A normal temperature mechanical perfusion solution containing taurine suitable for liver preservation for the elderly and application thereof

Abstract

The application relates to a room temperature mechanical perfusion solution containing taurine suitable for liver preservation for the elderly and application thereof. The room temperature mechanical perfusion solution is composed of a basic liquid component and taurine; wherein the concentration of taurine in the room temperature mechanical perfusion solution is 10-30 mmol / L; the basic liquid component contains red blood cells, and the hematocrit (HCT) is maintained at 10-30%. Through in-vitro experiment verification, the room temperature mechanical perfusion solution provided by the application can reduce the levels of liver aging-related biomarkers p16INK4a, p21, BCL-2, p53, SA-beta-gal, IL-6, IL-1beta and MMPs. In addition, the room temperature mechanical perfusion solution provided by the application can greatly improve organ utilization rate, expand available liver source, shorten waiting time, reduce early complications, improve long-term survival, reduce medical costs, has the advantages of convenient operation, easy popularization and remarkable social benefits, and the like.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and organ transplantation technology, and in particular to a taurine-containing room-temperature mechanical perfusion solution suitable for the preservation of elderly donor livers and its application. Background Technology

[0002] The demand for organ transplantation among patients with end-stage liver disease is increasing, and the shortage of young donors has forced the clinical use of extended-criteria donors (ECD) more and more, with elderly donors (usually referring to donors older than 65 years) accounting for a significant proportion (Tingle, SJ, et al., Machine perfusion in liver transplantation. The Cochrane Database of Systematic Reviews, 2023. 9(9): p. CD014685.; Vodkin, I. and A. Kuo, Extended Criteria Donors in Liver Transplantation. Clinics InLiver Disease, 2017. 21(2): p. 289-301.). However, elderly donors face the following serious challenges in clinical application: (1) Decreased cell reserve function: With age, the regenerative capacity of hepatocytes weakens, telomeres shorten, and fatty degeneration is often present. (2) Extremely poor stress tolerance: The liver of the elderly is highly sensitive to ischemia-reperfusion injury (IRI), and the risk of early graft dysfunction (EAD) and primary non-function (PNF) after transplantation is much higher than that of the younger donor liver.(3) Decreased metabolic and detoxification capabilities: Physiological functions such as bile synthesis, lactate clearance, and coagulation factor production deteriorate rapidly during ex vivo preservation (Tang, Q., C. Dong, and Q. Sun, Immune response associated with ischemia and reperfusion injury during organ transplantation. Inflammation Research : Official Journal of the European Histamine Research Society ... [et Al.], 2022. 71(12): p. 1463-1476.; Panconesi, R., et al., Mitochondrial injury during normothermic regional perfusion (NRP) and hypothermic oxygenated perfusion (HOPE) in a rodent model of DCD liver transplantation. EBioMedicine, 2023. 98: p. 104861.; Pollok, JM, et al., Enhanced recovery for liver transplantation: recommendations from the 2022 International LiverTransplantation Society consensus conference. The Lancet.). Gastroenterology &Hepatology, 2023. 8(1): p. 81-94.).

[0003] Currently, the commonly used liver preservation techniques in clinical practice mainly include conventional static cryopreservation (SCS) and normothermic mechanical perfusion (NMP). Conventional static cryopreservation (SCS) has long relied on a low-temperature environment of 0-4°C to reduce metabolism (Zhang, Y., et al., Avoiding Ischemia Reperfusion Injury in LiverTransplantation. Journal of Visualized Experiments: JoVE, 2020(166).). However, for elderly donor livers, the low-temperature environment not only fails to repair inherent cellular damage but also induces cold ischemia injury, leading to mitochondrial structural disintegration and lysosomal enzyme release. Therefore, normothermic mechanical perfusion (NMP) technology has emerged. NMP provides oxygen and nutrient substrates to the donor liver by simulating the physiological environment of the organ at 36-37°C in vitro, thus maintaining its metabolically active state (Hosgood, SA, et al., Normothermic machine perfusion versus static cold storage in donation after circulatory death kidney transplantation: a randomized controlled trial. Nature Medicine, 2023. 29(6): p. 1511-1519.; Hessheimer, AJ, et al., Normothermic regional perfusion vs. super-rapid recovery in controlled donation after circulatory death liver transplantation. Journal of Hepatology, 2019. 70(4): p. 658-665.).

[0004] Current NMP technology is mainly used to assess donor liver viability (such as monitoring bile production, lactate trends, etc.). In addition, by adding various protective agents, such as antioxidants (such as N-acetylcysteine), hormones, or vasodilators to the perfusion fluid, the aim is to improve the quality of donor liver preservation by reducing ischemia-reperfusion injury (IRI) (Lin, Y., et al., Assessing Donor Liver Quality and Restoring Graft Function in the Era of Extended Criteria Donors. Journal of Clinical and Translational Hepatology, 2023. 11(1): p. 219-230.; Martins, PN, et al., The role of normothermicmachine perfusion in liver transplantation. International Journal of Surgery (London, England), 2020. 82S: p. 52-60.). For example, patent CN113287602B discloses a room-temperature mechanical perfusion solution and a method for in vitro liver preservation. The room-temperature mechanical perfusion solution includes the following components: resveratrol, albumin, L-glutamine, insulin, hydrocortisone, heparin, penicillin, streptomycin, and Williamsmedium E. The active ingredient, resveratrol, is used to repair marginal donor livers such as those with DCD (degenerative disc herniation) and fatty liver. Another example is patent US10874098B2, which discloses an aqueous organ preservation solution, i.e., a mechanical perfusion solution, containing taurine, L-alanine, L-glutamine, and glutamate. This can improve the oxygenation preservation of organs and organ components and prevent damage to organs typically caused by ischemia, hypoxia, hyperoxia, energy depletion, hypothermia, and reperfusion injury. It is used to maintain the viability of organs and organ components. However, these solutions primarily focus on alleviating acute stress injury during transplantation.

[0005] However, the additives in existing normothermic mechanical perfusion fluids are mainly aimed at the transient oxidative stress and inflammatory response caused by ischemia-reperfusion, which is a relief of "symptoms".

[0006] However, there is still no treatment plan in place to achieve "functional de-aging" of the liver in the elderly through proactive intervention via the NMP platform. Summary of the Invention

[0007] Existing room temperature mechanical perfusion fluids mainly contain additives that target transient oxidative stress and inflammatory responses caused by ischemia-reperfusion, which are essentially symptom relief. The purpose of this invention is to overcome the limitation of room temperature mechanical perfusion fluids in passively defending against acute injury to the donor liver, and to provide a room temperature mechanical perfusion fluid containing taurine suitable for the preservation of elderly donor livers and its application.

[0008] This invention upgrades the ambient temperature mechanical perfusion scheme from simple "preservation" to "ex vivo intervention and repair" for the first time. Through the multi-target regulatory effect of taurine, it achieves a leapfrog improvement in the treatment of elderly donor livers, from "delaying damage" to "reversing aging".

[0009] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention proposes a room-temperature mechanical perfusion solution containing taurine suitable for the preservation of elderly donor livers, wherein the room-temperature mechanical perfusion solution is formed by adding taurine to a base solution; The concentration of taurine in the mechanical perfusion fluid at room temperature is 10-30 mmol / L; The base solution contains red blood cells, and the hematocrit (HCT) is maintained at 10-30%.

[0010] In one embodiment of the present invention, the concentration of taurine in the room temperature mechanical perfusion fluid is 10-20 mmol / L.

[0011] In one embodiment of the present invention, the hematocrit (HCT) in the basal solution is maintained at 15%-25%.

[0012] The present invention provides a taurine-containing room-temperature mechanical perfusion solution suitable for liver preservation in elderly donors. Before use, taurine is simply added to a base solution to prepare the solution. It is used with a room-temperature mechanical perfusion device and can be prepared on-site. In one embodiment of the invention, red blood cells serve as the oxygen-carrying medium.

[0013] In one embodiment of the present invention, the base fluid contains: 10-30 mL of red blood cells; 20-30 mL of DMEM culture medium; The final concentration of D-glucose is 0.5-2 g / L; The final concentration of sodium pyruvate is 0.5-2 g / L; Heparin 3000-6000 IU; Metronidazole 2-3 mL; Meropenem 20-30 mg; Hydrocortisone 1-2 mg; Prostaglandin 3000-6000 IU; Essential amino acids: Final concentration 30-60 mg / L.

[0014] Preferably, the base fluid contains: 15 mL of red blood cells; 25 mL of DMEM medium; The final concentration of D-glucose is 1 g / L; The final concentration of sodium pyruvate is 1 g / L; Heparin 5000 IU; Metronidazole 2.5 mL; Meropenem 25 mg; Hydrocortisone 1.25 mg; Prostaglandin 5000 IU; Essential amino acids: Final concentration 50 mg / L.

[0015] In one embodiment of the present invention, the essential amino acid is selected from one or more of lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, or valine.

[0016] In one embodiment of the present invention, the volume ratio of red blood cells to DMEM culture medium is 15:25.

[0017] In a more specific embodiment of the present invention, the preparation method of the base solution is as follows: 25 mL of DMEM containing 1 g / L D-glucose and 1 g / L sodium pyruvate is mixed with 15 mL of separated red blood cells, and 1.5 mL of compound amino acid injection, 1 mL of meropenem solution diluted with 25 mg / mL physiological saline (containing the main component meropenem), 2.5 mL of metronidazole, 1.25 mg of hydrocortisone, 5000 IU of heparin and 5000 IU of prostaglandin are added.

[0018] In one embodiment of the present invention, the prostaglandin is preferably prostaglandin E1.

[0019] Secondly, the present invention provides the application of the taurine-containing room-temperature mechanical perfusion solution suitable for the preservation of elderly donor livers, for non-therapeutic purposes of room-temperature mechanical perfusion preservation of isolated elderly donor livers.

[0020] In one embodiment of the present invention, the elderly donor liver is selected from rat liver or human liver.

[0021] Furthermore, the donor rat livers were aged rats older than 24 months and weighing more than 600g; the human livers were diseased livers extracted from donors older than 65 years.

[0022] In one embodiment of the present invention, the taurine-containing room-temperature mechanical perfusion solution suitable for liver preservation in elderly donors is used in conjunction with a room-temperature mechanical perfusion device, and the temperature of the room-temperature mechanical perfusion solution is maintained at 36-37°C.

[0023] In one embodiment of the present invention, the perfusion of the ambient temperature mechanical perfusion device adopts a portal vein single-pathway perfusion mode, the portal vein pressure is maintained at 7-15 mmHg, and the portal vein flow rate is controlled at 1.0-1.5 ml / kg / min according to the liver mass.

[0024] Using the ambient temperature mechanical perfusion solution of the present invention for non-therapeutic purposes, the isolated elderly donor liver can be preserved by ambient temperature mechanical perfusion, which can repair the isolated elderly donor liver, reduce the level of aging-related biomarkers, and achieve the effect of "aging-reversing donor liver".

[0025] In one embodiment of the present invention, aging-related biomarkers include p16INK4a, p21, BCL-2, p53, SA-β-gal, IL-6, IL-1β, and MMPs.

[0026] Thirdly, this invention also provides a multi-dimensional evaluation system for "aging-reversing donor livers", including metabolic indicators, secretory function, perfusion parameters, liver enzyme levels, and histological verification.

[0027] In one embodiment of the present invention, the metabolic indicator is: within 4 hours of perfusion, lactate (Lac) < 2.5 mmol / L; Secretory function: During perfusion, it continuously produces golden-yellow bile, with a bile output of 0.5-1 mL / h, and the bile pH value remains alkaline (pH>7.3); Infusion parameters: The infusion pressure was kept stable at 7-15 mmHg and the flow rate was kept at 1.0-1.5 ml / kg / min during infusion. Liver enzyme levels: During perfusion, the liver enzyme levels in the NMP (taurine) group were lower than those in the NMP-only group; Histological verification: HE staining results showed that the liver tissue structure was stable, no cellular edema was observed, and fatty changes were improved.

[0028] Taurine, a sulfur-containing amino acid, has multi-target effects, exhibiting various biological effects in vivo, including anti-oxidative stress, maintaining cell osmotic pressure, regulating calcium homeostasis, and promoting bile secretion. Taurine plays a significant role in inhibiting endoplasmic reticulum stress (ERS) and enhancing mitophagy, two key molecular mechanisms contributing to the decline of liver function in the elderly. By stabilizing the endoplasmic reticulum and lysosomes and promoting mitochondrial renewal, it can reverse the aging phenotype. Based on this, this invention proposes for the first time the introduction of taurine into a normothermic mechanical perfusion system, aiming to achieve in vitro functional repair of elderly donor livers through targeted intervention in the aforementioned aging-related pathways, providing a novel technical solution for expanding the clinical utilization rate of elderly donor livers.

[0029] The core objective of existing normothermic machine perfusion (NMP) technology is to "maintain" the original function of organs in an ex vivo state and avoid further damage. This invention upgrades NMP from passive "preservation" to active "ex vivo repair" for the first time, achieving functional remodeling of aging livers.

[0030] Unlike the non-specific protective agents (such as antioxidants and vasodilators) widely used in existing technologies, this invention precisely intervenes in the multidimensional molecular defects unique to elderly donor livers by introducing taurine into the perfusion fluid, including: Energy metabolism disorders: mitochondrial dysfunction and autophagic flux blockage; Protein homeostasis imbalance: abnormal endoplasmic reticulum stress (ERS) and unfolded protein response (UPR); Cell fate dysregulation: senescence-related cell cycle arrest (high expression of p16INK4a / p21) and apoptosis resistance (abnormal BCL-2). Genomic instability: DNA damage accumulation (γ-H2A.X); Systematic regulation of the aforementioned aging-related targets was achieved.

[0031] There is a lack of targeted interventions in "clearing senescent cells" or "reversing cellular senescence" during normothermic mechanical perfusion. Existing perfusion fluid additives mostly focus on basic nutritional supplementation or broad-spectrum antioxidants, lacking specific repair mechanisms for the "senescence-associated secretory phenotype (SASP)" and mitochondrial dynamics imbalance in aged livers. This application achieves systematic regulation of senescence-related targets by introducing taurine into the perfusion fluid.

[0032] This invention pioneers a synergistic mechanism of "temperature-controlled enhancement," innovatively utilizing the physiological temperature environment of 36-37°C (NMP) to endow hepatocytes with active metabolic capabilities. Under this context, the biological effects of taurine are fully activated: Temperature dependence: At room temperature, the efficiency of hepatocytes in taking up and metabolizing taurine is significantly higher than at low temperature; Synergistic Function: An active metabolic state provides energy support for energy-consuming processes such as mitochondrial autophagy and protein repair, maximizing the anti-aging effects of taurine.

[0033] Traditional static cold preservation (SCS) lacks objective organ assessment standards, relying solely on the subjective judgment of transplant surgeons to evaluate organ quality. This invention, for the first time, introduces a systematic combination of aging assessment indicators into the field of liver preservation, constructing a multi-dimensional "aging reversal" evaluation system. This overcomes the limitations of traditional techniques that only focus on conventional indicators such as liver enzymes and bile volume. Experiments conducted in this application revealed the following changes in aging donor livers after taurine perfusion at room temperature: Cell cycle arrest: downregulation of p16INK4a and p21 expression; DNA damage repair: attenuation of γ-H2A.X signaling; Apoptosis regulation: Normalization of BCL-2 expression; Age-associated secretory phenotype (SASP): clearance rate of pro-inflammatory factors such as IL-6 and IL-1β; Lysosomal activity: SA-β-gal staining negative.

[0034] The solution provided in this application achieves closed-loop verification from the molecular level to the tissue level, providing a solid scientific basis for the technical effects of this invention.

[0035] This invention utilizes the caudate lobe of senescent liver tissue discarded during clinical organ transplantation for in vitro validation. By directly demonstrating the anti-aging effect of taurine in isolated human liver tissue, a translational pathway from animal models to human clinical trials has been established, significantly reducing the technical risks of subsequent clinical trials.

[0036] Compared with the prior art, the present invention has the following beneficial effects: 1. Significantly reverses the liver aging phenotype mRNA level: Experiments have shown that after perfusion using the method of this invention, the mRNA expression levels of aging markers p16INK4a and p21 in aged liver tissue were significantly downregulated, and telomerase activity was partially restored.

[0037] Protein levels: Experiments have shown that after perfusion using the method of this invention, the protein translation levels of aging markers p16INK4a, p21, BCL-2, and H2A.X in aged liver tissue were significantly downregulated, and aging was reversed.

[0038] SA-β-gal staining: Liver tissue samples were collected before perfusion, 3 hours after perfusion, and 6 hours after perfusion to detect the expression level of the aging-related biomarker SA-β-gal and to evaluate the anti-aging efficacy.

[0039] 2. Improve organ utilization rate Currently, the proportion of livers discarded in clinical practice due to advanced age (>65 years) or unqualified functional assessment is as high as 20%-40%. This invention can restore a significant portion of these organs to the transplantation standard after in vitro repair, significantly expanding the source of usable donor livers and providing usable donor livers for patients with end-stage liver disease.

[0040] 3. Improve post-transplant clinical prognosis Reduce early complications: By mitigating ischemia-reperfusion injury (IRI) and inhibiting SASP-related inflammatory responses, the technology of this invention can significantly reduce the incidence of early graft dysfunction (EAD).

[0041] Improving long-term survival: Taurine's multiple protective effects—including anti-oxidative stress, repair of mitochondrial damage, promotion of bile acid metabolism, and improvement of liver microcirculation—work together to promote liver function reconstruction after transplantation, potentially extending recipient survival and improving survival rates.

[0042] 4. Significant economic and social benefits Reduced medical costs: Improved donor liver quality is directly linked to shorter ICU stay and total hospital stay for recipients after surgery, thereby effectively reducing the overall medical costs of a single transplant.

[0043] Easy to operate and easy to promote: Taurine, as a pharmaceutical excipient with well-defined components, stable properties, low price and high safety, does not require complicated preparation processes and can be seamlessly integrated into existing commercial NMP systems, possessing extremely high clinical translation potential and industrialization prospects.

[0044] Significant social benefits: By improving organ utilization and transplant prognosis, this invention is expected to save the lives of more patients with end-stage liver disease without increasing the source of donors, which has great public health significance. Attached Figure Description

[0045] Figure 1 A schematic diagram illustrating the changes in lactate levels in the livers of elderly patients during perfusion with taurine at different concentration gradients.

[0046] Figure 2 This is a schematic diagram of a room-temperature mechanical injection equipment.

[0047] Figure 3 This is a schematic diagram of metabolomic analysis of young blood and old blood, and young liver and old liver.

[0048] Figure 4 The graph shows the liver perfusion parameters, bile secretion, and metabolic function analysis of three groups of elderly patients in an in vitro perfusion experiment.

[0049] Figure 5 This is a schematic diagram illustrating the analysis of liver injury marker levels in elderly patients during three in vitro perfusion experiments.

[0050] Figure 6 This is a schematic diagram of the liver HE pathology assessment for elderly patients in group C.

[0051] Figure 7 This is a schematic diagram of the RT-qPCR detection results for three groups of in vitro perfusion experiments.

[0052] Figure 8 This is a schematic diagram of the WB detection results for three groups of in vitro perfusion experiments.

[0053] Figure 9 This is a schematic diagram of the IF test results for group C.

[0054] Figure 10 This is a schematic diagram of SA-β-gal staining in group C.

[0055] Figure 11 A schematic diagram comparing the SA-β-gal staining results of the three groups of experiments.

[0056] Figure 12 This is a schematic diagram of the WB assay results for taurine's ability to reverse cell senescence.

[0057] Figure 13 This is a schematic diagram of the detection results of H2A.X, a DNA damage marker for taurine's role in reversing cellular aging.

[0058] Figure 14 This is a schematic diagram showing the results of the synthesis and release of senescence-associated secretory phenotype (SASP) factors that taurine can use to reverse cellular senescence.

[0059] Figure 15 The study compared serum ALT, AST, LDH, and ALP levels between the control group and the taurine experimental group 1-7 days post-transplantation. Detailed Implementation

[0060] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0061] The ambient temperature mechanical perfusion device used in this invention can be a conventional ex vivo animal organ perfusion device. The ambient temperature mechanical perfusion system consists of a peristaltic pump, a hollow fiber oxygenator, a heat exchanger, a pressure sensor, and a self-circulating pipeline.

[0062] Temperature control: The temperature of the perfusion fluid in the system is maintained at the organ physiological temperature of 36-37°C through a heat exchanger.

[0063] Perfusion pressure: A single portal vein perfusion mode was used. The portal vein pressure was maintained at 7-15 mmHg, and the portal vein flow rate was controlled at 1.0-1.5 ml / kg / min based on liver weight.

[0064] Procedure for liver procurement and perfusion in elderly patients: The procedure for obtaining livers from aged rats is as follows: 1) Liver procurement and cryopreservation: (1) Animal anesthesia: Anesthesia was administered to aged rats. Anesthesia could be achieved by intraperitoneal injection of 3% sodium pentobarbital (30 mg / kg), with the animal gradually entering anesthesia approximately 5 minutes after injection; gas anesthesia could also be used. The depth of anesthesia needed to be adjusted according to the individual animal's response: if the depth of anesthesia was insufficient, an additional 0.1 ml of anesthetic could be added; if the anesthesia was too deep and caused respiratory depression, oxygen should be administered immediately (flow rate approximately 0.8 L / min) to relieve the condition. Subsequent surgical procedures could only be performed after the rats were fully anesthetized.

[0065] (2) Autologous blood collection and processing: Whole blood was collected from two aged rats and centrifuged at 2500 rpm for 10 minutes at 4℃. The upper plasma and lower red blood cells were separated and stored in a low-temperature environment for later use.

[0066] (3) Preoperative preparation: The rat was fixed in a supine position in a surgical tray, and medical gauze was pre-lined in the tray. The hair on the abdomen was removed with a shaver, and the skin of the surgical area was disinfected with 75% ethanol and povidone-iodine in sequence (applying in a spiral motion from the center of the abdomen to the periphery).

[0067] (4) Opening the abdomen and exposing the laparotomy: Make a cross-shaped incision along the midline of the abdomen to open the abdominal cavity. Use hemostatic forceps to flip the xiphoid process toward the head and fix it. Cover the surface of the liver with gauze moistened with physiological saline to keep it moist. Gently push the intestines toward the left side of the abdominal cavity with a moistened cotton swab and cover them with moist gauze to prevent water evaporation and damage to the intestinal tract.

[0068] (5) Liver detachment and vascular processing: Ligament transection: The falciform ligament and the left triangular ligament are cut, the tissue between the superior and inferior vena cava and the left diaphragmatic vein is freed, and the suture is passed through and ligated below the left diaphragmatic vein.

[0069] Liver rotation and exposure: Gently rotate the middle, left, right, and left lateral lobes of the liver towards the thoracic cavity using a moistened cotton swab, and cover and protect with moist gauze. Free and cut the ligaments around the papillary and caudate lobes. Use microforceps to grasp and cut the ligaments above the caudate lobe to fully expose the area.

[0070] Management of the esophageal venous plexus: Dissect the tissue surrounding the esophageal venous plexus, pass through sutures, and ligate.

[0071] Bile duct cannulation: Pass a suture about 1 cm below the bile duct bifurcation for later use. Make an oblique incision in the bile duct with scissors, insert a 24G indwelling needle, and ligate and secure it. Note: During the procedure, avoid separating the adipose tissue around the bile duct to protect the blood vessels supplying the bile duct and maintain bile duct blood supply.

[0072] Management of portal vein branches: Gently dissect the adipose tissue on the surface of the portal vein to the splenic vein confluence. Dissect the pyloric vein, pass it through a suture near the main trunk of the portal vein, and ligate it. Dissect and ligate the gastrosplenic branches and duodenal-pancreatic branches of the portal vein.

[0073] Treatment of hepatic artery branches: Ligate all arterial branches except the proper hepatic artery.

[0074] Systemic heparinization: 0.6 ml of heparinized saline (heparin concentration 166.7 U / ml) was injected via the inferior vena cava to achieve systemic anticoagulation.

[0075] Preparation of intubation access: Dissect the abdominal aorta below the left kidney, and then dissect upwards to the abdominal aorta above the hepatic artery for subsequent blood draw. Perform abdominal trunk cannulation and ligate for fixation.

[0076] Portal vein perfusion: After portal vein cannulation, immediately flush the liver with 20 ml of pre-cooled HTK solution (containing 25 U / ml heparin) at 4°C. During perfusion, continuously drip ice-cold saline onto the liver surface to achieve cooling and hydration.

[0077] Pre-excision transection: sequentially cut the bile duct, portal vein, and celiac trunk.

[0078] Liver excision: The superior and inferior vena cava are cut along the diaphragmatic ring, and the esophageal venous plexus is severed. The liver is gently removed with a moistened cotton swab and placed in UW preservation solution at 4°C for later use.

[0079] 2) Pre-filling and internal environment equilibration of the infusion system: The basal perfusion medium was DMEM (Thermo Fisher, 500 mL, USA) containing 1 g / L D-glucose and 1 g / L sodium pyruvate.

[0080] 5000 IU heparin (Chengdu Haitong Pharmaceutical, 2 mL: 12500 units, China), 2.5 mL metronidazole (Anhui Shuanghe Pharmaceutical, each 250 ml contains 0.5 g metronidazole and 2.25 g sodium chloride, China), 1 mL of 25 mg / ml saline diluted mefenamic acid solution (Zhongnuo Pharmaceutical, 0.5 g, China), 1.25 mg hydrocortisone (Liaoning Haisco Pharmaceutical, 40 mg, China), 5000 IU prostaglandin (Benxi Hengkang Pharmaceutical, 1 ml: 5 µg, China), and a compound amino acid injection rich in essential amino acids such as lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, and valine (Chenxin Pharmaceutical, 500 mL, China).

[0081] Take 25 mL of the above-mentioned basal perfusion culture medium and mix it with 15 mL of isolated rat erythrocytes. Then add 1.5 mL of compound amino acid injection rich in essential amino acids, 1 mL of mepiquat solution (500 mg mepiquat dissolved in 20 mL of physiological saline), 2.5 mL of metronidazole, 1.25 mg of hydrocortisone, 5000 IU of heparin and 5000 IU of prostaglandin in sequence.

[0082] Pre-filling and equilibration: Add the mixed red blood cells and basal perfusion solution to the NMP tubing system, start circulation and preheat to 36.5°C. Adjust the pH of the perfusion solution to 7.35-7.45 and the osmotic pressure to 280-320 mOsm / kg by supplementing with NaHCO3 solution, physiological saline or sterile water.

[0083] 3) Injection process: The donor liver is connected to the perfusion system, and the transition from cold storage temperature to room temperature is achieved by controlling the heat exchanger. After perfusion begins, 15 mmol / L taurine (the concentration of taurine in room temperature mechanical perfusion fluid) is added, maintaining a perfusion pressure of 7-15 mmHg and a flow rate of ≈1.0-1.5 mL / min / g, and perfusion is continued for 6-8 hours. Blood gas analysis is performed every 30 minutes during this period, and oxygen input and nutrient supplementation are adjusted in real time. Liver samples are collected 3 hours and 6 hours after the addition of taurine.

[0084] 3. Procedure for in vitro perfusion of liver tissue from elderly patients: This procedure uses human waste liver tissue (caudate lobe) for in vitro perfusion.

[0085] (1) Liver irrigation and intubation Irrigation and cannulation: After obtaining the caudate lobe of the liver, the liver is repaired, and cannulas are placed and fixed in the portal vein and common bile duct. Preservation: The donor liver should be preserved in renal preservation solution at 4°C and placed in an ice basin.

[0086] (2) Injection and repair: Pre-charge upon startup: Start the perfusion equipment to fill the perfusion pipeline with perfusion fluid, measure blood gas, and assess the initial state of the perfusion fluid.

[0087] Rewarming and infusion: The liver sample was rapidly perfused with 50 ml of 37°C physiological saline via a portal vein cannula to rewarm it. The liver sample was then placed into the perfusion machine, the portal vein cannula was connected, and the formal perfusion process began.

[0088] Infusion parameter settings: Physiological parameters during perfusion (such as flow rate, pressure, oxygenation target, etc.) can be set and adjusted with reference to relevant literature and clinical guidelines on human liver perfusion (Huang, C., et al., Prospective, single-centre, randomised controlled trial to evaluate the efficacy and safety of ischemia-free liver transplantation (IFLT) in the treatment of end-stage liver disease. BMJ Open, 2020. 10(5): p. e035374.;Guo, Z., et al., Arandomized-controlled trial of ischemia-free liver transplantation for end-stage liver disease. Journal of Hepatology, 2023. 79(2): p. 394-402.;Lascaris, B., VE de Meijer, and RJ Porte, Normothermic liver machineperfusion as a dynamic platform for regenerative purposes: What does the future have in store for us? Journal of Hepatology, 2022.). 77(3): p. 825-836.).

[0089] In the following embodiments, the parameters during the infusion process are as follows: Example 1: Preparation of Taurine-Fortified NMP Perfusion Solution (Suitable for Preservation of Elderly Liver Donors, Containing Taurine, at Room Temperature for Mechanical Perfusion) 1.1 Preparation of basic fluid: The perfusion fluid uses red blood cells as the oxygen-carrying medium, and the hematocrit (HCT) is maintained at 15%-25%.

[0090] In this embodiment, the preparation method of the base solution is as follows: 25 mL of DMEM containing 1 g / L D-glucose and 1 g / L sodium pyruvate is mixed with 15 mL of separated red blood cells, and 1.5 mL of compound amino acid injection, 1 mL of mepiquat chloride solution diluted with 25 mg / mL physiological saline, 2.5 mL of metronidazole, 1.25 mg of hydrocortisone, 5000 IU of heparin and 5000 IU of prostaglandin are added.

[0091] 1.2 Core Additive: Weigh taurine powder, dissolve it in physiological saline, and filter to remove bacteria.

[0092] 1.3 Final Concentration Setting: Before determining the final concentration of taurine, different concentration gradients (10, 20, 30 mmol / L) were set during in vitro perfusion. It was found that 10-30 mmol / L of taurine could benefit aging livers. Figure 1 As shown. Based on this study, in this embodiment, taurine was added to the above-mentioned base solution components before initiating perfusion, so that its final concentration was maintained at 15 mmol / L.

[0093] Example 2: In vitro repair experiment of livers from elderly donors 1. Donor sample selection: If the perfused liver is a rat liver, then the liver of an aged rat (>24 months old, weighing 600g) should be selected. If the liver to be perfused is a human liver, then a diseased liver removed from a donor whose age is >65 years old is selected.

[0094] 2. Experimental Groups: Group A (Standard NMP Group, Control): Liver of aged rats was perfused at room temperature using standard perfusion solution without taurine.

[0095] Group B (Young Blood perfusion of elderly livers): Elderly livers were perfused at room temperature using a standard perfusion solution containing young mouse erythrocytes.

[0096] Group C (Repair Group of this Invention, Taurine): Liver of aged rats was perfused at room temperature using a perfusion solution containing 15 mmol / L taurine.

[0097] 3. Injection process: The donor liver was connected to a pre-filled and balanced perfusion system. The heat exchanger was controlled to gradually and smoothly transition the organ from its cryopreservation temperature to physiological room temperature (36-37℃). After perfusion began, 15 mmol / L taurine was added, maintaining a perfusion pressure of 7-15 mmHg and a flow rate of approximately 1.0-1.5 mL / min / g for 6-8 hours. Blood gas analysis was performed every 30 minutes, and oxygen input and nutrient supplementation were adjusted in real time. Liver samples were collected 3 hours and 6 hours after taurine addition.

[0098] Among them, the infusion system is as follows Figure 2 As shown, it consists of a peristaltic pump, a hollow fiber oxygenator, a heat exchanger, a pressure sensor, and a self-circulating pipeline.

[0099] 4. Testing and verification of repair effectiveness It has been confirmed that elderly livers are deficient in taurine, and that taurine can reverse liver aging. (1) Confirmation of differences in taurine metabolism between elderly and young livers. Metabolomic analysis was performed on blood samples from young and elderly individuals, as well as liver samples from young and elderly individuals. The results are as follows: Figure 3 As shown, the taurine metabolism level in older livers is significantly lower than that in younger livers.

[0100] (2) Perfusion parameters, bile secretion and metabolic function (macroscopic functional evaluation) like Figure 4 As shown, the experimental results indicate that: Bile synthesis: such as Figure 4 As shown in Figure D, group C (the repair group of this invention) produced more bile during perfusion, and the liver had better bile synthesis function.

[0101] Lactic acid clearance: such as Figure 4 As shown in Figure C, the lactate level in group C rapidly decreased to below 2.5 mmol / L within 2 hours after the start of perfusion and remained at a low level, indicating that the mitochondrial oxidative phosphorylation function of hepatocytes was significantly restored.

[0102] Microcirculation perfusion: such as Figure 4 As shown in Figures A and B, group C had lower perfusion pressure and higher perfusion flow rate throughout the perfusion process, indicating that the liver microcirculation in the taurine treatment group was more stable, the damage was less severe, and the blood and oxygen supply to the cells was more sufficient.

[0103] (3) Levels of liver injury markers Liver enzyme levels: such as Figure 5 As shown, the liver enzyme levels in group C were lower than those in groups B and A, and even decreased in the later stages of perfusion, indicating that the taurine treatment group had a better protective effect on the liver and could repair liver damage during in vitro perfusion.

[0104] 5. Evaluation of HE pathology results HE staining: such as Figure 6 As shown in the HE staining results, aging liver is accompanied by steatosis, but as perfusion proceeds, taurine effectively repairs the steatotic liver.

[0105] 6. Determination of aging-related biomarkers (de-aging evaluation) (1) Detection by tissue biopsy and RT-qPCR: Liver tissue samples were collected before perfusion, 3 hours after perfusion, and 6 hours after perfusion to detect the expression levels of aging-related cell proliferation cycle markers p16INK4a and p21, and to evaluate the anti-aging efficacy.

[0106] p16INK4a, p21 expressions: such as Figure 7 As shown, 6 hours after perfusion, the expression level of p16 in group C tissue decreased by approximately 70% compared to before perfusion, while that in group B decreased by only approximately 30%. 6 hours after perfusion, the expression level of p21 in group C tissue decreased by approximately 50% compared to before perfusion, while that in group B decreased by only approximately 30%.

[0107] (2) By tissue biopsy and Western blot detection: p16INK4a, p21 expressions: such as Figure 8 As shown, the results are consistent with those of RT-qPCR: 6 hours after perfusion, the expression levels of p16 and p21 in group C tissues decreased significantly compared with those before perfusion.

[0108] (3) Through tissue biopsy and IF detection Liver tissue samples were collected before perfusion, 3 hours after perfusion, and 6 hours after perfusion to detect the expression levels of aging-related anti-apoptotic markers BCL-2 and cell cycle arrestin p53, and to evaluate the anti-aging efficacy.

[0109] BCL-2 and p53 expression: (e.g.) Figure 9 As shown, aging tissues exhibit cell proliferation cycle arrest and apoptosis arrest. Therefore, aging tissues show elevated levels of the anti-apoptotic marker BCL-2 and the cell cycle arrest protein p53. In contrast, liver tissue treated with taurine showed decreased levels of both BCL-2 and p53, further demonstrating its anti-aging effect.

[0110] (4) SA-β-gal staining: SA-β-gal is the most commonly used biomarker for detecting cellular senescence. In senescent cells, the content of lysosomes increases, leading to enhanced β-galactosidase activity. Liver tissue samples were collected before perfusion, 3 hours after perfusion, and 6 hours after perfusion to detect the expression levels of the senescence-related biomarker SA-β-gal and to evaluate the anti-aging efficacy.

[0111] The results are as follows Figure 10 , 11 As shown, the proportion of senescence-related β-galactosidase-positive cells in group C decreased significantly with the progress of taurine treatment, demonstrating that taurine effectively induced metabolic remodeling of senescent cells.

[0112] (5) H2A.X Expression: Liver tissue samples were collected before perfusion, 3 hours after perfusion, and 6 hours after perfusion to detect the expression level of H2A.X, a marker of aging-related DNA damage, and to evaluate the anti-aging efficacy. Figure 13 The results showed that as taurine treatment continued during perfusion, the expression level of the aging-related DNA damage marker H2A.X decreased significantly after 3 hours of perfusion, dropping to about 50% of the level before treatment in the aging liver.

[0113] (6) SASP phenotype inhibition: Significantly reduces the synthesis and release of aging-related secretory phenotype (SASP) factors such as pro-inflammatory cytokines (IL-6, IL-1β). For example... Figure 14 The results showed that, compared with the control group, the levels of aging-related secretory phenotypes (IL-1, IL-6, TNF-α, and TGF-β) in the treatment group with added taurine were significantly downregulated, while the SASP level in the untreated control group remained elevated, demonstrating the anti-aging effect of taurine.

[0114] By combining the above-mentioned multiple indicators, the degree of reversal of aging phenotypes in elderly livers after taurine perfusion was comprehensively evaluated from multiple dimensions such as cell cycle regulation, apoptosis resistance, DNA integrity and lysosomal activity.

[0115] 7. Cell validation Hep-G2 cells express p16INK4a and p21: Hep-G2 cell senescence was induced by doxorubicin at 25-50 ng / ml. After successfully inducing the senescent phenotype in the Hep-G2 cell line, taurine at 10-20 mmol / L was added for anti-aging treatment. Figure 12 As shown, the results of Western blot analysis demonstrate that taurine can also reverse the senescence state observed in the Hep-G2 cell line.

[0116] Example 3: Post-transplant prognostic assessment (simulating clinical outcomes) 1. Simulate orthotopic liver transplantation using the repaired rat donor liver: 2. Reperfusion injury markers: Comparison of serum ALT, AST, LDH, and ALP levels between the control and experimental groups 1-7 days post-transplantation. Figure 15 As shown, a 3vs3 rat liver transplantation was performed, and blood was collected from the tail vein on days 1, 2, 4, 6, and 7 post-transplantation for the detection of liver damage markers. The results showed that in the experimental group treated with taurine, ALT, AST, LDH, and ALP levels showed a transient increase on days 1-2 post-transplantation, followed by a significant decrease on days 3-4, and returned to normal levels within 7 days. In contrast, the liver enzyme damage markers in the untreated control group remained elevated, showing a decreasing trend on days 6-7 post-transplantation. Furthermore, one rat showed a persistently elevated liver enzyme damage marker level within one week post-transplantation, demonstrating the protective effect of taurine treatment on the liver of older rats.

[0117] 3. Survival rate: In the rat model, after repairing aged donor livers with taurine, the survival rates of rats in the simple perfusion group and the taurine perfusion group were compared. All rats in the control group and the experimental group survived within 7 days after transplantation.

[0118] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A taurine-containing room-temperature mechanical perfusion solution suitable for liver preservation in elderly donors, characterized in that, The ambient temperature mechanical infusion fluid is formed by adding taurine to the base fluid; The concentration of taurine in the mechanical perfusion fluid at room temperature is 10-30 mmol / L; The base solution contains red blood cells, and the hematocrit of the base solution is maintained at 10-30%. The basal solution was prepared from DMEM culture medium. The base fluid contains: 15 mL of red blood cells; 25 mL of DMEM medium; The final concentration of D-glucose is 1 g / L; The final concentration of sodium pyruvate is 1 g / L; Heparin 5000 IU; Metronidazole 2.5 mL; Meropenem 25 mg; Hydrocortisone 1.25 mg; Prostaglandin 5000 IU; Essential amino acids: Final concentration 50 mg / L.

2. The taurine-containing room-temperature mechanical perfusion solution suitable for liver preservation in elderly donors according to claim 1, characterized in that, The essential amino acid is selected from one or more of lysine, tryptophan, phenylalanine, methionine, threonine, isoleucine, leucine, or valine.

3. The application of the taurine-containing room-temperature mechanical perfusion solution for liver preservation in elderly donors, as described in claim 1 or 2, characterized in that... For non-therapeutic purposes, it is used for the mechanical perfusion preservation of isolated elderly donor livers at ambient temperature.

4. The application according to claim 3, characterized in that, The elderly donor livers were selected from rat livers or human livers.

5. The application according to claim 4, characterized in that, The donors of rat livers were aged rats older than 24 months and weighing more than 600g. Human liver refers to a diseased liver extracted from a donor aged >65 years.

6. The application according to claim 3, characterized in that, The aforementioned taurine-containing room-temperature mechanical perfusion solution, suitable for liver preservation in elderly donors, is used in conjunction with room-temperature mechanical perfusion equipment, with the temperature of the room-temperature mechanical perfusion solution maintained at 36-37°C.

7. The application according to claim 6, characterized in that, The perfusion of the ambient temperature mechanical perfusion equipment adopts the portal vein single-pathway perfusion mode, the portal vein pressure is maintained at 7-15 mmHg, and the portal vein flow rate is controlled at 1.0-1.5 ml / kg / min according to the liver weight.

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