Maintaining culture dish for myocardial slice

Abstract

The maintaining culture dish comprises a culture dish body provided with an inner cavity and a myocardial slice supporting device arranged in the inner cavity, the culture dish body is made of a flexible material, and the inner cavity is filled with a tissue culture solution; the culture dish is detachably connected with an intermittent stretching device; the myocardial slice supporting device is used for placing a myocardial slice and maintaining the middle part of the myocardial slice above the bottom wall of the inner cavity; the intermittent stretching device is used for driving the myocardial slice in the culture dish to generate periodic stretching according to a set frequency. The culture mode of the myocardial slice can be optimized, the survival rate and the growth quality of myocardial cells are improved, and a stable physiological structure is maintained; and a more reliable and accurate experimental platform is provided for biomedical research and clinical application.

Description

Technical Field

[0001] The invention relates to the technical field of in vitro cell culture, in particular to a maintenance culture dish for myocardial slices. Background Art

[0002] Cardiomyocytes have unique physiological and morphological characteristics, such as the inability to divide and proliferate, the tight connection between cells and a high degree of functional coordination; these characteristics make the culture of cardiomyocytes more complex than other types of cells. In biomedical research and clinical applications, the culture of myocardial slices is an important means to understand the mechanism of heart disease, develop treatment methods and evaluate drug effects; in particular, the precious living myocardial tissue in clinical practice is of great significance to the mechanism of disease occurrence and development, drug screening and new drug development.

[0003] Traditional myocardial slice culture methods have many shortcomings, such as difficult to control the culture environment, low cell survival rate, and unstable physiological structure and function of the slices.

[0004] The inventor knows of a soft cell culture dish (CN 116333882A) that can achieve radial tension and compression and out-of-plane bending, which includes a drive unit and a control unit; the drive unit includes a radial tension and compression driver and a bending driver; the control unit is used to perform pneumatic drive control on the radial tension and compression driver and the bending driver; the cells are located on the upper surface of the radial tension and compression driver; the radial tension and compression driver is arranged on the upper part of the bending driver; the radial tension and compression driver is evacuated or inflated to apply radial pressure to the cells; the bending driver is evacuated to drive the radial tension and compression driver to undergo bending deformation, and as the cells grow downward, the culture dish always remains in a state of covering the cells.

[0005] However, the above application has at least the following technical problems: Cell culture substrates cannot simulate the real scene required for cell growth (e.g., culture fluid), which is not conducive to cell growth and culture. Culture substrates cannot achieve cell fixation or position limitation, which is not conducive to subsequent experiments and observations. Summary of the invention

[0006] The purpose of the present invention is to provide a maintenance culture dish for myocardial slices, which can optimize the culture method of myocardial slices, improve the survival rate and growth quality of myocardial cells, maintain a stable physiological structure, and provide a more reliable and accurate experimental platform for biomedical research and clinical applications.

[0007] The present invention adopts the following technical solutions: A culture dish for maintaining myocardial slices comprises a culture dish with an inner cavity and a myocardial slice supporting device arranged in the inner cavity, wherein the culture dish is made of a flexible material and the inner cavity is filled with a tissue culture fluid; the culture dish is detachably connected to an intermittent stretching device; the myocardial slice supporting device is used to place the myocardial slices and maintain the middle of the myocardial slices above the bottom wall of the inner cavity, and the intermittent stretching device is used to drive the myocardial slices in the culture dish to produce periodic stretching according to a set frequency.

[0008] The myocardial slice supporting device comprises raised platforms symmetrically arranged at the left and right ends of the inner cavity, and the upper end surface of the raised platforms is higher than the bottom wall of the inner cavity of the culture dish.

[0009] It also includes a myocardial slice fixing device, which is used to fix the myocardial slice on the myocardial slice supporting device.

[0010] It also includes an electrical stimulation device, which is used to stimulate the myocardial slices with electric current according to a set frequency.

[0011] The intermittent stretching device adopts an electric-controlled stretching device; the electric-controlled stretching device is provided with a stretching arm that reciprocates periodically, and mounting holes corresponding to the stretching arm are arranged around the culture dish.

[0012] The myocardial slice fixing structure includes a negative pressure channel arranged in the myocardial slice supporting devices at both ends, and a plurality of pressure difference through holes penetrating the upper end surface of the myocardial slice supporting devices. The negative pressure channel is connected to the plurality of pressure difference through holes, and the plurality of pressure difference through holes are correspondingly closed at both ends of the myocardial slice.

[0013] The negative pressure channel utilizes flowing gas or liquid to achieve negative pressure at the pressure difference through hole.

[0014] The myocardial slice fixing structure also includes a circulation system, which includes a containing box provided with a containing cavity, the containing cavity is filled with tissue culture fluid, and a culture dish is arranged in the containing cavity; a pressure reducing pipeline connected to a micro liquid pump in the middle is provided on the containing box; the rear ends of the negative pressure channels in the myocardial slice supporting devices at both ends of the culture dish are connected, the front end of one of the negative pressure channels is connected to the liquid outlet of the micro liquid pump through the pressure reducing pipeline, and the front end of the other negative pressure channel is connected to the containing cavity.

[0015] The front and rear sides of the culture dish are provided with connecting holes at positions higher than the upper end surface of the myocardial slice supporting device, and the connecting holes connect the culture dish and the containing box to form a connecting vessel.

[0016] The intermittent stretching device comprises a water outlet pipe penetrating the bottom wall of the culture dish, the outlet of the water outlet pipe is located in the culture dish and lower than the upper end surface of the myocardial slice support device; the water inlet of the water outlet pipe is connected to the outlet of the micro liquid pump through a connecting pipe provided with an electric control valve.

[0017] The present invention provides a myocardial slice support device, which can make the myocardial slice suspended in the inner cavity, increase the contact area with the tissue culture fluid in the inner cavity, enhance the gas exchange effect, and reduce the probability of hypoxia caused by the myocardial slice adhering to the wall; the present invention provides an intermittent stretching device, which can drive the myocardial slice in the culture dish to produce periodic stretching according to a set frequency, so as to simulate the contraction and stretching state of myocardial tissue in the human body environment, maintain the normal physiological structure and function of the myocardial slice, and improve the survival rate of myocardial cells; Furthermore, the present invention can ensure the fixing effect of the two ends of the myocardial slice and the corresponding myocardial slice supporting device by providing a myocardial slice fixing device; Furthermore, the present invention is also provided with an electrical stimulation device, which can further simulate the normal physiological state of myocardial cells or tissues to achieve the effect of stimulating myocardial slices. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the structure of the myocardial slice of the present invention; Figure 2 It is a structural schematic diagram of the culture dish of the present invention; Figure 3 It is a structural schematic diagram of the elevated platform of the present invention; Figure 4 It is a structural schematic diagram of the negative pressure channel of the present invention; Figure 5 It is a schematic diagram of the structure of the pressure difference through hole of the present invention; Figure 6 It is a schematic structural diagram of the micro liquid pump of the present invention. DETAILED DESCRIPTION

[0019] Figures 1 to 6 As shown, the culture dish 2 for maintaining myocardial slices 1 of the present invention comprises a culture dish 2 provided with an inner cavity and a myocardial slice supporting device provided in the inner cavity, the culture dish 2 is made of a flexible material and the inner cavity is filled with tissue culture fluid; the culture dish 2 is detachably connected with an intermittent stretching device; The myocardial slice support device is used to place the two ends of the myocardial slice 1 and maintain the middle of the myocardial slice 1 above the bottom wall of the inner cavity, so that the myocardial slice 1 is suspended in the inner cavity, increasing the contact area with the tissue culture fluid in the inner cavity, enhancing the gas exchange effect, and reducing the probability of hypoxia caused by the myocardial slice 1 adhering to the wall; The intermittent stretching device is used to drive the myocardial slice 1 in the culture dish 2 to produce periodic stretching at a set frequency to simulate the contraction and stretching state of myocardial tissue in the human body environment, maintain the normal physiological structure and function of the myocardial slice 1, and improve the survival rate of myocardial cells.

[0020] In the present invention, the culture dish 2 made of a flexible material can produce a deformation effect in conjunction with an intermittent stretching device. The flexible material can be PDMS. PDMS has the advantages of being non-toxic and having good biocompatibility, and also has excellent ductility, resilience, durability and light transmittance, can ensure that the environment in the culture dish 2 is stable, and has the advantages of not damaging the myocardial slice 1, and being convenient for observing the myocardial cells and the myocardial slice 1 morphology, color, wall adhesion and other indicators at any time.

[0021] In order to facilitate supporting the two ends of the myocardial slice 1, in the present invention, the myocardial slice supporting device includes a raised platform 3 symmetrically arranged at the left and right ends of the inner cavity, and the upper end surface of the raised platform 3 is higher than the bottom wall of the inner cavity of the culture dish 2. When in use, the two ends of the myocardial slice 1 are respectively placed on the upper end surfaces of the raised platform 3 at the two ends of the inner cavity.

[0022] In order to further enhance the fixation effect of the myocardial slice 1, a myocardial slice fixation device is also provided in the present invention.

[0023] In this embodiment, the myocardial slice fixing device can use a needle row 4; the needle row 4 is used to pierce the two ends of the myocardial slice 1 until the lower part of the needle row 4 is inserted into the upper part of the raising platform 3, so that the two ends of the myocardial slice 1 are fixed to the corresponding raising platform 3, ensuring that the myocardial slice 1 is suspended in the inner cavity, increasing the contact area with the tissue culture fluid in the inner cavity, and enhancing the gas exchange effect.

[0024] In the present invention, in order to further simulate the normal physiological state of myocardial cells or tissues, an electric stimulation device is also provided. The electric stimulation device can use a power supply connected to electrodes 5, and the positive and negative electrodes 5 can be electrically connected to the row of needles 4 set on the raised platform 3 on one side. The power supply is used to stimulate the myocardial slice 1 with current through the electrodes 5 and the row of needles 4 according to the set frequency, so as to achieve the effect of stimulating the myocardial slice 1.

[0025] In the present invention, the intermittent stretching device can be an electrically controlled stretching device; the electrically controlled stretching device is provided with a stretching arm 6 that reciprocates periodically, and mounting holes corresponding to the stretching arms 6 are provided around the culture dish 2. In this embodiment, the electrically controlled stretching device is provided with two groups of stretching arms 6 symmetrically, and each group of stretching arms 6 includes two stretching rods symmetrically arranged front and back; four groups of stretching arm 6 mounting holes are correspondingly provided around the culture dish 2, and each stretching rod is respectively inserted into the corresponding mounting hole.

[0026] When in use, the two groups of stretching arms 6 perform periodic alternating movements toward and against each other at a set frequency and strength, so that the culture dish 2 produces corresponding periodic stretching and contraction deformations, and finally act on the myocardial slice 1 through the myocardial slice fixing device, thereby achieving the purpose of simulating the contraction and stretching state of myocardial tissue in the human body environment, thereby maintaining the normal physiological structure and function of the myocardial slice 1 and improving the survival rate of myocardial cells.

[0027] Considering that the needle row 4 can achieve a good fixation effect, it may cause certain damage and destruction to the morphology of the myocardial slice 1 and the internal myocardial cells, the present invention also designs a myocardial slice fixation device based on the negative pressure principle and an intermittent stretching device coordinated therewith.

[0028] In this embodiment, the myocardial slice fixing structure includes a negative pressure channel 7 arranged in the raised platforms 3 at both ends and a plurality of pressure difference holes 8 penetrating the upper end surface of the raised platforms 3. The negative pressure channel 7 and the plurality of pressure difference holes 8 are connected, and the two ends of the myocardial slice 1 correspond to the plurality of pressure difference holes 8 closed; that is, the negative pressure at the pressure difference holes 8 and the pressure of the tissue culture fluid can be used to fix the two ends of the myocardial slice 1, instead of fixing it with the row of needles 4.

[0029] During the working process, the inner cavity of the culture dish 2 is filled with static tissue culture fluid; by covering the myocardial slice 1 on the upper surface of a plurality of pressure difference through holes 8, at this time, the two ends of the myocardial slice 1 are subjected to the negative pressure at the pressure difference through holes 8 and the pressure of the tissue culture fluid, and will be tightly attached to the upper surface of the pressure difference through holes 8, thereby making the two ends of the myocardial slice 1 close to the corresponding upper end surface of the raising platform 3.

[0030] The negative pressure at the pressure difference through hole 8 can be achieved by using flowing gas or liquid in the negative pressure channel 7 .

[0031] Based on the tissue culture fluid required for the experiment, other media that may cause contamination of the myocardial slice 1 are discarded. In this embodiment, the tissue culture fluid is used to achieve the negative pressure in the negative pressure channel 7. The negative pressure channel 7 is filled with the tissue culture fluid, and the tissue culture fluid in the negative pressure channel 7 is made to flow, so that negative pressure is formed at the pressure difference through hole 8 in the negative pressure channel 7.

[0032] In order to enable the tissue culture fluid in the negative pressure channel 7 to circulate and be reused; in this embodiment, the myocardial slice fixing structure also includes a circulation system connected to the culture dish 2, and the circulation system includes a receiving box 9 provided with a receiving cavity, the receiving cavity is filled with tissue culture fluid, and the bottom surface of the receiving cavity is provided with a receiving groove adapted to the shape of the culture dish 2, and the culture dish 2 is arranged in the receiving groove and maintains a stable position. A receiving space for the tissue culture fluid is formed between the outer side of the culture dish 2 and the receiving cavity of the receiving box 9, and a pressure reduction pipeline 10 is provided at the lower part of the receiving box 9, and the pressure reduction pipeline 10 is connected to the pressure difference through hole 8 in the culture dish 2 through the negative pressure channel 7; the middle part of the pressure reduction pipeline 10 is connected to a micro liquid pump 11, and the micro liquid pump 11 is used to circulate the tissue culture fluid in the pressure reduction pipeline 10 and the negative pressure channel 7, so that negative pressure is generated at the pressure difference through hole 8 in the culture dish 2. The use of the micro liquid pump 11 belongs to the prior art and will not be repeated here.

[0033] In order to form a circulation of the depressurization pipeline 10, the negative pressure channel 7 and the tissue culture fluid in the accommodating chamber, in the present invention, the front and rear ends of the raised platforms 3 at the left and right ends of the inner chamber of the culture dish 2 extend to the inner wall of the inner chamber of the culture dish 2, and the openings of the negative pressure channel 7 at the rear ends of the two raised platforms 3 are connected through a U-shaped tube, and the opening of the negative pressure channel 7 at the front end of the raised platform 3 on one side is connected to the liquid outlet of the micro liquid pump 11 as a liquid inlet, and the opening of the negative pressure channel 7 at the front end of the raised platform 3 on the other side is connected to the accommodating chamber of the accommodating box 9 as a liquid outlet. Under the action of the micro liquid pump 11, the tissue culture fluid in the accommodating chamber is injected into the liquid inlet of the negative pressure channel 7 in the raised platform 3 on one side through the micro liquid pump 11, and flows into the accommodating chamber of the accommodating box 9 from the liquid outlet of the negative pressure channel 7 in the raised platform 3 on the other side through the U-shaped tube, thereby forming a circulation of the tissue culture fluid.

[0034] During the culture process of the myocardial slice 1, there may be a situation where both ends of the myocardial slice 1 cannot completely cover all the pressure difference through holes 8. Due to the negative pressure at the pressure difference through holes 8, when the pressure difference through holes 8 are exposed, the tissue culture fluid in the culture dish 2 will flow into the receiving chamber of the receiving box 9 through the negative pressure channel 7, causing the level of the tissue culture fluid in the culture dish 2 to drop. In order to replenish the tissue culture fluid in the culture dish 2 in time, in this embodiment, a connecting hole 12 is also opened at a position slightly higher than the raising platform 3 on the front and rear sides of the culture dish 2. The connecting hole 12 can make the culture dish 2 and the receiving box 9 form a connecting vessel, thereby maintaining the tissue culture fluid level in the culture dish 2 and the receiving box 9 higher than the myocardial slice 1, so that the myocardial slice 1 is always in the tissue culture fluid. Even if part of the tissue culture fluid in the culture dish 2 flows into the receiving chamber of the receiving box 9 through the negative pressure channel 7, due to the effect of the connecting hole 12, the tissue culture fluid in the receiving chamber of the receiving box 9 can quickly flow back to the culture dish 2 to maintain the liquid level in the culture dish 2.

[0035] In order to cooperate with the myocardial slice fixing device based on the negative pressure principle, in this embodiment, a special intermittent stretching device is also provided based on the tissue culture fluid required for the experiment. The micro liquid pump 11 used to generate negative pressure is used to achieve intermittent surge of the tissue culture fluid in the culture dish 2, and the fluid impact force generated during the surge is used to achieve intermittent stretching of the myocardial slice 1.

[0036] In this embodiment, the intermittent stretching device includes a water outlet pipe 13 passing through the bottom wall of the culture dish 2, the water outlet of the water outlet pipe 13 is located between the two raised platforms 3 in the culture dish 2 and is lower than the upper end surface of the raised platform 3; the water inlet of the water outlet pipe 13 is connected to the water outlet of the micro liquid pump 11 through a connecting pipe 15 provided with an electric control valve 14. The electric control valve 14 can control the intermittent conduction of the connecting pipe 15, so that the tissue culture fluid in the accommodating cavity is intermittently injected into the culture dish 2 through the water outlet pipe 13, and the tissue culture fluid is surged below the myocardial slice 1. The surging tissue culture fluid squeezes the middle position of the myocardial slice 1 to achieve intermittent stretching of the myocardial slice 1, thereby achieving the purpose of simulating the contraction and stretching state of the myocardial tissue in the human body environment, thereby maintaining the normal physiological structure function of the myocardial slice 1 and improving the survival rate of myocardial cells.

[0037] This embodiment is based on the tissue culture fluid required for the experiment, and discards other media that may cause contamination of the myocardial slice 1. The tissue culture fluid is used to fix the myocardial slice 1, and the intermittent stretching of the myocardial slice 1 is achieved by generating a surge of the tissue culture fluid, ultimately achieving a good simulation of the contraction and stretching state of the myocardial tissue in the human body environment.

Claims

1. A culture dish for maintaining myocardial slices, characterized in that: It comprises a culture dish with an inner cavity and a myocardial slice supporting device arranged in the inner cavity, wherein the culture dish is made of a flexible material and the inner cavity is filled with tissue culture fluid; the culture dish is detachably connected with an intermittent stretching device; the myocardial slice supporting device is used for placing myocardial slices and maintaining the middle of the myocardial slices above the bottom wall of the inner cavity, and the intermittent stretching device is used for driving the myocardial slices in the culture dish to produce periodic stretching according to a set frequency.

2. The culture dish for maintaining myocardial slices according to claim 1, characterized in that: The myocardial slice supporting device comprises raised platforms symmetrically arranged at the left and right ends of the inner cavity, and the upper end surface of the raised platforms is higher than the bottom wall of the inner cavity of the culture dish.

3. The culture dish for maintaining myocardial slices according to claim 1, characterized in that: It also includes a myocardial slice fixing device, which is used to fix the myocardial slice on the myocardial slice supporting device.

4. The culture dish for maintaining myocardial slices according to claim 1, characterized in that: It also includes an electrical stimulation device, which is used to stimulate the myocardial slices with electric current according to a set frequency.

5. The culture dish for maintaining myocardial slices according to claim 1, characterized in that: The intermittent stretching device adopts an electric-controlled stretching device; the electric-controlled stretching device is provided with a stretching arm that reciprocates periodically, and mounting holes corresponding to the stretching arm are arranged around the culture dish.

6. The culture dish for maintaining myocardial slices according to claim 3, characterized in that: The myocardial slice fixing structure includes a negative pressure channel arranged in the myocardial slice supporting devices at both ends, and a plurality of pressure difference through holes penetrating the upper end surface of the myocardial slice supporting devices. The negative pressure channel is connected to the plurality of pressure difference through holes, and the plurality of pressure difference through holes are correspondingly closed at both ends of the myocardial slice.

7. The culture dish for maintaining myocardial slices according to claim 6, characterized in that: The negative pressure channel utilizes flowing gas or liquid to achieve negative pressure at the pressure difference through hole.

8. The culture dish for maintaining myocardial slices according to claim 6, characterized in that: The myocardial slice fixing structure also includes a circulation system, which includes a containing box provided with a containing cavity, the containing cavity is filled with tissue culture fluid, and a culture dish is arranged in the containing cavity; a pressure reducing pipeline connected to a micro liquid pump in the middle is provided on the containing box; the rear ends of the negative pressure channels in the myocardial slice supporting devices at both ends of the culture dish are connected, the front end of one of the negative pressure channels is connected to the liquid outlet of the micro liquid pump through the pressure reducing pipeline, and the front end of the other negative pressure channel is connected to the containing cavity.

9. The culture dish for maintaining myocardial slices according to claim 8, characterized in that: The front and rear sides of the culture dish are provided with connecting holes at positions higher than the upper end surface of the myocardial slice supporting device, and the connecting holes connect the culture dish and the containing box to form a connecting vessel.

10. The culture dish for maintaining myocardial slices according to claim 8, characterized in that: The intermittent stretching device comprises a water outlet pipe penetrating the bottom wall of the culture dish, the outlet of the water outlet pipe is located in the culture dish and lower than the upper end surface of the myocardial slice support device; the water inlet of the water outlet pipe is connected to the outlet of the micro liquid pump through a connecting pipe provided with an electric control valve.

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