WO2021184999A1 - 基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝 - Google Patents

基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝 Download PDF

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WO2021184999A1
WO2021184999A1 PCT/CN2021/075804 CN2021075804W WO2021184999A1 WO 2021184999 A1 WO2021184999 A1 WO 2021184999A1 CN 2021075804 W CN2021075804 W CN 2021075804W WO 2021184999 A1 WO2021184999 A1 WO 2021184999A1
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plasma
outlet
blood
pressure monitor
peristaltic pump
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PCT/CN2021/075804
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English (en)
French (fr)
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施晓雷
任昊桢
王经琳
丁义涛
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南京鼓楼医院
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Priority to US17/434,766 priority Critical patent/US11400199B2/en
Publication of WO2021184999A1 publication Critical patent/WO2021184999A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3687—Chemical treatment
    • A61M1/3689—Chemical treatment by biological cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A61M1/1698—Blood oxygenators with or without heat-exchangers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A61M1/1621—Constructional aspects thereof
    • A61M1/1629—Constructional aspects thereof with integral heat exchanger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/14—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis
    • A61M1/16—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes
    • A61M1/26—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes and internal elements which are moving
    • A61M1/267—Dialysis systems; Artificial kidneys; Blood oxygenators ; Reciprocating systems for treatment of body fluids, e.g. single needle systems for hemofiltration or pheresis with membranes and internal elements which are moving used for pumping
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/34—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration
    • A61M1/3472—Filtering material out of the blood by passing it through a membrane, i.e. hemofiltration or diafiltration with treatment of the filtrate
    • A61M1/3486—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents
    • A61M1/3489—Biological, chemical treatment, e.g. chemical precipitation; treatment by absorbents by biological cells, e.g. bioreactor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3621—Extra-corporeal blood circuits
    • A61M1/3639—Blood pressure control, pressure transducers specially adapted therefor
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/3672—Means preventing coagulation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61M—DEVICES FOR INTRODUCING MEDIA INTO, OR ONTO, THE BODY; DEVICES FOR TRANSDUCING BODY MEDIA OR FOR TAKING MEDIA FROM THE BODY; DEVICES FOR PRODUCING OR ENDING SLEEP OR STUPOR
    • A61M1/00—Suction or pumping devices for medical purposes; Devices for carrying-off, for treatment of, or for carrying-over, body-liquids; Drainage systems
    • A61M1/36—Other treatment of blood in a by-pass of the natural circulatory system, e.g. temperature adaptation, irradiation ; Extra-corporeal blood circuits
    • A61M1/369—Temperature treatment
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/04—Flat or tray type, drawers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/34—Internal compartments or partitions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/10—Perfusion
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/26—Conditioning fluids entering or exiting the reaction vessel

Definitions

  • the invention relates to the technical field of bioartificial livers, in particular to a bioartificial liver based on iPSCs induced hepatocytes and a multilayer porous bioreactor.
  • Bioartificial liver is a new type of liver support system that uses human or animal-derived liver cells to temporarily or partially replace the failed liver function in the body, and can assist in the treatment of liver failure or related liver diseases.
  • BAL not only has the function of liver detoxification, but also can achieve liver synthesis, metabolism, secretion and other functions through the liver cells in the reactor, which is expected to fundamentally replace the functions of the liver.
  • BAL research has made great progress, and it has shown a certain liver support effect.
  • Human hepatocellular carcinoma cell lines such as C3A cells were used in the bioartificial liver system (ELAD) in the United States, but they have the defect of lower metabolic function.
  • the technical problem to be solved by the present invention is to provide a bioartificial liver based on iPSCs induced hepatocytes and a multi-layer porous bioreactor to effectively reduce the risk of heterogeneous virus infection and the risk of immune response in view of the above-mentioned shortcomings of the prior art.
  • the technical solution adopted by the present invention is: a bioartificial liver based on human iPSCs induced hepatocytes and a multilayer porous bioreactor, including a plasma separation/reinfusion cycle part and a cell reactor/plasma component Exchange the double circulation part, the two parts are connected to each other through the circulation pipeline;
  • the plasma separation/reinfusion circulation part includes blood input pipe, blood input peristaltic pump, heparin pump, plasma separation column, first pressure monitor and heater, which are connected in sequence by connecting pipes, between blood input pipe and blood input peristaltic pump
  • the connecting pipeline is provided with an exhaust pipe spring clip
  • the plasma separation column is provided with a blood inlet, a plasma outlet and a blood cell outlet, and the blood inlet is connected with the outlet of the heparin pump;
  • the cell reactor/plasma component exchange dual-circulation part includes a plasma input peristaltic pump connected in sequence to the plasma outlet through a connecting pipe, a semipermeable membrane exchange column set in a special incubator at 37°C, a plasma exchange peristaltic pump, a red blood cell pool, and a membrane Lung, multi-layer porous bioreactor, second pressure monitor and third pressure monitor.
  • the semipermeable membrane exchange column is provided with a lower plasma inlet, a lower plasma outlet, an upper plasma inlet and an upper plasma outlet, and the lower plasma inlet Connected with the outlet of the plasma input peristaltic pump, the lower plasma outlet is connected with the inlet of the plasma exchange peristaltic pump, human iPSCs induced hepatocytes are perfused in the multi-layer porous bioreactor, and the second pressure monitor is connected to the upper plasma inlet , The upper outlet of the plasma is connected to the third pressure monitor, the outlet of the third pressure monitor and the blood cell outlet are connected to the inlet of the first pressure monitor together, and the outlet of the first pressure monitor is connected to the heater and the blood output tube in sequence.
  • the plasma separation column is a membrane type plasma separator.
  • the semi-permeable membrane exchange column is a membrane-type plasma component separator, and the semi-permeable membrane has a pore size of 11 nm.
  • the red blood cell pool is a silicone rubber bag containing 110 mL of red blood cells, the front and rear ends of the silicone rubber bag are respectively provided with inlets and outlets, and the inlets and outlets are connected with connecting pipelines.
  • the membrane lung is a membrane oxygenator.
  • the connecting pipeline is made of medical grade polyvinyl chloride, wherein the outer diameter of the connecting pipeline connected with the blood input peristaltic pump and the plasma input peristaltic pump is 13mm, and the outer diameter of the other connecting pipelines is 5.7 mm.
  • the multi-layer porous bioreactor includes a tank body, a tank cover, and a multi-layer porous plate device.
  • the tank cover is threadedly connected with the upper tank mouth of the tank body, and is further sealed by a rubber ring sleeved on the upper tank mouth.
  • the upper surface of the tank lid is provided with a cell input hole and a culture solution outlet pipe, and the bottom of the tank body is provided with a culture solution introduction pipe.
  • the hole is equipped with a sealing cover, the membrane lung is connected to the culture solution introduction pipe through the connecting pipe, the second pressure monitor is connected to the culture solution outlet pipe through the connecting pipe, and the multilayer porous plate device is slidably connected up and down.
  • the inner wall of the tank is equipped with a sealing cover, the membrane lung is connected to the culture solution introduction pipe through the connecting pipe, the second pressure monitor is connected to the culture solution outlet pipe through the connecting pipe, and the multilayer porous plate device is slidably connected up and down.
  • the multi-layer porous plate device is a porous plate with at least one layer
  • the porous plate includes a plate, a groove, and a bottom support.
  • the groove and the bottom support are respectively ringed on the upper surface and the lower surface of the plate and The position is directly opposite, the bottom support of the upper porous plate is inserted into the groove of the lower porous plate, and the upper and lower adjacent layers of porous plates are detachably connected.
  • the surface of the plate is evenly distributed with vertical hollow holes.
  • the hollow hole penetrates the flat plate longitudinally, the bottom bracket is a hollow cylinder, and the side wall of the bottom bracket is provided with a horizontal hollow hole.
  • the tank body has a diameter of 126mm, a height of 160mm, and a wall thickness of 4mm.
  • the multi-layer porous plate device includes 50-60 layers of porous plates.
  • the plate of the porous plate is made of polycarbonate with a diameter of 120mm and a thickness of 1mm.
  • the groove and the bottom support are respectively ringed inside and outside 2 circles on the upper surface and the bottom surface of the flat plate, and each circle is provided with 6 or 8 or 11 grooves and the bottom support, the groove and the bottom support Evenly distributed on the outer periphery of the center of the plate, the grooves are cylindrical grooves with a diameter of 11 mm and a depth of 0.8 mm, the bottom bracket is a circular bottom bracket with a diameter of 11 mm and a height of 2 mm, the vertical hollow hole and the horizontal The hollow holes are all round holes with a diameter of 2mm.
  • the present invention has the following beneficial effects:
  • the present invention controls the blood flow of the bioartificial liver at 37°C through a special incubator at 37°C, maintains the temperature required for the best state of blood and cells, and provides oxygen required by the cells through the red blood cell pool and membrane lung, thereby ensuring that the blood and cells are all
  • the required temperature and oxygen supply provide the efficiency of bioartificial liver
  • the present invention adopts human-derived cells (human iPSCs induced hepato-like cells) to reduce the risk of heterogeneous virus infection;
  • the present invention can effectively adjust the number of cells and increase the effective exchange area through the adjustment of the number of layers of the multi-layer porous bioreactor.
  • the horizontal hollow holes of the bottom support uniformly radiate the flowing liquid into contact with the cells, reducing the damage to the cells caused by the flow rate.
  • Improve the liquid exchange efficiency and fully carry out the material exchange the outer surface of the plate is coated with collagen and polycarbonate to reduce the shear force, promote the cell adhesion 3D aggregation growth, and improve the biocompatibility; through the liquid in the multi-layer porous bioreactor
  • the internal reverse flow from bottom to top promotes the efficiency of liquid exchange; at the same time, the tank has a large volume and a large amount of carrying cells, which can meet clinical needs;
  • the present invention performs material exchange through a semi-permeable membrane exchange column, which effectively reduces the risk of immune reaction.
  • Figure 1 is a schematic diagram of the overall structure of the bioartificial liver of the present invention.
  • Figure 2 is a schematic diagram of the structure of the multilayer porous bioreactor in Figure 1 :;
  • Fig. 3 is a side view of the porous plate of the multilayer porous plate device of Fig. 2;
  • Fig. 4 is a top view of the porous plate of Fig. 3.
  • the reference signs are: blood input tube 1, blood input peristaltic pump 2, heparin pump 3, plasma separation column 4, blood inlet 4-1, plasma outlet 4-2, blood cell outlet 4-3, first pressure monitor 5. Heater 6. Exhaust pipe spring clip 7. Plasma input peristaltic pump 8. 37°C dedicated incubator 9. Semi-permeable membrane exchange column 10, plasma lower inlet 10-1, plasma lower outlet 10-2, plasma upper Side inlet 10-3, upper plasma outlet 10-4, plasma exchange peristaltic pump 11, red blood cell pool 12, membrane lung 13, multilayer porous bioreactor 14, tank 14-1, tank cover 14-2, multilayer porous Plate device 14-3, plate 14-31, groove 14-32, bottom support 14-33, vertical hollow hole 14-34, horizontal hollow hole 14-35, growth factor collagen 14-36, cell input hole 14- 4.
  • a bioartificial liver based on human iPSCs induced hepatocytes and a multilayer porous bioreactor including a plasma separation/reinfusion cycle part and a cell reactor/plasma component exchange dual cycle part, two Parts are connected to each other through circulation pipelines, and the connecting pipelines are made of medical grade polyvinyl chloride;
  • the plasma separation/reinfusion circulation part includes blood input tube 1, blood input peristaltic pump 2, heparin pump 3, plasma separation column 4, first pressure monitor 5, heater 6, and blood input tube 1 which are sequentially connected by connecting pipes.
  • An exhaust pipe spring clamp 7 is provided on the connecting pipeline between the blood input peristaltic pump 2 and the plasma separation column 4 is a membrane type plasma separator (available from Asahi Kasei Kuraray Medical Co., Ltd.), and the plasma separation column 4 is provided with blood The inlet 4-1, the plasma outlet 4-2 and the blood cell outlet 4-3, the blood inlet 4-1 is connected to the outlet of the heparin pump 3;
  • the cell reactor/plasma component exchange dual-circulation part includes a plasma input peristaltic pump 8 sequentially connected to the plasma outlet 4-2 through a connecting pipe, a semipermeable membrane exchange column 10 set in a special incubator 9 at 37°C, and a plasma exchange peristaltic pump.
  • the outer diameter of the pipeline is 13mm, and the outer diameters of other connecting pipelines are all 5.7mm.
  • the semipermeable membrane exchange column 10 is a membrane type plasma component separator with a semipermeable membrane pore size of 11nm (available from Kawasumi Chemical Industry Co., Ltd. ), the semipermeable membrane exchange column 10 is provided with a lower plasma inlet 10-1, a lower plasma outlet 10-2, an upper plasma inlet 10-3, and an upper plasma outlet 10-4.
  • the lower plasma inlet 10-1 and the plasma input The outlet of the peristaltic pump 8 is connected, and the lower plasma outlet 10-2 is connected to the inlet of the plasma exchange peristaltic pump 11.
  • the red blood cell pool 12 is a silicone rubber bag containing 110 mL of red blood cells. The front and rear ends of the silicone rubber bag are respectively provided with inlets and outlets.
  • the outlet of the plasma exchange peristaltic pump 11 and the inlet of the red blood cell pool 12 are connected through a connecting pipe, the membrane lung 13 is a membrane oxygenator (available from Weigao Group Co., Ltd.), and the outlet of the red blood cell pool 12 is connected to the inlet of the membrane lung 13
  • the pipelines are connected.
  • the multilayer porous bioreactor 14 includes a tank body 14-1, a tank cover 14-2 and a multilayer porous plate device 14-3.
  • the size of the tank body 14-1 is 126mm in diameter, 160mm in height, and 4mm in wall thickness.
  • the lid 14-2 is threadedly connected with the upper can mouth of the can body 14-1, and is further sealed by a rubber ring sleeved on the upper can mouth.
  • the inner wall (similar to the connection relationship between the pressing tablet and the inner wall of the washing tub), the upper surface of the tank cover 14-2 is provided with a cell input hole 14-4 and a culture solution outlet pipe 14-5, and the bottom of the tank 14-1 is provided with culture Liquid introduction tube 14-6, human iPSCs induced hepatocytes are input into the tank 14-1 through the cell input hole 14-4, the cell input hole 14-4 is equipped with a sealing cover, and the membrane lung 13 is connected and cultured through the connecting pipe
  • the liquid introduction pipe 14-6, the second pressure monitor 15 is connected to the culture solution outlet pipe 14-5 through the connecting pipe, and the multilayer porous plate device 14-3 is slidably connected up and down on the inner wall of the tank 14-1 (similar to a compression tablet
  • one end of the second pressure monitor 15 is connected to the culture solution outlet pipe 14-5 through a connecting pipe
  • the other end of the second pressure monitor 15 is connected to the upper plasma inlet 10-3 through a connecting pipe.
  • the plasma upper outlet 10-4 is connected to the third pressure monitor 16.
  • the outlet of the third pressure monitor 16 and the blood cell outlet 4-3 are connected to the inlet of the first pressure monitor 5, and the outlets of the first pressure monitor 5 are in turn
  • the heater 6 and the blood output tube 17 are connected and finally communicated with the human body.
  • the multi-layer porous plate device 14-3 is a porous plate with at least one layer.
  • the porous plate includes a plate 14-31, a groove 14-32 and a bottom bracket 14-33, and a groove 14-32 and a bottom bracket 14- 33 are respectively ringed on the upper surface and lower surface of the plate 14-31 and the positions are directly opposite.
  • the bottom bracket 14-33 of the upper porous plate is inserted into the groove 14-32 of the lower porous plate.
  • the surface of the plate 14-31 is evenly distributed with vertical hollow holes 14-34.
  • the vertical hollow holes 14-34 penetrate the plate 14-31 longitudinally.
  • the bottom bracket 14-33 is a hollow cylinder, and the bottom bracket 14-
  • the side wall of 33 is provided with horizontal hollow holes 14-35.
  • the multi-layer porous plate device 14-3 is composed of 50-60 layers of porous plates.
  • the plate 14-31 of the porous plate is a polycarbonate circular plate with a diameter of 120 mm and a thickness of 1 mm.
  • the outer surface of the polycarbonate circular plate is covered. Covered by a layer of growth factor collagen 14-36.
  • the groove 14-32 and the bottom bracket 14-33 are respectively ringed with two inner and outer rings on the upper surface and the lower surface of the flat plate 14-31, and each ring has 6 or 8 or 11 grooves 14- 32 and bottom bracket 14-33, the grooves 14-32 and bottom bracket 14-33 are evenly distributed on the outer periphery of the center of the plate 14-31, the groove 14-32 is a cylindrical groove with a diameter of 11mm and a depth of 0.8mm, the bottom bracket 14-33 is a circular bottom bracket with a diameter of 11mm and a height of 2mm.
  • the vertical hollow holes 14-34 and the horizontal hollow holes 14-35 are both circular holes with a diameter of 2mm.
  • the patient's blood will be taken out of the body when used by the patient, and then enter the bioartificial liver system through the blood input tube 1 ,
  • the plasma separation column 4 separates the blood cells in the blood from the plasma, and the separated plasma enters the 37°C dedicated incubator 9 through the plasma input peristaltic pump 8 and communicates with the semipermeable membrane exchange column 10, and enters the plasma of the semipermeable membrane exchange column 10
  • the peristaltic pump 11 for plasma exchange is connected to the red blood cell pool 12 and the membrane lung 13 and enters the multi-layer porous bioreactor 14 from the culture medium introduction tube 14-6.
  • the multi-layer multi-well plate device 14-3 is preferably composed of 50-60 layers of multi-well plates , And the number of layers is adjustable. Each layer of liquid can flow radially around the plate 14-31 through the horizontal hollow holes 14-35 on the side wall of the bottom support 14-33. The liquid is fully exchanged.
  • the plate 14-31 adopts the type induced by human iPSCs
  • the round plate of polycarbonate with good biocompatibility of the liver, and the outer surface is coated with a layer of growth factor collagen 14-36, which is conducive to cell aggregation and adhesion, and 3D culture can fully function, so that toxic substances in plasma can be carried out.
  • the culture solution outlet pipe 14-5 of the multi-layer porous bioreactor 14 is connected to the second pressure monitor 15 to connect the circulating liquid to the upper plasma inlet 10-3
  • the semipermeable membrane exchange column 10, the plasma upper outlet 10-4 of the semipermeable membrane exchange column 10 is connected to the third pressure monitor 16 and then enters and exits the 37°C dedicated incubator 9, and the blood cell outlet 4-3 of the plasma separation column 4 outputs After the blood cells are confluent, they are connected to the first pressure monitor 5 and the heater 6 and communicate with the human body through the blood output tube 17.
  • the bioartificial liver based on human iPSCs induced hepatocytes and multi-layer porous bioreactors of the present invention uses human-derived cells to avoid infection of heterologous viruses, significantly improve biocompatibility, promote 3D cell culture, and give full play to similar in vivo culture Function (After culture, albumin secretion is maintained at 70 ⁇ g/116 cells/day, ammonia clearance rate is maintained at 25 ⁇ mol/116 cells/day, urea synthesis rate is 10 ⁇ g/116 cells/day, glucose metabolism rate is 120 ⁇ mol/116 cells /Day); radiate the flowing liquid uniformly into contact with the cells, reduce the damage of the flow rate to the cells, improve the liquid exchange efficiency, and fully carry out the material exchange; the volume is large and the carrying cell volume is large, which meets the clinical needs (the carrying cell volume reaches 1111); Material exchange through the semi-permeable membrane of the semi-permeable membrane exchange column 10 can effectively reduce the immune response (the semi-permeable membrane has a pore size of 11 nm
  • the levels of IgG and IgM immune antibodies have not increased or decreased significantly. , Showing good virus and immune safety); 37°C dedicated incubator 9, red blood cell pool 12 and membrane lung 13 ensure the temperature and oxygen supply required by blood and cells, and provide bioartificial liver performance.

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Abstract

基于人iPSCs诱导类肝细胞及多层多孔生物反应器（14）的生物人工肝，血浆分离/回输循环部分包括顺序相连的血液输入管（1）、排气管弹簧夹（7）、血液输入蠕动泵（2）、肝素泵（3）、血浆分离柱（4）、第一压力监测仪（5）和加热器（6），细胞反应器/血浆成分交换双循环部分包括与血浆出口（4-2）顺序相连的血浆输入蠕动泵（8）及37℃专用培养箱（9）内的半透膜交换柱（10）、血浆置换蠕动泵（11）、红细胞池（12）、膜肺（13）、多层多孔生物反应器（14）、第二压力检测仪（15）、第三压力监测仪（16），第三压力监测仪（16）的出口与血细胞出口（4-3）一并接入第一压力监测仪（5）的入口，再依次连接加热器（6）和血液输出管（17）。通过两循环部分配合可维持细胞所需的温度和供氧，同时提高液体交换效率和生物相容性，满足临床需求，同时可降低异种病毒感染风险及免疫反应风险。

Description

基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝 技术领域
本发明涉及生物人工肝技术领域，尤其涉及一种基于iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝。
背景技术
生物人工肝(bioartificial liver，BAL)是利用人源性或者动物源性肝脏细胞暂时或者部分替代体内的衰竭的肝脏功能，可以协助治疗肝衰竭或者相关肝脏疾病的一种新型肝脏支持系统。BAL不仅具有肝脏解毒功能，而且可通过反应器内肝细胞达到肝脏合成、代谢、分泌等功能，有望在根本上代替肝脏的功能。近年来BAL研究得到较大进展，并显示了其一定的肝脏支持作用。人类的肝癌细胞系如C3A细胞曾在美国的生物人工肝系统(ELAD)中使用，却有代谢功能较低的缺陷，而实验证明C3A的来源细胞HepG2实际并没有对ALF小鼠的治疗效果；猪来源的原代肝细胞也曾被用在国外的AMC-BAL以及HepatAssist系统；这些细胞来源在免疫源性上被人诟病(在临床应用后在体内发现猪抗性的IgG)，并具有动物来源病毒感染的风险。但是，要想完全达到体外肝脏支持替代作用，满足临床治疗需要，还有诸多问题需要解决。因此，建立高效、安全的BAL支持系统，以及功能肝细胞的获得，是临床目前亟需解决的难题。
发明内容
本发明所要解决的技术问题是针对上述现有技术的不足，提供一种基于iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝，可有效降低异种病毒感染风险和免疫反应风险。
为实现上述技术目的，本发明采取的技术方案为：一种基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝，包括血浆分离/回输循环部分和细胞反应器/血浆成分交换双循环部分，两部分通过循环管路互相连接；
血浆分离/回输循环部分包括通过连接管路顺序相连的血液输入管、血液输入蠕动泵、肝素泵、血浆分离柱、第一压力监测仪和加热器，血液输入管与血液输入蠕动泵之间连接管路上设有排气管弹簧夹，血浆分离柱上设有血液入口、血浆出口和血细胞出口，血液入口与肝素泵的出口相连通；
细胞反应器/血浆成分交换双循环部分包括通过连接管路与血浆出口顺序相连的血浆输入蠕动泵及设置于37℃专用培养箱内的半透膜交换柱、血浆置换蠕动泵、红细胞池、膜肺、多层多孔生物反应器、第二压力监测仪和第三压力监测仪，半透膜交换柱上设有血浆下入口、血 浆下侧出口、血浆上侧入口和血浆上出口，血浆下入口与血浆输入蠕动泵的出口相连通，血浆下侧出口与血浆置换蠕动泵的入口相连通，人iPSCs诱导类肝细胞灌注在多层多孔生物反应器中，第二压力监测仪连通血浆上侧入口，血浆上出口连通第三压力监测仪，第三压力监测仪的出口与血细胞出口一并接入第一压力监测仪的入口，第一压力监测仪的出口依次连接加热器和血液输出管。
进一步地，所述血浆分离柱为膜型血浆分离器。
进一步地，所述半透膜交换柱为膜型血浆成分分离器，其半透膜孔径为11nm。
进一步地，所述红细胞池为装有110mL红细胞的硅橡胶袋，所述硅橡胶袋的前后端分别设有进出口，所述进出口与连接管路相连接。
进一步地，所述膜肺为膜式氧合器。
进一步地，所述连接管路采用医用级聚氯乙烯制成，其中，与血液输入蠕动泵和血浆输入蠕动泵相连的连接管路外径为13mm，其他处连接管路的外径均为5.7mm。
进一步地，所述多层多孔生物反应器包括罐体、罐盖和多层多孔平板装置，所述罐盖与罐体的上罐口螺纹连接，并通过上罐口套设的橡胶圈进一步密封，所述罐盖的上表面设置有细胞输入孔和培养液导出管，所述罐体的底部设置有培养液导入管，人iPSCs诱导类肝细胞通过细胞输入孔输入罐体内，所述细胞输入孔上配设有密封盖，所述膜肺通过连接管路连通培养液导入管，所述第二压力监测仪通过连接管路连通培养液导出管，所述多层多孔平板装置上下滑动连接在罐体的内壁。
进一步地，所述多层多孔平板装置为至少一层的多孔平板，所述多孔平板包括平板、凹槽和底托，所述凹槽和底托分别环设于平板的上表面和下表面且位置正对，通过上多孔平板的底托插嵌在下多孔平板的凹槽中，上下相邻层多孔平板之间可拆卸连接，所述平板的表面均布有竖直镂空孔，所述竖直镂空孔纵向贯穿平板，所述底托为空心圆柱体，底托的侧壁设有水平镂空孔。
进一步地，所述罐体尺寸为直径126mm，高160mm，壁厚4mm，所述多层多孔平板装置包括50～60层多孔平板，所述多孔平板的平板采用直径120mm、厚1mm的聚碳酸酯圆形平板，所述聚碳酸酯圆形平板的外表面包被一层生长因子胶原。
进一步地，所述凹槽和底托在平板的上表面和下表面上下对应地分别环设内外2圈，每圈设6个或8个或11个凹槽和底托，凹槽和底托在平板中心的外周均匀分布，所述凹槽为直径11mm、深0.8mm的圆柱形凹槽，所述底托为直径11mm、高2mm的圆环形底托，所述竖直 镂空孔和水平镂空孔均为直径2mm的圆孔。
与现有技术相比，本发明的有益效果在于：
1)本发明通过37℃专用培养箱控制生物人工肝血液流动全程37℃，保持了血液、细胞最佳状态所需温度，通过红细胞池、膜肺提供细胞所需氧气，从而保证血液、细胞所需的温度和供氧，提供生物人工肝效能；
2)本发明采用人源性细胞(人iPSCs诱导类肝细胞)可降低异种病毒感染风险；
3)本发明通过多层多孔生物反应器的层数调整可有效调节细胞数量、增加有效交换面积，通过底托的水平镂空孔将流动液体辐射式均匀与细胞接触，降低流速对细胞的损伤，提高液体交换效率，充分进行物质交换；通过平板外表面生长因子胶原被覆聚碳酸酯包被降低剪切力、促进细胞黏附3D聚集生长、提高生物相容性；通过液体在多层多孔生物反应器内由下至上逆向流动促进液体交换效率；同时罐体容积大、承载细胞量多，可满足临床需求；
4)本发明通过半透膜交换柱进行物质交换，有效降低免疫反应风险。
附图说明
图1为本发明的生物人工肝整体结构示意图；
图2为图1的多层多孔生物反应器结构示意图：；
图3为图2多层多孔平板装置的多孔平板侧视图；
图4为图3的多孔平板俯视图。
其中的附图标记为：血液输入管1、血液输入蠕动泵2、肝素泵3、血浆分离柱4、血液入口4-1、血浆出口4-2、血细胞出口4-3、第一压力监测仪5、加热器6、排气管弹簧夹7、血浆输入蠕动泵8、37℃专用培养箱9、半透膜交换柱10、血浆下入口10-1、血浆下侧出口10-2、血浆上侧入口10-3、血浆上出口10-4、血浆置换蠕动泵11、红细胞池12、膜肺13、多层多孔生物反应器14、罐体14-1、罐盖14-2、多层多孔平板装置14-3、平板14-31、凹槽14-32、底托14-33、竖直镂空孔14-34、水平镂空孔14-35、生长因子胶原14-36、细胞输入孔14-4、培养液导出管14-5、培养液导入管14-6、第二压力监测仪15、第三压力监测仪16、血液输出管17。
具体实施方式
为了使本领域技术领域人员更好地理解本发明的技术方案，下面结合附图对本发明的实施例作进一步详细描述。
如图1～4所示，一种基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝， 包括血浆分离/回输循环部分和细胞反应器/血浆成分交换双循环部分，两部分通过循环管路互相连接，连接管路采用医用级聚氯乙烯制成；
血浆分离/回输循环部分包括通过连接管路顺序相连的血液输入管1、血液输入蠕动泵2、肝素泵3、血浆分离柱4、第一压力监测仪5和加热器6，血液输入管1与血液输入蠕动泵2之间连接管路上设有排气管弹簧夹7，血浆分离柱4为膜型血浆分离器(可购自旭化成可乐丽医疗株式会社)，血浆分离柱4上设有血液入口4-1、血浆出口4-2和血细胞出口4-3，血液入口4-1与肝素泵3的出口相连通；
细胞反应器/血浆成分交换双循环部分包括通过连接管路与血浆出口4-2顺序相连的血浆输入蠕动泵8及设置于37℃专用培养箱9内的半透膜交换柱10、血浆置换蠕动泵11、红细胞池12、膜肺13、多层多孔生物反应器14、第二压力监测仪15和第三压力监测仪16，其中，与血液输入蠕动泵2和血浆输入蠕动泵8相连的连接管路外径为13mm，其他处连接管路的外径均为5.7mm，半透膜交换柱10为膜型血浆成分分离器，其半透膜孔径为11nm(可购自川澄化学工业株式会社)，半透膜交换柱10上设有血浆下入口10-1、血浆下侧出口10-2、血浆上侧入口10-3和血浆上出口10-4，血浆下入口10-1与血浆输入蠕动泵8的出口相连通，血浆下侧出口10-2与血浆置换蠕动泵11的入口相连通，红细胞池12为装有110mL红细胞的硅橡胶袋，硅橡胶袋的前后端分别设有进出口，血浆置换蠕动泵11出口与红细胞池12进口通过连接管路相连通，膜肺13为膜式氧合器(可购自威高集团有限公司)，红细胞池12出口与膜肺13入口通过连接管路相连通，多层多孔生物反应器14包括罐体14-1、罐盖14-2和多层多孔平板装置14-3，罐体14-1尺寸为直径126mm，高160mm，壁厚4mm，罐盖14-2与罐体14-1的上罐口螺纹连接，并通过上罐口套设的橡胶圈进一步密封，多层多孔平板装置14-3上下滑动连接在罐体14-1的内壁(类似压衣片和洗衣桶内壁的连接关系)，罐盖14-2的上表面设置有细胞输入孔14-4和培养液导出管14-5，罐体14-1的底部设置有培养液导入管14-6，人iPSCs诱导类肝细胞通过细胞输入孔14-4输入罐体14-1内，细胞输入孔14-4上配设有密封盖，膜肺13通过连接管路连通培养液导入管14-6，第二压力监测仪15通过连接管路连通培养液导出管14-5，多层多孔平板装置14-3上下滑动连接在罐体14-1的内壁(类似压衣片和洗衣桶内壁的连接关系)，第二压力监测仪15一端通过连接管路连接培养液导出管14-5，第二压力监测仪15另一端通过连通管路连接血浆上侧入口10-3，血浆上出口10-4连通第三压力监测仪16，第三压力监测仪16的出口与血细胞出口4-3一并接入第一压力监测仪5的入口，第一压力监测仪5的出口依次连接加热器6和血液输出管17并最终与人体连通。
实施例中，多层多孔平板装置14-3为至少一层的多孔平板，多孔平板包括平板14-31、凹槽14-32和底托14-33，凹槽14-32和底托14-33分别环设于平板14-31的上表面和下表面且位置正对，通过上多孔平板的底托14-33插嵌在下多孔平板的凹槽14-32中，上下相邻层多孔平板之间可拆卸连接，平板14-31的表面均布有竖直镂空孔14-34，竖直镂空孔14-34纵向贯穿平板14-31，底托14-33为空心圆柱体，底托14-33的侧壁设有水平镂空孔14-35。
进一步地，多层多孔平板装置14-3由50～60层多孔平板组成，多孔平板的平板14-31采用直径120mm、厚1mm的聚碳酸酯圆形平板，聚碳酸酯圆形平板的外表面包被一层生长因子胶原14-36。
进一步地，凹槽14-32和底托14-33在平板14-31的上表面和下表面上下对应地分别环设内外2圈，每圈设6个或8个或11个凹槽14-32和底托14-33，凹槽14-32和底托14-33在平板14-31中心的外周均匀分布，凹槽14-32为直径11mm、深0.8mm的圆柱形凹槽，底托14-33为直径11mm、高2mm的圆环形底托，竖直镂空孔14-34和水平镂空孔14-35均为直径2mm的圆孔。
使用本发明的基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝时，首先通过多层多孔生物反应器14的细胞输入孔14-4将人iPSCs诱导类肝细胞通过无菌操作缓慢输注罐体14-1内，然后打开37℃专用培养箱9培养4小时，待细胞稳定粘附，病人使用时，将患者血液引出体外后，通过血液输入管1进入生物人工肝系统，血浆分离柱4将血液中的血细胞与血浆分离，分离后的血浆经过血浆输入蠕动泵8进入37℃专用培养箱9内与半透膜交换柱10连通，进入半透膜交换柱10的血浆通过血浆置换蠕动泵11与红细胞池12、膜肺13相连接并由培养液导入管14-6进入多层多孔生物反应器14，多层多孔平板装置14-3优选50～60层多孔平板组成，且层数可调，每层液体可以通过底托14-33侧壁的水平镂空孔14-35呈放射状流入平板14-31周围，液体充分交换，平板14-31采用与人iPSCs诱导的类肝细生物相容性较好的聚碳酸酯圆形平板，且外表面包被一层生长因子胶原14-36，有利于细胞聚集成团黏附，3D培养充分发挥功能，从而可进行血浆中有毒物质与多层多孔生物反应器14中有效成分物质交换，多层多孔生物反应器14的培养液导出管14-5与第二压力监测仪15连接将循环液体通过血浆上侧入口10-3连接至半透膜交换柱10，半透膜交换柱10的血浆上出口10-4与第三压力监测仪16连接后出入37℃专用培养箱9，与血浆分离柱4的血细胞出口4-3输出的血细胞汇合后连接第一压力监测仪5、加热器6后经过血液输出管17与人体相连通。
本发明的基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝采用人源性细 胞，避免异种病毒感染，显著提高生物相容性，促进细胞3D培养，充分发挥类似在体培养功能(培养后白蛋白分泌保持在70μg/116个细胞/天，氨清除率保持在25μmol/116个细胞/天，尿素合成率为10μg/116个细胞/天，葡萄糖代谢率为120μmol/116细胞/天)；将流动液体辐射式均匀与细胞接触，降低流速对细胞的损伤，提高液体交换效率，充分进行物质交换；容积大并且承载细胞量多，满足临床需求(承载细胞量达到1111)；通过半透膜交换柱10的半透膜进行物质交换，有效降低免疫反应(半透膜孔径为11nm，未检测出HBV、HIV等病毒，IgG，IgM免疫抗体水平未出现明显的升高或下降，显示了良好的病毒和免疫安全性)；37℃专用培养箱9、红细胞池12和膜肺13保证血液、细胞所需的温度和氧供，提供生物人工肝效能。
以上仅是本发明的优选实施方式，本发明的保护范围并不仅局限于上述实施例，凡属于本发明思路下的技术方案均属于本发明的保护范围。应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理前提下的若干改进和润饰，应视为本发明的保护范围。

Claims (10)

  1. 一种基于人iPSCs诱导类肝细胞及多层多孔生物反应器的生物人工肝，其特征在于，包括血浆分离/回输循环部分和细胞反应器/血浆成分交换双循环部分，两部分通过循环管路互相连接；
    所述血浆分离/回输循环部分包括通过连接管路顺序相连的血液输入管(1)、血液输入蠕动泵(2)、肝素泵(3)、血浆分离柱(4)、第一压力监测仪(5)和加热器(6)，所述血液输入管(1)与血液输入蠕动泵(2)之间连接管路上设有排气管弹簧夹(7)，所述血浆分离柱(4)上设有血液入口(4-1)、血浆出口(4-2)和血细胞出口(4-3)，所述血液入口(4-1)与肝素泵(3)的出口相连通；
    所述细胞反应器/血浆成分交换双循环部分包括通过连接管路与血浆出口(4-2)顺序相连的血浆输入蠕动泵(8)及设置于37℃专用培养箱(9)内的半透膜交换柱(10)、血浆置换蠕动泵(11)、红细胞池(12)、膜肺(13)、多层多孔生物反应器(14)、第二压力监测仪(15)和第三压力监测仪(16)，所述半透膜交换柱(10)上设有血浆下入口(10-1)、血浆下侧出口(10-2)、血浆上侧入口(10-3)和血浆上出口(10-4)，所述血浆下入口(10-1)与血浆输入蠕动泵(8)的出口相连通，所述血浆下侧出口(10-2)与血浆置换蠕动泵(11)的入口相连通，人iPSCs诱导类肝细胞灌注在多层多孔生物反应器(14)中，所述第二压力监测仪(15)连通血浆上侧入口(10-3)，所述血浆上出口(10-4)连通第三压力监测仪(16)，所述第三压力监测仪(16)的出口与血细胞出口(4-3)一并接入第一压力监测仪(5)的入口，所述第一压力监测仪(5)的出口依次连接加热器(6)和血液输出管(17)。
  2. 根据权利要求1所述的生物人工肝，其特征在于：所述血浆分离柱(4)为膜型血浆分离器。
  3. 根据权利要求2所述的生物人工肝，其特征在于：所述半透膜交换柱(10)为膜型血浆成分分离器，其半透膜孔径为11nm。
  4. 根据权利要求3所述的生物人工肝，其特征在于：所述红细胞池(12)为装有110mL红细胞的硅橡胶袋，所述硅橡胶袋的前后端分别设有进出口，所述进出口与连接管路相连接。
  5. 根据权利要求4所述的生物人工肝，其特征在于：所述膜肺(13)为膜式氧合器。
  6. 根据权利要求1所述的生物人工肝，其特征在于：所述连接管路采用医用级聚氯乙烯制成，其中，与血液输入蠕动泵(2)和血浆输入蠕动泵(8)相连的连接管路外径为13mm，其他处连接管路的外径均为5.7mm。
  7. 根据权利要求6所述的生物人工肝，其特征在于：所述多层多孔生物反应器(14)包括罐体(14-1)、罐盖(14-2)和多层多孔平板装置(14-3)，所述罐盖(14-2)与罐体(14-1)的 上罐口螺纹连接，并通过上罐口套设的橡胶圈进一步密封，所述罐盖(14-2)的上表面设置有细胞输入孔(14-4)和培养液导出管(14-5)，所述罐体(14-1)的底部设置有培养液导入管(14-6)，人iPSCs诱导类肝细胞通过细胞输入孔(14-4)输入罐体(14-1)内，所述细胞输入孔(14-4)上配设有密封盖，所述膜肺(13)通过连接管路连通培养液导入管(14-6)，所述第二压力监测仪(15)通过连接管路连通培养液导出管(14-5)，所述多层多孔平板装置(14-3)上下滑动连接在罐体(14-1)的内壁。
  8. 根据权利要求7所述的生物人工肝，其特征在于：所述多层多孔平板装置(14-3)为至少一层的多孔平板，所述多孔平板包括平板(14-31)、凹槽(14-32)和底托(14-33)，所述凹槽(14-32)和底托(14-33)分别环设于平板(14-31)的上表面和下表面且位置正对，通过上多孔平板的底托(14-33)插嵌在下多孔平板的凹槽(14-32)中，上下相邻层多孔平板之间可拆卸连接，所述平板(14-31)的表面均布有竖直镂空孔(14-34)，所述竖直镂空孔(14-34)纵向贯穿平板(14-31)，所述底托(14-33)为空心圆柱体，底托(14-33)的侧壁设有水平镂空孔(14-35)。
  9. 根据权利要求8所述的生物人工肝，其特征在于：所述罐体(14-1)尺寸为直径126mm，高160mm，壁厚4mm，所述多层多孔平板装置(14-3)包括50～60层多孔平板，所述多孔平板的平板(14-31)采用直径120mm、厚1mm的聚碳酸酯圆形平板，所述聚碳酸酯圆形平板的外表面包被一层生长因子胶原(14-36)。
  10. 根据权利要求9所述的生物人工肝，其特征在于：所述凹槽(14-32)和底托(14-33)在平板(14-31)的上表面和下表面上下对应地分别环设内外2圈，每圈设6个或8个或11个凹槽(14-32)和底托(14-33)，凹槽(14-32)和底托(14-33)在平板(14-31)中心的外周均匀分布，所述凹槽(14-32)为直径11mm、深0.8mm的圆柱形凹槽，所述底托(14-33)为直径11mm、高2mm的圆环形底托，所述竖直镂空孔(14-34)和水平镂空孔(14-35)均为直径2mm的圆孔。
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