Kidney in-vitro low-temperature preservation and rewarming reperfusion method, preservation solution and perfusion solution

Abstract

The invention provides a kidney in-vitro low-temperature preservation and rewarming reperfusion method, preservation fluid and perfusion fluid, and belongs to the technical field of biomedical engineering.The method comprises the steps that an isolated organ sample is placed in deep supercooling preservation fluid, an oil layer is added to the surface of the isolated organ sample to isolate air, and deep supercooling preservation is conducted for 1-7 days under the condition that the deep supercooling temperature is minus 10 DEG C; after the preservation is finished, connecting the isolated organ sample into a perfusion system for rewarming and reperfusion: transitioning from a deep supercooling temperature of-10 DEG C to a set temperature, heating by stages, gradually increasing the perfusion pressure, and supplying oxygen at the same time; wherein perfusate used for reperfusion is cell-free perfusate, and the perfusate is based on low-sugar DMEM (Dulbecco Modified Eagle Medium) and supplements nutrient substances and creatinine. According to the invention, cell damage and oxidative stress caused by rapid rewarming to organs can be reduced to a certain extent.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and particularly relates to a method for extracorporeal hypothermic preservation, rewarming and reperfusion of kidneys, a preservation solution and a perfusion solution. Background Art

[0002] Kidney transplantation is the most effective treatment for patients with end-stage renal disease, which can significantly restore renal function and improve the quality of life of patients. However, the shortage of donors remains the main bottleneck in organ transplantation worldwide. Among the available kidney donors, the effect of organ preservation plays a decisive role in the success rate of transplantation surgery and the long-term function after transplantation. Therefore, how to maximize the utilization of existing kidney sources under the condition of donor shortage and ensure the transplantation success rate is the key problem to be solved urgently at present.

[0003] 1. Existing mainstream kidney preservation technologies and limitations. Currently, the most commonly used kidney preservation method in clinics is static cold storage. Its operating principle is to immerse the kidney in a preservation solution (such as UW solution) in a low-temperature environment of about 4°C, and extend the preservation time by reducing the organ metabolism level. Since static cold storage has simple steps, low equipment requirements, and relatively low economic costs, it is still the most commonly used preservation technology in kidney transplantation surgeries worldwide.

[0004] However, static cold storage has the following defects and deficiencies: 1) Limited preservation time: Generally, it is recommended that the kidney preservation time does not exceed 24 hours under 4°C cold storage conditions. After exceeding this time limit, significant damage to kidney function and tissue structure will occur; 2) It may lead to cell edema and accumulation of metabolic products: In a low-temperature environment, although the overall metabolic rate decreases, there are still some metabolic activities, and the interruption of kidney blood supply leads to the inability to remove metabolic wastes in time, ultimately resulting in ischemia-reperfusion injury; 3) It is difficult to meet the needs of long-distance transportation and organ sharing: With the increasing demand for cross-regional and even cross-country allocation of organs, traditional static cold storage is difficult to support long-distance transportation due to the limitation of preservation time.

[0005] To break through the preservation time limit of static cold storage, researchers have gradually started to explore the "supercooling" technology below 0°C. Theoretically, when the temperature decreases by 10°C, the organ metabolic rate can be reduced by about 50%. Therefore, lower temperatures can further slow down ischemic injury and extend the preservation time. However, the formation of ice crystals below 0°C will cause irreversible damage to cell and tissue structures, which is the core problem that must be solved in supercooling preservation.

[0006] 2. Technical difficulties faced during rewarming and reperfusion processes. Regardless of the cryopreservation method used, the kidney needs to be rewarmed to near physiological temperature before transplantation and then transplanted into the recipient. The traditional approach is to rapidly warm the refrigerated kidney and directly perfuse it with physiological solution. However, during the sudden changes in temperature and vascular perfusion pressure, severe reperfusion injury is highly likely to occur. Additionally, how to accurately evaluate the functional status of the kidney after rewarming and promote its functional recovery as much as possible is also an urgent problem to be solved clinically. For this reason, normothermic machine perfusion technology has received attention. At a physiological temperature of 37°C, by perfusing nutrients, oxygen, and specific drugs to simulate the in vivo environment, the kidney can recover some physiological functions in vitro and thereby evaluate its suitability for transplantation. However, if only normothermic machine perfusion technology is used, when the organ is directly heated from 4°C or even lower to 37°C, it will still face severe temperature shock and sudden changes in vascular pressure.

[0007] In summary, the existing kidney preservation technologies mainly include 4°C static cold storage, which has limited preservation time and cannot well meet the current demand for cross-regional allocation of organs; while supercooling preservation can significantly reduce the metabolic level and extend the preservation time, but still faces the difficult-to-break bottleneck of "ice crystal nucleation"; at the same time, how to achieve a smooth transition between low temperature and normal temperature, reduce reperfusion injury, and accurately evaluate kidney function is also a major difficulty in clinical application. Summary of the Invention

[0008] To overcome the above problems, the present invention proposes a method for cryopreservation, rewarming, and reperfusion of kidneys in vitro, a preservation solution, and a perfusion solution. Through the concept of controlled oxygenation rewarming, during the heating process from low temperature to normal temperature, the temperature and perfusion pressure are gradually increased, and appropriate oxygenation is provided, which can reduce cell damage and oxidative stress caused by rapid rewarming to a certain extent.

[0009] To achieve the above objectives, the present invention adopts the following technical solutions: In a first aspect, a method for cryopreservation, rewarming, and reperfusion of organs in vitro includes: Placing an ex vivo organ sample in a supercooling preservation solution and adding an oil layer on the surface to isolate air, and performing supercooling preservation at a supercooling temperature of -10°C for 1 - 7 days; After the preservation is completed, connecting the ex vivo organ sample to a perfusion system for rewarming and reperfusion: when transitioning from the supercooling temperature of -10°C to the set temperature, using staged heating and gradually increasing the perfusion pressure, while supplying oxygen; among them, the perfusion solution used for reperfusion is a cell-free perfusion solution, and the perfusion solution is based on low-glucose DMEM and supplemented with nutrients and creatinine.

[0010] As a further improvement of the present invention, the temperature of the supercooling preservation solution is 4°C, and the kidney is completely immersed in the bottom.

[0011] As a further improvement of the present invention, the oil layer is paraffin oil, and the paraffin oil floats on the surface of the deeply supercooled preservation solution.

[0012] As a further improvement of the present invention, the composition of the deeply supercooled preservation solution includes:

[0013] Wherein, it is measured based on the total volume of the deeply supercooled preservation solution being 1 L; Before use, the deeply supercooled preservation solution is dissolved with pure water and the pH is adjusted to 7.35 - 7.4.

[0014] As a further improvement of the present invention, the perfusion system is of the flow control type, adopts a closed-loop operation mode, and uses a peristaltic pump to transport the perfusion fluid; the membrane oxygenator in the loop is connected to a gas flow meter, and Carbogen gas is used to oxygenate the perfusion fluid discharged from the renal vein, and then it is transported to the renal artery, thereby realizing continuous perfusion of the kidney; By adjusting the flow rate of the peristaltic pump, the perfusion pressure is made 20 - 100 mmHg.

[0015] As a further improvement of the present invention, the composition of the perfusion fluid for rewarming and reperfusion includes:

[0016] Wherein, it is measured based on the total volume of the perfusion fluid being 450 - 550 mL.

[0017] As a further improvement of the present invention, during the rewarming and reperfusion process, perfusion fluid and urine samples are collected at intervals and stored at -80°C; The concentrations of glucose, lactate, electrolytes in the perfusion fluid or urine are also measured, as well as the partial pressure of oxygen, partial pressure of carbon dioxide, pH value and osmotic pressure; the concentrations of aspartate aminotransferase, lactate dehydrogenase and uric acid in the perfusion fluid are determined, as well as the creatinine concentration in the perfusion fluid and urine.

[0018] As a further improvement of the present invention, the rewarming and reperfusion stage includes two stages: controlled oxygenated rewarming and normothermic mechanical perfusion: Controlled oxygenated rewarming: The initial perfusion temperature is set at 4°C, and then it is gradually increased to 10°C, 25°C and 37°C at 15 minutes, 30 minutes and 60 minutes respectively; As the temperature increases, the arterial perfusion pressure is also adjusted accordingly: it is 20 - 40 mmHg at 4°C, 40 - 80 mmHg at 25°C, and finally adjusted to the physiological range of 80 - 100 mmHg at 37°C; Normothermic mechanical perfusion: Maintain continuous perfusion at 37°C for 1 hour, and the perfusion pressure is 80 - 100 mmHg.

[0019] Second aspect, a deep supercooling preservation solution for cryopreserving organs in vitro, comprising:

[0020] wherein, based on the total volume of the deep supercooling preservation solution being 1 L for measurement; Third aspect, the present invention provides a perfusion solution for rewarming and reperfusion, for rewarming and reperfusing organs in vitro, comprising:

[0021] wherein, based on the total volume of the perfusion solution being 450 - 550 mL for measurement.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The deep supercooling technology proposed by the present invention can inhibit the ice crystal nucleation of the solution by using the preservation solution and adding an oil layer on the surface at an environment as low as -10°C to achieve more stable supercooling preservation; meanwhile, combined with controlled oxygenation rewarming, the kidney is gradually heated from -10°C to 37°C; then, normal temperature mechanical perfusion is used to simulate the in-vivo physiological environment to evaluate and restore the kidney function in real time. Experiments prove that under this comprehensive technology, not only can the kidney preservation time be significantly extended to 96 hours (compared with the traditional static cold storage which can only maintain for 24 hours), but also the performance of the organ after rewarming in terms of oxidative stress, cell damage markers, and energy metabolism is better than or equivalent to the prior art level, providing a more flexible and reliable technical solution for kidney transplantation, especially for remote organ allocation. Description of the Drawings

[0023] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0024] Figure 1 is the research design process. a, Schematic diagram of the sample temperature change in the scheme. b, Cold flushing (removing blood) of the rat kidney. c, Deep supercooling (DSC) preservation of the rat kidney. d, Mechanical perfusion system (main components are marked in the figure). e, Appearance of the rat kidney preserved by DSC before and after rewarming and reperfusion.

[0025] Figure 2 shows the electrolytes and perfusion parameters during rewarming and reperfusion. a - d, Perfusion solution electrolytes (potassium, sodium, calcium, and chloride ion concentrations). e, pH value of the perfusion solution. f, Osmotic pressure of the perfusion solution. g, Oxygen partial pressure of the perfusion solution. h, Carbon dioxide partial pressure of the perfusion solution. i, Perfusion pressure.

[0026] Figure 3 shows renal function and metabolic indicators. a, Lactic acid production. b, Glucose consumption. c, Uric acid production. d, Total urine volume during controlled oxygenated rewarming and normothermic machine perfusion. e, Adenosine triphosphate (ATP) content in renal tissue. f, Total creatinine clearance (CrCl).

[0027] Figure 4 They are indicators of kidney injury, oxidative stress, and antioxidant defense. a, Aspartate aminotransferase (AST) level in perfusion fluid. b, Lactate dehydrogenase (LDH) level in perfusion fluid. c, Increase in kidney weight after rewarming and reperfusion. d, Urinary protein level. e, Urinary glucose level. f, Level of kidney injury molecule-1 (KIM-1) in urine. g, Reactive oxygen species (ROS) level in renal tissue. h, Superoxide dismutase (SOD) activity in renal tissue.

[0028] Figure 5 They are for histological analysis. a, Renal cortex (HE staining). b, Renal medulla (HE staining). c, Kidney injury score (n = 5 for each group; the scoring criteria are detailed in the Materials and Methods section). Black, blue, green, and yellow arrows indicate lumen dilation, glomerular injury, vacuolization, and necrosis or exfoliation of renal tubular epithelial cells, respectively. d, Renal cortex (Masson trichrome staining). e, Renal medulla (Masson trichrome staining). f, Collagen volume fraction in renal tissue (n = 4 for each group). Detailed implementation manners

[0029] To make the technical problems, technical solutions, and beneficial effects to be solved by this application clearer and more understandable, the following further details this application in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not used to limit this application.

[0030] In this application, the term "and / or" describes the association relationship of associated objects and indicates that there can be three relationships. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Here, A and B can be singular or plural. The character " / " generally represents an "or" relationship between the front and back associated objects.

[0031] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following" or its similar expressions refer to any combination of these items, including any combination of single item(s) or plural item(s). For example, "at least one of a, b, or c", or, "at least one of a, b, and c" can both represent: a, b, c, a - b (i.e., a and b), a - c, b - c, or a - b - c, where a, b, and c can be single or multiple respectively.

[0032] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not imply the sequence of execution. Some or all of the steps may be executed in parallel or sequentially. The execution sequence of each process should be determined according to its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0033] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "the", and "said" used in the embodiments of the present application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0034] The weights of the relevant components mentioned in the specification of the embodiments of the present application not only can refer to the specific contents of each component, but also can represent the proportional relationship of the weights between each component. Therefore, as long as the contents of the relevant components in the specification of the embodiments of the present application are enlarged or reduced in proportion, they are within the scope disclosed in the specification of the embodiments of the present application. Specifically, the mass described in the specification of the embodiments of the present application can be mass units well-known in the chemical engineering field such as μg, mg, g, kg, etc.

[0035] The present invention provides an in vitro organ cryopreservation and rewarming reperfusion method, including: Placing the ex vivo organ sample in a deeply supercooled preservation solution, and adding an oil layer on the surface to isolate air, and performing deeply supercooled preservation for 1 - 7 days at the deeply supercooled temperature of -10°C; After the preservation is completed, connecting the ex vivo organ sample to a perfusion system for rewarming reperfusion: when transitioning from the deeply supercooled temperature of -10°C to the set temperature, heating in stages and gradually increasing the perfusion pressure, and simultaneously supplying oxygen; wherein, the perfusion fluid used for reperfusion is a cell-free perfusion fluid, and the perfusion fluid is based on low-glucose DMEM and supplemented with nutrients and creatinine.

[0036] To better understand the present invention, the present invention will be described in detail below in conjunction with specific embodiments, but the content of the present invention is not limited to the following embodiments.

[0037] Example 1 Step 1. Materials and methods Step 1.1 Experimental animals The kidney samples used in this embodiment were taken from male Sprague-Dawley (SD) rats weighing 200 - 250 grams. These SD rats were provided by the Animal Center of Xi'an Jiaotong University. All animals received humane care in accordance with the guidelines of the Experimental Animal Use and Management Committee of Xi'an Jiaotong University.

[0038] Step 1.2 Research design This method first flushes the donor kidney with UW solution at 4°C to remove blood and terminate warm ischemia (this process takes about 20 minutes). Subsequently, the kidney is excised and placed in a deep supercooling preservation solution (the composition is shown in Table 1), and an oil layer is added on the surface to isolate air, achieving deep supercooling preservation at -10°C (1 - 7 days). After the preservation is completed, the kidney is connected to a mechanical perfusion system for rewarming and reperfusion. Through controlled oxygenated rewarming (1 hour), the temperature is gradually increased and the vascular perfusion pressure is adjusted, and finally, kidney function assessment is carried out under normothermic mechanical perfusion (1 hour) ( Figure 1 a).

[0039] Figure 1 shows the research design process. a, Schematic diagram of the sample temperature change in the protocol. b, Cold flushing (removing blood) of the rat kidney. c, Deep supercooling (DSC) preservation of the rat kidney. d, Mechanical perfusion system (the main components are marked in the figure). e, Appearance of the DSC-preserved rat kidney before and after rewarming and reperfusion. COR, Controlled oxygenated rewarming; NMP, Normothermic mechanical perfusion; DSC, Deep supercooling preservation; UW, University of Wisconsin solution; SIB, Synthetic ice inhibitor (1,4-cyclohexanediol is used in this example); PEG, Polyethylene glycol; PVA, Polyvinyl alcohol; PO, Paraffin oil.

[0040] Step 1.3 Acquisition of the kidney In this example, kidney samples are obtained through an animal dissection procedure. Before the experiment, SD rats are fasted for 8 hours. The rats are anesthetized by intraperitoneal injection of sodium pentobarbital (10%, 0.3 ml / 100 g). After routine abdominal disinfection, a median abdominal incision is made to ensure complete exposure of the kidneys. Subsequently, 1 mL of heparin sodium (3500 IU / mL) is injected into the inferior vena cava, and the renal artery, vein, and ureter of the donor kidney are dissected. Then, the proximal and distal ends of the inferior vena cava and the abdominal aorta are ligated.

[0041] Since the residual blood in the organ will change the composition and properties of the preservation solution, thus affecting the organ preservation effect, cold perfusion is required to remove the blood in the kidney and terminate warm ischemia. A catheter is inserted into the abdominal aorta, and the kidney is perfused with UW solution (25 ml, 4°C). The incision of the inferior vena cava serves as the outflow channel for the solution, and the perfusion continues until the color of both kidneys turns white ( Figure 1 b). After the perfusion is completed, the kidney is quickly excised and placed in cold PBS buffer to remove excess adipose tissue.

[0042] Step 1.4 Deep supercooling preservation of the kidney After clearing the blood, the kidney samples were placed in a 50 ml centrifuge tube containing 5 ml of deeply supercooled preservation solution (Table 1; the solution was at 4 °C), and the kidneys were completely immersed at the bottom. Then, approximately 2 ml of paraffin oil was slowly added along the inner wall of the centrifuge tube to float on the surface of the deeply supercooled preservation solution, thus isolating air and preventing ice crystal formation. Since factors such as air bubbles in the deeply supercooled preservation solution may also cause heterogeneous ice crystal nucleation, pretreatment including removing air bubbles was required. Finally, the centrifuge tube was transferred to a refrigerator set at the target temperature for deeply supercooled preservation ( Figure 1 c).

[0043] Table 1. Composition of the deeply supercooled preservation solution

[0044] After dissolving with pure water, the pH was adjusted to 7.4 using sodium hydroxide.

[0045] Step 1.5 Setup of the in vitro mechanical perfusion system The perfusion system ( Figure 1 d) was of the flow control type, operating in a closed-loop mode, and using a peristaltic pump to deliver the perfusion fluid. The membrane oxygenator in the circuit was connected to a gas flow meter, and Carbogen gas (a mixture of 95% O 2 and 5% CO 2 ) was used to oxygenate the perfusion fluid discharged from the renal vein at a flow rate of 1 L / min and then deliver it to the renal artery, thus achieving continuous perfusion of the kidney. The arterial perfusion catheter was connected to a multi-channel physiological signal acquisition system for monitoring parameters such as perfusion pressure and temperature. By adjusting the flow rate of the peristaltic pump, the perfusion pressure could be maintained within the range of 20 - 100 mmHg. The organ chamber was placed in a constant-speed programmable temperature-controlled bath to precisely adjust the perfusion temperature.

[0046] Step 1.6 Experimental group setup All rat kidneys were perfused with UW solution to clear the blood and randomly assigned to one of the following five groups: 1. Fresh group: The kidneys were not refrigerated or otherwise treated and were immediately connected to the mechanical perfusion system for controlled oxygenation rewarming and normothermic mechanical perfusion treatment, serving as the fresh control group.

[0047] 2. SCS group: The kidneys were statically refrigerated in UW solution at 4 °C for 24 hours, serving as the standard control group.

[0048] 3. DSC-D1 group: The kidneys were preserved in the deeply supercooled preservation solution at -10 °C (composition shown in Table 1) for 24 hours.

[0049] 4. DSC-D4 group: The kidneys were preserved in the deeply supercooled preservation solution at -10 °C for 96 hours.

[0050] 5. DSC - D7 group: The kidneys were stored in a deep supercooling preservation solution at -10°C for 168 hours.

[0051] After the storage, the kidneys in groups 2 - 5 were all connected to a mechanical perfusion system for 2 - hour controlled oxygenated rewarming and normothermic mechanical perfusion ( Figure 1 e). In this example, the conditions of controlled oxygenated rewarming and normothermic mechanical perfusion for all groups were kept consistent. There were no significant differences among the groups in terms of warm ischemia time, duration of controlled oxygenated rewarming and normothermic mechanical perfusion, and initial kidney weight.

[0052] Step 1.7 Rewarming and reperfusion method (controlled oxygenated rewarming and normothermic mechanical perfusion) The rewarming and reperfusion stage included two stages: controlled oxygenated rewarming (1 hour) and normothermic mechanical perfusion (1 hour), and the total duration was set to 2 hours.

[0053] Controlled oxygenated rewarming: The initial perfusion temperature was set at 4°C, and then gradually increased to 10°C, 25°C, and 37°C at 15 minutes, 30 minutes, and 60 minutes respectively. Meanwhile, as the temperature increased, the arterial perfusion pressure was also adjusted accordingly: 20 - 40 mmHg at 4°C, 40 - 80 mmHg at 25°C, and finally adjusted to 80 - 100 mmHg within the physiological range at 37°C.

[0054] Normothermic mechanical perfusion: Maintain a continuous perfusion at 37°C for 1 hour, with a perfusion pressure of 80 - 100 mmHg to simulate the surgical transplantation stage.

[0055] The perfusion fluid used for reperfusion was a cell - free perfusion fluid (Table 2). The perfusion fluid was based on low - glucose DMEM and supplemented with various nutrients and drugs to promote renal function recovery. Creatinine was also added to the perfusion fluid to evaluate the creatinine clearance ability of the kidneys.

[0056] Table 2. Composition of the perfusion fluid

[0057] Step 1.8 Determination of perfusion fluid, urine, and tissue - related indicators During the rewarming and reperfusion process, perfusion fluid and urine samples were collected every 30 minutes and stored at -80 °C for subsequent analysis. Glucose, lactate, electrolyte concentrations, as well as partial pressure of oxygen, partial pressure of carbon dioxide, pH value, and osmotic pressure in the perfusion fluid or urine were measured using a blood gas analyzer. Aspartate aminotransferase, lactate dehydrogenase, and uric acid concentrations in the perfusion fluid, as well as creatinine concentration in the perfusion fluid and urine, were determined using an automatic biochemical analyzer. In this example, creatinine clearance rate (CrCl) was used as an indicator of glomerular filtration rate, and its calculation formula was: CrCl = creatinine (urine) × urine flow rate / creatinine (perfusion fluid). Urinary protein concentration was measured using an enhanced BCA protein quantification kit according to the manufacturer's instructions. The level of the renal tubular injury marker kidney injury molecule 1 (KIM-1) was determined using an ELISA kit for rat KIM-1. Kidney tissue samples were collected immediately after the completion of rewarming and reperfusion, and relevant indicators were determined according to the instructions of the reagent supplier. The energy status of the kidney tissue was obtained by measuring the ATP content. The tissue oxidative stress status was obtained by measuring the ROS level. The tissue antioxidant defense ability was obtained by measuring the superoxide dismutase activity. To monitor potential edema formation, the kidney weight was measured before and after rewarming and reperfusion, and the weight increase rate was calculated as: (weight increase / initial weight × 100%).

[0058] Step 1.9 Histology After the experiment, kidney samples were rapidly collected and fixed in 4% paraformaldehyde solution for subsequent histological examination. The morphological integrity of the renal parenchyma was evaluated by hematoxylin-eosin (HE) staining. The HE staining scoring standard was used to evaluate four aspects on a 5-point scale: tubular dilation, vacuolization, glomerular injury, and tubular epithelial cell exfoliation. The scoring criteria were as follows: 0 = no injury; 1 = injury < 10%; 2 = 10% to 25%; 3 = 25% to 50%; 4 = 50% to 75%; 5 => 75%.

[0059] For each kidney, 10 non-overlapping cortical fields were randomly selected at 100× magnification for blind scoring. The average score of the four evaluated aspects was calculated as the average tissue injury score for each kidney.

[0060] The collagen deposition in the kidney tissue was evaluated by Masson trichrome staining, and the collagen volume fraction was used as a quantitative index of fibrosis. For each kidney, 10 non-overlapping cortical fields were randomly selected at 100× magnification for measurement. The calculation formula for the collagen volume fraction was: (collagen fiber area / total tissue area) × 100%.

[0061] Step 1.10 Data analysis: All values are presented as mean ± standard deviation. Statistical analysis and graph plotting were performed using GraphPad Prism software. Intergroup differences were evaluated using one-way or two-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0062] Step 2. Results of functional characterization Step 2.1 Electrolytes and perfusion parameters Figure 2 shows the electrolytes and perfusion parameters during rewarming and reperfusion. a-d, Electrolytes in the perfusion fluid (potassium, sodium, calcium, and chloride ion concentrations). e, pH value of the perfusion fluid. f, Osmotic pressure of the perfusion fluid. g, Partial pressure of oxygen in the perfusion fluid. h, Partial pressure of carbon dioxide in the perfusion fluid. i, Perfusion pressure.

[0063] Values are expressed as mean ± standard deviation, with n = 5 in each group. * p < 0.05, ** p < 0.01 compared with the fresh group; ns, no significant difference. SCS, static cold storage for 24 hours; DSC-D1 / D4 / D7, deep supercooling preservation for 24, 96, and 168 hours.

[0064] Regarding the electrolyte concentrations in the perfusion fluid, after 2 hours of rewarming and reperfusion, the concentrations of K⁺, Na⁺, and Cl⁻ increased in each group ( Figure 2 a, b, d), while the Ca²⁺ concentration decreased ( Figure 2 c). Only the K⁺ ( Figure 2 a, p = 0.0016) and Na⁺ concentrations ( Figure 2 b, p = 0.0080) in the DSC-D7 group were significantly higher than those in the Fresh group. No significant differences were observed between the other groups. The pH value of the perfusion fluid remained similar among the groups but gradually decreased in the later stage of rewarming and reperfusion (90 - 120 minutes). Although the pH decrease in the DSC-D7 group was more obvious, the intergroup difference was not statistically significant ( Figure 2 e). The osmotic pressure of the perfusion fluid increased in each group during reperfusion, and only the osmotic pressure in the DSC-D7 group was significantly higher than that in the Fresh group at 120 minutes ( Figure 2 f, p = 0.0182). In terms of gas exchange, the partial pressure of oxygen was relatively stable in each group ( Figure 2 g), while the partial pressure of carbon dioxide gradually decreased ( Figure 2 h), and there were no significant differences between the groups. The perfusion pressure gradually increased in each group, which was consistent with the requirements of controlled oxygenated rewarming and normothermic machine perfusion, and no significant differences were observed between the groups ( Figure 2 i).

[0065] Step 2.2 Renal function and metabolic indicators Figure 3 shows renal function and metabolic indices. a, Lactic acid production. b, Glucose consumption. c, Uric acid production. d, Total urine output during controlled oxygenthermic reperfusion and normothermic machine perfusion. e, Adenosine triphosphate (ATP) content in renal tissue. f, Total creatinine clearance (CrCl).

[0066] Values are expressed as mean ± standard deviation, n = 5 per group. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001 vs. Fresh group; # p<0.05, ## p<0.01, ### p<0.001, #### p<0.0001 vs. SCS group; ns, no significant difference. SCS, static cold storage for 24 h; DSC-D1 / D4 / D7, deep supercooling preservation for 24, 96, and 168 h.

[0067] Lactic acid levels gradually accumulated during reperfusion ( Figure 3 a). Compared with the Fresh group, the lactic acid concentration in the DSC-D7 group was significantly higher at 90 min (p = 0.0071) and 120 min (p = 0.006), while no significant differences were observed between the other groups. Uric acid concentrations showed a steady upward trend in all groups ( Figure 3 c). There was no significant difference between the DSC-D1 group and the Fresh group, and the DSC-D4 group was similar to the SCS group. However, the uric acid concentration in the DSC-D7 group was significantly lower than that in the SCS group (p values at 30, 60, 90, and 120 min were 0.0151, 0.0361, 0.0123, and 0.0023, respectively). In addition, all groups continuously consumed glucose during rewarming reperfusion ( Figure 3 b), and the consumption rate gradually increased, indicating ongoing metabolic activity. Compared with the Fresh group, the glucose consumption rate in the DSC-D7 group was significantly lower at 30 min (p = 0.0002), but significantly higher than that in the SCS group at 120 min (p = 0.0019). No significant differences were observed between the other groups. During rewarming reperfusion, the total urine output in the Fresh group was significantly higher than that in all cold storage groups ( Figure 3 d, p<0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0209 vs. DSC-D1). The urine output in the DSC-D1 group was significantly higher than that in the SCS group (p = 0.0127), while there was no significant difference between the DSC-D4 and DSC-D7 groups and the SCS group. The ATP content in the kidneys of the Fresh group was significantly higher than that in all cold storage groups ( Figure 3e, p < 0.0001). The ATP level in the DSC-D1 group was significantly higher than that in the SCS group (p < 0.0001). Although the ATP content in the DSC-D4 group was slightly higher than that in the SCS group, the difference was not statistically significant. The ATP content in the DSC-D7 group was significantly lower than that in the SCS group (p = 0.0233). The creatinine clearance rate was the highest in the Fresh group ( Figure 3 f), significantly higher than all other groups (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0004 vs. DSC-D1). The creatinine clearance rate in the DSC-D1 group was significantly higher than that in the SCS group (p = 0.0054), while there was no significant difference between the DSC-D4 and DSC-D7 groups and the SCS group.

[0068] Step 2.3 Kidney injury, oxidative stress, and oxidative defense indicators Figure 4 were used as kidney injury, oxidative stress, and antioxidant defense indicators. a, Aspartate aminotransferase (AST) level in the perfusion fluid. b, Lactate dehydrogenase (LDH) level in the perfusion fluid. c, Increase in kidney weight after rewarming and reperfusion. d, Urinary protein level. e, Urinary glucose level. f, Level of kidney injury molecule-1 (KIM-1) in urine. g, Reactive oxygen species (ROS) level in kidney tissue. h, Superoxide dismutase (SOD) activity in kidney tissue. Values are expressed as mean ± standard deviation, n = 5 per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the Fresh group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, compared with the SCS group; ns, no significant difference. SCS, static cold storage for 24 hours; DSC-D1 / D4 / D7, deep supercooling preservation for 24, 96, and 168 hours.

[0069] Regarding the injury markers, aspartate aminotransferase in the perfusion fluid ( Figure 4 a) and lactate dehydrogenase ( Figure 4b) Horizontally, it gradually accumulated in each group during reperfusion. The aspartate aminotransferase level in the DSC-D1 group was comparable to that in the Fresh group and was significantly lower than that in the SCS group at 90 minutes (p = 0.0097) and 120 minutes (p < 0.0001). In contrast, the aspartate aminotransferase level in the DSC-D7 group was significantly higher than that in the SCS group at 90 minutes (p = 0.0199) and 120 minutes (p < 0.0001). For lactate dehydrogenase, the level in the DSC-D1 group was similar to that in the Fresh group, and the levels in the DSC-D1 group and DSC-D4 group were significantly lower than those in the SCS group at 90 minutes and 120 minutes (p < 0.0001). In contrast, there was no significant difference between the DSC-D7 group and the SCS group. The changes in kidney weight indicated that varying degrees of edema occurred in each group after reperfusion ( Figure 4 c). The weight increase in the Fresh group was the lowest (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0351 vs. DSC-D1). The weight increase in the DSC-D1 group was significantly lower than that in the SCS group (p = 0.0001), while the weight increase in the DSC-D7 group was significantly higher than that in the SCS group (p = 0.0044). Proteins and glucose were detectable in the urine ( Figure 4 d, e). The urinary protein (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0119 vs. DSC-D1) and glucose levels (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0129 vs. DSC-D1) in the Fresh group were the lowest among all groups. The urinary protein (p < 0.0001) and glucose levels (p < 0.0001) in the DSC-D1 group were significantly lower than those in the SCS group, while the urinary protein (p < 0.0001) and glucose levels (p = 0.0003) in the DSC-D7 group were significantly higher than those in the SCS group. The urinary KIM-1 levels ( Figure 4 f) indicated tubular injury in each group. The KIM-1 level in the Fresh group was the lowest (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.031 vs. DSC-D1). The KIM-1 level in the DSC-D1 group was significantly lower than that in the SCS group (p = 0.0242), while the level in the DSC-D7 group was significantly higher than that in the SCS group (p = 0.0002). The levels of reactive oxygen species reflecting oxidative stress ( Figure 4g) Significantly lower in the Fresh group than in other groups (p < 0.0001 vs. SCS and DSC-D7; p = 0.0437 vs. DSC-D1; p = 0.0004 vs. DSC-D4). The level of reactive oxygen species in the DSC-D1 group was significantly lower than that in the SCS group (p = 0.0061), while there was no significant difference between the DSC-D7 group and the SCS group. The superoxide dismutase activity reflecting the antioxidant defense ability ( Figure 4 h) Significantly higher in the Fresh group than in other groups (p < 0.0001 vs. SCS, DSC-D4 and DSC-D7; p = 0.016 vs. DSC-D1). The superoxide dismutase activity in the DSC-D1 group was significantly higher than that in the SCS group (p < 0.0001), while the superoxide dismutase activity in the DSC-D7 group was significantly lower than that in the SCS group (p = 0.0069). Except for the level of lactate dehydrogenase in the perfusion fluid, the DSC-D4 group showed no significant difference compared with the SCS group in the above indexes.

[0070] Step 2.4 Histological analysis Figure 5 For histological analysis. a, Renal cortex (HE staining). b, Renal medulla (HE staining). c, Renal injury score (n = 5 in each group; the scoring criteria are detailed in the Materials and Methods section). Black, blue, green and yellow arrows indicate lumen dilation, glomerular injury, vacuolization and necrosis or exfoliation of renal tubular epithelial cells, respectively. d, Renal cortex (Masson trichrome staining). e, Renal medulla (Masson trichrome staining). f, Collagen volume fraction of renal tissue (n = 4 in each group). Black triangles indicate collagen deposition. The scale bar is 50 μm. Values are expressed as mean ± standard deviation. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, compared with the Fresh group; # p < 0.05, ## p < 0.01, ### p < 0.001, #### p < 0.0001, compared with the SCS group; ns, no significant difference. SCS, static cold storage for 24 hours; DSC-D1 / D4 / D7, deep supercooling preservation for 24, 96 and 168 hours; HE, hematoxylin-eosin staining.

[0071] After 2 hours of rewarming and reperfusion treatment, microscopic examination of HE-stained renal tissue sections showed that acute tubular injury was present in all groups, manifested as tubular dilation, vacuolization, and necrosis and exfoliation of renal tubular epithelial cells, which was consistent with ischemia-reperfusion injury ( Figure 5). The injury score results showed that the injury level in the Fresh group was significantly lower than that in other groups (p < 0.0001 vs. SCS, DSC-D4, and DSC-D7; p = 0.0387 vs. DSC-D1). The injury level in the DSC-D1 group was significantly lower than that in the SCS group (p < 0.0001). Although the injury score in the DSC-D4 group was lower than that in the SCS group, the difference was not statistically significant. The injury level in the DSC-D7 group was significantly higher than that in the SCS group (p = 0.0027). The results of Masson's trichrome staining showed that collagen fiber deposition was present in the renal tissues of all groups ( Figure 5 ), and it was quantified by calculating the collagen volume fraction. The results showed that the collagen volume fraction in the Fresh group was comparable to that in the DSC-D1 group, but significantly lower than that in other groups (p < 0.0001 vs. SCS and DSC-D7; p = 0.0132 vs. DSC-D4). Compared with the SCS group, the collagen volume fraction in the DSC-D1 group and the DSC-D4 group was significantly decreased (p < 0.0001), while no significant difference was observed between the DSC-D7 group and the SCS group.

[0072] Example 2 Example 2 uses the same method as Example 1, and the main difference is the component dosage of the deep supercooling preservation solution and the perfusion solution, which are as follows: The deep supercooling preservation solution, used for low-temperature preservation of organs in vitro, includes:

[0073] Among them, it is measured based on the total volume of the deep supercooling preservation solution being 1 L; The perfusion solution for rewarming and reperfusion, used for rewarming and reperfusion of organs in vitro, includes:

[0074] Among them, it is measured based on the total volume of the perfusion solution being 450 mL.

[0075] Example 3 Example 3 uses the same method as Example 1, and the main difference is the component dosage of the deep supercooling preservation solution and the perfusion solution, which are as follows: The deep supercooling preservation solution, used for low-temperature preservation of organs in vitro, includes:

[0076] Among them, it is measured based on the total volume of the deep supercooling preservation solution being 1 L; The perfusion solution for rewarming and reperfusion, used for rewarming and reperfusion of organs in vitro, includes:

[0077] Among them, the total volume of the perfusion fluid is measured as 550 mL.

[0078] Example 4 Example 4 uses the same method as Example 1. The main difference is the different component dosages of the deep supercooling preservation solution and the perfusion fluid, which are specifically as follows: The deep supercooling preservation solution is used for low-temperature preservation of organs in vitro and includes:

[0079] Among them, the total volume of the deep supercooling preservation solution is measured as 1 L; The perfusion fluid for rewarming and reperfusion is used for rewarming and reperfusion of organs in vitro and includes:

[0080] Among them, the total volume of the perfusion fluid is measured as 520 mL.

[0081] Examples 2 to 4 also achieved the purpose of low-temperature preservation and rewarming and reperfusion through experiments.

[0082] Among them, the units of the present invention are all proportional units, not specific limitations, and can be enlarged or reduced in proportion according to actual needs.

[0083] In summary, through a series of targeted technical improvements, the present invention provides a complete and clear-step solution for the three core problems of "how to effectively and stably supercool the kidney at -10°C", "how to safely rewarm to reduce reperfusion injury", and "how to truly evaluate and promote the recovery of kidney function". The deep supercooling technology makes up for the deficiency of the traditional 4°C static cold storage in the organ preservation time limit; the controlled oxygenation rewarming overcomes the perfusion shock and oxidative stress problems brought by rapid warming; the normothermic mechanical perfusion provides a more accurate organ function evaluation method and further repair support.

[0084] From the time delay of the existing technology of 4°C static cold storage to the -10°C deep supercooling of the present invention, it can be seen that the present invention successfully realizes a long-term stable supercooling preservation state of the kidney at -10°C by using the deep supercooling preservation solution and covering an oil layer on the solution surface to isolate air and inhibit ice crystal nucleation. The advantages are as follows: 1. Lower temperature: Compared with 4°C, -10°C can further reduce the metabolic rate of the kidney (according to research, for every 10°C decrease, about 50% of the metabolic consumption can be reduced), thereby significantly reducing energy consumption and metabolite accumulation and extending the acceptable ischemic time limit of the organ.

[0085] 2. Effectively avoid ice crystal damage: During conventional freezing, ice crystals can pierce cell membranes and damage organelles, leading to irreversible tissue damage. The present invention minimizes ice nucleation and ice crystal growth through oil layer isolation and the addition of ice inhibitors, enabling tissues to maintain their original stable state in sub-zero environments.

[0086] 3. Significantly extend the preservation time: Under the same or similar operation difficulty and cost conditions as traditional SCS, the preservation duration of kidneys is extended from 24 hours to 96 hours and above, providing greater flexibility for long-distance organ transportation or delayed transplantation.

[0087] If the temperature is between 0°C and 4°C, the energy consumption and metabolite accumulation caused by residual metabolism are relatively fast; when the temperature is further reduced to -10°C, the metabolic rate decreases significantly; at the same time, by inhibiting ice crystal formation, cell structure damage in the traditional cryopreservation mode is avoided; thus, the goal of "significantly extending the preservation time + maximizing the maintenance of tissue activity" is achieved.

[0088] The present invention also realizes the reduction from rapid rewarming to controlled oxygenated rewarming. When transitioning from a deeply supercooled temperature (-10°C) to body temperature (about 37°C), staged warming is adopted and the perfusion pressure is gradually increased, while oxygen supply (95% O 2 and 5% CO 2 ) is carried out, that is, in the way of "gradually increasing temperature + gradually increasing pressure". The advantages are as follows: 1. Prevent "temperature shock": A sudden increase in temperature can lead to microcirculation disorders, damage to capillary endothelial cells, and enhanced oxidative stress. Controlled oxygenated rewarming enables kidney cells and blood vessels to have a smoother adaptation process to temperature changes through multi-step warming.

[0089] 2. Alleviate "reperfusion injury": Gradually increasing the perfusion pressure can avoid high-pressure shocks on the blood vessel wall in a short time, protect the vascular endothelial structure, and reduce the sudden generation of reactive oxygen species.

[0090] 3. Optimize tissue repair conditions: Controlled oxygenated rewarming can enable the organ to obtain sufficient oxygen supply during the rewarming process, ensure the gradual recovery of mitochondrial function, and reduce further damage that may be caused by energy depletion during the rewarming period.

[0091] The essence of the reperfusion process is to restore blood supply (nutrient supply) and oxygen, which leads to a sudden increase in inflammatory mediators, free radicals, etc.; sudden high temperature and high perfusion pressure will superimpose and amplify the above stress responses; staged warming and gradually increasing pressure can enable biological membranes, organelles, and vascular endothelium to repair and adapt within a controllable range, avoiding "explosive" damage.

[0092] The present invention further realizes the authenticity and accuracy from the difficult static cold storage evaluation to normal temperature machine perfusion. After completing the controlled oxygenation and rewarming, the kidney is placed in a 37°C normal temperature machine perfusion system to simulate the normal physiological environment in vivo for 1-hour ex vivo function evaluation and repair. The advantages are as follows: 1. More accurate function evaluation: At near-physiological temperature and pressure, the indexes such as creatinine clearance rate, urine production, and metabolites of the kidney are closer to the in-vivo situation, and the evaluation results are more valuable for clinical transplantation decisions.

[0093] 2. Promote kidney function recovery: The normal temperature machine perfusion process provides an environment with continuous oxygen, nutrients, and "elution" of metabolic wastes, which can help renal cells repair the slight damage caused by the ischemic period and supercooling period, laying a good foundation for subsequent transplantation.

[0094] 3. Reduce unforeseen damage: If the organ is judged directly in the low-temperature state or non-perfusion state, it is easy to underestimate or overestimate its actual function, and this evaluation deviation is avoided in the normal temperature machine perfusion stage.

[0095] The physiological functions of the kidney (such as filtration, reabsorption, and secretion) highly depend on blood perfusion and appropriate temperature; only when various indexes are measured under an environment close to the true physiological environment in vivo can it be judged whether the organ can effectively perform its functions after transplantation; meanwhile, during NMP, the kidney can still continuously repair the potential damage existing during donor acquisition or cryopreservation, improving the transplantation success rate.

[0096] The solution of the present invention has overall synergistic effects and clinical applicability. Low-temperature extended preservation + stable inhibition of ice crystals + gradual rewarming + normal temperature function evaluation form a complete and coherent organ protection and evaluation plan; the improvement points of each link are interconnected: deep supercooling puts the kidney into a low-temperature metabolic inhibition state, controlled oxygenation rewarming provides support for safe rewarming, and normal temperature machine perfusion ultimately realizes the "recovery + evaluation" of kidney function.

[0097] It also has clinical applicability. The operation process is similar to conventional static cold storage, the difference lies in the deep supercooling preservation solution and surface oil seal treatment, and the supporting equipment (cryopreservation box, machine perfusion system, oxygenator, etc.) is relatively mature; it is operable for the personnel in the transplantation department without the need for expensive or overly complex equipment transformation; it provides technical guarantee for the long-distance transportation and delayed transplantation of scarce organs.

[0098] The application scope includes: 1. Kidney preservation and transplantation: The present invention can be used in clinical kidney transplantation, especially in cases where there is a shortage of kidney donors, long-distance transportation is required, or delayed transplantation is needed. It can effectively extend the kidney preservation time and ensure that the kidney can recover a relatively high function after rewarming, and is applicable to kidney transplantation with cross-regional and long-time transportation. 2. Organ preservation research: In addition to the kidney, by combining deep supercooling and controlled oxygenation rewarming techniques, the preservation method of the present invention can be extended to the cryopreservation and rewarming reperfusion research of other organs (such as the liver, heart, lungs, etc.), providing a reference for the development of preservation and transplantation techniques for other types of organs. 3. Animal experiment research: This method is also applicable to the kidney preservation in various animal experiments, especially in the research involving organ survival and function recovery in experimental models, and has potential experimental research value.

[0099] The present invention can also: 1. Reduce organ waste: By extending the preservation time, reduce the organ abandonment caused by short preservation period and improve the organ utilization rate. 2. Lower transportation costs: Simplify the transportation process and reduce the temperature control equipment and related costs required for organ transportation. 3. Improve the transplantation success rate: Improve the kidney preservation effect, increase the transplantation success rate, and reduce related medical costs. 4. Promote the development of related industries: Promote the technological progress and industrialization of industries such as cryopreservation, cold chain logistics, and transplantation equipment.

[0100] Furthermore, it can: 1. Improve the overall level of transplantation technology: Extending the organ preservation time can increase the transplantation success rate and shorten the waiting time of patients. 2. Promote the coordinated development of industries: The deep supercooling and rewarming techniques will drive the development of supporting industries such as preservation solutions, cryogenic equipment, and mechanical perfusion systems, and promote the technological upgrading of the upstream and downstream industrial chains. 3. Stimulate the research and development in the fields of biomedicine and related technologies: The present invention provides new ideas and directions for the research and development in related fields such as stem cell therapy, artificial organs, and tissue engineering in terms of cryobiology and reperfusion technology.

[0101] The above content is only to illustrate the technical idea of the present invention and cannot be used to limit the protection scope of the present invention. Any modification made on the basis of the technical solution according to the technical idea proposed by the present invention falls within the protection scope of the claims of the present invention.

Claims

1. A method for cryopreservation and rewarming and reperfusion of an organ in vitro, characterized in that: include: The isolated organ samples are placed in a deep supercooled preservation solution, and an oil layer is added on the surface to isolate the air. The samples are stored at a deep supercooled temperature of -10°C for 1-7 days. After the preservation is completed, the isolated organ samples are connected to the perfusion system for rewarming and reperfusion: when transitioning from the deep supercooling temperature of -10°C to the set temperature, staged warming is adopted and the perfusion pressure is gradually increased, and oxygen is supplied at the same time; among them, the perfusion fluid used for reperfusion is a cell-free perfusion fluid, which is based on low-sugar DMEM and supplemented with nutrients and creatinine.

2. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The temperature of the deep supercooled preservation solution was 4°C, and the kidney was completely immersed at the bottom.

3. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The oil layer is paraffin oil, which floats on the surface of the deep supercooled preservation solution.

4. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The deep supercooling storage solution comprises: Among them, the total volume of deep supercooled preservation solution is measured as 1L; The deep supercooling storage solution is dissolved in pure water before use and the pH is adjusted to 7.35-7.

4.

5. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The perfusion system is of flow control type, adopts closed-circuit operation mode, and uses a peristaltic pump to deliver the perfusion fluid; the membrane oxygenator in the loop is connected to the gas flow meter, and uses carbogen gas to oxygenate the perfusion fluid discharged from the renal vein, and then delivers it to the renal artery, thereby achieving continuous perfusion of the kidney; The perfusion pressure was adjusted to 20-100 mmHg by adjusting the flow rate of the peristaltic pump.

6. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The rewarming and reperfusion perfusion solution comprises: The total volume of the perfusion fluid is measured at 450-550 mL.

7. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: During the rewarming and reperfusion process, perfusate and urine samples were collected at intervals and stored at −80°C; Glucose, lactate, and electrolyte concentrations, as well as oxygen tension, carbon dioxide tension, pH, and osmolarity were also measured in the perfusate or urine; aspartate aminotransferase, lactate dehydrogenase, and uric acid concentrations were determined in the perfusate, and creatinine concentrations were determined in the perfusate and urine.

8. The method for hypothermic preservation and rewarming and reperfusion of an organ in vitro according to claim 1, characterized in that: The rewarming and reperfusion stage includes two stages: controlled oxygenation rewarming and normothermic mechanical perfusion: Controlled oxygenation rewarming: the initial perfusion temperature was set at 4°C, which was then gradually increased to 10°C, 25°C, and 37°C at 15, 30, and 60 minutes, respectively; As the temperature rises, the arterial perfusion pressure is adjusted accordingly: 20-40 mmHg at 4°C, 40-80 mmHg at 25°C, and finally to the physiological range of 80-100 mmHg at 37°C; Normothermic mechanical perfusion: maintain continuous perfusion at 37°C for 1 hour with a perfusion pressure of 80-100 mmHg.

9. A deep supercooled preservation solution for cryopreservation of in vitro organs, characterized in that: include: The total volume of the deep supercooled storage solution is measured as 1L.

10. A rewarming and reperfusion perfusion solution for rewarming and reperfusion of an organ in vitro, characterized in that: include: The total volume of the perfusion fluid is measured at 450-550 mL.

Images