Electrical stimulation device for cell culture

Abstract

The invention relates to an electrical stimulation device for cell culture, the electrical stimulation device comprises at least one electrical stimulation chamber, the electrical stimulation chamber comprises an upper cover, a conductive cell culture platform and a plate-shaped structure with holes; a lead and an electrode pair consisting of a positive electrode and a negative electrode are fixed on the upper cover, and the lead is communicated with the electrode pair; a conductive clamp is fixed on the conductive cell culture platform, and the cell culture platform is communicated with the electrode pair through the conductive clamp; the perforated plate-shaped structure is used for containing a culture medium and accommodating the cell culture platform; and the upper cover and the hole structure form a closed environment required by cell culture. According to the electrical stimulation device, the cell platform serves as a conducting medium, direct use of a culture medium as the conducting medium is avoided, chemical reaction of current and the culture medium is relieved, release of harmful substances is reduced, growth of cells is facilitated, and the effect of the current is improved.

Description

Technical Field

[0001] The present invention belongs to the field of cell engineering, and particularly relates to an electric stimulation device for cell culture. Background Art

[0002] In the field of cell engineering, electric stimulation is an effective way to regulate cell behavior. For example, electric stimulation can promote the regeneration of stem cells (such as tendon stem cells).

[0003] However, the current electric stimulation devices have certain deficiencies, mainly including two aspects. On the one hand, most electric stimulation devices directly use cell culture medium as the conductive medium, and the current may chemically react with the culture medium to generate substances harmful to cells. On the other hand, most electric stimulation devices have complex structures, which are not conducive to observing the growth state of cells and will also affect subsequent operations such as staining and collecting cells.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The present invention is to solve the problems that the existing electric stimulation devices are prone to chemical reactions with the culture medium and / or are not convenient for observing and collecting cells during the process of electrically stimulating cells. To solve the above technical problems, the present invention provides the following technical solutions:

[0006] In a first aspect, the present invention provides an electric stimulation device, which includes at least one electric stimulation chamber. The electric stimulation chamber includes an upper cover, a conductive cell culture platform, and a perforated plate-like structure;

[0007] The upper cover is fixed with a wire and an electrode pair composed of a positive electrode and a negative electrode, and the wire is connected to the electrode pair;

[0008] The conductive cell culture platform is fixed with conductive clips, and the cell culture platform is connected to the electrode pair through the conductive clips;

[0009] The perforated plate-like structure is used for containing the culture medium and accommodating the cell culture platform;

[0010] The upper cover and the perforated plate-like structure form a closed environment required for cell culture.

[0011] In some embodiments, the electric stimulation device includes a plurality of the electric stimulation chambers. In some specific embodiments, the circuit between the plurality of electric stimulation chambers is a parallel circuit.

[0012] In some embodiments, the wire is fixed to the upper cover by welding, and the welding method is preferably soldering.

[0013] In some embodiments, the electrode pair is fixed by punching holes in the upper cover and bonding, preferably, resin glue is used for bonding and fixing.

[0014] In some embodiments, the material of the electrode pair is a stainless steel electrode.

[0015] In some embodiments, the distance between the positive electrode and the negative electrode of the electrode pair is 20 - 30 mm, preferably 25 mm.

[0016] In some embodiments, the diameter of the positive electrode is 1 - 3 mm, preferably 2 mm.

[0017] In some embodiments, the diameter of the negative electrode is 1 - 3 mm, preferably 2 mm.

[0018] In some embodiments, the positive electrode and / or the negative electrode is in an "L" shape.

[0019] In some specific embodiments, the total length of the "L" - shaped electrode is 25 - 35 mm, preferably 30 mm, wherein the length of the vertical end perpendicular to the upper cover is 5 - 15 mm, preferably 10 mm, and the length of the parallel end parallel to the upper cover is 15 - 25 mm, preferably 20 mm.

[0020] In some specific embodiments, the vertical end of the "L" - shaped electrode includes an exposed end above the upper cover, and the exposed end is used as the connection point of the wire. Optionally, the length of the exposed end is 3 - 7 mm, preferably 5 mm.

[0021] In some specific embodiments, the distance between the parallel end of the "L" - shaped electrode and the bottom of the hole structure is 4 - 6 mm, preferably 5 mm.

[0022] In some embodiments, the clamping thickness of the conductive clip is 0.8 - 1.3 mm, preferably 1.1 mm.

[0023] In some embodiments, the upper end of the conductive clip is fixed to the electrode pair (for example, fixed to the parallel end of the "L" - shaped electrode), and the lower end of the conductive clip is fixed to the cell culture platform.

[0024] In some embodiments, the conductive cell culture platform is conductive glass, preferably ITO conductive glass.

[0025] In some embodiments, the side length of the cell culture platform is 20 - 30 mm, preferably 25 mm; the sheet resistance is 7 - 12 Ω, preferably 10 Ω; the thickness is 0.8 - 1.3 mm, preferably 1.1 mm.

[0026] In some embodiments, the pore size of the pore structure is 30 - 40 mm, preferably 35 mm.

[0027] In some embodiments, the pore structure and the upper cover are modified from a cell culture well plate. Optionally, the cell culture well plate is a 6-well plate, a 12-well plate or a 24-well plate.

[0028] In some embodiments, the electrical stimulation device further includes a power supply, preferably a constant voltage DC power supply.

[0029] In some embodiments, the constant voltage DC power supply is connected to the electrical stimulation chamber.

[0030] In some embodiments, the constant voltage DC power supply provides a minimum voltage output of 5 - 15 mV; preferably provides a minimum voltage output of 10 mV.

[0031] In some embodiments, the trunk current of the electrical stimulation device is 15 - 25 μA, preferably 20 μA.

[0032] In some embodiments, the electrical stimulation device is used for cell culture, for example, the culture of stem cells, preferably for the culture of tendon stem cells.

[0033] In a second aspect, a method for culturing cells using the electrical stimulation device of the present invention is provided. The method includes:

[0034] Cells are inoculated on the conductive cell culture platform;

[0035] A culture medium is added to the perforated plate-like structure, and the liquid level of the culture medium is higher than the cell culture platform but lower than the electrode pair; and,

[0036] When needed, the power supply is connected to generate a stimulating current.

[0037] In some embodiments, the cells are stem cells, preferably tendon stem cells.

[0038] In some embodiments, the cell culture environment is 37 °C and 5% CO2 concentration.

[0039] In some embodiments, the culture medium is replaced every 2 - 3 days.

[0040] In some embodiments, the culture medium is a tendon induction medium.

[0041] In a third aspect, an application of the electrical stimulation device of the present invention in promoting cell proliferation, migration and / or differentiation ability is further provided.

[0042] In some embodiments, the cells are stem cells, preferably tendon stem cells, and the differentiation is preferably the differentiation of tendon stem cells into tendon cells.

[0043] In some embodiments, the culture medium used in the application is a tendon induction medium.

[0044] Beneficial effects

[0045] The present invention includes at least one or more of the following beneficial effects:

[0046] (1) The electrostimulation device of the present invention forms a circuit loop using electrode pairs, conductive clips, and a conductive cell culture platform. Taking the cell culture platform as a conductive medium, electrostimulation is performed on the cells inoculated on the platform, thereby avoiding directly using the culture medium as a conductive medium, reducing the chemical reaction between the current and the culture medium, reducing the release of harmful substances, being beneficial to cell growth, and improving the effect of current action.

[0047] (2) The material of the conductive cell culture platform of the electrostimulation device of the present invention is preferably conductive glass, especially ITO conductive glass. The ITO conductive glass sheet has good light transmittance, which is beneficial to observing the cell growth state and facilitating operations such as cell staining and collection.

[0048] (3) The electrostimulation device of the present invention can be prepared on the basis of a cell culture well plate, and the raw materials are cheap and easily available.

[0049] (4) The electrostimulation device and / or method of the present invention is suitable for the culture of stem cells, preferably the culture of tendon stem cells. Description of the drawings

[0050] Figure 1 is the upper cover of the electrostimulation device, and the "L"-shaped electrode pair is fixed to the inner side of the upper cover;

[0051] Figure 2 is the upper cover connected with a conductive cell culture platform. The upper cover is a cell culture cover, and the conductive cell culture platform is conductive glass;

[0052] Figure 3 is Figure 2 the side view of the upper cover shown and each component;

[0053] Figure 4 is the circuit diagram of the electrostimulation device after being connected to the power supply, and the electrostimulation chambers are in parallel circuits;

[0054] Figure 5 is the SEM image of the conductive side of the conductive glass at different magnification;

[0055] Figure 6Current-voltage relationship diagrams of the conductive glass in the dry state (left) and after being immersed in DMEM medium for 14 days (right);

[0056] Figure 7 Current-voltage relationship diagram of the electrode in the dry state. Detailed implementation mode

[0057] To better understand the present invention, specific embodiments will be given to further illustrate the present invention. However, it should be understood that the described embodiments are exemplary embodiments, and the present invention can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to be able to more thoroughly understand the present invention and to be able to fully convey the scope of the present invention to those skilled in the art.

[0058] As described above, the object of the present invention is to provide a novel electrical stimulation device that avoids direct chemical reactions between current and the culture medium. The device uses a conductive cell culture platform and conductive clips fixed to the platform to communicate with the electrode pair to form a circuit, thereby electrically stimulating the cells inoculated on the platform.

[0059] In a preferred technical solution of the present invention, the electrical stimulation device includes at least one electrical stimulation chamber, and the electrical stimulation chamber includes an upper cover, a conductive cell culture platform, and a perforated plate-like structure; a wire and an electrode pair composed of a positive electrode and a negative electrode are fixed on the upper cover, and the wire is connected to the electrode pair; a conductive clip is fixed on the conductive cell culture platform, and the cell culture platform is connected to the electrode pair through the conductive clip; the perforated plate-like structure is used to hold the culture medium and accommodate the cell culture platform; the upper cover and the perforated plate-like structure form a closed environment required for cell culture.

[0060] Upper cover

[0061] The upper cover is arranged above the perforated plate-like structure, and it cooperates with the perforated plate-like structure to provide a closed environment for cell growth. A wire and an electrode pair are fixed on the upper cover. In some embodiments, the wire can be fixed above the upper cover by welding (such as soldering). The electrode pair is arranged below the upper cover. In some embodiments, the electrode pair is fixed by using an adhesive (such as resin glue) after drilling holes in the upper cover. The wire is connected to the positive electrode and the negative electrode of the electrode pair. The wire can also be connected to a power source (such as a regulated DC power source) or other circuit units.

[0062] In some embodiments, the distance between the positive electrode and the negative electrode of the electrode pair is 20 - 30 mm, preferably 25 mm. The diameters of the positive and negative electrodes are 1 - 3 mm, preferably 2 mm.

[0063] The shapes of the positive and negative electrodes can be "L"-shaped, which include a parallel end parallel to the upper cover and a vertical end perpendicular to the upper cover. More specifically, the total length of the "L"-shaped electrode is 25 - 35 mm, preferably 30 mm. Among them, the length of the vertical end is 5 - 15 mm, preferably 10 mm, and the length of the parallel end is 15 - 25 mm, preferably 20 mm. More specifically, the vertical end further includes an exposed end exposed from the upper cover, which can be used as a connection point for a wire, and the length of the exposed end is 3 - 7 mm, preferably 5 mm. In some embodiments, the material of the electrode pair is stainless steel.

[0064] Conductive cell culture platform

[0065] A conductive cell culture platform is used to support the growth of cells and the transmission of current. The cell culture platform is fixed with conductive clips, and the conductive clips can be connected to the electrode pair, so as to realize the connection between the electrode pair - conductive clip - conductive cell culture platform. The current uses the conductive cell culture platform as a conductive medium, reducing the chemical reaction between the current and the culture medium, which is beneficial to cell growth. At the same time, the conductive cell culture platform is used for inoculating cells to ensure stable contact between the current and the cells, and can improve the effect of the electrical stimulation.

[0066] In some cases, the upper end of the conductive clip is fixed to the electrode pair (for example, the parallel end of the "L"-shaped electrode), and the lower end of the conductive clip is fixed to the cell culture platform.

[0067] In some cases, the conductive cell culture platform is conductive glass, preferably ITO conductive glass. Conductive glass (such as ITO conductive glass) has good light transmittance, which is beneficial to observing the cell growth state and facilitating operations such as cell staining and collection.

[0068] In some cases, the side length of the cell culture platform is 20 - 30 mm, preferably 25 mm; the sheet resistance is 7 - 12 Ω, preferably 10 Ω; the thickness is 0.8 - 1.3 mm, preferably 1.1 mm.

[0069] In some cases, the clamping thickness of the conductive clip is 0.8 - 1.3 mm, preferably 1.1 mm.

[0070] In some cases, the height of the conductive clip can be adjusted by the height of the plate-like structure with holes, so that the cell culture platform fits the bottom of the conductive cell culture platform to facilitate cell growth.

[0071] Plate-like structure with holes

[0072] The perforated plate-like structure is used to hold the culture medium and accommodate the cell culture platform. In some embodiments, the pore structure and the upper cover are modified from a cell culture well plate. Optionally, the cell culture well plate is a 6-well plate, a 12-well plate or a 24-well plate. In some embodiments, the pore diameter of the pore structure is 30-40 mm, preferably 35 mm.

[0073] Power supply

[0074] In some solutions, the electrical stimulation device further includes a power supply, or is connected to an external power supply through a wire. The power supply is preferably a constant voltage DC power supply. In some embodiments, the constant voltage DC power supply provides a minimum voltage output of 5-15 mV; preferably provides a minimum voltage output of 10 mV. In some embodiments, the main circuit current of the electrical stimulation device is 15-25 μA, preferably 20 μA.

[0075] The circuit relationship between each electrical stimulation chamber

[0076] The electrical stimulation device of the present invention may further include a plurality of the electrical stimulation chambers. In some specific embodiments, the circuits between the plurality of electrical stimulation chambers are parallel circuits.

[0077] Usage method

[0078] In some aspects, using the electrical stimulation device of the present invention to stimulate and / or culture cells, it includes:

[0079] Cells are inoculated on the conductive cell culture platform;

[0080] The culture medium is added to the perforated plate-like structure, and the liquid level of the culture medium is higher than the cell culture platform but lower than the electrode pair; and,

[0081] When needed, connect the power supply to generate a stimulation current.

[0082] In some embodiments, the cells are stem cells, preferably tendon stem cells.

[0083] In some embodiments, the culture environment of the cells is 37 °C and 5% CO2 concentration.

[0084] In some embodiments, the culture medium is replaced every 2-3 days.

[0085] In some embodiments, the culture medium is a tendon induction medium.

[0086] Application

[0087] In some aspects, the electrical stimulation device of the present invention is used to promote cell proliferation, migration and / or differentiation.

[0088] In some embodiments, the cells are stem cells, preferably tendon stem cells, and the differentiation is preferably the differentiation of tendon stem cells into tendon cells.

[0089] In some embodiments, the culture medium used in the application is a tendon induction medium.

[0090] Example 1

[0091] This example provides an electrical stimulation device (multi-electrical stimulation chamber) with a cell culture lid, conductive glass, and cell culture well plate as the upper lid, conductive cell culture platform, and perforated plate-like structure:

[0092] Cell culture lid (i.e., the upper lid): It is set above the cell culture bottom plate and is used to provide a sealed environment for cell growth. In addition, it is used to fix the wires and electrode pairs. Optionally, the cell culture lid is a 6-well plate, corresponding to a 6-well plate cell culture well plate, and the diameter of each well is about 35 mm.

[0093] Wires: They are set above the cell culture lid, connected to the positive and negative poles of the electrode pair, forming a closed loop with the regulated DC power supply, and the circuits between the electrical stimulation chambers are in parallel and do not affect each other. The parallel connection ensures that the voltage and current parameters in each culture well are the same. The wires are fixed on the electrodes by soldering.

[0094] Electrode pair: It is set below the cell culture lid. As Figure 1 shown, a pair of L-shaped stainless steel rods (10 mm vertical end, 20 mm parallel end) are fixed on the cell culture lid, spaced 25 mm apart, and sealed and reinforced with epoxy resin. The length of the exposed part of the vertical end is about 5 mm, serving as the wire connection point, and is connected to the positive and negative poles of the regulated DC power supply through the circuit unit to form a closed loop. There are multiple cell culture wells in the cell well plate, and each well corresponds to a pair of electrodes. Multiple electrode pairs are connected in parallel with each other and form a closed loop with the regulated DC power supply through the power interface. Each pair of electrode pairs is connected to a square conductive glass (i.e., the conductive cell culture platform) through a pair of conductive clips. The conductive glass is placed in the culture well of the cell well plate, and the current conducts from the positive pole of the electrode pair to the negative pole through the conductive glass to form a loop. As the connection point of the circuit unit. Preferably, the distance between the parallel ends of the electrode pair and the bottom of the cell culture well plate is about 5 mm. The upper end of the conductive clip is fixed to the parallel end of the electrode pair, and the lower end of the conductive clip is connected to the conductive glass.

[0095] Conductive clip: Connects the electrode pair and the conductive glass. Optionally, the clamping thickness is 1.1 mm.

[0096] Conductive glass (conductive cell culture platform): Used for cell seeding, supporting cell growth and current transmission. The conductive glass is placed parallelly in each cell culture well. Optionally, the side length of the conductive glass is 25 mm, the sheet resistance is 10 Ω, and the thickness is 1.1 mm.

[0097] Cell culture well plate (plate-like structure with holes): Used to accommodate the conductive glass and provide culture medium.

[0098] Regulated DC power supply: Provides a constant voltage, and is connected in parallel with each cell culture well through a circuit unit. To minimize the heat generation effect of the current, a minimum voltage output of 10 mV is provided by the regulated DC power supply, and the trunk current in the circuit unit of the electrical stimulation device is about 20 μA.

[0099] As Figures 2-3 shown, the conductive glass is connected to the electrode pair through a conductive clip, so that each culture well is directly connected to the regulated DC power supply, and the current forms a path from the negative electrode through the conductive glass to the positive electrode. By connecting the conductive glass (4) to the electrode pair (2) through the conductive clip (3), the current uses the conductive glass (4) as the conductive medium, reducing the chemical reaction between the current and the culture medium, which is beneficial to cell growth. The conductive glass (4) is used for cell seeding, ensuring stable contact between the current and the cells, and can improve the effect of the electrical stimulation. The height of the conductive clip (3) can be adjusted according to the height of the cell culture well plate, so that the conductive glass (4) fits the bottom of the culture well of the cell culture well plate for cell growth.

[0100] Figure 4 Shows the connection of the circuit unit. The regulated DC power supply provides electrical energy for the electrical stimulation device. Each pair of electrodes is connected in parallel, and the circuits do not affect each other, and can also ensure that the electrical stimulation parameters of each circuit are consistent. Before seeding cells, connect the device to measure the trunk current value of the circuit. The minimum output voltage of the regulated DC power supply is 10 mV, and the trunk current in the circuit unit of the electrical stimulation device is about 20 μA at this time.

[0101] Test Example 1 Performance Test of Electrical Stimulation Device

[0102] To observe the microscopic structure of the conductive glass of the electrical stimulation device described in the present invention, a scanning electron microscope was used to observe the surface morphology of the conductive side of the conductive glass at different magnifications; to illustrate the high conductivity and long-term stability of the device, the current-voltage relationship curves of the conductive glass in the dry state and after being soaked in DMEM culture medium for two weeks were tested. Since the electrode pair does not directly contact the culture medium, the current-voltage relationship of the electrode in the dry state was tested.

[0103] The results show that:

[0104] (1) The surface of the conductive glass is overall smooth and flat, without obvious irregular morphology, seeFigure 5 。

[0105] (2) The current-voltage images of the conductive glass in the dry state and after being immersed in DMED medium for 14 days have an obvious linear relationship. The change of the conductive glass before and after soaking in the medium is not significant, and it still has a good conductivity. See Figure 6 。

[0106] (3) The current-voltage image of the stainless steel electrode in the dry state has an obvious linear relationship and has a good conductivity. See Figure 7 。

[0107] Conclusion:

[0108] The cell culture platform (conductive glass) of the electrostimulation device described in the present invention is beneficial to the growth and migration of cells. The transparent conductive glass does not affect the observation of cell staining results; the electrostimulation device has a good conductivity and good stability.

Claims

1. An electrical stimulation device, characterized in that, The electrostimulation device includes at least one electrostimulation chamber, and the electrostimulation chamber includes an upper cover, a conductive cell culture platform, and a perforated plate-like structure; The upper cover is fixed with a wire and an electrode pair composed of a positive electrode and a negative electrode, and the wire is in communication with the electrode pair; A conductive clip is fixed on the conductive cell culture platform, and the cell culture platform is in communication with the electrode pair through the conductive clip; The perforated plate-like structure is used to hold the culture medium and accommodate the cell culture platform; The upper cover and the perforated plate-like structure form a closed environment required for cell culture.

2. The electrical stimulation device according to claim 1, wherein The electrostimulation device includes a plurality of the electrostimulation chambers, and the circuits between the plurality of electrostimulation chambers are parallel circuits.

3. The electrical stimulation device according to claim 1 or 2, characterized in that, The wire is fixed to the upper cover by welding, and the welding method is preferably soldering.

4. The electrical stimulation device according to claim 1 or 2, characterized in that, The electrode pair is fixed by punching holes in the upper cover and bonding, and preferably, a resin adhesive is used for bonding and fixing.

5. The electrical stimulation device according to claim 1 or 2, characterized in that, The clamping thickness of the conductive clip is 0.8 - 1.3 mm, preferably 1.1 mm.

6. The electrical stimulation device according to claim 1 or 2, characterized in that, The conductive cell culture platform is conductive glass, preferably ITO conductive glass; preferably, the side length of the conductive glass is 20 - 30 mm, the sheet resistance is 7 - 12 Ω, and the thickness is 0.8 - 1.3 mm; more preferably, the side length of the conductive glass is 25 mm, the sheet resistance is 10 Ω, and the thickness is 1.1 mm.

7. The electrical stimulation device according to claim 1 or 2, characterized in that, The aperture of the pore structure is 30 - 40 mm, preferably 35 mm.

8. The electrical stimulation device according to claim 1 or 2, characterized in that, The electrostimulation device further includes a power source, preferably a constant voltage DC power source.

9. A method for electrostimulating cells using the electrostimulation device according to any one of claims 1 - 8, the method comprising: Inoculating cells on the conductive cell culture platform; Adding the culture medium to the perforated plate-like structure, wherein the liquid level of the culture medium is higher than the cell culture platform but lower than the electrode pair; and, When needed, connecting the power source to generate a stimulating current.

10. The method according to claim 9, wherein The cells are stem cells, preferably tendon stem cells.

Images