阅读说明：本技术 细胞培养孔板及安装有细胞培养孔板的细胞培养在线检测装置 (Cell culture orifice plate and cell culture on-line detection device provided with same ) 是由 王慧锋 顾震 田晓丽 颜秉勇 周家乐 沈斌 于 2021-07-23 设计创作，主要内容包括：本发明涉及生物化学检测技术领域,公开了一种集成电学刺激与测量技术的细胞培养孔板,包括孔板底座,在底座上设置若干孔洞,孔洞底部设置电极体,所述电极体包括第一电极和第二电极,第一电极和第二电极均向对侧形成多条支极,支极间隔排列,每个支极均由重复规则结构组成,相邻支极间的图案呈交错排列,排布后的电极体形状与孔洞底部形状相匹配,所述若干孔洞阵列排布,每个孔洞中的第一电极和第二电极引出至孔板底座的两端。本发明还公开了所述细胞培养孔板配套的细胞培养在线检测装置。本发明实现对细胞培养过程中细胞状态及生长情况的控制和更精确的检测。(The invention relates to the technical field of biochemical detection, and discloses a cell culture pore plate integrating electrical stimulation and measurement technology, which comprises a pore plate base, wherein a plurality of pores are formed in the base, an electrode body is arranged at the bottom of each pore, the electrode body comprises a first electrode and a second electrode, a plurality of branch electrodes are formed on the opposite sides of the first electrode and the second electrode, the branch electrodes are arranged at intervals, each branch electrode is composed of a repeated regular structure, patterns between adjacent branch electrodes are staggered, the shape of the arranged electrode body is matched with the shape of the bottom of each pore, the plurality of pores are arranged in an array mode, and the first electrode and the second electrode in each pore are led out to two ends of the pore plate base. The invention also discloses a cell culture online detection device matched with the cell culture pore plate. The invention realizes the control and more accurate detection of the cell state and the growth condition in the cell culture process.)

1. A cell culture well plate, comprising: the electrode body comprises a first electrode and a second electrode, wherein the first electrode and the second electrode are connected by a wire to form a plurality of branch electrodes towards opposite sides, the branch electrodes are arranged at intervals to form an interdigital form, each branch electrode is a geometric pattern unit structure which is repeatedly arranged, patterns between adjacent branch electrodes are arranged in a staggered mode, the shape of the electrode body after arrangement is matched with the shape of the bottom of each hole, the holes are arranged in an array mode, and the first electrode and the second electrode of each hole are led out to interfaces at two ends of the hole plate base.

2. The cell culture well plate of claim 1, wherein: the number of the branch poles is 16 to 100; the hole array is 16-384 holes.

3. The cell culture well plate of claim 1, wherein: the electrode body structure is processed at the bottom of the hole through a magnetron sputtering or evaporation process, the outer layer of the electrode body is a conductive material with the thickness of 10nm to 500nm, and the conductive material on the outer layer of the electrode body is one of gold, platinum, rhodium, ruthenium, silver chloride, polypyrrole or poly 3, 4-ethylenedioxythiophene.

4. The cell culture well plate of claim 1, wherein: cells are cultured in an adherent way at the bottom of the hole, and the individual cells cover the electrode body area and span between the first electrode and the second electrode during the culture process.

5. The cell culture on-line detection device is characterized in that: the cell culture device comprises a base and a cell culture pore plate, wherein a pore plate groove is arranged on the base, the cell culture pore plate is arranged in the pore plate groove, a control circuit is arranged in the base, an electrode body on the cell culture pore plate is connected to the control circuit, the control unit comprises a main control circuit, a signal reference source, a multiplexer and a signal measurement circuit, the main control circuit controls a reference signal source to generate excitation signals, the multiplexer is respectively connected to each first electrode on the cell culture pore plate, the multiplexer connects the signal input end of the signal measurement circuit with one of the first electrodes under the control of the main control circuit, the signal output end of the signal measurement circuit is connected with all the second electrodes, the signal reference source is connected with the signal measurement circuit, and the signal measurement circuit conditions the output signals of the signal reference source and outputs the excitation signals to all the second electrodes, and the first electrode selected by the multiplexer forms a loop for applying electric stimulation to the cells or measuring the electrical properties of the cells and monitoring the growth state of the cells.

6. The on-line cell culture detection device according to claim 5, wherein: the control unit periodically generates pulses of 10mV to 10V voltage amplitude, 1Hz to 1kHz frequency, and 1% to 50% duty cycle on the second electrode, which pulses modulate the arrangement and state of the cells on the electrode surface.

7. The on-line cell culture detection device according to claim 5, wherein: the control unit measures the current amplitude and phase change data on the electrode, performs normalization processing to serve as an index of cell growth state, and analyzes the change of the index along with time to obtain cell growth trend information.

8. The on-line cell culture detection device according to claim 5, wherein: the excitation signal output to the second electrode is a voltage signal with waveforms of sine waves, square waves, triangular waves, pulses and the like, and the amplitude of the excitation signal output to the second electrode is 1mV to 150 mV.

9. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: the control unit further comprises a calibration circuit, the calibration circuit is connected to the signal measurement circuit through the multiplexer, the control circuit stores calibration data, and the signal measurement circuit calibrates the signal measurement result through the signal calibration circuit and stores the calibration result for compensating subsequent measurement data.

10. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: the control circuit is also connected with a chip in-place detection circuit, the chip in-place detection circuit is connected to the cell culture pore plate, and the loading state of the cell culture pore plate is detected and fed back to the control circuit.

11. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: the main control circuit is also connected to a storage unit, and the storage unit is used for storing detection data.

12. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: the main control circuit is connected with the upper computer software through the communication circuit, sends the data collected by the measuring circuit to the upper computer, and receives the instruction sent by the upper computer to execute the operation.

13. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: and the main control circuit is also connected with a status indicator lamp.

14. The cell culture online detection device according to any one of claims 5 to 8, characterized in that: the cell culture plate is characterized in that a turnover cover is arranged on the pore plate groove, when the turnover cover is closed, the cell culture pore plate is fixed in the pore plate groove and the turnover cover, and a detachable transparent observation window is arranged above the cell culture pore plate.

Technical Field

The invention relates to the technical field of biochemical detection, in particular to a cell culture pore plate for cell growth online detection and a cell culture online detection device provided with the cell culture pore plate.

Background

The development of drug development and cell therapy technologies has made an urgent need for on-line, in-situ, non-destructive and continuous cell detection technologies for quantitative analysis of cell growth state, growth tendency and number in cell culture processes. The technology can be widely used for drug screening, drug toxicity analysis, antitumor drug killing efficiency analysis, stem cell culture process optimization, stem cell differentiation state monitoring, CarT cell treatment method verification and the like.

Conventional cell viability assays require labeling of the cells and light intensity measurements after incubation are performed to obtain the desired data, namely CI (known as CellIndex). This results in a long experimental flow, and only a few or even one data can be obtained in one experiment, which is inefficient, and the data analysis needs to be performed through multiple experiments to meet the requirements of the data amount of the graph drawing and the data analysis.

The measurement of the growth state of cells based on their electrical properties is one of the most important cell on-line detection methods. Generally, the technique obtains electrical properties of the cell by applying an excitation voltage, for example, by measuring the resistance, capacitance, or impedance of the cell. At present, the existing technology usually utilizes the effect of cells on the surface impedance of an electrode by culturing the cells on the surface of the electrode to obtain cell growth information. The method generally adopts linear interdigital electrodes for measurement, and the used excitation voltage is a sine waveform with fixed frequency, so when different types of cells are measured by the method, the detection sensitivity achieved by the method is different due to the difference of cell orientation, size and shape, namely the design of the interdigital electrode unit in a single electrode body branch is difficult to meet the high-sensitivity detection of all specified cell growth.

Obviously, in order to achieve higher sensitivity of measurement on specific cells, parameters such as the shape, the spacing, the material and the thickness of the interdigital electrode need to be improved or redesigned, and an instrument system with high accuracy and stability is developed to measure the cell growth on the surface of the electrode, so as to obtain a cell growth state dynamic change curve, thereby being widely used in cell growth experiments.

Disclosure of Invention

The invention aims to solve the problems and provides a cell culture pore plate and a cell culture online detection device, which realize the control of cell growth and the detection of cell growth state in the cell culture process.

The technical scheme adopted by the invention is as follows:

a cell culture pore plate is characterized by comprising a pore plate base, wherein a plurality of holes are formed in the base, electrode bodies are arranged at the bottoms of the holes and comprise first electrodes and second electrodes, the first electrodes and the second electrodes form a plurality of branch electrodes towards opposite sides, the branch electrodes are arranged at intervals to form an interdigital form, each branch electrode is formed by a geometric pattern unit structure which is repeatedly arranged and is connected by a lead, patterns between adjacent branch electrodes are arranged in a staggered mode, the shape of the electrode bodies after arrangement is matched with the shape of the bottoms of the holes, the holes are arranged in an array mode, and the first electrodes and the second electrodes of each hole are led out to interfaces at two ends of the pore plate base.

Further, the number of the branch poles is 16 to 100; the hole array is 16-384 holes.

Further, the cross section of the hole is one of a circle, a square, a rectangle or a hexagon, the bottom of the hole is made of one of glass, quartz, polytetrafluoroethylene, polyethylene terephthalate, polymethyl methacrylate and polyvinyl chloride, the electrode body structure is processed at the bottom of the hole through a magnetron sputtering or evaporation process, the outer layer of the electrode body is made of a conductive material with the thickness of 10nm to 500nm, and the conductive material of the outer layer of the electrode body is one of gold, platinum, rhodium, ruthenium, silver chloride, polypyrrole or poly 3, 4-ethylenedioxythiophene.

Further, the cells are cultured in an adherent way at the bottom of the hole, and the individual cells cover the electrode body area and span between the first electrode and the second electrode during the culture process.

An on-line detection device for cell culture is characterized by comprising a base and a cell culture pore plate, wherein a pore plate groove is arranged on the base, the cell culture pore plate is arranged in the pore plate groove, a control circuit is arranged in the base, an electrode body on the cell culture pore plate is connected to the control circuit, the control unit comprises a main control circuit, a signal reference source, a multiplexer and a signal measurement circuit, the main control circuit controls a reference signal source to generate an excitation signal, the multiplexer is respectively connected to each first electrode on the cell culture pore plate, the multiplexer connects the signal input end of the signal measurement circuit with one of the first electrodes under the control of the main control circuit, the signal output end of the signal measurement circuit is connected with all the second electrodes, the signal reference source is connected with the signal measurement circuit, and the signal measurement circuit conditions the output signal of the signal reference source, and outputting the excitation signals to all the second electrodes to form a loop with the first electrodes selected by the multiplexer, and applying electrical stimulation to the cells or measuring the electrical properties of the cells to monitor the growth state of the cells.

Further, the control unit periodically generates pulses of 10mV to 10V voltage amplitude, 1Hz to 1kHz frequency, and 1% to 50% duty cycle on the second electrode, the pulses adjusting the arrangement and state of the cells on the electrode surface.

Furthermore, the control unit measures the current amplitude and phase change data of the electrode, performs normalization processing to be used as a cell growth state index, and analyzes the change of the cell growth state index along with time to obtain cell growth trend information.

Further, the excitation signal output to the second electrode is a voltage signal having a waveform of a sine wave, a square wave, a triangular wave, a pulse, or the like, and the amplitude of the excitation signal output to the second electrode is 1mV to 150 mV.

Furthermore, the control unit also comprises a calibration circuit, the calibration circuit is connected to the signal measurement circuit through the multiplexer, the control circuit stores calibration data, and the signal measurement circuit calibrates the signal measurement result through the signal calibration circuit and stores the calibration result for compensating subsequent measurement data.

Furthermore, the control circuit is also connected with a chip in-place detection circuit, the chip in-place detection circuit is connected to the cell culture pore plate, and the loading state of the cell culture pore plate is detected and fed back to the control circuit.

Further, the main control circuit is also connected to a storage unit, and the storage unit is used for storing the detection data.

Furthermore, the main control circuit is connected with the upper computer software through the communication circuit, sends the data collected by the measuring circuit to the upper computer, and receives the instruction sent by the upper computer to execute the operation.

Furthermore, a status indicator lamp is connected to the main control circuit.

Further, set up flip on the orifice plate groove, when flip closed, will the cell culture orifice plate is fixed in orifice plate groove and flip set up the transparent observation window of detachable above the cell culture orifice plate.

The invention has the beneficial effects that:

(1) the electrode design of the cell culture pore plate can carry out parametric design by adjusting the width and the angle of a structural unit aiming at cells with specific size, shape and physical and chemical properties so as to improve the detection sensitivity;

(2) the electrodes adopt a design of staggered arrangement of a rhombic or hexagonal unit structure, which is beneficial to improving the detection sensitivity of cells with different sizes and spatial orientations;

(3) the geometric shape of the electrode is beneficial to the processing of various processes including photoetching, laser etching, sputtering and the like;

(4) the cell culture online detection device selects a channel through a multiplexer, and can measure any channel in single detection;

(5) modulating cell growth by applying an electrical stimulation signal on the cell;

(6) and carrying out automatic calibration compensation on the measurement result through a calibration circuit.

Drawings

FIG. 1 is a schematic perspective view of a cell culture well plate according to the present invention;

FIG. 2 is a schematic plan view of a circular electrode;

FIG. 3 is an enlarged view of the bending portion of the branch pole corresponding to FIG. 2;

FIG. 4 is a schematic plan view of an electrode of rectangular shape;

FIG. 5 is a diamond-shaped enlarged view of the pole corresponding to FIG. 4;

FIG. 6 is a schematic plan view of a diamond shaped electrode;

FIG. 7 is a schematic plan view of a circular shaped electrode;

FIG. 8 is an enlarged hexagonal view of a pole corresponding to FIG. 7;

FIG. 9 is a schematic perspective view of an in-line cell culture detection apparatus;

FIG. 10 is a schematic perspective view of the cell culture on-line measuring device with the lid open;

FIG. 11 is a block diagram of a control unit of the on-line cell culture detecting apparatus;

FIG. 12 is a graph of the growth of cells in multiple channels before and after intervention.

Detailed Description

The following describes in detail embodiments of the cell culture well plate and the cell culture on-line detection device according to the present invention with reference to the accompanying drawings.

Referring to fig. 1, a cell culture well plate 1 includes a well plate base 2, a plurality of wells 3 are disposed on the base 2, and the number of the wells 3 is 16 to 384 wells, generally 16, 48, 96, 384, etc. in an array.

Referring to fig. 2, fig. 2 is a bottom shape of the holes 3 in a form, an electrode body 4 is disposed at the bottom of each hole 3, the electrode body 4 includes a first electrode 5 and a second electrode 6, the first electrode 5 and the second electrode 6 are formed into a plurality of branch electrodes 7 toward opposite sides, and the number of the branch electrodes 7 is preferably 16 to 100, and is determined according to the size of the cultured cells. The branch poles 7 are arranged at intervals, each branch pole 7 is of a zigzag structure, the shape of the electrode body 4 after arrangement is matched with the shape of the bottom of the hole 3, and the electrode body 4 is also circular according to the circular hole 3 in the figure.

Referring to fig. 3, the zigzag bending width of the branch pole 7 is d, the bending angle ω is, and the specific values of the width d and the angle ω are determined according to the actual cell type, so that the contact position between the cell growth process and the branch pole 7 can be optimally matched.

Referring to fig. 4, fig. 4 shows the shape of the electrode at the bottom of the rectangular hole 3. The first electrode 5 and the second electrode 6 are enclosed into a rectangular shape, the branch poles 7 are 16 and are formed by connecting a plurality of rhombuses, and the rhombuses on the adjacent branch poles 7 are staggered.

Referring to fig. 5, in the corresponding diamond parameters, ω is 133 °, and d is 100 μm.

Referring to fig. 6, fig. 6 shows the bottom electrode shape of the diamond shaped holes 3. The first electrode 5 and the second electrode 6 surround a rhombus shape, the branch poles 7 are 20, the branch poles 7 are arranged from the top to the bottom of the rhombus electrode, the branch poles 7 are formed by connecting a plurality of rhombuses, and the rhombuses on the adjacent branch poles 7 are staggered. Corresponding to the diamond parameters in fig. 5, ω is 70 °, d is 50 μm.

Two electrodes of the electrode body 4 are respectively led out to one end of the pore plate base 2, two electrodes of the plurality of holes 3 are respectively arranged at two ends of the pore plate base 2, and the led-out wires at two ends of the pore plate base 2 are arranged into contacts on the lower surface of the pore plate base 2.

On this basis, the cross-sectional shape of the hole 3 may be circular, square, rectangular or hexagonal. Referring to fig. 7 and 8, the first electrode 5 and the second electrode 6 are enclosed into a circular shape, the branch poles 7 are 16 and are formed by connecting a plurality of regular hexagons, and the regular hexagons on the adjacent branch poles 7 are staggered. The spacing between adjacent regular hexagons in each direction is equal. The circumscribed circle diameter r of the regular hexagon is set differently according to different cells.

The bottom of the hole 3 can be made of glass, polyethylene terephthalate, polymethyl methacrylate, polyvinyl chloride and other materials, the electrode body structure is processed at the bottom of the hole 3 through a magnetron sputtering or evaporation process, the outer layer of the electrode body is made of a conductive material with the thickness of 10nm to 500nm, and the conductive material of the outer layer of the electrode body is gold, platinum, rhodium, ruthenium, silver chloride, polypyrrole or poly 3, 4-ethylenedioxythiophene.

When measuring, the cells can be cultured at the bottom of the hole 3 in an adherent way, and most of the cells individually cover the electrode body area and span between the first electrode 5 and the second electrode 6 in the culture process by adjusting the angle and the width of the repeated regular structural unit of the branch electrode.

Referring to fig. 9 and 10, the cell culture well plate 1 is installed in the cell culture on-line detection device 8. The cell culture online detection device 8 is characterized in that a pore plate groove 9 is formed in a base of the cell culture online detection device 8, a cell culture electrode channel pore plate 1 is arranged in the pore plate groove 9, electrode contacts are arranged at two ends of the pore plate groove 9, and after the cell culture pore plate 1 is installed, contacts at two ends of a pore plate base 2 are in contact electrical connection with the electrode contacts at two ends of the pore plate groove 9 and used for electrically connecting electrodes in the cell culture pore plate 1 with the cell culture online detection device 8.

A turning cover 10 is arranged on the pore plate groove 9, when the turning cover 10 is closed, the cell culture pore plate 1 is sealed in the pore plate groove 9 and the turning cover 10, and a transparent observation window is arranged above the turning cover 10. After cells and relevant culture auxiliary materials are added into the cell culture pore plate 1, the turning cover 10 is closed, and the whole cell culture online detection device 8 is placed into an incubator for culture and measurement.

Referring to fig. 11, a control circuit is arranged in the base, electrode contacts at two ends of the pore plate groove are connected to a control unit, the control unit comprises a main control circuit, a signal reference source, a multiplexer and a signal measurement circuit, the main control circuit controls a reference signal source to generate an excitation signal, the multiplexer is connected to electrodes on the cell culture pore plate, the multiplexer is connected with the signal measurement circuit, and the signal measurement circuit measures signals of an electrode passage selected by the multiplexer.

The control unit further comprises a calibration circuit, the calibration circuit is connected to the signal measurement circuit through the multiplexer, the control circuit stores calibration data, and the signal measurement circuit calibrates a signal measurement result through the signal calibration circuit. The control circuit is also connected with a chip in-place detection circuit, the chip in-place detection circuit is connected to the cell culture pore plate, and the loading state of the cell culture pore plate is detected and fed back to the control circuit.

The main control circuit is also connected to a storage unit, and the storage unit is used for storing detection data. The main control circuit is connected to the upper computer software through a communication circuit.

The master control circuit controls the reference signal source to generate an excitation signal with a certain frequency, waveform, amplitude and phase. The method comprises the following steps: sinusoidal, pulsed, triangular, and square wave. The signal measuring circuit amplifies and outputs the excitation signal, simultaneously monitors the output signal and the current signal passing through the cell culture pore plate, and obtains cell response signals under different frequencies in a frequency scanning mode in single measurement for subsequent analysis.

The channel is selected by the multiplexer, the contact probe is connected with each sensing electrode of the cell culture pore plate, and the measurement can be carried out on any channel in a single detection. The multiplexer can connect the signal measuring circuit with the calibration circuit for automatic calibration of the signal measuring result; the calibration data is stored by the master control circuit and is used for compensating the result of the subsequent actual measurement. The chip in-place detection is used for detecting whether the sensing array chip is correctly loaded in real time.

The status indicator lamp is used for indicating the current equipment status including standby, operation, fault and calibration.

The main control circuit is communicated with an upper computer through a communication circuit, and the upper computer sends the measurement result to upper computer software in real time through an interface 11 on the cell culture online detection device 8; if the communication of the upper computer software is interrupted in the running process, the measured data is stored in a storage unit of the equipment and is sent to the upper computer after the communication is recovered.

The measurement process is as follows: the electrode body on the cell culture pore plate is connected to the control circuit, the control circuit can apply electrical stimulation to cells or measure the electrical properties of the cells, the control unit comprises a main control circuit, a signal reference source, a multiplexer and a signal measuring circuit, the main control circuit controls a reference signal source to generate excitation signals, the multiplexer is respectively connected to each first electrode on the cell culture pore plate, the multiplexer connects the signal input end of the signal measuring circuit with one of the first electrodes under the control of the main control circuit, the signal output end of the signal measuring circuit is connected with all the second electrodes, the signal reference source is connected with the signal measuring circuit, the signal measuring circuit conditions the output signals of the signal reference source and outputs the excitation signals to all the second electrodes, and a loop is formed with the first electrode selected by the multiplexer, so that cell electrical stimulation or cell signal measurement is realized.

The purpose of the electrical stimulation of the cells is to modulate the arrangement and state of the cells on the electrode surface by external action, which is represented by the continuous generation of a series of pulses with a voltage amplitude of 10mV to 10V, a frequency of 1Hz to 1kHz and a duty cycle of 1% to 50% with a certain period.

The cell signal measurement aims at estimating the number of cells attached to the bottom of a pore plate by measuring the total electrical quantity of the cells covered on the electrodes so as to estimate the change trend of cell growth, the cell signal measurement principle is that a series of excitation signals with different frequencies are output on a second electrode, the current amplitude and phase change on a selected first electrode are measured simultaneously, signals in an initial cell culture state are used as reference, normalization processing is carried out on the measured current amplitude and phase change data to be used as cell growth state indexes, the cell growth state indexes are recorded at certain time intervals, and a 'cell growth state index-time' curve is drawn to visualize the cell growth trend.

The control unit controls the excitation signal output to the second electrode to be a voltage signal having waveforms of a sine wave, a square wave, a triangular wave, a pulse and the like, and the amplitude of the excitation signal output to the second electrode is 1mV to 150 mV.

The visualization of the cell growth trend can be used for optimizing the stem cell culture process, analyzing the killing effect of cancer cells of anti-cancer drugs, inhibiting the microorganism by antibiotics and monitoring the in-vitro culture condition of functional cells.

In application, the cell to be detected and the culture solution are added into a pore plate of the cell culture online detection device, then the cell culture online detection device is placed into an incubator, the growth, adaptability and behavior of the cell are studied in real time, the invasion and migration of the cell are monitored, the cytotoxic reaction caused by a wide molecular target is monitored, the process of differentiating the stem cell into the somatic cell is captured in real time, the adhesion and the extension of the cell are continuously monitored, and the like. Can be used for cancer immunotherapy.

Referring to fig. 12, fig. 12 is a SKOV3 ovarian cancer tumor cell killing experiment. 20000 tumor cells are respectively paved at the bottom of the cell culture pore plate one day in advance, and the target ratio is paved in the corresponding pore plate 2.5: 1 and 5: 1 with CAR-T cells, for a total of 4 groups, wherein 2.5: 1 is 50000 cells, 5: 1 is 100000 cells. Curves 1 and 2 show that the effective target ratio is 2.5: 1 and 5: 1 blank control NC-T experimental group; curves 3 and 4 show that the effective target ratio is 2.5: 1 and 5: 1 CAR-T cell panel. The experimental results show that the CAR-T cells can remarkably kill tumor cells.

It can be seen from the figure that cells grew normally before killing cancer cells, and at 1200 min, CAR-T cells were added to the CAR-T cell experimental group channels, and then the cell growth tendency (Cellindex) of cancer cells in the channels decreased, while the cells of the blank control group were not affected.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.