Electrode device, cell culture kit, and cell culture method using cell culture kit

Abstract

To provide an electrode device in which an electrode is made of a material other than platinum and electrical stimulation is given to all wells of a well plate for cell culture in which the electrode is arranged.SOLUTION: The electrode device 1 includes a first electrode member 10 in which n electrodes (except that the electrodes are made of platinum) e1 to en are disposed on an elongated first substrate 11, and a second electrode member 20 in which n electrodes (except that the electrodes are made of platinum) are disposed on an elongated second substrate 21. In the electrode device, a circuit is formed such that only one of the first end side region 14 of the first substrate and the third end side region 24 of the second substrate becomes a positive electrode to which a current from an external power supply is supplied, the current supplied to the positive electrode passes through all of the n electrodes disposed on the first substrate and the n electrodes disposed on the second substrate, and only one of the second end side region 15 of the first substrate and the fourth end side region 25 of the second substrate becomes a negative electrode for returning the current to the external power supply.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The disclosure in this application relates to an electrode device for applying electrical stimulation to cells cultured in a cell culture well plate, a cell culture kit including the electrode device, and a method for culturing cells using the cell culture kit. [Background technology]

[0002] Skeletal muscle atrophy is a condition characterized by a decrease in muscle protein content, muscle fiber diameter, and contractile force generation. Diseases that cause skeletal muscle atrophy include hereditary muscle diseases such as muscular dystrophy, inflammatory muscle diseases such as polymyositis and dermatomyositis, immobility due to bedriddenness, and aging. Because muscle atrophy worsens quality of life, the development of anti-atrophy substances is highly desirable.

[0003] In the development of anti-atrophy substances, muscle atrophy models in which skeletal muscle cells are cultured are generally used. FIG. 1 of Non-Patent Document 1 describes a device that combines a device for culturing skeletal muscle cells to form skeletal muscle tissue with electrodes for applying electrical stimulation to the cultured skeletal muscle tissue. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] K. Yamamoto et al., “Screening of anti-atrophic peptides by using photo-cleavable peptide array and 96-well scale contractile human skeletal muscle atrophy models”, Biotechnol Bioeng. 2022;119:2196-2205.DOI:10.1002 / bit.28125 Summary of the Invention [Problem to be solved by the invention]

[0005] The device described in Non-Patent Document 1 involves fabricating a cell culture device with pillars (see Figure 1(a)), placing the fabricated cell culture device in each well of a 96-well plate, forming skeletal muscle tissue between a pair of pillars (see Figure 1(c)), and inserting electrodes (see Figure 1(b)) from above the 96-well plate to electrically stimulate the skeletal muscle tissue (see Figure 1(c)).

[0006] However, the present inventors have found that the invention described in Non-Patent Document 1 (1) Platinum electrodes are expensive, and prolonged electrical stimulation of platinum electrodes can cause electrolysis of the water that makes up the culture medium, which can hinder long-term cell culture. (2) The electrodes shown in Figure 1(b) are designed to allow electricity to be applied to each electrode pair inserted into each well, but are not suitable for experiments in which electrical stimulation is applied to multiple wells simultaneously. We have discovered that new problems such as the following occur.

[0007] The disclosure of the present application has been made to solve the above problems. (1) Making the electrodes out of materials other than platinum; (2) By devising a circuit design on the substrate on which the electrodes are placed, it is possible to connect the positive and negative poles to an external power source, thereby passing current through all of the placed electrodes at once and providing electrical stimulation to all of the wells in which the electrodes are placed. We have now discovered that the above problem can be solved.

[0008] That is, the purpose of the disclosure in this application is to provide an electrode device for applying electrical stimulation to cells cultured in a cell culture well plate, a cell culture kit including the electrode device, and a method for culturing cells using the cell culture kit. [Means for solving the problem]

[0009] The disclosure of the present application relates to an electrode device, a cell culture kit, and a method for culturing cells using the cell culture kit, as described below.

[0010] (1) An electrode device for applying electrical stimulation to cells cultured in a cell culture well plate, the electrode device comprising: a first electrode member in which n electrodes (excluding the electrodes being made of platinum) are arranged on an elongated first substrate; a second electrode member in which n electrodes (excluding the case where the electrodes are made of platinum) are arranged on an elongated second substrate; Including, n is an integer of 2 or more, and the number of electrodes arranged on the first substrate is the same as the number of electrodes arranged on the second substrate; One end of the first substrate is defined as a first end, and the other end is defined as a second end; one end of the second substrate is defined as a third end and the other end is defined as a fourth end; The n electrodes arranged on the first base material are defined as 1st to nth electrodes in order from the first end portion side, The n electrodes arranged on the second base material are defined as 1st to nth electrodes in order from the third end side, A combination of the same electrode arranged on the first base material and the second base material is defined as a 1st to nth electrode pair, A first end side region of the first substrate is defined as a first end side region of the first electrode, and a second end side region of the first substrate is defined as a second end side region of the n-th electrode, a third end side region of the second substrate from the first electrode to a third end side, and a fourth end side region of the second substrate from the n-th electrode to a fourth end side, When it is assumed that the first electrode member and the second electrode member are arranged substantially parallel to each other so that the first end and the third end face each other and the second end and the fourth end face each other, and that a current flows between two electrodes constituting each of the 1st to nth electrode pairs, The first substrate and the second substrate include: Only one of the first end side region and the third end side region serves as a positive electrode to which a current is supplied from an external power supply, a current supplied to the positive electrode passes through all of the n electrodes arranged on the first substrate and the n electrodes arranged on the second substrate; Only one of the second end side region and the fourth end side region serves as a negative pole for current to return to an external power supply. The circuit is formed as follows: Electrode device. (2) When the first end side region is a positive electrode, a circuit is formed on the first substrate so that a current flows through all electrodes from the positive electrode to the n-th electrode; the fourth end region is a negative electrode, and a circuit is formed in the second substrate so that a current flows through all electrodes from the negative electrode to a first electrode; When the third end side region is a positive electrode, a circuit is formed on the second substrate so that a current flows through all electrodes from the positive electrode to the n-th electrode; The second end side region is a negative electrode, and a circuit is formed in the first substrate so that a current flows through all electrodes from the negative electrode to the first electrode. The electrode device according to (1) above. (3) When the first end side region is a positive electrode, a circuit is formed on the first substrate so that a current flows between the positive electrode and a first electrode, and a circuit is formed so that a current flows between an even-numbered electrode and an odd-numbered electrode; a circuit is formed on the second substrate so that a current flows between odd-numbered electrodes and even-numbered electrodes; When the third end side region is a positive electrode, a circuit is formed on the second substrate so that a current flows between the positive electrode and a first electrode, and a circuit is formed so that a current flows between an even-numbered electrode and an odd-numbered electrode; A circuit is formed on the first substrate so that a current flows between odd-numbered electrodes and even-numbered electrodes. The electrode device according to (1) above. (4) When the number of electrodes arranged on the first base material and the second base material is even, When the first end side region is a positive pole, The negative electrode is the second end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode, When the third end side region is a positive electrode, The negative electrode is the fourth end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode. The electrode device according to (3) above. (5) When the number of electrodes arranged on the first base material and the second base material is odd, When the first end side region is a positive pole, the negative electrode is the fourth end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode; When the third end side region is a positive electrode, The negative electrode is the second end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode. The electrode device according to (3) above. (6) The electrode is a carbon electrode. The electrode device according to any one of (1) to (5) above. (7) The first end and the third end are connected by a first connecting member, the second end and the fourth end are connected by a second connecting member, The first and second connecting members are non-conductive and have the same length. The electrode device according to any one of (1) to (6) above. (8) The number of electrodes arranged on the first substrate and the second substrate is 6, 8, or 12. The electrode device according to any one of (1) to (7) above. (9) The cell culture well plate further includes a frame that can be fixed to the outer periphery of the cell culture well plate, and the frame includes: the first electrode member and the second electrode member are sized so that the first end-side region, the second end-side region, the third end-side region, and the fourth end-side region abut against each other when the first electrode member and the second electrode member are arranged substantially parallel to each other, At least the frame portion where the positive electrode and the negative electrode come into contact is made of a non-conductive material and circuitized for connection to an external power source; or Made of conductive material The electrode device according to any one of (1) to (8) above. (10) An electrode device according to any one of (1) to (9) above; a cell culture device; A cell culture kit comprising: The cell culture device comprises: A third substrate; n pillar holders formed to protrude from the third base material and for holding pillars; Including, the number of the n pillar holding portions is the same as the number of the n electrodes, A pair of pillars that serve as scaffolds for cultured cells are formed on each of the pillar holders, The third base material has a size such that it can be placed between the first electrode member and the second electrode member when the first electrode member and the second electrode member are placed on a cell culture well plate so that the first electrode member and the second electrode member are substantially parallel to each other. Cell culture kit. (11) The cell culture well plate further includes a frame that can be fixed to the outer periphery of the cell culture well plate, and the frame includes: the first electrode member and the second electrode member are sized so that the first end-side region, the second end-side region, the third end-side region, and the fourth end-side region abut against each other when the first electrode member and the second electrode member are arranged substantially parallel to each other, The frame portion where the positive electrode and the negative electrode come into contact is made of a non-conductive material and circuitized for connection to an external power source; or Made of conductive material The cell culture kit according to (10) above. (12) A method for culturing cells using a cell culture kit, the method being carried out using the cell culture kit according to (10) or (11), In one well of a separately prepared cell culture well plate, the first electrode member and the second electrode member are arranged in a cell culture well plate so that one electrode of the first electrode member and one electrode of the second electrode member are arranged; the cell culture device is placed between the first electrode member and the second electrode member so that the pair of pillars formed in the pillar holder of the cell culture device are immersed in the culture medium in one well. The preparation process and a cell culture step of culturing cells in the wells; an electrical application step of applying an electrical stimulus to the cells being cultured during at least a portion of the cell culture step; Contains Culture method. [Effects of the Invention]

[0011] The electrode device disclosed in this application is made of a material other than platinum. Therefore, electrolysis is less likely to occur compared to platinum electrodes. Furthermore, the electrode device disclosed in this application has a circuit configuration that allows current to flow through all of the placed electrodes at once by connecting the positive and negative poles to an external power source. Therefore, electrical stimulation can be applied to all wells in which electrodes are placed, improving experimental efficiency despite the simple configuration. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a schematic diagram of a conventional electrode device and cell culture device described in Non-Patent Document 1. As shown in FIG. [Figure 2] FIG. 2 is a schematic top view of the electrode device 1 according to the first embodiment. [Figure 3] FIG. 3 is a cross-sectional view of the electrode device 1 according to the first embodiment. [Figure 4] FIG. 4 is a schematic top view of the electrode device 1 according to the first embodiment. [Figure 5] FIG. 5 is a schematic top view of the electrode device 1 according to the first embodiment. [Figure 6] FIG. 6 is a schematic top view of the electrode device 1 according to the first embodiment. [Figure 7] FIG. 7 is a top view showing an outline of an optional configuration example that can be adopted by the electrode device 1 according to the first embodiment. [Figure 8] FIG. 8 is a schematic top view of a frame 40 included in the electrode device 1 according to the second embodiment. [Figure 9] FIG. 9 is a top view and a cross-sectional view showing a schematic diagram of a cell culture device 50. [Figure 10] FIG. 10 is a top view and a cross-sectional view showing an outline of the positional relationship when the first electrode member 10, the cell culture device 50, and the second electrode member 20 are arranged on a cell culture well plate 60. As shown in FIG. [Figure 11] FIG. 11 is a photograph substituted for a drawing, which is a photograph of the electrode device produced in Example 1. [Figure 12] FIG. 12 is a photograph used as a drawing, which is a photograph of the frame produced in Example 1. [Figure 13] FIG. 13 is a photograph substituted for a drawing, which is a photograph of the cell culture device produced in Example 1. [Figure 14] FIG. 14 is a graph showing the results of Example 2, in which the contractile force of muscle tissue was increased by applying electrical stimulation to cultured cells. DETAILED DESCRIPTION OF THE INVENTION

[0013] The electrode device, cell culture kit, and cell culture method using the cell culture kit disclosed in the present application are described in detail below. Note that the position, size, range, etc. of each component shown in the drawings may not represent the actual position, size, range, etc., for ease of understanding. Therefore, the disclosure of the present application is not necessarily limited to the position, size, range, etc., disclosed in the drawings. Note that in this specification, components having similar functions are assigned the same or similar symbols. Repeated descriptions of components assigned the same or similar symbols may be omitted.

[0014] In addition, in this specification, (1) A numerical range expressed using "~" means a range that includes the numbers written before and after "~" as the lower and upper limits. (2) Numerical values, numerical ranges, and qualitative expressions (e.g., expressions such as "same" and "the same") indicate numerical values, numerical ranges, and properties that include errors generally accepted in the relevant technical field. (3) When describing something as "approximately ____ shape," it includes not only the exact ____ shape, but also a shape that can be understood as roughly ____ shape. This is interpreted as:

[0015] (First embodiment of the electrode device) An electrode device 1 according to a first embodiment will be described with reference to Figures 2 to 6. Figure 2 is a top view showing an outline of the electrode device 1 according to the first embodiment. Figure 3 is a cross-sectional view taken along the arrow XX in Figure 2A. The electrode device 1 includes a first electrode member 10 and a second electrode member 20.

[0016] The first electrode member 10 has n electrodes e1 to en arranged on an elongated first base material 11. The second electrode member 20 has n electrodes e1 to en arranged on an elongated second base material 21. The number of electrodes arranged on the first base material 11 is the same as the number of electrodes arranged on the second base material 21.

[0017] There are no particular limitations on the material for forming the electrodes e1 to en, as long as it is an electrode material other than platinum and does not easily cause electrolysis of the water that constitutes the culture medium. Examples of the material include, but are not limited to, carbon and gold.

[0018] The number of electrodes, n, is an integer equal to or greater than 2. Examples of n include, but are not limited to, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and 20. Among currently commercially available cell culture well plates, 6 × 8 (48-well) plates and 8 × 12 (96-well) plates have relatively large numbers of wells. When using these cell culture well plates, the number of electrodes, n, is preferably selected from 6, 8, and 12. To simultaneously apply electrical stimulation to a large number of cultured cells, a cell culture well plate with an arbitrary number of electrodes can be fabricated. Alternatively, it is also possible to simultaneously apply electrical stimulation to a predetermined number (even or odd) of cells in a commercially available well plate. Therefore, the number of electrodes can be appropriately determined depending on the number of wells in the row and column directions of the cell culture well plate to be used and which wells are to be used.

[0019] As shown in FIG. 2A, one end of the first substrate 11 is defined as the first end 12, the other end as the second end 13, one end of the second substrate 21 as the third end 22, and the other end as the fourth end 23. The n electrodes arranged on the first substrate 11 are defined as the first to n-th electrodes (e1 to en) in order from the first end 12 side, and the n electrodes arranged on the second substrate 21 are defined as the first to n-th electrodes (e1 to en) in order from the third end 22 side. The combinations of the same electrodes arranged on the first substrate 11 and the second substrate 21 are defined as the first to n-th electrode pairs. In the example shown in FIG. 2A, electrode e1 of the first substrate and electrode e1 of the second substrate 21 constitute the first electrode pair, and electrode en of the first substrate and electrode en of the second substrate 21 constitute the n-th electrode pair.

[0020] The first substrate 11 has a first end 12 side from the first electrode (e1) defined as a first end side region 14, and a second end 13 side from the nth electrode (en) defined as a second end side region 15. The second substrate 21 has a third end 22 side from the first electrode (e1) defined as a third end side region 24, and a fourth end 23 side from the nth electrode defined as a fourth end side region 25.

[0021] Next, the circuit configuration of the electrode device 1 according to the first embodiment will be described. The circuit is configured such that the first electrode member 10 and the second electrode member 20 are arranged substantially parallel to each other, with the first end 12 and the third end 22 facing each other and the second end 13 and the fourth end 23 facing each other, and assuming that a current flows between the two electrodes constituting each of the 1st to nth electrode pairs, the first substrate 11 and the second substrate 12 are provided with: (1) Only one of the first end side region 14 and the third end side region 24 serves as a positive electrode P to which current is supplied from an external power source. (2) The current supplied to the positive electrode P passes through all of the n electrodes (e1 to en) arranged on the first substrate 11 and the n electrodes (e1 to en) arranged on the second substrate 21, (3) Only one of the second end side region 15 and the fourth end side region 25 becomes the negative pole N for the current to return to the external power supply. It is sufficient if the structure is formed as follows.

[0022] An example of the circuit configuration will be described in more detail with reference to FIGS. 2 and 3. In the example shown in FIG. 2A, the first end-side region 14 is a positive electrode P. In the example shown in FIG. 2A, a circuit c is formed in the first substrate 11 so that a current flows between the positive electrode P and the first electrode e1, and between even-numbered electrodes and odd-numbered electrodes (e2-e3, e4-e5, etc. in the example shown in FIG. 2A). In the second substrate 21, a circuit c is formed so that a current flows between odd-numbered electrodes and even-numbered electrodes (e1-e2, e3-e4, etc. in the example shown in FIG. 2A). Then, as shown in FIG. 2, when the number of electrodes arranged on the first substrate 11 and the second substrate 21 is even, the negative electrode N becomes the second end-side region 15, and a circuit c is formed so that a current flows between the negative electrode and the nth electrode (en).

[0023] The electrode device 1 according to the first embodiment is arranged so that each of the first to nth electrode pairs is immersed in the medium in each well of a cell culture well plate. More specifically, the electrode pairs e1-e1, e2-e2, e3-e3, and so on are immersed in different wells as shown in FIG. 2A. The medium is conductive. Therefore, as shown by the arrows in FIG. 2B, current supplied to the positive electrode P flows from the positive electrode P to the electrode e1 of the first electrode member 10 via the circuit c, from the electrode e1 of the first electrode member 10 via the medium to the electrode e1 of the second electrode member 20, from the electrode e1 of the second electrode member 20 via the circuit c to the electrode e2 of the second electrode member 20, from the electrode e2 of the second electrode member 20 via the medium to the electrode e2 of the first electrode member 10, and so on. The current then flows through the circuit c and the medium. Finally, the circuit c formed in the second end-side region 15 becomes the negative electrode N, and the current returns to the external power source.

[0024] The circuit c is not particularly limited as long as it is configured so that a current flows between the electrodes connected to the circuit c. For example, as shown in FIGS. 2 and 3, the circuit c may be formed by using an insulating material for the first substrate 11 and the second substrate 21 and laminating a conductive material thereon. The insulating material and the conductive material may be materials commonly used in the field of printed circuit boards. In the example shown in FIG. 3, the circuit c is formed on the upper surface of the first substrate 11 (the surface opposite the cell culture well plate when placed on the cell culture well plate). Alternatively, the circuit c may be formed on the lower surface of the first substrate 11 (the surface facing the cell culture well plate when placed on the cell culture well plate), or on both the upper and lower surfaces of the first substrate 11. Although not shown, the circuit c of the second substrate 21 may also be formed in the same manner as the circuit c of the first substrate 11. Furthermore, if the circuit c is exposed, there is a risk of electric shock. Therefore, after forming the circuit c, the circuit c portion may be optionally covered with an insulating material, except for the portion required for connection to an external power source.

[0025] As described above, the electrode device 1 according to the first embodiment is placed so as to be immersed in the medium of a cell culture well plate. Therefore, the size of the electrodes is preferably such that the electrode pair can be placed in the well plate. Furthermore, there is no particular limitation on the shape of the electrodes as long as they can be placed in the wells. Examples include, but are not limited to, rod-shaped and plate-shaped. The diameter of a well in a commercially available 96-well plate is approximately 7 mm, although this varies depending on the manufacturer. The diameter of a well in a commercially available 48-well plate is approximately 10 mm, although this varies depending on the manufacturer. The size and shape of the electrodes can be appropriately adjusted so that the electrode pair can be placed at a predetermined distance in the wells when the first electrode member 10 and the second electrode member 20 are placed in the wells.

[0026] Furthermore, commercially available cell culture well plates have wells of the same size arranged in a row. Therefore, it is preferable that the electrodes e1 to en have the same size and shape and are arranged at approximately equal intervals on the first substrate 11 and the second substrate 21. When electrodes of the same size and shape are arranged at approximately equal intervals, the same electrical stimulus is applied to the cells cultured in each well, making it possible to make the experimental conditions between each well the same. Alternatively, the size and shape of the electrodes e1 to en may be different, and they may not be arranged at equal intervals on the first substrate 11 and the second substrate 21, as long as each electrode pair can be arranged in the well.

[0027] (Other examples of circuit configurations) The example shown in Fig. 2 is one example of a circuit configuration. Other circuit configuration examples will be described with reference to Figs. 4 to 6. Figs. 4 to 6 are diagrams mainly for explaining the circuit configuration examples. To simplify the drawings, reference numerals are omitted for components that are not necessary for explaining the circuit configuration, but the configurations indicated by the omitted reference numerals are the same as the configuration shown in Fig. 2.

[0028] (Part 1) In the example shown in FIG. 2, the first end-side region 14 is the positive pole P, but in the example shown in FIG. 4, the third end-side region 24 is the positive pole P. When the third end-side region 24 is the positive pole P, the negative pole N becomes the fourth end-side region 25. As shown in FIG. 4, in the second substrate 21, a circuit c is formed so that a current flows between the positive pole P and the first electrode e1, a circuit c is formed so that a current flows between the even-numbered electrodes and the odd-numbered electrodes, and a circuit is formed so that a current flows between the negative pole N and the n-th electrode. In addition, in the first substrate 11, a circuit c is formed so that a current flows between the odd-numbered electrodes and the even-numbered electrodes.

[0029] (Part 2) 2 and 4, an even number of electrodes are arranged on the first substrate 11 and the second substrate 21. Fig. 5 shows a circuit configuration in which an odd number of electrodes are arranged on the first substrate 11 and the second substrate 21.

[0030] 5A shows an example in which the first end side region 14 is the positive pole P. In this case, the negative pole N becomes the fourth end side region 25, and a circuit is formed so that a current flows between the negative pole N and the nth electrode en. FIG. 5B shows an example in which the third end side region 24 is the positive pole P. In this case, the negative pole N becomes the second end side region 15, and a circuit is formed so that a current flows between the negative pole N and the nth electrode en.

[0031] (Part 3) In the examples shown in Figures 2 to 5, the current supplied to the positive electrode P flows in one stroke to the negative electrode N (hereinafter, this may be referred to as a "series arrangement"). On the other hand, Figure 6 shows an example in which the current supplied to the positive electrode P flows in parallel between the two electrodes constituting each electrode pair arranged in each well (hereinafter, this may be referred to as a "parallel arrangement").

[0032] 6A shows an example in which the first end side region 14 is the positive pole P and the fourth end side region 25 is the negative pole N. In the example shown in Fig. 6A, a circuit c is formed on the first substrate 11 so that a current flows to all electrodes from the positive pole P to the n-th electrode, and a circuit c is formed on the second substrate 21 so that a current flows to all electrodes from the negative pole N to the first electrode e1.

[0033] As described above, the electrode device 1 shown in FIG. 6 is arranged so that each electrode pair is immersed in the culture medium in each well of a cell culture well plate. The culture medium is conductive. Therefore, as shown by the arrows in FIG. 6B, a current supplied to the positive electrode P flows from the positive electrode P to the electrode en of the first electrode member 10 via the circuit c. On the other hand, the current flowing through the electrodes e1 to en of the first electrode member 10 flows through the culture medium to the electrodes e1 to en of the second electrode member 20 on the other side in the same well. The current flowing through the electrodes e1 to en of the second electrode member 20 via the circuit c returns to the external power source from the negative electrode N formed in the fourth end-side region 25.

[0034] When the third end side region 24 is the positive pole P, the second end side region 15 becomes the negative pole, and the direction of the current flowing between the first electrode member 10 and the second electrode member 20 is opposite to that in the example shown in Fig. 6B, but other than this, the example is the same as the example shown in Fig. 6. Therefore, the illustration of the case where the third end side region 24 is the positive pole P is omitted.

[0035] The electrode device 1 according to the first embodiment described above has the following advantages. (1) The electrode device 1 is made of a material other than platinum. Therefore, compared to platinum electrodes, electrolysis of water constituting the culture medium is less likely to occur. Furthermore, while the application of electrical stimulation for a long period of time can hinder cell culture, the electrode device 1 according to the first embodiment is made of a material that is less likely to cause electrolysis of water, and therefore can provide electrical stimulation to cultured cells for a long period of time. (2) By connecting the positive and negative electrodes to an external power source, it is possible to pass current through all of the placed electrodes at once. This means that electrical stimulation can be applied to all of the wells in which the electrodes are placed, improving experimental efficiency despite the simple configuration. (3) In the example shown in Figures 2 and 4, electrode devices 1 can be placed in all wells of a cell culture well plate by simply preparing two types of electrode members each having a number of electrodes corresponding to the number of wells in the row or column direction of the cell culture well plate. On the other hand, in the example shown in Figures 5 and 6, the first electrode member 10 and the second electrode member 20 are essentially the same member when turned upside down. Therefore, in the example shown in Figures 5 and 6, electrode devices 1 can be placed in all wells of a cell culture well plate by simply preparing one type of electrode member, thereby reducing manufacturing costs and improving user convenience. (4) It is possible to manufacture an electrode device in which the same number of electrode pairs as the number of wells are formed on a substrate corresponding to the row and column dimensions of a cell culture well plate so that electrode pairs can be inserted into all wells of the well plate. However, in such a case, even if one of the electrodes is damaged or soiled, repair is required, and depending on the extent of the damage or soiling, the entire electrode device may become unusable. On the other hand, as described in (3) above, the electrode device 1 disclosed in the present application allows electrode pairs to be inserted into all wells of a cell culture well plate using one or two types of electrode members. Therefore, even if an electrode is damaged, it can be quickly replaced by preparing one or two types of electrode members in advance.

[0036] (Optional additional configuration examples that can be adopted by the electrode device 1 according to the first embodiment) Next, an optional configuration example that can be adopted by the electrode device 1 according to the first embodiment will be described. Fig. 7 is a diagram showing an outline of the optional configuration example. Note that, for the sake of simplicity, reference numerals are omitted for components that are not necessary for the description of the optional configuration example, but the configurations indicated by the omitted reference numerals are the same as those shown in Fig. 2.

[0037] 7, the first end 12 and the third end 22 are connected by a first connecting member 31, and the second end 13 and the fourth end 23 are connected by a second connecting member 32. The first connecting member 31 and the second connecting member 32 are non-conductive and have the same length.

[0038] When the electrode device 1 according to the embodiment optionally includes the first connecting member 31 and the second connecting member 32, the following effects are achieved. (1) Even the electrode device 1 shown in Figures 2 to 6 can be placed on a cell culture well plate so that the distance between the first electrode member 10 and the second electrode member 20 is the same during use. However, if the electrode device 1 is provided with the first connecting member 31 and the second connecting member 32, the distance between the first electrode member 10 and the second electrode member 20 is always the same. Therefore, when placed on a cell culture well plate, the conditions for the electrical stimulation applied to each well are the same, and the placement operation is easier.

[0039] The electrode device 1 according to the first embodiment and optional additional configuration examples described above are merely examples of the electrode device 1, and are not limited to these examples. Other configurations may be added or removed within the scope of the technical concept disclosed in this application. For example, in the example shown in FIG. 7, two electrode members are connected using a connecting member. Alternatively, four electrode members may be connected using a connecting member. In this case, the electrode device 1 can be placed in two rows or two columns of wells of a cell culture well plate at once.

[0040] Furthermore, when the electrode device 1 is arranged in parallel as shown in FIG. 6 , instead of forming a circuit c out of a conductive material on the first substrate 11 formed of an insulating material, the first substrate 11 itself may be formed of a conductive material, and one of the first end region 14 or the second end region 15 may be coated with an insulating material for insulation. Similarly, the second substrate 21 may be formed of a conductive material, and one of the third end region 24 or the fourth end region 25 may be coated with an insulating material. Furthermore, when the electrode device 1 is arranged in series as shown in FIGS. 2 to 5 , the first substrate 11 may be formed by alternately joining conductive and non-conductive materials, and the first substrate 11 itself may form a circuit. By arranging electrodes at the junctions between the conductive and non-conductive materials, current can be passed between the two electrodes arranged at both ends of the conductive material. Similarly to the first substrate 11, the second substrate 21 may also form a circuit by alternately joining conductive and non-conductive materials. In this specification, the "circuit" formed on the first substrate 11 and the second substrate 21 means both the "conductive material" formed on the first substrate 11 and the second substrate 21 formed from an insulating material, and the "first substrate 11 and the second substrate 21 themselves formed from a conductive material."

[0041] (Electrode device 1 according to the second embodiment) An electrode device 1 according to a second embodiment will be described with reference to Fig. 8. The electrode device 1 according to the second embodiment includes a frame body 40 for arranging any of the electrode devices 1 described in the first embodiment.

[0042] In the example shown in FIG. 8 , the frame 40 is made of a non-conductive material. The non-conductive material is not particularly limited, and may be made of a non-conductive resin or the like. The frame 40 is sized so that the first end region 14, the second end region 15, the third end region 24, and the fourth end region 25 abut against each other when the first electrode member 10 and the second electrode member 20 are arranged substantially parallel to each other. Circuits for connecting to an external power source are formed on the frame 40 at least in the areas where the positive electrode P and the negative electrode N abut. In the example shown in FIG. 8 , a positive-side circuit 41 for supplying current from an external power source to the positive electrode P and a negative-side circuit 42 are arranged on the frame 40. While FIG. 8 only shows a top view to facilitate understanding of the arrangement of the circuit c, the circuit c is formed around the entire periphery of the first substrate 11 or on the underside of the first substrate 11 in the first end region 14, which is the positive electrode P, to electrically connect to the positive-side circuit 41. Similarly, the second end side region 15, which is the negative pole N, is electrically connected to the negative side circuit 42, so that a circuit c is formed in the second end side region 15 around the entire periphery of the first substrate or on the underside of the second substrate 21. The third end side region 24 and the fourth end side region 25 do not have a conductive circuit c. Therefore, even if the third end side region 24 and the fourth end side region 25 are disposed on the positive side circuit 41 and the negative side circuit 42, the circuit configuration is not affected. In the example shown in FIG. 8, a single positive side circuit 41 and a single negative side circuit 42 can simultaneously apply current to the first electrode member 10 and the second electrode member 20 disposed on the frame 40.

[0043] 8, one positive side circuit 41 and one negative side circuit 42 are arranged as described above, but alternatively, the positive side circuit 41 and the negative side circuit 42 may be divided into two or more circuits. When the positive side circuit 41 and the negative side circuit 42 are divided, there is an effect that the electrical stimulation conditions can be changed for each unit into which the cell culture well plate is divided.

[0044] Although not shown, two opposing sides of frame 40 may be formed from a conductive material, and the other two opposing sides may be formed from a non-conductive material. In this case, one side formed from a conductive material functions as plus-side circuit 41 in Fig. 8, and the other side formed from a conductive material functions as minus-side circuit 42 in Fig. 8.

[0045] (Examples of optional additional configurations that can be adopted by the frame body 40) Referring to FIG. 8, an optional configuration example that can be adopted by the frame 40 will be described. (Engagement part) The frame 40 may have an engagement portion formed thereon to facilitate positioning of the first electrode member 10 and the second electrode member 20. While not shown in detail, FIG. 8 illustrates a groove 43 as an example of an engagement portion. When the groove 43 is formed, the positive side circuit 41 and the negative side circuit 42 may be disposed along the groove 43. The formation of the groove 43 facilitates positioning of the first electrode member 10 and the second electrode member 20 by fitting the first end-side region 14, the second end-side region 15, the third end-side region 24, and the fourth end-side region 25 into the groove 43. Furthermore, although not shown in the drawings, the frame 40 may have a protrusion, and the first electrode member 10 and the second electrode member 20 may have a recess into which the protrusion can be inserted, thereby forming an engagement portion between the protrusion and the recess. Alternatively, the frame 40 may have a recess, and the first electrode member 10 and the second electrode member 20 may have a protrusion. Similarly to the groove 43, when the engagement portion is formed by a convex portion and a concave portion, the positioning of the first electrode member 10 and the second electrode member 20 can be easily performed.

[0046] (Part that engages with the cell culture well plate) The frame 40 may be formed with an engagement portion 44 for engaging with the cell culture well plate without misalignment. As shown in the view in the Y direction in FIG. 8 (the view surrounded by the dotted line), the engagement portion 44 is formed so as to protrude from the frame 40 toward the cell culture well plate. When the engagement portions 44 are formed, they may be formed at at least two diagonal corners of the frame 40, and more preferably at four corners of the frame 40. Forming the engagement portions 44 on the frame 40 reduces the risk of the frame 40 being misaligned when placed on the cell culture well plate. As a result, there is an effect of reducing the risk of misalignment between the well and the electrodes of the first electrode member 10 and the second electrode member 20 immersed in the medium in the well.

[0047] (Embodiment of cell culture kit) The cell culture kit according to the embodiment includes an electrode device 1 and a cell culture device 50. The electrode device 1 is the same as the electrode device 1 according to any of the above-described embodiments. Therefore, detailed description of the electrode device 1 will be omitted to avoid redundancy.

[0048] The cell culture device 50 will be described in detail with reference to FIG. 9. FIG. 9A is a schematic top view of the cell culture device 50, and FIG. 9B is a cross-sectional view taken along the line XX in FIG. 9A. The cell culture device 50 includes a third substrate 51 and a pillar holder 52. The pillar holder 52 is formed to protrude from the third substrate 51, and each pillar holder 52 has a pair of pillars 53 formed thereon, which serve as a scaffold for the cultured cells to form tissues. A flange 53a is formed at the tip of the pillar 53 to prevent the cultured cell tissue from falling off the pillar 53. The third substrate 51 has n pillar holders 52 formed thereon. The number of pillar holders 52 is the same as the number of electrodes of the first electrode member 10 and the second electrode member 20.

[0049] In the example shown in FIG. 9 , the pillar holding portion 52 is formed in a generally concave shape. Forming the pillar holding portion 52 in a generally concave shape has the effect of providing an environment suited to the purpose of the cells to be cultured in the well, for example by filling the concave portion with hydrogel when inserting the cell culture kit 50 into a cell culture well plate. In this case, it is preferable that the generally concave-shaped portion of the pillar holding portion 52 is inserted into the well in addition to the pillars 53, and in this case, the pillar holding portion 52 may be described as a portion inserted into the well. When inserting the pillar holding portion 52 into the well, the length by which it protrudes from the third base material 51 may be set appropriately.

[0050] Note that the example shown in FIG. 9 is one example of the pillar holding portion 52 and is not limited thereto. The pillar holding portion 52 may be solid and have no recesses. In that case, as long as the pillar 53 is inserted into the well, the pillar holding portion 52 may or may not be inserted into the well. The length of the pillar holding portion 52 protruding from the third substrate 51 may be set appropriately depending on whether the pillar holding portion 52 is inserted into the well or not. Furthermore, the outer shape of the pillar holding portion (insertion portion) 52 (the outer shape of the pillar holding portion 52 shown in FIG. 9A) is not particularly limited as long as it can hold the pillar 53 inserted into the well in a suitable position, and examples of such shapes include a circle, an ellipse, and a polygon with four or more sides.

[0051] The third substrate 51 is sized so that it can be placed between the first electrode member 10 and the second electrode member 20 when the first electrode member 10 and the second electrode member 20 are placed on a cell culture well plate so that the first electrode member 10 and the second electrode member 20 are approximately parallel to each other.

[0052] Referring to FIG. 10, a schematic diagram of the positional relationship when a first electrode member 10, a cell culture device 50, and a second electrode member 20 are arranged on a cell culture well plate 60 is shown. FIG. 10A is a schematic top view of the cell culture device 50 arranged between the first electrode member 10 and the second electrode member 20 (however, the cell culture well plate is not shown). FIGS. 10B to 10D are diagrams showing the first electrode pair e1 and the insertion portion 52 inserted into a well 61 of the cell culture well plate 60, with FIG. 10B being a schematic top view, FIG. 10C being a schematic view as viewed from the X direction of FIG. 10A, and FIG. 10D being a schematic view as viewed from the Y direction of FIG. 10A. Note that, in FIGS. 10B to 10D, components unnecessary for explanation are omitted to allow for a more detailed understanding of the positional relationship when the electrodes and insertion portion 52 are inserted into the well 61. 10C and 10D are not accurate cross-sectional views, but show a combination of configurations that appear in a cross section viewed from the X direction or Y direction.

[0053] As shown in Figures 10A to 10D, when using the cell culture kit disclosed in the present application, the cell culture device 50 is inserted into the well from above. Then, an electrode e1 is placed across a pair of pillars 53, using the pillars 53 as a scaffold, to sandwich the cultured cell tissue. Then, pillars 53 and electrodes e1 are placed in each well, similar to Figures 10B to 10D. Therefore, currents under the same conditions can be applied simultaneously to the cells cultured in each well.

[0054] As shown in FIG. 10 , the cell culture device 50 can be fabricated by any method as long as it can form n pillar holders 52, each having a pillar 53, on a third substrate 51. For example, a mold designed to have the desired number of pillar holders 52 and the desired size is fabricated, and resin is poured into the mold and solidified. The resin is preferably non-conductive, and examples include plastics such as cycloolefin polymer (COP), polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), polycarbonate (PC), and rigid polyethylene, as well as silicone. When flanges 53 a are optionally provided on the pillars 53, the flanges 53 a may be attached to the pillars 53 of the cell culture device 50 fabricated using the mold. During use, the flanges 53 a are positioned below the pillars 53 in the direction of gravity, as shown in FIG. 10 . This prevents the cultured cell tissue from falling off by clinging to the pillars 53. Alternatively, the pillars 53 may be made thicker downward in the direction of gravity to make it difficult for the cell tissue to fall off the pillars 53. Furthermore, the surface of the pillars 53 may be made uneven to make it difficult for the cell tissue to fall off. In the cell culture device 50 disclosed in the present application, the cultured cell tissue is cultured so that it clings to the pillars 53 that serve as scaffolds, and the cell tissue is basically held between the pillars 53. Therefore, a configuration such as a flange for making it difficult for the cell tissue to fall off the pillars 53 is merely an optional additional configuration. The cell culture device 50 may also be produced using a 3D printer. Note that the cultured cells may be observed under a microscope during an experiment. Therefore, the cell culture device 50 is preferably made of a highly transparent resin. Furthermore, an engagement portion similar to the "(engagement portion)" of the above-described "(electrode device 1 according to the second embodiment)" may be optionally formed between the cell culture device 50 and the frame body 40.

[0055] The cell culture device described in Non-Patent Document 1 is formed so as to be inserted into each well of a cell culture well plate. On the other hand, the cell culture device 50 disclosed in the present application is designed so that the first electrode member 10 and the second electrode member 20 are inserted from above into each well of a cell culture well plate. Therefore, unlike the cell culture device described in Non-Patent Document 1, the cell culture device 50 disclosed in the present application can arrange a pillar holder 52, on which a pair of pillars 53 is arranged, in each of multiple wells. Furthermore, the orientation of the cell culture device 50 disclosed in the present application when arranged in a well differs from that of Non-Patent Document 1. Therefore, the cell culture device 50 disclosed in the present application is a novel device different from that of Non-Patent Document 1. Therefore, the cell culture device 50 may be provided as a cell culture kit together with the electrode device 1, or the cell culture device 50 may be provided alone. When the cell culture device 50 is provided alone, the number n of pillar holders 52 formed on the third substrate 51 may be selected from the possible number n of electrodes (e.g., an integer between 2 and 20). Furthermore, it is desirable that the number n of pillar holders 52 be selected from 6, 8, or 12 in consideration of the number of wells in a commercially available well plate for cell culture.

[0056] When cell culture is performed using the cell culture kit according to the embodiment, the following effects are achieved. (1) In the invention described in Non-Patent Document 1, a cell culture device needs to be placed in each well. This takes time, and if the orientation of the cell culture device placed in the well is different, the experimental conditions may differ. On the other hand, the cell culture device disclosed in the present application can place the cell culture devices 50 in the wells at once by forming the same number of pillar holders 52 as the number of wells in the row or column direction of the cell culture well plate in advance. Therefore, the time required for placing the cell culture devices 50 can be saved. Furthermore, if the pillar holders 52 of the pillars 53 have the same shape, the conditions for the cell culture devices placed in the wells can be easily made the same. (2) In the cell culture kit according to the embodiment, for example, the spacing between the electrodes of the first electrode member 10 and the second electrode member 20, the width of the third substrate 51 of the cell culture device 50, the spacing between the pillars 53, etc. can be designed in advance. Therefore, the positional relationship between the pillar holder 52, the pair of pillars 53, and the electrode pair e1 arranged in each well can all be made the same. In this case, the experimental conditions in each well, including the electrical stimulation conditions, can all be made the same. (3) On the other hand, although not shown, the cell culture kit according to the embodiment can change the positional relationship of the electrode e1 with respect to the pair of pillars 53, the length of the electrode e1 inserted into the well, the pillar holder 52, and the pillar 53 for each well. Therefore, by using the cell culture kit according to the embodiment, it is possible to easily carry out experiments under different conditions, such as by gradually changing the location where the cultured cells are electrically stimulated for each well. (4) Assume a case where a flange 53a is optionally provided. As shown in FIG. 1(c), the cell culture device described in Non-Patent Document 1 is positioned in the well with the flange facing upward. Therefore, the cell tissue cultured on the pair of pillars as a scaffold cannot be prevented from migrating in the direction of gravity. On the other hand, in the cell culture device 50 according to the embodiment, as shown in FIGS. 10C and 10D, the flange 53a is positioned toward the bottom of the well during use. Therefore, the flange 53a can prevent the cell tissue cultured on the pair of pillars as a scaffold from migrating in the direction of gravity and falling off. (5) Even if either the electrode device 1 or the cell culture device 50 constituting the cell culture kit is damaged or soiled, it can be easily replaced. Furthermore, when the frame 40 is used, the electrode device 1 and / or the cell culture device 50 to be replaced can be easily attached to and detached from the frame 40.

[0057] (Embodiment of the culture method) The culture method according to the embodiment is carried out using the cell culture kit described above. The cell culture kit used in the culture method is the same as that of the cell culture kit according to the embodiment described above. Therefore, a detailed description of the cell culture kit will be omitted to avoid redundancy.

[0058] A culture method according to an embodiment will be described with reference to Fig. 10. The culture method includes a preparation step, a cell culture step, and an electricity application step.

[0059] In the preparation step, a well 61 of a separately prepared well plate 60 for cell culture is The first electrode member 10 and the second electrode member 20 are arranged on the cell culture well plate 60 so that one electrode of the first electrode member 10 and one electrode of the second electrode member 20 are arranged, The cell culture device 50 is placed between the first electrode member 10 and the second electrode member 20 so that a pair of pillars 53 formed on the pillar holding portion 52 of the cell culture device 50 are immersed in the culture medium in one well.

[0060] In the cell culture step, cells are cultured in the wells 61. The culture medium may be selected appropriately depending on the cells to be cultured. Examples of cells used in the culture method disclosed in the present application include cells that form cellular tissue between a pair of pillars 53 when cultured. Examples of such cells include, but are not limited to, skeletal muscle cells, cardiac muscle cells, and smooth muscle cells. The exemplified cells may be cells collected from tissue or cells obtained by induction from various stem cells.

[0061] In the electricity application step, the positive pole P and negative pole N of the electrode device 1 form a circuit with an external power source to apply an electrical stimulus to the cells being cultured during at least a portion of the cell culture step. The applied voltage and / or current may be set appropriately depending on the type of cell and the purpose of the experiment. In the electricity application step, the positive pole P and negative pole N of the electrode device 1 may be connected to the external power source directly or via the above-mentioned frame 40. In this case, the frame 40 may be engaged with a separately prepared cell culture well plate 60 before the above-mentioned preparation step is performed.

[0062] The culture method according to the embodiment has the following effects in addition to the effects of the cell culture kit according to the embodiment described above. (1) Electrical stimulation can be applied to cultured cells at the desired timing and duration. For example, when skeletal muscle tissue is created using skeletal muscle cells, the effects of exercise can be mimicked by applying electrical stimulation. (2) Materials other than platinum are used to form the electrodes, which reduces the risk of the cultured cells being affected by electrolysis of the water that makes up the culture medium.

[0063] The following examples are provided to specifically explain the embodiments disclosed in the present application, but these examples are merely for the purpose of explaining the embodiments and are not intended to limit or restrict the scope of the invention disclosed in the present application. [Example]

[0064] Example 1 [Fabrication of electrode device 1] Conductive aluminum square bars were used as the first and second substrates. Eight 1.5 mm diameter through-holes were drilled at equal intervals in the aluminum square bar (1.8 × 1.8 × 79 mm), and one side was protected with insulating heat-shrink tubing. 1.3 mm diameter carbon rods (PSR-2B13, manufactured by KOKUYO) were cut to 14 mm lengths and inserted into the through-holes in the aluminum square bars. They were then fixed in place using a highly conductive adhesive (TK Paste, manufactured by Kaken Tech Co., Ltd.). Figure 11A shows a photograph of the aluminum square bar with the through-holes, and Figure 11B shows a photograph of the fabricated electrode device 1.

[0065] (Fabrication of the frame 40) A 0.8 mm thick acrylic plate was cut to fit the periphery of the wells of a 96-well plate (CELLSTAR, manufactured by GREINER). After cutting a 3 mm thick aluminum plate, 24 grooves for fitting the electrode device 1 were created. The grooves were of two sizes (2.5 mm wide and 2 mm deep), and were alternately created at 2.5 mm and 1.5 mm intervals. The side of the electrode device 1 wrapped with insulating heat-shrink tubing was inserted into the 2.5 mm wide and deep grooves, while the side without insulating heat-shrink tubing was inserted into the 2 mm wide and deep grooves. Another 0.8 mm thick aluminum plate was cut to the same shape as the aluminum plate with the grooves to serve as a cover to prevent the electrode device 1 from being removed. Figure 12 shows a photograph of the frame 40 (bottom of Figure 12) and a photograph of the cover (top of Figure 12) of the fabricated frame 40.

[0066] (Fabrication of cell culture devices) A Teflon (registered trademark) mold was designed to allow eight pillar holders 52 to protrude from the third substrate 51. PDMS (Sylgard 184, manufactured by Dow Corning Toray Co., Ltd.) mixed at a monomer:catalyst ratio of 10:1 was poured into the mold and degassed. A transparent acrylic square rod (2 mm square) was embedded in the third substrate 51 and heat-treated at 70°C for 1 hour to solidify the PDMS. After removal from the mold, a separately prepared, roughly disk-shaped flange 53a was bonded to the top of the pillar 53 with PDMS and then heat-treated at 70°C for 1 hour to fix the flange 53a to the pillar 53. Note that the transparent acrylic square rod was used for reinforcement purposes and is not an essential component of the cell culture device 50. Figure 13 shows a photograph of the fabricated cell culture device 50.

[0067] <Example 2> [Embodiment of the culture method] Sterilized cell culture devices were immersed in a 0.2% aqueous solution of Pluronic F-127 (P2443, Sigma-Aldrich) and left at 4°C for 24 hours. Human skeletal muscle cells, Hu5 / KD3 (gift from the National Center for Geriatrics and Gerontology), were resuspended in growth medium at a concentration of 4.0 × 10^6 cells / mL to prepare a cell suspension. The growth medium consisted of Dulbecco's Modified Eagle Medium (DMEM, 08458-16, Nacalai Tesque) containing 20% ​​fetal bovine serum, 2 mM L-glutamine, 2% Ultroser G (15950-017, Sartorius), and 1% penicillin-streptomycin. Next, a sol solution consisting of the cell suspension, Figrinogen (F8630, Sigma-Aldrich), 2x DMEM (5919, Nissui Pharmaceutical), Matrigel (354234, Corning), and Thrombin (T4648, Sigma-Aldrich) was prepared on an ice-cooled aluminum block. The cell culture device was removed from the solution, and 12 μL of the sol solution was placed in the recesses of the pillar holders while cooling on ice. This was then allowed to stand at 37°C for 20 minutes to produce a cell-filled gel. The cell-filled gel was then cultured in tissue growth medium supplemented with 2% tranexamic acid.

[0068] The frame prepared in Example 1 was placed in a 96-well plate, and the first and second electrode members of the electrode device prepared in Example 1 were fitted into the grooves of the frame. 250 μL of tissue growth medium was poured into each well of the 96-well plate, and the cell culture device was placed between the first and second electrode members with the cell-containing gel facing downward. Culture was performed at 37°C and in a 5% CO2 environment. After two days of culture, the medium was replaced with tissue differentiation medium. DMEM containing 2% horse serum, 2% tranexamic acid, and 1% penicillin was used as the tissue differentiation medium. The medium was replaced every two days. Starting on day 4 of differentiation, myotubes were cultured with electrical stimulation for 48 hours. A C-PACE electrical stimulator was used (voltage 0.5 V / mm, frequency 1 Hz or 30 Hz, pulse width 1 ms). At 30 Hz, stimulation was cycled between 1 second on and 29 seconds off, ensuring the total number of stimulations was the same across conditions.

[0069] The contractile force of muscle tissue constructed by culturing cell-filled gels was measured before the start of electrical stimulation on day 4 of differentiation and after 48 hours of electrical stimulation on day 6 of differentiation. The electrical stimulation conditions for measuring contractile force were 4 mm interelectrode distance, 20 V (4 V / mm), 30 Hz, and 2 ms pulse width. The flange 53a was observed under electrical stimulation using an inverted microscope (CKX53, Olympus) equipped with a 10x objective lens and photographed with a microscope digital camera (DP21, Olympus). Images were taken before and after electrical stimulation, and the displacement of the flange 53a was measured using the image analysis software ImageJ. Contractile force F was calculated from the displacement using the formula (F = 3πER^4δ / 4L^3), where E = 1.7 MN / m^2, R = 0.25 mm, L = 4 mm, and displacement distance = δ.

[0070] The results are shown in Figure 14. It was confirmed that contractile force was significantly increased by applying electrical stimulation to cells cultured by the culture method disclosed in the present application. [Industrial Applicability]

[0071] The electrode device, cell culture kit, and cell culture method using the cell culture kit disclosed in the present application can easily apply electrical stimulation to cultured cells. Therefore, the electrode device, cell culture kit, and cell culture method using the cell culture kit can be used for the development of medicines using cultured cells, and are therefore useful for drug discovery in medical institutions, universities, companies, research institutes, etc. [Explanation of symbols]

[0072] 1...electrode device, 10...first electrode member, 11...first substrate, 12...first end, 13...second end, 14...first end side region, 15...second end side region, 20...second electrode member, 21...second substrate, 22...third end, 23...fourth end, 24...third end side region, 25...fourth end side region, 31...first connecting member, 32...second connecting member, 40...frame, 41...positive side circuit, 42...negative side circuit, 43...groove portion, 44...engagement portion, 50...cell culture device, 51...third substrate, 52...pillar holding portion, 53...pillar, 53a...flange, 60...cell culture well plate, 61...well, e1 to en...electrode, P...positive electrode, N...negative electrode

Claims

1. An electrode device for applying electrical stimulation to cells cultured in a cell culture well plate, the electrode device comprising: A first elongated substrate is provided with n electrodes (except that the electrodes are made of platinum). a first electrode member on which the first electrode member is disposed; A second elongated substrate is provided with n electrodes (except that the electrodes are made of platinum). a second electrode member on which the first electrode 12 is disposed; Including, n is an integer of 2 or more, and the number of electrodes arranged on the first substrate is the same as the number of electrodes arranged on the second substrate; One end of the first substrate is defined as a first end, and the other end is defined as a second end; one end of the second substrate is defined as a third end and the other end is defined as a fourth end; The n electrodes arranged on the first base material are defined as 1st to nth electrodes in order from the first end side, The n electrodes arranged on the second base material are defined as 1st to nth electrodes in order from the third end side, Combinations of the same electrodes arranged on the first substrate and the second substrate are defined as 1st to nth electrode pairs, A first end side region of the first substrate is defined as a first end side region of the first electrode, and a second end side region of the first substrate is defined as a second end side region of the n-th electrode, a third end side region of the second substrate from the first electrode to a third end side, and a fourth end side region of the second substrate from the nth electrode to a fourth end side, When it is assumed that the first electrode member and the second electrode member are arranged substantially parallel to each other so that the first end and the third end face each other and the second end and the fourth end face each other, and that a current flows between two electrodes constituting each of the first to nth electrode pairs, The first substrate and the second substrate include: Only one of the first end side region and the third end side region serves as a positive electrode to which a current is supplied from an external power supply, a current supplied to the positive electrode passes through all of the n electrodes arranged on the first substrate and the n electrodes arranged on the second substrate; Only one of the second end side region and the fourth end side region serves as a negative pole for current to return to an external power supply. The circuit is formed as follows: Electrode device.

2. When the first end side region is a positive electrode, a circuit is formed on the first substrate so that a current flows through all electrodes from the positive electrode to the n-th electrode; the fourth end region is a negative electrode, and a circuit is formed on the second substrate so that a current flows through all electrodes from the negative electrode to a first electrode; When the third end side region is a positive electrode, a circuit is formed on the second substrate so that a current flows through all electrodes from the positive electrode to the n-th electrode; The second end side region is a negative electrode, and a circuit is formed on the first substrate so that a current flows through all electrodes from the negative electrode to the first electrode. The electrode device of claim 1 .

3. When the first end side region is a positive electrode, a circuit is formed on the first substrate so that a current flows between the positive electrode and a first electrode, and a circuit is formed so that a current flows between an even-numbered electrode and an odd-numbered electrode; a circuit is formed on the second substrate so that a current flows between odd-numbered electrodes and even-numbered electrodes; When the third end side region is a positive electrode, a circuit is formed on the second substrate so that a current flows between the positive electrode and a first electrode, and a circuit is formed so that a current flows between an even-numbered electrode and an odd-numbered electrode; A circuit is formed on the first substrate so that a current flows between odd-numbered electrodes and even-numbered electrodes. The electrode device of claim 1 .

4. When the number of electrodes arranged on the first substrate and the second substrate is an even number, When the first end side region is a positive electrode, the negative electrode is the second end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode; When the third end side region is a positive electrode, The negative electrode is the fourth end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode. The electrode device of claim 3 .

5. When the number of electrodes arranged on the first substrate and the second substrate is odd, When the first end side region is a positive electrode, the negative electrode is the fourth end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode; When the third end side region is a positive electrode, The negative electrode is the second end side region, and a circuit is formed so that a current flows between the negative electrode and the n-th electrode. The electrode device of claim 3 .

6. The electrode is a carbon electrode. The electrode device of claim 1 .

7. the first end and the third end are connected by a first connecting member, the second end and the fourth end are connected by a second connecting member, The first and second connecting members are non-conductive and have the same length. The electrode device of claim 1 .

8. The number of electrodes arranged on the first substrate and the second substrate is 6, 8, or 12. The electrode device of claim 1 .

9. The cell culture well plate further includes a frame that can be fixed to the outer periphery of the cell culture well plate, and the frame includes: the first electrode member and the second electrode member are sized to abut against each other when the first electrode member and the second electrode member are arranged substantially parallel to each other, and At least the frame portion where the positive electrode and the negative electrode come into contact is made of a non-conductive material and circuitized for connection to an external power source; or Made of conductive material 9. An electrode device according to any one of claims 1 to 8.

10. An electrode device according to any one of claims 1 to 8; a cell culture device; A cell culture kit comprising: The cell culture device comprises: A third substrate; n pillar holders formed to protrude from the third base material and for holding pillars; Including, the number of the n pillar holding portions is the same as the number of the n electrodes, A pair of pillars that serve as scaffolds for cultured cells are formed on each of the pillar holders, The third base material has a size such that it can be placed between the first electrode member and the second electrode member when the first electrode member and the second electrode member are placed in a cell culture well plate so that the first electrode member and the second electrode member are substantially parallel to each other. Cell culture kit.

11. The cell culture well plate further includes a frame that can be fixed to the outer periphery of the cell culture well plate, and the frame includes: the first electrode member and the second electrode member are sized to abut against each other when the first electrode member and the second electrode member are arranged substantially parallel to each other, and The frame portion where the positive electrode and the negative electrode come into contact is made of a non-conductive material and circuitized for connection to an external power source; or Made of conductive material The cell culture kit according to claim 10.

12. A method for culturing cells using a cell culture kit, the method being carried out using the cell culture kit according to claim 10, In one well of a separately prepared cell culture well plate, the first electrode member and the second electrode member are arranged in a cell culture well plate so that one electrode of the first electrode member and one electrode of the second electrode member are arranged; the cell culture device is placed between the first electrode member and the second electrode member so that the pair of pillars formed in the pillar holder of the cell culture device are immersed in the culture medium in one well. The preparation process and a cell culture step of culturing cells in the wells; an electrical application step of applying an electrical stimulus to the cells being cultured during at least a portion of the cell culture step; Contains Culture method.

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