CN113621514A

CN113621514A - Cell atrial fibrillation model stimulation culture system

Info

Publication number: CN113621514A
Application number: CN202110806510.8A
Authority: CN
Inventors: 刘弟世闻; 赵庆彦; 杨媚; 潘玉苗
Original assignee: Wuhan University WHU
Current assignee: Wuhan University WHU
Priority date: 2021-07-16
Filing date: 2021-07-16
Publication date: 2021-11-09
Anticipated expiration: 2041-07-16
Also published as: CN113621514B

Abstract

The invention provides a cell atrial fibrillation model stimulation culture system, which belongs to the technical field of medical prediction models and comprises a culture dish unit, a base unit and a host unit, wherein the base unit is arranged below the culture dish unit, the host unit is electrically connected with the base unit, the culture dish unit comprises a plurality of culture dishes of which the bottoms are provided with culture dish electrodes, the base unit comprises a plurality of bases provided with the base electrodes, the top ends of the culture dish electrodes are contacted with a culture medium, the bottom ends of the culture dish electrodes are contacted with the base electrodes, the base electrodes are respectively connected with base leads, and direct current pulses output by the host unit are transmitted to the culture medium arranged in the culture dish through the base leads, the base electrodes and the culture dish electrodes in sequence. The invention can avoid the direct contact of the culture medium with the outside, realize that the external electrode provides experimental pulse under the condition of not contacting the culture medium, and improve the safety and reliability of the experiment.

Description

Cell atrial fibrillation model stimulation culture system

Technical Field

The invention relates to the technical field of medical prediction models, in particular to a cell atrial fibrillation model stimulation culture system.

Background

Atrial Fibrillation (AF) is common arrhythmia in clinic and is one of diseases seriously harming human health, foreign research suggests that the prevalence rate of people is about 0.9%, domestic research shows that the total prevalence rate of AF in China is 0.77%, the incidence rate of AF is in a trend of increasing year by year, and AF patients in China are about 1000 ten thousand at present. Complications such as stroke and heart failure caused by AF thromboembolism seriously affect the quality of life of a patient and even endanger life. The pathophysiological mechanism of AF is complex, and no effective method for radically treating AF exists at present although treatment schemes such as radio frequency ablation operation, medicines and the like exist clinically. Therefore, intensive research on the mechanism of AF is particularly important.

In basic studies, large animals such as dogs or pigs are usually implanted with pacemakers and then rapidly paced to simulate pathological AF models. However, because of the need of an X-ray machine, a pacemaker, a sterile operating room, corresponding personnel allocation and the like, the molding cost is greatly increased, a plurality of laboratories which do not have the conditions have great difficulty in researching AF, and the AF molecular mechanism cannot be deeply researched based on a large animal model.

The cell experiment can overcome the corresponding problems, so that the intensive research on AF from the cell level is very important. At present, common laboratories have essentially no technical capability to make models of cellular AF. Therefore, the traditional method is usually adopted, a culture dish is punched, the electrodes are placed in a culture medium and then are externally connected with relevant instruments such as cell stimulation and the like, the process is quite complicated, cells are easy to pollute, and for some precious samples, huge risks are faced, and experimental failure is easily caused.

Disclosure of Invention

Aiming at the defects in the prior art, the cell atrial fibrillation model stimulation culture system provided by the invention is compatible with mainstream culture dishes such as a 6-hole plate, a middle dish and a big dish, and the culture dish electrode is arranged at the bottom of the culture dish to be in contact with the base electrode, so that an external electrode can provide an experiment pulse under the condition of not contacting a culture medium, and the experiment safety and reliability are improved.

In order to achieve the above purposes, the technical scheme is as follows:

a cell atrial fibrillation model stimulation culture system comprises:

the culture dish unit comprises a plurality of culture dishes, each culture dish is internally provided with a culture medium, and the bottom of each culture dish is provided with a plurality of culture dish electrodes which are in contact with the culture medium; the categories of the plurality of culture dishes include a 6-well plate, a medium dish, and a large dish;

the base unit comprises a plurality of bases matched with the structure of the culture dish unit, each base is used for placing a culture dish, a plurality of base electrodes are arranged on each base, the top end of each base electrode is contacted with the bottom end of one culture dish electrode, and the bottom end of each base electrode is connected with a base lead;

and the host unit comprises a plurality of groups of pulse direct current subsystems, and each group of pulse direct current subsystems are used for providing direct current pulses for the culture medium through a plurality of base leads arranged on a corresponding base.

Preferably, when the type of the culture dish is a 6-well plate, 12 culture dish electrodes are arranged at the bottom of the culture dish, and 2 culture dish electrodes are respectively arranged at each hole of the 6-well plate;

when the type of the culture dish is a medium dish, 2 culture dish electrodes are arranged at the bottom of the medium dish;

when the type of the culture dish is a large dish, 2 culture dish electrodes are arranged at the bottom of the culture dish;

each base is provided with 12 base electrodes.

Preferably, six holes of the 6-hole plate are distributed in two rows and three columns;

a first row of 6-well plates is provided with a first well, a second well, and a third well in sequence;

a fourth hole, a fifth hole and a sixth hole are sequentially arranged in the second row of the 6-hole plate;

the first and fourth apertures are disposed adjacent to each other.

Preferably, 12 base electrodes on the base are distributed in a four-row three-column form;

a first electrode corresponding to the first hole, a second electrode corresponding to the second hole and a third electrode corresponding to the third hole are sequentially arranged in a first row on the base;

a fourth electrode corresponding to the first hole, a fifth electrode corresponding to the second hole and a sixth electrode corresponding to the third hole are sequentially arranged on the second row of the base;

a seventh electrode corresponding to the fourth hole, an eighth electrode corresponding to the fifth hole and a ninth electrode corresponding to the sixth hole are sequentially arranged on the third row of the base;

a tenth electrode corresponding to the fourth hole, an eleventh electrode corresponding to the fifth hole and a twelfth electrode corresponding to the sixth hole are sequentially arranged on the fourth row of the base;

all set up the slip track at second electrode and eleventh electrode department, the slip track sets up along the direction of being listed as, and the distance between the relative both ends of two slip tracks matches with the aperture of well ware, and the distance between the both ends that two spouts are in opposite directions matches with the aperture of big ware, and every slip track's both ends all are equipped with the draw-in groove that is used for the unable adjustment base electrode.

Preferably, the aperture of the 6-well plate, the middle dish, and the large dish is 35mm, 60mm, and 100mm, respectively.

Preferably, the culture dish electrode is a carbon electrode, and an iron layer is wrapped at the contact part of the bottom of the culture dish electrode and the base electrode;

the base electrode adopts a copper electrode, and the contact part of the top of the base electrode and the culture dish electrode is wrapped with a gamma magnet layer.

Preferably, the cell atrial fibrillation model stimulation culture system further comprises:

the top cover unit comprises a plurality of top covers matched with the culture dish unit, each top cover is used for covering a culture dish, and a plurality of top cover electrodes and high-temperature disinfection pieces are arranged on each top cover;

the pulsed dc subsystem is also used to provide dc pulses to the culture medium through the cap electrode.

Preferably, the top cover electrode is a carbon electrode.

Preferably, the direct current pulse output by the pulse direct current subsystem comprises regular pulses and irregular pulses;

the proportion of irregular pulse is 0-99%, and the step length is 1%;

the duration range of the direct current pulse is 1-20ms, and the step length is 1 ms;

the voltage adjustable range of the direct current pulse is 0-24V, and the step length is 0.1V;

the frequency range of the direct current pulse is 0-20Hz, and the step length is 0.1 Hz.

The invention has the beneficial effects that: set up culture dish electrode and base electrode on two faces that culture dish and base contacted each other respectively, the direct current pulse of pulse direct current subsystem output loops through base electrode and culture dish electrode conduction to the built-in culture medium of culture dish to avoid culture medium direct and external contact, realize that external electrode provides experimental pulse under the condition of contactless culture medium, improve experiment security and reliability.

Drawings

Fig. 1 is a schematic structural diagram of a 6-well plate in the embodiment of the present invention.

FIG. 2 is a schematic structural diagram of a middle dish according to an embodiment of the present invention.

FIG. 3 is a schematic structural diagram of a boat in an embodiment of the invention.

Fig. 4 is a schematic structural diagram of the embodiment of the invention when the base is fitted with the 6-hole plate.

FIG. 5 is a bottom schematic view of a 6-well plate fitted with a base according to an embodiment of the present invention.

FIG. 6 is a schematic diagram of the structure of the second electrode and the eleventh electrode when the base is fitted to the boat in the embodiment of the present invention.

FIG. 7 is a bottom view of the base adapted to the boat in accordance with one embodiment of the present invention.

FIG. 8 is a schematic diagram of the structure of the second electrode and the eleventh electrode when the base is adapted to the boat in the embodiment of the present invention.

FIG. 9 is a bottom view of the base adapted to a boat in accordance with an embodiment of the present invention.

FIG. 10 is a schematic diagram of the structure of the culture dish electrode in the embodiment of the present invention.

FIG. 11 is a schematic structural diagram of a base electrode according to an embodiment of the present invention.

FIG. 12 is a circuit diagram of a pulsed DC subsystem according to an embodiment of the present invention.

Fig. 13 is a circuit schematic diagram of a minimum circuit of the single chip microcomputer in the embodiment of the invention.

Fig. 14 is a circuit schematic diagram of a power supply circuit of a single chip microcomputer in the embodiment of the invention.

Fig. 15 is a schematic circuit diagram of a download port of a single chip microcomputer according to an embodiment of the present invention.

Fig. 16 is a schematic circuit diagram of a serial lcd interface of a single chip in the embodiment of the present invention.

FIG. 17 is a circuit diagram of a pulse generating circuit according to an embodiment of the present invention.

FIG. 18 is a circuit diagram of an ACDC circuit according to an embodiment of the present invention.

Fig. 19 is a circuit schematic diagram of a power supply circuit of a 3.3V single chip microcomputer in the embodiment of the invention.

FIG. 20 is a schematic circuit diagram of a 24V pulse power supply circuit according to an embodiment of the present invention.

Reference numerals:

1-6 pore plates; 2-medium vessel; 3-big dish; 4-a base; 5-a culture dish electrode; 6-a base electrode; 7-a first hole; 8-a second well; 9-a third aperture; 10-fourth well; 11-fifth hole; 12-sixth hole; 13-a first electrode; 14-a second electrode; 15-a third electrode; 16-a fourth electrode; 17-a fifth electrode; 18-a sixth electrode; 19-a seventh electrode; 20-an eighth electrode; 21-ninth electrode; 22-tenth electrode; 23-an eleventh electrode; 24-a twelfth electrode; 25-a sliding track; 26-base leads; 27-an iron layer; 28-gamma magnet layer.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific examples described herein are intended to be illustrative only and are not intended to be limiting. Moreover, all other embodiments that can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort belong to the protection scope of the present invention.

The present invention will be described in further detail with reference to the accompanying drawings and examples.

As shown in fig. 1 to 9, the present invention provides a cell atrial fibrillation model stimulation culture system, which includes a culture dish unit, a base unit disposed below the culture dish unit, and a host unit electrically connected to the base unit. The culture dish unit includes that a plurality of bottoms are equipped with the culture dish of culture dish electrode 5, and the base unit includes a plurality of bases 4 that are equipped with base electrode 6. 5 tops of culture dish electrode and culture medium contact, the bottom contacts with the top of base electrode 6, base wire 26 is connected to the bottom of base electrode 6, and the direct current pulse of host computer unit output loops through base wire 26, base electrode 6 and culture dish electrode 5 and conducts to the built-in culture medium of culture dish to avoid the culture medium direct and external contact, realize that external electrode provides experimental pulse under the condition of contactless culture medium, improve experiment security and reliability.

Specifically, the culture dish is synthesized from high-purity polystyrene and coated with polylysine, and each culture dish in the culture dish unit is filled with a culture medium and provided with a plurality of culture dish electrodes 5 contacting the culture medium at the bottom. The categories of the plurality of culture dishes include a 6-well plate 1, a middle dish 2, and a large dish 3.

Further, 6 orifice plates 1, medium dishes 2 and big dishes 3 can be compatible with same base 4, and if the base unit is provided with 6 bases, then the whole base unit can be compatible with 6 orifice plates 1 simultaneously, or compatible with 6 medium dishes 2 through the position of moving base electrode 6, or compatible with 6 big dishes 3 through the position of moving base electrode 6, and the corresponding host unit is provided with 6 pulse direct current subsystems to output direct current pulses to 6 bases respectively. According to the requirement of culturing cells, the number and the structural size of the culture dishes in the culture dish unit, the number and the structural size of the bases 4 in the base unit and the number and the output of the pulse direct current subsystems of the host unit can be adjusted according to the requirement. For example, a base system similar to the 6-well plate 1 compatible with the medium dish 2 or the large dish 3 can be individually designed according to the requirement of culturing cells.

As shown in FIG. 1, when the type of the dish is a 6-

well plate

1, 12 dish electrodes 5 are provided at the bottom of the plate, and 2 dish electrodes 5 are provided at each well of the 6-well plate 1. The aperture of the 6-hole plate 1 is 35 mm.

As shown in FIG. 2, when the type of the culture dish is the

middle dish

2, 2 culture dish electrodes 5 are provided on the bottom thereof. The aperture of the middle dish 2 is 60 mm.

As shown in FIG. 3, when the type of the culture dish is a

large dish

3, 2 dish electrodes 5 are provided on the bottom thereof. The aperture of the large dish 3 is 100 mm.

As shown in fig. 4 to 5, 7, and 9, the base 4 is provided with 12 base electrodes 6.

Further, with continued reference to fig. 1, the six holes of the 6-hole plate 1 are distributed in two rows and three columns.

The first row of the 6-well plate 1 is provided with a first well 7, a second well 8, and a third well 9 in that order.

The fourth well 10, the fifth well 11, and the sixth well 12 are provided in this order in the second row of the 6-well plate 1.

The first aperture 7 and the fourth aperture 10 are arranged adjacently.

Further, as shown in fig. 4 to 9, 12 base electrodes 6 on the base 4 are distributed in four rows and three columns.

The first row on the base 4 is provided with a first electrode 13 in a position corresponding to the first hole 7, a second electrode 14 in a position corresponding to the second hole 8, and a third electrode 15 in a position corresponding to the third hole 9 in this order.

A second row on the base 4 is provided in sequence with a fourth electrode 16 corresponding to the position of the first hole 7, a fifth electrode 17 corresponding to the position of the second hole 8, and a sixth electrode 18 corresponding to the position of the third hole 9.

The seventh electrode 19 corresponding to the position of the fourth hole 10, the eighth electrode 20 corresponding to the position of the fifth hole 11, and the ninth electrode 21 corresponding to the position of the sixth hole 12 are sequentially arranged in the third row on the base 4.

The tenth electrode 22 corresponding to the position of the fourth hole 10, the eleventh electrode 23 corresponding to the position of the fifth hole 11, and the twelfth electrode 24 corresponding to the position of the sixth hole 12 are sequentially arranged in the fourth row on the base 4.

The second electrode 14 and the eleventh electrode 23 are both provided with a sliding rail 25, the sliding rails 25 are arranged along the row direction, the distance between the two opposite ends of the two sliding rails 25 is matched with the aperture of the middle dish 2, the distance between the two opposite ends of the two sliding chutes is matched with the aperture of the big dish 3, and the two ends of each sliding rail 25 are provided with clamping grooves for fixing the base electrode 6. Namely, the second electrode 14 and the eleventh electrode 23 slide along the sliding rail 25 to the inside to the clamping groove to be matched with the medium dish 2, and slide along the sliding rail 25 to the outside to be matched with the large dish 3.

Further, as shown in fig. 10, the above-mentioned culture dish electrode 5 is a carbon electrode, and the contact portion of the bottom thereof with the base electrode 6 is covered with an iron layer 27. As shown in FIG. 11, the base electrode 6 is a copper electrode, and a gamma magnet layer 28 and an iron layer 27 are wrapped around the contact portion of the top of the copper electrode and the petri dish electrode 5.

Further, the cell atrial fibrillation model stimulation culture system further comprises:

and a lid unit (not shown) including a plurality of lids fitted to the dish unit, each lid being for covering a dish, each lid being provided with a plurality of lid electrodes and a high-temperature sterilization member. The top cover unit can be compatible with a 6-hole plate 1, a middle dish 2 and a big dish 3 which are commonly used in the market, and a high-temperature disinfection piece arranged on the top cover is used for self disinfection. Whether the petri dish is covered with the cap is determined according to experimental requirements, for example, if a sterile petri dish is used, the petri dish may not be covered with the cap.

The top electrode (not shown) is a carbon electrode.

The structure of the top cover is similar to that of the

base

4, and 12 top cover electrodes matched with the culture dish electrodes 5 in position can be arranged on the top cover.

Further, the dc pulse output by the pulsed dc subsystem includes regular pulses and irregular pulses.

The proportion of irregular pulse is 0-99%, and the step length is 1%.

The duration range of the direct current pulse is 1-20ms, and the step length is 1 ms.

The voltage adjustable range of the direct current pulse is 0-24V, and the step length is 0.1V.

Further, as shown in fig. 12, a schematic circuit diagram of a pulse dc subsystem is shown, where the pulse dc subsystem includes a power supply circuit module a, a single-chip microcomputer control module B, and a pulse transmitting module C.

The power supply circuit module A mainly has the function of providing power for the singlechip control module B and the pulse transmitting module C.

Specifically, the voltage-reducing circuit is used for converting 220V alternating current into 24V direct current, the 24V direct current has two purposes, one is converted into 3.3V output after 5V voltage reduction and used for driving a singlechip control module B, and the other is directly output by the 24V direct current and used for driving a pulse transmitting module C.

The 1 power supply circuit module A comprises 6 pulse power supply circuits, the 6 pulse generating circuits can be independently controlled and can respectively output pulse voltages with different frequencies to the same culture dish, and the pulse mode is also determined according to different forms of PWM waves.

The singlechip control module B mainly has the functions of adjusting and controlling the output frequency, the output voltage and the display control operation of the pulse transmitting module C.

Specifically, the singlechip control module B is modified by a conventional singlechip, and is externally provided with a liquid crystal interface and a programming interface. The single chip microcomputer outputs two PWM (Pulse width modulation) waves, one is output to the 6-path Pulse transmitting module C through an IO port named as PWM1-36 to control a Pulse switch, and the other is used for disclosing the Pulse power supply voltage through the IO port 1-path power supply circuit module A named as DAC 1-6. That is, 36 switches are independent, and the voltage value is 6 switches independent.

The pulse emitting module C has the main function of emitting a stimulus waveform, and the waveform output is connected with the base system through the base lead 26.

Specifically, the power supply circuit module A provides a 24V constant voltage, the single chip microcomputer controls the constant voltage direct current to be converted into a stimulation pulse which can be adjusted in a range of 0-24V in the pulse transmitting module C, the single chip microcomputer can also control the adjusting frequency, and the pulse transmitting module C connects the adjusted pulse direct current to the base electrode 6 through the base lead 26, so that a closed circuit is formed on the base to complete output.

The 1 pulse transmitting module C includes 6 pulse generating circuits.

As shown in fig. 13, which is a schematic circuit diagram of a minimum circuit of a single chip, the output PWM of the single chip may be replaced by a low-cost single chip, which includes 36 IO ports named as PWM1-36 and 6 IO ports named as DAC1-6, pins PCB7-PCB11 are reserved 4 interfaces, pin BOOT0 is grounded, pin NRST is connected to a midpoint between resistor R1 and capacitor C6, resistor R1 is connected to 33V input, and capacitor C6 is grounded.

As shown in fig. 14, which is a schematic circuit diagram of a power circuit of a single chip microcomputer, the 33V output is grounded through 5 capacitors.

As shown in fig. 15, it is a schematic circuit diagram of the download port of the single chip microcomputer.

As shown in fig. 16, the schematic circuit diagram of the serial lcd interface of the single chip microcomputer can be modified according to practical applications.

As shown in fig. 17, which is a schematic circuit diagram of a pulse generating circuit, the 6 pulse generating circuits in the same pulse transmitting module C have the same circuit structure. One input pin of the optocoupler in the figure is connected with 33V input through a resistor R91, and the other input pin of the optocoupler is connected with an IO pin of the singlechip output PWM 1. An output pin of the optical coupler is connected with an IO pin of a single chip microcomputer output DAC6 through a resistor R89, a grid electrode of a P-channel enhancement type MOS tube Q36 is connected with the output pin, a drain electrode is connected with a resistor R89, a source electrode is connected with an electrode plate load P2, and the electrode plate load P2 is further connected with the other output pin of the optical coupler.

In the figure, a P-channel enhancement type MOS tube Q36 is used as a power switch for outputting pulses with different frequencies.

Fig. 18 is a schematic circuit diagram of an ACDC circuit for converting 220V ac to 24V dc.

As shown in fig. 19, it is a schematic circuit diagram of a power circuit of a 3.3V single chip microcomputer.

As shown in fig. 20, which is a schematic circuit diagram of a 24V pulse power supply circuit, a 20V-1.23V programmable voltage source is used for pulse excitation power supply, the duty ratio is configured to be 30% -70%, and the PWM amplitude is 3.3V.

The present invention is not limited to the above-described embodiments, and it will be apparent to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and such modifications and improvements are also considered to be within the scope of the present invention. Those not described in detail in this specification are within the skill of the art.

Claims

1. A cell atrial fibrillation model stimulation culture system is characterized by comprising:

2. The cell atrial fibrillation model-stimulated culture system of claim 1, wherein when the culture dish is a 6-well plate, 12 culture dish electrodes are arranged at the bottom of the culture dish, and 2 culture dish electrodes are respectively arranged at each hole of the 6-well plate;

each base is provided with 12 base electrodes.

3. The cell atrial fibrillation model-stimulated culture system of claim 2, wherein the six wells of the 6-well plate are distributed in two rows and three columns;

the first and fourth apertures are disposed adjacent to each other.

4. The cell atrial fibrillation model stimulation and culture system of claim 3, wherein the 12 base electrodes on the base are distributed in four rows and three columns;

5. The cell atrial fibrillation model-stimulated culture system of claim 1, wherein the aperture of each of the 6-well plate, the middle dish and the large dish is 35mm, 60mm and 100 mm.

6. The cell atrial fibrillation model stimulation culture system of claim 1, wherein the culture dish electrode is a carbon electrode, and the contact part of the bottom of the culture dish electrode and the base electrode is wrapped with an iron layer;

7. The cell atrial fibrillation model stimulation culture system of claim 1, wherein the cell atrial fibrillation model stimulation culture system further comprises:

8. The cell atrial fibrillation model-stimulated culture system of claim 7, wherein the top cover electrode is a carbon electrode.

9. The cell atrial fibrillation model stimulation culture system of claim 1, wherein the direct current pulses output by the pulsed direct current subsystem comprise regular pulses and irregular pulses;

the proportion of irregular pulse is 0-99%, and the step length is 1%;