A maintenance culture dish for myocardial slices

Abstract

The present invention discloses a maintenance culture dish for myocardial slices, which includes a culture dish provided with an inner cavity and a myocardial slice support device arranged in the inner cavity. The culture dish is made of a flexible material and the inner cavity is filled with tissue culture medium; the culture dish is detachably connected with an intermittent stretching device; the myocardial slice support device is used to place the myocardial slice and maintain the middle part of the myocardial slice above the bottom wall of the inner cavity, and the intermittent stretching device is used to drive the myocardial slice in the culture dish to generate periodic stretching according to a set frequency. The present invention can optimize the culture method of myocardial slices, improve the survival rate and growth quality of myocardial cells, and maintain a stable physiological structure; it provides a more reliable and accurate experimental platform for biomedical research and clinical applications.

Description

Technical Field

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

[0002] Cardiomyocytes have unique physiological and morphological characteristics, such as: the characteristics of non - division and proliferation, the tight junction characteristics between cells, and high 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 mechanisms of heart diseases, develop treatment methods, and evaluate the effects of drugs; especially precious living myocardial tissues in clinical practice, which are of great significance for understanding the mechanisms of disease occurrence and development, drug screening, and new drug research and development.

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

[0004] A soft cell culture dish (CN 116333882A) known to the inventors that can achieve radial tension - compression and out - of - plane bending includes a driving unit and a control unit; the driving unit includes a radial tension - compression driver and a bending driver; the control unit is used to perform pneumatic driving control on the radial tension - compression driver and the bending driver; cells are located on the upper surface of the radial tension - compression driver; the radial tension - compression driver is arranged above the bending driver; air is pumped out or inflated to the radial tension - compression driver, thereby applying radial pressure to the cells; air is pumped out from the bending driver, driving the radial tension - compression driver to bend and deform, and as the cells grow downward, the culture dish always maintains a state of covering the cells.

[0005] However, the above application has at least the following technical problems:

[0006] The cell culture medium material cannot simulate the real scenario required for cell growth (such as culture solution), which is not conducive to cell growth and culture. The culture medium material cannot fix or limit the cells, which is not conducive to subsequent experiments and observations. Summary of the Invention

[0007] 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 cardiomyocytes, and maintain a stable physiological structure; and provide a more reliable and accurate experimental platform for biomedical research and clinical applications.

[0008] The present invention adopts the following technical solutions:

[0009] A maintenance culture dish for myocardial slices, comprising a culture dish provided with an inner cavity and a myocardial slice support device arranged in the inner cavity. The culture dish is made of a flexible material and the inner cavity is filled with tissue culture medium; the culture dish is detachably connected with an intermittent stretching device; the myocardial slice support device is used for placing myocardial slices and maintaining the middle part 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 generate periodic stretching according to a set frequency.

[0010] The myocardial slice support device includes raised platforms symmetrically arranged at the left and right ends of the inner cavity, and the upper end surfaces of the raised platforms are higher than the bottom wall of the inner cavity of the culture dish.

[0011] It further includes a myocardial slice fixing device, and the myocardial slice fixing device is used for fixing the myocardial slices on the myocardial slice support device.

[0012] It further includes an electrical stimulation device, and the electrical stimulation device is used for performing current stimulation on the myocardial slices according to the set frequency.

[0013] The intermittent stretching device adopts an electronically controlled stretching device; the electronically controlled stretching device is provided with a stretching arm that moves periodically back and forth, and mounting holes corresponding to the stretching arm are arranged around the culture dish.

[0014] The myocardial slice fixing structure includes a negative pressure channel arranged in the myocardial slice support devices at both ends, and a number of differential pressure through holes penetrating the upper end surface of the myocardial slice support device. The negative pressure channel is communicated with the number of differential pressure through holes, and both ends of the myocardial slices correspondingly seal the number of differential pressure through holes.

[0015] Negative pressure at the differential pressure through holes is achieved by using flowing gas or liquid in the negative pressure channel.

[0016] The myocardial slice fixing structure further includes a circulation system, and the circulation system includes a containing box provided with a containing cavity. The containing cavity is filled with tissue culture medium, and the culture dish is arranged in the containing cavity; a pressure reducing pipeline with a micro liquid pump connected in the middle is arranged on the containing box; the rear ends of the negative pressure channels in the myocardial slice support devices at both ends of the culture dish are communicated, and 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 communicated with the containing cavity.

[0017] Communication holes are opened at positions on the front and rear sides of the culture dish where the upper end surfaces are higher than those of the myocardial slice support device, and the communication holes communicate the culture dish with the containing box to form a communicating vessel.

[0018] The intermittent stretching device includes a water outlet pipe penetrating through the bottom wall of the culture dish. The water outlet of the water outlet pipe is located inside 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 water outlet of the micro liquid pump through a connecting pipeline provided with an electronically controlled valve.

[0019] By providing a myocardial slice support device, the present invention can suspend the myocardial slice in the inner cavity, increasing its contact area with the tissue culture medium in the inner cavity, enhancing the gas exchange effect, and reducing the probability of hypoxia caused by the adhesion of the myocardial slice to the wall. By providing an intermittent stretching device, the present invention can drive the myocardial slice in the culture dish to generate periodic stretching according to a set frequency, so as to simulate the contraction and stretching states 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.

[0020] Furthermore, by providing a myocardial slice fixing device, the present invention can ensure the fixing effect between both ends of the myocardial slice and the corresponding myocardial slice support device.

[0021] 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, achieving the effect of stimulating the myocardial slice. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic structural view of the myocardial slice of the present invention;

[0023] Figure 2 is a schematic structural view of the culture dish of the present invention;

[0024] Figure 3 is a schematic structural view of the high platform of the present invention;

[0025] Figure 4 is a schematic structural view of the negative pressure channel of the present invention;

[0026] Figure 5 is a schematic structural view of the differential pressure through hole of the present invention;

[0027] Figure 6 is a schematic structural view of the micro liquid pump of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0028] Figures 1 to 6 As shown, the maintenance culture dish 2 for the myocardial slice 1 of the present invention includes a culture dish 2 provided with an inner cavity and a myocardial slice support device arranged in the inner cavity. The culture dish 2 is made of a flexible material and the inner cavity is filled with a tissue culture medium; the culture dish 2 is detachably connected with an intermittent stretching device;

[0029] The myocardial slice support device is used to place both ends of the myocardial slice 1 and maintain the middle part 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 its contact area with the tissue culture medium in the inner cavity, enhancing the gas exchange effect, and reducing the probability of hypoxia caused by the adhesion of the myocardial slice 1 to the wall.

[0030] The intermittent stretching device is used to drive the myocardial slice 1 in the culture dish 2 to generate periodic stretching according to a set frequency, so as to simulate the contraction and stretching states 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.

[0031] In the present invention, the culture dish 2 made of a flexible material can cooperate with the intermittent stretching device to produce a deformation effect. The flexible material can be PDMS. PDMS has the advantages of being non-toxic and having good biocompatibility. At the same time, it has excellent ductility, resilience, durability and light transmittance, which can ensure the stability of the environment in the culture dish 2, and has the advantages of no damage to the myocardial slice 1 and being convenient for observing indicators such as the morphology, color and adherence of myocardial cells and the myocardial slice 1 at any time.

[0032] In order to facilitate the support of both ends of the myocardial slice 1, in the present invention, the myocardial slice support device includes raised platforms 3 symmetrically arranged at the left and right ends of the inner cavity. The upper end surface of the raised platform 3 is higher than the bottom wall of the inner cavity of the culture dish 2. During use, both ends of the myocardial slice 1 are respectively placed on the upper end surfaces of the raised platforms 3 at both ends of the inner cavity.

[0033] In order to further enhance the fixing effect of the myocardial slice 1, a myocardial slice fixing device is also provided in the present invention.

[0034] In this embodiment, the myocardial slice fixing device can adopt a row of needles 4; the row of needles 4 are respectively passed through both ends of the myocardial slice 1 until the lower part of the row of needles 4 is inserted into the upper part of the raised platform 3, so that both ends of the myocardial slice 1 are fixed to the corresponding raised platform 3, ensuring that the myocardial slice 1 is suspended in the inner cavity, increasing the contact area with the tissue culture solution in the inner cavity, and enhancing the gas exchange effect.

[0035] In the present invention, in order to further simulate the normal physiological state of myocardial cells or tissues, an electric stimulation device is also added. The electric stimulation device can adopt a power supply connected with electrodes 5. The positive and negative electrodes 5 can be electrically connected to the row of needles 4 arranged on one side of the raised platform 3. By using the power supply to apply current stimulation to the myocardial slice 1 through the electrodes 5 and the row of needles 4 according to the set frequency, the effect of stimulating the myocardial slice 1 can be achieved.

[0036] In the present invention, the intermittent stretching device can adopt an electronically controlled stretching device; the electronically controlled stretching device is provided with a stretching arm 6 that moves periodically in a reciprocating manner. Installation holes corresponding to the stretching arm 6 are arranged around the culture dish 2. In this embodiment, two sets of stretching arms 6 are symmetrically arranged on the left and right of the electronically controlled stretching device. Each set of stretching arms 6 includes two stretching rods symmetrically arranged in the front and back; four sets of stretching arm installation holes corresponding to the stretching arms 6 are arranged around the culture dish 2, and each stretching rod is respectively inserted into the corresponding installation hole.

[0037] During use, the two sets of stretching arms 6 perform periodic alternating movements towards and away from each other at a set frequency and intensity, causing the culture dish 2 to correspondingly undergo periodic stretching and contraction deformations. Finally, through the myocardial slice fixing device, it acts on the myocardial slice 1, achieving the purpose of simulating the contraction and stretching states 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.

[0038] Considering that although the needle array 4 can achieve a good fixing effect, it may cause certain damage and destruction to the morphology of the myocardial slice 1 and the internal myocardial cells. In the present invention, a myocardial slice fixing device based on the negative pressure principle and an intermittent stretching device cooperating therewith are also designed.

[0039] In this embodiment, the myocardial slice fixing structure includes a negative pressure channel 7 provided in the raised platforms 3 at both ends and a plurality of differential pressure through holes 8 penetrating through the upper end surface of the raised platforms 3. The negative pressure channel 7 is communicated with the plurality of differential pressure through holes 8, and both ends of the myocardial slice 1 correspondingly seal the plurality of differential pressure through holes 8; thus, the negative pressure at the differential pressure through holes 8 and the pressure of the tissue culture solution can be used to fix both ends of the myocardial slice 1 to replace the needle array 4 for fixing.

[0040] During the working process, the inner cavity of the culture dish 2 is filled with static tissue culture solution; by covering the myocardial slice 1 on the upper surface of the plurality of differential pressure through holes 8, at this time, both ends of the myocardial slice 1 are subjected to the negative pressure at the differential pressure through holes 8 and the pressure of the tissue culture solution, and will be tightly attached to the upper surface of the differential pressure through holes 8, so that both ends of the myocardial slice 1 are tightly attached to the upper end surface of the corresponding raised platforms 3.

[0041] The negative pressure at the differential pressure through holes 8 can be achieved by using flowing gas or liquid in the negative pressure channel 7.

[0042] Based on the tissue culture solution necessary for the experiment and abandoning other media that may cause contamination of the myocardial slice 1, in this embodiment, the tissue culture solution is used to achieve the negative pressure in the negative pressure channel 7. The negative pressure channel 7 is filled with the tissue culture solution, and the tissue culture solution in the negative pressure channel 7 is made to flow, thereby forming a negative pressure at the differential pressure through holes 8 in the negative pressure channel 7.

[0043] In order to enable the tissue culture medium in the negative pressure channel 7 to circulate and be reused; in this embodiment, the myocardial slice fixing structure further includes a circulation system connected to the culture dish 2. The circulation system includes a containing box 9 provided with a containing cavity. The containing cavity is filled with the tissue culture medium. The bottom surface of the containing cavity is provided with a containing groove adapted to the shape of the culture dish 2. The culture dish 2 is arranged in the containing groove and maintains a stable position. A containing space for the tissue culture medium is formed between the outer side surface of the culture dish 2 and the containing cavity of the containing box 9. A pressure reduction pipeline 10 is arranged at the lower part of the containing box 9. The pressure reduction pipeline 10 is communicated with the pressure difference through hole 8 in the culture dish 2 through the negative pressure channel 7. A micro liquid pump 11 is connected in the middle of the pressure reduction pipeline 10. The micro liquid pump 11 enables the tissue culture medium in the pressure reduction pipeline 10 and the negative pressure channel 7 to circulate, so as to generate negative pressure 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 elaborated here.

[0044] In order to form a circulation of the tissue culture medium in the pressure reduction pipeline 10, the negative pressure channel 7 and the containing cavity, in the present invention, the front and rear ends of the raised platforms 3 at the left and right ends of the inner cavity of the culture dish 2 extend to the inner wall of the inner cavity of the culture dish 2. The openings of the negative pressure channels 7 at the rear ends of the two raised platforms 3 are communicated through a U-shaped pipe. The opening of the negative pressure channel 7 at the front end of one side of the raised platform 3 serves as the liquid inlet and is communicated with the liquid outlet of the micro liquid pump 11. The opening of the negative pressure channel 7 at the front end of the other side of the raised platform 3 serves as the liquid outlet and is communicated with the containing cavity of the containing box 9. Under the action of the micro liquid pump 11, the tissue culture medium in the containing cavity is injected into the liquid inlet of the negative pressure channel 7 in one side of the raised platform 3 through the micro liquid pump 11, and flows into the containing cavity of the containing box 9 from the liquid outlet of the negative pressure channel 7 in the other side of the raised platform 3 through the U-shaped pipe, thus forming a circulation of the tissue culture medium.

[0045] Since during the culture process of the myocardial slice 1, it is possible that 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 openings of the pressure difference through holes 8 are exposed, the tissue culture medium in the culture dish 2 will flow into the containing cavity of the containing box 9 through the negative pressure channel 7, resulting in a decrease in the liquid level of the tissue culture medium in the culture dish 2. In order to timely supplement the tissue culture medium in the culture dish 2, in this embodiment, communication holes 12 are also opened at positions slightly higher than the raised platforms 3 on the front and rear side surfaces of the culture dish 2. The communication holes 12 can make the culture dish 2 and the containing box 9 form a communicating vessel, so as to maintain the liquid levels of the tissue culture medium in the culture dish 2 and the containing box 9 higher than the myocardial slice 1, and enable the myocardial slice 1 to always be located in the tissue culture medium. Even if part of the tissue culture medium in the culture dish 2 flows into the containing cavity of the containing box 9 through the negative pressure channel 7, due to the function of the communication holes 12, the tissue culture medium in the containing cavity of the containing box 9 can quickly flow back into the culture dish 2 to maintain the liquid level height in the culture dish 2.

[0046] To cooperate with the myocardial slice fixing device based on the negative pressure principle adopted, in this embodiment, a special intermittent stretching device is also provided based on the tissue culture solution necessary for the experiment. By using the micro liquid pump 11 used to generate negative pressure, the intermittent surging of the tissue culture solution in the culture dish 2 is realized, and the intermittent stretching of the myocardial slice 1 is realized by using the fluid impact force generated during the surging.

[0047] In this embodiment, the intermittent stretching device includes a water outlet pipe 13 penetrating through the bottom wall of the culture dish 2. The water outlet of the water outlet pipe 13 is located between 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 as to intermittently inject the tissue culture solution in the accommodating cavity into the culture dish 2 through the water outlet pipe 13, and realize the surging of the tissue culture solution below the myocardial slice 1. The surging tissue culture solution squeezes the middle position of the myocardial slice 1 to realize the intermittent stretching of the myocardial slice 1, so as to achieve the purpose of simulating the contraction and stretching states 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.

[0048] Based on the tissue culture solution necessary for the experiment, this embodiment abandons other media that may cause contamination of the myocardial slice 1, uses the tissue culture solution to fix the myocardial slice 1, realizes the intermittent stretching of the myocardial slice 1 by generating the surging of the tissue culture solution, and finally well realizes the simulation of the contraction and stretching states of myocardial tissue in the human body environment.

Claims

1. A culture dish for maintaining myocardial slices, characterized in that: The invention comprises a myocardial slice fixing device, a culture dish provided with an inner cavity, and a myocardial slice supporting device provided 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 with an intermittent stretching device; the myocardial slice supporting device is used to place the myocardial slice and maintain the middle of the myocardial slice above the bottom wall of the inner cavity, and the intermittent stretching device is used to drive the myocardial slice in the culture dish to produce periodic stretching according to a set frequency; The myocardial slice fixing device is used to fix the myocardial slice on the myocardial slice supporting device; The myocardial slice fixing device comprises 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 two ends of the myocardial slice are correspondingly closed with the plurality of pressure difference through holes; the myocardial slice fixing device also comprises a circulation system, the circulation system comprises a containing box provided with a containing cavity, the containing cavity is filled with tissue culture fluid, and the culture dish is arranged in the containing cavity; a pressure reducing pipeline connected to a micro liquid pump in the middle is arranged 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 negative pressure channel 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; the intermittent stretching device comprises a water outlet pipe penetrating the bottom wall of the culture dish, the water outlet of the water outlet pipe is located in the culture dish and is lower than the upper end surface of the myocardial slice supporting device; the water inlet of the water outlet pipe is connected to the water outlet of the micro liquid pump through a connecting pipe provided with an electric control valve.

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 an electrical stimulation device, which is used to stimulate the myocardial slices with electric current according to a set frequency.

4. The culture dish for maintaining myocardial slices according to claim 1, characterized in that: The negative pressure channel utilizes flowing liquid to achieve negative pressure at the pressure difference through hole.

5. The culture dish for maintaining myocardial slices according to claim 1, 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.

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