An electrical stimulation cell culture device

Abstract

The present invention provides an electrical stimulation cell culture device, comprising a dish body, a dish cover covering the upper part of the dish body and a support frame below the dish body, wherein the dish body comprises a dish body inner wall conductive layer and a dish body outer wall power generation layer interconnected with each other, wherein the conductive layer and the power generation layer are conductive materials; the support frame comprises a base, a support part and a friction unit hinged to the base through a connecting rod, wherein the friction unit comprises a magnetic force area, an insulating material area and is sleeved with the connecting rod through a through hole. The culture device has the advantages of low cost, simple operation and no influence on the internal environment of the culture box.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to an electrical stimulation cell culture device. Background Art

[0002] Bioelectricity is widely present in the human body and plays an indispensable role in maintaining normal biological functions. For example, many biological processes including embryogenesis, wound healing, tissue repair and remodeling, and normal biological growth are regulated by endogenous bioelectric currents, while nerves, muscles, and glandular tissues transmit electrical signals / pulses through internal electric fields. Studies have shown that endogenous electric fields in the body can regulate biological processes by controlling organ asymmetry through the transport of ions and macromolecules, membrane voltage, and cell division axis. Bioelectric signals enter epigenetic and transcriptional cascades, triggering changes in cell properties such as proliferation, differentiation, migration, shape changes, gene expression, and apoptosis.

[0003] Specifically, biophysical changes in the electric field can be triggered on the cell surface, affecting membrane protein functions such as enzyme activity (Na+ / K+ATPase and Ca2+ATPase), membrane receptor complexes, and ion transport channels by changing the charge distribution (i.e., conformation) on biomolecules. Based on this principle, in vitro simulation of in vivo bioelectric stimulation has been widely used to affect excitable cells such as nerves, muscle cells, skeletal muscles, and heart cells. It also plays an important role in non-excitable cells such as stem cells and osteoblasts. In addition, it also plays a very important role in improving the efficiency of plasmid transfection.

[0004] However, current in vitro simulation of in vivo bioelectric stimulation culture devices mainly rely on connecting power supply devices with stimulation current to the culture system. These devices have complex devices such as electrodes, connecting wires, electrical control systems and power supplies. Such devices are either integrated into the incubator, which is a complex engineering method with high costs; or the electrical control system is externalized through connecting wires, which affects the airtightness of the incubator and is complicated to operate.

[0005] Therefore, developing a cell culture device that is low-cost, easy to operate, and does not affect the internal environment of the incubator will be extremely beneficial to the promotion of in vitro electrical stimulation cell culture technology. Summary of the invention

[0006] The object of the present invention is to provide an electrical stimulation cell culture device, which has the advantages of low cost, simple operation, and no impact on the internal environment of the culture box.

[0007] When developing the electrical stimulation cell culture device referred to in the present invention, we believe that the source of electrical stimulation is crucial. After studying many electrical stimulation sources, we found electrostatic pulses. The phenomenon of friction electrification can be seen everywhere in life. The mechanical energy can be converted into electrical energy by using the principles of friction electrification and electrostatic induction, and it can be used as a power source. For example, the technologies referred to in Chinese patent publications CN203445807U, CN205051599U, CN104052327A, etc. convert various forms of mechanical energy into electrical energy through different structural designs. Through reasonable design, this device made based on the principles of friction electrification and electrostatic induction can be used to generate a pulse electrical signal with controllable frequency and adjustable output size, and it can be applied to the field of biomedical cell biology technology. This method can first avoid other materials such as electrodes from contacting cells or cell culture media, greatly reducing the possibility of cell contamination, and there is no need to connect to an external controller through a connecting line, maintaining the airtightness and stability of the culture environment, which is beneficial to the promotion and application of electrical stimulation cell culture technology.

[0008] Specifically, the technical solution of the present invention is: an electric stimulation cell culture device, comprising a dish body, a dish cover covering the upper part of the dish body, and a support frame under the dish body, wherein the dish body comprises a dish body inner wall conductive layer and a dish body outer wall power generation layer interconnected, and the connection mode can be built-in wire connection or mutually extended connection. The conductive layer and the power generation layer are conductive materials. According to the principle of friction electrification, when the conductive material is rubbed by the insulating material, the conductive material is easy to lose electrons and thus generate an electrostatic effect; the support frame comprises a base, a support part, and a friction unit hinged to the base through a connecting rod, and the friction unit comprises a magnetic area, an insulating material area, and is sleeved with the connecting rod through a through hole.

[0009] When in use, the entire device can be disinfected. The culture medium and cells to be cultured are poured into the dish of the assembled and disinfected culture device, and the dish is covered and placed in the incubator. When electrical stimulation is required, a magnetic device is set in the area directly opposite to the magnetic area outside the incubator. Based on the principle of repulsion of the same level, the existence of the magnetic device causes the magnetic area of ​​the friction unit to squeeze toward the dish body, and the insulating material of the friction unit sticks to the power generation layer. At this time, the magnetic device is moved back and forth periodically along the direction of the connecting rod, and then the magnetic area drives the friction unit to rub back and forth periodically on the power generation layer to generate electrostatic pulses, which are transmitted to the cells in the culture medium through the conductive layer, stimulating the cells and affecting cell characteristics, such as proliferation, apoptosis, differentiation, membrane protein changes, etc. The culture device can be used for the culture of any cell line. After the electrical stimulation is over, it is only necessary to remove the external magnetic field. After the magnetic force is lost, the friction unit is separated from the contact with the power generation layer due to gravity.

[0010] Preferably, the conductive layer and the power generation layer are non-ferromagnetic conductive materials, and the conductive materials include metal materials, semiconductor materials, and conductive polymer materials. These materials can lose electrons and conduct electricity after friction with insulating materials. In order to avoid being attracted by the magnetic field of the friction unit, if the conductive material is a metal material, ferromagnetic materials such as iron metal should be avoided. Among non-ferromagnetic metal materials, copper has higher conduction efficiency and more moderate cost, and metallic copper material is more preferred.

[0011] When copper material is selected, the dish body can be made by electroplating, which has a simple process and high efficiency.

[0012] Preferably, the insulating material area of ​​the friction unit is polytetrafluoroethylene nylon material. The insulating material is a material that is non-conductive under the allowable voltage, including nylon, polyoxymethylene, polyethylene oxide, polyimide, polyvinylidene fluoride, polystyrene, polymethyl methacrylate, polyvinyl chloride, polydimethylsiloxane, polytetrafluoroethylene, etc. Since polytetrafluoroethylene has high safety and strong electron-accepting ability, as a preferred solution, the friction unit is bonded and fixed to the magnetic area by adhesive.

[0013] Preferably, the power generation layer is located at the bottom outside the dish body, and the friction unit is located below the dish body. This structure is conducive to the assembly of the device, and when in use, the magnetic field can be changed below the bottom of the incubator.

[0014] Preferably, the power generation layer is connected to the conductive layer via a through hole, and the connection via the through hole uses less raw materials and has lower costs.

[0015] Preferably, the power generation layer is located on the outer wall of the dish body, and the friction unit is located on one side of the dish body. This structure is relatively easy to operate, and the magnetic field change operation can be performed on the side wall corresponding to the incubator and the magnetic zone, and the operating space is larger and freer.

[0016] Preferably, the power generation layer extends inward from the outer wall to communicate with the conductive layer. Since the outer side of the dish body side wall is a friction electrification area, the process is simpler to directly extend the power generation layer inward to communicate with the conductive layer.

[0017] Preferably, the magnetic region is a permanent magnet, such as but not limited to rare earth permanent magnet material, ferrite permanent magnet material, aluminum nickel cobalt permanent magnet material, etc. The permanent magnet has stronger magnetic force and more obvious polarity.

[0018] The support frame is any non-ferromagnetic material with a supporting function, that is, it is not attracted by an external magnetic field, such as but not limited to aluminum alloy supports, stainless steel supports, various plastic supports, etc. Of course, in order to reduce the loss of charge, it is best to use a non-conductive plastic support.

[0019] Implementing the embodiments of the present invention has the following beneficial effects:

[0020] The electrical stimulation cell culture device of the present invention has a simple structure and does not need to be connected to an external controller via a connecting wire, thereby maintaining the airtightness and stability of the culture environment, which is beneficial to the promotion and application of the electrical stimulation cell culture technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 is a schematic structural diagram of an electrical stimulation cell culture device described in Example 1;

[0022] Figure 2 It is an exploded view of parts of an electrical stimulation cell culture device described in Example 1;

[0023] Figure 3 is a cross-sectional view of an electrical stimulation cell culture device described in Example 1;

[0024] Figure 4 is a schematic structural diagram of an electrical stimulation cell culture device described in Example 2;

[0025] Figure 5 It is an exploded view of parts of an electrical stimulation cell culture device described in Example 2;

[0026] Figure 6 is a cross-sectional view of an electrical stimulation cell culture device described in Example 2;

[0027] Figure 7 is a voltage curve diagram of pulsed electrical stimulation output by the electrical stimulation cell culture device described in Example 3;

[0028] Figure 8 is a current curve diagram of pulsed electrical stimulation output by the electrical stimulation cell culture device described in Example 3;

[0029] Fig. 9 It is a comparison chart of the transfection efficiency of the experimental group and the control group in Example 3. DETAILED DESCRIPTION

[0030] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0031] Embodiment 1:

[0032] See also Figure 1-Figure 3As shown, an electrical stimulation cell culture device of this embodiment includes a dish body 01, a dish cover 02 covering the upper part of the dish body 01 and a support frame 03 below the dish body 01, wherein the dish body 01 includes a conductive layer 011 on the inner wall of the dish body 01 and a power generation layer 012 on the outer wall of the dish body 01 which are interconnected, wherein the conductive layer 011 and the power generation layer 012 are conductive materials; wherein the support frame 03 includes a base 031, a support portion 032 and a friction unit 04 hinged to the base 031 through a connecting rod 033, wherein the friction unit 04 includes a magnetic region 041, an insulating material region 042 and is sleeved with the connecting rod 033 through a through hole 043, and the friction unit 04 can slide on the connecting rod 033 through a sleeved relationship.

[0033] Furthermore, the conductive layer 011 and the power generation layer 012 are made of non-ferromagnetic conductive materials.

[0034] Furthermore, the insulating material area 042 of the friction unit 04 is made of polytetrafluoroethylene material.

[0035] Furthermore, the power generation layer 012 is located at the outer bottom of the dish body, and the friction unit 04 is located below the dish body 01.

[0036] Furthermore, the power generation layer 012 is connected to the conductive layer 011 through the through hole 013 .

[0037] Furthermore, the magnetic region 041 is a permanent magnet.

[0038] Embodiment 2:

[0039] See also Figure 4-Figure 6 As shown, an electrically stimulated cell culture device of this embodiment is different from that of embodiment 1 in that the power generation layer 012 is located on the outer wall of the dish body 01, and correspondingly, the friction unit 04 is located on one side of the dish body 01. When this embodiment is used, a magnetic field opposite to the magnetic region 041 is applied to the outer side of the incubator corresponding to the friction unit 04, so that the friction unit 04 can be pushed toward the side wall of the dish body 01, and the insulating material region 042 of the friction unit 04 contacts the power generation layer 012. At this time, the magnetic field outside the incubator is moved up and down along the direction of the connecting rod 033, and the friction unit 04 can be driven to slide up and down under the action of magnetic traction, thereby generating static electricity on the power generation layer through the movement of the friction unit 04.

[0040] Furthermore, the power generation layer 012 extends inward from the outer side wall and communicates with the conductive layer 011 .

[0041] Embodiment 3:

[0042] A human non-small cell lung cancer cell line A549 was selected for culture, and the cells and culture medium were added to the electric stimulation culture device shown in Example 1, an experimental group, and a control group, an ordinary culture dish, and cultured and expanded in an incubator. When the cell density reached 60%-80%, a green fluorescent protein (GFP) eukaryotic expression plasmid packaged in lipo2000 liposomes was added, and a periodically variable magnetic field was added to the bottom of the incubator. The frequency and intensity of the variable magnetic field were adjusted so that the magnet drove the friction material to produce periodic reciprocating motion and rubbed with the bottom surface of the culture dish, and the friction was used to generate electrical and electrostatic induction to generate electrical stimulation to the cells in the culture dish; the electrical stimulation was stopped after 1 hour, and the changes in transfection efficiency were compared at different time points after transfection. The transfection efficiency was detected by flow cytometry, and it was observed that the cell culture dish with electric stimulation function could improve the transfection efficiency and shorten the transfection time.

[0043] The friction material of the electrical stimulation culture device is nylon film; the conductive material is copper film; the magnet is a strong neodymium iron boron magnet with a diameter of 10 mm and a thickness of 2 mm; the frequency of the changing magnetic field is 3.3 Hz and the intensity is 2000 Gs. Figure 7-8 As shown, the voltage generated by the culture device during the magnetic field change process is a 130V pulse voltage, which can output a peak current of 6 microamperes.

[0044] like Fig. 9 As shown, the plasmid transfection efficiency of the experimental group and the control group was measured 6 hours, 12 hours, 24 hours, and 48 hours after culture, and it was found that with the increase of culture time, the transfection rate of the experimental group was significantly higher than that of the control group.

[0045] The experimental results of the above embodiments prove that the electrical stimulation cell culture device of the present invention can generate stable pulse electrical stimulation by periodically changing the external magnetic field. On the basis of ensuring the electrical stimulation effect, because no other materials such as electrodes contact cells or cell culture medium, the possibility of cell contamination is greatly reduced; at the same time, there is no need to connect to the external controller through a connecting wire, which maintains the airtightness and stability of the culture environment. The above advantages can greatly help promote the electrical stimulation culture technology.

[0046] The above is a preferred embodiment of the present invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. An electrical stimulation cell culture device, characterized in that: It comprises a dish body, a dish cover covering the upper part of the dish body and a support frame under the dish body, wherein the dish body comprises a dish body inner wall conductive layer and a dish body outer wall power generation layer which are interconnected, and the conductive layer and the power generation layer are made of conductive materials; the support frame comprises a base, a support part and a friction unit hinged to the base through a connecting rod, and the friction unit comprises a magnetic area, an insulating material area and is sleeved with the connecting rod through a through hole; The conductive layer and the power generation layer are made of non-ferromagnetic conductive materials, and the magnetic region is a permanent magnet.

2. The electrical stimulation cell culture device according to claim 1, characterized in that: The insulating material area of ​​the friction unit is made of polytetrafluoroethylene material.

3. The electrical stimulation cell culture device according to claim 1, characterized in that: The power generation layer is located at the outer bottom of the dish body, and the friction unit is located below the dish body.

4. The electrical stimulation cell culture device according to claim 3, characterized in that: The power generation layer is connected with the conductive layer through the through hole.

5. The electrical stimulation cell culture device according to claim 1, characterized in that: The power generation layer is located on the outer side wall of the dish body, and the friction unit is located on one side of the dish body.

6. The electrical stimulation cell culture device according to claim 5, characterized in that: The power generation layer extends inward from the outer side wall and communicates with the conductive layer.

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