Cell culture membrane and measurement method

Abstract

To provide a cell culture membrane that enables precise measurement of the pH in the vicinity of cells.SOLUTION: A cell culture membrane includes: a membrane body having a first face and a second face located on a side opposite to the first face; 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; and a polyaniline film provided to overlap the metal film. The membrane body has a plurality of holes, and each of the plurality of holes has a mortar shape and opens at least in the first 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 or the amount of a specific substance in the culture solution have been measured (for example, Patent Document 1). Patent Document 1 describes a cell culture chip equipped with a pH sensor, which is a physical property of the culture solution. In this cell culture chip, the pH sensor is placed above the well in which the cells are cultured. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2004-113092 A Summary of the Invention [Problem to be solved by the invention]

[0004] In the above-mentioned cell culture chip, since cells are cultured on the bottom surface of the well, there may be cases where the pH measured by the pH sensor does not reflect the pH in the vicinity of the cells. [Means for solving the problem]

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

[0006] (1) According to one 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 opposite to the first surface, a metal membrane including at least one of a first metal membrane formed on the first surface and a second metal membrane formed on the second surface, and a polyaniline membrane formed on the metal membrane, the membrane body having a plurality of holes, each of which is cone-shaped and opens at least on the first surface. According to this embodiment, when cells are cultured on the polyaniline membrane, the polyaniline membrane comes into contact with or is close to the cells, and therefore the metal membrane is used as an electrode and the polyaniline membrane is used as a proton-responsive membrane, thereby enabling accurate measurement of pH in the vicinity of the cells. (2) In the cell culture membrane of the above embodiment, each of the holes may be a blind hole that does not open to the second surface and has an inner periphery that defines the hole, 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 covering the inner periphery, and the polyaniline film has a polyaniline inner periphery covering the metal inner periphery. According to this embodiment, the area of ​​the polyaniline film that is in contact with or close to the cells that have entered each hole can be increased, thereby further improving the measurement accuracy of pH near the cells. (3) In the cell culture membrane of the above embodiment, each of the holes may be a through hole penetrating from the first surface to the second surface, and 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, the metal film may not include the second metal film but may include the first metal film, the first metal film may have a metal inner periphery formed to cover the inner periphery, and the polyaniline film may have a polyaniline inner periphery formed to cover the metal inner periphery. According to this embodiment, 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 cellular state closer to the state in a living body can be created. (4) In the cell culture membrane of the above embodiment, each of the holes is a through hole penetrating from the first surface to the second surface, and has 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, 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 by covering the inner periphery and a metal blocking part blocking the second opening, the polyaniline film has a polyaniline inner periphery formed by covering the metal inner periphery and the metal blocking part, and the metal blocking part may have a crack. According to this embodiment, cells cultured on the first surface and 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 a living body. (5) In the cell culture membrane of the above embodiment, the plurality of holes may have a first average pore size on the first surface that is larger than a second average pore size on the second surface. This embodiment increases the likelihood that cells can enter the holes from the first openings and be cultured in the pore-entered state without passing through the second openings. (6) 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. (7) 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. (8) In the cell culture membrane of the above embodiment, the metal membrane may include a first metal layer mainly made of gold (Au). According to this embodiment, the first metal layer mainly made of gold can be used as the metal membrane. (9) In the cell culture membrane of the above embodiment, the metal membrane may further include a second metal layer mainly made of platinum (Pt) and a third metal layer mainly made of titanium (Ti). According to this embodiment, a laminated film of a first metal layer mainly made of gold, a second metal layer mainly made of platinum, and a third metal layer mainly made of titanium can be used as the metal membrane. (10) A method for measuring pH using the cell culture membrane of the above embodiment may include a first step of immersing the cell culture membrane in a culture solution to culture cells on the polyaniline film of the cell culture membrane, a second step of inserting a reference electrode into the culture solution, using the metal film as a working electrode, and measuring a potential difference between the reference electrode and the working electrode, and a third step of determining the pH of the culture solution using the measured potential difference. According to this embodiment, when cells are cultured on the cell culture membrane, it is possible to measure pH using the metal film as an electrode. [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 pH. [Figure 4] FIG. 1 shows a measurement system assembled for measuring pH. [Diagram 5] FIG. 2 is a diagram illustrating an oxidation-reduction reaction of conductive polyaniline. [Figure 6] A graph showing the relationship between pH and potential difference. [Figure 7] Schematic diagram of co-culture using a cell culture membrane. [Figure 8] FIG. 2 shows embodiments (C1) to (C3) of the cell culture membrane. [Figure 9] 1A to 1C are diagrams showing embodiments (C4) to (C6) of the cell culture membrane. [Figure 10] 14A-14C show embodiments (C7) and (C8) of the cell culture membrane. [Figure 11]FIG. 13 is a diagram showing another embodiment of the culture vessel. [Figure 12] SEM images of membranes produced at each stage of the cell culture membrane manufacturing process. [Figure 13] SEM image of a cell culture membrane on which cells are cultured. [Figure 14] FIG. 1 is a diagram explaining the chemical reaction that occurs when ammonium chloride is added to an aqueous solution containing cells. [Figure 15] Measurement results of the open circuit potential when ammonium chloride is added to PBS. [Figure 16] SEM image of a cell culture membrane on which cells are cultured. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0008] A. Embodiment: A-1. Cell culture membrane composition: 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, a metal membrane 30, and a polyaniline membrane 40. In this embodiment, the cell culture membrane 10 is made of polyurethane. The metal membrane 30 is formed 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 third metal layer 33 mainly made of titanium (Ti) from the top to the bottom. The third metal layer 33 is disposed between the second metal layer 32 and the membrane body 20, thereby improving the adhesion between the first metal layer 31 and the second metal layer 32 and the membrane body 20. As another embodiment of the metal membrane 30, the metal membrane 30 may be composed of only the first metal layer 31 mainly made of gold (Au). Since the cell culture membrane 10 comprises the metal membrane 30 and the polyaniline membrane 40, after cells are cultured on the polyaniline membrane 40, the pH of the culture solution 305 described below can be measured by using the metal membrane 30 as an electrode and the polyaniline membrane 40 as a proton responsive membrane.

[0009] The cell culture membrane 10 has a membrane body 20 having a first surface 21 and a second surface 22 located on the opposite side to the first surface 21, and a plurality of through holes 23 as a plurality of holes 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.

[0010] Each through-hole 23 is cone-shaped. 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 the case of a through-hole, the shape in which the second hole diameter Db is smaller than the first hole diameter Da is included in the cone-shaped shape. Furthermore, both the case in which the shape of the inner periphery 23c is a curved surface and the case in which it is a flat surface are included in the cone-shaped shape. 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 below, when co-culture is performed using the cell culture membrane 10, after the first cells 301 are seeded on the cell culture membrane 10, 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 migration of the first cells 301 through the through-holes 23 to the surface opposite to the surface on which they were seeded may be referred to as "wandering around."

[0011] 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.

[0012] The metal film 30 includes a first metal film MF1 formed to overlap 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.

[0013] The polyaniline film 40 has a polyaniline inner peripheral portion 40c. The polyaniline inner peripheral portion 40c is formed to cover the metal inner peripheral portion 30c. Since the metal film 30 and the polyaniline film 40 are formed to cover the inner peripheral portion 23c, it is possible to further improve the measurement accuracy of pH in the vicinity of the cell 300, as described later.

[0014] A-2. Manufacturing method of cell culture membrane: FIG. 2 is a flow chart showing the manufacturing process of the cell culture membrane 10. As shown in FIG. 2, in step S10, a membrane body 20 formed 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 different first hole diameters Da and second hole diameters Db can be easily manufactured. Also, for example, the membrane can be manufactured as described in Japanese Patent No. 6343492.

[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 third metal layer 33 is 1 minute. Note that the sputtering time is not limited to the above.

[0016] In step S16, the metal film 30 is ashed. Specifically, in step S16, the surface of the metal film 30 is irradiated with oxygen plasma. This makes it possible to impart hydrophilicity to the surface of the metal film 30.

[0017] In step S18, a polyaniline film 40 is formed on the metal film 30. Specifically, the polyaniline film 40 is formed by electrolytically polymerizing aniline on the metal film 30. This results in a conductive polyaniline film 40. In this embodiment, the polyaniline film 40 is formed using a potential scanning method, but the polyaniline film 40 may also be formed using a constant potential method or a constant current method. Since the metal film 30 is made hydrophilic in step S16, which is performed before step S18, the inner periphery 23c of the through hole 23 can be modified with polyaniline.

[0018] In step S20, 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 and the polyaniline film formed on the substrate SB are separated from the film body 20. Therefore, the second opening 23b of the film body 20 is not blocked by the metal film.

[0019] A-3. How to measure pH: Fig. 3 is a flow chart showing the procedure of a measurement method for measuring the pH of a culture medium 305 after cells are cultured on the cell culture membrane 10. Fig. 4 is a diagram showing a measurement system 90 that is assembled when measuring pH.

[0020] 3, in the culture step of step S30, 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.

[0021] As shown in FIG. 4, the culture vessel 80 has a cell culture membrane 10 and a cylindrical member 50. The cylindrical member 50 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 50. This allows the culture vessel 80 to function as a cylindrical vessel with the cell culture membrane 10 as the bottom surface. A conductor 70 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 50 is 8 mm.

[0022] In step S30 as the first process, with the culture vessel 80 placed in the resin vessel 60, a culture solution 305 is poured inside the cylindrical member 50, and the cells 300 are seeded and cultured.

[0023] In step S32 as the second process in Fig. 3, the potential difference is measured. As shown in Fig. 4, the measurement system 90 includes a potential meter 93 and a reference electrode RE. A conductor 70 electrically connected to the metal film 30 is electrically connected to the potential meter 93. In the measurement, the metal film 30 functions as a working electrode WE. The reference electrode RE is electrically connected to the potential meter 93 via the conductor 70. Specifically, in step S32, the reference electrode RE is inserted into the culture solution 305, and the potential meter 93 measures the potential difference between the metal film 30 functioning as the working electrode WE and the reference electrode RE.

[0024] In step S34 as the third process in Fig. 3, the pH of the culture solution 305 is calculated using the measured potential difference. Specifically, the pH is calculated using a relational expression between pH and potential difference calculated in advance. Since the cell 300 is in contact with or in close proximity to the polyaniline film 40 and the metal film 30, the pH near the cell 300 can be measured with high accuracy. Specifically, an approximation formula can be used as the relational expression between pH and potential difference. The pH may also be calculated using a map showing the relationship between the range of potential difference and the pH applied to each range.

[0025] A-4. pH measurement principle: Fig. 5 is a diagram illustrating the oxidation-reduction reaction of conductive polyaniline. As shown in Fig. 5, polyaniline reversibly adds and removes protons. Therefore, the potential of an electrode modified with conductive polyaniline changes depending on the pH.

[0026] FIG. 6 is a diagram showing the relationship between pH and potential difference. FIG. 6 shows the results of an experiment performed by the inventors using a cell culture membrane 10. The cell culture membrane 10 was placed in a buffer solution with a known pH, and the open circuit potential (OCP), which is the potential difference between the metal film 30 functioning as the working electrode WE and the reference electrode RE, was measured. The relationship between pH and open circuit potential was obtained by measuring the potential difference for buffer solutions with various pH values. An acetate buffer solution was used as a buffer solution with a pH of 4 to 5. A phosphate buffer solution was used as a buffer solution with a pH of 6 to 8. The polyaniline film 40 was prepared by a potential scanning method, and the number of sweeps was 15. The measurement was performed on the cell culture membrane 10 on which cell culture was not performed and the cell culture membrane 10 on which cell culture was performed. "Without cells" in FIG. 6 indicates the cell culture membrane 10 on which cell culture was not performed. "With cells" in FIG. 6 indicates the cell culture membrane 10 on which cell culture was performed.

[0027] It is expected that the lower the pH of the buffer solution, the more the protonation of polyaniline progresses and the higher the potential of the working electrode WE, and therefore the larger the potential difference between the working electrode WE and the reference electrode RE. As shown in FIG. 6, it was confirmed that the open circuit potential is proportional to the pH, and the lower the pH, the higher the open circuit voltage. Therefore, by using the cell culture membrane 10 to obtain a relationship between pH and open circuit voltage in advance and measuring the open circuit voltage of the culture solution 305 with an unknown pH, the pH of the culture solution 305 can be obtained.

[0028] B. Other forms of culture methods: 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 cells can also be cultured on each of both sides of the cell culture membrane 10. Furthermore, in this case, co-culture can also be performed in which two or more different types of cells are cultured on each of both sides of the cell culture membrane 10.

[0029] FIG. 7 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.

[0030] The cell culture using the cell culture membrane 10 is not limited to the above. The same type of cells 300 may be cultured on the first surface 21 and the second surface 22, or two or more types of cells 300 may be cultured on each of the first surface 21 and the second surface 22.

[0031] C. Other embodiments of the cell culture membrane: 8 is a diagram showing embodiments (C1) to (C3) 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.

[0032] (C1) As shown in "C1" of FIG. 8, the cell culture membrane 110 has a membrane body 20, a metal membrane 30, and a polyaniline membrane 40. 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. The polyaniline membrane 40 has a polyaniline inner peripheral portion 40c formed by covering the metal inner peripheral portion 30c and the metal blocking portion 30d.

[0033] 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 first metal layer 31 is 9 minutes. The sputtering time of the second metal layer 32 is 2 minutes. The sputtering time of the third metal layer 33 is 2 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 film body 20 is removed from the substrate SB in step S20, the metal film 30 and the polyaniline film 40 formed on the substrate SB are not separated from the film body 20 and remain bonded to the film body 20.

[0034] 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.

[0035] (C2) As shown in "C2" of Fig. 8, the cell culture membrane 210 has a membrane body 20, a metal membrane 30, and a polyaniline membrane 40. The cell culture membrane 210 differs from the cell culture membrane 10 shown in Fig. 1 in that the polyaniline membrane 40 has a polyaniline blocking portion 40d that blocks the second opening 23b. According to this cell culture membrane 210, the area of ​​the polyaniline membrane 40 that is in contact with or close to the cell 300 that has entered inside the polyaniline inner peripheral portion 40c can be increased, so that the measurement accuracy of the pH near the cell 300 can be further improved.

[0036] (C3) As shown in "C3" of FIG. 8, the cell culture membrane 310 has a membrane body 20, a metal film 30, and a polyaniline film 40. The membrane body 20 does not have a through hole 23, but has a plurality of holes 24 that are non-through holes, which is different from the cell culture membrane 10 shown in FIG. 1. The holes 24 are cone-shaped. In the case of non-through holes, the following shapes (A) and (B) are included in the cone-shaped shape. Shape (A) refers to a shape in which the inner circumferential surface that defines the hole is roughly hemispherical, with the apex being the point where the distance between this inner circumferential surface and the second surface is the shortest. Shape (B) refers to a shape in which the cross-sectional area of ​​the cross section of the inner circumferential surface parallel to the first surface at the position where the distance between the inner circumferential surface that defines the hole and the second surface is the shortest is smaller than the area of ​​the opening of the hole in the first surface. The hole 24 is defined by the inner circumferential portion 23d. The metal film 30 has a metal inner peripheral portion 30f that covers the inner peripheral portion 23d. The polyaniline film 40 has a polyaniline inner peripheral portion 40e that covers the metal inner peripheral portion 30f. This cell culture membrane 310 can increase the area of ​​the polyaniline film 40 that is in contact with or close to the cell 300 that has entered inside the polyaniline inner peripheral portion 40e, thereby further improving the measurement accuracy of the pH near the cell 300.

[0037] 9 is a diagram showing embodiments (C4) to (C6) which are other embodiments of the cell culture membrane 10. The common point between the embodiments (C4) to (C6) is that the polyaniline film 40 does not penetrate into the through-holes 23 or the holes 24. The same components as those in the above embodiments are denoted by the same reference numerals, and detailed explanations will be omitted as appropriate.

[0038] (C4) As shown in "C4" in Fig. 9, a cell culture membrane 410 has a membrane body 20, a metal film 30, and a polyaniline film 40. The membrane body 20 and the metal film 30 have the same structure as the cell culture membrane 10 shown in Fig. 1, and therefore a description thereof will be omitted. The polyaniline film 40 differs from the cell culture membrane 10 shown in Fig. 1 in that it does not enter the through-hole 23 and is formed so as to block the first opening 23a.

[0039] (C5) As shown in "C5" in Fig. 9, a cell culture membrane 510 has a membrane body 20, a metal film 30, and a polyaniline film 40. The membrane body 20 and the metal film 30 have the same structure as the cell culture membrane 210 shown in Fig. 8, and therefore a description thereof will be omitted. The polyaniline film 40 differs from the cell culture membrane 210 shown in Fig. 8 in that it does not enter the through-hole 23 and is formed so as to block the first opening 23a.

[0040] (C6) As shown in "C6" in Fig. 9, a cell culture membrane 610 has a membrane body 20, a metal film 30, and a polyaniline film 40. The membrane body 20 and the metal film 30 have the same structure as the cell culture membrane 310 shown in Fig. 8, and therefore a description thereof will be omitted. The polyaniline film 40 differs from the cell culture membrane 310 shown in Fig. 8 in that it does not enter the through-hole 23 and is formed so as to block the first opening 23a.

[0041] As described above, the cell culture membrane 10 shown in Fig. 1 is made hydrophilic by ashing in step S16 of the manufacturing process shown in Fig. 2. In contrast, step S16 is not performed in the manufacturing process of the embodiments (C4) to (C6) shown in Fig. 8. As a result, the polyaniline film 40 is formed so as not to enter the through-holes 23 or holes 24 but to block the through-holes 23 or holes 24. Regardless of whether the polyaniline film 40 enters the through-holes 23 or holes 24, the polyaniline film 40 is formed on the metal film 30, and thus the pH of the culture solution 305 can be measured.

[0042] 10 is a diagram showing embodiment (C7) and embodiment (C8) which are other embodiments of the cell culture membrane 10. The same components as those in the above-mentioned embodiments are given the same reference numerals, and detailed explanations will be omitted as appropriate.

[0043] (C7) As shown in "C7" of FIG. 10, the cell culture membrane 710 has a membrane body 20, a metal film 30, and a polyaniline film 40. 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 710 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 substrate SB is changed in position 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.

[0044] (C8) As shown in "C8" of FIG. 10, the cell culture membrane 810 has a membrane body 20, a metal film 30, and a polyaniline film 40. The metal film 30 includes a first metal film MF1 and a second metal film MF2. The second metal film MF2 is formed on the second surface 22 of the membrane body 20. Unlike the above embodiment (C7), the first metal film MF1 and the second metal film MF2 are formed on a part of the membrane body 20. In the "plan view" of "C8" of FIG. 10, the first metal film MF1 is shown by a solid line, and the second metal film MF2 is shown by a broken line. The first metal film MF1 and the second metal film MF2 are formed at positions apart from each other in the membrane surface direction. As a result, the first metal film MF1 and the second metal film MF2 are not electrically connected to each other. This cell culture membrane 810 is produced in the same manner as in embodiment (C7). However, in step S14, the metal film 30 is partially formed by masking so that the metal film 30 is formed on a desired portion. According to this embodiment, when different cells are cultured on the first surface 21 and the second surface 22 of the cell culture membrane 810, the pH of each of the cells can be measured.

[0045] D. Other embodiments of the culture vessel: FIG. 11 is a diagram showing another embodiment of the culture vessel 80. The same components as those in the above embodiment are given the same reference numerals, and detailed explanations are omitted as appropriate. As shown in FIG. 11, the culture vessel 180 of this embodiment is made of resin and has a cylindrical shape. A cell culture membrane 10 is attached to the axial end of a cylindrical member 50. In the culture process, the cell culture membrane 10 is immersed in a well 52 containing a culture solution 305, and culture is performed.

[0046] According to the embodiment described above, the cell culture membrane 10 includes the membrane body 20 having a plurality of through-holes 23, the metal membrane 30, and the polyaniline membrane 40 formed on the metal membrane 30. The metal membrane 30 includes a first metal film MF1. Each of the plurality of through-holes 23 is open at least to the first surface 21. As a result, when cells 300 are cultured on the polyaniline membrane 40, the metal membrane 30 is used as an electrode and the polyaniline membrane 40 is used as a proton responsive membrane, thereby enabling accurate measurement of pH in the vicinity of the cells.

[0047] Moreover, the first metal film MF1 has a metal inner peripheral portion 30c formed to cover the inner peripheral portion 23c. The polyaniline film 40 has a polyaniline inner peripheral portion 40c formed to cover the metal inner peripheral portion 30c. Therefore, the area of ​​the polyaniline film 40 that is in contact with or close to the cell 300 that has entered each through-hole 23 can be increased, and the pH measurement accuracy can be further improved. Furthermore, when the first cell 301 is cultured on the first surface 21 and the 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 second opening 23b, and therefore a cellular state closer to the state in the living body can be created.

[0048] 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.

[0049] The measurement method includes step S30 as a first step of culturing cells 300 on the metal film 30, step S32 as a second step of measuring the potential difference, and step S34 as a third step of determining the pH. This makes it possible to measure the pH using the metal film 30 as an electrode when the cells 300 are cultured on the cell culture membrane 10.

[0050] According to another embodiment (C3), the cell culture membrane 10 has holes 24 that are non-through holes. The first metal film MF1 has a metal inner peripheral portion 30f that covers the inner peripheral portion 23d that defines the holes 24. The polyaniline film 40 has a polyaniline inner peripheral portion 40e that covers the metal inner peripheral portion 30f. This makes it possible to increase the area of ​​the polyaniline film 40 that is in contact with or close to the cells 300 that have entered each hole 24, thereby further improving the accuracy of pH measurement.

[0051] According to another embodiment (C1), the metal film 30 has a metal blocking portion 30d that blocks the second opening 23b. The metal blocking portion 30d has a crack 30e. Therefore, 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. Since the size of the crack 30e is smaller than the size of the second opening 23b, it is expected that a cellular state closer to the state in vivo can be created.

[0052] E. Working Example: E-1. Preparation of cell culture membrane: Fig. 12 shows SEM (Scanning Electron Microscope) images of membranes produced in each step of the manufacturing process of the cell culture membrane 10. The "metal film surface" in Fig. 12 is an SEM image of the membrane surface after the metal film 30 is formed in step S14 shown in Fig. 2. As shown in the "metal film surface" in Fig. 12, the metal film 30 is formed along the inner periphery 23c of the through-hole 23.

[0053] "Polyaniline film surface" in Fig. 12 is an SEM image of the film surface after the polyaniline film 40 is formed in step S18 shown in Fig. 2. The right image of "polyaniline film surface" in Fig. 12 is an enlarged image of the left image. "Polyaniline film cross section" in Fig. 12 is an SEM image of the cross section of the film after the polyaniline film 40 is formed in step S18 shown in Fig. 2.

[0054] As shown in the "polyaniline film cross section" of FIG. 12, the polyaniline film 40 is formed along the inner periphery 23c of the through-hole 23. As shown in the right diagram of the "polyaniline film surface" of FIG. 12, for some of the through-holes 23 among all the through-holes 23 formed in the film body 20, the polyaniline film 40 may be formed so as to close the opening of the through-hole 23 without entering the through-hole 23. Even if the polyaniline film 40 does not enter the through-hole 23 for all the through-holes 23 formed in the film body 20, the measurement accuracy of pH can be improved by the polyaniline film 40 that is formed by entering the through-hole 23. As described above, whether the polyaniline film 40 enters the through-hole 23 can be controlled by the presence or absence of the ashing process in step S16.

[0055] E-2.Culture results: Fig. 13 is an SEM image of the cell culture membrane 10 after cells were cultured on the cell culture membrane 10. The left image in Fig. 13 is an SEM image after one day of culture. The right image in Fig. 13 is an SEM image after three days of culture. As shown in Fig. 13, cells 300 could be cultured well using the cell culture membrane 10.

[0056] E-3. pH measurement during cell culture: FIG. 14 is a diagram illustrating the chemical reaction that occurs when ammonium chloride (NH4Cl) is added to an aqueous solution containing cells.

[0057] As shown in "outside the cell" in Figure 14, when ammonium chloride is added to the aqueous solution, ammonium ions (NH4 + ) and hydroxide ion (OH- ) reacts with ammonia (NH3) to produce ammonia. Therefore, the hydroxide ion is converted into a proton (H + ) and the aqueous solution becomes acidic.

[0058] As shown in Figure 14, ammonium "outside the cell" passes through the cell membrane and moves "inside the cell." As shown in Figure 14 "inside the cell," ammonium reacts with protons to produce ammonium ions. As a result, protons are reduced relative to hydroxide ions, and the inside of the cell becomes alkaline.

[0059] Figure 15 shows the results of measuring the open circuit potential when ammonium chloride was added to phosphate-buffered saline (PBS). As shown in Figure 15, the change over time in the open circuit potential was measured when ammonium chloride was added to the culture solution for (Sample A) a cell culture membrane 10 in which cells were cultured, and (Sample B) a cell culture membrane 10 in which cells were not cultured. The solutions used and the culture conditions for Sample A were as follows: solution Solution A: PBS (pH7.4) Solution B: 10mM NH4Cl in PBS Solution C: PBS Culture conditions Cell: GFP-HUVEC Culture days: 5 days

[0060] Fig. 15 is an SEM image of the cell culture membrane 10 (sample A) observed after cells were cultured on the cell culture membrane 10. As shown in Fig. 15, the cells could be cultured well.

[0061] As shown in Fig. 15, for each of (Sample A) and (Sample B), the cell culture membrane 10 was immersed in PBS of pH 7.4, and the open circuit potential was measured for 100 seconds. 100 seconds after the start of the measurement, ammonium chloride was added to a concentration of 10 mM, and the open circuit potential was measured for 100 seconds. 100 seconds after the start of the measurement, the solution in which the cell culture membrane 10 was immersed was replaced with PBS, and the open circuit potential was measured for 100 seconds.

[0062] 15, in (Sample A), when ammonium chloride was added, the open circuit potential increased. When the PBS was then replaced, the open circuit potential decreased. Therefore, it is understood that the cell culture membrane 10 can measure the pH near the cells with high accuracy.

[0063] 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]

[0064] 10,110,210,310,410,510,610,710,810...cell culture membrane, 20...membrane body, 21...first surface, 22...second surface, 23...through hole, 23a...first opening, 23b...second opening, 23c, 23d...inner circumference, 24...hole, 30...metal membrane, 30c, 30f...metal inner circumference, 30d...metal blocking portion, 30e...crack, 31...first metal layer, 32...second metal layer, 33...third metal layer, 40...poly Aniline film, 40c, 40e... polyaniline inner periphery, 40d... polyaniline blocking portion, 50, 150... cylindrical member, 60... resin container, 70... conducting wire, 80... culture container, 90... measurement system, 93... potential measuring device, 300... cell, 301... first cell, 302... second cell, 305... culture medium, 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; a metal film including at least one of a first metal film formed on the first surface and a second metal film formed on the second surface; a polyaniline film formed on the metal film, The cell culture membrane, wherein the membrane body has a plurality of holes, each of the plurality of holes being cone-shaped and opening at least on the first surface.

2. 2. The cell culture membrane according to claim 1 , Each of the plurality of holes is a blind hole that does not open to the second surface and has an inner periphery that defines the hole, 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 portion covering the inner periphery portion, The polyaniline film has a polyaniline inner periphery covering the metal inner periphery.

3. 2. The cell culture membrane according to claim 1 , each of the plurality of holes is a through hole penetrating from the first surface to the second surface, and has a first opening portion opening to the first surface, a second opening portion opening to the second surface, and an inner periphery connecting the first opening portion and the second opening portion; 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 portion formed to cover the inner periphery portion, The polyaniline film has a polyaniline inner periphery formed covering the metal inner periphery.

4. 2. The cell culture membrane according to claim 1 , each of the plurality of holes is a through hole penetrating from the first surface to the second surface, and has a first opening portion opening to the first surface, a second opening portion opening to the second surface, and an inner periphery connecting the first opening portion and the second opening portion; 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 portion formed to cover the inner periphery portion and a metal closing portion closing the second opening portion, the polyaniline film has a polyaniline inner peripheral portion formed to cover the metal inner peripheral portion and the metal closing portion, The cell culture membrane, wherein the metal closure portion has a crack.

5. The cell culture membrane according to claim 3 or 4, The plurality of pores have a first average pore size at the first surface that is larger than a second average pore size at the second surface.

6. The cell culture membrane according to any one of claims 1 to 4, The cell culture membrane, wherein the membrane body is formed of polyurethane.

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

8. 2. The cell culture membrane according to claim 1 , The metal membrane comprises a first metal layer consisting essentially of gold (Au),

9. 9. The cell culture membrane according to claim 8, The metal membrane is a cell culture membrane, further comprising a second metal layer consisting essentially of platinum (Pt) and a third metal layer consisting essentially of titanium (Ti).

10. A method for measuring pH using the cell culture membrane according to claim 1, A first step of culturing cells on the polyaniline film of the cell culture membrane by immersing the cell culture membrane in a culture solution; a second step of inserting a reference electrode into the culture solution, using the metal film as a working electrode, and measuring a potential difference between the reference electrode and the working electrode; A third step of determining the pH of the culture solution using the measured potential difference.

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