and described. However, the suction method is not limited to the pipette 31. The cell culture chip 1 of the present invention can adopt other general methods in which the tip of the suction device is arranged on one well side (for example, the first well 10) and the culture solution 30 stored in the well is sucked.

[0089] 〈5〉In the above embodiment, the cell culture chip 1 in which a pair of wells (10, 20) are connected by the chamber 7 has been described. However, in the cell culture chip 1 of the present invention, the number of wells and the number of chambers are not limited.

[0090] Figs. 12A to 12D are plan views schematically showing the cell culture chip 1 of another embodiment in accordance with Fig. 1.

[0091] The cell culture chip 1 shown in Fig. 12A has three wells (10, 20, 41) formed in series, and chambers (7, 7) are formed so as to connect between the respective wells. The cell culture chip 1 shown in Fig. 12B has three wells (10, 20, 41) formed. However, unlike the cell culture chip 1 shown in Fig. 12A, the chamber 7 connecting the well 10 and the well 20 and the chamber 7 connecting the well 10 and the well 41 are interconnected.

[0092] The cell culture chip 1 shown in Fig. 12C includes four wells (10, 20, 41, 42), and the chamber 7 connecting the well 10 and the well 20, the chamber 7 connecting the well 10 and the well 41, and the chamber 7 connecting the well 10 and the well 42 are each independently formed.

[0093] The cell culture chip 1 shown in Fig. 12D includes six wells (10, 20, 41, 42, 43, 44), and chambers 7 are formed so as to connect adjacent wells. However, all the wells (10, 20, 41, 42, 43, 44) are connected in series in a ring shape by each chamber 7.

[0094] Also in the cell culture chip 1 shown in each of FIGS. 12A to 12D, the wells (10, 20, 41, 42, 43, 44) have a shape in which the capillary force is lower than the capillary force of the chamber 7. For examples of more specific structures, since they are common to the above-described embodiments, the description thereof is omitted.

[0095] 〈6〉In the cell culture chip 1 described with reference to FIG. 2 above, the communication wells (19, 29) have been described as communicating in the Y direction between each well (10, 20) and the chamber 7. However, the communication wells (19, 29) may be formed so as to communicate in a direction parallel to the main surface 5a of the base portion 5 (for example, the X direction) between each well (10, 20) and the chamber 7. That is, the end portion of the chamber 7 is not disposed directly below the wells (10, 20), and the communication wells (19, 29) may be formed so as to communicate the wells (10, 20) and the chamber 7 by extending also in the X direction.

Explanation of Signs

[0096] 1: Cell culture chip 3: Bottom 5: Base portion 5a: Main surface of the base portion 7: Chamber 7a: Culture chamber 7b, 7c: Communication channels 10: First well 10a: Opening surface of the first well 10b: Opening diameter of the first well 10c: Inner wall of the first well 11: Reduced diameter region 19: Communication well 20: Second well 20a: Opening surface of the second well 29: Communication well 30, 30a: Culture solution 31: Pipette 41, 42, 43, 44: Wells 100: Conventional microchannel chip 101: Substrate 102: Resin film 110: Groove 111: Inlet 112: Outlet 120: Pipette 130, 130a: Culture solution

Claims

1. A method for using a cell culture chip, comprising: The cell culture chip includes: A bottom; A base portion formed on the upper surface of the bottom; A first well formed by opening the base portion in a first direction from a part of the main surface, which is the surface on the side opposite to the bottom of the base portion, toward the bottom; A second well formed by opening the base portion in the first direction at a position spaced from the first well in a second direction parallel to the main surface; A tubular chamber that connects the first well and the second well in the second direction by a region sandwiched between the bottom and the base portion; The first well has a shape in which the capillary force of the first well is lower than the capillary force of the chamber; Step (a) of filling the first well and the chamber with a culture solution; Step (b) of exposing the bottom surface of the first well without causing the culture solution in the chamber to flow into the first well by sucking the culture solution in the first well with a predetermined suction force; The first well has a reduced-diameter region in a position closer to the bottom than the main surface of the base portion, and the opening diameter continuously decreases without increasing as it approaches the bottom; The cell culture chip has a communication well that is continuously formed with the first well in the first direction and connects the reduced-diameter region of the first well and the chamber in the first direction; The communication well uses the bottom as the bottom surface, and the opening diameter is narrower than the reduced-diameter region of the first well. A method for using a cell culture chip.

2. The method for using a cell culture chip according to claim 1, wherein step (b) is performed until the culture solution stored in the first well is completely sucked.

3. The method for using a cell culture chip according to claim 1 or 2, further comprising step (c) of filling the first well with a new culture solution after step (b).

4. The first well has an inner wall formed of the base portion, The method for using a cell culture chip according to any one of claims 1 to 3, wherein the inner wall includes a curved surface or a plane non-parallel to the main surface in the reduced-diameter region of the first well.

5. The method for using a cell culture chip according to any one of claims 1 to 4, wherein the second well has a shape in which the capillary force of the second well is lower than the capillary force of the chamber.

6. The method for using a cell culture chip according to claim 5, wherein the second well has a reduced diameter region that continuously decreases without increasing the opening diameter as it approaches the bottom at a position closer to the bottom than the main surface of the base portion.

Images