CN112375679B - 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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- CN112375679B CN112375679B CN202011283806.8A CN202011283806A CN112375679B CN 112375679 B CN112375679 B CN 112375679B CN 202011283806 A CN202011283806 A CN 202011283806A CN 112375679 B CN112375679 B CN 112375679B
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- 230000007246 mechanism Effects 0.000 claims abstract description 23
- 238000004140 cleaning Methods 0.000 claims abstract description 3
- 230000022131 cell cycle Effects 0.000 claims description 19
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 12
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- 210000004027 cell Anatomy 0.000 description 35
- 230000000694 effects Effects 0.000 description 14
- 210000001612 chondrocyte Anatomy 0.000 description 7
- 102000043136 MAP kinase family Human genes 0.000 description 6
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- 230000001737 promoting effect Effects 0.000 description 4
- 125000004122 cyclic group Chemical group 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
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- 102000004169 proteins and genes Human genes 0.000 description 3
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- 102000008186 Collagen Human genes 0.000 description 2
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- 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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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
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- C12N2527/00—Culture process characterised by the use of mechanical forces, e.g. strain, vibration
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Abstract
The invention relates to a cell periodic pressure electric applying device and a cell periodic pressure electric applying method, comprising the following steps: a magnetic cover slip arranged on the cells and comprising a cover slip body and a first magnetic conduction sheet arranged in the cover slip body; the pressure applying mechanism is used for carrying the cell culture dish and comprises a carrying plate, an electromagnetic generating sheet arranged in the carrying plate, a variable resistor electrically connected with the electromagnetic generating sheet and a second magnetic conduction sheet arranged on the upper carrying plate and the lower carrying plate; and the arbitrary waveform generator is electrically connected with the pressure applying mechanism. When pressure is applied to cells, firstly cleaning the magnetic cover glass, then placing the magnetic cover glass in a cell culture dish, covering the cells, and placing the cell culture dish on a pressure applying mechanism; and then the pressure applying mechanism is connected with an arbitrary waveform generator, and the magnitude and the frequency of the periodical pressure are adjusted by adjusting the field intensity and the frequency of the magnetic field, so that the periodical pressure with various parameters is generated.
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 a cell periodic pressure electric applying method.
Background
Cell mechanics (cell mechanics) is a leading-edge field of biomechanics and is also an important component of tissue engineering. It relates to the research of mechanical properties of cell membrane and cytoskeleton under the action of mechanical load, such as elastic constant, viscoelasticity and adhesive force, and the influence of mechanical factors on cell growth, development, maturation, proliferation, senility and death, etc. and its mechanism research.
Adherent cells grown in ordinary petri dishes cannot be directly subjected to any periodic mechanical pressure with current technology. Various periodic pressure generating devices reported in the literature all need to culture cells on special media, such as Bioflex plates, cell climbing plates and the like, or the pressure generating devices are huge, complex in structure and high in price, or the cells need to be moved out of an incubator for stress application, so that stress cannot be applied for a long time.
In order to solve the above-mentioned technical problems, the inventors have previously invented a mechanical cell cycle pressure applying device, in which a cell slide is covered with a cover slip, and the application of cycle pressure is completed by the oscillating pressure generated by the drop falling vertically on the slide. Although mechanical periodic pressure application is achieved, the entire device is exposed to air during the pressure application process, and there are a number of disadvantages: 1) Easy pollution; 2) The climbing process is complex; 3) The whole device has larger volume and occupies precious space resources in the incubator; 4) The pressure parameters can only be estimated roughly, and are inaccurate; 5) The device needs high-temperature high-pressure disinfection, and the preparation time is long; 6) A large amount of PBS or culture fluid is consumed.
Disclosure of Invention
The invention aims to solve the problems, improves on the prior mechanical cell cycle pressure applying device, and provides a simple cell cycle pressure electric applying device and a simple cell cycle pressure electric applying method.
In a first aspect of the invention, there is provided a cell cycle pressure electrokinetic application device comprising: a magnetic cover slip arranged on the cells and comprising a cover slip body and a first magnetic conduction sheet arranged in the cover slip body; the pressure applying mechanism is used for carrying the cell culture dish and comprises a carrying plate, an electromagnetic generating sheet arranged in the carrying plate, a variable resistor electrically connected with the electromagnetic generating sheet and a second magnetic conduction sheet arranged on the upper carrying plate and the lower carrying plate; and the arbitrary waveform generator is electrically connected with the pressure applying mechanism.
Preferably, in the cell cycle pressure electric applying device provided by the invention, the magnetic cover glass and the first magnetic conduction sheet are both round, the diameter of the first magnetic conduction 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 cell cycle pressure electric applying device provided by the invention, the first magnetic conductive sheet and the second magnetic conductive sheet are iron sheets, and the electromagnetic generating sheet is an electromagnetic coil or an electromagnet.
Preferably, in the cell cycle pressure electric applying 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 cell cycle pressure electric applying device provided by the invention, the number of the electromagnetic generating sheets is a plurality of the electromagnetic generating sheets, and the electromagnetic generating sheets are arranged in a matrix in the bearing plate.
Preferably, in the cell cycle pressure electric applying device provided by the invention, the size of the second magnetic conductive sheet is at least equal to the size of the first magnetic conductive sheet. When the magnetic cover glass is placed, the first magnetic conduction sheet corresponds to the second magnetic conduction sheet.
In a second aspect of the invention, there is provided a method of applying periodic pressure to a cell, comprising the steps of:
A. placing the magnetic cover glass in a cell culture dish after cleaning treatment, covering the cell culture dish, 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 generation sheet is electrified has an attraction effect on the magnetic cover glass, cells growing on the wall are arranged below the cover glass, and the cover glass moves towards the electromagnetic coil under the effect of the magnetic field, so that the pressure effect is exerted on the cells below the cover glass. The magnitude and frequency of the periodical pressure can be infinitely adjusted by adjusting the field intensity and frequency of the magnetic field controlled by the arbitrary waveform generator, so that the periodical pressure with various parameters is generated, and the influence and mechanism of the periodical pressure with different intensities on the cell biological behaviors are studied.
The beneficial guarantee and effect of the invention are as follows:
the device can directly apply periodic pressure with arbitrary waveform, frequency and intensity to adherent cells in a common culture dish. Can be used for applying the periodic pressure of the adherent cells in any culture dish or culture plate and researching the biological effects and mechanisms of the periodic pressure on the growth, differentiation, apoptosis, migration and the like of the cells, and has wide market prospect.
Compared with a mechanical pressure applying device, the device has the advantages of stepless adjustable parameters, uniform pressure and accurate action effect; compared with the electric pressure applying device in the prior art, the electric pressure applying device is low in cost, and the three 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 adopted for experiments, the process is simple, special treatment is not required for cells in the culture dish for adherent culture, and the periodic pressure application process can be realized by only putting a cover glass and a connecting device;
the current guided by the waveform generator is loaded on the electromagnetic coil to generate a periodically-changing magnetic field, so that the cover glass with the iron-containing sheet has a periodic attraction effect, and the cover glass is made of a rigid material, thereby generating uniform periodic pressure on cells adhered to the lower surface of the cover glass. The random waveform generator can generate stepless adjustable frequency, current and waveform (sine wave, square wave, triangular wave, irregular wave and the like), so that the device can generate periodic pressure with random size, frequency and mode;
the device has smaller volume, can be integrally placed in the cell culture box, does not influence the growth environment of cells, and therefore, the duration of applying pressure to the cells is not limited. Meanwhile, the device is not limited to the periodical pressure application process of single-layer adherent cells, the cover glass is placed on various cell supports, and the periodical pressure can still be transferred to cells growing in the supports through the transfer of the supports.
Drawings
FIG. 1 is a schematic diagram of a cell cycle pressure electrokinetic applicator in accordance with an embodiment of the present invention;
FIG. 2 is a schematic view of a magnetic cover slip in an embodiment of the invention;
FIG. 3 is a schematic view of a pressure applying mechanism according to an embodiment of the present invention;
FIG. 4 is the effect on CollagenII and promotion of inflammatory factor MMP-13 expression in rat chondrocytes after application of cyclic pressure;
FIG. 5 is the effect on MAPK signaling pathways in rat chondrocytes after application of cyclic pressure;
FIG. 6 is the effect on MAPK signaling pathways in rat osteoblasts after application of cyclic 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 may be prepared by literature procedures. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
Example 1: cell periodic pressure electric applying device
According to fig. 1, the cell cycle pressure electric applying apparatus 100 includes three parts of a magnetic cover slip 1, a pressure applying mechanism 2, and an arbitrary waveform generator 3. The magnetic cover glass 1 is provided 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 the arbitrary waveform generator 3 outside the cell culture chamber.
As shown in fig. 1 and 2, the magnetic cover slip 1 is circular and is placed on cells in a cell culture dish or a porous cell culture plate when in use, and comprises a cover slip body 11 and a first magnetic conductive sheet 12 built into the cover slip body. The cover glass body is the same as the existing cover glass in material, the first magnetic conduction 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 taking of the magnetic cover glass 1, it is provided with various dimensions, and is slightly smaller than the inner diameter of the plate 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 second magnetic conductive sheets 24 provided on the upper and lower carrier plates.
The carrier plate 21 is made of transparent plastic; the electromagnetic generating sheet 22 is an electromagnetic coil or an electromagnet, one or more of which is/are suitable for a cell culture dish, and a plurality of which are arranged in a matrix in a bearing plate and are suitable for a porous cell culture plate; the variable resistor 23 is connected with the electromagnetic generation sheet 22 and is used for controlling current and generating magnetic field gradient; the second magnetic conductive sheet 24 is also an iron sheet, and is respectively disposed on the upper and lower surfaces of the carrier plate 21, and has a size at least equal to that of the first magnetic conductive sheet 12.
The dimensions and properties of the electromagnets used in this example are shown in table 1:
table 1 summary of electromagnet size and performance
The arbitrary waveform generator 3 is similar to the prior art, and is provided with a built-in power amplifier, and is electrically connected with the pressure applying mechanism to supply power to the arbitrary waveform generator. The random waveform generator can generate stepless adjustable frequency, current and waveform (sine wave, square wave, triangular wave, irregular wave and the like), so that the whole device can generate periodic pressure with random size, frequency and mode.
When the device is adopted to apply periodic pressure to cells, the magnetic cover glass is placed in a cell culture dish after being cleaned, the magnetic cover glass is covered on the cells, and the cell culture dish is placed on a pressure applying mechanism; and then the pressure applying mechanism is connected with an arbitrary waveform generator, and the field intensity and frequency of the magnetic field are adjusted by adjusting a knob on the waveform generator, so that the magnitude and frequency of the periodic pressure are adjusted, and the periodic pressure with various parameters is generated.
The magnetic field generated after the electromagnetic generation sheet is electrified has an attraction effect on the magnetic cover glass, cells growing on the wall are arranged below the cover glass, and the cover glass moves towards the electromagnetic coil under the effect of the magnetic field, so that the pressure effect is exerted on the cells below the cover glass. The magnitude and frequency of the periodical pressure can be infinitely adjusted by adjusting the field intensity and frequency of the magnetic field controlled by the arbitrary waveform generator, so that the periodical pressure with various parameters is generated, and the influence and mechanism of the periodical pressure with different intensities on the cell biological behaviors are studied.
Example 2: effect verification
1. Effects on rat chondrocytes
After the pressure with different intensity is acted on the rat chondrocytes for 1h, the culture is continued for three days, and the total cell protein is extracted for Western-blot detection, and the result is shown in FIG. 4: the low intensity pressure (4 Hz, sine wave, peak 8 v) promotes the synthesis of chondrocyte Collagen II, while the high intensity pressure (4 Hz, sine wave, peak 15 v) promotes the expression of inflammatory factor MMP-13 while reducing its expression of Collagen II.
2. Promoting MAPK signaling pathway of rat chondrocytes
After the pressure with different intensity is acted on the rat chondrocyte for 0.5h, the total cell protein is extracted for Western-blot detection, and the result is shown in FIG. 5: both low intensity pressure (4 Hz, sine wave, peak 8 v) and high intensity pressure (4 Hz, sine wave, peak 15 v) activate MAPK signaling pathways, promoting phosphorylation of ERK and JNK.
3. Promoting MAPK signaling pathway of rat osteoblasts
After the pressure with different intensity is acted on the rat osteoblast for 0.5h, the total cell protein is extracted for Western-blot detection, and the result is shown in FIG. 6: both low intensity pressure (4 Hz, sine wave, peak 8 v) and high intensity pressure (4 Hz, sine wave, peak 15 v) activate MAPK signaling pathways, promoting phosphorylation of ERK and JNK.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (7)
1. A cell cycle pressure electrokinetic applicator comprising:
a magnetic cover slip arranged on the cells and comprising a cover slip body and a first magnetic conduction sheet arranged in the cover slip body;
the pressure applying mechanism is used for carrying the cell culture dish and comprises a carrying plate, an electromagnetic generation sheet arranged in the carrying plate, a variable resistor electrically connected with the electromagnetic generation sheet and a second magnetic conduction sheet arranged on the upper carrying plate and the lower carrying plate;
and the arbitrary waveform generator is electrically connected with the pressure applying mechanism.
2. The cell cycle pressure electrokinetic applicator of claim 1, wherein:
the magnetic cover glass and the first magnetic conduction sheet are round, and the diameter of the first magnetic conduction sheet is 1/7-1/5 of the diameter of the magnetic cover glass.
3. The cell cycle pressure electrokinetic applicator of claim 1, wherein:
the first magnetic conductive sheet and the second magnetic conductive sheet are iron sheets, and the electromagnetic generating sheet is an electromagnetic coil or an electromagnet.
4. The cell cycle pressure electrokinetic applicator of claim 1, wherein:
wherein the power of the electromagnetic generating sheet is 2.5W, the suction force is 2KC, and the jump voltage is 12V and 24V.
5. The cell cycle pressure electrokinetic applicator of claim 1, wherein:
the number of the electromagnetic generating sheets is a plurality of the electromagnetic generating sheets, and the electromagnetic generating sheets are arranged in the bearing plate in a matrix mode.
6. The cell cycle pressure electrokinetic application device according to claim 1, wherein the cell cycle pressure electrokinetic device comprises,
the size of the second magnetic conduction sheet is at least equal to that of the first magnetic conduction sheet.
7. A method of applying periodic pressure to cells using the electric applicator of any one of claims 1 to 6, comprising the steps of:
A. placing the magnetic cover glass in a cell culture dish after cleaning treatment, covering the cell culture dish, 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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| CN202011283806.8A CN112375679B (en) | 2020-11-17 | 2020-11-17 | Cell periodic pressure electric applying device and applying method |
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| CN112375679B true CN112375679B (en) | 2024-04-09 |
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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 |
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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 |
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