Cell device and method for controlling insulin secretion

Abstract

To provide a cell device that allows quicker induction of insulin secretion and suppression of insulin secretion from the initiation of operation, and also to provide a method for controlling insulin secretion.SOLUTION: A cell device 1 comprises: a culture vessel 2; a medium 3 and pancreatic β-cells 4 accommodated in the culture vessel 2; a working electrode 5, to which the pancreatic β-cells 4 adhere and which contains PEDOT:PzSS; a counter electrode 6 provided so as to be in contact with the medium 3; and a potential-application section 8 that applies a potential to the pancreatic β-cells 4 by means of the working electrode 5 and the counter electrode 6.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a cellular device comprising pancreatic beta cells and a method for controlling insulin secretion. [Background technology]

[0002] In recent years, it has become possible to create a variety of functional cells using ES cells and iPS cells, and cell devices using these functional cells are also being investigated.

[0003] For example, Non-Patent Document 1 discloses that human pancreatic β cells were cultured on a porous membrane, platinum electrodes were placed on either side of the porous membrane, and an electric pulse stimulus was applied to the human pancreatic β cells, thereby controlling insulin secretion in real time. In particular, it was confirmed that the amount of insulin secreted reached its maximum after 10 minutes of applying the electric pulse stimulus for 40 minutes. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Krzysztof Krawczyk1, et al., “Electrogenetic cellular insulin release for real-time glycemic control in type 1 diabetic mice” Science 368, 993-1001 (2020) Summary of the Invention [Problem to be solved by the invention]

[0005] An object of the present invention is to provide a cell device that allows for more rapid insulin secretion from the start of operation and a method for inducing insulin secretion in pancreatic β cells. [Means for solving the problem]

[0006] The cell device of the present invention comprises a culture vessel, culture medium and pancreatic beta cells contained in the culture vessel, a working electrode containing PEDOT:PSS and to which the pancreatic beta cells adhere, a counter electrode provided in contact with the culture medium, and a potential application unit that applies a potential to the pancreatic beta cells via the working electrode and the counter electrode.

[0007] The cell device may further include a reference electrode provided so as to be in contact with the culture medium.

[0008] In the cell device, the working electrode may further contain tetraethyl orthosilicate (TEOS).

[0009] Furthermore, the insulin secretion control method of the present invention may include an induction step of inducing insulin secretion from the pancreatic beta cells in any of the cell devices by applying an electric potential to the pancreatic beta cells so as to open voltage-dependent calcium channels possessed by the pancreatic beta cells. In addition, the insulin secretion control method of the present invention may include an inhibition step of suppressing insulin secretion from the pancreatic beta cells by applying an electric potential to the pancreatic beta cells in any of the cell devices so as to close the voltage-dependent calcium channels possessed by the pancreatic beta cells. [Effects of the Invention]

[0010] According to the cell device of the present invention, the pancreatic β cells adhere to the working electrode, and thus insulin secretion can be rapidly induced by applying a potential. Similarly, the insulin secretion induction method can also rapidly induce insulin secretion. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 is a schematic diagram showing a pancreatic β-cell in a glucose-unstimulated state. [Figure 2] FIG. 2 is a schematic diagram showing a pancreatic β-cell under glucose stimulation. [Figure 3]FIG. 3 is a schematic cross-sectional view showing the general structure of the cell device. [Figure 4] FIG. 4 is an enlarged schematic diagram showing the working electrode of the cell device of FIG. 3 and the pancreatic β cells adhered thereto in the state where no potential is applied. [Figure 5] 5 is an enlarged schematic diagram showing the working electrode of the cell device of FIG. 3 and pancreatic β cells adhered thereto while a potential is being applied. [Figure 6] FIG. 6 is a schematic diagram showing the arrangement of a culture vessel, electrodes, a potentiostat, and a microscope in the example. [Figure 7] FIG. 7(a) is a luminescence image of the cell before application of a potential in Experiment 1, and FIG. 7(b) is a diagram showing the state of the cell at this time. [Figure 8] FIG. 8(a) is a luminescence image taken of the luminescence of a cell during application of a potential in Experiment 1, and FIG. 8(b) is a diagram showing a schematic diagram of the state of the cell at this time. [Figure 9] Figure 9 shows luminescence images of cells taken before application of a potential in Experiment 2 (nicardipine addition). [Figure 10] Figure 10 shows images of the luminescence of cells during application of a potential in Experiment 2 (nicardipine addition). [Figure 11] FIG. 11 is a graph showing the relative luminescence intensity in Experiment 1, where 30 seconds before the start of potential application is set to zero seconds and the luminescence intensity at zero seconds is set to 1. [Figure 12] FIG. 12 is a graph showing the relative luminescence intensity in Experiment 2, where 30 seconds before the start of potential application is set to zero seconds and the luminescence intensity at zero seconds is set to 1. [Figure 13] FIG. 13 is a graph showing the relative luminescence intensity in Experiment 3, where the luminescence intensity at 0 seconds is set to 1 and the time 20 seconds before the application of the constant potential is set to zero seconds. [Figure 14] FIG. 14 is a graph showing the relative luminescence intensity of the buffer solution after application of a potential in Experiment 4, with the luminescence intensity of the buffer solution before application of a potential set to 1. [Figure 15]FIG. 15 shows bright field and luminescence images before and after potential application using a working electrode containing 48 wt % PEDOT:PSS from Experiment 5. [Figure 16] FIG. 16 shows bright-field and luminescence images before and after application of a potential using a working electrode containing 37 wt % PEDOT:PSS in Experiment 5. [Figure 17] FIG. 17 shows bright field and luminescence images before and after potential application using a working electrode containing 30 wt % PEDOT:PSS from Experiment 5. [Figure 18] Figure 18 shows the TEOS concentration in the mixed solution when forming the working electrode (top row), a photograph of the slide glass after applying the mixed solution once and before drying (middle row), and a bright-field image after cell culture (bottom row). [Figure 19] Figure 19 shows luminescence photographs taken 17.3 seconds (natural potential), 39.0 seconds (natural potential + 500 mV), and 49.8 seconds (natural potential) after the start of observation in a potential application experiment using a cell device (TEOS concentration in the mixed solution during working electrode formation was 2.5 v / v%), as well as a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 20] Figure 20 shows luminescence photographs taken 17.3 seconds (natural potential), 39.0 seconds (natural potential + 500 mV), and 49.8 seconds (natural potential) after the start of observation in a potential application experiment using a cell device (TEOS concentration in the mixed solution during working electrode formation was 5 v / v%), as well as a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 21] This figure shows luminescence photographs taken 17.3 seconds (natural potential), 39.0 seconds (natural potential + 500 mV), and 49.8 seconds (natural potential) after the start of observation in a potential application experiment using a cell device (TEOS concentration in the mixed solution during working electrode formation was 7.5 v / v%), as well as a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 22]This shows luminescence photographs taken 17.3 seconds (natural potential), 39.0 seconds (natural potential + 500 mV), and 49.8 seconds (natural potential) after the start of observation in a potential application experiment using a cell device (TEOS concentration in the mixed solution during working electrode formation was 10 v / v%), as well as a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 23] Figure 23 shows luminescence photographs taken 19.5 seconds (natural potential), 78.3 seconds (natural potential +150 mV), and 86.7 seconds (natural potential) after the start of observation in a potential application experiment using a cell device, a graph of the electrode potential, and a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 24] Figure 24 shows luminescence photographs taken 19.5 seconds (natural potential), 78.3 seconds (natural potential +200 mV), and 86.7 seconds (natural potential) after the start of observation in a potential application experiment using a cell device, a graph of the electrode potential, and a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 25] Figure 25 shows luminescence photographs taken 19.5 seconds (natural potential), 36.8 seconds (natural potential + 150 mV), 41.2 seconds (natural potential + approximately 200 mV), and 70.8 seconds (natural potential + approximately 500 mV) after the start of observation in a potential application experiment using LSV (Linear Sweep Voltammetry) on a cell device, a graph of the electrode potential, and a graph of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1. [Figure 26] Figure 26 shows bright-field images of cultured cells in a cell membrane potential evaluation experiment using Fluovolt membrane potential dye, fluorescent photographs at each electrode potential, a graph of the electrode potential, and a graph of relative fluorescence intensity, with the fluorescence intensity at the start of observation set to 1. [Figure 27] Figure 27 shows graphs of the electrode potential and relative luminescence intensity when the natural potential was measured for 10 seconds (0 to 10 seconds), the natural potential was applied for 10 seconds (10 to 20 seconds), then a natural potential of +500 mV was applied for 2 seconds (20 to 22 seconds), and finally the circuit was disconnected (i.e., the application of potential was stopped) (from 22 seconds onwards). [Figure 28]Figure 28 shows graphs of the electrode potential and relative luminescence intensity when the natural potential was measured for 10 seconds (0 to 10 seconds), the natural potential was applied for 10 seconds (10 to 20 seconds), and then a natural potential of +500 mV was applied for 2 seconds and then the natural potential was applied for 30 seconds, alternating three times each (20 to 116 seconds), and then a natural potential of +500 mV was continuously applied (from 116 seconds onwards). [Figure 29] Figure 28 is a graph of the electrode potential and a graph of the relative luminescence intensity when the natural potential was measured for 10 seconds (0 to 10 seconds), the natural potential was applied for 10 seconds (10 to 20 seconds), then the natural potential of +500 mV was applied for 30 seconds (20 to 50 seconds), and the natural potential was applied for 10 seconds (50 to 60 seconds). [Figure 30] FIG. 30 shows graphs of the electrode potential and the relative luminescence intensity when the culture was continued for three days after the experiment in FIG. 29 and a similar potential was applied. DETAILED DESCRIPTION OF THE INVENTION

[0012] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The following description of preferred embodiments is merely exemplary in nature and is not intended to limit the present invention, its application, or its uses.

[0013] (Pancreatic beta cells) Pancreatic β cells secrete insulin in response to an increase in glucose concentration through a mechanism called glucose-stimulated insulin secretion (GSIS).

[0014] Glucose-responsive insulin secretion will be explained with reference to Figures 1 and 2. Figures 1 and 2 are schematic diagrams showing the mechanism of insulin secretion in pancreatic β cells, with Figure 1 showing the glucose-unstimulated state and Figure 2 showing the glucose-stimulated state.

[0015] As shown in Figure 1, pancreatic beta cells have ATP-sensitive potassium channels and voltage-gated calcium channels. When not stimulated, potassium ions diffuse across the cell membrane from the open ATP-sensitive potassium channels. As a result, the outside of the cell membrane is positive and the inside is negative, maintaining a constant potential difference (cell membrane potential). In this state, the voltage-gated calcium channels are closed.

[0016] Pancreatic beta cells also contain the transmembrane protein GLUT2 transporter. As shown in Figure 2, when the glucose concentration outside the beta cells increases, glucose molecules move into the cells via GLUT2. When glucose is metabolized via glycolysis or the TCA cycle, the ratio of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) increases within the beta cells. In response to this increase in the ATP ratio, ATP-sensitive potassium channels close, preventing potassium ions from diffusing out of the cells. This results in an increase in the cell membrane potential (depolarization). This potential change opens voltage-dependent calcium channels, allowing calcium ions to flow into the cells. As a result, insulin-containing vesicles move to the cell membrane, and insulin is secreted by exocytosis.

[0017] (Cell Devices) The structure of the cell device will be described with reference to Fig. 3. Fig. 3 is a schematic cross-sectional view showing the general structure of the cell device 1.

[0018] The cell device 1 comprises a culture vessel 2, a culture medium 3 and pancreatic beta cells 4 contained in the culture vessel 2, a working electrode 5 to which the pancreatic beta cells 4 adhere, a counter electrode 6 and a reference electrode 7 arranged in contact with the culture medium 3, and a potential application unit 8 that applies a potential to the pancreatic beta cells 4 via the working electrode 5 and the counter electrode 6.

[0019] The culture vessel 2 may have any structure as long as it does not interfere with the culture of pancreatic β cells 4 and insulin secretion. In this embodiment, the culture vessel 2 has a substrate 21 that forms the bottom, a cylindrical vessel body 22 that forms the side, and a lid 23 that forms the top. As shown in FIG. 3 , a working electrode 5 is disposed inside the culture vessel 2, particularly at the bottom of the culture vessel 2, and therefore the culture vessel 2 and working electrode 5 may collectively be referred to as a "culture unit."

[0020] The medium 3 may be any liquid that can maintain the pancreatic β cells 4 in a state in which they can secrete insulin when an electric potential is applied. For example, the medium 3 preferably has a composition suitable for culturing mammalian cells (salts, amino acids, vitamins, sugars, buffers, etc.). Furthermore, the pancreatic β cells 4 may be surrounded by a buffer solution, for example, for operational testing of the cell device 1, short-term storage, or cleaning. Therefore, the medium broadly includes buffer solutions.

[0021] As described above, pancreatic β cells 4 are cells that secrete insulin in response to the cell membrane potential. In the present invention, pancreatic β cells 4 may be derived from humans or from other mammals. Furthermore, there is no limitation on whether or not the pancreatic β cells 4 have been artificially genetically modified. Pancreatic β cells 4 are adhesive cells, and at least a portion of them adhere to the working electrode 5. To adhere the pancreatic β cells 4 to the working electrode 5, a culture medium 3 is added to a culture vessel 2 having a working electrode 5 at its bottom, and the pancreatic β cells 4 are seeded and cultured. Culture conditions that are suitable for the pancreatic β cells 4 include temperature, carbon dioxide concentration, and the type and concentration of antibiotics.

[0022] The working electrode 5 is formed on the substrate 21 and contains poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS). PEDOT:PSS is a conductive polymer that is hydrophilic and biocompatible. Therefore, pancreatic beta cells 4 can be attached and cultured on the working electrode 5. Furthermore, PEDOT:PSS is easy to process and has high transparency to visible light, which has the advantage of facilitating observation, such as checking the state of the cells. In other words, it is preferable that the working electrode 5 be translucent.

[0023] The working electrode 5 may contain components other than PEDOT:PSS, provided that the objective of the present invention, i.e., controlling insulin secretion by applying a potential, is not deviated from. The working electrode 5 preferably contains 30% by weight or more of PEDOT:PSS, and may be composed solely of PEDOT:PSS. Components other than PEDOT:PSS can be selected from materials commonly used for electrodes, provided that the working electrode 5 is biocompatible, preferably translucent. The content of such components can also be determined based on the same considerations. The working electrode 5 may have a multilayer structure containing a PEDOT:PSS-containing layer, and in this case, it is preferable that the top layer (the surface in contact with pancreatic β cells) be a PEDOT:PSS-containing layer from the viewpoint of biosynthesis.

[0024] An example of another component contained in the working electrode 5 is tetraethylorthosilicate (TEOS). TEOS can increase the adhesiveness of the working electrode 5 to the substrate 21. Furthermore, when the working electrode 5 contains TEOS, the cell viability and adhesiveness of the cells to the working electrode 5 are improved. Therefore, when the working electrode 5 has a multilayer structure, it is preferable that a layer containing PEDOT:PSS and TEOS is provided in at least one of the bottom layer in contact with the substrate 21 and the top layer in contact with the pancreatic β cells.

[0025] The working electrode 5 may be formed on the entire bottom surface of the culture vessel 2, or may be formed on only a part of it.

[0026] The working electrode 5 can be formed by dropping a PEDOT:PSS-containing solution onto a substrate 21 such as glass, applying it evenly using a coater, and then heating and drying it. The thickness of the working electrode 5 can be adjusted by repeating the application and heating and drying processes. The PEDOT:PSS-containing solution may also be referred to as a PEDOT:PSS dispersion. The solvent may be either water or an organic solvent, or a mixture of these. Known solvents used in forming electrodes using PEDOT:PSS, such as DMSO and isopropanol, can be used as the organic solvent.

[0027] The PEDOT:PSS-containing solution may contain TEOS. The PEDOT:PSS-containing solution is prepared, for example, by mixing TEOS with a PEDOT:PSS stock solution (aqueous solution), optionally adding and mixing DMSO. In this case, it is preferable that the volume Vp of the PEDOT:PSS stock solution and the volume Vt of the TEOS satisfy the relationship Vt / (Vp+Vt)×100≦10. When the volumes of the PEDOT:PSS stock solution and the TEOS satisfy this relationship, it becomes easy to apply the PEDOT:PSS-containing solution uniformly, that is, to obtain a working electrode 5 with a uniform thickness. Furthermore, it is preferable that the Vt / (Vp+Vt)×100 is 2.5≦Vt / (Vp+Vt)×100. When the TEOS concentration satisfies this range, cell viability and adhesion are improved. It is also preferable to mix TEOS with PEDOT:PSS so that the molar ratio of SiO2 / EDOT is 4.4 / 1 to 19 / 1, which is the same in the completed electrode.

[0028] The counter electrode 6 may be configured so that a potential sufficient to stimulate insulin secretion can be applied to the pancreatic β cells 4 between the counter electrode 6 and the working electrode 5 .

[0029] The reference electrode 7 employs a configuration commonly used for reference electrodes, such as an Ag / AgCl reference electrode. The reference electrode 7 provides a reference potential for application of a potential between the working electrode 5 and the counter electrode 6. In other words, the potential application unit 8 (described below) applies a voltage between the working electrode 5 and the counter electrode 6 so that a predetermined potential is applied to the working electrode 5 based on the potential of the reference electrode 7. In this embodiment, the cell device 1 is a three-electrode device having a working electrode, a counter electrode, and a reference electrode, but may also be a two-electrode system that includes a working electrode and a counter electrode but omits the reference electrode.

[0030] The potential application unit 8 is connected to the working electrode 5, the counter electrode 6, and the reference electrode 7, and can apply a potential to the pancreatic beta cells 4 via the working electrode 5 and the counter electrode 6, with the potential of the reference electrode 7 as a reference. The potential application unit 8 is also connected to an input unit (not shown). The input unit can receive instructions from an operator or output instructions to the potential application unit 8 to turn the potential application on / off and to specify the magnitude of the potential when certain conditions are met. The magnitude of the potential applied to the working electrode 5 changes the cell membrane potential of the pancreatic beta cells 4, thereby inducing or suppressing insulin secretion.

[0031] (Method for controlling insulin secretion using a cellular device) The operation of the cellular device 1, i.e., the method for controlling insulin secretion, will be described with reference to Figures 4 and 5. Figures 4 and 5 are enlarged schematic diagrams of the working electrode 5 and the pancreatic β cells 4 adhered thereto in the cellular device of this embodiment. Figure 4 shows a state in which no exocytosis is occurring and no induced potential, described below, is applied, while Figure 5 shows a state in which an induced potential is applied. Note that Figures 4 and 5 omit illustrations of the pancreatic β cells other than the cell membrane, insulin, insulin granules, and voltage-dependent calcium channels. In the cell device 1, insulin secretion from pancreatic β cells can be induced by applying an induction potential, and insulin secretion from pancreatic β cells can be suppressed by applying an inhibitory potential. The possible mechanism of such control is described below, but the present invention is not limited thereto.

[0032] As shown in Figure 4, when no glucose is added to the medium and no induced potential is applied, the voltage-dependent calcium channels are closed. When an induced potential is applied using the working electrode 5 and the counter electrode 6, as shown in Figure 5, the cell membrane potential increases (depolarization), causing the voltage-dependent calcium channels to open and calcium ions to flow into the cells. As mentioned above, the influx of calcium ions causes insulin secretion. The induced potential is a potential that depolarizes the membrane potential of pancreatic β cells, i.e., a potential at which voltage-dependent calcium channels open. For example, it is preferable to apply a potential exceeding the natural potential of the working electrode 5 (i.e., the potential at which oxidation and reduction reactions are balanced on the working electrode) +150 mV as the induced potential. More specifically, insulin secretion may be induced by applying a natural potential of +180 mV or more, or +200 mV or more. The electrode potential can directly affect the membrane potential; for example, a natural potential of +200 mV can produce a membrane potential change of approximately 40 mV, the same level as typical insulin secretion. The potential may be an AC potential, a DC constant potential, or a potential sweep (LSV: Linear Sweep Voltammetry). For example, in the case of an AC potential, the potential applied to the reference electrode 7 may have a waveform whose maximum value is equal to or greater than the induced potential. Alternatively, a constant potential equal to or greater than the induced potential may be applied. In the case of a sweep, the potential may be swept up to the induced potential. The present invention is not limited to these specific values. The natural potential may be measured before, preferably immediately before, the application of the potential.

[0033] "Induction" of insulin secretion may mean either causing insulin to be secreted from pancreatic beta cells that are not secreting insulin, or increasing the amount of insulin secreted from pancreatic beta cells that are secreting insulin.

[0034] Furthermore, an inhibitory potential can be applied to the pancreatic β cells 4 to perform an inhibitory step of suppressing insulin secretion from the pancreatic β cells 4. The inhibitory potential is a potential that reduces the cell membrane potential to the extent that voltage-dependent calcium channels are closed. In other words, the inhibitory potential repolarizes the cell membrane potential of the pancreatic β cells, thereby closing the voltage-dependent calcium channels and suppressing insulin secretion. The inhibitory potential is lower than the induced potential, for example, at or below the rest potential +150 mV, and more specifically, is near the rest potential or the rest potential. As with the induced potential, the potential may be applied as either an AC potential or a DC constant potential, or may be a potential sweep. "Suppression" of insulin secretion means reducing the amount of insulin secreted compared to when no natural potential is applied (including not secreting insulin). For example, "suppression" includes both reducing the amount of insulin secreted from pancreatic β cells that are already secreting insulin, and reducing insulin secretion below normal levels even when a factor that stimulates insulin secretion from pancreatic β cells acts.

[0035] According to this embodiment, the working electrode 5 contacts the pancreatic β cells 4, allowing for more reliable application of a potential to each cell. As a result, it is believed that it is possible to more quickly and accurately control the on / off of insulin secretion. In the aforementioned Non-Patent Document 1, a porous membrane is placed between platinum electrodes, and an electric pulse stimulus is applied to the cells on the porous membrane. In this document, the maximum amount of insulin secretion was confirmed after 10 minutes of application of an electric pulse stimulus for 40 minutes. In contrast, according to this embodiment, as shown in the examples, insulin secretion can be confirmed immediately after application of the potential, and the secretion amount reaches its maximum after 10 seconds. Thus, according to this embodiment, application of a potential enables rapid insulin secretion. Furthermore, according to this embodiment, the length of insulin secretion time, i.e., the secretion amount, can be changed by switching between application of an induction potential and application of an inhibitory potential.

[0036] As described above, the cell device 1 of this embodiment can perform either the step of inducing or the step of suppressing insulin secretion, or both.

Example

[0037] <A. Insulin secretion induction by potential application> 1. Subculture of iGL cells The cells, medium, etc. used for the culture are as follows. Cells: Rat pancreatic β cell line iGL cells (Cosmo Bio) Medium: 50 mL (45 mL of RPMI1640, 2.5 mL of 5% FBS, 500 μL of 10 μM sodium pyruvate, 500 μL of 500 μM monothioglycerol, 1.5 mL of 6.7 mg / mL G-418 sulfate) Dish: NUNC EASYDISH Dish 100mm (Thermo SCIEMTIFIC) Trypsin-EDTA solution: 0.25 w / v% trypsin - 1 mM EDTA, Fujifilm Wako Pure Chemical Corporation iGL cells are a cell line established from the rat pancreatic β cell line INS-1E as the parental strain to constitutively express a fusion protein of human insulin and secreted Gaussia luciferase (GLase) (Insulin-Glase). iGL cells can simply and sensitively measure insulin secretion by utilizing the luminescence reaction of GLase.

[0038] The subculture was performed as follows. After removing the medium of the cells cultured in the dish, 5 mL of Phosphate Buffered Saline (PBS) was added for washing. 500 μL of trypsin-EDTA solution was added and left standing at 37°C for 5 minutes to detach the cells from the dish. 5 mL of medium was put into the dish to stop the reaction of trypsin, and then the cell clumps were loosened by pipetting. The cell suspension was transferred to a 50 mL centrifuge tube and centrifuged at 300 rpm for 5 minutes at room temperature. After removing the supernatant, 1 mL of medium was added and suspended again. 1.0×10 6 ~~2.0×10 6The cells were seeded onto a new dish and 10 mL of medium was added. The seeded cells were cultured at 37°C in a 5% CO2 atmosphere. Every 3 or 4 days, the medium was removed and washed with PBS, after which 10 mL of new medium was added. The above procedure was repeated on the 7th day after the start of culture for subculture.

[0039] In the above and following procedures, a clean bench was used when adding a medium or a drug to the cells.

[0040] 2. Cell Device Fabrication (Creation of a culture unit for cell devices) (1) Preparation Example 1 2 mL of PEDOT:PSS stock solution (Heraeus, Clevios PH1000) was mixed with 100 μL of dimethyl sulfoxide (DMSO). Approximately 30 μL of the above mixed solution was dropped onto a 22 mm square glass slide (Matsunami Glass), and then coated onto a flat surface using a non-wire bar coater Select-Roller (52 μm / wet). After coating, the mixture was heated and dried on a hot plate at 120°C for 5 minutes. The coating and heating and drying process was repeated five times to form a working electrode. In this way, a culture unit consisting of a culture vessel and a working electrode was produced.

[0041] Next, an acrylic ring with an inner diameter of 18 mm, a height of 7 mm, and a thickness of 2 mm was attached to the PEDOT:PSS-coated surface of the glass slide using Bond Ultra Multipurpose S·U.

[0042] Conductive wires were attached to the PEDOT:PSS-coated surface outside the acrylic ring using CONDUCTIVE EPOXY (Chemtronics, CW2400).

[0043] The culture vessel thus prepared was sterilized by UV irradiation for approximately 17 hours.

[0044] (2) Preparation Examples 2 to 4 A PEDOT:PSS stock solution (Heraeus, Clevios PH1000) was filtered using a syringe filter (Advantech, DISMIC, 25CS045AN, pore size 45 μm). The filtered material was used as the PEDOT:PSS stock solution in the following procedure.

[0045] Mixture solutions A to C were prepared by stirring the following materials overnight. Mixture solution A (PEDOT:PSS concentration 48 wt%): 950 μL of PEDOT:PSS stock solution, 50 μL of TEOS, and 50 μL of dimethyl sulfoxide (DMSO). Mixture solution B (PEDOT:PSS concentration 37 wt%): 925 μL of PEDOT:PSS stock solution, 75 μL of TEOS, and 50 μL of DMSO; Mixture C (PEDOT:PSS concentration 30 wt%): 900 μL of PEDOT:PSS stock solution, 100 μL of TEOS, and 50 μL of DMSO Approximately 30 μL of each of mixed solutions A, B, and C was dropped onto separate 22 mm square glass slides (Matsunami Glass Co., Ltd.), and then coated onto a flat surface using a non-wire bar coater Select-Roller (52 μm / wet). After coating, the mixture was heated and dried on a hot plate at 100°C for 30 minutes. This coating and heating and drying process was repeated five times.

[0046] Thereafter, an acrylic ring and a lead wire were attached to prepare a culture vessel in the same manner as in Preparation Example 1. The obtained culture vessel was sterilized by UV irradiation for approximately 17 hours. Culture vessels prepared using mixed solution A, mixed solution B, and mixed solution C were used for culturing and testing iGL cells as Preparation Examples 2 to 4, respectively.

[0047] (Cultivation of iGL cells in a culture vessel for cell devices) The above-mentioned culture vessel was placed in a NUNC Dish 35 mm with a lid. 5.0 × 10 subcultured iGL cells were added. 5 The cells were seeded in a culture vessel (inside an acrylic ring), 900 μL of medium was added, and the cells were cultured at 37°C in a 5% CO2 atmosphere for 4 days.

[0048] 3. Potential application (1) Test on Preparation Example 1 (Luminescence observation of live cell response to applied electric potential) The following instruments and devices were used to observe the induction of insulin secretion by applying a voltage.

[0049] Microscope: Inverted research microscope IX71 (OLYMPUS), 20x objective lens Camera: An EM-CCD camera (ImagEM C9100-13, Hamamatsu Photonics) was connected to the microscope. During observation, the camera was set to the highest sensitivity and the exposure time was set to 500 msec.

[0050] Camera control and image analysis software: HCImage (Hamamatsu Photonics) Potentiostat: Electrochemical measurement system HZ-7000 (Hokuto Denko) Electrodes: A three-electrode system was used, using the above-mentioned PEDOT:PSS electrode as the working electrode, a platinum wire rolled into a circle with a diameter of 4 mm as the counter electrode, and Ag / AgCl as the reference electrode.

[0051] For luminescence observation, the culture vessel was placed on the observation stage of the microscope, and the tips of the salt bridge and counter electrode connected to the reference electrode were immersed in Krebs-Ringer Bicarbonate (KRB)-Buffer (Sigma-Aldrich) inside the culture vessel, as shown in Figure 6. Furthermore, the various electrodes (working electrode, reference electrode, and counter electrode) were connected to a potentiostat. The microscope was placed inside the darkroom, and the potentiostat was placed outside the darkroom.

[0052] (Experiment 1: Application of a sinusoidal potential) After culturing iGL cells for 4 days, the medium was removed from the culture vessel and washed twice with 1 mL of KRB-Buffer (Sigma-Aldrich). 500 μL of KRB-Buffer containing 10 μg / mL Coelenterazine (a type of luciferin, a luminescent substrate; manufactured by Cosmo Bio) was added to the washed iGL cells.

[0053] Immediately, the culture vessel and electrodes were arranged and connected as shown in Figure 6, the darkroom was closed to ensure darkness, and then photography and potential application were performed. Specifically, the natural potential was measured for 1 second, and that natural potential was applied for 1 second. Subsequently, a sinusoidal potential of +0 mV to +500 mV, 10 Hz, was applied to the reference electrode for 60 seconds.

[0054] (Experiment 2: Addition of voltage-sensitive calcium channel inhibitors) Before adding the luminescent substrate, nicardipine hydrochloride (Fujifilm Wako) was added to inhibit the function of voltage-sensitive calcium channels during voltage application experiments. iGL cells were cultured in 500 μL of KRB-Buffer containing 0.5 μL of 2 mM nicardipine hydrochloride at 37°C in a 5% CO2 atmosphere for 30 minutes. The nicardipine-containing KRB-Buffer was then removed, and the cells were washed twice with 1 mL of fresh nicardipine-free KRB-Buffer. Subsequently, a voltage was applied and observations were performed using the same procedure as in Experiment 1.

[0055] (Experiment 3: Application of constant potential) Instead of the potential application conditions in Experiment 1, the natural potential was measured for 1 second, and after applying that natural potential for 1 second, a constant potential of +500 mV relative to the natural potential was applied for 10 seconds. Except for this, potential application and photography were carried out in the same manner as in Experiment 1.

[0056] (Experiment 4: Measurement of extracellular (buffered) insulin levels) As described above, iGL cells were cultured for 4 days in a culture vessel for cell devices.

[0057] Immediately before the start of the experiment, the cells were washed twice with 1 mL of KRB-Buffer, and 700 μL of KRB-Buffer was added. Immediately thereafter, the culture vessel and electrodes were arranged and connected as shown in Figure 6.

[0058] Immediately before applying the potential, 200 μL of KRB-Buffer was sampled. Then, a sinusoidal potential was applied as in Experiment 1. Immediately after the potential application was completed, 200 μL of KRB-Buffer was sampled. The sampled KRB-Buffer was stored at 4°C until immediately before luminescence measurement.

[0059] Luminescence measurements were performed as follows: 50 μL of the collected culture buffer solution was placed in a 96-well plate (flat-bottom, black). Next, 50 μL of KRB-Buffer containing 10 μg / mL Coelenterazine was added. After addition, the mixture was mixed by pipetting for 5 seconds, and luminescence measurements were immediately performed using a Spectra MAX M5 multimode plate reader (Molecular Devices) (measurement wavelength: 488 nm, integration: 500 msec, interval: 20 sec). The amount of insulin luminescence was evaluated using the integrated luminescence values ​​measured immediately after the start of measurement through 1 minute after the start of measurement. For each culture device, the amount of insulin luminescence after potential application was evaluated relative to the amount of insulin luminescence in the culture buffer collected immediately before potential application, which was set to 1.

[0060] (2) Test on Preparation Examples 2 to 4 (Experiment 5) Using the same equipment and facilities as those used in Preparation Example 1, the following tests were carried out on the cell devices having cell containers of Preparation Examples 2 to 4.

[0061] The natural potential was measured for 1 second, and the natural potential was applied for 1 second, after which a constant potential of +500 mV was applied for 10 seconds, and the natural potential was further applied for 50 seconds.

[0062] Bright field images and luminescence photographs before application of the potential and after application of the natural potential for 50 seconds are shown in FIGS.

[0063] 4.Results (1) Experiments 1-4 The results of Experiment 1 will be explained with reference to Figures 7 and 8. Figure 7(a) is an image of the luminescence of a cell before application of a potential, and Figure 7(b) is a diagram that schematically illustrates the state of the cell at that time. Figure 8(a) is an image of the luminescence of a cell during application of a potential, and Figure 8(b) is a diagram that schematically illustrates the state of the cell at that time. The application of the sine wave potential began when the counter (minutes:seconds) displayed in the upper right corner of Figures 7(a) and 8(a) reached "00:30.00" and ended when it reached "01:30.00." In other words, Figure 8(a) is an image taken approximately 31 seconds after the start of application of the sine wave potential.

[0064] As shown in Figure 7(a), before application of the voltage, the cells were observed to be luminescent. As mentioned above, a fusion protein of insulin and luciferase is expressed in iGL cells. The added luminescent substrate is taken up into the cells by concentration diffusion. As a result, it is thought that the cells emitted light due to a reaction between the luciferase in the fusion protein and the luminescent substrate inside the cells (Figure 7(b)).

[0065] On the other hand, as shown in Figure 8(a), the cells became dark while the potential was being applied. This is thought to be because the application of the potential increased the cell membrane potential, opening voltage-dependent calcium channels, which resulted in the release of the fusion protein outside the cells, preventing the fusion protein from reacting with the luminescent substrate that had entered the cells (Figure 8(b)). There was no change in the bright-field image before and after the application of the potential. Therefore, the difference between Figures 7(a) and 8(a) is thought to be due to the secretion of the fusion protein by the action of voltage-dependent calcium channels, rather than due to the outflow of the fusion protein due to cell rupture.

[0066] The results of Experiment 2 are explained with reference to Figures 9 and 10. Figure 9 shows images of the cell luminescence before and during potential application, respectively. Similar to Figures 7(a) and 8(a), the application of the sinusoidal potential began when the counter (minutes:seconds) displayed in the upper right corner reached "00:30.00" and ended when it reached "01:30.00." In other words, Figure 10, like Figure 8(a), shows an image taken approximately 31 seconds after the application of the sinusoidal potential. As can be seen from these images, the application of nicardipine hydrochloride did not alter the luminescence intensity of the cells. This is likely because nicardipine, a voltage-gated calcium channel inhibitor, prevented the voltage-gated calcium channels from opening even when the cell membrane potential changed, preventing calcium ions from entering the cells and the secretion of the fusion protein.

[0067] Figure 11 shows the results of Experiment 1, and Figure 12 shows the results of Experiment 2. The horizontal axis represents the time (zero seconds), which is 30 seconds before the start of the sinusoidal potential application, and the vertical axis represents the relative luminescence intensity, with the luminescence intensity at zero seconds being 1. As shown in Figure 11, without nicardipine, the luminescence intensity began to decrease immediately after the potential application, and a clear decrease was observed after 4 seconds. Furthermore, it remained almost constant after 10 seconds. This indicates that the fusion protein was secreted rapidly upon potential application. On the other hand, as shown in Figure 12, the relative luminescence intensity did not decrease in the presence of nicardipine, indicating that the change in relative luminescence intensity in Figure 11 was due to the opening of voltage-dependent calcium channels.

[0068] The results of Experiment 3 will be explained with reference to Figure 13. Figure 13 is a graph in which the horizontal axis represents time, with 20 seconds before the application of the constant potential being set to zero seconds, and the vertical axis represents the relative luminescence intensity, with the luminescence intensity at zero seconds being set to 1. As shown in Figure 13, even with the application of the constant potential, the luminescence intensity began to decrease immediately after application, and a clear decrease was observed after about 4 seconds. After 10 seconds, it further decreased to 0.6. Thus, the fusion protein was rapidly secreted even with the application of the constant potential.

[0069] Regarding the results of Experiment 4, an explanation will be given with reference to FIG. 14. FIG. 14 is a graph showing the amount of luminescence using the buffer solution before applying the potential as 1 and the relative luminescence intensity as the amount of luminescence using the buffer solution after applying the potential. As shown in FIG. 14, the relative luminescence intensity of the buffer solution after applying the potential has increased, indicating that the fusion protein has been secreted extracellularly.

[0070] As described above, it was found that cell culture and evaluation observation of insulin secretion induction are possible with a cell device having PEDOT:PSS prepared only by coating on glass and heat drying as a working electrode. And it was shown that insulin secretion induction can be promptly performed by applying a potential by directly culturing on the electrode.

[0071] (2) Experiment 5 As shown in FIGS. 15 to 17, in the cell devices of Production Examples 2 to 4 as well, insulin secretion by applying a potential was observed through cell luminescence, similar to Production Example 1. Since there was no change in the bright-field image before and after applying the potential, it is considered that the change in the amount of cell luminescence is due to the secretion of the fusion protein by the action of the voltage-dependent calcium channel, rather than the outflow of the fusion protein due to cell rupture.

[0072] Thus, it was possible to fabricate a cell device having an electrode containing PEDOT:PSS and TEOS only by coating the raw material solution on glass and heat drying. It was shown that cell culture and evaluation observation of insulin secretion induction are possible with this cell device, and insulin secretion induction can be promptly performed by applying a potential by directly culturing on the electrode.

[0073] <B. TEOS Concentration in Working Electrode Formation (Production Examples 2 to 6)> 1. Fabrication of Cell Device Culture vessels for Preparation Examples 5 and 6 were further prepared in the same manner as Preparation Example 2, except that the amounts of TEOS and PEDOT:PSS stock solution in the mixed solution were changed as shown in Table 1. The "TEOS concentration (v / v%)" is shown in Table 1, where Vp is the volume of the PEDOT:PSS stock solution and Vt is the volume of TEOS, Vt / (Vp+Vt) × 100, along with the molar ratio of SiO2 / EDOT. As shown in Table 1 below, in Preparation Examples 2 to 6, the TEOS concentration in the mixed solution ranged from 2.5 to 12.5 (v / v%).

[0074] [Table 1]

[0075] In these culture vessels, iGL cells were cultured in the same manner as in Preparation Example 2 to obtain cell devices.

[0076] 2. Potential application The cell devices of Preparation Examples 2 to 5 (TEOS concentration 2.5 to 10% v / v) were subjected to spontaneous potential measurement for 10 seconds, and the spontaneous potential was applied for 10 seconds. After that, a constant potential of spontaneous potential +500 mV was applied for 20 seconds, and the spontaneous potential was then applied for another 10 seconds. Observation and measurement of luminescence intensity were started from the start of spontaneous potential application.

[0077] Figures 19 to 22 show luminescence photographs taken 17.3 seconds (restoring potential), 39.0 seconds (restoring potential +500 mV), and 49.8 seconds (restoring potential) after the start of observation, as well as graphs of the relative luminescence intensity, with the luminescence intensity at the start of observation set to 1.

[0078] 3.Results Figure 18 shows the TEOS concentration in the mixed solution during the formation of the working electrode (upper), a photograph of the dried slide glass after applying the mixed solution five times (middle), and a bright-field image after cell culture (lower). As shown in the middle photograph, the mixed solution with a TEOS concentration of 2.5 to 10 (v / v%) could be uniformly coated. On the other hand, when the TEOS concentration increased up to 12.5 (v / v%), uneven coating occurred. Thus, it was found that in order to obtain a uniform working electrode, the TEOS concentration is preferably 10 (v / v%) or less.

[0079] Furthermore, when cells were cultured on these devices (lower part of Figure 18), within the range of 10 (v / v%) or less that could be uniformly coated, the higher the TEOS concentration, the higher the cell viability and adhesion rate.

[0080] Also, as shown in Figures 19 to 22, in all cell devices of Preparation Examples 2 to 5 (TEOS concentration 2.5 to 10 v / v%), disappearance of luminescence due to the application of a natural potential of +500 mV was observed. That is, insulin secretion due to potential application was observed in all cell devices.

[0081] <C. Relationship between potential height and insulin secretion> Using the cell device of Preparation Example 3, after measuring the natural potential for 10 seconds, a constant potential was applied with the natural potential for 10 seconds, the natural potential +150 or natural potential +200 mV for 60 seconds, and the natural potential for 10 seconds. Observation and measurement of the luminescence intensity were started from the start of applying the natural potential.

[0082] Figures 23 to 24 show the luminescence photographs, the graph of the electrode potential, and the graph of the relative luminescence intensity with the luminescence intensity at the start of observation set to 1, respectively, 19.5 seconds after the start of observation (natural potential), 78.3 seconds after (natural potential +150 or 200 mV), and 86.7 seconds after (natural potential).

[0083] As shown in Figure 23, disappearance of luminescence was not observed with the application of a potential of natural potential +150 mV, suggesting that insulin secretion did not occur.

[0084] On the other hand, as shown in Fig. 24, when a potential of +200 mV of natural potential was applied, the emission disappeared, indicating that insulin secretion had occurred. From this, it became clear that the applied potential required for insulin secretion is between 150 and 200 mV.

[0085] <Insulin Secretion by D.LSV> Using the cell device of Production Example 3, after measuring the natural potential for 10 seconds (0 to 10 seconds), the natural potential was applied for 10 seconds (10 to 20 seconds), and then the potential was increased to +500 mV of the natural potential at 10 mV / sec for 50 seconds by LSV (20 to 70 seconds). Thereafter, electrode potential measurement was performed for 20 seconds (70 to 90 seconds).

[0086] Fig. 25 shows the luminescence photographs, the graph of the electrode potential, and the graph of the relative luminescence intensity with the luminescence intensity at the start of observation set to 1, respectively, 19.5 seconds after the start of observation (natural potential), 36.8 seconds after (natural potential +150 mV), 41.2 seconds after (natural potential + about 200 mV), and 70.8 seconds after (natural potential + about 500 mV).

[0087] As shown in Fig. 25, even when the potential was continuously increased, the relative luminescence intensity did not decrease at natural potential +150 mV, and started to decrease when it reached natural potential +200 mV. The applied potential required for insulin secretion was between natural potential +150 and natural potential +200 mV.

[0088] <E. Influence of Applied Potential on Cell Membrane Potential> Using the Fluovolt membrane potential dye (Thermo Fisher Scientific), the influence of the applied potential on the cell membrane potential was confirmed. The Fluovolt membrane potential dye shows a rapid fluorescence change in response to a change in the membrane potential.

[0089] In the cell device of Production Example 3, while observing the change in membrane potential using the Fluovolt membrane potential dye, the applied potential was increased by 100 mV each time. That is, (1) 0 - 10 seconds: natural potential measurement, 10 - 20 seconds: natural potential application, (2) 20 - 40 seconds: natural potential + 100 mV application, (3) 40 - 60 seconds: natural potential + 200 mV application, (4) 60 - 80 seconds: natural potential + 300 mV application, (5) 80 - 100 seconds: natural potential + 400 mV application, (6) 100 - 120 seconds: natural potential + 500 mV application, (7) 120 - 140 seconds: natural potential application.

[0090] Figure 26 shows a bright-field image of cultured cells, fluorescence photographs of (1) - (7) above, a graph of electrode potential, and a graph of relative fluorescence intensity with the fluorescence intensity at the start of measurement taken as 1.

[0091] As shown in the graph of Figure 26, the relative fluorescence intensity increased as the electrode potential increased. Specifically, when a potential of natural potential + 200 mV was applied, the fluorescence intensity increased by about 10%. Since the Fluovolt membrane potential dye shows a fluorescence change of about 25% per 100 mV change in membrane potential, the change in membrane potential due to the application of natural potential + 200 mV is推测 to be about +40 mV (rising from -70 mV to -30 mV).

[0092] <F. Inhibition of insulin secretion by application of natural potential> Using the same experimental system as in <C.> above, as shown in Figure 27, the natural potential was measured for 10 seconds (0 - 10 seconds), the natural potential was applied for 10 seconds (10 - 20 seconds), then a natural potential + 500 mV was applied for 2 seconds (20 - 22 seconds), and finally the circuit was disconnected (i.e., the potential application was stopped) (22 seconds -). As a result, the relative luminescence intensity continued to decrease even after the potential application was stopped. That is, it was found that insulin secretion continues without stopping even after the potential application is stopped.

[0093] As shown in Figure 28, the rest potential was measured for 10 seconds (0–10 seconds), the rest potential was applied for 10 seconds (10–20 seconds), and then a rest potential of +500 mV was applied for 2 seconds, followed by a 30-second rest potential application, alternating three times (20–116 seconds). The rest potential of +500 mV was then applied continuously (from 116 seconds onward). The relative luminescence intensity dropped sharply after the first application of the rest potential of +500 mV, but this drop was suppressed by subsequent application of the rest potential. The second application of the rest potential of +500 mV also decreased the relative luminescence intensity, although the change was smaller than in the first application. The subsequent application of the rest potential also suppressed this drop. The third application of the rest potential of +500 mV also decreased the relative luminescence intensity, although the change was even smaller than in the second application. The subsequent application of the rest potential also suppressed this drop. Finally, the relative luminescence intensity gradually decreased with continuous application of a resting potential of +500 mV. Thus, insulin secretion was suppressed by applying a resting potential to the insulin-secreting pancreatic beta cells.

[0094] Furthermore, as shown in Figures 29 and 30, after the induction of insulin secretion, it was possible to induce insulin secretion again after continuing the culture. First, as shown in Figure 29, the natural potential was measured for 10 seconds (0 to 10 seconds), the natural potential was applied for 10 seconds (10 to 20 seconds), then the natural potential of +500 mV was applied for 30 seconds (20 to 50 seconds), and the natural potential was applied for 10 seconds (50 to 60 seconds).The relative luminescence intensity decreased during the application of the natural potential of +500 mV.

[0095] After the experiment in Figure 29, the culture was continued for three days, and the luminescence intensity was measured again under the same conditions. That is, the rest potential was measured for 10 seconds (0-10 seconds), the rest potential was applied for 10 seconds (10-20 seconds), a rest potential of +500 mV was applied for 30 seconds (20-50 seconds), and the rest potential was applied for 10 seconds (50-60 seconds). As a result, as shown in Figure 30, the relative luminescence intensity decreased with the application of a rest potential of +500 mV. As described above, after insulin was released by the application of a potential, insulin was resynthesized by the culture, and further application of a potential was able to induce insulin secretion.

[0096] As shown in the various experiments above, insulin secretion was induced by application of an induction potential exceeding the resting potential +150 mV, and insulin secretion continued even after application of the induction potential was stopped, but insulin secretion could be suppressed by application of an inhibitory potential lower than the induction potential. These results suggest that application of an induction potential opens voltage-sensitive calcium channels, and the resulting influx of calcium into the cells triggers insulin secretion, and while the channels remain open under this condition, application of an inhibitory potential closes the channels, stopping calcium influx into the cells and ultimately suppressing insulin secretion. [Industrial Applicability]

[0097] The cell device and method for inducing insulin secretion of the present invention may be applicable to medical devices.

Claims

1. A culture vessel; a culture medium and pancreatic β cells contained in the culture vessel; a working electrode to which the pancreatic beta cells adhere and which contains PEDOT:PSS; A counter electrode provided so as to be in contact with the culture medium; a potential application unit that applies a potential to the pancreatic β cells by the working electrode and the counter electrode; A cellular device comprising:

2. Further comprising a reference electrode provided in contact with the culture medium. The cell device of claim 1 .

3. The working electrode further contains tetraethyl orthosilicate. The cell device of claim 1 .

4. The cell device according to any one of claims 1 to 3, further comprising an inducing step of inducing insulin secretion from the pancreatic β cells by applying a potential to the pancreatic β cells so as to open voltage-dependent calcium channels in the pancreatic β cells. A method for controlling insulin secretion.

5. The cell device according to any one of claims 1 to 3, further comprising a suppression step of suppressing insulin secretion from the pancreatic β cells by applying a potential to the pancreatic β cells so as to close voltage-dependent calcium channels in the pancreatic β cells. A method for controlling insulin secretion.

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