Lung graft preservation method based on oil seal low-temperature static preservation

Abstract

The invention provides a lung graft preservation method based on oil seal low-temperature static preservation. The lung graft preservation method comprises the following steps: S1, transferring a collected lung graft into a tissue preservation solution; s2, covering the surface of the tissue preserving fluid with a layer of paraffin oil or mineral oil to form a covering layer; and S3, controlling the temperature of the tissue preservation liquid at 0-10 DEG C, and keeping the lung graft to be preserved in a static cold preservation environment. The method is simple, convenient, efficient and low in cost, has transformation potential, and solves the problems of short preservation time, heavy low-temperature damage, high PGD occurrence rate, complex operation and the like in the prior art.

Description

Technical Field

[0001] The present invention relates to the technical field of lung transplant preservation, and in particular to a method for cold static preservation of lung transplants, and more particularly to a lung transplant preservation method based on oil-sealed low-temperature static preservation. Background Art

[0002] With the increasing incidence of end-stage lung disease, lung transplantation has become the only effective means of improving patient survival and quality of life. However, lung graft preservation remains a key technical bottleneck limiting its widespread application. Currently, the most widely used method for lung graft preservation in clinical practice is cold static preservation (CSP) at 4°C. This method relies on a dextran-containing tissue preservation solution (such as Perfadex solution) to temporarily inhibit metabolic processes and reduce the risk of ischemia-reperfusion injury (IRI). Although this method is simple and inexpensive, it has a short preservation window (approximately 6 hours) and often causes primary graft dysfunction (PGD) during lung graft reperfusion, significantly limiting the safety and effectiveness of lung transplantation. To overcome these issues, an ex vivo lung perfusion (EVLP) system has been developed to dynamically assess lung graft perfusion and function. While this method can effectively prolong preservation time and reduce the incidence of PGD, it is expensive, complex, and has limited applicability to a limited population, making it difficult to commercialize.

[0003] Furthermore, studies have shown that hypothermic, hypoxic environments can induce a metabolically inhibited state in cells, thereby alleviating cold ischemic injury to a certain extent. However, directly exposing lung transplants to hypoxia can easily induce sudden stress, leading to mitochondrial damage, ROS accumulation, and cell apoptosis. Therefore, developing a novel preservation strategy that allows for progressive hypoxia and activates metabolic protective pathways without increasing operational complexity is an urgent challenge in the field of organ preservation. Summary of the Invention

[0004] The present invention aims to provide a lung transplant preservation method based on oil-sealed low-temperature static storage. This method is simple, efficient, low-cost, and has translational potential. It solves the problems of the prior art, such as short storage time, severe cryogenic damage, high incidence of PGD, and complex operation.

[0005] The present invention is achieved through the following technical solutions: The present invention provides a lung transplant preservation method based on oil-sealed low-temperature static preservation, comprising the following steps: S1, the harvested lung graft was transferred into tissue preservation solution; S2, covering the surface of the tissue preservation solution with a layer of paraffin oil or mineral oil to form a covering layer; S3, control the temperature of the tissue preservation solution at 0-10°C and keep the lung graft in a static cold storage environment.

[0006] Preferably, in S1, the tissue preservation solution is Perfadex solution.

[0007] Preferably, in S1, the lung transplant is a lung from a small animal, a large animal or a human.

[0008] Preferably, in S2, the thickness of the covering layer is 1-3 cm.

[0009] Preferably, in S2, the paraffin oil is medical grade paraffin oil.

[0010] Furthermore, in S2, the purity of the paraffin oil is ≥99%.

[0011] Furthermore, in S3, the storage time is 12-48 hours.

[0012] Preferably, the lung transplant preservation method based on oil-sealed low-temperature static preservation further comprises: S4, gradually removing the covering layer after the preservation is completed.

[0013] Compared with the prior art, the present invention has the following beneficial effects: The present invention utilizes paraffin or mineral oil to coat the surface of the tissue preservation solution, forming a physical seal that effectively restricts oxygen diffusion, thereby establishing a progressively hypoxic microenvironment. This progressively hypoxic microenvironment continuously activates the HIF-1α signaling pathway within the lung graft, initiating cellular self-protection programs such as metabolic reprogramming, oxidative stress relief, and cell death inhibition, thereby enhancing the lung graft's tolerance to cold storage. Furthermore, this progressively hypoxic microenvironment promotes glutamate and glycolysis metabolic pathways, inhibits the TCA cycle and fatty acid oxidation, and helps cells maintain ATP supply and survival in a more energy-efficient manner at low temperatures. Consequently, the oil-sealed, low-temperature static preservation method of the present invention, without the aid of any perfusion system, can effectively extend the cold storage time of lung grafts to 48 hours while maintaining good lung function indicators (such as compliance and gas exchange capacity), significantly reducing the incidence of post-transplant PGD. Furthermore, compared to dynamic perfusion systems like EVLP, the oil-sealed, low-temperature static preservation method of the present invention requires only a layer of paraffin oil on the surface of the tissue preservation solution, requiring minimal equipment, facilitating standardized operation and widespread adoption. This method has been systematically validated on rat, pig, and human discarded clinical donor lungs, demonstrating excellent organ protection and possessing high potential for clinical translation and cross-species applicability. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0015] Figure 1 Schematic diagram comparing the storage method of the present invention and the 4°C static cold storage method.

[0016] Figure 2 Comparison of functional indicators of rat lung transplants under different preservation methods evaluated on the EVLP platform; among them, a) is peak airway pressure, b) is plateau airway pressure, c) is dynamic compliance, d) is static compliance, e) is oxygenation index (P / F), and f) is wet-to-dry weight ratio (W / D).

[0017] Figure 3 The results of H&E staining, TUNEL staining, and molecular expression of lung allografts after 48 hours of cold storage under different preservation methods; among them, a) is the tissue structure of H&E staining, b) is the TUNEL staining result, c) is the ROS intensity in the lung allograft, d) is the SOD level in the lung allograft, and e) is the expression levels of Caspase-3 and HMGB1 proteins.

[0018] Figure 4 Transmission electron micrographs of lung transplants after 48 hours of cold storage under different preservation methods.

[0019] Figure 5 This is a KEGG enrichment analysis of lung allograft metabolomics after 48 hours of cold storage and its association with the HIF-1α pathway in Example 1 of the present invention. (a) shows the enrichment of HIF-1α-related amino acid metabolic pathways; (b) shows the level of glutamate in lung allografts; (c) shows the level of glucose in lung allografts; and (d) shows the level of pyruvate in lung allografts.

[0020] Figure 6 Western blot and immunohistochemical analysis of lung allografts after cold storage under different preservation methods. (a) HIF-1α protein expression levels in each group; (b) HIF-1α protein levels in the PO group as storage time increases; (c) PDK4 expression levels in lung allografts by immunohistochemical staining.

[0021] Figure 7 are the oxidative stress levels of cells after preservation in paraffin oil (PO) or mineral oil (MO).

[0022] Figure 8 The tissue structures of large animal lung transplants after cold preservation using the preservation method of the present invention are shown in Figures a) and b) respectively. DETAILED DESCRIPTION

[0023] The following describes the embodiments of the present invention through specific examples. Those skilled in the art will readily understand the other advantages and benefits of the present invention from the disclosure herein. The present invention may also be implemented or applied through various other specific embodiments, and the details in this specification may be modified or altered based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] It should be noted that the process equipment or devices not specifically specified in the following embodiments are all conventional equipment or devices in the art.

[0025] It should be noted that the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions. For example, a process, method, system, product, or apparatus comprising a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to these processes, methods, products, or apparatuses. Furthermore, unless otherwise specified, the numbering of each method step is merely a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the invention. Changes or adjustments to their relative relationships, without substantially changing the technical content, should also be considered within the scope of the invention.

[0026] The present invention provides a cold static preservation method based on sealing the surface of tissue preservation fluid with paraffin oil. This method involves covering the surface of the tissue preservation fluid with a layer of non-toxic, anhydrous, and immiscible paraffin oil or mineral oil at 0-10°C, thereby forming a closed microenvironment by covering the surface of the tissue preservation fluid with the paraffin oil or mineral oil.

[0027] Paraffin oil has the following notable characteristics: (1) Physical barrier effect: Paraffin oil forms a uniform covering layer of 1-3 cm on the surface of tissue preservation solution, effectively isolating the direct contact between the gas phase and the liquid phase, and reducing the oxidative stress response caused by oxygen exposure; (2) Low toxicity and high stability: Paraffin oil itself is non-toxic and chemically stable, and will not react with tissue preservation fluid or cause damage to lung transplants; (3) Dynamic adaptability: The covering effect of paraffin oil can be adjusted according to the needs of tissue preservation fluid, which can ensure the normal fluidity of the fluid and meet the metabolic contact needs of the lung transplant.

[0028] Specifically, the present invention provides a lung transplant preservation method based on oil-sealed low-temperature static storage, comprising the following steps: S1, Lung graft collection and pretreatment: After the lung graft is collected, it is quickly placed in tissue preservation solution.

[0029] The tissue preservation solution may be a Perfadex solution containing protective components. No exogenous amino acids are added to the tissue preservation solution.

[0030] S2, paraffin oil or mineral oil covering: evenly cover the surface of the tissue preservation solution with a layer of paraffin oil or mineral oil. The thickness of the covering layer should be controlled between 1-3 cm to ensure that the surface of the tissue preservation solution is completely covered.

[0031] A paraffin or mineral oil overlay creates a stable sealing barrier without unduly inhibiting gas exchange.

[0032] The paraffin oil or mineral oil used in the present invention can be medical grade paraffin oil with a purity of ≥99%.

[0033] S3, cooling and static storage: The temperature of the tissue preservation solution is controlled at 4°C, and the lung graft is kept in a static cold storage environment for 12-48 hours.

[0034] During cold storage, the sealing effect of paraffin oil or mineral oil dynamically regulates the expression of HIF-1α, promotes metabolic reprogramming of lung transplants, and reduces ROS generation and inflammatory response.

[0035] S4, transplantation preparation and reperfusion: After preservation is completed, the covering layer is gradually removed and the oxygen concentration is slowly restored during transplantation to reduce cellular stress response.

[0036] By controlling the rate of oxygen recovery during reperfusion, the adaptability and functional integrity of the lung graft can be effectively improved.

[0037] The present invention provides a lung transplant preservation method based on oil-sealed low-temperature static storage. Paraffin oil or mineral oil is used to cover the surface of the tissue preservation solution to form a physical seal, effectively restricting oxygen diffusion and thus establishing a progressively hypoxic microenvironment. The present invention has discovered that this progressively hypoxic microenvironment can induce activation of the lung transplant's endogenous hypoxia-inducible factor (HIF-1α) during storage, thereby inducing a series of metabolic reprogramming events and enhancing the lung transplant's tolerance to hypothermic hypoxia stress, including: (1) Upregulating PDK4 expression, inhibiting PDH activity, and reducing TCA cycle flux; (2) Enhance glycolysis and glutamate synthesis; (3) Improve the ability to resist oxidative stress (reduce ROS and increase SOD activity); (4) Alleviate cell apoptosis and necrosis (reduction of Caspase-3 and HMGB1); (5) Improve lung compliance and oxygenation capacity, and reduce the risk of pulmonary edema and PGD.

[0038] Furthermore, the present invention has verified the broad applicability and tissue-protective effects of this method through animal experiments (rats and pigs) and studies on discarded lung samples from clinical settings, demonstrating that the preservation method described herein is suitable for lung donation from small and large animals, as well as humans. Compared to dynamic perfusion techniques such as EVLP, this method does not require an external circulatory system, is less expensive, and is simpler to operate, making it suitable for clinical cold chain transportation and organ sharing processes. Therefore, this invention provides a novel lung transplant preservation method with potential for clinical translation, providing important technical support for improving the success rate of lung transplantation and expanding lung donor resources.

[0039] The lung transplant of the present invention can be used for ex vivo lung perfusion or in situ lung transplantation before or after preservation.

[0040] The paraffin oil and mineral oil of the present invention are both purchased from Merck & Co., Ltd. The product number of the paraffin oil is 76235, and the CAS number is 8012-95-1; the product number of the mineral oil is M5904, and the CAS number is 8042-47-5.

[0041] Example 1 like Figure 1 As shown, the lung transplant preservation method based on oil-sealed low-temperature static preservation in this embodiment includes the following steps: (1) Lung transplant acquisition: The rat lung grafts were processed according to standard procedures, including anesthesia, endotracheal intubation, chest opening, and perfusion with tissue preservation solution. While the airway remained ventilated, the pulmonary artery was flushed with a low-temperature Perfadex solution, completing the left lung harvest. The pulmonary veins were trimmed to form a left atrial cuff, preserving the structure and creating the lung graft.

[0042] (2) Storage method of cold static oil seal: After harvest, the lung graft was immediately placed in a tissue preservation solution (Perfadex solution) precooled to 4°C and secured to a fixture using a tether. A 2-cm-thick layer of paraffin oil was placed over the surface of the tissue preservation solution. This layer physically sealed the tissue preservation solution, restricting oxygen exchange and inducing a gradual decrease in oxygen content within the storage environment, thereby creating a progressively hypoxic microenvironment. The lung graft was stored in this closed storage environment at 4°C for 48 hours, without the need for ventilation or external perfusion equipment, making it easy to operate.

[0043] Comparative Example 1 (1) Lung transplant acquisition: The rat lung grafts were processed according to standard procedures, including anesthesia, endotracheal intubation, chest opening, and perfusion with tissue preservation solution. While the airway remained ventilated, the pulmonary artery was flushed with a low-temperature Perfadex® solution, completing the left lung harvest. The pulmonary veins were trimmed as needed to form a left atrial cuff, preserving the structure and creating the lung graft.

[0044] (2) 4℃ static cold storage method: The harvested lung grafts were immediately placed in a tissue preservation solution (Perfadex solution) precooled to 4°C and stored at 4°C for 48 hours.

[0045] The lung transplants after the storage period of Example 1 (PO group) and the lung transplants after the storage period of Comparative Example 1 (CSP group) were evaluated as follows: 1) Extracorporeal lung perfusion: Connect the patient to an extracorporeal lung perfusion (EVLP) system, gradually raise the temperature to 37°C, perform perfusion and ventilation for 2 hours, and monitor airway pressure, lung compliance, and oxygenation capacity (PaO2 / FiO2, abbreviated as P / F) in real time.

[0046] 2) Wet-to-dry weight ratio (W / D): assesses the degree of pulmonary edema.

[0047] 3) Histomorphological analysis: including H&E staining, TUNEL staining, immunohistochemistry, transmission electron microscopy, etc., to observe tissue structure, edema, inflammatory cell infiltration, mitochondrial morphology, etc.

[0048] 4) Molecular biology testing: Western blot or immunohistochemistry was used to detect the expression of HIF-1α, PDK4, SOD, Caspase-3, HMGB1 and other proteins.

[0049] 5) Metabolomics analysis: Assess changes in metabolic pathways, especially the levels of metabolites such as glutamate, lactate, and glucose, to infer the state of metabolic reprogramming.

[0050] Figure 2Comparison of functional indicators of rat lung transplants under different preservation methods in Example 1 and Comparative Example 1 evaluated on the EVLP platform, including peak airway pressure, airway platform pressure, dynamic compliance, static compliance, oxygenation index, and wet-to-dry weight ratio. Figure 2 It can be seen that compared with the CSP group, the PO group had higher peak airway pressure and airway plateau pressure, better dynamic compliance and static compliance, higher oxygenation index, and lower wet-to-dry weight ratio, indicating that the functional indicators of the lung grafts preserved by the preservation method of the present invention (PO group) were significantly better than those preserved by the 4°C static cold preservation method (CSP group).

[0051] Figure 3 The results of H&E staining of the tissue structure, TUNEL staining and molecular expression of lung transplants after 48 hours of cold storage under different storage methods in Example 1 and Comparative Example 1 are shown. The Ctrl group is a blank control group, i.e., fresh lung transplants after collection. Figure 3 As can be seen in a) and b), the lung grafts in the PO group had intact structures and reduced cell apoptosis, while the CSP group showed obvious edema, inflammatory cell infiltration, and structural damage. Figure 3 As can be seen from Figures c), d), and e), compared with the CSP group, the ROS of the lung transplants in the PO group was reduced and the SOD activity was increased, that is, the anti-oxidative stress ability of the PO group was relatively higher; the Caspase-3 and HMGB1 of the lung transplants in the PO group were reduced, that is, cell apoptosis and necrosis were relatively less. Figure 3 The results showed that compared with the static cold storage method at 4°C, the storage method of the present invention can better maintain the structural integrity of the lung graft.

[0052] Figure 4 The transmission electron micrographs of rat lung transplants under different preservation methods in Example 1 and Comparative Example 1 are shown. Figure 4 It can be seen that the mitochondrial structure of the Ctrl group was clear, the mitochondrial structure of the PO group was well preserved, and the mitochondria of the CSP group showed obvious swelling and cristae rupture.

[0053] Figure 5 This is the KEGG enrichment analysis of the metabolomics of lung transplants after 48 hours of cold storage in Example 1. Among them, the enrichment of the metabolism and production of various amino acids (such as glutamate and alanine) is highly correlated with the metabolic changes of amino acids caused by activation of the HIF-1α pathway. The results show that the metabolic pathways in the PO group are significantly enriched and highly overlap with the downstream metabolic pathways of HIF-1α.

[0054] Figure 6 The Western blot and immunohistochemical analysis results of lung transplants after cold storage in Example 1 and Comparative Example 1 are shown. Figure 6 As can be seen in b), the expression of HIF-1α protein in the PO group gradually increased with the extension of storage time. Figure 6 As can be seen in a) and c), compared with the CSP group, the HIF-1α protein expression in the lung transplants of the PO group was upregulated, accompanied by increased pyruvate dehydrogenase kinase 4 (PDK4) and inhibition of the pyruvate dehydrogenase complex (PDH), supporting the theory of metabolic reprogramming mechanism described in the present invention.

[0055] Example 2 The lung transplant preservation method of this embodiment based on oil-sealed low-temperature static preservation includes the following steps: (1) Lung transplant acquisition: The rat lung grafts were processed according to standard procedures, including anesthesia, endotracheal intubation, chest opening, and perfusion with tissue preservation solution. While the airway remained ventilated, the pulmonary artery was flushed with a low-temperature Perfadex solution, completing the left lung harvest. The pulmonary veins were trimmed to form a left atrial cuff, preserving the structure and creating the lung graft.

[0056] (2) Storage method of cold static oil seal: After harvest, the lung grafts were immediately placed in a tissue preservation solution (Perfadex solution) precooled to 4°C. A 2-cm-thick layer of mineral oil was placed over the surface of the tissue preservation solution. This layer formed a physical seal, restricting oxygen exchange and inducing a gradual decrease in oxygen content within the storage environment, thereby creating a progressively hypoxic microenvironment. The lung grafts were stored in this closed storage environment at 4°C for 48 hours, without the need for ventilation or external perfusion equipment, making it easy to operate.

[0057] Figure 7 The results show that both paraffin oil and mineral oil can exert an anti-oxidative stress effect during cold storage, and PO has a more significant anti-oxidative stress effect than MO.

[0058] Example 3 The lung transplant preservation method of this embodiment based on oil-sealed low-temperature static preservation includes the following steps: (1) Lung transplant acquisition: The pig lung grafts were processed according to standard operating procedures, including anesthesia, endotracheal intubation, chest opening, and perfusion of tissue preservation solution. While the airway remained ventilated, the pulmonary artery was flushed with a low-temperature Perfadex solution, and the left lung was harvested to obtain the lung graft.

[0059] (2) Storage method of cold static oil seal: After harvest, the lung grafts were immediately placed in tissue preservation solution precooled to 4°C. A 3-cm-thick layer of paraffin oil was placed over the surface of the tissue preservation solution. This layer physically sealed the tissue, restricting oxygen exchange and inducing a gradual decrease in oxygen content within the storage environment, thereby creating a progressively hypoxic microenvironment. The lung grafts were stored in this closed environment at 4°C for 48 hours, without the need for ventilation or external perfusion equipment, making it easy to operate.

[0060] Example 4 The lung transplant preservation method of this embodiment based on oil-sealed low-temperature static preservation includes the following steps: (1) Lung transplant acquisition: Obtain human lungs that are discarded in clinical practice and obtain lung transplants.

[0061] (2) Storage method of cold static oil seal: After harvest, the lung grafts were immediately placed in a tissue preservation solution (Perfadex solution) precooled to 4°C. A 3-cm-thick layer of paraffin oil was placed over the surface of the tissue preservation solution. This layer physically sealed the tissue, restricting oxygen exchange and inducing a gradual decrease in oxygen content within the storage environment, thereby creating a progressively hypoxic microenvironment. The lung grafts were stored in this sealed environment at 4°C for 48 hours, without the need for ventilation or external perfusion equipment, making it easy to operate.

[0062] Figure 8 Middle (a) is the H&E stained tissue structure of the pig lung after preservation in Example 3. It can be seen that the lung graft structure is intact without obvious edema. Figure 8 (b) shows the H&E-stained histological structure of the discarded human lung from Example 4 after preservation. It can be seen that the lung graft is structurally intact, with no significant edema. This result demonstrates that the method of the present invention has a good protective effect on both porcine and human lungs, indicating that the method of the present invention is suitable for cold preservation of lung grafts from large animals.

Claims

1. A lung transplant preservation method based on oil-sealed low-temperature static storage, characterized in that: The following steps are involved: S1, the harvested lung graft was transferred into tissue preservation solution; S2, covering the surface of the tissue preservation solution with a layer of paraffin oil or mineral oil to form a covering layer; S3, control the temperature of the tissue preservation solution at 0-10°C and keep the lung graft in a static cold storage environment.

2. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: In S1, the tissue preservation solution is Perfadex solution.

3. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: In S1, the lung transplant is a lung from a small animal, a large animal or a human.

4. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: In S2, the cover layer thickness is 1-3 cm.

5. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: In S2, the paraffin oil is medical grade paraffin oil.

6. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 5, characterized in that: In S2, the purity of paraffin oil is ≥99%.

7. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: The storage time is 12-48 hours.

8. The lung transplant preservation method based on oil-sealed low-temperature static storage according to claim 1, characterized in that: Also includes: S4, after the saving is completed, gradually remove the covering layer.

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