WO2016015371A1 - Bio-artificial liver system - Google Patents

Abstract

A bio-artificial liver system comprises three parts, namely, a plasma separation/blood transfusion cycle, a bioreactor cycle, and a cell renewing system. The three parts are in communication through a pipeline. The bioreactor cycle comprises a bio-artificial liver reactor set consisting of one or more bio-artificial liver reactors. Outer space of the bio-artificial liver reactors is filled with L-02 cells, so that the therapeutic effect and safety are better. The cell renewing system is used for renewing the L-02 cells. The therapeutic cells in the reactors can be renewed timely in the therapeutic process, continuous and stable therapeutic effect is ensured, interruption of the therapy to replace the bio-artificial liver reactors is not required, the therapeutic time is shortened, the whole system can be manually operated or full-automatically controlled by means of a PLC, frequent sampling and detection are eliminated for medical staff, real-time monitoring is achieved, and the system is highly efficient, safe and well practical and is applicable to industrial production.

Description

Bioartificial liver system Technical field

The invention relates to the field of bioartificial liver technology, in particular to a bioartificial liver system.

Background technique

China is the country with the most serious liver disease in the world. The number of new cases of liver failure is more than 1 million per year. About 500,000 patients with liver failure each year die due to lack of effective treatment. There is only one chance of cure, namely liver transplantation, but due to the difficulty of liver source, long waiting time, or no chance to get liver donor, most patients have no chance of survival. Therefore, there is an urgent need for an artificial liver system to maintain the life of the patient, waiting for a liver transplant opportunity, or a chance for the patient's own liver to recover.

However, because the traditional artificial liver (ie, physical artificial liver) is extremely expensive, most patients can not afford it, and the curative effect is very limited, which can not significantly reduce the mortality of liver failure, so the clinical application is limited. In recent years, the addition of active hepatocytes or hepatocyte-like cells to the artificial liver system to improve the efficacy has become a hot spot in the world. The artificial liver based on the hepatocyte filling reactor is called bio-artificial liver. , BAL), a large number of studies have proved that its efficacy is significantly better than physical artificial liver, can significantly reduce the mortality rate of liver failure, significantly prolong survival, for the liver failure of patients with acute liver failure and chronic liver failure patients waiting for the liver The transplant created the conditions.

The existing bioartificial liver system has the same structure, and the bioartificial liver reactor is the core. The reactor is mainly composed of a hollow fiber reactor, which is filled with a certain amount of hepatocytes or hepatocytes (such as human liver cells, Immortalized liver cells, liver cancer cell lines, pig liver cells, etc.). These technologies have two major problems that limit the possibility of clinical application: First, the above cell sources have major defects, such as the former two or the source of cells is difficult or unable to increase in large scale, so it can not be promoted and applied; Poor liver function and safety (cancer or transfection of animal viruses) limits clinical application. Second, the structure of the artificial liver system is unreasonable, because after using for a period of time, the cells in the reactor will gradually die due to the influence of toxins, and the efficiency of the bioreactor to purify the blood decreases with the extension of the working time, in order to maintain the curative effect, at intervals. A reactor with fresh cells needs to be replaced. Bioartificial liver reactors are costly and may require replacement of multiple bioartificial liver reactors in one treatment. The cost of treatment is high; if not replaced It is difficult to ensure the efficacy when the replacement is small; the system needs to be shut down during the replacement, which increases the waiting time of the patient, prolongs the treatment time, and completely replaces the risk of system contamination by manual operation.

Summary of the invention

The object of the present invention includes two aspects. On the one hand, it overcomes the defects of high cost, pollution and safety risks in the structure of the existing bioartificial liver system, and provides a safe and reliable bioartificial liver system to realize uninterrupted treatment; On the one hand, it is used in combination with a bioartificial liver reactor filled with L-02 cells to overcome the defects of low efficacy and poor safety of existing cell sources. L-02 cells have the advantages of good liver function, strong value-adding and safety. The bioartificial liver system of the invention achieves complete improvement in cell and structure, has better curative effect, makes the damaged liver more likely to regenerate, or provides more effective support for patients with liver failure until it can be found for transplantation. Donor organ.

In order to achieve the above object, the bioartificial liver system designed by the invention comprises three parts: a plasma separation/blood reinfusion cycle, a bioreactor cycle, and a cell renewal system, and the three parts are connected through a pipeline;

The plasma separation/blood recirculation cycle includes a serially connected blood input port, a blood pump, a plasma separation column, a blood mixer, and a blood return port; the plasma separation column is provided with a blood inlet, a plasma output port, and a blood cell outlet. The blood inlet is connected to the blood pump, the blood cell outlet is connected to the inlet of the blood mixer, and the blood mixer outlet is connected to the blood return port;

The bioreactor cycle includes a bioartificial liver reactor set sequentially connected to a plasma output port, an inlet of a cell filter; the outlet of the cell filter is also in communication with an inlet of the blood mixer;

The bioartificial liver reactor group comprises one or more biociprocal liver reactors connected in parallel; the bioartificial liver reactor is provided with a plasma inlet, a plasma outlet, a cell inlet and a waste liquid discharge port; and a plasma of the bioartificial liver reactor The inlet is connected to the plasma outlet of the plasma separation column through a shut-off valve, and the plasma outlet is connected to the inlet of the cell filter; the cell inlet of the bioartificial liver reactor is connected to the cell renewal system, and the waste liquid discharge port of the bioartificial liver reactor is connected. Can be connected to a waste storage tank;

The cell renewal system includes a cell eluent tank, a cell suspension tank, a constant temperature water bath, and an infusion pump. The cell eluent tank and the cell suspension tank are connected to the input end of the infusion pump through a three-way valve, and the output ends of the infusion pump are respectively passed through the control valve and the cells of each bioartificial liver reactor. The mouth is connected; the cell suspension tank is placed in a constant temperature water bath.

Preferably, the bioartificial liver system further includes a biochemical analyzer for detecting a biochemical indicator, and the first detection port and the second detection port are respectively disposed before the plasma inlet of the bioartificial liver reactor and after the plasma outlet. The first detection port and the second detection port are connected to the detection end of the biochemical analyzer.

Preferably, a heparin pump is further disposed at the blood inlet or the blood pump, and a pressure pump is disposed between the plasma outlet and the first detection port of the biochemical analyzer. The pressure pump is used to ensure the normal operating pressure of the bioartificial liver reactor.

Preferably, a warmer and/or a glucose pump is provided between the plasma outlet and the first detection port of the biochemical analyzer. They are used to ensure plasma temperature into the bioartificial liver reactor and to add sugar to the plasma.

Preferably, an oxygenator is further disposed between the plasma outlet and the first detection port of the biochemical analyzer.

Preferably, a booster pump is further provided at the outlet of the blood mixer, and an acceleration pump is further provided at the plasma outlet of the bioartificial liver reactor. The booster pump can recirculate blood by pressurization. If the booster pump is not provided, it is necessary to ensure that the outlet of the blood mixer and the blood return port have a certain height difference, so that the blood can be returned by gravity.

Preferably, the plasma inlet of the bioartificial liver reactor is provided with a temperature sensor and a pressure sensor, and a temperature sensor is further provided at the plasma outlet.

Preferably, a discharge valve is provided at the waste liquid discharge port of the bioartificial liver reactor.

Preferably, the exogenous hepatocytes in the bioartificial liver reactor are L-02 cells.

Preferably, the bioartificial liver system further comprises a programmable logic controller for controlling valves, pumps, and various electrically controllable instruments in the bioartificial liver system. Add programmable logic controllers for fully automated processing.

Preferably, the bioartificial liver reactor is a nonwoven fabric reactor, a bacterial cellulose microcarrier reactor, a bacterial cellulose reactor, a hollow fiber reactor, a microcarrier reactor, and the above is not intended to limit the invention.

None of the prior art bioartificial liver systems can be widely applied in clinical practice. The inventors of the present invention found that this is because human liver cells are extremely limited in source, and human liver cells have short survival periods and cannot be propagated in vitro, so they cannot be widely used. Application; and other various alternative cells have serious shortages It traps the application. The L-02 cells used in the invention have important characteristic proteins and enzymes of normal liver cells, good liver function, strong proliferation ability and good safety. These characteristics indicate that L-02 cells are a superior and unique source of bioartificial hepatocytes, superior to any of the cell sources currently under development at home and abroad. L-02 cells not only have good liver function, excellent immortalization in vitro culture and reproduction ability, can be easily mass-produced in culture, and have not been found to be carcinogenic by many animal experiments.

The invention has the advantages of improved structure: the cell regeneration system is used for timely replacement of hepatocytes for the bioartificial liver reactor running in the system, and the bioartificial liver reactor and the intermittent treatment are not needed, and the treatment time is shortened, and the whole system can be used. PLC (Programmable Logic Controller) performs full automatic control, eliminating the frequent sampling and testing work of medical staff, real-time monitoring, high efficiency and safety, and it is used with L-02 cell line with good liver function, which has good practicability. Suitable for industrial production.

DRAWINGS

Figure 1 is a schematic view showing the structure of a bioartificial liver system of the present invention.

2 is a schematic enlarged view of the bioartificial liver reactor group of FIG. 1.

In the figure, blood input port 1, blood pump 2, plasma separation column 3, blood inlet 3.1, plasma output port 3.2, blood cell outlet 3.3, bioartificial liver reactor group 4, bioartificial liver reactor 5, plasma inlet 5.1, plasma Outlet 5.2, cell inlet 5.3, waste vent 5.4, cell filter 6, blood mixer 7, booster pump 8, blood return port 9, warmer 10, glucose pump 11, aerator 12, cell wash The liquid removal tank 13, the cell suspension tank 14, the first detection port 15, the second detection port 16, the biochemical analyzer 17, the temperature sensor 18, the infusion pump 19, the pressure sensor 20, the three-way valve 21, the heparin pump 22 Acceleration pump 23, constant temperature water bath 24, pressure pump 25, shut-off valve A1, shut-off valve A2, shut-off valve An, control valve B1, control valve B2, control valve Bn, discharge valve C1, discharge valve C2, release Discharge valve Cn.

detailed description

The invention is further described in detail below with reference to the drawings and specific embodiments.

As shown in Figures 1 and 2, the bioartificial liver system designed by the present invention includes plasma separation/blood back The three parts of the circulation cycle, the bioreactor cycle, and the cell renewal system are connected through the pipeline;

The plasma separation/blood reinfusion cycle includes a serially connected blood input port 1, a heparin pump 22, a blood pump 2, a plasma separation column 3, a blood mixer 7, a booster pump 8 and a blood return port 9; on the plasma separation column 3 There is a blood inlet 3.1, a plasma outlet 3.2 and a blood cell outlet 3.3, a blood inlet 3.1 is connected to the blood pump 2, a blood cell outlet 3.3 is connected to the inlet of the blood mixer 7, and an outlet of the blood mixer 7 is connected to the blood return port 9. A booster pump 8 is also provided at the outlet of the blood mixer 7 to facilitate returning blood.

The bioreactor cycle includes a glucose pump 11 sequentially connected to the plasma outlet port 3.2, a pressurizing pump 25, a warmer 10, an aerator 12, an automatic biochemical analyzer 17, a bioartificial liver reactor set 4, and a cell filter 6. At the inlet, the outlet of the cell filter 6 is also in communication with the inlet of the blood mixer 7;

The bioartificial liver reactor set 4 includes one or more bio-initiated liver reactors 5 in parallel; the exogenous hepatocytes in the bioartificial liver reactor 5 are L-02 cells.

The bioartificial liver reactor 5 is provided with a plasma inlet 5.1, a plasma outlet 5.2, a cell inlet 5.3 and a waste liquid discharge port 5.4; the plasma inlets 5.1 of the two bioartificial liver reactors 5 pass through the shut-off valve A1, the shut-off valve A2, ... The shutoff valve An is connected to the plasma outlet 3.2 of the plasma separation column 3, and the plasma outlet 5.2 is connected to the inlet of the cell filter 6; after the plasma outlet of the bioartificial liver reactor group 4 is merged, an acceleration pump 23 is also provided.

The cell renewal system comprises a cell eluent tank 13, a cell suspension tank 14, a constant temperature water bath 24 and an infusion pump 19, and the cell suspension tank 14 is placed in a constant temperature water bath 24, and the cell eluent tank 13 and the cells are mixed. The suspension tank 14 is connected to the input end of the infusion pump 19 through a three-way valve 21, and the output end of the infusion pump 19 passes through the control valve B1, the control valve B2, ... the control valve Bn and the cells of each bioartificial liver reactor 5, respectively. The inlet 5.3 is connected, and the waste liquid discharge port 5.4 of the bioartificial liver reactor 5 is provided with a discharge valve C1, a discharge valve C2, a discharge valve Cn, and each discharge valve can be connected through a pipe to output liquid to waste liquid. Collect containers.

The bioartificial liver system further includes a biochemical analyzer 17 for detecting biochemical indicators. The first detection port 15 is provided before the plasma inlet 5.1 of the bioartificial liver reactor 5, and the second detection port 16 is provided after the plasma outlet 5.2. The first detection is performed. The mouth 15 and the second detecting port 16 are connected to the detecting end of the biochemical analyzer 17.

A warmer 10 is provided between the plasma outlet port 3.2 and the first detecting port 15 of the biochemical analyzer 17 The pump 25, the glucose pump 11, and the aerator 12 are provided.

The plasma inlet 5.1 of the bioartificial liver reactor 5 is provided with a temperature sensor 18 and a pressure sensor 20, and a temperature sensor 18 is also provided at the plasma outlet 5.2.

The bioartificial liver system further includes a programmable logic controller (not shown) for controlling valves, pumps, and various electrically controllable instruments in the bioartificial liver system. In this embodiment, the programmable logic controller and the shutoff valve are respectively , control valve, bleed valve, blood pump 2, booster pump 8, warmer 10, glucose pump 11, aerator 12, biochemical analyzer 17, temperature sensor 18, infusion pump 19, pressure sensor 20, three The valve 21 and the heparin pump 22 are electrically connected to control the above device.

The embodiment of the present invention facilitates the description of the working process of the bioartificial liver reactor group 4 by two parallel bio-artificial liver reactors 5. The number of bio-artificial liver reactors 5 can be adjusted according to actual needs.

When the above bioartificial liver system is used, L-02 cells cultured in a large-scale common culture method can be used to harvest active cells with an activity rate of 98% or more. Then, it is aseptically operated in a biological cabinet, and an appropriate amount of liver cells are perfused and retained in the bioartificial liver reactor 5 and the cell suspension tank 14, and the inlet and outlet are closed, and the bioartificial liver reactor 5 and the cell suspension tank 14 are closed. Store in a sterile bag and store at 0~4 °C until use. The bioartificial liver reactor 5 and the cell suspension tank 14 are taken out during the pre-treatment installation, aseptically operated and installed in the order shown, and the patient's arterial blood inlet and the venous return port are connected to prepare for treatment.

At the beginning of treatment, after the blood of the patient or the experimental animal is taken out of the body, the blood input port 1 enters the bioartificial liver system, and the whole blood is separated into plasma and blood cell components through the plasma separation column 3, wherein the plasma enters the bioartificial liver reactor group. The separated blood cell components and the detoxified plasma in the bioartificial liver reactor group are recombined at the blood mixer 7 and returned to the patient or the experimental animal through the blood return port 9. When the phase is running, the shutoff valve A1 is cut off. The valves A2, ... are closed, the control valve B1, the control valve B2, ... the control valve Bn is closed, and the infusion pump 19 is de-energized.

During the treatment, the biochemical analyzer 17 monitors the bilirubin, ammonia, and creatinine in the plasma of the bioartificial liver reactor group 4 by using the first detection port 15 and the second detection port 16 located at both ends of the bioartificial liver reactor group 4. Changes in biochemical indicators such as urea nitrogen, liver enzymes, and albumin. When the efficiency of the bioartificial liver reactor group 4 was found to decrease, it was judged that the L-02 cells as exogenous hepatocytes in the bioartificial liver reactor 5 were depleted of necrosis. First, the rate of the blood pump 2 and the booster pump 8 is lowered by the programmable logic controller to drop Low blood flow rate, controlling one or more shut-off valves to close for hepatocyte replacement in a portion of the bioartificial liver reactor 5, for example, shut-off valve A1 is closed, and the bioartificial liver reactor 5 connected thereto stops supplying plasma, and control The valve B1 and the bleed valve C1 are opened, the three-way valve 21 is first communicated only with the cell eluent reservoir 13, the pump 19 starts to work, and the cell eluate in the cell eluent tank 13 such as physiological saline is pumped into the living organism. The artificial liver reactor 5 performs internal flushing, so that the dead hepatocytes are detached from the waste liquid discharge port 5.4; then, the three-way valve 21 is disconnected from the cell eluent tank 13, and communicates with the cell suspension tank 14. The fresh cells in the cell suspension tank 14 are pumped into the bioartificial liver reactor 5 by the pump 19 to complete the replacement and replacement of the cells. After the completion, the drain valve C1 and the control valve B1 are closed, and the infusion pump 19 is powered off. The shut-off valve A1 is opened to restart the operation of the bio-artificial liver reactor 5, which has been detoxified.

Then, in the same order, the other bio-artificial liver reactor 5 shut-off valve, control valve, and bleed valve are used to complete the replacement and replacement of the cells in the other bio-artificial liver reactors 5, and then the rate of the blood pump 2 and the booster pump 8 is restored. normal. This process can be done manually by hand or by programmable logic controller or manual operation. The advantage is that there is no need to treat patients with intermittent liver disease, and there is no need to replace the bioartificial liver reactor, saving cost and time, especially if When the logic controller is controlled, the circulating plasma temperature and pressure change can be monitored in real time, the temperature is increased by the warmer 10, the glucose pump 11 is supplemented with glucose, the aerator 12 is aerated, the biochemical analyzer 17 is automatically sampled and detected, and the data alarm is transmitted. Achieve all the monitoring, sampling and detection, and automatic replacement of liver cell replacement in the bioartificial liver reactor, saving a lot of medical manpower.

Claims (10)

A bioartificial liver system characterized by comprising: a plasma separation/blood reinfusion cycle, a bioreactor cycle, and a cell renewal system; the three parts are connected through a pipeline; The plasma separation/blood reinfusion cycle includes a serially connected blood input port (1), a blood pump (2), a plasma separation column (3), a blood mixer (7), and a blood return port (9); The plasma separation column (3) is provided with a blood inlet (3.1), a plasma outlet (3.2) and a blood cell outlet (3.3), a blood inlet (3.1) connected to the blood pump (2), a blood cell outlet (3.3) and a blood mixer. The inlet of (7) is connected, and the outlet of the blood mixer (7) is connected to the blood return port (9); The bioreactor cycle comprises a bioartificial liver reactor set (4) sequentially connected to a plasma outlet (3.2), an inlet of a cell filter (6); the outlet of the cell filter (6) is also mixed with blood The inlet of the device (7) is connected; The bioartificial liver reactor group (4) comprises one or more bio-initiated liver reactors (5) connected in parallel; the bioartificial liver reactor (5) is provided with a plasma inlet (5.1), a plasma outlet (5.2), Cell inlet (5.3) and waste vent (5.4); plasma inlet (5.1) of bioartificial liver reactor (5) through the shut-off valves (A1, A2, ... An) and plasma separation column (3) plasma The outlet (3.2) is connected and the plasma outlet (5.2) is connected to the inlet of the cell filter (6); The cell renewal system comprises a cell eluent tank (13), a cell suspension tank (14), a constant temperature water bath (24), and an infusion pump (19), the cell eluent tank (13) and the cell mixture The suspension tank (14) is connected to the input end of the infusion pump (19) through a three-way valve (21), and the output ends of the infusion pump (19) are respectively passed through control valves (B1, B2, ... Bn) and each The cell inlet (5.3) of the bioartificial liver reactor (5) is connected; the cell suspension tank (14) is placed in a constant temperature water bath (24). The bioartificial liver system according to claim 1, further comprising a biochemical analyzer (17) for detecting biochemical indicators, said plasma inlet (5.1) of said bioartificial liver reactor (5) and plasma outlet ( 5.2) A first detecting port (15) and a second detecting port (16) are respectively disposed, and the first detecting port (15) and the second detecting port (16) are connected to the detecting end of the biochemical analyzer (17). The bioartificial liver system according to claim 1, characterized in that the blood input port (1) or the blood pump (2) is further provided with a heparin pump (22), the plasma output port (3.2) and biochemistry. A pressure pump (25) is disposed between the first detection ports (15) of the meter (17). The bioartificial liver system according to claim 1, characterized in that a warming device (10) and/or a temperature increasing device (10) is provided between the plasma outlet (3.2) and the first detecting port (15) of the biochemical analyzer (17). Glucose pump (11). The bioartificial liver system according to claim 1, characterized in that an oxygenator (12) is further disposed between the plasma outlet (3.2) and the first detection port (15) of the biochemical analyzer (17). The bioartificial liver system according to claim 1, characterized in that the outlet of the blood mixer (7) is further provided with a booster pump (8), the plasma outlet of the bioartificial liver reactor (5) ( An acceleration pump (23) is also available at 5.2). The bioartificial liver system according to claim 1, characterized in that the plasma inlet (5.1) of the bioartificial liver reactor (5) is provided with a temperature sensor (18) and a pressure sensor (20), and a plasma outlet ( There is also a temperature sensor (18) at 5.2). The bioartificial liver system according to any one of claims 1 to 4, characterized in that the waste liquid discharge port (5.4) of the bioartificial liver reactor (5) is provided with a discharge valve (C1, C2, ... Cn). ). The bioartificial liver system according to any one of claims 1 to 3, wherein the exogenous hepatocytes in the bioartificial liver reactor (5) are L-02 cells. The bioartificial liver system according to any one of claims 1-9, further comprising a programmable logic controller for controlling valves, pumps, and respective electrically controllable instruments in the bioartificial liver system.

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