Donor heart preservation method based on crystal cardioplegic solution and UW solution and application of donor heart preservation method

Abstract

The invention discloses a donor heart preservation method based on a crystal cardioplegic solution (CC solution) and a UW solution and application of the donor heart preservation method, and belongs to the field of biomedical engineering. The method comprises the following steps: (a) preparing CC liquid; (b) perfusing CC liquid through the aorta at the pressure of 150mmHg for 3-5 minutes to cause cardiac arrest; (c) taking out the heart, and performing secondary perfusion with 2000ml of 4 DEG C UW solution for 8-12 minutes; and (d) immersing the heart into a UW solution for cold preservation, wherein the cold ischemia time is less than or equal to 8 hours. In combination with a CC liquid perfusion step, the protection effect of the cold preservation liquid is remarkably improved by reducing the metabolic rate of cardiac cells; through double perfusion of CC liquid and UW liquid, optimization of metabolic balance and effective inhibition of inflammatory response are realized in the cold storage process of the heart; the invention has the functions of prolonging cold ischemia time and keeping heart cells, and provides a new technical means for long-time cold preservation.

Description

Technical Field

[0001] The invention belongs to the field of biomedical engineering, and in particular relates to a donor heart preservation method based on crystalloid cardioplegia solution and UW solution and application thereof. Background Art

[0002] Heart transplantation is an effective treatment for patients with advanced heart failure, but donor hearts are scarce and the length of cold ischemia time (CIT) directly affects the success rate of heart transplantation. Studies have shown that long-term preservation of the heart under cold ischemia conditions can lead to a series of problems such as cell metabolism disorders, energy depletion, oxidative damage, and inflammatory response, which in turn leads to acute cardiac dysfunction (PGD), which seriously affects the recovery of heart function after transplantation and the survival rate of patients.

[0003] At present, cold preservation technology mostly uses UW preservation solution (The University of Wisconsin solution), which can provide effective cold ischemia protection, but its ability to prolong the cold ischemia time is still limited, and the protective effect on the heart often cannot meet clinical needs under high-risk conditions (such as long cold ischemia time, poor heart quality, etc.). In addition, the existing cold preservation method fails to fully consider the metabolic regulation, oxidative stress and immune response of heart cells, and still has the disadvantages of complex operation and unstable effect.

[0004] Therefore, developing a cardiac cold ischemia protection method that can prolong the cold ischemia time and provide stronger protection has become a key technical issue that needs to be urgently solved in the field of heart transplantation. Summary of the invention

[0005] In order to solve the above technical problems, the present invention proposes the idea of ​​combining crystalloid cardioplegia (CC) with traditional UW preservation solution to reduce the energy consumption of cardiac cells, slow down the accumulation of metabolites, inhibit the inflammatory response during cardiac preservation, and significantly improve the survival rate of the heart during cold ischemia and the recovery effect of cardiac function after transplantation.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a liquid composition for heart preservation, which comprises crystalloid cardioplegia solution (CC solution) and UW solution; the CC solution comprises: 470-480 ml of compound electrolyte, 5-7 ml of 10% potassium chloride, 9-11 ml of 50% glucose, 7-8 ml of 5% sodium bicarbonate and 2-3 ml of heparin; and the UW solution comprises 1800-2200 ml of 4°C UW solution.

[0008] The present invention also provides a method for preserving a donor heart, comprising using the liquid composition.

[0009] The present invention also provides an application of the liquid composition or the donor heart preservation method in the preservation of an ex vivo donor heart.

[0010] Furthermore, the method for using the liquid composition in the application is to first double-perfuse with CC solution and UW solution, and then store it with UW solution.

[0011] Furthermore, the application specifically includes the following steps:

[0012] (a) Prepare CC solution according to the formula;

[0013] (b) CC solution was perfused through the aorta at a pressure of 140-160 mmHg for 3-5 minutes to stop the heart and cool it down;

[0014] (c) The heart was removed and perfused again with UW solution for 8-12 minutes;

[0015] (d) The heart was preserved in UW solution with a cold ischemia time of ≤8 h.

[0016] Furthermore, the formula of the CC solution is: 475 ml of compound electrolyte, 6 ml of 10% potassium chloride, 10 ml of 50% glucose, 7.5 ml of 5% sodium bicarbonate and 2.5 ml of heparin.

[0017] Furthermore, in step (b), the perfusion pressure of the CC solution is 150 mmHg.

[0018] Furthermore, in step (b), the aortic root pressure is maintained at 60-100 mmHg.

[0019] Furthermore, the UW solution is 2000 ml of 4° C. UW solution.

[0020] Furthermore, the cold ischemia time is 4-8 hours.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) Prolonging the cold ischemia time: The combination of CC solution and UW preservation solution can significantly prolong the cold ischemia time and reduce the waste of donor hearts;

[0023] (2) Improve the preservation state of the heart: CC solution can significantly reduce the metabolic burden after cardiac arrest, while UW preservation solution further stabilizes the cell state and improves the recovery effect after cold storage;

[0024] (3) Reducing acute cardiac dysfunction after transplantation: Experimental data show that the incidence of PGD after transplantation of hearts preserved using the method of the present invention is lower and the heart recovers better;

[0025] (4) Simple and efficient operation: Compared with traditional heart preservation methods, the CC+UW solution combination is simpler to operate and can provide a more stable protective effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a comparison of metabolite consumption between the CC+UW group and the UW group in Comparative Example 2 of the present invention;

[0027] Figure 2 The changes in the phosphorylation levels of key molecules in the NF-κB pathway in Example 1 of the present invention;

[0028] Figure 3 The cardiac electrophysiological function recovery data after cold storage in Example 2 of the present invention;

[0029] Figure 4 The results of the correlation analysis between cold ischemia time and survival rate in Example 3 of the present invention;

[0030] Figure 5 These are the baseline characteristics of the donor and the acceptor in Example 3 of the present invention. DETAILED DESCRIPTION

[0031] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or substitutions made to the method, steps or conditions of the present invention are within the scope of the present invention. The reagents, products and instruments used in the following examples can all be obtained commercially, and the methods used in the examples are consistent with the conventional methods unless otherwise specified.

[0032] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0033] The technical solution of the present invention combines crystalloid cardioplegia (CC) with traditional UW preservation solution to reduce the energy consumption of cardiac cells, slow down the accumulation of metabolites, inhibit the inflammatory response during cardiac preservation, and significantly improve the survival rate of the heart during cold ischemia and the recovery effect of cardiac function after transplantation.

[0034] The specific steps are as follows:

[0035] (1) Preparation of crystalloid cardioplegia solution

[0036] 475ml compound electrolyte (Baxter Medical, National Medicine Standard No. H20000475) + 6ml 10% potassium chloride + 10ml 50% glucose + 7.5ml 5% sodium bicarbonate + 2.5ml heparin. The percentages are all mass percentages. Pay attention to the accuracy of the drug dosage during preparation, keep the preparation process sterile, and store the crystalloid cardioplegia solution at 4℃ after preparation.

[0037] (2) Crystalloid cardioplegia solution perfusion donor heart

[0038] After the donor is disinfected, skin incised, chest opened, and the aorta completely blocked, cold crystalloid cardioplegia solution is perfused, and the perfusion pressure and perfusion time are monitored and recorded. The pressure in the pressure band is always maintained at 150 mmHg by the assistant, and the pressure at the aortic root is 60-100 mmHg. At the same time, the pressure of the aortic root and the left ventricle is sensed to ensure perfusion and avoid high tension in the left ventricle. The cold crystalloid perfusion is completed in 3-5 minutes. Before removing the donor heart, it should be ensured that it has completely stopped beating and is soft to the touch. The donor heart is quickly cooled with ice chips and removed.

[0039] Cold crystalloid cardioplegia perfusion can rapidly induce cardiac arrest, immediately reduce cardiac energy consumption, reduce oxidative stress, slow down cellular metabolic processes, and provide better conditions for subsequent cold preservation.

[0040] (3) UW preservation solution (Bridge to Life, specification: 1000ml) is perfused into the donor heart

[0041] After the donor heart is removed, it is placed in a three-layer sterile plastic bag and perfused again through the aorta with 2000ml of 4℃ UW solution for 8-12 minutes. UW preservation solution has a good low-temperature protection effect, can provide a suitable cold ischemic environment for the heart, and further maintain the function of heart cells by controlling ion balance and providing nutrients.

[0042] (4) Preservation of donor heart with UW preservation solution

[0043] After the heart perfusion is completed, the donor heart is then immersed in 4°C UW solution and placed in a low-temperature storage box for cold storage. During the cold storage period, the UW solution protects the heart tissue and delays cell damage until the heart is transplanted into the recipient.

[0044] Through the above method, the present invention can significantly prolong the cold ischemia time, maintain the cell function of the heart in a low temperature environment, and at the same time reduce the accumulation of metabolites and reduce the risk of post-transplant acute heart failure (PGD).

[0045] Example 1 Application of CC+UW solution in cold storage of pig heart

[0046] Experimental design: 10 healthy adult pigs were selected and randomly divided into two groups. The experimental group used CC solution and UW preservation solution for cold preservation (see the specific steps above for the method), and the control group used only traditional UW preservation solution. The cold ischemia time was set to 0, 4, and 8 hours.

[0047] Experimental procedures: After anesthesia, the animals underwent sternotomy to expose the heart and great vessels. After aortic clamping, cardiac arrest was induced by perfusion of 1000 ml of 4°C crystalloid cardioplegia (CC+UW group) or UW solution (UW group). The left and right atria were quickly cut to reduce cardiac pressure. Then the pulmonary veins, superior and inferior vena cava, pulmonary artery and aorta were cut, while maintaining a perfusion pressure of 60-100 mmHg. The donor heart was quickly cooled with ice chips. After removal, the donor heart was placed in a three-layer sterile plastic bag and flushed again with 2000 mL of 4°C UW solution through the aortic root (perfusion time: 8-12 minutes). Subsequently, the donor heart was immersed in low-temperature UW solution for static cold storage for 0 hours, 4 hours or 8 hours.

[0048] The experimental results are as follows Figure 2 The results showed that the CC+UW group significantly reduced the phosphorylation levels of pro-inflammatory proteins p65 (Ser529), IKK-α, IKK-β and RelB, and increased the phosphorylation of anti-inflammatory protein XIAP, indicating that it effectively alleviated the inflammatory response by inhibiting the activity of core molecules of the NFκB pathway. In the 8-hour cold storage group, CC+UW inhibited the activation of inflammation-related pathways such as PDGF, VEGF, MAPK, AKT and mTOR, and reduced the inflammatory cascade caused by ischemia-reperfusion injury. By inhibiting the p53 pathway and apoptosis-related signals, the risk of cardiomyocyte death was reduced. The CC+UW group still maintained a low level of inflammation after 8 hours of cold storage, supporting its application in long-term donor heart preservation.

[0049] Example 2 Application of CC+UW solution in cold storage of rat heart and evaluation of its effect

[0050] Experimental design: Healthy adult rats were selected and divided into an experimental group (CC+UW group, the method also refers to the specific steps above) and a control group (UW group). The cardiac electrical conduction, ventricular pressure changes and cardiac recovery of each group were evaluated to compare the effects of CC+UW group and traditional UW preservation solution during cold storage.

[0051] Experimental procedures: Rats were anesthetized and opened for systemic anticoagulation. After the ascending aorta was dissected, a perfusion needle was inserted and the ascending aorta was fixed to the needle to prevent air from entering the ascending aorta. The heart was stopped by perfusing about 10 mL of 4°C UW solution (UW group) or crystalloid cardioplegia (CC+UW group) with initial cardioplegia. Then, the heart was flushed with 10 mL of cold UW solution again after cardiac arrest. Then, the heart and the perfusion needle were placed in a centrifuge tube containing 4°C UW solution and kept in an ice-water mixture for 8 hours. After the cold storage ended, the UW solution was replaced with room-temperature oxygen-enriched KH solution (NaCl 119 mM, NaHCO3 25 mM, KCl 4 mM, KH2PO4 1.2 mM, MgCl2·6H2O 1 mM, CaCl2·2H2O 1.8 mM, D-glucose 10 mM), and the heart was perfused slowly until peristalsis occurred. Then, the hearts were transferred to a low-pressure perfusion system and perfused with normothermic oxygen-enriched KH solution for 15 min, followed by transfer to a constant-pressure perfusion system for rewarming to resume perfusion. The control hearts were perfused with KH solution directly after isolation. Simultaneously, epicardial activation mapping was performed on the left and right atrial surfaces using two 64-electrode multi-electrode arrays. ECG electrodes were placed in the right atrium and at the apex of the heart. Electrophysiological signals and left ventricular pressure were recorded for the different groups under sinus rhythm and 6 Hz, S1, and S2 stimulation. Data were recorded using a multichannel system (EMS64-USB-1003; MappingLab Ltd.) and analyzed using EmapScope 5.0 software (MappingLab Ltd.).

[0052] The experimental results are as follows Figure 3As shown, the results showed that the CC+UW group showed a greater pulse pressure change after 8 hours (48.7mmHg vs.29.3mmHg, P<0.05), showing its advantage in maintaining cardiac ejection function. The CC+UW-8 hour group showed a similar electrical conduction pattern to the control group, but the electrical conduction velocity of the CC+UW-8 hour group was significantly faster than that of the UW group (left ventricle: 0.8mm / ms vs.0.4mm / ms; right ventricle: 0.8mm / ms vs.0.6mm / ms, P<0.05). At the same time, the conduction diffusion degree and sinus recovery time of the CC+UW group were similar to those of the control group, while the conduction diffusion and recovery time of the UW group were significantly increased (13.1 minutes vs.15.4 minutes, P<0.001), suggesting that CC+UW can better maintain electrophysiological function during cold storage. The QRS and QT intervals in the CC+UW group were shorter than those in the UW group (QRS: 13.6ms vs. 19.8ms, QT: 76.8ms vs. 98.9ms, P<0.001), indicating that the CC+UW solution can better maintain cardiac electrophysiological stability during cold storage. Based on the analysis of left ventricular pulse pressure changes, electrical conduction velocity, and heart rhythm, the CC+UW solution significantly improved the functional recovery level of the rat heart, especially in terms of cardiac ejection function and electrophysiological stability after cold storage.

[0053] Compared with the traditional UW solution, CC+UW solution has obvious advantages in static cold preservation of rat hearts. CC+UW solution can not only improve the ejection function of the heart, but also significantly increase the electrical conduction velocity, shorten the heart rhythm recovery time, and reduce the occurrence of arrhythmias. These results show that the application of CC+UW solution in cold preservation of donor hearts has important clinical potential, especially in the case of prolonged cold ischemia, which can improve the survival rate and functional recovery of heart transplants.

[0054] Example 3 Effect of CC+UW on mid-term survival rate of heart transplantation by prolonging cold ischemia time

[0055] In order to verify the advantages of the above CC+UW combination in cold preservation of donor hearts with prolonged cold ischemia time, a retrospective cohort analysis of heart transplant data from Wuhan Union Hospital and the United States Heart Transplant Registry (UNOS) was conducted. The analysis subjects were data from 360 patients who underwent heart transplantation using CC+UW preservation solution at Wuhan Union Hospital between January 1, 2019 and December 31, 2023, and data from 13,164 patients who used traditional UW solution in the UNOS database between January 2005 and December 2021.

[0056] Patients with a history of previous organ transplantation, those who were dependent on mechanical circulatory support, those who underwent multivisceral transplantation, or those who lacked complete donor and survival data were excluded from this study. The primary endpoint of the study was 5-year all-cause mortality after heart transplantation. The survival rates of the two groups of heart transplant patients under different cold ischemia time (CIT) were compared and compared using the Kaplan-Meier survival analysis method, and the Log-rank test was used for statistical evaluation. Figure 4 The basic characteristics of the acceptor and donor are shown in Figure 5 shown.

[0057] Research results: The cold ischemia time of donors in the CC+UW group of Wuhan Union Hospital was significantly longer than that in the UNOS-UW group. Specific data showed that the proportion of cold ischemia time exceeding 4 hours in the CC+UW group was higher, while the cold ischemia time of the UNOS group using traditional UW solution was concentrated in a shorter time range.

[0058] Survival analysis showed that there was no significant difference in the 5-year survival rate between the two groups. However, when the effect of cold ischemia time on survival was further analyzed, it was found that in the UNOS-UW group, when the cold ischemia time exceeded 3.3 hours, the survival rate after heart transplantation was significantly reduced; while when CC+UW was used in Wuhan United Hospital, the risk increased significantly when the cold ischemia time exceeded 5.8 hours. This shows that CC+UW can effectively prolong the cold ischemia time without significantly affecting the survival rate after transplantation.

[0059] The relationship between cold ischemia time and mortality risk was further evaluated by Cox regression analysis. The results showed that the CC+UW group was able to maintain a lower mortality risk and significantly improve the 5-year survival rate compared with the UW group even with a longer cold ischemia time.

[0060] In order to eliminate the influence of differences in the donor organ allocation system and recipient diseases, the data of 281 donors using CC+UW were extracted from the UNOS database and matched with the UNOS-UW group through PSM. The results after matching showed that the 5-year survival rate of the CC+UW group was significantly higher than that of the UW group (86.7% vs. 76.8%, P = 0.0034). This difference indicates that CC+UW can significantly improve the mid-term survival rate, especially in donor heart transplantation with a longer cold ischemia time, the advantage is more obvious.

[0061] Cox regression analysis showed that CC+UW could effectively reduce the risk of death caused by prolonged cold ischemia time compared with traditional UW solution, and the cardiac recovery index (DRI) and immune response score (IMPACT) of patients in the CC+UW group were better than those in the traditional UW group, further proving the advantage of the CC+UW group in improving the mid-term survival rate of heart transplantation.

[0062] CC+UW can not only effectively prolong the cold ischemia time, but also significantly improve the mid-term survival rate of heart transplantation. Compared with the traditional UW solution, CC+UW provides a wider use window for heart transplantation, especially in the case of long cold ischemia time, CC+UW has shown significant clinical advantages.

[0063] Comparative Example 1

[0064] This comparative example adopts the existing technology of cold storage of heart, that is, using 4℃ modified St.Thomas solution (K + 20mmol / L) perfusion to stop the heart, after the donor heart is taken out, it is rinsed with cold saline, followed by perfusion with 1000ml of 4℃ UW solution, and then immersed in 4℃ saline for low-temperature storage. This existing technology can make the cold ischemia time of the donor heart 3 hours. After the cold ischemia of the donor heart exceeds 3 hours, the risk of metabolite depletion increases significantly, and the 5-year survival rate of transplant patients decreases significantly. It is less suitable for donor hearts that require long-term transportation and marginal donors.

[0065] The technology in the embodiment of the present invention uses 4℃ cold crystalloid cardioplegia to stop the heart. After the donor heart is taken out, it is perfused again with 2000ml 4℃ UW solution for 8-12 minutes. The donor heart is then stored in a 4℃ UW solution environment. CC rapid cardioplegia reduces warm ischemia time, and UW solution continuously inhibits metabolism, prolonging the storage time. The technology of the present invention can prolong the cold ischemia time of the donor heart to more than 8 hours, and the clinical application safety time is up to 5.8 hours ( Figure 4 The technology of the present invention reduces the consumption of metabolites by 60% ( Figure 1 ), which can delay energy depletion, protect mitochondrial function, and reduce the risk of reperfusion injury. The technology of the present invention can increase the left ventricular systolic pressure by 66% (48.7mmHg) and accelerate the electrical conduction velocity to 0.8mm / ms ( Figure 3 ), the recovery of donor heart function is enhanced, the ejection capacity and electrophysiological stability are improved, and the risk of postoperative arrhythmia is reduced. In terms of clinical prognosis, the 5-year survival rate of patients is increased to 86.7%, and the survival rate remains high after cold ischemia for more than 6 hours, and the survival rate is significantly improved. The technology of the present invention is particularly suitable for long-term transportation or marginal donors, expanding the utilization rate of the donor pool.

[0066] Comparative Example 2

[0067] This comparative example adopts UW solution perfusion cardioplegia and UW solution preservation technology.

[0068] The results showed that compared with the method in this comparative example, the experimental group using CC+UW solution in the embodiment of the present invention had better recovery of transplanted heart function after cold ischemia, especially the recovery of ATP level and metabolic markers. The CC+UW method stopped the heart from contracting within seconds.

[0069] Since the left ventricle (LV) plays a major role in cardiac recovery after cold storage, a quantitative metabolomics approach was used to further validate the effects of the above two methods on LV metabolite levels. Figure 1 The results showed that the CC+UW method significantly reduced the consumption of D-glucose during SCS (1.110^6 vs. 0.410^6 ng / g at 0 h, 1.510^6 vs. 0.310^6 ng / g at 8 h, P<0.05). Analysis of D-glucose metabolic intermediates showed that the glycolytic flux was reduced in the CC+UW group compared with the UW group. Intermediate metabolites such as F6P (0.710^6 vs. 1.210^6 ng / g at 0 hour, P<0.05), FBP (0.510^5 vs. 6.610^5 ng / g at 0 hour, P<0.05), DAP (0.210^4 vs. 2.210^4 ng / g at 0 hour, P<0.05), G3P (0.210^4 vs. 0.810^4 ng / g at 0 hour, P<0.05), 3-PGA (2.110^3 vs. 8.010^3 ng / g at 0 hour, P<0.05) and 2-PGA (0.310^4 vs. 1.010^4 ng / g at 0 hour, P<0.05) were at lower levels in the CC+UW group, and their levels then gradually returned to those of the UW group. The levels of 1,3-BPGA and PEP were lower in the CC+UW group.

[0070] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A liquid composition for heart preservation, characterized in that: The liquid composition includes crystalloid cardioplegia solution (CC solution) and UW solution; the CC solution formula is: 470-480ml of compound electrolytes, 5-7ml of 10% potassium chloride, 9-11ml of 50% glucose, 7-8ml of 5% sodium bicarbonate and 2-3ml of heparin; the UW solution is 1800-2200ml of 4°C UW solution.

2. A method for preserving a donor heart, characterized in that: The method comprises using the liquid composition of claim 1.

3. Use of the liquid composition according to claim 1 or the donor heart preservation method according to claim 2 in the preservation of an ex vivo donor heart.

4. The use according to claim 3, characterized in that: The method of using the liquid composition in the application is to first double-perfuse with CC solution and UW solution, and then store it with UW solution.

5. The use according to claim 4, characterized in that: The application specifically comprises the following steps: (a) Prepare CC solution according to the formula; (b) CC solution was perfused through the aorta at a pressure of 140-160 mmHg for 3-5 minutes to stop the heart and cool it down; (c) The heart was removed and perfused again with UW solution for 8-12 minutes; (d) The heart was preserved in UW solution with a cold ischemia time of ≤8 h.

6. The use according to claim 5, characterized in that: The formula of the CC solution is: 475 ml of compound electrolyte, 6 ml of 10% potassium chloride, 10 ml of 50% glucose, 7.5 ml of 5% sodium bicarbonate and 2.5 ml of heparin.

7. The use according to claim 5, characterized in that: The perfusion pressure of CC solution in step (b) is 150 mmHg.

8. The use according to claim 5, characterized in that: In step (b), the aortic root pressure is maintained at 60-100 mmHg.

9. The use according to claim 5, characterized in that: The UW solution is 2000 ml of 4° C. UW solution.

10. The use according to claim 5, characterized in that: The cold ischemia time is 4-8 hours.

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