Cell culture membrane and measurement method

Abstract

To provide a cell culture membrane that enables precise electrical measurement for evaluating the culture condition after performing cell culture.SOLUTION: A cell culture membrane includes: a membrane body having a first face, a second face located on a side opposite to the first face, and plural through holes penetrating from the first face to the second face; and a metal film including at least one of a first metal film provided to overlap the first face or a second metal film provided to overlap the second face. The plural through holes have a first average hole diameter at the first face larger than a second average hole diameter at the second face.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present disclosure relates to cell culture membranes and measurement methods. [Background technology]

[0002] Conventionally, in order to evaluate the state of cell culture, the physical properties of the culture solution and the amount of a specific substance in the culture solution have been measured (for example, Patent Document 1). Patent Document 1 describes a device for measuring transepithelial electrical resistance using electrodes. Measurements performed using electrodes in cell culture include, in addition to transepithelial electrical resistance, the impedance and pH of the culture solution, and the amount of glucose. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2020-146015 A Summary of the Invention [Problem to be solved by the invention]

[0004] Since the above measurements are performed on the culture medium, measurement errors may be large depending on the arrangement of the electrodes. [Means for solving the problem]

[0005] The present disclosure can be realized in the following forms.

[0006] (1) According to a first embodiment of the present disclosure, a cell culture membrane is provided. The cell culture membrane includes a membrane body having a first surface and a second surface located on the opposite side to the first surface, the membrane body having a plurality of through holes penetrating from the first surface to the second surface, and a metal membrane including at least one of a first metal film formed on the first surface and a second metal film formed on the second surface, the plurality of through holes having a first average pore size on the first surface larger than a second average pore size on the second surface. According to this embodiment, when cells are cultured on the metal membrane, in a measurement using the metal membrane as an electrode, the metal membrane comes into contact with or close to the cells, so that the accuracy of the measurement of the culture state near the cells can be improved. In addition, when different types of cells are cultured on the first surface and the second surface, the cells cultured on the first surface and the cells cultured on the second surface can come into contact with each other through the through holes, so that a state of the cells closer to the state in the living body can be created. (2) In the cell culture membrane of the above embodiment, each of the plurality of through-holes may have a first opening opening on the first surface, a second opening opening on the second surface, and an inner periphery connecting the first opening and the second opening, and the metal film may not include the second metal film but may include the first metal film, and the first metal film may have a metal inner periphery formed to cover the inner periphery. According to this embodiment, the area of ​​the metal film in contact with or close to the cells entering each through-hole can be increased, thereby further improving the measurement accuracy. (3) In the cell culture membrane of the above embodiment, the first metal film may have a metal blocking portion that blocks the second opening, and the metal blocking portion may have a crack. According to this embodiment, the cells cultured on the first surface and the cells cultured on the second surface can contact each other through the crack, and the size of the crack is smaller than the size of the second opening, so that it is expected to create a cellular state closer to the state in vivo. (4) In the cell culture membrane of the above embodiment, the membrane body may be formed of polyurethane. According to this embodiment, it is possible to easily prepare a membrane body having a plurality of through-holes whose first average pore size is larger than the second average pore size in the second surface. (5) In the cell culture membrane of the above embodiment, the second average pore size may be 7 μm or less. According to this embodiment, when cells having a size of about 10 μm are cultured, the cells cultured on either the first surface or the second surface can be prevented from migrating to the other of the first surface or the second surface through the through-holes. (6) In the cell culture membrane of the above embodiment, the metal membrane may include a first metal layer mainly composed of either gold (Au) or platinum (Pt). According to this embodiment, either the first metal layer mainly composed of gold or the first metal layer mainly composed of platinum can be used as the metal membrane. (7) The cell culture membrane of the above embodiment may further include a titanium layer disposed between the first metal layer and the membrane body, the titanium layer being mainly made of titanium (Ti). According to this embodiment, the titanium layer can improve the adhesion of the first metal layer to the membrane body. Therefore, the measurement accuracy can be improved. (8) In the cell culture membrane of the above embodiment, the metal membrane may include a first metal layer mainly made of gold (Au) and a second metal layer mainly made of platinum (Pt) and disposed between the first metal layer and the membrane body. This embodiment can improve the measurement accuracy. (9) In the cell culture membrane of the above embodiment, the metal membrane may further include a titanium layer disposed between the second metal layer and the membrane body, the titanium layer being mainly made of titanium (Ti). According to this embodiment, the titanium layer can improve the adhesion of the first metal layer and the second metal layer to the membrane body. Therefore, the measurement accuracy can be improved. (10) The measurement method using the cell culture membrane of the above embodiment may include a first step of immersing the cell culture membrane in a culture solution and culturing cells on the metal film of the cell culture membrane, and a second step of electrochemically measuring the glucose concentration in the culture solution using the metal film as a working electrode. According to this embodiment, when cells are cultured on the cell culture membrane, it is possible to measure the glucose concentration using the metal film as an electrode. (11) A measurement method using the cell culture membrane of the above embodiment may include a first step of immersing the cell culture membrane in a culture solution and culturing cells on the metal film of the cell culture membrane, and a second step of performing electrochemical impedance measurement in the culture solution using the metal film as a working electrode. According to this embodiment, when cells are cultured on the cell culture membrane, electrochemical impedance measurement using the metal film as an electrode can be realized. The present disclosure can be realized in various forms, and can be realized in the form of, for example, a method for producing a cell culture membrane in addition to the above cell culture membrane. [Brief description of the drawings]

[0007] [Figure 1] Schematic diagram of a cross section of a cell culture membrane. [Diagram 2] 1 is a flowchart showing a process for producing a cell culture membrane. [Diagram 3] 4 is a flowchart showing the steps of a method for measuring a glucose concentration. [Figure 4] FIG. 1 is a diagram showing the configuration of a measurement system. [Diagram 5] 4 shows the results of measuring the relationship between glucose concentration and current value. [Figure 6] Schematic diagram of co-culture using a cell culture membrane. [Figure 7] 4 is a flowchart showing the steps of a measurement method for measuring impedance. [Figure 8] FIG. 1 shows a measurement system for performing impedance measurements using three electrodes. [Figure 9] FIG. 13 shows another embodiment of the cell culture membrane. [Figure 10] SEM image of a cell culture membrane on which cells are cultured. [Figure 11] FIG. 13 shows the measurement results of glucose concentration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] A. Embodiment: A-1. Cell culture membrane: FIG. 1 is a schematic diagram of a cross section of a cell culture membrane 10. The cell culture membrane 10 includes a membrane body 20 and a metal membrane 30. In this embodiment, the cell culture membrane 10 is formed of polyurethane. The metal membrane 30 is configured by sequentially stacking a first metal layer 31 mainly made of gold (Au), a second metal layer 32 mainly made of platinum (Pt), and a titanium layer 33 mainly made of titanium (Ti) from the top to the bottom. That is, the titanium layer 33 is disposed between the second metal layer 32 and the membrane body 20. By disposing the titanium layer 33 between the second metal layer 32 and the membrane body 20, the adhesion between the first metal layer 31 and the second metal layer 32 and the membrane body 20 can be improved.

[0009] As another embodiment of the metal film 30, the metal film 30 may be composed of only the first metal layer. In this case, the first metal layer may be either a layer mainly made of gold or a layer mainly made of platinum (Pt). In this case, the metal film 30 may further include a titanium layer 33 between the first metal layer and the film body 20. By providing the titanium layer 33, the adhesion between the first metal layer 31 and the film body 20 can be improved. In this embodiment, the metal film 30 includes the first metal layer 31, the second metal layer 32, and the titanium layer 33 stacked in this order. As another embodiment, the titanium layer 33 may not be included, and the metal film 30 may include only the first metal layer 31 and the second metal layer 32. By providing the metal film 30, the cell culture membrane 10 can perform various electrical measurements by culturing cells on the metal film 30 and then using the metal film 30 as an electrode.

[0010] The membrane body 20 has a first surface 21, a second surface 22 located on the opposite side to the first surface 21, and a plurality of through holes 23 formed in the membrane body 20. The plurality of through holes 23 are holes penetrating from the first surface 21 to the second surface 22. Each through hole 23 has a first opening 23a opening to the first surface 21, a second opening 23b opening to the second surface 22, and an inner periphery 23c connecting the first opening 23a and the second opening 23b. By having the second opening 23b, the cell culture membrane 10 can bring different types of cells into contact with each other when different types of cells are cultured on the first surface 21 and the second surface 22, respectively.

[0011] The hole diameter of the through-hole 23 on the first surface 21 is different from the hole diameter on the second surface 22. The hole diameter of the through-hole 23 on the first surface 21 is called the first hole diameter Da, and the hole diameter of the through-hole 23 on the second surface 22 is called the second hole diameter Db. The first hole diameter Da is larger than the second hole diameter Db. In addition, the first average hole diameter, which is the average value of the first hole diameters Da of the multiple through-holes 23, is different from the second average hole diameter, which is the average value of the second hole diameters Db on the second surface 22. The first average hole diameter is larger than the second average hole diameter. As a result, as described later, the cells 300 can be cultured in a state in which they are inserted inside the through-holes 23. The second average hole diameter is preferably 7 μm or less. As a result, as described later, when co-culture is performed using the cell culture membrane 10, after the first cells 301 are seeded on the cell culture membrane 10, the migration of the first cells 301 through the through-holes 23 to the surface opposite to the surface on which they were seeded can be suppressed. In the following description, the movement of first cell 301 through through-hole 23 to the surface opposite to the surface on which it was seeded may be referred to as "wandering around."

[0012] The first average hole diameter is a value obtained by shining light on the first surface 21 facing upward with the second surface 22 facing downward, and observing the first surface 21 facing upward with a microscope. Since light is not reflected from the through-holes 23, the through-holes 23 are visually recognized as black. Specifically, the circle-equivalent diameters of all the through-holes 23 observed in a specific field of view are measured, and the average of the measured values ​​is obtained. The circle-equivalent diameter refers to the diameter of a perfect circle equivalent to the area of ​​the through-holes 23. The second average hole diameter is also a value obtained in the same manner. In other words, the second average hole diameter is a value obtained by shining light on the second surface 22 facing upward with the first surface 21 facing downward, and observing the second surface 22 facing upward with a microscope.

[0013] The metal film 30 includes a first metal film MF1 formed over the first surface 21. The first metal film MF1 has a metal inner periphery portion 30c. The metal inner periphery portion 30c is formed to cover the inner periphery portion 23c. Since the metal film 30 is formed to cover the inner periphery portion 23c, it is possible to further improve the accuracy of electrical measurements made in the vicinity of the cell 300, as described below.

[0014] A-2. Manufacturing method of cell culture membrane: Fig. 2 is a flow chart showing the steps of manufacturing the cell culture membrane 10. As shown in Fig. 2, in step S10, a membrane body 20 made of polyurethane is produced. Specifically, water vapor is supplied to uncured polyurethane raw material formed in a thin plate shape on a substrate SB, and the uncured polyurethane raw material is foamed and cured to produce the membrane. According to this manufacturing method, the through-holes 23 are formed by foaming, so that the cell culture membrane 10 having a first hole diameter Da and a second hole diameter Db different from each other can be easily manufactured.

[0015] In step S14, a metal film 30 is formed on the first surface 21 of the film body 20 by sputtering. Specifically, the film body 20 formed on the substrate SB is placed together with the substrate SB into a vacuum chamber, and metal is deposited on the first surface 21 to form the metal film 30. In this embodiment, the sputtering time of the first metal layer 31 is 3 minutes. The sputtering time of the second metal layer 32 is 1 minute. The sputtering time of the titanium layer 33 is 1 minute. Note that the sputtering time is not limited to the above.

[0016] In step S16, the film body 20 is removed from the substrate SB, and this manufacturing process is completed. In the process of removing the film body 20 from the substrate SB, the metal film formed on the substrate SB is separated from the film body 20. Therefore, the second opening 23b of the film body 20 is not blocked by the metal film.

[0017] A-3.How to measure glucose: Fig. 3 is a flow chart showing the procedure of a measurement method for measuring the glucose concentration in a culture solution after cells are cultured on the cell culture membrane 10. Fig. 4 is a diagram showing the configuration of a measurement system 90 assembled when measuring the glucose concentration. Glucose is metabolized by cells 300. Therefore, in the culture process of cells 300, the culture state can be evaluated by measuring the glucose concentration in the culture solution.

[0018] 3, in the culture step of step S20, cells 300 are cultured on a cell culture membrane 10. In this embodiment, the culture is performed using a culture vessel 80 including the cell culture membrane 10.

[0019] As shown in FIG. 4, the culture vessel 80 has a cell culture membrane 10 and a cylindrical member 40. The cylindrical member 40 is made of resin and has a cylindrical shape. The cell culture membrane 10 is attached to the central position in the axial direction of the cylindrical member 40. The cylindrical member 40 is placed in a resin container 50 for use. A conductor 60 is electrically connected to the metal film 30 of the cell culture membrane 10. In this embodiment, the thickness of the cell culture membrane 10 is 5 μm. In this embodiment, the inner diameter of the cylindrical member 40 is 8 mm.

[0020] In step S20 as a first process shown in Fig. 3, the culture vessel 80 shown in Fig. 4 is placed in a resin vessel 50, and a culture solution 305 is poured inside the cylindrical member 40, and cells 300 are seeded and cultured. The culture solution 305 contains glucose.

[0021] As shown in Fig. 3, in step S22, the value of a current flowing through the metal film 30 is measured. As shown in Fig. 4, the measurement system 90 includes a potentiostat 91, a counter electrode CE, and a reference electrode RE. A conductor 60, which is electrically connected to the metal film 30, is electrically connected to the potentiostat 91. In the measurement, the metal film 30 functions as a working electrode WE. The counter electrode CE and the reference electrode RE are each electrically connected to the potentiostat 91 via the conductor 60.

[0022] In step S22, the pH of the surface of the working electrode WE is adjusted to alkaline by pretreatment, and then the potential difference between the metal film 30 functioning as the working electrode WE and the reference electrode RE is controlled to a predetermined potential difference using a potentiostat 91. Specifically, the pretreatment is a process for generating hydroxide ions in the vicinity of the working electrode WE by causing an electrolysis reaction on the working electrode WE.

[0023] As shown in FIG. 3, in step S24, the glucose concentration in the culture solution 305 is calculated using the measured current value. Specifically, the glucose concentration is calculated using a relational expression between the glucose concentration and the current value, which is previously calculated. Since the cell 300 is in contact with or close to the metal film 30, the glucose concentration near the cell 300 can be measured with high accuracy. Specifically, an approximation formula can be used as the relational expression between the glucose concentration and the current value. The glucose concentration may also be calculated using a map showing the relationship between the range of the current value and the glucose concentration applied to each range. Steps S22 and S24 are also referred to as the second step. As described above, in the second step, the glucose concentration is measured electrochemically in the culture solution 305 using the metal film 30 as the working electrode.

[0024] A-4. Glucose concentration measurement principle: Regarding the electrode reaction of glucose at a gold electrode under alkaline conditions, Tomas Jufik (formal notation is shown in Table 1) et al., Nanostructured gold deposited in gelatin template applied for electrochemical assay of glucose in serum, Electrochimica Acta 188 (2016) 277-285 (Reference 1) has proposed. [Table 1]

[0025] According to the scheme proposed in Reference 1, as the potential of the working electrode where the electrode reaction takes place increases in the positive direction during the forward scan of cyclic voltammetry, step I, step II, and step III are carried out in this order. According to the proposal in Reference 1, in step I, glucose is adsorbed onto the electrode surface after hydrogen atoms are removed. In step II, glucose adsorbed onto the electrode surface is oxidized to a gluconolactone intermediate adsorbed onto the electrode surface. In step III, the gluconolactone intermediate is desorbed from the electrode surface to become gluconolactone.

[0026] Referring to the cyclic voltammogram in Reference 1, the inventors set the potential of the working electrode WE to the potential at which steps I and II are performed, applied a voltage to an aqueous solution containing glucose, and examined the current value of the current flowing through the working electrode WE.

[0027] Figure 5 shows the results of measuring the relationship between glucose concentration and current value. Specifically, multiple aqueous solutions with various glucose concentrations were prepared, and a reference electrode, a counter electrode, and a working electrode were placed in each aqueous solution, and measurements were performed using a potentiostat. The measurement conditions were as follows: Reference pole:Ag / AgCl(sat.KCL) Counterpole: Pt Working electrode: Au (φ3mm) Preconditioning: -1.6V, 1 second Applied potential: 0.3V The applied potential of 0.3 V is the potential of the working electrode where the above steps I and II are performed. A voltage was applied between the working electrode and the reference electrode so that the potential of the working electrode was 0.3 V by measuring the voltage between the reference electrode and the working electrode, and the current value of the current flowing between the working electrode and the counter electrode 2 seconds after the start of current flow was measured. The number of samples was 3. The pretreatment is a process for making the surface of the working electrode alkaline. Specifically, an electrolytic reaction is caused on the working electrode to generate hydroxide ions near the working electrode.

[0028] As shown in FIG. 5, it can be seen that there is a correlation in which the higher the glucose concentration, the larger the current value. The higher the glucose concentration in the aqueous solution, the greater the number of electrons released by the oxidation reaction. For this reason, it is expected that the higher the glucose concentration, the larger the current value. And this experiment proved that the higher the glucose concentration, the larger the current value. In this way, by determining in advance the relational expression between the glucose concentration and the current value, the glucose concentration of the aqueous solution can be determined using the measured current value of the aqueous solution.

[0029] B. Other embodiments of the culture method: 3, the cell culture using the cell culture membrane 10 is not limited to the case where cells are cultured on one side of the cell culture membrane 10, but may be cultured on each of both sides of the cell culture membrane 10. Furthermore, in this case, co-culture may be performed in which two or more different types of cells are cultured on each of both sides of the cell culture membrane 10.

[0030] FIG. 6 is a schematic diagram of a case where a first cell 301 and a second cell 302 of a different type from the first cell 301 are co-cultured on a cell culture membrane 10. The cell culture membrane 10 is attached, for example, to the end of an insert housed in a well. In a first step of co-culture using the cell culture membrane 10, the first cell 301 is seeded and cultured on the second surface 22 of the cell culture membrane 10 in a state in which the second surface 22 of the cell culture membrane 10 is arranged so as to face upward in the vertical direction. In the next second step, the second cell 302 is seeded and cultured on the metal membrane 30 in a state in which the first surface 21 of the cell culture membrane 10 is arranged so as to face upward in the vertical direction. In the culture, the second cell 302 enters the through hole 23. Thus, the first cell 301 and the second cell 302 can come into contact with each other. For example, by using epithelial cells as the first cells 301 and vascular endothelial cells as the second cells 302, a three-dimensional model closer to a living organism can be produced.

[0031] Note that cell culture using the cell culture membrane 10 is not limited to the above. The same type of cells may be cultured on the first surface 21 and the second surface 22, or two or more types of cells may be cultured on each of the first surface 21 and the second surface 22. Also, cells may be cultured only on the second surface 22.

[0032] C. Other embodiments of the measurement method: (C1) Electrochemical impedance measurement using three electrodes: FIG. 7 is a flow chart showing the procedure of the measurement method for measuring impedance. FIG. 8 is a diagram showing a measurement system 390 for performing electrochemical impedance measurement using three electrodes. The same configurations and processing steps as those in the above embodiment are given the same reference numerals, and detailed explanations are omitted as appropriate. As shown in FIG. 8, the measurement system 390 includes a potentiostat 91, a counter electrode CE, and a reference electrode RE. The counter electrode CE and the reference electrode RE are electrically connected to the potentiostat 91. The conductor 60 electrically connected to the metal film 30 is electrically connected to the potentiostat 91. The counter electrode CE and the reference electrode RE are placed in a culture solution 305 placed inside a cylindrical member 140. The cylindrical member 140 used in this embodiment is made of resin and has a cylindrical shape. The cell culture membrane 10 is attached to the axial end of the cylindrical member 140.

[0033] 7, in step S30 as a first step, the cell culture membrane 10 is immersed in a culture solution 305, and cells 300 are cultured on the metal film 30 of the cell culture membrane 10. In step S32 as a second step, electrochemical impedance measurement is performed using the metal film 30 as a working electrode in the culture solution 305. In step S32, the potential difference between the working electrode WE and the reference electrode RE is controlled using a potentiostat 91 so as to be a predetermined potential difference.

[0034] Specifically, the impedance of the current path connecting the working electrode WE and the counter electrode CE is obtained. In step S32, an analysis by electrochemical impedance spectroscopy (EIS) may be further performed. This makes it possible to obtain information on the interface state of the working electrode WE.

[0035] D. Other embodiments of the cell culture membrane: 9 is a diagram showing embodiments (D1) to (D3) which are other embodiments of the cell culture membrane 10. The same components as those in the above embodiments are given the same reference numerals, and detailed explanations will be omitted as appropriate.

[0036] (D1) As shown in "D1" in Fig. 9, the cell culture membrane 110 has a membrane body 20 and a metal membrane 30. The metal membrane 30 includes a first metal membrane MF1. The first metal membrane MF1 differs from the cell culture membrane 10 shown in Fig. 1 in that it has a metal blocking portion 30d that blocks the second opening 23b of the membrane body 20. The metal blocking portion 30d has a crack 30e.

[0037] The cell culture membrane 110 is produced in the same manner as in the manufacturing process shown in FIG. 2. However, the sputtering time in step S14 is set longer than the sputtering time of the cell culture membrane 10. Specifically, the sputtering time of the titanium layer 33 is 2 minutes. The sputtering time of the second metal layer 32 is 2 minutes. The sputtering time of the first metal layer 31 is 9 minutes. As a result, the film thickness of the metal film 30 of the cell culture membrane 110 is thicker than the film thickness of the metal film 30 of the cell culture membrane 10. Therefore, when the membrane body 20 is removed from the substrate SB in step S16, the metal film formed on the substrate SB is not separated from the membrane body 20 and remains bonded to the membrane body 20.

[0038] The cracks 30e are gaps smaller than 3 μm. Since the cracks 30e are formed in the cell culture membrane 110, the first cell 301 and the second cell 302 can be brought into contact with each other through the cracks 30e when co-culture is performed. It is expected that the cracks 30e can reproduce the role of minute pores in the basement membrane. Therefore, it is expected that the culture using the cell culture membrane 110 can reproduce a cell state closer to the state in the living body.

[0039] (D2) As shown in "D2" of FIG. 9, the cell culture membrane 210 has a membrane body 20 and a metal film 30. The metal film 30 includes a first metal film MF1 and a second metal film MF2. The second metal film MF2 is formed by overlapping with the second surface 22 of the membrane body 20. The cell culture membrane 210 is produced in the same manner as the manufacturing process shown in FIG. 2. However, in step S14, after a metal is deposited on the first surface 21 to form the metal film 30, the position of the membrane body 20 is changed on the substrate SB so that the second surface 22 faces upward, and then a metal is deposited on the second surface 22 to form the second metal film MF2.

[0040] (D3) As shown in "D3" of FIG. 9, the cell culture membrane 310 has a membrane body 20 and a metal membrane 30. The metal membrane 30 includes a first metal membrane MF1 and a second metal membrane MF2. Unlike the above (D2), the first metal membrane MF1 and the second metal membrane MF2 are formed on a part of the membrane body 20. In the "plan view" of "D3" of FIG. 9, the first metal membrane MF1 is indicated by a solid line, and the second metal membrane MF2 is indicated by a broken line. The first metal membrane MF1 and the second metal membrane MF2 are formed at positions apart from each other in the membrane surface direction. As a result, the first metal membrane MF1 and the second metal membrane MF2 are not electrically connected. The cell culture membrane 310 is produced in the same manner as in the embodiment (D2). However, in step S14, the metal membrane 30 is partially formed by masking so that the metal membrane 30 is formed on a desired portion. According to this embodiment, when different cells 300 are cultured on the first surface 21 and the second surface 22 of the cell culture membrane 310, the impedance of each of the cells 300 can be measured with high accuracy.

[0041] The cell culture membrane 10 is not limited to the above. The cell culture membrane 10 may not include the first metal film MF1, but may include the second metal film MF2. The first metal film MF1 may not have the metal inner periphery 30c. The cell culture membrane 10 has the metal film 30 on the membrane surface of the membrane body 20, so that electrical measurement can be realized using the metal film 30 as an electrode.

[0042] According to the embodiment described above, the cell culture membrane 10 includes the membrane body 20 and the metal membrane 30. The metal membrane 30 includes the first metal membrane MF1. The membrane body 20 has a plurality of through holes 23. The plurality of through holes 23 have a first average pore size on the first surface 21 that is larger than a second average pore size on the second surface 22. Therefore, when the cells 300 are cultured on the metal membrane 30 of the cell culture membrane 10, the metal membrane 30 comes into contact with or is close to the cells 300, so that the accuracy of the measurement of the culture state near the cells 300 can be improved. In addition, since the cells 300 can enter each through hole 23, when different types of cells 300 are cultured on each of the first surface 21 and the second surface 22, the cells cultured on the first surface 21 and the cells cultured on the second surface 22 can come into contact with each other through the through holes 23, so that a state of the cells closer to the state in the living body can be created.

[0043] Each through-hole 23 has a first opening 23a opening to the first surface 21, a second opening 23b opening to the second surface 22, and an inner periphery 23c connecting the first opening 23a and the second opening 23b. The first metal film MF1 has a metal inner periphery 30c. This makes it possible to increase the area of ​​the metal film 30 that is in contact with or close to the cell 300 that has entered each through-hole 23, thereby further improving the measurement accuracy.

[0044] Moreover, the membrane body 20 is formed of polyurethane. Therefore, it is possible to easily fabricate the membrane body 20 having a plurality of through-holes 23 in which the first average pore size is larger than the second average pore size. Moreover, the second average pore size is 7 μm or less. Therefore, when culturing cells 300 with a size of about 10 μm, it is possible to suppress the wrapping around of the cells 300.

[0045] Moreover, the metal film 30 includes a first metal layer 31 mainly made of gold and a second metal layer 32 mainly made of platinum. The second metal layer 32 is disposed between the first metal layer 31 and the film body 20. This can improve the measurement accuracy. Furthermore, the metal film 30 includes a titanium layer 33. The titanium layer 33 is disposed between the second metal layer 32 and the film body 20. This can improve the adhesion of the first metal layer 31 and the second metal layer 32 to the film body 20.

[0046] The measurement method also includes step S20 as a first step of culturing cells 300 on the metal film 30, and step S22 and step S24 as a second step of measuring the glucose concentration. This makes it possible to realize measurement of the glucose concentration, including electrical measurement using the metal film 30 as an electrode, when cells 300 are cultured on the metal film 30 of the cell culture membrane 10.

[0047] According to another embodiment (D1), the first metal film MF1 has a metal blocking portion 30d that blocks the second opening 23b. The metal blocking portion 30d has a crack 30e. As a result, when a first cell 301 is cultured on the first surface 21 and a second cell 302 is cultured on the second surface 22, the first cell 301 and the second cell 302 can come into contact with each other through the crack 30e, so that a cellular state closer to the state in vivo can be created.

[0048] Moreover, according to another embodiment (C1), there is provided a step S30 as a first step of culturing cells 300 on the metal film 30, and a step S32 as a second step of performing electrochemical impedance measurement. As a result, when cells 300 are cultured on the metal film 30 of the cell culture membrane 10, it is possible to realize impedance measurement using the metal film 30 as an electrode.

[0049] E. Working Example: E-1.Culture results: 10 is a SEM (Scanning Electron Microscope) image of the cell culture membrane 10 observed after cells were cultured on the metal film 30 of the cell culture membrane 10. The culture was performed under two conditions: "Condition 1" in which the cell culture membrane 10 was not collagen-coated, and "Condition 2" in which the cell culture membrane 10 was collagen-coated. The culture conditions were as follows: Cell: GFP-HUVEC (Angio-Proteomie) Culture medium: Endothelial Cell Growth Medium (Promocell, model number: C-22111) Seeding density: 2.0×10 4 cells / cm 2 (Condition 1) Number of culture days: 2 days (Condition 2) Number of culture days: 4 days As shown in FIG. 10, cells could be cultured well in both cases where collagen coating was performed and where collagen coating was not performed.

[0050] E-2. Measurement of glucose concentration in spheroids: FIG. 11 is a diagram showing the measurement results of the glucose concentration for Samples 1 to 4. Sample 1 (Without) is PBS with a glucose concentration of 5 mM. Sample 2 (Supernatant) is a medium supernatant after culturing spheroids in PBS with a glucose concentration of 5 mM using a culture substrate different from the cell culture membrane 10. Sample 3 (Living) is a medium after culturing spheroids in PBS with a glucose concentration of 5 mM using the cell culture membrane 10. Sample 4 (Fixed) was prepared in the following procedure. First, the cells of the spheroids cultured using a culture substrate different from the cell culture membrane 10 were fixed using 4% paraformaldehyde-phosphate buffer. Then, the fixed spheroids were transferred to the cell culture membrane 10, and the cell culture membrane 10 with the fixed spheroids was placed in PBS with a glucose concentration of 5 mM, and the collected PBA was used as Sample 4 (Fixed). The cells used in Samples 2 to 4 were MCF-7 (10,000 cells), and the culture period was 3 days. The glucose concentrations of Samples 1 to 4 were measured under the following conditions. Pretreatment: -1.6V, 1s Working electrode potential: 0.3V

[0051] As shown in FIG. 11, the glucose concentration of sample 3 is about 2 mM, which is decreased from 5 mM. This is because the cells consumed glucose through metabolism. In sample 2, the glucose concentration also decreased because the cells consumed glucose through metabolism, but the amount of decrease was not reflected in the measurement result. This is considered to be because, in the process of putting the culture medium supernatant into the cylindrical member 40 of the measurement system 90 for measurement, the glucose concentration became almost uniform throughout the aqueous solution, and the amount of decrease in the glucose concentration became the same as the measurement error. As can be seen by comparing sample 2 and sample 3, in sample 3, minute changes in the glucose concentration near the cells can be measured. With this cell culture membrane 10, it was confirmed that the metal membrane 30 is in contact with or close to the cells, so that the glucose concentration near the cells can be accurately measured.

[0052] The present disclosure is not limited to the above-mentioned embodiment, and can be realized in various configurations without departing from the spirit of the present disclosure. For example, the technical features of the embodiments corresponding to the technical features in each aspect described in the Summary of the Invention can be appropriately replaced or combined to solve some or all of the above-mentioned problems or to achieve some or all of the above-mentioned effects. Furthermore, if a technical feature is not described as essential in this specification, it can be appropriately deleted. [Explanation of symbols]

[0053] 10,110,210,310...cell culture membrane, 20...membrane body, 21...first surface, 22...second surface, 23...through hole, 23a...first opening, 23b...second opening, 23c...inner circumference, 30...metal membrane, 30c...metal inner circumference, 30d...metal blocking portion, 30e...crack, 31...first metal layer, 32...second metal layer, 33...titanium layer, 40,140...cylindrical member , 42...well, 50...resin container, 60...conductor, 80...culture container, 90,390...measurement system, 91...potentiostat, 300...cell, 301...first cell, 302...second cell, 305...culture medium, CE...counter electrode, Da...first hole diameter, Db...second hole diameter, MF1...first metal film, MF2...second metal film, RE...reference electrode, SB...substrate, WE...working electrode

Claims

1. A membrane body having a first surface and a second surface located opposite to the first surface, the membrane body having a plurality of through holes penetrating from the first surface to the second surface; a metal film including at least one of a first metal film formed overlying the first surface and a second metal film formed overlying the second surface; A cell culture membrane, wherein the plurality of through holes have a first average pore size on the first surface that is larger than a second average pore size on the second surface.

2. 2. The cell culture membrane according to claim 1 , Each of the plurality of through holes has a first opening that opens to the first surface, a second opening that opens to the second surface, and an inner periphery that connects the first opening and the second opening, the metal film does not include the second metal film but includes the first metal film, The first metal film has a metal inner periphery formed covering the inner periphery.

3. The cell culture membrane according to claim 2, the first metal film has a metal closing portion that closes the second opening, The cell culture membrane, wherein the metal closure portion has a crack.

4. 2. The cell culture membrane according to claim 1 , The cell culture membrane, wherein the membrane body is formed of polyurethane.

5. 2. The cell culture membrane according to claim 1 , The second average pore size is 7 μm or less.

6. 2. The cell culture membrane according to claim 1 , The metal membrane comprises a first metal layer consisting essentially of either gold (Au) or platinum (Pt).

7. 7. The cell culture membrane according to claim 6, The cell culture membrane further includes a titanium layer disposed between the first metal layer and the membrane body, the titanium layer being mainly composed of titanium (Ti).

8. 2. The cell culture membrane according to claim 1 , The metal film is a first metal layer consisting essentially of gold (Au); A cell culture membrane comprising: a second metal layer consisting essentially of platinum (Pt), the second metal layer being disposed between the first metal layer and the membrane body.

9. 9. The cell culture membrane according to claim 8, The metal film is The cell culture membrane further includes a titanium layer disposed between the second metal layer and the membrane body, the titanium layer being mainly composed of titanium (Ti).

10. A measurement method using the cell culture membrane according to claim 1, A first step of immersing the cell culture membrane in a culture solution and culturing cells on the metal film of the cell culture membrane; a second step of electrochemically measuring the glucose concentration in the culture solution using the metal film as a working electrode.

11. A measurement method using the cell culture membrane according to claim 1, A first step of immersing the cell culture membrane in a culture solution to culture cells on the metal film of the cell culture membrane; a second step of performing electrochemical impedance measurement in the culture solution using the metal film as a working electrode.

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