Support system for preserving in-vitro liver through bidirectional driving of liver and stem cell tank

Abstract

The invention provides a support system for preserving an in-vitro liver through bidirectional driving of a liver and stem cell tank, which comprises an organ preserving device, a first circulating pipeline and a second circulating pipeline, and the first circulating pipeline and the second circulating pipeline are mutually independent; the organ storage device is used for storing the in-vitro liver; the first circulating pipeline is provided with a power part and a circulating purification unit, one end of the first circulating pipeline is connected with the inferior vena cava of the in-vitro liver in the organ storage device, and the other end of the first circulating pipeline is connected with the portal vein of the in-vitro liver; the second circulating pipeline is provided with a circulating pump and a bioreactor inoculated with mesenchymal stem cells, one end of the second circulating pipeline is connected with the inferior vena cava of the in-vitro liver in the organ preservation device, and the other end of the second circulating pipeline is connected with the hepatic artery of the in-vitro liver. The in-vitro liver is perfused through two channels, the purposes of detoxifying and supplementing essential factors can be achieved at the same time, and the in-vitro preservation time of the in-vitro liver can be effectively prolonged.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of organ preservation, in particular to a support system for bidirectional driving of a liver and stem cell tank to preserve an ex vivo liver. BACKGROUND

[0002] Liver transplantation is the most effective treatment for patients with end-stage liver disease. With the continuous improvement of liver transplantation technology, the problem of liver shortage is increasingly prominent, and the quality requirements for donor livers are gradually increasing. Therefore, how to expand the source of donor livers and ensure the quality of donor livers is a difficult problem to be solved.

[0003] In recent years, mesenchymal stem cells have been increasingly applied to organ transplantation for liver function repair, immunosuppression, etc. However, most of the researches use the method of injecting mesenchymal stem cells in vivo, which endangers the life safety of patients due to the tumorigenicity of stem cells. SUMMARY

[0004] The application aims to provide a support system for bidirectional driving of a liver and stem cell tank to preserve an ex vivo liver, which can effectively improve the quality of the ex vivo liver and prolong the preservation time of the ex vivo liver.

[0005] In order to achieve the above-mentioned purpose, the application provides the following technical solutions: A support system for bidirectional driving of a liver and stem cell tank to preserve an ex vivo liver, comprising an organ preservation device, a first circulation pipeline and a second circulation pipeline, wherein the first circulation pipeline and the second circulation pipeline are independent of each other. The organ preservation device is used for preserving an ex vivo liver. The first circulation pipeline is provided with a power component and a circulation purification unit, one end of the first circulation pipeline is connected with the inferior vena cava of the ex vivo liver placed in the organ preservation device, and the other end of the first circulation pipeline is connected with the portal vein of the ex vivo liver. The second circulation pipeline is provided with a circulation pump and a bioreactor inoculated with mesenchymal stem cells, one end of the second circulation pipeline is connected with the inferior vena cava of the ex vivo liver placed in the organ preservation device, and the other end of the second circulation pipeline is connected with the hepatic artery of the ex vivo liver.

[0006] Further provided is that the bioreactor is a hollow fiber bioreactor, the hollow fiber bioreactor comprises a hollow fiber inner cavity and an outer cavity sleeved outside the hollow fiber inner cavity, and the outer cavity is inoculated with mesenchymal stem cells.

[0007] Further provided is that the end of the second circulation pipeline connected with the inferior vena cava of the ex vivo liver is connected to the bottom of the hollow fiber bioreactor, and the top of the hollow fiber bioreactor is connected with the hepatic artery of the ex vivo liver.

[0008] Further arrangement: the circulating purification unit comprises a biological purifier, a liver cell carrier area and a perfusion liquid channel are arranged in the biological purifier, the liver cell carrier area is filled with liver cell carriers loaded with liver cells, and the liver cell carrier area and the perfusion liquid channel are physically isolated by a hollow fiber membrane.

[0009] Further arrangement: the biological purifier is provided with a wave-shaped support tube for supporting the hollow fiber membrane, and the top of the biological purifier is provided with an inlet connected with the perfusion liquid channel, and the bottom of the biological purifier is provided with an outlet connected with the perfusion liquid channel.

[0010] Further arrangement: the power component comprises a peristaltic pump arranged at the inlet of the biological purifier, and the peristaltic pump controls the perfusion flow rate of the perfusion liquid to be 5-15 mL / min.

[0011] Further arrangement: the inner wall of the biological reactor is provided with an elastic membrane covering the liver cell carrier area, the Young's modulus of the elastic membrane ranges from 0.1 MPa to 10 MPa, and the elastic membrane is connected with a pneumatic diaphragm pump for driving it to generate periodic air pressure fluctuations.

[0012] Further arrangement: the organ preserver comprises a preservation cabin and a support platform for supporting the isolated liver, the preservation cabin is provided with a inferior vena cava connector, a portal vein connector, a hepatic artery connector and a common bile duct connector connected with the inferior vena cava, the portal vein, the hepatic artery and the common bile duct of the isolated liver respectively, and the inferior vena cava connector is provided with two interfaces connected with the first circulating pipeline and the second circulating pipeline respectively.

[0013] Further arrangement: the common bile duct connector of the preservation cabin is connected with a bile collection bag.

[0014] Further arrangement: the first circulating pipeline and the second circulating pipeline are both provided with a monitoring mechanism, and the monitoring mechanism comprises pressure monitoring, flow monitoring, bubble monitoring, temperature monitoring and pH monitoring.

[0015] Compared with the prior art, the scheme of the present application has the following advantages: 1. In the liver and stem cell tank bidirectional driving support system for preserving isolated liver involved in the present application, two circulating pipelines are adopted to provide energy supply for the isolated liver organ, wherein the first circulating pipeline provided with a circulating purification unit can simulate the metabolism detoxification and synthesis function of the liver, and the second pipeline provided with a hollow fiber biological reactor inoculated with mesenchymal stem cells can effectively improve the functional state of the isolated liver, enhance the tissue repair ability, and ensure the quality of the isolated liver, thereby reducing the occurrence of complications after isolated liver transplantation, reducing the economic burden of patients, and providing effective guarantee for the life safety of patients.

[0016] 2. In the support system for bidirectional driving of the liver and stem cell tank for preservation of the isolated liver, two hollow fiber membranes are used to isolate the biological purifier to form three areas of the liver cell carrier area-perfusion liquid channel-liver cell carrier area, and the perfusion liquid channel is arranged in a wave shape, which can enhance the turbulent flow effect of the perfusion liquid input into the biological purifier, increase the contact area of the perfusion liquid with the liver cells, and improve the mass transfer efficiency. The hollow fiber membranes can be maintained in a wave shape by arranging wave-shaped support pipes to ensure the mechanical stability of the hollow fiber membranes and the liver cell carrier area and the perfusion liquid channel.

[0017] 3. In the support system for bidirectional driving of the liver and stem cell tank for preservation of the isolated liver, an elastic membrane is used to apply pressure to the liver cells to simulate the respiratory movement in the body, simulate the indirect mechanical stimulation of the diaphragm contraction on the liver, activate the mechanical sensitive ion channels of the liver cells, promote the cytoskeleton reorganization and metabolic activity, and effectively prolong the life cycle of the in vitro liver cell culture.

[0018] Additional aspects and advantages of the present application will be in part apparent and in part pointed out hereinafter in the description of the application, which will be understood in light of the following description and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0019] The above and / or additional aspects and advantages of the present application will become apparent and be readily understood from the following description, taken in conjunction with the accompanying drawings, in which: Figure 1 The structure schematic diagram of the support system for bidirectional driving of the liver and stem cell tank for preservation of the isolated liver according to the present application is shown in the figure. Figure 2 The structure schematic diagram of the combined biological artificial liver system for driving the liver cell function according to the present application is shown in the figure. Figure 3 The structure schematic diagram of the combined biological artificial liver system for driving the liver cell function according to the present application is shown in the figure.

[0020] In the figure, 1, organ preserver; 11, preservation box; 12, supporting platform; 2, first circulation pipeline; 21, power element; 22, liquid supplementing device; 23, first oxygenator; 24, biological purifier; 241, liver cell carrier area; 242, perfusion liquid channel; 243, inlet; 244, outlet; 25, bilirubin adsorber; 26, resin blood perfusion device; 3, second circulation pipeline; 31, circulation pump; 32, bioreactor; 4, bile collection bag; 5, plasma separator; 6, arterial pipeline; 61, delivery pump; 62, drug input device; 7, mixer; 8, venous pipeline. DETAILED DESCRIPTION

[0021] Embodiments of the present application are described below in detail, examples of which are shown in the accompanying drawings, wherein the same or similar notations represent the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the accompanying drawings are exemplary, only for explaining the present application, and cannot be interpreted as a limitation on the present application.

[0022] The present application relates to a liver, stem cell tank bidirectional driving support system for preserving ex vivo liver, please see Figure 1 , provides a kind of ex vivo liver cleaning and detoxification of liver, can guarantee ex vivo liver to the greatest extent not to be destroyed, provide health guarantee for the preservation of ex vivo liver.

[0023] It should be noted that in the liver blood circulation system, portal vein is the functional blood vessel of liver, which inputs the substances absorbed from gastrointestinal into liver. The portal vein is divided into left and right branches at the hepatic portal, which enters the left and right lobes of liver respectively, and then repeatedly branches between liver lobules to form interlobular veins. The interlobular veins branch into small branches, called terminal portal microveins, which run between adjacent two liver lobules. The branches of terminal portal microveins are connected with blood sinusoids to input portal vein blood into liver lobules. In addition, hepatic artery blood is rich in oxygen, which is the nutritional blood vessel of liver. The branches of hepatic artery run with the branches of portal vein, and are sequentially divided into interlobular arteries and terminal hepatic microarteries, which finally also enter blood sinusoids. The interlobular arteries also branch into small branches to supply capsule, interstitium and bile duct. Therefore, the liver blood sinusoids contain mixed blood of portal vein and hepatic artery. The blood of liver blood sinusoids flows from the periphery of lobule to the center, and is collected into central vein. The endothelium of central vein is not smooth muscle, but only has a small amount of connective tissue. A number of central veins converge into sublobular vein, which runs alone in the interlobular connective tissue, has a larger diameter and a thicker wall. The sublobular vein further converges into 2-3 hepatic veins, which enter inferior vena cava after leaving liver. According to the physiological structure of liver, perfusion liquid is perfused through portal vein, biliary tract and hepatic artery of liver, and macrophages and other immune cells, blood and harmful substances containing a large amount of toxins inside liver are discharged through the upper and lower ends of inferior vena cava, so that the toxins, blood and harmful substances in each corner of liver can be completely removed.

[0024] Please see Figure 1 , the liver, stem cell tank bidirectional driving support system for preserving ex vivo liver includes organ preserver 1, first circulation pipeline 2 and second circulation pipeline 3. The organ preserver 1 is used for preserving ex vivo liver. The two ends of the first circulation pipeline 2 are respectively connected with two different interfaces of the organ preserver 1. The two ends of the second circulation pipeline 3 are respectively connected with two different interfaces of the organ preserver 1. The first circulation pipeline 2 and the second circulation pipeline 3 are two independent circulation pipelines.

[0025] Specifically, the organ preserver 1 comprises a preservation box 11 and a supporting platform 12 for supporting the isolated liver organ, the preservation box 11 is provided with a plurality of connecting pipes, the plurality of connecting pipes comprises an inferior vena cava connecting pipe, a portal vein connecting pipe, a hepatic artery connecting pipe and a common bile duct connecting pipe which are respectively connected with the inferior vena cava, the portal vein, the hepatic artery and the common bile duct of the isolated liver organ, the inferior vena cava connecting pipe is provided with two interfaces for connecting the first circulation pipeline 2 and the second circulation pipeline 3 respectively, so as to ensure that the first circulation pipeline 2 and the second circulation pipeline 3 are two independent circulation pipelines.

[0026] The first circulation pipeline 2 is connected with the inferior vena cava connecting pipe and the portal vein connecting pipe at two ends respectively, so as to be connected to the inferior vena cava and the portal vein of the isolated liver organ, the first circulation pipeline 2 is provided with a power member 21, and the possible selection of the power member 21 includes a circulation pump and a peristaltic pump, so as to provide power for the perfusion solution flowing from the inferior vena cava to the portal vein in the first circulation pipeline 2. The first circulation pipeline 2 is connected with a liquid supplementing device 22, the liquid supplementing device 22 can supplement the perfusion solution lost due to evaporation, leakage or sampling in real time, so as to ensure the stability of the total amount of liquid in the circulation pipeline, avoid the situation that the perfusion pressure is reduced due to insufficient liquid amount, and also add electrolyte, buffer or medicine to the first circulation pipeline 2, so as to maintain the osmotic pressure, pH value and anticoagulation performance of the perfusion solution, and guarantee the metabolic demand of the isolated liver. The perfusion solution flows into the liver sinusoidal system of the isolated liver organ through the first circulation pipeline 2 connecting the inferior vena cava and the portal vein, and along with the continuous perfusion of the perfusion solution, the macrophages and other immune cells containing a large amount of toxins in the liver, blood and harmful substances are discharged from the inferior vena cava.

[0027] Further, the first circulation pipeline 2 is also provided with a circulation purification unit, the circulation purification unit comprises a biological purifier 24, the biological purifier 24 is provided with a liver cell carrier area 241 and a perfusion solution channel 242, the liver cell carrier area 241 is filled with liver cell carriers loaded with liver cells, and the liver cell carrier area 241 and the perfusion solution channel 242 are physically isolated by a hollow fiber membrane.

[0028] In this embodiment, a polyurethane foam carrier with 90% porosity and gradient pore size is used. Before loading hepatocytes, the carrier is washed with ethanol or PBS, and pre-coated with collagen or laminin to increase subsequent adhesion to hepatocytes. Additionally, plasma treatment or surface grafting with hydrophilic groups can reduce hydrophobicity to further improve hepatocyte adhesion efficiency. While pre-treating the carrier, a hepatocyte suspension is prepared. Animal-derived liver tissue is obtained and minced. The liver tissue is digested with collagenase (type IV), filtered through a sieve, and centrifuged to remove cell debris. Hepatocytes are purified using Percoll density gradient centrifugation (1.035-1.110 g / mL). Cells from the middle and lower interfacial layers are collected and resuspended in DMEM / F12 medium containing 10% fetal bovine serum. The cells are pipetted into a single-cell suspension, and the concentration is precisely adjusted to 10 using a hemocytometer or flow cytometry. 7– 10 8 Hepatocytes / L. Then, using a dynamic perfusion method, the hepatocyte suspension is perfused into the carrier at a flow rate of 0.5-2 mL / min to allow the hepatocytes to adhere to the carrier. Alternatively, static culture can be used, where the hepatocyte suspension is directly added to the carrier, and gravity sedimentation allows the hepatocytes to enter the carrier pores. This yields a hepatocyte-loaded carrier, which is then seeded into the hepatocyte carrier area 241 of the bio-purifier 24.

[0029] Hepatocytes located in the hepatocyte carrier region 241 exchange substances with the perfusion fluid passing through the perfusion fluid channel 242 via a hollow fiber membrane. The pore size of the hollow fiber membrane is 0.3-20 μm. Toxins in the perfusion fluid diffuse through the hollow fiber membrane to the hepatocytes via a concentration gradient. At the same time, hepatocyte metabolites (such as albumin) can diffuse back from the hepatocyte side into the perfusion fluid, thereby detoxifying the isolated liver, reducing the metabolic burden on the isolated liver, and the hepatocytes in the bioreactor 32 can simulate the liver's synthetic function, secreting albumin, coagulation factors, etc., thereby maintaining the physiological environment of the isolated liver, buying time for the repair of the isolated liver or subsequent transplantation, and improving organ survival rate.

[0030] The hollow fiber membrane of the present application maintains a wavy arrangement through the wavy support tube arranged in the biological purifier to ensure the mechanical stability among the hollow fiber membrane, the hepatocyte carrier 241 area and the perfusion liquid channel 242. In one embodiment, two hollow fiber membranes are arranged in parallel and in a wavy shape to divide the internal space of the biological purifier 24 into three areas, the area between the two hollow fiber membranes is the perfusion liquid channel 242, and the other two areas outside the two hollow fiber membranes are the hepatocyte carrier areas 241. The wavy channel can enhance the turbulent effect of the perfusion liquid, increase the contact area between the perfusion liquid and the hepatocytes, and improve the mass transfer efficiency. In other embodiments, the hollow fiber membrane can also be in a cylindrical structure and sleeved outside the wavy support tube, or in a spiral shape and wrapped outside the wavy support tube, so that the perfusion liquid channel 242 is formed inside the hollow fiber membrane, and the hepatocyte carrier area 241 is located outside the hollow fiber membrane.

[0031] The biological purifier 24 is provided with an inlet 243 at the top thereof communicating with the top of the perfusion liquid channel 242, and correspondingly, the bottom of the biological purifier 24 is provided with an outlet 244 communicating with the bottom of the perfusion liquid channel 242. The power member 21 of the present embodiment is selected as a peristaltic pump, which is arranged at the inlet 243 of the biological purifier 24 and controls the perfusion flow rate of the perfusion liquid to be 5-15 mL / min. This flow rate range can simulate the physiological blood flow dynamic state of the liver sinusoidal space, ensure the full contact between the hepatocytes and the plasma, improve the mass exchange efficiency, and ensure the detoxification and biosynthesis effect of the hepatocytes on the perfusion liquid. At the same time, the flow rate of 5-15 mL / min can avoid shear force damage to the hepatocytes, effectively stabilize the microenvironment of the hepatocytes, reduce the risk of hepatocyte edema and necrosis, and prolong the life cycle of the hepatocytes in the biological reactor 32.

[0032] In addition, the inner wall of the biological purifier 24 is also provided with an elastic membrane covering the hepatocyte carrier area 241. The present application uses the elastic membrane to press the hepatocytes to simulate the respiratory movement in vivo, simulate the indirect mechanical stimulation of the diaphragm contraction on the liver, activate the mechanical sensitive ion channels of the hepatocytes, and promote the reorganization of the cytoskeleton and the improvement of the metabolic activity.

[0033] The elastic membrane is connected with a pneumatic device for driving it to generate periodic air pressure fluctuations. The pneumatic device of the embodiment adopts a pneumatic diaphragm pump to adjust the air pressure to simulate the change of chest pressure under different breathing depths, to simulate the mechanical stress of diaphragm movement on the liver under the breathing state, so that the liver cells are subjected to a periodic compression-dilation stress close to that in the body, stimulate the promotion of liver cell CYP450 enzyme activity, promote the secretion of albumin, growth factors and other secretions, and approach the physiological level in the body. In addition, the elastic membrane of the embodiment is made of polydimethylsiloxane (PDMS), and its Young's modulus is 0.2-1.0MPa, which is close to the elasticity of liver tissue. At the same time, a carbon nanotube / graphene conductive network is integrated on the elastic membrane, and the resistance change has a linear relationship with the mechanical change. The elastic modulus of the elastic membrane will change when it is pressed, so that the mechanical stress received by the liver cells can be monitored in real time. Specifically, by measuring the change of the resistance value through the sensor, the mechanical stress received by the elastic membrane can be inversely deduced in real time and quantitatively. Since the Young's modulus of PDMS is close to that of liver tissue, it can be considered that the stress change monitored by the sensor can directly reflect the mechanical stress received by the liver cells. The operator can adjust the pneumatic device to change the static pressure or shear force on the liver cells in time. Dynamic stress regulation can promote the expression of polar proteins in liver cells and enhance metabolic activity.

[0034] The application sets an elastic membrane in the biological purifier 24, and the liver cell carriers in the liver cell carrier area 241 can be attached to the elastic membrane. The mechanical stress of the liver organ under the respiratory state in the body is simulated by driving the elastic membrane with an external pneumatic device, which can effectively improve the enzyme activity of the liver and provide a more realistic metabolic microenvironment for the artificial liver system, so as to prolong the life cycle of the liver cells in vitro. At the same time, mechanical stress can promote the proliferation and regeneration of liver cells and enhance the biological synthesis function.

[0035] In addition, the first oxygenator 23 is also arranged at the inlet 243 of the biological purifier 24, and the biological purifier 24 is supplied with oxygen through the first oxygenator 23 to maintain the oxygen partial pressure required for liver cell metabolism, avoid hypoxic injury, and promote the detoxification and biological synthesis function of liver cells.

[0036] Therefore, the perfusion fluid of the isolated liver is detoxified by the circulating purification unit, which reduces the risk of toxin accumulation of the isolated liver. At the same time, the liver cells in the biological purifier 24 can also secrete anti-inflammatory factors and eliminate pro-inflammatory factors, which can reduce the inflammatory response of the isolated liver and reduce the oxidative damage of the isolated liver. By pre-removing toxins and verification factors from the isolated liver, the incidence of primary non-function after transplantation can be reduced, and the probability of complications can be reduced.

[0037] The second circulation pipeline 3 is connected with the inferior vena cava connector and the hepatic artery connector respectively, so as to be connected with the inferior vena cava and the hepatic artery of the isolated liver organ. A circulation pump 31 is arranged on the second circulation pipeline 3, so that the liquid flow direction of the second circulation pipeline 3 is from the inferior vena cava to the hepatic artery. Meanwhile, a bioreactor 32 inoculated with mesenchymal stem cells is arranged on the second circulation pipeline 3.

[0038] The bioreactor 32 of the present application is a hollow fiber bioreactor, which comprises a hollow fiber inner cavity (not shown in the figure) and an outer cavity (not shown in the figure). The mesenchymal stem cells are inoculated in the outer cavity. The hollow fiber inner cavity can form a porous filtration support for cell adhesion. The in vivo cell growth mode is similar, so as to facilitate the adhesion and culture of the mesenchymal stem cells in the outer cavity. Moreover, the input end of the hollow fiber bioreactor is located at the bottom, so that the nutrient solution can be delivered from bottom to top, and the mesenchymal stem cells can easily accumulate with each other to form a layer with multiple layers of cells. The mesenchymal stem cells can secrete cytokines (such as IL-10 and TGF-β), growth factors (vascular endothelial growth factor and hepatocyte growth factor), exosomes and proteases, etc. The cytokines can diffuse through the semi-permeable membrane of the hollow fiber inner cavity into the perfusion solution, which has a good inhibitory effect on the inflammatory response of the liver and reduces the immune damage of the isolated liver. The growth factors penetrate into the perfusion solution and return to the isolated liver, which can activate the proliferation of the progenitor cells in the isolated liver and differentiate into functional hepatocyte-like cells, so as to accelerate the repair of the isolated liver. Meanwhile, the mesenchymal stem cells deliver miRNA and other substances through exosomes, which can regulate the oxidative stress and energy metabolism of the isolated liver, enhance the survival rate of hepatocytes, and then achieve the purpose of prolonging the preservation time of the isolated liver.

[0039] In addition, the bile duct connector of the preservation box 11 is connected with a bile collection bag 4. Since the isolated liver organ is continuously perfused with perfusion solution, the bile can be flushed out through the biliary system, so that the bile in the isolated liver organ can be removed, and the bile can be recycled for parameter index detection.

[0040] The first circulation pipeline 2 and the second circulation pipeline 3 are both provided with monitoring mechanisms, so as to monitor the circulation in real time. Specifically, the monitoring mechanisms include pressure monitoring, flow monitoring, bubble monitoring, temperature monitoring and pH monitoring, etc. The experimental personnel can master the changes of the biochemical indexes of the isolated liver in real time, so as to ensure the functional quality of the isolated liver.

[0041] The first circulation channel 2 and the second circulation channel 3 of this application achieve a synergistic effect mechanism. The circulation purification unit of the first circulation channel 2 directly simulates the detoxification and synthesis functions of the liver, directly removing toxins produced during the metabolism of the isolated liver. Simultaneously, hepatocytes, while synthesizing albumin, clotting factors, and other substances in the bio-purifier 24, participate in drug metabolism, reducing the metabolic burden on the isolated liver. The second circulation channel 3, through the mesenchymal stem cell bioreactor 32, secretes cytokines and growth factors to promote hepatocyte proliferation, inhibit inflammation, and enhance tissue repair capabilities. Furthermore, the dual-channel mechanism formed by the first circulation channel 2 and the second circulation channel 3 can simulate the blood supply characteristics of the hepatic artery and portal vein, optimizing the oxygenation and nutrient supply of the isolated liver, improving hepatocyte survival rate, and effectively extending the in vitro preservation time of the isolated liver.

[0042] It should also be noted that the first circulation pipeline 2 and the second circulation pipeline 3 of this application are respectively equipped with circulation pumps 31, so that the flow rates of the first circulation pipeline 2 and the second circulation pipeline 3 can be adjusted according to the actual perfusion requirements to avoid damage to the isolated liver organ during the perfusion process.

[0043] The perfusion solution of this application can be formulated according to different conditions of the isolated liver organ to ensure that the isolated liver organ can maintain a state close to its physiological state to ensure its normal function. For example, if the isolated liver organ is in a constant state of metabolic energy consumption, the oxygen content of the perfusion solution can be maintained at a level close to or higher than the physiological level to meet metabolic needs; the perfusion solution can also be designed to have a sufficient concentration of high-energy components to provide an energy source for the isolated liver organ; bile salts can also be added to the perfusion solution to assist the isolated liver organ in producing bile.

[0044] In summary, the bidirectional driven support system for preserving ex vivo livers using a liver and stem cell container of this application employs two circulation pipelines to provide energy to the ex vivo liver organ. The first circulation pipeline 2, equipped with a circulation purification unit, can simulate the metabolic detoxification and synthesis functions of the liver, while the second pipeline, equipped with a hollow fiber bioreactor 32 inoculated with mesenchymal stem cells, can effectively improve the functional state of the ex vivo liver, enhance tissue repair capabilities, and ensure the quality of the ex vivo liver, thereby reducing the occurrence of complications after ex vivo liver transplantation, alleviating the economic burden on patients, and providing an effective guarantee for the safety of patients' lives.

[0045] Furthermore, both the first circulation line 2 and the second circulation line 3 of this application can be used independently as an artificial liver system directly connected to the patient. The hollow fiber bioreactor on the second circulation line 3, inoculated with mesenchymal stem cells, is suitable for early-stage inflammatory conditions in liver disease. The cytokines and growth factors secreted by the paracrine function of mesenchymal stem cells can effectively inhibit liver inflammation, thereby reducing immune damage to the patient's liver. When the patient's liver disease progresses to the middle or late stages, the liver's ability to synthesize albumin is severely impaired, coagulation disorders exist, and metabolic and detoxification functions are impaired. In this case, the circulation purification unit on the first circulation line 2 can be used to detoxify the patient's blood, effectively removing accumulated toxins such as ammonia, bilirubin, and endotoxins, improving hepatic encephalopathy and systemic inflammatory responses, regulating electrolyte balance, alleviating acidosis and azotemia, and buying time for hepatocyte regeneration. Furthermore, the hepatocytes in the circulation purification unit can synthesize albumin, maintain plasma colloid osmotic pressure, supplement essential amino acids, and promote the recovery of residual hepatocyte function.

[0046] Therefore, this application also relates to a combined bioartificial liver system that drives and enhances hepatocyte function; please refer to [link to relevant documentation]. Figure 2 It includes a plasma separator 5 and a first circulation pipeline 2. The plasma separator 5 includes a blood inlet, a plasma outlet and a blood cell outlet. The first circulation pipeline 2 is connected between the plasma outlet and the blood cell outlet, and a mixer 7 is provided between the blood cell outlet and the output end of the first circulation pipeline 2. The blood inlet and the mixer 7 are respectively connected to the patient's artery and vein. The blood inlet of the plasma separator 5 is connected to the patient's artery through an arterial pipeline 6, and the mixer 7 is connected to the patient's vein through a venous pipeline 8.

[0047] The arterial line 6 is equipped with a delivery pump 61, which provides power for transporting the patient's blood from the patient's body to the plasma separator 5. The arterial line 6 also has a drug inlet 62, which, in possible embodiments, can be a syringe or other quantitative or timed device to inject medication into the arterial line 6. In this embodiment, the medication inlet 62 can be an anticoagulant to prevent blood clotting. Because the arterial line 6 has a drug inlet 62, there is no need to install a separate fluid replacement device on the first circulation line 2, simplifying the device structure.

[0048] The plasma separator 5 separates the patient's blood into plasma and blood cells through centrifugation. The plasma enters the first circulation line 2 from the plasma outlet, while the separated blood cells enter the mixer 7 from the blood cell outlet. This first circulation line 2 is the same as the one described above that includes a circulation purification unit. A peristaltic pump on the first circulation line 2 delivers the plasma to the biological purifier 24 at a target speed. The plasma comes into contact with hepatocytes cultured in the biological purifier 24, and through a semi-permeable membrane, the endogenous toxins such as ammonia and bilirubin in the plasma are metabolically removed. At the same time, the hepatocytes synthesize and secrete substances such as albumin and clotting factors, thereby replenishing the plasma with essential substances such as albumin and clotting factors.

[0049] Plasma flows through the perfusion channel 242 of the biological purifier 24. The perfusion channel 242 is set as a wave-shaped channel. The wave-shaped channel extends the length of the plasma flow path through the meandering path, prolongs the contact time with hepatocytes, improves the efficiency of toxin removal and the efficiency of metabolite synthesis. The wave-shaped channel can also reduce the mechanical damage to hepatocytes caused by high-speed flow, while maintaining sufficient shear force to promote the diffusion of substances between plasma and hepatocytes.

[0050] Meanwhile, a first oxygenator 23 is provided at the inlet 243 of the biological purifier 24. Before entering the biological purifier 24, the plasma flows through the first oxygenator 23 at a rate of 15 ml / min, which increases the oxygen partial pressure of the plasma, meets the high oxygen consumption of hepatocytes, effectively maintains the activity of hepatocytes, and enhances the activity of hepatocytes under high oxygen conditions, effectively improving the metabolic clearance rate of ammonia and bilirubin.

[0051] After purification by the biological purifier 24, the plasma enters the mixer 7 and mixes with the blood cells. The mixer 7 adopts a spiral turbulent flow design, which allows the plasma and blood cells to be uniformly mixed within 3-5 seconds. The mixed whole blood is then reinfused into the patient through the venous tubing 8, maintaining the hematocrit within the physiological range of 35-45%. The patient's blood undergoes extracorporeal circulation to remove harmful substances such as bilirubin, ammonia, and endotoxins from the plasma, significantly reducing blood ammonia levels and bilirubin concentrations. Furthermore, hepatocytes secrete cytokines that reduce the patient's systemic inflammatory response, while albumin and coagulation factors synthesized by hepatocytes effectively correct hypoalbuminemia and coagulation disorders, reducing the risk of bleeding.

[0052] Furthermore, in a further preferred embodiment, please refer to Figure 3The first circulation pipeline 2 can also be connected in series with a bilirubin adsorbent 25 and a resin hemoperfusion device 26. This allows the plasma separated from the plasma separator 5 to undergo preliminary detoxification via the bilirubin adsorbent 25 and resin hemoperfusion device 26. The bilirubin adsorbent 25 can further specifically adsorb bilirubin, while the resin hemoperfusion device 26, relying on van der Waals forces and molecular sieve action, can adsorb medium to large molecular toxins in the plasma, such as inflammatory mediators, TNF-α, and IL-6. The plasma then enters the biological purifier 24. After preliminary detoxification, the burden on the hepatocytes within the biological purifier 24 is reduced, allowing them to further perform their biosynthetic functions based on further detoxification.

[0053] In addition, this embodiment is equipped with monitoring mechanisms on both the first circulation line 2 and the arterial line 6. The monitoring mechanisms include pressure monitoring, flow monitoring, bubble monitoring, temperature monitoring and pH monitoring. Through real-time monitoring, dangerous situations such as line blockage, needle tip adhesion, coagulation and air entry can be detected and dealt with early, avoiding serious complications. It can also help to assess the stability of the patient's arteriovenous fistula function, provide a basis for the maintenance of vascular access, and is also the foundation for ensuring the efficacy of blood purification.

[0054] The first circulation tubing 2 of this application can be used not only for ex vivo liver preservation but also as an extracorporeal artificial liver directly connected to the patient. By culturing hepatocytes to simulate the liver's detoxification and metabolic synthesis functions, and by incorporating an elastic membrane in the bio-purifier 24 to simulate the mechanical stress of the diaphragm on hepatocytes during respiration, and by regulating the flow rate of perfusion fluid or blood through the bio-purifier 24, it simulates the in vivo hepatocyte culture environment, thereby increasing the lifespan of ex vivo hepatocytes and enhancing their detoxification efficiency and biosynthetic capacity.

[0055] The above description is only a partial embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container, characterized in that, It includes an organ preserver, a first circulation tubing, and a second circulation tubing, the first and second circulation tubing being independent of each other; The organ preserver is used to preserve an ex vivo liver; The first circulation pipeline is equipped with a power unit and a circulation purification unit. One end of the first circulation pipeline is connected to the inferior vena cava of the isolated liver placed in the organ preserver, and the other end is connected to the portal vein of the isolated liver. The second circulation pipeline is equipped with a circulation pump and a bioreactor inoculated with mesenchymal stem cells. One end of the second circulation pipeline is connected to the inferior vena cava of the isolated liver placed in the organ preserver, and the other end is connected to the hepatic artery of the isolated liver.

2. The support system for bidirectionally driven preservation of ex vivo liver using a liver and stem cell container as described in claim 1, characterized in that, The bioreactor is a hollow fiber bioreactor, which includes a hollow fiber inner lumen and an outer lumen sleeved outside the hollow fiber inner lumen, and mesenchymal stem cells are seeded in the outer lumen.

3. The bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container as described in claim 2, characterized in that, One end of the second circulation pipeline, which connects to the inferior vena cava of the isolated liver, is connected to the bottom of the hollow fiber bioreactor, and the top of the hollow fiber bioreactor is connected to the hepatic artery of the isolated liver via the second circulation pipeline.

4. The support system for bidirectionally driven preservation of ex vivo liver using a liver and stem cell container as described in claim 1, characterized in that, The circulating purification unit includes a biological purifier, which contains a hepatocyte carrier area and a perfusion fluid channel. The hepatocyte carrier area is filled with hepatocyte carriers loaded with hepatocytes, and the hepatocyte carrier area and the perfusion fluid channel are physically isolated by a hollow fiber membrane.

5. The bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container as described in claim 4, characterized in that, The biological purifier is equipped with a corrugated support tube for supporting the hollow fiber membrane, and the top of the biological purifier is provided with an inlet connected to the perfusion fluid channel, and the bottom of the biological purifier is provided with an outlet connected to the perfusion fluid channel.

6. The bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container as described in claim 4, characterized in that, The power unit includes a peristaltic pump located at the inlet of the biological purifier, which controls the infusion flow rate of the perfusion fluid to be 5-15 mL / min.

7. The bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container as described in claim 4, characterized in that, The inner wall of the bioreactor is provided with an elastic membrane covering the hepatocyte carrier region. The elastic membrane has a Young's modulus ranging from 0.1 to 10 MPa. The elastic membrane is connected to a pneumatic diaphragm pump for driving it to generate periodic air pressure fluctuations.

8. The support system for bidirectional preservation of ex vivo liver using a liver and stem cell container according to claim 1, characterized in that, The organ preserver includes a preservation chamber and a support platform for supporting the excised liver. The preservation chamber is equipped with inferior vena cava connectors, portal vein connectors, hepatic artery connectors, and common bile duct connectors that are respectively connected to the inferior vena cava, portal vein, hepatic artery, and common bile duct of the excised liver. The inferior vena cava connector has two interfaces for connecting to the first circulation pipeline and the second circulation pipeline, respectively.

9. The bidirectional drive support system for preserving ex vivo livers using a liver and stem cell container as described in claim 8, characterized in that, The common bile duct of the storage box is connected to a bile collection bag.

10. The support system for bidirectionally driven preservation of ex vivo liver using a liver and stem cell container according to claim 1, characterized in that, Both the first and second circulation pipelines are equipped with monitoring mechanisms, which include pressure monitoring, flow monitoring, bubble monitoring, temperature monitoring, and pH monitoring.

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