Isolated heart perfusion system

Abstract

The utility model relates to the technical field of isolated heart perfusion, and provides an isolated heart perfusion system which comprises a perfusion liquid container, a perfusion tube and a connector, the first end of the perfusion tube is connected with the perfusion liquid container, and the second end of the perfusion tube is connected with the connector; the connector is used for being inserted into the aorta of the isolated heart, and a groove is formed in the outer wall face of the connector. According to the isolated heart perfusion system, the groove is formed in the outer wall surface of the connector, and after the connector is inserted into the aorta of the isolated heart, the position of the groove can be touched across the aorta to determine the insertion depth of the connector, so that the problem that the insertion depth of the connector into the aorta cannot be guaranteed is solved, and the connector is prevented from being inserted too deep or too shallow.

Description

Technical Field

[0001] The present application relates to the technical field of isolated heart perfusion, and in particular to an isolated heart perfusion system. Background Art

[0002] The isolated heart perfusion system is a system used to perform perfusion experiments on isolated animal hearts. The animal heart is removed from the body and placed in a specific physiological substitute fluid. The aorta of the isolated heart is connected to the isolated heart perfusion system, and the isolated heart is perfused with the perfusion fluid to decompose the heart tissue and obtain myocardial cells, thereby realizing the study of cardiovascular diseases.

[0003] Typically, an isolated heart perfusion system includes a perfusion tube and a connector, wherein the connector is connected to the end of the perfusion tube. During perfusion, the connector is inserted into the aorta of the isolated heart. During this process, the depth of insertion of the connector into the aorta cannot be guaranteed. If the insertion is shallow, the heart is likely to slip off the connector, and if the insertion is deep, the heart is likely to be damaged. Utility Model Content

[0004] In view of this, an embodiment of the present application provides an ex vivo heart perfusion system to solve the problem that the depth of the connector inserted into the aorta cannot be guaranteed.

[0005] An embodiment of the present application proposes an isolated heart perfusion system, comprising a perfusion liquid container, a perfusion tube and a connector, wherein the first end of the perfusion tube is connected to the perfusion liquid container, and the second end of the perfusion tube is connected to the connector; the connector is used to be inserted into the aorta of the isolated heart, and a groove is provided on the outer wall surface of the connector.

[0006] The isolated heart perfusion system provided in the embodiment of the present application provides a groove on the outer wall surface of the connector. After the connector is inserted into the aorta of the isolated heart, the position of the groove can be touched through the aorta to determine the insertion depth of the connector, thereby solving the problem that the insertion depth of the connector into the aorta cannot be guaranteed, thereby avoiding the connector from being inserted too deeply or too shallowly. Moreover, when the aorta is fixed to the connector with silk thread, the silk thread can be tied to the groove, so that the aorta is not easy to slip off the connector, thereby ensuring the stability of the isolated heart suspended on the connector.

[0007] In some embodiments, the connector is a needle.

[0008] In some embodiments, the perfusion liquid container is located above the connecting head, and the height difference between the perfusion liquid container and the connecting head is 80-100 cm.

[0009] In some embodiments, the perfusate container includes a first container for storing a perfusion buffer and a second container for storing an enzyme buffer, and the first container and the second container share the perfusion tube.

[0010] In some embodiments, a defoaming chamber is provided on the perfusion tube.

[0011] In some embodiments, the isolated heart perfusion system further comprises a thermostatic container through which the perfusion tube passes.

[0012] In some embodiments, the portion of the perfusion tube located within the constant temperature container is a spiral structure.

[0013] In some embodiments, the isolated heart perfusion system further comprises a constant temperature water tank, a water inlet pipe and a water return pipe, and the constant temperature water tank is connected to the constant temperature container via the water inlet pipe and the water return pipe.

[0014] In some embodiments, the constant temperature container is arranged horizontally.

[0015] In some embodiments, the isolated heart perfusion system also includes a three-way stopcock, a first port of the three-way stopcock is connected to the second end of the perfusion tube, a second port of the three-way stopcock is connected to the connector, and a third port of the three-way stopcock is used to connect a pressure sensor or a syringe.

[0016] The above description is only an overview of the technical solution of the present application. In order to more clearly understand the technical means of the present application, it can be implemented in accordance with the contents of the specification. In order to make the above and other purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or conventional technical descriptions will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0018] Figure 1 is a schematic diagram of the structure of an isolated heart perfusion system provided in some embodiments of the present application;

[0019] Figure 2 yes Figure 1 A schematic diagram of the structure of the connector of the isolated heart perfusion system shown;

[0020] Figure 3 is a schematic diagram of removing bubbles from an isolated heart and a connector provided in some embodiments of the present application;

[0021] Figure 4 It is a schematic diagram of the change in flow rate of the perfusion buffer provided in some embodiments of the present application.

[0022] The meanings of the marks in the figure are:

[0023] 10. Isolated heart perfusion system;

[0024] 11. perfusion liquid container; 111. first container; 112. second container;

[0025] 12. perfusion tube; 121. first end; 122. second end; 123. defoaming chamber;

[0026] 13. Connector; 131. Groove;

[0027] 14. Constant temperature container;

[0028] 15. Constant temperature water tank; 151. Water inlet pipe; 152. Water return pipe;

[0029] 16. three-way stopcock; 161. first port; 162. second port; 163. third port;

[0030] 17. Beaker;

[0031] 18. Syringe;

[0032] 20. Isolated heart;

[0033] 30. Needle-nosed pliers;

[0034] 40. Micro tweezers. DETAILED DESCRIPTION

[0035] The following embodiments of the technical solution of the present application are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present application, and are therefore only used as examples, and cannot be used to limit the scope of protection of the present application.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which this application belongs; the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" in the specification and claims of this application and the above-mentioned figure descriptions and any variations thereof are intended to cover non-exclusive inclusions.

[0037] In the description of the embodiments of the present application, the technical terms "first", "second", etc. are only used to distinguish different objects, and cannot be understood as indicating or implying relative importance or implicitly indicating the number, specific order or primary and secondary relationship of the indicated technical features. In the description of the embodiments of the present application, the meaning of "multiple" is more than two, unless otherwise clearly and specifically defined.

[0038] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0039] In the description of the embodiments of the present application, the term "and / or" is only a description of the association relationship of the associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. In addition, the character " / " in this article generally indicates that the associated objects before and after are in an "or" relationship.

[0040] In the description of the embodiments of the present application, the term "multiple" refers to more than two (including two). Similarly, "multiple groups" refers to more than two groups (including two groups), and "multiple pieces" refers to more than two pieces (including two pieces).

[0041] In the description of the embodiments of the present application, the orientations or positional relationships indicated by technical terms such as "center", "longitudinal", "lateral", "length", "width", "thickness", "up", "down", "front", "back", "vertical", "horizontal", "top", "bottom", "inside" and "outside" are based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the embodiments of the present application and simplifying the description, and do not indicate or imply that the referred device or element must have a specific orientation, be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the embodiments of the present application.

[0042] In the description of the embodiments of the present application, unless otherwise clearly specified and limited, technical terms such as "installed", "connected", "connected", "fixed" and the like should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium, and it can be the internal connection of two elements or the interaction relationship between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present application can be understood according to the specific circumstances.

[0043] The embodiments of the present application provide an isolated heart perfusion system, specifically a Langendorff perfusion system. Figure 1 and Figure 2 The isolated heart perfusion system 10 includes a perfusion liquid container 11, a perfusion tube 12 and a connector 13. The first end 121 of the perfusion tube 12 is connected to the perfusion liquid container 11, and the second end 122 of the perfusion tube 12 is connected to the connector 13; the connector 13 is used to be inserted into the aorta of the isolated heart 20, and a groove 131 is provided on the outer wall surface of the connector 13.

[0044] The perfusion liquid container 11 is used to store the perfusion liquid, wherein the shape of the perfusion liquid container 11 is not limited in this application, as long as it can store the perfusion liquid. For example, the perfusion liquid container 11 can be a plastic centrifuge tube, a measuring cylinder, a beaker, etc.

[0045] The perfusion tube 12 is used to lead the perfusion liquid in the perfusion liquid container 11 to the connector 13, so that the perfusion liquid enters the isolated heart 20 through the connector 13; optionally, the perfusion tube 12 is a soft tube, such as a plastic tube, a silicone tube, etc.

[0046] The perfusion tube 12 has a first end 121 and a second end 122. Optionally, the first end 121 is higher than the second end 122. The connection position of the first end 121 and the perfusion liquid container 11 is not limited in this application. It can be understood that the first end 121 can be connected to the bottom of the perfusion liquid container 11. During perfusion, the perfusion liquid in the perfusion liquid container 11 can flow directly into the perfusion tube 12; or the first end 121 can also be connected to the top of the perfusion liquid container 11, that is, the first end 121 is inserted into the perfusion liquid container 11 from the top of the perfusion liquid container 11. During perfusion, the siphon principle is used to make the perfusion liquid in the perfusion liquid container 11 flow into the perfusion tube 12.

[0047] The present application does not limit the connection method between the second end 122 of the perfusion tube 12 and the connector 13. It can be understood that the connector 13 can be directly connected to the second end 122. For example, the connector 13 is connected to the second end 122 by means of threaded connection, clamping, fastener connection, interference connection, etc.; or, the connector 13 can also be connected to the connector 13 by other structures. For example, the other structure can be a three-way stopcock 16.

[0048] The connector 13 serves to connect the isolated heart 20 and the perfusion tube 12 , and has a hollow passage. During perfusion, the perfusion liquid in the perfusion liquid container 11 enters the isolated heart 20 through the perfusion tube 12 and the connector 13 .

[0049] Since the connector 13 is provided with a groove 131 , it can be felt by hand. After the connector 13 is inserted into the aorta of the isolated heart 20 , the position of the groove 131 can be touched through the aorta to determine the insertion depth of the connector 13 .

[0050] It can be understood that the groove 131 can be an annular groove, and two or three or more grooves 131 can also be arranged at intervals along the circumference of the connecting head 13.

[0051] There is no limitation on the manufacturing method of the groove 131 . For example, the groove 131 can be formed by clamping the outer wall of the connector 13 with needle-nosed pliers 30 .

[0052] It should be noted that the position of the groove 131 is determined according to the length of the aorta of the experimental mouse. For example, for an adult mouse (8 to 12 weeks), the distance between the groove 131 and the end of the connector 13 is about 2 mm.

[0053] The isolated heart perfusion system 10 provided in the embodiment of the present application is provided with a groove 131 on the outer wall surface of the connector 13. After the connector 13 is inserted into the aorta of the isolated heart 20, the position of the groove 131 can be touched through the aorta to determine the insertion depth of the connector 13, thereby solving the problem that the insertion depth of the connector 13 into the aorta cannot be guaranteed, thereby avoiding the connector 13 from being inserted too deeply or too shallowly. Moreover, when the aorta is fixed to the connector 13 with a silk thread, the silk thread can be tied to the groove 131, so that the aorta is not easy to slip off the connector 13, thereby ensuring the stability of the isolated heart 20 suspended on the connector 13.

[0054] Please also refer to Figure 1 and Figure 2 In some embodiments, the connector 13 is a needle.

[0055] The diameter of the needle should be close to the diameter of the aorta of the isolated heart 20. For example, for adult mice (8-12 weeks), an 18G flat-tip needle can be used. According to international standards, the outer diameter of an 18G needle is about 1.2 mm, and the larger the number before G, the smaller the outer diameter of the needle. Of course, depending on the diameter of the aorta, a 16G flat-tip needle with an outer diameter of about 1.6 mm can also be used; or a 20G flat-tip needle with an outer diameter of about 0.9 mm can also be used.

[0056] Since the needle is a standard part, it is easy to obtain; moreover, the end of the needle has a thread, which is convenient for connecting with the perfusion tube 12.

[0057] In other embodiments, the connector 13 may be a connecting tube, one end of which is connected to the second end 122 of the perfusion tube 12 , and the other end of which is inserted into the isolated heart 20 .

[0058] Please refer to Figure 1 In some embodiments, the perfusion liquid container 11 is located above the connector 13, and a height difference H between the perfusion liquid container 11 and the connector 13 is 80-100 cm.

[0059] The perfusion liquid container 11 can be directly above the connector 13, or can be obliquely above the connector 13, as long as the height difference H between the perfusion liquid container 11 and the connector 13 is 80-100 cm. The height difference H is calculated based on the coronary artery perfusion pressure of mice and rats, that is, different height differences and different coronary artery perfusion pressures correspond to different sizes of experimental mice.

[0060] It can be understood that the height difference H between the perfusion liquid container 11 and the connecting head 13 can be 80 cm, 90 cm, 100 cm, etc.

[0061] By designing the perfusion liquid container 11 and the connector 13 to have the above-mentioned height difference, that is, using gravity as the perfusion pressure, the perfusion liquid in the perfusion liquid container 11 can flow into the isolated heart 20 through the perfusion tube 12 and the connector 13 under the action of gravity. Since the isolated heart 20 has a higher tension in the undigested state, and the completely digested isolated heart 20 has a lower tension due to extracellular hydrolysis, when gravity is used as the perfusion pressure, the perfusion pressure of the isolated heart 20 will not change with the progress of the digestion process, which is more in line with the biological characteristics of the heart.

[0062] In other embodiments, a peristaltic pump may be provided on the perfusion tube 12 , in which case the perfusion liquid container 11 may be at the same height as the connector 13 ; or, the perfusion liquid container 11 may be lower than the connector 13 ; or, the perfusion liquid container 11 may be higher than the connector 13 .

[0063] Please refer to Figure 1 In some embodiments, the perfusion liquid container 11 includes a first container 111 for storing a perfusion buffer and a second container 112 for storing an enzyme buffer, and the first container 111 and the second container 112 share a perfusion tube 12 .

[0064] Optionally, the first container 111 and the second container 112 are independently provided, that is, the first container 111 and the second container 112 are not connected to each other. In this design, the first container 111 and the second container 112 do not affect each other.

[0065] Optionally, the first container 111 and the second container 112 are both plastic centrifuge tubes.

[0066] The first container 111 and the second container 112 share the perfusion tube 12. It can be understood that when perfusion buffer is used for perfusion, the first end 121 of the perfusion tube 12 is inserted into the first container 111, and when enzyme buffer is used for perfusion, the first end 121 of the perfusion tube 12 is inserted into the second container 112; or, the first end 121 of the perfusion tube 12 is provided with a first branch tube and a second branch tube, the first branch tube is connected to the first container 111, and the second branch tube is connected to the second container 112, wherein valves are respectively provided on the first branch tube and the second branch tube.

[0067] Since the first container 111 and the second container 112 share the perfusion tube 12 , the number of perfusion tubes 12 can be reduced and the structure of the isolated heart perfusion system 10 can be simplified.

[0068] In other embodiments, the perfusate container 11 has two storage chambers, one of which is used to store a perfusion buffer and the other is used to store an enzyme buffer.

[0069] Please refer to Figure 1 In some embodiments, a defoaming chamber 123 is provided on the perfusion tube 12 .

[0070] The defoaming chamber 123 is similar to the dripping funnel of an infusion set. The diameter of the defoaming chamber 123 is larger than the diameter of the perfusion tube 12. After the perfusion liquid enters the defoaming chamber 123, the liquid falls and the air floats, so as to achieve the purpose of removing bubbles.

[0071] By adopting the above technical solution, bubbles are eliminated after the perfusion liquid passes through the defoaming chamber 123, thereby preventing bubbles from entering the isolated heart 20 along with the perfusion liquid and causing coronary artery air embolism, thereby leading to myocardial cell death.

[0072] Please refer to Figure 1 In some embodiments, the isolated heart perfusion system 10 further includes a thermostatic container 14 through which the perfusion tube 12 passes.

[0073] Since the perfusion tube 12 passes through the constant temperature container 14, the constant temperature container 14 can heat the perfusion liquid in the perfusion tube 12 so that the perfusion liquid enters the isolated heart 20 at a suitable temperature, thereby ensuring the vitality of the myocardial cells.

[0074] In other embodiments, the constant temperature container 14 may not be included, and the isolated heart perfusion system 10 performs the perfusion operation on the isolated heart 20 in an environment with a suitable temperature.

[0075] Please refer to Figure 1 In some embodiments, the isolated heart perfusion system 10 includes a constant temperature container 14, and the portion of the perfusion tube 12 in the constant temperature container 14 is a spiral structure.

[0076] Since the portion of the perfusion pipe 12 in the constant temperature container 14 is a spiral structure, the heat exchange area can be increased, which is conducive to the temperature of the perfusion liquid in the perfusion pipe 12 reaching the set temperature. Optionally, the constant temperature container 14 is a long strip structure.

[0077] In other embodiments, the portion of the perfusion tube 12 in the constant temperature container 14 may be a reciprocating structure, that is, the portion of the perfusion tube 12 in the constant temperature container 14 is arranged in an S shape. Optionally, the constant temperature container 14 is a flat structure.

[0078] In some embodiments, the isolated heart perfusion system 10 further includes a constant temperature water tank 15 , a water inlet pipe 151 and a water return pipe 152 , and the constant temperature water tank 15 is connected to the constant temperature container 14 via the water inlet pipe 151 and the water return pipe 152 .

[0079] The constant temperature water tank 15 is used to heat water and keep it at a set temperature. It can be understood that the constant temperature water tank 15 can be a constant temperature water bath.

[0080] Optionally, the constant temperature container 14 is a long strip structure, and the constant temperature container 14 has a first end and a second end. The end of the water inlet pipe 151 away from the constant temperature water tank 15 is connected to the first end of the constant temperature container 14, and the end of the water return pipe 152 away from the constant temperature water tank 15 is connected to the second end of the constant temperature container 14, that is, the water inlet pipe 151 and the water return pipe 152 are respectively connected to the two ends of the constant temperature container 14.

[0081] Optionally, the portion of the perfusion tube 12 located in the constant temperature container 14 flows from the first end of the constant temperature container 14 to the second end of the constant temperature container 14 .

[0082] In other embodiments, the constant temperature container 14 , the water inlet pipe 151 and the water return pipe 152 may not be provided, and part of the perfusion pipe 12 may be directly placed in the constant temperature water tank 15 .

[0083] Please refer to Figure 1 In some embodiments, the constant temperature container 14 is arranged horizontally.

[0084] It should be noted that the horizontal arrangement of the constant temperature container 14 does not mean an absolute horizontal arrangement, but also includes an approximately horizontal arrangement.

[0085] By adopting the above technical solution, compared with arranging the constant temperature container 14 vertically, the flow rate of the perfusion liquid can be slowed down to extend the heat exchange time, which is further conducive to the perfusion liquid temperature in the perfusion tube 12 reaching the set temperature.

[0086] In other embodiments, when the heat exchange area is large enough, the constant temperature container 14 may be arranged vertically or tilted so that the temperature of the perfusion liquid in the perfusion tube 12 can reach the set temperature.

[0087] In some embodiments, the isolated heart perfusion system 10 also includes a three-way stopcock 16, the first port 161 of the three-way stopcock 16 is connected to the second end 122 of the perfusion tube 12, the second port 162 of the three-way stopcock 16 is connected to the connector 13, and the third port 163 of the three-way stopcock 16 is used to connect a pressure sensor or a syringe 18.

[0088] Optionally, the three-way stopcock 16 is a medical three-way stopcock. It can be understood that by screwing the stopcock, the first port 161, the second port 162 and the third port 163 can be connected, or only the second port 162 and the third port 163 can be connected.

[0089] Since the first port 161 of the three-way stopcock 16 is connected to the second end 122 of the perfusion tube 12 , the second port 162 of the three-way stopcock 16 is connected to the connector 13 , that is, the connector 13 is connected to the second end 122 of the perfusion tube 12 through the three-way stopcock 16 .

[0090] When the third port 163 is connected to a pressure sensor, the pressure sensor can monitor the perfusion pressure; when the third port 163 is connected to a syringe 18 , drugs can be supplemented through the syringe 18 .

[0091] In other embodiments, when it is not necessary to monitor the perfusion pressure and replenish the medicine, the three-way stopcock 16 may not be provided, that is, the connector 13 is directly connected to the second end 122 of the perfusion tube 12 .

[0092] In the embodiment of the present application, the steps of obtaining myocardial cells of an isolated heart 20 using the isolated heart perfusion system 10 are as follows:

[0093] S1. Heart removal: Cut the aortic arch of the heart as close to the innominate artery as possible and remove the heart from the mouse chest cavity; under a stereo microscope, place the isolated heart 20 in a culture dish with perfusion buffer at a temperature of 4°C.

[0094] S2. Intubation: Select a suitable needle according to the diameter of the aorta. For example, the diameter of the aorta of an adult mouse (8-12 weeks) is similar to that of an 18G flat-head needle. Connect the needle to a syringe 18 with perfusion buffer and fix the syringe 18 with a bracket. Before intubation, two points need to be determined: one is that the isolated heart 20 is completely immersed in the perfusion buffer of the culture dish; the other is to push the syringe 18 to eliminate bubbles that may appear in the needle. During intubation, insert the needle into the aorta of the isolated heart 20, and use a silk thread to tie the aorta to the needle so that the isolated heart 20 is suspended on the needle. This process should be completed within two minutes as much as possible, and the insertion depth of the needle should be appropriate, not lower than the aortic valve or enter the left ventricle, and try to avoid bubbles entering the aorta.

[0095] S3. Bubble removal: Bubbles entering the needle or aorta can cause coronary air embolism and lead to myocardial cell death, so bubbles need to be removed. Figure 3 As shown, the perfusion buffer is poured into the beaker 17, the syringe 18 is removed, and the isolated heart 20 connected to the needle is placed in the beaker 17. If there are bubbles in the aorta or the needle, the isolated heart 20 and the needle will float in an upright position. Two micro forceps 40 are used to guide and remove the bubbles in the aorta or the needle (usually visible to the naked eye). The sign of successful debubbling is that there are no visible bubbles at the top of the needle, and, when no external force is applied, the isolated heart 20 and the needle will sink to the bottom of the beaker 17.

[0096] S4. Connecting system: connect the syringe 18 to the third port 163, open the three-way stopcock 16, and use the negative pressure of the syringe 18 to produce a siphon effect so that the perfusion buffer in the first container 111 flows downward. When a stable perfusion buffer flows out from the second port 162, connect the needle with the isolated heart 20 to the second port 162.

[0097] S5. Gravity perfusion: After the perfusion buffer is perfused under gravity for 3 minutes, the first end 121 of the perfusion tube 12 is moved to the second container 112 containing the enzyme buffer. 2+ , but not in the perfusion buffer, so when the enzyme buffer just enters the coronary artery, it will cause a sudden increase in myocardial tension. Therefore, the phenomenon we will observe is, Figure 4 As shown, the speed of the liquid dripping from the bottom of the isolated heart 20 suddenly slows down from the first beaker 17 to the second beaker 17, and the occurrence of this phenomenon indicates that the aorta cannulation and perfusion are successful.

[0098] Traditional methods often use peristaltic pumps as the power source for perfusion buffer. Peristaltic pumps maintain a constant flow rate, which may cause excessive pressure in the early stages of digestion and damage myocardial cells. Using gravity as the perfusion pressure is more in line with the biological characteristics of the heart. Since the heart has a higher tension in the undigested state, and the fully digested heart has a lower tension due to extracellular hydrolysis, when gravity is used as the perfusion pressure, the perfusion pressure of the heart will not change as the digestion process progresses.

[0099] According to the analysis of the experimental results, the proportion of rod-shaped cardiomyocytes isolated by gravity perfusion (83.10%±3.27%, mean±SD) was significantly higher than that by peristaltic pump (70.97%±3.98%, mean±SD).

[0100] S6. Digestion termination criteria: The degree of digestion of the heart has a great impact on the results of subsequent experiments. Insufficient or excessive digestion will lead to unreliable experimental results. For example, in the field of cardiac regeneration, the number counting and ploidy evaluation of cardiomyocytes require strict digestion standards to ensure the consistency and accuracy of the experiment. Animals of different species, ages, and physiological or pathological states (such as physiological adult mice, physiological aged mice, surgical model pathological mice, or gene knockout or overexpression mice) have different degrees of fibrosis and ECM composition, and the optimal digestion time is bound to be different. Therefore, it is particularly important to develop a simple and effective method to judge the degree of digestion.

[0101] In the isolated heart perfusion system 10 provided in the embodiment of the present application, by observing the change in the velocity of the droplets flowing out from the bottom of the isolated heart 20, we can easily determine when to stop digestion. Figure 4 As shown, at the beginning of perfusion, the perfusion buffer enters the isolated heart 20, and the flow rate is maintained at about 5 mL / min, and the flow rate does not change. Once the enzyme buffer enters the isolated heart 20, the flow rate suddenly slows down to about 2 ml / min, which can be easily identified by the naked eye, such as Figure 4 The first beaker 17 to the second beaker 17 shown. After a few minutes, as the extracellular matrix of the isolated heart 20 is hydrolyzed, the tension of the isolated heart 20 decreases, and the droplet flow rate gradually increases, as shown in FIG. Figure 4 The second beaker 17 to the third beaker 17 shown. Finally, when the isolated heart 20 is completely digested, the flow rate is suddenly increased, such as Figure 4 The third beaker 17 to the fourth beaker 17 shown may be due to a rupture in the coronary system or atrial system (not less than 25 ml / min).

[0102] Through observation, a shorter digestion time (about 3 minutes shorter than the standard time, 56.47% ± 3.13%, mean ± SD) leads to a decrease in cardiomyocyte viability. There is no significant difference in cardiomyocyte viability between a longer digestion time (3 minutes longer than the standard time, 77.30% ± 5.43%, mean ± SD) and a standard digestion time (83.47% ± 3.17%, mean ± SD). Although appropriately extending the digestion time does not affect the viability of cardiomyocytes, overdigestion will inevitably occur, affecting the physiological state of cardiomyocytes. This method is simple and easy to use, can well regulate the digestion termination time, and is suitable for various situations of mice.

[0103] S7. Multiple natural gravity sedimentation: Before gravity sedimentation, tear the digested isolated heart 20 into small pieces, and then pipette several times until no large pieces of myocardial tissue are visible. Use three 15ml centrifuge tubes, add 10ml of stop buffer to each centrifuge tube, and add CaCl2 solution, adjusted to 100, 400 and 900um Ca respectively.2+ Concentration. With gradient Ca 2+ The liquid stop buffer needs to be replaced every 15 minutes. Since cardiomyocytes are much larger than other cardiomyocytes, gravity will cause the cardiomyocytes to sink to the bottom faster. At the same time, due to the aggregation effect, highly active cardiomyocytes aggregate. Afterwards, flow cytometry can detect the purity of cardiomyocytes before sequencing. The proportion of cardiomyocytes detected before purification was only 63.8% ± 4.1% (mean ± SD), and the proportion of cardiomyocytes detected after purification reached 96.8% ± 0.4% (mean ± SD). Cardiomyocyte nuclei were stained with Hoechst 33342. Under the microscope, we can see rod-shaped cardiomyocytes with sharp membrane edges and intact nuclei. The results showed that the cardiomyocytes had good viability (83.10% ± 3.27%, mean ± SD), purity (96.83% ± 0.40%, mean ± SD) and cell count (0.9~1.2×10 6 ) has been well optimized for the purity and viability of cardiomyocytes, which can meet the requirements of single-cell sequencing and improve the stability and accuracy of experimental results, which is of great significance for cardiovascular disease research.

[0104] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit them. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. These modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present application, and should all be included in the protection scope of the present application.

Claims

1. An isolated heart perfusion system, characterized in that: It includes a perfusion liquid container, a perfusion tube and a connector. The first end of the perfusion tube is connected to the perfusion liquid container, and the second end of the perfusion tube is connected to the connector. The connector is used to be inserted into the aorta of an isolated heart, and a groove is provided on the outer wall of the connector.

2. The isolated heart perfusion system according to claim 1, wherein: The connecting head is a needle.

3. The isolated heart perfusion system according to claim 1, wherein: The perfusion liquid container is located above the connector, and the height difference between the perfusion liquid container and the connector is 80-100 cm.

4. The isolated heart perfusion system according to claim 1, wherein: The perfusion liquid container includes a first container for storing a perfusion buffer and a second container for storing an enzyme buffer, and the first container and the second container share the perfusion tube.

5. The isolated heart perfusion system according to claim 1, wherein: The perfusion pipe is provided with a defoaming chamber.

6. The isolated heart perfusion system according to any one of claims 1 to 5, wherein: The isolated heart perfusion system further comprises a constant temperature container, and the perfusion tube passes through the constant temperature container.

7. The isolated heart perfusion system according to claim 6, wherein: The portion of the perfusion tube located in the constant temperature container is a spiral structure.

8. The isolated heart perfusion system according to claim 6, wherein: The isolated heart perfusion system further comprises a constant temperature water tank, a water inlet pipe and a water return pipe, wherein the constant temperature water tank is connected to the constant temperature container via the water inlet pipe and the water return pipe.

9. The isolated heart perfusion system according to claim 6, wherein: The constant temperature container is arranged horizontally.

10. The isolated heart perfusion system according to any one of claims 1 to 5, characterized in that: The isolated heart perfusion system also includes a three-way stopcock, a first port of the three-way stopcock is connected to the second end of the perfusion tube, a second port of the three-way stopcock is connected to the connector, and a third port of the three-way stopcock is used to connect to a pressure sensor or a syringe.

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