CN112375679A - Cell periodic pressure electric applying device and applying method - Google Patents
Cell periodic pressure electric applying device and applying method Download PDFInfo
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- CN112375679A CN112375679A CN202011283806.8A CN202011283806A CN112375679A CN 112375679 A CN112375679 A CN 112375679A CN 202011283806 A CN202011283806 A CN 202011283806A CN 112375679 A CN112375679 A CN 112375679A
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- 230000000737 periodic effect Effects 0.000 title claims abstract description 45
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- 238000004113 cell culture Methods 0.000 claims abstract description 24
- 230000007246 mechanism Effects 0.000 claims abstract description 23
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/02—Electrical or electromagnetic means, e.g. for electroporation or for cell fusion
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Abstract
The invention relates to a cell periodic pressure electric applying device and an applying method, comprising the following steps: the magnetic cover glass is arranged on the cells and comprises a cover glass body and a first magnetic conduction sheet arranged in the cover glass body; the pressure applying mechanism is used for bearing the cell culture dish and comprises a bearing plate, an electromagnetic generating sheet arranged in the bearing plate, a variable resistor electrically connected with the electromagnetic generating sheet and a second magnetic conductive sheet arranged on the upper bearing plate and the lower bearing plate; and the arbitrary waveform generator is electrically connected with the pressure applying mechanism. When pressure is applied to the cells, the magnetic cover glass is cleaned and then placed in a cell culture dish to cover the cells, and the cell culture dish is placed on the pressure applying mechanism; then the pressure applying mechanism is connected with an arbitrary waveform generator, and the magnitude and the frequency of the periodic pressure are adjusted by adjusting the field intensity and the frequency of the magnetic field, so as to generate the periodic pressure with various parameters.
Description
Technical Field
The invention belongs to the technical field of cell culture, and particularly relates to a cell periodic pressure electric applying device and an applying method.
Background
Cell mechanics (cell mechanics) is a leading field of biomechanics and is also an important component of tissue engineering. It relates to the research of the cell membrane and cytoskeleton deformation, elastic constant, viscoelasticity, adhesion and other mechanical properties under the action of mechanical load, and the research of the influence of mechanical factors on cell growth, development, maturation, proliferation, senility, death and other mechanisms.
Adherent cells grown in ordinary culture dishes cannot directly apply any periodic mechanical pressure with current technology. Various periodic pressure generating devices reported in the literature at present need to culture cells on special media, such as Bioflex plates, cell crawl sheets and the like, or the pressure generating devices are large, complex in structure and expensive, or the cells need to be removed from an incubator for stress application, so that stress cannot be applied for a long time.
In order to solve the above technical problems, the inventors have previously invented a mechanical cell periodic pressure applying apparatus, which covers a cell slide with a cover glass, and applies periodic pressure by using oscillation pressure generated when a droplet drops vertically onto the cover glass. Although the mechanical periodic pressure application is realized, the whole device is exposed to air in the pressure application process, and the device has the following defects: 1) the pollution is easy; 2) the process of climbing the film is complex; 3) the whole device has larger volume and occupies valuable space resources in the incubator; 4) the pressure parameter can only be roughly estimated, and is inaccurate; 5) the device needs high-temperature and high-pressure sterilization, and the preparation time is long; 6) consuming large amounts of PBS or culture medium.
Disclosure of Invention
The invention aims to solve the problems, improves on the basis of the mechanical cell periodic pressure applying device in the past, and provides a simple cell periodic pressure electric applying device and an applying method.
In a first aspect of the present invention, there is provided a cell cycling pressure electric application device comprising: the magnetic cover glass is arranged on the cells and comprises a cover glass body and a first magnetic conduction sheet arranged in the cover glass body; the pressure applying mechanism is used for bearing the cell culture dish and comprises a bearing plate, an electromagnetic generating sheet arranged in the bearing plate, a variable resistor electrically connected with the electromagnetic generating sheet and a second magnetic conductive sheet arranged on the upper bearing plate and the lower bearing plate; and the arbitrary waveform generator is electrically connected with the pressure applying mechanism.
Preferably, in the electric cell periodic pressure applying device provided by the invention, the magnetic cover glass and the first magnetic conductive sheet are both circular, the diameter of the first magnetic conductive sheet is 1/7-1/5 of the diameter of the magnetic cover glass, and the weight of the cover glass is kept unchanged as much as possible.
Preferably, in the electric cell periodic pressure application device provided by the present invention, the first magnetic conductive sheet and the second magnetic conductive sheet are both iron sheets, and the electromagnetic generating sheet is an electromagnetic coil or an electromagnet.
Preferably, in the electric cell periodic pressure application device provided by the invention, the power of the electromagnetic generating sheet is 2.5W, the suction force is 2KC, and the jump voltage is 12V and 24V.
Preferably, in the electric cell periodic pressure application device provided by the present invention, the number of the electromagnetic generating sheets is plural, and the electromagnetic generating sheets are arranged in a matrix in the carrier plate.
Preferably, in the electric cell periodic pressure applying device provided by the invention, the size of the second magnetic conductive sheet is at least equal to that of the first magnetic conductive sheet. When the magnetic cover glass is placed, the first magnetic conductive sheet and the second magnetic conductive sheet are corresponding.
In a second aspect of the invention, there is provided a method of applying periodic pressure to a cell, comprising the steps of:
A. cleaning the magnetic cover glass, placing the cleaned magnetic cover glass in a cell culture dish, covering the cell culture dish on the cells, and placing the cell culture dish on a pressure applying mechanism;
B. the pressure applying mechanism is connected with an arbitrary waveform generator, and the magnitude and the frequency of the periodic pressure are adjusted by adjusting the field intensity and the frequency of the magnetic field, so that the periodic pressure with various parameters is generated.
The principle of the invention is as follows: the magnetic field generated after the electromagnetic generating sheet is electrified has an attraction effect on the magnetic cover glass, the cells growing adherently are arranged below the cover glass, and under the action of the magnetic field, the cover glass moves towards the electromagnetic coil, so that the pressure effect is exerted on the cells below the electromagnetic coil. By adjusting the field intensity and frequency of the control magnetic field of the arbitrary waveform generator, the magnitude and frequency of the periodic pressure can be infinitely adjusted, so that the periodic pressure of various parameters is generated, and the influence and mechanism of different intensity pressures on the cell biological behavior are researched.
The invention has the following beneficial guarantee and effects:
the device can directly apply periodic pressure with any waveform, frequency and intensity to adherent cells in a common culture dish. Can be used for applying periodic pressure to adherent cells in any culture dish or culture plate and researching the biological effects and mechanisms of the periodic pressure on cell growth, differentiation, apoptosis, migration and the like, and has wide market prospect.
Compared with a mechanical pressure applying device, the device has the advantages that the parameters can be infinitely adjusted, the pressure is uniform, and the action effect is accurate; compared with the electric pressure applying device in the prior art, the device has low cost, and the core components including the electromagnetic coil, the waveform generator and the cover glass containing the iron sheet can be purchased or customized, so that the cost is low.
In addition, when the device is used for carrying out experiments, the process is simple, cells cultured by adherence in a culture dish do not need to be specially treated, and the periodic pressure applying process can be carried out only by arranging a cover glass and connecting the device;
the current led out by the waveform generator is loaded on an electromagnetic coil to generate a periodically changing magnetic field, and the periodically changing magnetic field has periodic attraction effect on the cover glass containing the iron sheets, and the cover glass is made of a rigid material, so that uniform periodic pressure on cells attached to the lower wall of the cover glass is generated. Because the arbitrary waveform generator can generate stepless adjustable frequency, current and waveform (sine wave, square wave, triangular wave, irregular wave and the like), the device can generate periodic pressure with any size, frequency and mode;
the device has small volume, can be integrally placed in a cell culture box, does not influence the growth environment of cells, and therefore, the time for applying pressure to the cells is not limited. Meanwhile, the device is not limited to the periodic pressure application process of the monolayer adherent cells, the cover glass is placed on various cell scaffolds, and the periodic pressure can be still transmitted to the cells growing in the scaffolds through the transmission of the scaffolds.
Drawings
FIG. 1 is a schematic structural diagram of an electromotive cell-cycling pressure applying device according to an embodiment of the present invention;
FIG. 2 is a schematic structural view of a magnetic cover glass in an embodiment of the present invention;
FIG. 3 is a schematic structural view of a pressure applying mechanism in the embodiment of the present invention;
FIG. 4 is a graph showing the effect on Collagen II and on promoting the expression of inflammatory factor MMP-13 in rat chondrocytes after application of periodic pressure;
FIG. 5 is a graph of the effect on MAPK signaling pathways in rat chondrocytes following application of periodic pressure;
FIG. 6 is a graph showing the effect on MAPK signaling pathways in rat osteoblasts following application of periodic pressure.
Detailed Description
The present invention will now be described in detail with reference to examples and drawings, but the practice of the invention is not limited thereto.
The reagents and starting materials used in the present invention are commercially available or can be prepared according to literature procedures. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.
Example 1: cell periodic pressure electric applying device
According to fig. 1, the cell periodic pressure electric application apparatus 100 includes three parts of a magnetic cover glass 1, a pressure application mechanism 2, and an arbitrary waveform generator 3. The magnetic cover glass 1 is set in the cell culture apparatus 200, the cell culture apparatus 200 is placed on the pressure applying mechanism 2, and the pressure applying mechanism 2 is electrically connected to an arbitrary waveform generator 3 outside the cell culture chamber.
As shown in fig. 1 and fig. 2, the magnetic cover glass 1 is circular, and is placed on cells in a cell culture dish or a multi-well cell culture plate, and includes a cover glass body 11 and a first magnetic conductive sheet 12 embedded in the cover glass body. The cover glass body is made of the same material as the existing cover glass, the first magnetic conductive sheet 12 is an iron sheet, the diameter D is 1/7-1/5 of the diameter D of the magnetic cover glass, and the weight of the cover glass is kept unchanged as much as possible.
In this embodiment, in order to facilitate the placement and the removal of the magnetic cover glass 1, the magnetic cover glass is provided with various dimensions, and is slightly smaller than the inner diameter of the hole of the culture dish or the cell culture plate so as to be suitable for different cell culture devices.
As shown in fig. 1 and 3, the pressure applying mechanism 2 includes a carrier plate 21, an electromagnetic generating sheet 22 built in the carrier plate, a variable resistor 23 electrically connected to the electromagnetic generating sheet, and a second magnetic conductive sheet 24 provided on the upper and lower carrier plates.
The carrier plate 21 is made of transparent plastic; the electromagnetic generating sheets 22 are one or more electromagnetic coils or electromagnets, one is suitable for the cell culture dish, and a plurality of electromagnetic generating sheets are arranged in the bearing plate in a matrix manner and are suitable for the porous cell culture plate; the variable resistor 23 is connected with the electromagnetic generating sheet 22 and used for controlling current and generating magnetic field gradient; the second magnetic conductive sheet 24 is also an iron sheet, and is respectively arranged on the upper and lower surfaces of the bearing plate 21, the size of the second magnetic conductive sheet is at least equal to that of the first magnetic conductive 12 sheets, and when the magnetic cover glass is placed, the first magnetic conductive sheet and the second magnetic conductive sheet are corresponding.
The dimensions and properties of the electromagnets used in this example are shown in table 1:
TABLE 1 summary of electromagnet dimensions and Performance
The arbitrary waveform generator 3 is the same as the prior art, and has a built-in power amplifier electrically connected to the pressure applying mechanism to supply power thereto. The arbitrary waveform generator can generate stepless adjustable frequency, current and waveform (sine wave, square wave, triangular wave, irregular wave and the like), and the whole device can generate periodic pressure with arbitrary size, frequency and mode.
When the device is used for applying periodic pressure to cells, the magnetic cover glass is placed in a cell culture dish after being cleaned and covered on the cells, and the cell culture dish is placed on the pressure applying mechanism; and then the pressure applying mechanism is connected with an arbitrary waveform generator, the field intensity and the frequency of a magnetic field are adjusted by adjusting a knob on the waveform generator, and the size and the frequency of the periodic pressure are adjusted, so that the periodic pressure with various parameters is generated.
The magnetic field generated after the electromagnetic generating sheet is electrified has an attraction effect on the magnetic cover glass, the cells growing adherently are arranged below the cover glass, and under the action of the magnetic field, the cover glass moves towards the electromagnetic coil, so that the pressure effect is exerted on the cells below the electromagnetic coil. By adjusting the field intensity and frequency of the control magnetic field of the arbitrary waveform generator, the magnitude and frequency of the periodic pressure can be infinitely adjusted, so that the periodic pressure of various parameters is generated, and the influence and mechanism of different intensity pressures on the cell biological behavior are researched.
Example 2: effect verification
1. Effect on rat chondrocytes
After the rat chondrocytes are subjected to 1 hour of pressure action by using different intensities, the rat chondrocytes are cultured for three days, and the total cell proteins are extracted for Western-blot detection, and the results are shown in a figure 4: low-intensity pressure (4Hz, sine wave, peak 8v) promotes synthesis of chondrocyte Collagen II, while high-intensity pressure (4Hz, sine wave, peak 15v) promotes expression of inflammatory factor MMP-13 while reducing expression of Collagen II.
2. Promoting MAPK signal pathway of rat chondrocytes
After rat chondrocytes are acted for 0.5h by using pressures with different intensities, total cell proteins are extracted and detected by Western-blot, and the results are shown in a figure 5: both low (4Hz, sine wave, peak 8v) and high (4Hz, sine wave, peak 15v) intense pressures activate MAPK signaling pathways, promoting ERK and JNK phosphorylation.
3. Promoting MAPK signaling pathway in rat osteoblasts
After the rat osteoblasts are acted by pressure with different intensities for 0.5h, total cell protein is extracted and detected by Western-blot, and the results are shown in FIG. 6: both low (4Hz, sine wave, peak 8v) and high (4Hz, sine wave, peak 15v) intense pressures activate MAPK signaling pathways, promoting ERK and JNK phosphorylation.
While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.
Claims (7)
1. An electromotive cell-cycling pressure application device, comprising:
the magnetic cover glass is arranged on the cells and comprises a cover glass body and a first magnetic conduction sheet arranged in the cover glass body;
the pressure applying mechanism is used for bearing the cell culture dish and comprises a bearing plate, an electromagnetic generating sheet arranged in the bearing plate, a variable resistor electrically connected with the electromagnetic generating sheet and a second magnetic conductive sheet arranged on the upper bearing plate and the lower bearing plate;
and the arbitrary waveform generator is electrically connected with the pressure applying mechanism.
2. The cell cycling pressure motorized applicator of claim 1, wherein:
the magnetic cover glass and the first magnetic conductive sheet are both circular, and the diameter of the first magnetic conductive sheet is 1/7-1/5 of the diameter of the magnetic cover glass.
3. The cell cycling pressure motorized applicator of claim 1, wherein:
the first magnetic conductive sheet and the second magnetic conductive sheet are both iron sheets, and the electromagnetic generating sheet is an electromagnetic coil or an electromagnet.
4. The cell cycling pressure motorized applicator of claim 1, wherein:
wherein the power of the electromagnetic generating sheet is 2.5W, the suction force is 2KC, and the jumping voltage is 12V and 24V.
5. The cell cycling pressure motorized applicator of claim 1, wherein:
the number of the electromagnetic generating sheets is multiple, and the electromagnetic generating sheets are arranged in the bearing plate in a matrix mode.
6. The electro-kinetic cell-cycling pressure-applying device according to claim 1,
the size of the second magnetic conductive sheet is at least equal to that of the first magnetic conductive sheet.
7. A method of applying a cyclic pressure to a cell using the motorized applicator of any one of claims 1 to 6, comprising the steps of:
A. cleaning the magnetic cover glass, placing the cleaned magnetic cover glass in a cell culture dish, covering the cell culture dish on the cells, and placing the cell culture dish on a pressure applying mechanism;
B. the pressure applying mechanism is connected with an arbitrary waveform generator, and the magnitude and the frequency of the periodic pressure are adjusted by adjusting the field intensity and the frequency of the magnetic field, so that the periodic pressure with various parameters is generated.
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| CN202011283806.8A CN112375679B (en) | 2020-11-17 | 2020-11-17 | Cell periodic pressure electric applying device and applying method |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114456934A (en) * | 2022-03-02 | 2022-05-10 | 邹玉良 | Intelligent automatic culture apparatus for tissue culture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483075A (en) * | 2000-12-22 | 2004-03-17 | �Ϻ���ͨ��ѧ | Culturing tissue using magnetically generated mechanical stresses |
| CN106467891A (en) * | 2016-09-22 | 2017-03-01 | 苏州百源基因技术有限公司 | A kind of gas supply device being applied to instrument for extracting nucleic acid and instrument for extracting nucleic acid |
| CN207047255U (en) * | 2017-04-05 | 2018-02-27 | 中国人民解放军第二军医大学 | A kind of cell cycle pressure applying means |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1483075A (en) * | 2000-12-22 | 2004-03-17 | �Ϻ���ͨ��ѧ | Culturing tissue using magnetically generated mechanical stresses |
| CN106467891A (en) * | 2016-09-22 | 2017-03-01 | 苏州百源基因技术有限公司 | A kind of gas supply device being applied to instrument for extracting nucleic acid and instrument for extracting nucleic acid |
| CN207047255U (en) * | 2017-04-05 | 2018-02-27 | 中国人民解放军第二军医大学 | A kind of cell cycle pressure applying means |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114456934A (en) * | 2022-03-02 | 2022-05-10 | 邹玉良 | Intelligent automatic culture apparatus for tissue culture |
| CN114456934B (en) * | 2022-03-02 | 2024-01-26 | 上海碧博生物医药工程有限公司 | Intelligent automatic culture device for tissue culture |
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