Novel organ cryopreservation liquid based on PVP (Polyvinyl Pyrrolidone) and containing fluorocarbon emulsion

Abstract

The invention belongs to the technical field of biomedical materials, and particularly relates to a novel organ cryopreservation solution based on PVP (Polyvinyl Pyrrolidone) and containing fluorocarbon emulsion. The invention discloses an organ preserving fluid which takes KPS-1 as a basis, polyvinylpyrrolidone (PVP) as a main anti-freezing component, fluorocarbon emulsion as an oxygen carrying agent and ATP and glucose as energy donors. In addition, the cleaning agent contains the surfactant, trehalose and other components, does not contain DMSO, and is non-toxic. Compared with the traditional UW liquid which can be preserved for 24 hours at 4 DEG C, the oxygen carrying capacity is remarkably improved, ischemia reperfusion injury is reduced, the UW liquid can be preserved for a long time at the ultralow temperature of-80 DEG C, and meanwhile sufficient energy is provided for maintaining metabolism of isolated organs. The cryopreservation time of tissues and organs such as in-vitro liver and kidney can be remarkably prolonged, and the preservation effect is superior to that of UW liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical materials, and in particular relates to a novel organ cryopreservation solution based on PVP-containing fluorocarbon emulsion. Background Art

[0002] Traditional cold storage has always been the gold standard for organ preservation because of its convenient operation and good effect in preserving standard donor organs. UW solution invented by the University of Wisconsin is recognized as the standard preservation solution for ex vivo organs such as liver and kidney, and is widely used in the international organ transplant community. However, with the full development of organ donation after the death of Chinese citizens, the preservation and maintenance of donor organs have been given higher requirements, and the standard donors and organs from cardiac death have been expanded. The tolerance of donor organs to cold and warm ischemic damage is poor, and traditional cold storage alone has limited effect on the preservation of such organs. In addition, according to a survey report released by the Global Organ Donation and Transplantation Statistics Association (GODT) in 2016, a total of about 1.2 million people worldwide need organ transplantation treatment due to end-stage organ failure. Due to the shortage of donor organ sources, the actual number of transplants is less than 10%. One important reason is that the time from acquisition to final transplantation exceeds the shelf life of the organ.

[0003] The currently common 4°C cryopreservation method has a maximum preservation time of only 36 hours, depending on the organ type. This method mainly reduces organ damage by inhibiting organ metabolism and enzyme decomposition. Studies have shown that for every 10°C drop in temperature, the organ metabolic rate will be halved; at 4°C, the metabolic rate is only 10% to 12% of the original. Ultra-low temperature environments, such as -80°C and below, can significantly reduce cell enzyme activity and chemical activity, slow down cell function, while maintaining their cell structure, and theoretically preserve ex vivo organs for an unlimited period of time. But in fact, cells still maintain a low metabolic level in an ultra-low temperature environment, and UW solution cannot provide sufficient oxygen to maintain cell metabolism. The hypoxic environment will lead to insufficient energy, causing damage to cell function and structure, and limiting the length of time that ex vivo organs can be preserved in vitro. In addition, hypoxia is part of ischemia-reperfusion injury, which can cause adverse effects such as intracellular calcium ion disorder, inflammatory response, and cell apoptosis, further aggravating ex vivo organ damage. During the cooling and rewarming process, cells or tissues will be damaged by factors such as freezing, melting, and changes in osmotic pressure of the solution. Among them, the formation and growth of ice crystals in low temperature environments will cause irreversible damage to biological tissues, which is the main factor leading to the loss of cell vitality. Finally, due to the ongoing metabolic activities, excessive hydrogen ions will be produced, and the imbalance of the preservation fluid homeostasis will also cause certain damage to the transplanted organs.

[0004] Therefore, it is of significant clinical value to develop a new organ preservation fluid that contains non-toxic ice-control materials, oxygen-carrying components and energy substances to prolong the in vitro preservation time of donor organs while reducing the damage caused by preservation. Summary of the invention

[0005] In one aspect, the present invention provides an organ preservation solution, comprising: a fluorocarbon compound, a surfactant, and polyvinyl pyrrolidone.

[0006] In some embodiments, the organ preservation solution further contains one or more of trehalose, ATP and heparin.

[0007] In some embodiments, the organ preservation solution does not contain DMSO.

[0008] In some embodiments, the organ preservation solution also contains a buffer; in some embodiments, the buffer is one or more of KPS-1, UW solution, and HTK solution; in some embodiments, the fluorocarbon compound is one or more of perfluorooctyl bromide, perfluorodecane bromide, perfluorodecalin, perfluorotripropylamine, perfluoromethylcyclohexylpiperidine, perfluorotributylamine, perfluorodichlorooctane, and perfluorobutylethylene; in some embodiments, the surfactant is one or more of soybean phosphatidylcholine, egg yolk phosphatidylcholine, and hydrogenated phosphatidylcholine.

[0009] In one aspect, the present invention provides an organ preservation solution, wherein each 100 ml of the organ preservation solution comprises:

[0010] Perfluorodecalin: 0.5-1.0mL

[0011] Soy phosphatidylcholine: 0.5-1.0g

[0012] PVP: 2-10g

[0013] Trehalose: 1.71-2.50g

[0014] ATP: 0.05-0.10g

[0015] Heparin: 1000-1500 U

[0016] KPS-1: Make up to 100 mL.

[0017] Polyvinylpyrrolidone (PVP) is one of the few synthetic polymers with good biocompatibility. It is obtained by polymerizing the monomer N-vinylpyrrolidone (NVP). As a non-toxic polymer, PVP was used as a colloidal plasma substitute during World War II. In addition, PVP also plays an important protective role in the cryopreservation of tissues and cells, with the following characteristics:

[0018] Inhibit the formation of extracellular ice crystals: PVP, as a large molecular weight non-permeable cryoprotectant, can form hydrogen bonds with extracellular sugars in a low temperature environment, thereby inhibiting the formation of extracellular ice crystals and effectively protecting the physiological structure and function of isolated organs under low temperature conditions.

[0019] Inhibit the formation of intracellular ice crystals: PVP can dehydrate cells, lower the freezing point and inhibit the extracellular salt concentration, which can effectively inhibit the formation of ice crystals in the early stage of crystallization, thereby reducing the amount of intracellular ice, stabilizing the cell membrane and protecting cells from freezing damage.

[0020] Antioxidant effect: Reactive oxygen species (ROS) generated in ultra-low temperature environments will attack the unsaturated fatty acids in the mitochondrial membrane, change the membrane permeability, and cause the Ca2+ in the mitochondria to 2+ Outflow, mitochondrial DNA damage, resulting in decreased mitochondrial membrane potential, uncoupling of oxidative phosphorylation, failure of ATP synthesis, and excessive ROS production. Excessive ROS continue to damage the membrane system, forming a vicious cycle. PVP can effectively weaken this cycle and significantly improve cell antioxidant capacity, cell membrane integrity, and mitochondrial activity.

[0021] Polymer surface activity: The lactam on the PVP molecule is hydrophilic. After oxygen provides electrons to the pyrrolidone ring, the entire molecule becomes positive and can disperse and adsorb the suspension, maintain the stability of the solution, and reduce surface tension.

[0022] High biocompatibility: When organs are stored in vitro, the materials used must have extremely high biocompatibility. PVP's low immunogenicity and good biocompatibility make it possible to be used in organ cryopreservation.

[0023] Furthermore, another substance, perfluorodecalin, is a non-toxic, non-flammable, thermally stable, and non-bioaccumulative oxygen carrier. Perfluorodecalin, as an artificial blood substitute, is also known as artificial red blood cells. It is a fluorine-containing organic compound with the following properties:

[0024] Physical properties: Perfluorodecalin is a colorless, odorless, non-toxic liquid with low surface tension and high stability, which helps to maintain a stable state in the body. Studies have shown that after topical application of supersaturated oxygen emulsion (SSOE) containing perfluorodecalin to the eyeballs of mice for 1 hour, no eyelid irritation or redness, conjunctival congestion or increased secretions, corneal opacity or edema were detected after 4 hours, 24 hours, and 7 days.

[0025] High oxygen dissolving capacity: Perfluorodecalin can dissolve a large amount of oxygen and transfer oxygen to surrounding tissues by diffusion. In SSOE, the oxygen partial pressure can exceed 600 mmHg, while the oxygen partial pressure in air is 160 mmHg. It can be seen that perfluorodecalin is an excellent oxygen carrier. In the mouse model of ocular alkali burn, the survival rate of corneal cells in the SSOE-treated group (36.0±3.0%) was higher than that in the untreated group (17.2±4.5%), indicating that perfluorodecalin can reduce cell death caused by hypoxia.

[0026] The above two characteristics determine that perfluorodecalin can be used as a safe and stable oxygen-carrying compound to increase the oxygen content in organ preservation fluids. Even in ultra-low temperature environments, it can still provide sufficient oxygen to organs, thereby solving the ischemia-reperfusion injury caused by hypoxia during cryopreservation of transplanted organs and the insufficient energy supply caused by hypoxia.

[0027] Furthermore, since the ex vivo organs still maintain a low level of energy metabolism under ultra-low temperature conditions, energy substances such as ATP and glucose are added to the organ preservation solution to ensure the energy supply of the ex vivo kidneys, maintain their metabolism, and reduce cell damage.

[0028] Furthermore, since cell metabolism produces hydrogen ions, as a feasible embodiment, sodium hydroxide can be added to the buffer to adjust the pH of the organ preservation solution to about 7.4, so that the organ preservation solution is weakly alkaline to maintain the homeostasis of the internal environment of the isolated organ.

[0029] On the one hand, the present invention provides the use of the organ preservation solution in the storage or transportation of organ samples at minus 90-70 degrees Celsius; in some embodiments, the organ samples are stored or transported at minus 80 degrees Celsius.

[0030] In some embodiments, the organ sample is stored or transported at -90-70 degrees Celsius for 5-10 days; in some embodiments, the organ sample is stored or transported at -90-70 degrees Celsius for 7 days; in some embodiments, the organ sample is stored or transported at -80 degrees Celsius for 7 days.

[0031] In some embodiments, the organ is derived from humans or animals; preferably, the organ is liver or kidney.

[0032] In one aspect, the present invention provides a method for preparing the organ preservation solution, comprising the following steps:

[0033] Dissolving and mixing the fluorocarbon compound and the surfactant to obtain a solution 1;

[0034] Dissolve and mix PVP, trehalose, ATP, heparin and KPS-1 to obtain solution 2;

[0035] Solution 1 and solution 2 are mixed and homogenized using a high-pressure homogenizer to form a stable O / W emulsion to obtain solution 3;

[0036] Place the nozzle of the oxygen tank deep into the solution 3 to ensure continuous bubbles, and inflate for at least 15 minutes to obtain the preservation solution after sterilization.

[0037] In one aspect, the present invention provides a method for using the organ preservation solution, comprising the following steps:

[0038] The isolated organ is completely immersed in the organ preservation solution, cooled to -90-70 degrees Celsius at a rate of 2°C / min, and then preserved at -90-70 degrees Celsius for 5-10 days.

[0039] Compared with the existing organ preservation solution, the present invention has the following beneficial effects:

[0040] 1. The organ preservation solution of the present invention can freeze the liver and kidney for 7 days at -80°C, and the rats can survive for a long time after transplantation;

[0041] 2. The organ preservation solution of the present invention uses PVP as the main antifreeze component, does not contain DMSO, has no toxic effects, and has good safety;

[0042] 3. PVP can inhibit the formation of ice crystals inside and outside cells at the same time, maintaining the normal structure and function of isolated organs;

[0043] 4. The organ preservation solution of the present invention is added with oxygen-carrying substances, which significantly increases the oxygen content in the organ preservation solution, reduces the hypoxic damage of the cryopreserved organs, and can be more widely used in the cryopreservation of various organs such as the liver and kidney;

[0044] 5. The organ preservation solution of the present invention is added with energy substances to provide sufficient energy for the metabolism of the isolated organ;

[0045] 6. The organ preservation solution of the present invention has good biocompatibility and can significantly improve the quality of cryopreservation of ex vivo organs;

[0046] 7. The organ preservation solution of the present invention has a simple composition and is easy to prepare. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 The present invention is a comparison of the organ preservation solution of the present invention and the traditional UW solution in the preservation of liver organs (ALT).

[0048] Figure 2 The present invention is a comparison of the organ preservation solution of the present invention and the traditional UW solution in the preservation of liver organs (AST).

[0049] Figure 3The present invention is a comparison of the organ preservation solution of the present invention and the traditional UW solution in the preservation of kidney organs (CK).

[0050] Figure 4 The present invention is a comparison of the organ preservation solution of the present invention and the traditional UW solution in the preservation of kidney organs (LDH).

[0051] Figure 5 These are photos of the effects of the organ preservation solution of the present invention and the traditional UW solution on kidneys after kidney transplantation in Application Example 1; left: kidneys of rats in the UW group; right: kidneys of rats in the fluorocarbon group.

[0052] Figure 6 This is a comparison of the effects of the organ preservation solution of the present invention and the traditional UW solution on transplanted kidneys after preservation (HE staining); wherein, the left picture is the kidney of rats in the UW group; and the right picture is the kidney of rats in the fluorocarbon group.

[0053] Figure 7 This is a comparison of the effects of combination 1 and combination 2 on the transplanted kidney after preservation (HE staining) in the comparative example; the left picture is the kidney of the rat in combination 1; the right picture is the kidney of the rat in combination 2. DETAILED DESCRIPTION

[0054] The technical solution of the present invention is further described below by specific embodiments, which do not limit the protection scope of the present invention. Some non-essential modifications and adjustments made by others based on the concept of the present invention still fall within the protection scope of the present invention.

[0055] In the following examples, the organ preservation solution formulation composition and content are as follows:

[0056] Perfluorodecalin (Aladdin P169442-25g): 2.0% (w / v)

[0057] Soybean phosphatidylcholine (Aladdin L105734-25g): 1.0% (w / v)

[0058] PVP (Aladdin P110608-100g): 5.0% (w / v)

[0059] Trehalose (Inokai A52221): 1.71% (w / v)

[0060] ATP (SolarbioA9130-5g): 0.10% (w / v)

[0061] Heparin sodium (Kingclone RS6505-5×10ml): 1250U

[0062] KPS-1 (Jiangen KPS-1): make up to 100 mL.

[0063] In the embodiment, the concentration unit "w / v" of each component can be converted to g / ml. For example: 2.0% w / v = 2.0g / 100ml = 0.020g / ml. Since the components added to the organ preservation solution include solid (such as PVP) and liquid (such as perfluorodecalin), one way to express the concentration is w / v.

[0064] Preparation process of organ preservation solution: perfluorodecalin and soybean phosphatidylcholine are dissolved and mixed to obtain solution 1. PVP, trehalose, ATP, heparin and KPS-1 that are easily soluble in water are dissolved and mixed to obtain solution 2. Solution 1 and solution 2 are mixed and homogenized with a high-pressure homogenizer to form a stable O / W emulsion to obtain solution 3. The nozzle of the oxygen tank is deeply inserted into solution 3 to ensure continuous bubbles, and the solution is inflated for at least 15 minutes. After sterilization, the preservation solution is obtained.

[0065] Example 1

[0066] Study on the preservation effect of the organ preservation solution of the present invention on isolated kidney

[0067] 300g healthy adult Sprague-Dawley (SD) male rats were selected as donors, and the left kidney was harvested for the experiment. The rats were anesthetized with chloral hydrate and then opened the abdomen. A 0.7mm x 19mm (24G x 0.75) specification indwelling needle was used to construct a renal perfusion system through the renal artery. The groups were as follows:

[0068] 1. UW liquid group: The isolated kidney was completely immersed in UW liquid (Leagene qgbcy-1000ml) and placed on ice. A peristaltic pump was used to adjust the perfusion flow rate to 5-10mL / min and then circulated for perfusion for 30 minutes. After the perfusion was completed, 1 ml of perfusion liquid was taken for testing, recorded as UW1. The isolated kidney was placed in new UW liquid and stored at 4°C for 24 hours. After the storage was completed, 1 ml of UW liquid was taken for testing, recorded as UW2;

[0069] 2. New organ preservation solution group (fluorocarbon group): The isolated kidney was completely immersed in the new organ preservation solution and placed on ice. The perfusion flow rate was adjusted to 5-10 mL / min using a peristaltic pump and then circulated for perfusion for 30 minutes. After the perfusion was completed, 1 mL of the perfusion solution was taken for testing, which was recorded as fluorocarbon 1. The isolated kidney was placed in a new new organ preservation solution, and the temperature was quickly cooled to -80°C at a rate of 2°C / min using a programmed cooling device, and frozen for 7 days. After the preservation was completed, it was immediately placed in a 37°C water bath to dissolve, and 1 mL of the thawed new organ preservation solution was taken for testing, which was recorded as fluorocarbon 2.

[0070] The results show that: Figure 1As shown in Figure 2, after the isolated kidneys were stored in UW solution at 4°C for 24 hours and in the new organ preservation solution at -80°C for 7 days, the CK and LDH levels in Fluorocarbon 2 did not increase significantly compared with UW2, and the CK level in Fluorocarbon 2 was even slightly lower than that in UW2. In addition, the CK and LDH levels in UW1 and Fluorocarbon 1 were low and there was no significant difference.

[0071] Conclusion: The novel organ preservation solution described in the present invention has a similar perfusion effect on the kidney as UW solution. However, the novel organ preservation solution significantly reduces the storage temperature of the isolated kidney and prolongs the storage time of the isolated kidney. The preservation effect is not significantly different from that of UW, indicating that the novel organ preservation solution described in the present invention has a better preservation ability for the kidney.

[0072] Example 2

[0073] Study on the preservation effect of the organ preservation solution of the present invention on isolated liver

[0074] The livers of 300g healthy adult Sprague-Dawley (SD) male rats were selected as donors for the experiment. The rats were anesthetized with chloral hydrate and then opened the abdomen. A 0.7mm x 19mm (24G x 0.75) indwelling needle was used to construct a liver perfusion system through the aorta. The groups were as follows:

[0075] 1. UW liquid group: The isolated liver was completely immersed in UW liquid (Leagene qgbcy-1000ml) and placed on ice. A peristaltic pump was used to adjust the perfusion flow rate to 5-10mL / min and then circulated for perfusion for 30 minutes. After the perfusion, 1mL of perfusion liquid was taken for testing, recorded as UW1. The isolated liver was placed in new UW liquid and stored at 4°C for 24 hours. After the storage, 1mL of UW liquid was taken for testing, recorded as UW2;

[0076] 2. New organ preservation solution group (fluorocarbon group): The ex vivo liver was completely immersed in the new organ preservation solution and placed on ice. The perfusion flow rate was adjusted to 5-10 mL / min using a peristaltic pump and then circulated for perfusion for 30 minutes. After the perfusion was completed, 1 mL of the perfusion solution was taken for testing, which was recorded as fluorocarbon 1. The ex vivo liver was placed in a new new organ preservation solution, and the temperature was rapidly cooled to -80°C at a rate of 2°C / min using a programmed cooling device, and frozen for 7 days. After the preservation was completed, it was immediately placed in a 37°C water bath to dissolve, and 1 mL of the dissolved new organ preservation solution was taken for testing, which was recorded as fluorocarbon 2.

[0077] The results show that: Figure 3As shown in Figure 4, after the isolated liver was stored in UW solution at 4°C for 24 hours and in the novel organ preservation solution at -80°C for 7 days, the ALT and AST levels in Fluorocarbon 2 were much lower than those in UW2. In addition, the ALT and AST levels in UW1 and Fluorocarbon 1 were low and there was no significant difference.

[0078] Conclusion: The novel organ preservation solution described in the present invention has a similar perfusion effect on the liver as UW solution. However, the novel organ preservation solution significantly reduces the preservation temperature of the isolated liver, prolongs the preservation time of the isolated liver, and has a better preservation effect than UW solution, indicating that the novel organ preservation solution described in the present invention has a better preservation ability for the kidney.

[0079] Application Example 1

[0080] Using the above-mentioned Example 1, the kidney is cryopreserved and then transplanted

[0081] Since the urinary system structure of male rats is relatively simple, and female rats are easily interfered by the uterus during ureteral reconstruction, and the blood vessels of the left kidney are longer than those of the right kidney in anatomy, which is convenient for surgical operation, 300g healthy adult SD male rats were selected as donors and recipients, and the left kidney was collected for the experiment.

[0082] Rats were anesthetized with chloral hydrate and then opened to the abdomen. A 0.7 mm x 19 mm (24G x 0.75) indwelling needle was used to construct a renal perfusion system via the renal artery. The groups were as follows:

[0083] 1. UW solution group: The isolated kidney was completely immersed in UW solution and placed on ice. A peristaltic pump was used to adjust the perfusion flow rate to 5-10 mL / min and then circulated for perfusion for 30 minutes. After the perfusion, the isolated kidney was placed in new UW solution and stored at 4°C for 24 hours. After the storage, the isolated kidney was perfused with new UW solution on ice for 10 minutes and then placed at 4°C for standby use.

[0084] 2. New organ preservation solution group (fluorocarbon group): The ex vivo kidney was completely immersed in the new organ preservation solution and placed on ice. The perfusion flow rate was adjusted to 0.7mm x 19mm (24G x 0.75) mL / min using a peristaltic pump and then circulated for perfusion for 30 minutes. After the perfusion, the ex vivo kidney was placed in a new new organ preservation solution, and the temperature was rapidly cooled to -80°C at a rate of 2°C / min using a program cooling device, and frozen for 7 days. After the preservation was completed, it was immediately placed in a 37°C water bath to dissolve, and then the ex vivo kidney was perfused on ice with a new new organ preservation solution for 10 minutes, and then placed at 4°C for use.

[0085] Kidney transplant process:

[0086] 1. Kidney removal: Use an electronic balance to weigh the rats, and inject 10% chloral hydrate intraperitoneally at 0.004 mL / g to anesthetize the rats. Depilate the surgical area of ​​the rat abdomen. Enter the abdomen layer by layer, and push the rat intestines to the right side of the surgeon to fully expose the surgical field. Free the abdominal aorta, free the left renal artery and vein, peel off the peritoneum covering the left kidney, free and cut the left ureter, and ligate the left adrenal vein. Insert the indwelling needle from the level of the inferior mesenteric artery of the abdominal aorta and fix it properly, and inject 5X heparin saline containing 500 U of heparin to heparinize the rat. Ligate the abdominal aorta horizontally above the left renal artery and inject 10 mL of lactated Ringer's solution. Use an electric knife to cut off the branches of the abdominal aorta, such as the superior and inferior mesenteric arteries, from the root. Cut off the kidney to be transplanted from the distal end of the indwelling needle entrance, the proximal end of the abdominal aorta ligation, and the renal vein near the inferior vena cava.

[0087] 2. Transplantation: Weigh the rats using an electronic balance, and anesthetize the rats by intraperitoneal injection of 10% chloral hydrate at 0.004 mL / g. Depilate the surgical area of ​​the rat abdomen. Enter the abdomen layer by layer, push the rat intestines to the right side of the surgeon, and fully expose the surgical field. Free the left renal artery and vein, peel off the peritoneum covering the left kidney, and free and cut the left renal ureter. Clamp the left renal artery and vein with a vascular clamp, sever the left renal artery and vein from the distal end of the left renal artery and vein vascular clamp, and remove the original left kidney. Place the kidney to be transplanted into the abdominal cavity of the rat, anastomose the left renal vein and left renal artery end to end, open the left renal artery and vein vascular clamp, and allow the transplanted kidney to be perfused in vivo. End-to-end anastomosis of the ureter. Return the rat intestine to its original position and close the abdomen layer by layer. The transplant model is constructed.

[0088] The results showed that after the rat kidneys were frozen at -80℃ for one week and thawed, the kidneys were still alive, and after kidney transplantation, the kidneys turned pink and produced urine, which was no significant difference from the UW group rats. Figure 5 As shown in Figure 2, both groups of rats survived for a long time. Figure 6 As shown, after two weeks of survival, the transplanted kidneys of rats were pathologically stained, and there was no significant difference in the kidney cell structure and morphology in the fluorocarbon group compared with the transplanted kidneys in the UW group.

[0089] Conclusion: Compared with UW solution, the new organ preservation solution containing PVP and perfluorodecalin has a lower storage temperature, longer storage time and no difference in preservation effect in rat kidney preservation.

[0090] Comparative Example

[0091] Combination 1: Use a preservation solution with other antifreeze agents, replacing PVP with dimethyl sulfoxide.

[0092] Combination 2: Use a preservation solution containing other sugars and replace trehalose with sucrose.

[0093]

[0094]

[0095] Both rats in combination 1 and combination 2 died within 5 days after transplantation (transplantation and pathological section staining procedures were the same as those in application example 1). Pathological examination showed that some renal tubular epithelial cells in the glomerular cortex and medulla of the kidneys of combination 1 were necrotic, with cell shrinkage and dark staining; some glomeruli in the cortical area of ​​the kidney tissue of combination 2 were atrophic, with a decrease in the number of intraglomerular capillaries, and a slight expansion of the glomerular capsule. Some renal tubular epithelial cells in the glomerular cortex and medulla were necrotic, with cell shrinkage and dark staining, as shown in Figure 1. Figure 7 .

Claims

1. An organ preservation solution, characterized in that: include: Fluorocarbons, surfactants, and polyvinylpyrrolidone.

2. The organ preservation solution according to claim 1, characterized in that: It also contains one or more of trehalose, ATP and heparin.

3. The organ preservation solution according to claim 1, characterized in that: The organ preservation solution does not contain DMSO.

4. The organ preservation solution according to claim 1, characterized in that: The organ preservation solution also contains a buffer solution; Preferably, the buffer is one or more of KPS-1, UW solution, and HTK solution; Preferably, the fluorocarbon compound is one or more of perfluorooctyl bromide, perfluorodecane bromide, perfluorodecalin, perfluorotripropylamine, perfluoromethylcyclohexylpiperidine, perfluorotributylamine, perfluorodichlorooctane, and perfluorobutylethylene; Preferably, the surfactant is one or more of soybean phosphatidylcholine, egg yolk phosphatidylcholine, and hydrogenated phosphatidylcholine.

5. An organ preservation solution, characterized in that: Each 100 ml of organ preservation solution includes: Perfluorodecalin: 0.5-1.0mL; Soy phosphatidylcholine: 0.5-1.0g; PVP: 2-10g; Trehalose: 1.71-2.50g; ATP: 0.05-0.10g; Heparin sodium: 1000-1500U; KPS-1: Make up to 100 mL.

6. Use of the organ preservation solution according to any one of claims 1 to 5 in the storage or transportation of organ samples at -90 to -70 degrees Celsius; Preferably, the organ sample is stored or transported at minus 80 degrees.

7. The use according to claim 6, characterized in that The organ sample is stored or transported at -90-70 degrees Celsius for 5-10 days; Preferably, the organ sample is stored or transported at -90-70 degrees Celsius for 7 days; Preferably, the organ sample is stored or transported at minus 80 degrees for 7 days.

8. The use according to claim 6, characterized in that The organ is derived from human or animal; Preferably, the organ is liver or kidney.

9. The method for preparing the organ preservation solution according to any one of claims 1 to 5, characterized in that: The following steps are involved: Dissolving and mixing the fluorocarbon compound and the surfactant to obtain a solution 1; Dissolve and mix PVP, trehalose, ATP, heparin and KPS-1 to obtain solution 2; Solution 1 and solution 2 are mixed and homogenized using a high-pressure homogenizer to form a stable O / W emulsion to obtain solution 3; Place the nozzle of the oxygen tank deep into the solution 3 to ensure continuous bubbles, and inflate for at least 15 minutes to obtain the preservation solution after sterilization.

10. A method for using the organ preservation solution according to any one of claims 1 to 5, characterized in that: The following steps are involved: The isolated organ is completely immersed in the organ preservation solution, cooled to -90-70 degrees Celsius at a rate of 2°C / min, and then preserved at -90-70 degrees Celsius for 5-10 days.

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