Artificial liver, artificial liver culture system, and method for manufacturing an artificial liver

Abstract

The present invention provides an artificial liver, an artificial liver culture system, and a method for manufacturing an artificial liver, which improve the engraftment of liver organoids, enable suitable culture of the cell groups constituting hepatic lobules, and have functions closer to those of a living liver after manufacturing. [Solution] The artificial liver 10 comprises a first layer 110, 210 made of porous ceramic, through which a first channel for perfusion of culture medium or blood flow is formed along one direction, and the first channel is filled with liver organoids or liver spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells.

Description

Technical Field

[0001] The present invention relates to an artificial liver, an artificial liver culture system, and a method for manufacturing an artificial liver.

Background Art

[0002] In recent years, as a treatment method for organs and tissues that have fallen into dysfunction or defect, the development of hybrid artificial organs (also referred to as artificial organs) that combine cultured cells and biocompatible materials and regenerative medicine technologies has attracted attention.

[0003] For example, the fundamental treatment method for liver diseases such as liver failure is liver transplantation, but the shortage of organ providers (donors) is a major problem, and the development of artificial livers is required. However, it is difficult to replace complex and diverse liver functions only by artificial means, and recently, artificial livers that utilize hepatocytes themselves have attracted attention.

[0004] An artificial liver performs treatment by causing a biological side circuit that draws and circulates blood from a liver failure patient and an artificial liver module side circuit that circulates plasma and performs metabolism and detoxification on the artificial liver module side to exchange substances through a plasma separator. Such an artificial organ module is insufficient to use dispersed cells. That is, in the monolayer culture method that has been conventionally used for cell culture, the loss and decline of cell functions cannot be avoided, and it is important to construct and use a tissue body similar to a living tissue.

[0005] From such a viewpoint, recently, culture methods for organoid-like tissue bodies (organoids) such as cell spheroids and cylindrical tissue bodies (cylindroids) have been newly established, and high-function expression of cells under culture and long-term function maintenance are becoming achievable (for example, Patent Document 1).

[0006] Among tissues and organs where three-dimensional structure is important, the liver in particular has zoning (functional multilayer structure) as the foundation supporting its complex and diverse functions. In particular, a research group led by Professor Takanori Takebe of the Graduate School of Medicine, Osaka University, has recently succeeded in creating organoids with zoning present in living livers from human pluripotent stem cells (i-PS cells) for the first time in the world (Non-Patent Literature 1). The liver is the command center of overall metabolism in the body, responsible for the metabolism of nutrients and drugs and the processing of waste products. The liver is a special organ with spatial specificity called zoning, in which hepatocytes, arranged from the portal vein to the central vein, perform different functions depending on their position. Thanks to the existence of this specificity, sometimes contradictory effects (for example, promotion or inhibition of hepatocyte proliferation by TGF-β) occur in each zone within the liver, allowing the liver to exert its metabolic functions in a complex and complementary manner as a whole. In other words, the construction of normal zoning in the liver is an indispensable element for maintaining homeostasis throughout the body.

[0007] Furthermore, in the liver after partial hepatectomy, compensatory proliferation of residual hepatocytes is synchronously induced to compensate for the quantitative loss of the organ, and this proliferation stops when the liver weight recovers to its original weight. Various cytokines and cell growth factors that induce or stop hepatocyte proliferation have been identified as being involved in this process, but it has been found that hemodynamics flowing within the hepatic sinusoids, specifically changes in shear stress on endothelial cells, are deeply involved in the generation of these liver regeneration and its cessation signals.

[0008] Furthermore, the complex and diverse functions of the liver are largely due to the unique workings of hepatic sinusoidal endothelial cells, which are in close contact with the blood through their cribriform plate structure. Hepatocytes directly take in oxygen and plasma components through the pores of the cribriform plate of hepatic sinusoidal endothelial cells, while simultaneously secreting metabolic products and waste products.

[0009] On the other hand, unlike other vascular endothelial cells, hepatic sinusoidal endothelial cells lack a basement membrane. A space called the space of Disse exists between hepatic sinusoidal endothelial cells and hepatocytes, and the space of Disse mediates the exchange of substances such as nutrients, oxygen, and hormones between the sinusoids and hepatocytes. The space of Disse plays an important role in the uptake of nutrients, oxygen, and hormones by hepatocytes. It also plays an important role in the uptake of proteins supplied from the sinusoids into the space of Disse by hepatocytes, and it also regulates the flow rate of blood by releasing substances produced by hepatocytes into the space of Disse. Thus, the space of Disse is an important structure that mediates the exchange of substances between hepatocytes and sinusoids in the liver, and its formation is a very important factor in the overall organ structure of the liver. [Prior art documents] [Patent Documents]

[0010] [Patent Document 1] Japanese Patent Publication No. 2004-24069 [Non-patent literature]

[0011] [Non-Patent Document 1] Hasan Al Reza,et al.,Multi-zonal liver organoids from human pluripotent stem cells,Nature,volume 641,pages 1258-1267(2025) [Overview of the project] [Problems that the invention aims to solve]

[0012] Currently, organoid culture, a three-dimensional culture technology, remains confined to drug discovery research. The reason for this is the inability to scale up the culture to tissues and organs. The biggest bottleneck is the construction of vascular structures, and there have been challenges in cell engineering and tissue engineering in that it cannot be applied to subjects such as the liver, where the function and structure of tissues and organs are fine and complex. Specifically, it has been difficult to directly reconstruct the complex structure of hepatic lobules, which are composed of cell groups such as hepatic sinusoidal endothelial cells, hepatocytes, hepatic stellate cells, and Kupffer cells, in an artificial liver.

[0013] Thus, there has been a need to improve the engraftment of liver organoids and to cultivate the cell populations that make up liver lobules in a suitable manner.

[0014] The present invention has been made in view of these circumstances, and aims to provide an artificial liver, an artificial liver culture system, and a method for manufacturing an artificial liver that can improve the engraftment of liver organoids, allow for the suitable culture of cell groups constituting hepatic lobules, and have functions closer to those of a living liver after production. [Means for solving the problem]

[0015] To solve the above problems, the present invention employs the following means. The present invention provides an artificial liver comprising a first layer made of porous ceramic, through which a first channel for perfusation of culture medium or blood flow is formed along one direction, wherein the first channel is filled with liver organoids or liver spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells.

[0016] The artificial liver of the present invention comprises a first layer made of porous ceramic, through which a first channel for perfusion of culture medium or blood flow is formed along one direction. The first channel is filled with liver organoids or liver spheroids (hereinafter also simply referred to as "liver organoids") whose surfaces are coated with hepatic sinusoidal endothelial cells. Thus, in the present invention, the surface of the liver organoids or liver spheroids is coated with hepatic sinusoidal endothelial cells. Even if the three-dimensional positional relationship between cell groups such as hepatocytes and hepatic stellate cells separated by hepatic sinusoidal endothelial cells and the blood flow containing red blood cells is changed, the overall liver function is not impaired. Therefore, the surface of the liver organoids is coated with hepatic sinusoidal endothelial cells in order to enhance and promote intercellular interactions with hepatic sinusoidal endothelial cells, which are responsible for the proliferation, growth, and suppression control of hepatic parenchymal cells and other cells. This makes it easier to control changes in hemodynamics applied to hepatic sinusoidal endothelial cells, specifically the so-called "mechanical homeostasis" such as shear stress caused by blood flow applied to endothelial cells. This enables the optimal supply of oxygen, cell growth factors, and cytokines to the target cell population to be proliferated and grown, thereby improving the engraftment of liver organoids. This allows for the optimal culture of the cell populations that make up the liver lobules. Furthermore, the manufactured artificial liver can mimic and realize the detoxification function of harmful substances and the metabolic regulation function of nutrients that are inherent in a living liver with a high degree of reproducibility, and has functions that are closer to those of a living organ.

[0017] Furthermore, the first layer is made of porous ceramic and has a narrow first channel formed in the pores of the ceramic. The first channel is configured to be filled with coated liver organoids by flowing a culture medium containing coated liver organoids through it. In this case, since the cross-sectional area of ​​the first channel is small, it is expected that the frequency and contact area of ​​the coated liver organoids with the wall of the first channel will increase. This allows the coated liver organoids to adhere well to the first channel. Moreover, since the first layer is made of highly biocompatible ceramic, it is ultimately possible to transplant the artificial liver into the living body.

[0018] The artificial liver further comprises a second layer laminated on the first layer, with a second channel formed along the same direction through which perfluorocarbons flow or bile is discharged, and a third layer made of porous ceramic laminated on the second layer, with a third channel formed along the same direction through which culture medium or blood flow is perfused, wherein the third channel is filled with bile duct organoids or bile duct spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells. ru.

[0019] The artificial liver of the present invention comprises a second layer laminated on a first layer, with a second channel formed in one direction through which perfluorocarbons flow or bile is discharged. Furthermore, the artificial liver of the present invention comprises a third layer made of porous ceramic, laminated on the second layer, with a third channel formed in one direction through which culture medium or blood flow is perfused. The third channel is filled with bile duct organoids or bile duct spheroids (hereinafter also simply referred to as "bile duct organoids") whose surfaces are coated with hepatic sinusoidal endothelial cells. In other words, the artificial liver of the present invention can constitute a bile duct-like structure with bile discharge channels and bile duct organoids. Therefore, since the artificial liver of the present invention includes hepatic parenchymal cells, hepatic stellate cells, and all other morphological features of hepatic lobules, including bile duct-like structures and bile channels, it can be configured as an artificial liver having functions equivalent to those of a living liver.

[0020] Furthermore, the third layer is made of porous ceramic and has a narrow third channel formed in the pores of the ceramic. The third channel is configured so that, for example, a culture medium containing coated bile duct organoids is passed through the third channel, filling it with coated bile duct organoids. In this case, because the cross-sectional area of ​​the third channel is small, it is expected that the frequency and contact area of ​​the coated bile duct organoids with the wall of the third channel will increase. This allows the coated bile duct organoids to adhere well to the third channel. In addition, since the third layer is made of highly biocompatible ceramic, it is ultimately possible to transplant the artificial liver into the living body.

[0021] In the above artificial liver, a three-layer structure composed of a first layer, a second layer, and a third layer may be repeatedly laminated a plurality of times in this order.

[0022] In the present invention, a three-layer structure composed of a first layer, a second layer, and a third layer is repeatedly laminated a plurality of times in this order. Therefore, the artificial liver of the present invention has a three-dimensional structure including elements configured in layers. Thereby, the artificial liver of the present invention can improve the reproducibility of the structurally and functionally complex living liver.

[0023] The above artificial liver In , The first, second, and third layers are composed of a plurality of members divided in the stacking direction, and the plurality of members are in the direction opposite to the one direction They can slide against each other configuration It was done may also be. Alternatively, the artificial liver may comprise a plurality of units formed by attaching a pair of end caps to both ends of the laminate of the first, second, and third layers, and the plurality of units may be connected via the end caps.

[0024] In the present invention, the artificial liver has a structure that can be extended in the direction opposite to the one direction. Therefore, when subculture of the liver organoid becomes necessary, by extending the layer structure of the artificial liver in the direction opposite to the one direction, an additional area for subculture of the liver organoid can be secured. Thereby, automatic subculture can be enabled. Therefore, it is possible to reduce the pipetting operation, the labor of reseeding into a new container, and the risk of contamination during long-term subculture by hand. Also, both cost and time can be suppressed. Further, by configuring the artificial liver to be extendable in multiple stages, only the area necessary as appropriate during subculture can be extended, enabling long-term automatic subculture.

[0025] For example, the first layer, the second layer, and the third layer are each configured by combining a plurality of members divided in the stacking direction, and these plurality of members are configured to be slidable with respect to each other in the direction opposite to the one direction. Thereby, the first layer, the second layer, and the third layer can have a structure that can be extended in the direction opposite to the one direction. Alternatively, an artificial liver unit can be formed by attaching a pair of end caps to both ends of a laminate consisting of the first, second, and third layers, and then automatically subculturing by placing multiple units adjacent to each other in a clean bench and connecting the end caps of each unit. By connecting the first channel via the end caps, it becomes easy to add more units, and as a result, it becomes unnecessary to enzymatically dismantle tissue organoids one by one, thus significantly reducing cumbersome operations such as pipetting and the risk of contamination.

[0026] Furthermore, the present invention provides an artificial liver culture system comprising the artificial liver described above, a metabolite measurement unit for measuring metabolites produced from the artificial liver, and a culture medium flow rate control unit for controlling the flow rate of the culture medium perfused to the artificial liver, wherein the culture medium flow rate control unit provides feedback control of the flow rate of the culture medium based on the measurement results measured by the metabolite measurement unit.

[0027] In the artificial liver culture system of the present invention, the culture medium flow rate control unit provides feedback control of the culture medium flow rate based on the measurement results obtained by the metabolite measurement unit. This allows the environment within the artificial liver to be automatically adjusted to conditions suitable for culturing liver organoids. Therefore, liver organoids and the like can be cultured effectively. Specifically, metabolites that act as flow rate control factors include various growth factors (HGF, EGF, etc.), cytokines (TGF-β, etc.), and NO synthase.

[0028] Furthermore, the flow rate of the culture medium may be adjusted at the discretion of the culturist, regardless of the measurement results from the metabolite measurement unit.

[0029] The artificial liver culture system described above may include a culture medium flow rate measuring unit that measures the flow rate of the culture medium perfusing the artificial liver, and the culture medium flow rate control unit may control the flow rate of the culture medium based on the flow rate measurement value obtained by the culture medium flow rate measuring unit.

[0030] In this invention, the culture medium flow rate control unit controls the flow rate of the culture medium based on the flow rate measurement value obtained by the culture medium flow rate measuring unit. This makes it possible to control the flow rate of the culture medium perfusing the artificial liver based on the actual flow rate of the culture medium perfusing the artificial liver. Therefore, the flow rate of the culture medium perfusing the artificial liver can be controlled with high precision.

[0031] The artificial liver culture system described above includes an oxygen supply control unit that controls the amount of oxygen supplied to the artificial liver, and the oxygen supply control unit may control the amount of oxygen supplied based on an algorithm that has been set in advance in accordance with the artificial liver.

[0032] In this invention, the oxygen supply control unit controls the oxygen supply based on a preset algorithm corresponding to the artificial liver. This optimizes the timing and amount of oxygen supply to the artificial liver, and automatically adjusts the oxygen concentration inside the artificial liver to a level suitable for culturing liver organoids. Therefore, liver organoids can be cultured effectively.

[0033] Furthermore, the present invention includes a first layer formation step of forming a first layer made of porous ceramic in which a first channel through which a culture medium or blood flow perfuses is formed along one direction, and a first layer filling step of filling the first layer with liver organoids or liver spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells. A second layer formation step is to form a second layer which is laminated on the first layer and in which a second channel through which perfluorocarbons flow or bile is discharged is formed along the one direction; a third layer formation step is to form a third layer made of porous ceramic which is laminated on the second layer and in which a third channel through which culture medium or blood flow is perfused is formed along the one direction; and a third layer filling step is to fill the third layer with bile duct organoids or bile duct spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells. The present invention provides a method for manufacturing an artificial liver having [a specific characteristic]. [Effects of the Invention]

[0034] The artificial liver and its manufacturing method of the present invention enable the optimal supply of oxygen, cell growth factors, and cytokines to the target cell population to be proliferated and grown, thereby improving the engraftment of liver organoids. This allows for the favorable culture of the cell population constituting the hepatic lobules. Furthermore, the manufactured artificial liver can mimic and realize the detoxification function of harmful substances and the metabolic regulation function of nutrients inherent in a living liver with a high degree of reproducibility, and thus possesses functions closer to those of a living organ. [Brief explanation of the drawing]

[0035] [Figure 1] This is a diagram showing the configuration of an artificial liver culture system according to one embodiment of the present invention. [Figure 2] This is a front view of a laminate constituting an artificial liver according to one embodiment of the present invention. [Figure 3] This is a longitudinal cross-sectional view of Figure 2. [Figure 4] This is a partial longitudinal cross-sectional view of part III in Figure 3. [Figure 5] This is a partial longitudinal cross-sectional view of section IV in Figure 3. [Figure 6] This is a photograph showing the cribriform plate structure of hepatic sinusoidal endothelial cells. [Figure 7] This is a schematic diagram comparing the sizes of hepatocytes, HSECs, pores, and red blood cells. [Figure 8] This is a longitudinal cross-section of an artificial liver. [Figure 9] This is a circuit diagram of an artificial liver culture system according to one embodiment of the present invention. [Figure 10] This is a cross-sectional view of the end cap when it is cut along line AA in Figure 2. [Figure 11] This is a cross-sectional view of the end cap when cut along the BB line in Figure 2. [Figure 12] This is a longitudinal cross-section of the end cap. [Figure 13] This is a partial longitudinal cross-sectional view showing the connection portion of the end cap to the second layer of the laminate. [Figure 14] This is a schematic diagram showing the internal structure of the first layer in a laminate. [Modes for carrying out the invention]

[0036] An embodiment of the artificial liver, artificial liver culture system, and method for producing the artificial liver according to the present invention will be described below with reference to the drawings.

[0037] [Artificial liver culture system] Figure 1 shows the configuration of the artificial liver culture system 1 according to this embodiment. The artificial liver culture system 1 of this embodiment comprises an artificial liver 10, an oxygen column 20, a liver circulation line 30, an oxygen line 31, a biliary circulation line 32, and a control unit 50. The artificial liver culture system 1 is a system for culturing the artificial liver 10. In manufacturing the artificial liver 10, for example, normal cells are extracted from the liver of a liver disease patient outside the artificial liver culture system 1, and organoids or spheroids are formed based on these normal cells. Alternatively, organoids or spheroids are formed based on i-PS cells outside the artificial liver culture system 1. The formed organoids or spheroids are engrafted onto the artificial liver 10 and cultured, and the artificial liver 10 is determined to be complete when the number of organoids or spheroids has grown to a sufficient amount to ensure that the artificial liver 10 can guarantee the function of a normal living liver after connection to a living organism. The artificial liver culture system 1 is a system that can semi-automatically determine the timing of completion based on measurements of changes in blood flow dynamics, etc. The completed artificial liver 10 is removed from the artificial liver culture system 1 and transplanted into the living body.

[0038] The artificial liver 10 is connected to a hepatic circulation line 30, an oxygen line 31, and a biliary circulation line 32. The hepatic circulation line 30 is fluidly connected to the first channel 114 of the artificial liver 10 (see Figures 3-5). The oxygen line 31 is connected to the second channel 124 of the artificial liver 10 (see Figures 2 and 3). The biliary circulation line 32 is connected to the third channel 134 of the artificial liver 10 (see Figures 3 and 5). Details of the structure of the artificial liver 10 will be described later.

[0039] The oxygen column 20 is a gas exchange column and is equipped with a flow channel consisting of a bundle of numerous gas-permeable hollow fiber tubes, such as silicon hollow fiber tubes. The oxygen column 20 is configured to selectively supply oxygen, nitric oxide, or both from an external source. Perfluorocarbons (PFCs) flow through the flow channel of the oxygen column 20 during the culture of the artificial liver 10 and when it is transplanted into a living organism.

[0040] The liver circulation line 30 is connected to the first channel inlet 11 and the first channel outlet 12 of the artificial liver 10. The liver circulation line 30 circulates culture medium (perfusion medium) or living blood flow from the first channel inlet 11 towards the first channel outlet 12. During the culture of the artificial liver 10, culture medium flows through the liver circulation line 30. After the artificial liver 10 is connected to the living body, living blood flow flows through the liver circulation line 30. The flow rate of culture medium or living blood flowing through the liver circulation line 30 is controlled by the opening of a valve (not shown in Figure 1) and the operation of a pump.

[0041] The oxygen line 31 is connected to the second channel's first opening 13 and the second channel's second opening 14 of the artificial liver 10. During the culture of the artificial liver 10, perfluorocarbon solution flows through the oxygen line 31 from the second channel's first opening 13 towards the second channel's second opening 14. After the artificial liver 10 is connected to the living body, the second channel's second opening 14 is closed, and bile is secreted from the second channel 124 towards the second channel's first opening 13 (i.e., in the opposite direction to the flow of the perfluorocarbon solution). The flow rate of the perfluorocarbon solution flowing through the oxygen line 31 is controlled by the operation of a pump (not shown in Figure 1).

[0042] The biliary circulation line 32 is connected to the third channel inlet 15 and the third channel outlet 16 of the artificial liver 10. The biliary circulation line 32 circulates culture medium (perfusion medium) or living blood flow from the third channel inlet 15 towards the third channel outlet 16. Culture medium flows through the biliary circulation line 32 while the artificial liver 10 is being cultured. After the artificial liver 10 is connected to the living body, living blood flow flows through the biliary circulation line 32. The flow rate of culture medium or living blood flowing through the biliary circulation line 32 is controlled by the opening of a valve (not shown in Figure 1) and the operation of a pump.

[0043] The control unit 50 manages the operation of the artificial liver culture system 1. The control unit 50 includes a metabolite measuring unit 51, a culture medium flow rate control unit 52, a culture medium flow rate measuring unit 53, and an oxygen supply amount control unit 54. The control unit 50 is composed of, for example, a CPU (Central Processing Unit), RAM (Random Access Memory), ROM (Read Only Memory), and a computer-readable storage medium. A series of processes for realizing various functions are stored in the storage medium in the form of a program, for example. The CPU reads this program into the RAM and performs information processing and calculations to realize the various functions. The program may be pre-installed in the ROM or other storage medium, provided in a state where it is stored in a computer-readable storage medium, or distributed via wired or wireless communication. Computer-readable storage media include magnetic disks, magneto-optical disks, CD-ROMs, DVD-ROMs, semiconductor memory, etc.

[0044] The metabolite measurement unit 51 measures metabolites produced from the artificial liver 10. Specifically, a metabolite measuring device (not shown) is connected near the outlet 12 of the first flow path in the liver circulation line 30. Metabolites are measured in the culture medium flowing through the connection point. Specifically, metabolites include various growth factors (HGF, EGF, etc.), cytokines (TGF-β, etc.), NO synthase, etc., which are factors that cause flow control.

[0045] The culture medium flow rate control unit 52 controls the flow rate of the culture medium perfusing the artificial liver 10. Specifically, the culture medium flow rate control unit 52 controls the flow rate of the culture medium circulating in the liver circulation line 30 by controlling the opening of a valve (not shown in Figure 1) and the operation of a pump. In particular, the culture medium flow rate control unit 52 provides feedback control of the culture medium flow rate based on the measurement results measured by the metabolite measurement unit 51. In this way, the environment inside the artificial liver is automatically adjusted to conditions suitable for culturing liver organoids. Note that the culture medium flow rate may be adjusted at the discretion of the culturer, regardless of the measurement result data from the metabolite measurement unit 51.

[0046] The culture medium flow rate measuring unit 53 measures the flow rate of the culture medium that perfuses the artificial liver 10. Specifically, the culture medium flow rate measuring unit 53 measures the flow rate of the culture medium flowing through any point in the liver circulation line 30. The culture medium flow rate control unit 52 controls the flow rate of the culture medium to be perfused to the artificial liver 10 based on the flow rate measurement value measured by the culture medium flow rate measuring unit 53.

[0047] Furthermore, the metabolite measurement unit 51, the culture medium flow rate control unit 52, and the culture medium flow rate measurement unit 53 may also perform the same control on the culture medium flowing through the biliary circulation line 32 as they do on the culture medium flowing through the hepatic circulation line 30.

[0048] The oxygen supply control unit 54 controls the amount of oxygen supplied to the artificial liver 10. The oxygen supply control unit 54 controls the amount of oxygen supplied based on an algorithm pre-set for the artificial liver 10. Specifically, the oxygen supply control unit 54 controls the pressure of gaseous oxygen supplied to the oxygen column 20 to control the amount of oxygen supplied to the perfluorocarbon solution flowing through the oxygen line 31. The above algorithm predicts the oxygen consumption according to the growth status of the HSEC liver organoids 61 (see Figures 3 to 5) and HSEC bile duct organoids 71 ​​(see Figures 3 and 5) cultured in the artificial liver 10 and calculates the amount of oxygen to be supplied.

[0049] [Artificial liver] Next, the laminate 100 constituting the artificial liver 10 according to this embodiment will be described using Figures 2 to 8. Figure 2 is a front view of the laminate 100 constituting the artificial liver 10 according to this embodiment. Figure 3 is a longitudinal cross-sectional view of Figure 2. Figure 4 is a partial longitudinal cross-sectional view of part III of Figure 3. Figure 5 is a partial longitudinal cross-sectional view of part IV of Figure 3.

[0050] As shown in Figure 2, the laminate 100 constituting the artificial liver 10 comprises, from the radially inner side outwards, the first layer (1-1 layer 110), the second layer (1-2 layer 120), the third layer (1-3 layer 130), and the first layer (2-1 layer 210), in that order. Although not shown in Figure 2, the radially outer side of the 2-1 layer 210 is also provided with the second layer (2-2 layer 220) and the third layer (2-3 layer 230), in that order. The first layer (1-1 layer 110) is cylindrical, and the other layers are cylindrical. In this embodiment, the structure is made by repeating the first to third layers in this order twice, but it may be made by repeating it only once, or by repeating it three or more times.

[0051] The first layer 110 is made of porous ceramic, and when the artificial liver 10 is viewed from the front, it has numerous small holes 111 that penetrate in one direction. As shown in Figure 2, the cross-sectional shape of each small hole 111 is square, but the shape is not particularly limited. Each small hole 111 is separated by a lattice-shaped wall portion 112. The outer surface of the wall portion 112 is surrounded by a cylindrical peripheral wall 113.

[0052] As shown in Figures 3-5, a first channel 114 is formed along the inside of the small pore 111, through which culture medium or blood flow perfuses. Specifically, during the culture of the artificial liver 10, culture medium perfuses the first channel 114. When the artificial liver 10 is connected to (transplanted) a living organism, blood flow from the portal vein system or the hepatic artery system perfuses the first channel 114. Note that the peripheral wall 113 is omitted from the illustration in Figures 3-5.

[0053] The first channel 114 is filled with liver organoids 61 whose surface is coated with hepatic sinusoidal endothelial cells (HSEC). Specifically, the HSEC liver organoids 61 are attached to the wall 112 of the first channel 114. The HSEC liver organoids 61 are tissue in which the cell group constituting the liver organoid is surrounded (coated) by hepatic sinusoidal endothelial cells 62. The cell group constituting the liver organoid is hepatocytes 63, hepatic stellate cells 64, and Kupffer cells 65. Liver spheroids may be used instead of liver organoids.

[0054] Figure 6 is a photograph showing the cribriform plate structure of hepatic sinusoidal endothelial cells. As shown in Figure 6, hepatic sinusoidal endothelial cells have a cribriform plate structure in which multiple pores are formed.

[0055] Figure 7 is a schematic diagram comparing the sizes of hepatocytes, HSECs (hepatic sinusoidal endothelial cells), pores, and red blood cells. Although the size of hepatocytes varies considerably, it is approximately 20 μm. The size of hepatic sinusoidal endothelial cells is 10-15 μm wide and 25-50 μm long. The diameter of the pores in hepatic sinusoidal endothelial cells is 0.097-0.132 μm on average. The size of red blood cells is 7-8 μm. Although liver organoids contain multiple hepatic stellate cells and Kupffer cells in addition to hepatocytes, the size of liver organoids is estimated to be 150-200 μm. When liver organoids are coated with hepatic sinusoidal endothelial cell organoids from the outside, the overall size of the HSEC liver organoid becomes approximately 200-230 μm.

[0056] Coating of liver organoids with hepatic sinusoidal endothelial cells 62 is performed by co-culturing liver organoids that have been pre-embedded in Matrigel with hepatic sinusoidal endothelial cells 62. Alternatively, the liver organoids embedded in Matrigel may be attached to the wall 112 of the first channel 114, and then the culture medium containing hepatic sinusoidal endothelial cells 62 may be perfused into the first channel 114.

[0057] Matrigel is a gel composed of components such as laminin, collagen IV nitrate, and heparan sulfate proteoglycan, and also contains growth factors. Matrigel is an ECM protein-containing preparation developed by Corning International Ltd. The inclusion of heparan sulfate proteoglycan is thought to be related to the fact that heparan sulfate proteoglycan is a ligand for EGF and FGF. The presence of heparan sulfate proteoglycan and its intercellular junctions enable the liver to perform its complex and diverse functions.

[0058] The first-second layer 120 is stacked radially outside the first-first layer 110. The first-second layer 120 has a structure sandwiched between oxygen-permeable membranes 121, and a cavity for the second channel 124 is formed inside along one direction. Perfluorocarbon flows through the second channel 124 or bile is discharged through it. Specifically, during the culture of the artificial liver 10, oxygen-containing perfluorocarbon is perfused through the second channel 124. When the artificial liver 10 is connected to (transplanted) a living organism, the second channel 124 is connected to the extrahepatic bile duct. At that time, bile flows in the opposite direction to that shown in Figure 1 (towards the gallbladder). Perfluorocarbon flows through the second channel 124 or bile is discharged only through the dashed circle area that overlaps with the first-second layer 120 shown in Figure 2, and there is no inflow or outflow from other parts. For example, a polydimethylsiloxane (PDMS) membrane can be used as the oxygen-permeable membrane 121. The biggest problem in conventional tissue and organ perfusion cultures—ensuring a stable supply of high-concentration oxygen over the long term—can be solved, for example, by using polydimethylsiloxane (PDMS) membranes in this way.

[0059] The first to third layers 130 are stacked radially on the outer side of the first to second layers 120. The first to third layers 130 are made of porous ceramic, and when the artificial liver 10 is viewed from the front, numerous small holes 131 are formed that penetrate in one direction. As shown in Figure 2, the cross-sectional shape of each small hole 131 is square, but the shape is not particularly limited. Each small hole 131 is separated by a lattice-shaped wall portion 132. The wall portion 132 is sandwiched between cylindrical peripheral walls 133. Inside the small holes 131, a third channel 134 is formed along one direction through which culture medium or blood flow perfuses. Specifically, when the artificial liver 10 is cultured, culture medium perfuses the third channel 134. When the artificial liver 10 is connected to (transplanted) a living organism, the third channel 134 is connected to merge with the first channel 114, and blood flow circulates. Furthermore, the culture medium or blood flow perfuses the third channel 134 only through the dashed circle area that overlaps with the first to third layers 130 shown in Figure 2, and there is no inflow or outflow from any other part.

[0060] As shown in Figures 3 and 5, the third channel 134 is filled with bile duct organoids 71 ​​whose surface is coated with hepatic sinusoidal endothelial cells (HSEC bile duct organoids). Specifically, the HSEC bile duct organoids 71 ​​are attached to the wall 132 of the third channel 134. The HSEC bile duct organoids 71 ​​are tissue in which bile duct organoids 72 are surrounded (coated) by hepatic sinusoidal endothelial cells 62. Note that the surrounding wall 133 is omitted from the illustration in Figures 3 and 5.

[0061] Coating of bile duct organoids with hepatic sinusoidal endothelial cells 62 is performed by co-culturing the bile duct organoids and hepatic sinusoidal endothelial cells 62 beforehand. Alternatively, the bile duct organoids may be attached to the wall 132 of the third channel 134, and then the culture medium containing hepatic sinusoidal endothelial cells 62 may be perfused into the third channel 134. Bile duct spheroids may be used instead of bile duct organoids.

[0062] The 2-1 layer 210 is stacked on the radially outer side of the 1-3 layer 130. The structure of the 2-1 layer 210 is the same as that of the 1-1 layer 110, except that it is cylindrical, so a detailed explanation is omitted. The 2-2 layer 220 and the 2-3 layer 230, which will be described later, are also the same as the 1-2 layer 120 and the 1-3 layer 130, respectively.

[0063] The first layer 110, 210, the second layer 120, 220, and the third layer 130, 230 may each be constructed by combining multiple members divided in the stacking direction, and the multiple members may be configured to slide against each other in the opposite direction to the aforementioned one direction (for example, the left direction in Figure 1). This makes it possible to create a structure in which the laminate 100 can be stretched in the opposite direction to the one direction. Therefore, it is possible to create a structure in which the artificial liver 10 can be stretched in multiple stages.

[0064] Figure 8 is a longitudinal cross-sectional view of the artificial liver. An end cap 80 is provided at one end of the laminate 100, covering that end. Due to the flow rate of the fluids flowing through the channels of each layer, the end of the 1-1 layer 110 protrudes further toward the end cap 80 than the 1-2 layer 120, and the end of the 1-2 layer 120 protrudes further toward the end cap 80 than the 1-3 layer 130. That is, at the end of the laminate 100, a stepped structure is formed as each layer is shifted toward the opposite side of the end cap 80 as it moves radially outward. On the other hand, the end of the end cap 80 on the laminate 100 side protrudes further toward the 1-3 layer 130 than the part facing the 1-2 layer 120, and the part facing the 1-2 layer 120 protrudes further toward the part facing the 1-1 layer 110.

[0065] Similar to each layer of the laminate 100, the end cap 80 is also constructed by combining multiple divided members, and the multiple members are configured to slide against each other in directions opposite to one direction. This allows the end cap 80 to stretch in the opposite direction to one direction in accordance with the stretching of the laminate 100. As a result, the artificial liver 10 has a structure that can stretch in the opposite direction to one direction. Furthermore, it is possible to construct the artificial liver 10 in a way that allows it to be extended in the opposite direction to the one described above, using a method other than those described above. Specifically, a unit of the artificial liver 10 can be formed by attaching a pair of end caps 80, 80' (described later) to both ends of a laminate 100 formed by stacking the first layer 110, 210, the second layer 120, 220, and the third layer 130, 230. Multiple units can be placed adjacent to each other in a clean bench, and the end caps 80, 80' of each unit can be connected (connecting the end cap 80 of one unit to the end cap 80' of the other unit) to enable automatic subculturing. By connecting the first channel 114 via the end caps 80, 80', it becomes easy to add units. As a result, it is no longer necessary to enzymatically disintegrate tissue organoids each time, which significantly reduces cumbersome operations such as pipetting and the risk of contamination.

[0066] Next, Figure 9 will be shown to illustrate the circuit diagram of the artificial liver culture system 1 of the present invention. Figure 9 is a circuit diagram of the artificial liver culture system 1 according to this embodiment. The same reference numerals are used for the components described above, and detailed explanations are omitted. Also, for the sake of the drawing, the stepped structure of the end caps 80, 80' on the laminate 100 side is omitted in Figure 9 as shown in Figure 8.

[0067] As shown in Figure 9, the laminate 100 is entirely surrounded by a pair of end caps 80, 80' from both sides. These end caps 80, 80' are joined at their opposing ends. The liver circulation line 30 is connected to the first flow channel inlet 11 of the end cap 80 and the first flow channel outlet 12 of the end cap 80'. The liver circulation line 30 is connected to the first flow channel 114 in fluidic communication. The liver circulation line 30 is provided with a liver circulation line valve 301 and a liver circulation line pump 302. The flow rate of culture medium or biological blood flowing through the liver circulation line 30 is controlled by the opening of the liver circulation line valve 301 and the operation of the liver circulation line pump 302. The culture medium flow rate control unit 52 controls the flow rate of culture medium to perfuse the artificial liver 10 by controlling the opening of the liver circulation line valve 301 and the operation of the liver circulation line pump 302.

[0068] A space of a predetermined size (diffusion section 115) is provided between the inner circumferential surface of the end cap 80 and the laminate 100. Similarly, a space of a predetermined size (converging section 116) is provided between the inner circumferential surface of the end cap 80' and the laminate 100. The first channel 114 is formed to pass through the first layers 110 and 210 from the first channel inlet 11 via the diffusion section 115 and communicate with the first channel outlet 12 via the converging section 116. That is, the culture medium flowing in from the first channel inlet 11 is distributed into the first layers 110 and 210 via the diffusion section 115. The distributed culture medium merges via the converging section 116. Here, as shown in Figure 14, the first layer 110 is constructed by gradually increasing the density of the small pores 111 within the first layer 110 in three stages, from the first channel inlet 11 side to the first channel outlet 12 side, and also by gradually increasing the diameter of the small pores 111. As a result, the first channel 114 is configured with a high-pressure region 117, a medium-pressure region 118, and a low-pressure region 119 in that order from the inlet 11 to the outlet 12. This allows the growth of liver organoids to begin from the downstream side of the blood flow. The first layer 210 is configured similarly. The liver has the property of detecting its own tissue loss and producing and secreting growth factors and cytokines, and stopping growth when the liver weight returns to its original weight. By pre-configuring the ceramic density in this way to create high-pressure region 117, medium-pressure region 118, and low-pressure region 119 that resist blood flow, it becomes easier to successfully grow and culture cells in the column for a long period of time.

[0069] The liver possesses spatial specificity called zonation (a functional multilayer structure), in which cells perform different functions depending on their location from the portal vein to the central vein. A research group led by Professor Takanori Takebe of the Graduate School of Medicine, Osaka University, has succeeded in creating an organoid with zonation present in the living liver from human pluripotent stem cells (i-PS cells) for the first time in the world (Non-Patent Literature 1). When zonation is formed, each region exerts sometimes contradictory functions, and each region plays a complex and complementary role in metabolism. Zonation in the liver is the foundation that supports the diversity of liver function, and it is known that if there is a disease, each zone develops a defense response against the damage according to the disease, and the construction of normal liver zonation is an indispensable element for maintaining homeostasis of the entire body. In this embodiment, as described in paragraph

[0068] of this specification, high-pressure region 117, medium-pressure region 118, and low-pressure region 119 of resistance to blood flow are structurally set in advance along the direction from the portal vein to the central vein. In other words, a three-stage blood flow pressure anomaly structure is provided, which is an important element not only for controlling the normal proliferation of hepatocytes as described in paragraph

[0068] , but also for performing a protective response against liver disease.

[0070] Furthermore, the portions of the second layer 120, 220 and the third layer 130, 230 that come into contact with the diffusion section 115 and the convergence section 116 are closed off at both ends. This prevents fluid from the diffusion section 115 from flowing into the internal flow channels of the second layer 120, 220 and the third layer 130, 230, and prevents fluid from flowing from the internal flow channels into the convergence section 116.

[0071] The oxygen line 31 is connected to the first opening 13 of the second flow path of the end cap 80 and the second opening 14 of the second flow path of the end cap 80'. The oxygen line 31 is connected to the second flow path 124 in fluidic communication. An oxygen line pump 312 is provided with the oxygen line 31. The amount of oxygen supplied to the perfluorocarbon solution flowing through the oxygen line 31 is controlled by the pressure of the gaseous oxygen supplied to the oxygen column 20. The oxygen supply control unit 54 controls the pressure of the gaseous oxygen supplied to the oxygen column 20 to control the amount of oxygen supplied to the perfluorocarbon solution flowing through the oxygen line 31.

[0072] The end caps 80, 80' and the second layers 120, 220 are connected by a hollow pipe section 125. As a result, the second flow path 124 is formed to pass from the first opening 13 of the second flow path in one end cap 80, through the pipe section 125, through the second layers 120, 220, and then through the pipe section 125 in the other end cap 80' to communicate with the second opening 14 of the second flow path.

[0073] The biliary circulation line 32 is connected to the third channel inlet 15 of the end cap 80 and the third channel outlet 16 of the end cap 80'. The biliary circulation line 32 is connected to the third channel 134 in fluidic communication. The biliary circulation line 32 is equipped with a biliary circulation valve 321 and a biliary circulation pump 322. The flow rate of culture medium or biological blood flowing through the biliary circulation line 32 is controlled by the opening of the biliary circulation valve 321 and the operation of the biliary circulation pump 322. The culture medium flow rate control unit 52 controls the flow rate of culture medium to perfuse the artificial liver 10 by controlling the opening of the biliary circulation valve 321 and the operation of the biliary circulation pump 322.

[0074] The end caps 80, 80' and the third layers 130, 230 are connected by a hollow pipe section 135. As a result, the third flow path 134 is formed to communicate with the third flow path outlet 16, passing from the third flow path inlet 15 in one end cap 80, through the pipe section 135, through the third layers 130, 230, and through the pipe section 135 in the other end cap 80'.

[0075] As shown in Figure 9, O-rings 81 are provided at appropriate locations, such as near the pipe sections 125 and 135, in the artificial liver 10 to seal off fluid leakage.

[0076] Next, the internal structure of the end cap 80 will be explained in detail with reference to Figures 10-13. Figure 10 is a cross-sectional view of the end cap 80 when cut along line AA in Figure 2 (a cross-section along a plane tilted 60° counterclockwise with respect to the vertical direction). Figure 11 is a cross-sectional view of the end cap 80 when cut along line BB in Figure 2 (a cross-section along a plane tilted 60° clockwise with respect to the vertical direction). Figure 12 is a longitudinal cross-sectional view of the end cap (a cross-sectional view along a plane with respect to the vertical direction). Figure 13 is a partial longitudinal cross-sectional view showing the joint between the second layer of the laminate 100 and the end cap.

[0077] As shown in Figure 10, in a cross-section along a plane tilted 60° counterclockwise with respect to the vertical direction, third channel inlets 15 are formed at both the upper and lower ends of the end cap 80, and the third channel 134 communicates from the third channel inlets 15 to the pipe section 135. On the other hand, the second channel 124 does not communicate with the outside of the end cap 80 in this cross-section.

[0078] As shown in Figure 11, in a cross-section along a plane tilted 60° clockwise with respect to the vertical direction, the second flow path first opening 13 is formed at both the upper and lower ends of the end cap 80, and the second flow path 124 communicates from the second flow path first opening 13 to the pipe section 125. On the other hand, the third flow path 134 does not communicate with the outside of the end cap 80 in this cross-section.

[0079] As shown in Figure 12, in a cross-section along a plane with respect to the vertical direction, neither the second channel 124 nor the third channel 134 communicates with the outside of the end cap 80.

[0080] As shown in Figure 13, an opening 82 is provided on the inner circumferential surface of the end cap 80, into which the pipe portion 125 is fitted. The opening 82 also has an enlarged diameter portion 83 that widens toward the pipe portion 125. An O-ring 81 is provided between the end cap 80 and the pipe portion 125 in the enlarged diameter portion 83 to seal the gap.

[0081] The end cap 80' is designed to correspond to the end cap 80 and has a similar structure. The ends of the end caps 80 and 80' are each provided with threaded sections (not shown), allowing them to be connected to each other via these threaded sections.

[0082] [Method for manufacturing an artificial liver] Next, a method for manufacturing an artificial liver according to this embodiment will be described. The following describes, as an example, a method for manufacturing an artificial liver 10 by forming the circuit of the artificial liver culture system 1 shown in Figure 9.

[0083] [First layer formation process] In the first layer formation step, a porous ceramic 1-1 layer (first layer) 110 is formed, in which a first channel 114 through which culture medium or blood flow perfuses is formed along one direction.

[0084] [Second layer formation process] In the second layer formation process, the first-second layer (second layer) 120 is laminated radially outward of the first-first layer 110. The first-second layer 120 has a structure sandwiched between oxygen permeable membranes 121, and a cavity for the second channel 124 is formed inside along one direction.

[0085] [Third layer formation process] In the third layer formation process, the first to third layers (third layer) 130 are laminated radially outward of the first to second layers 120. The first to third layers 130 are made of porous ceramic and have a third channel 134 formed in one direction through which the culture medium or blood flow perfuses.

[0086] Subsequently, the 2-1 layer (1st layer) 210, the 2-2 layer (2nd layer) 220, and the 2-3 layer (3rd layer) 230 are stacked in the same manner as described above to form the laminate 100.

[0087] [End cap installation process] After the formation of each layer is complete, a pair of end caps 80 and 80' are attached to both ends of each layer. The pair of end caps 80 and 80' are fitted together from both ends of the laminate 100 and joined to each other.

[0088] [Circuit formation process] Once the end caps are attached, the circuit of the artificial liver culture system 1 is formed. The liver circulation line 30 is connected to the first channel inlet 11 of the end cap 80 and the first channel outlet 12 of the end cap 80'. The liver circulation line 30 is connected to the first channel 114 in fluidic communication. The liver circulation line 30 is equipped with a liver circulation line valve 301 and a liver circulation line pump 302. The flow rate of culture medium or biological blood flowing through the liver circulation line 30 is controlled by the opening of the liver circulation line valve 301 and the operation of the liver circulation line pump 302.

[0089] An oxygen line 31 is connected to the first opening 13 of the second flow path of the end cap 80 and the second opening 14 of the second flow path of the end cap 80'. An oxygen line pump 312 is provided for the oxygen line 31. The perfluorocarbon solution flowing through the oxygen line 31 is controlled by the pressure of the gaseous oxygen supplied to the oxygen column 20.

[0090] A biliary circulation line 32 is connected to the third channel inlet 15 of the end cap 80 and the third channel outlet 16 of the end cap 80'. The biliary circulation line 32 is connected to the third channel 134 in fluidic communication. The biliary circulation line 32 is equipped with a biliary circulation line valve 321 and a biliary circulation line pump 322. The flow rate of culture medium or biological blood flowing through the biliary circulation line 32 is controlled by the opening of the biliary circulation line valve 321 and the operation of the biliary circulation line pump 322.

[0091] [First layer filling process] The 1-1 layer 110 is filled with liver organoids (HSEC liver organoids 61) whose surfaces are coated with hepatic sinusoidal endothelial cells 62. The coating of the liver organoids with hepatic sinusoidal endothelial cells 62 is performed by co-culturing the cell population constituting the liver organoids with the hepatic sinusoidal endothelial cells 62 in advance. Alternatively, the cell population may be attached to the wall portion 112 of the first channel 114, and then the culture medium containing hepatic sinusoidal endothelial cells 62 may be perfused into the first channel 114.

[0092] For co-culturing, the conditions described in, for example, "Ultramorphological Characteristics of Cultured Hepatic Sinusoidal Endothelial Cells and Their Interrelationship with Cultured Hepatocytes" by Satoshi Shakado, The Japanese Society of Hepatology, Vol. 9, No. 12, published December 25, 1988, pp. 1571-1578, can be applied.

[0093] Specifically, after adjusting the cell count with William's medium E (hereinafter referred to as "WE") containing 10% calf serum, insulin, dexamethasone, and antibiotics, liver organoids are first cultured for 1-2 hours in a dish containing Matrigel. Then, hepatic sinusoidal endothelial cells are added and co-culture is performed. The number of hepatic sinusoidal endothelial cells added is adjusted to be approximately 10 times the amount of liver organoids.

[0094] The HSEC liver organoid 61 is attached to the wall 112 of the first channel 114 by flowing the culture medium containing the HSEC liver organoid 61 into the first channel 114 at an appropriate flow rate and perfusion pressure. Since it is known that increasing the perfusion pressure reduces the number of pores in sinusoidal endothelial cells and increases their diameter, the perfusion pressure should be kept below 10 cmH2O.

[0095] [Third layer filling process] After forming layers 1-3 130, bile duct organoids (HSEC bile duct organoids) 71 whose surfaces are coated with hepatic sinusoidal endothelial cells 62 are packed into layers 1-3 130. The coating of the bile duct organoids with hepatic sinusoidal endothelial cells 62 is performed by co-culturing the bile duct organoids 72 and hepatic sinusoidal endothelial cells 62 beforehand. Alternatively, the bile duct organoids 72 may be attached to the wall 132 of the third channel 134, and then the culture medium containing hepatic sinusoidal endothelial cells 62 may be perfused into the third channel 134. In the case of co-culturing, the conditions are the same as in the first layer packing step, except that bile duct organoids are used instead of liver organoids. The attachment of the HSEC bile duct organoids 71 ​​to the wall 132 of the third channel 134 can also be done in the same way as in the first layer packing step.

[0096] Once all processes are complete, start culturing the artificial liver 10. Culturing the artificial liver 10 should be carried out as appropriate according to standard conditions. Also, extend the artificial liver 10 as needed during subculturing. Continue culturing the artificial liver 10, and when the HSEC liver organoids 61 and HSEC bile duct organoids 71 ​​have proliferated to a sufficient quantity to ensure that the artificial liver 10 can guarantee the normal function of a living liver after connection to a living organism, the artificial liver 10 will be considered complete.

[0097] [How to use an artificial liver] Once the culture of the artificial liver 10 is complete, the artificial liver 10 is connected to the living organism as needed. Specifically, the completed artificial liver 10 is removed from the artificial liver culture system 1 and transplanted into the living organism. First, the culture is completed with the perfluorocarbon solution flowing through the second channel 124. Next, the perfusion of culture medium to the first channel 114 of the artificial liver 10 is blocked, and the portal vein system blood vessels and the hepatic artery system blood vessels are connected to the first channel inlet 11 of the end cap 80 and the first channel outlet 12 of the end cap 80'. At this time, the perfusion of culture medium to the third channel 134 and the supply of oxygen to the oxygen column 20 are continued. In addition, the nitric oxide (NO) gas supply circuit to the oxygen column 20 is opened so that oxygen and nitric oxide can be supplied to the oxygen column 20 in parallel at any time. After the connection to the portal vein and hepatic artery is completed, changes in the function of the artificial liver 10 (e.g., changes in indicators such as ALT) are observed for a while to determine whether or not NO supply measures are necessary. Once the stable maintenance of liver function in the artificial liver 10 is confirmed, the extrahepatic bile duct is connected to the first opening 13 of the second channel of the end cap 80, and the third channel inlet 15 of the end cap 80 and the third channel outlet 16 of the end cap 80' are connected to merge with the first channel 114. After confirming the stable maintenance of liver function again, the artificial liver 10 is finally transplanted into the patient's body. This makes it possible to perform liver transplantation with minimal exposure of the artificial liver 10 to ischemia, even if the surgery takes a little longer. Conversely, when separating the artificial liver 10 from the patient's own liver and starting its culture outside the body, almost the same measures can be taken. The second opening 14 of the second channel is closed. When the artificial liver 10 is connected to the living body, the living body's blood flow is circulated through the first layer 110, 210 and the third layer 130, 230. The second layer 120, 220 is connected to the extrahepatic bile duct, and bile flows into the gallbladder.

[0098] Furthermore, the cells used in the manufacture of the artificial liver 10 can be, for example, human-derived cells. Specifically, normal cells extracted from the livers of patients with liver disease can be used. Alternatively, organoids created using i-PS cells can also be used.

[0099] As described above, this embodiment provides the following effects and advantages. The artificial liver 10 of this embodiment includes a first layer 110 made of porous ceramic, through which a first channel 114 for perfusation with culture medium or blood flow is formed along one direction. The first channel is filled with liver organoids or liver spheroids (hereinafter also simply referred to as "liver organoids") 61 whose surfaces are coated with hepatic sinusoidal endothelial cells 62. Thus, in this embodiment, the surface of the liver organoids or liver spheroids is coated with hepatic sinusoidal endothelial cells 62. Even if the three-dimensional positional relationship between the cell groups such as hepatocytes 63 and hepatic stellate cells 64 separated by hepatic sinusoidal endothelial cells 62 and the blood flow containing red blood cells is changed, there is no impediment to the overall liver function. Therefore, the surface of the liver organoids is coated with hepatic sinusoidal endothelial cells 62 in order to enhance and promote intercellular interactions with hepatic sinusoidal endothelial cells 62, which are responsible for the proliferation, growth, and suppression control of hepatic parenchymal cells and other cells. This makes it easier to control the so-called "mechanical homeostasis," specifically the changes in hemodynamics applied to hepatic sinusoidal endothelial cells 62, such as shear stress caused by blood flow to the endothelial cells. This enables the optimal supply of oxygen, cell growth factors, and cytokines to the target cell group to be proliferated and grown, thereby improving the engraftment of liver organoids. This allows for the optimal culture of the cell groups that make up the hepatic lobules. Furthermore, the manufactured artificial liver 10 can mimic and realize the detoxification function of harmful substances and the metabolic regulation function of nutrients that are inherent in a living liver with a high degree of reproducibility, and has functions closer to those of a living organ.

[0100] Furthermore, the first layers 110 and 210 are made of porous ceramic and have narrow first channels 114 formed in the small pores 111 of the ceramic. The first channels 114 are configured to be filled with coated liver organoids 61 by flowing a culture medium containing coated liver organoids 61 through them. In this case, since the cross-sectional area of ​​the first channels 114 is small, it is expected that the frequency and contact area of ​​the coated liver organoids 61 with the wall portion 112 of the first channels 114 will increase. This allows the coated liver organoids 61 to adhere well to the first channels 114. Moreover, since the first layers 110 and 210 are made of highly biocompatible ceramic, it is ultimately possible to transplant the artificial liver 10 into the living body.

[0101] The artificial liver 10 of this embodiment includes second layers 120 and 220, which are laminated on the first layers 110 and 210 and have a second channel 124 formed in one direction through which perfluorocarbons flow or bile is discharged. Furthermore, the artificial liver 10 of this embodiment includes a third layer 130 and 230 made of porous ceramic, which is laminated on the second layers 120 and 220 and has a third channel 134 formed in one direction through which culture medium or blood flow is perfused. The third channel 134 is filled with bile duct organoids or bile duct spheroids (hereinafter also simply referred to as "bile duct organoids") 71 whose surface is coated with hepatic sinusoidal endothelial cells 62. In other words, in the artificial liver 10 of this embodiment, a bile duct-like structure can be formed by the bile discharge channel and the bile duct organoids. Therefore, the artificial liver 10 of this embodiment includes not only hepatic parenchymal cells and hepatic stellate cells, but also the entire morphology of the hepatic lobule, including bile duct-like structures and bile flow paths, and can be configured as an artificial liver 10 having functions equivalent to those of a living liver.

[0102] Furthermore, the third layers 130 and 230 are made of porous ceramic and have narrow third channels 134 formed in the small pores 131 of the ceramic. The third channels 134 are configured such that, for example, a culture medium containing coated bile duct organoids 71 ​​is flowed through the third channels 134, thereby filling it with coated bile duct organoids 71. In this case, since the third channels 134 have a small cross-sectional area, it is expected that the frequency and contact area of ​​the coated bile duct organoids 71 ​​with the wall portion 132 of the third channels 134 will increase. This allows the coated bile duct organoids 71 ​​to adhere well to the third channels 134. In addition, since the third layers 130 and 230 are made of highly biocompatible ceramic, it is ultimately possible to transplant the artificial liver 10 into the living body.

[0103] In this embodiment, a three-layer structure consisting of a first layer 110, 210, a second layer 120, 220, and a third layer 130, 230 is repeatedly stacked in this order. Therefore, the artificial liver 10 of this embodiment has a three-dimensional structure comprising layered elements. As a result, the artificial liver 10 of this embodiment can improve the reproducibility of a structurally and functionally complex living liver.

[0104] In this embodiment, the artificial liver 10 may have a structure that can be extended in a direction opposite to one direction. Therefore, when subculturing of liver organoids becomes necessary, the layered structure of the artificial liver 10 can be extended in a direction opposite to one direction to secure an additional area for subculturing the liver organoids. This makes automated subculturing possible. Consequently, the effort involved in pipetting, reseeding into new containers, and the risk of contamination during manual long-term subculturing can be reduced. Costs and time can also be reduced. Furthermore, by configuring the artificial liver 10 to be extendable in multiple stages, only the necessary area can be extended as needed during subculturing, enabling long-term automated subculturing.

[0105] For example, the first layer 110, 210, the second layer 120, 220, and the third layer 130, 230 can be constructed by combining multiple members that are divided in the stacking direction, and these multiple members can be configured to slide against each other in opposite directions. This allows the first layer 110, 210, the second layer 120, 220, and the third layer 130, 230 to be stretchable in opposite directions.

[0106] In the artificial liver culture system 1 of this embodiment, the culture medium flow rate control unit 52 provides feedback control of the culture medium flow rate based on the measurement results measured by the metabolite measurement unit 51. This allows the environment inside the artificial liver 10 to be automatically adjusted to conditions suitable for culturing liver organoids. Therefore, liver organoids and the like can be cultured effectively. Specifically, metabolites that act as flow rate control factors include various growth factors (HGF, EGF, etc.), cytokines (TGF-β, etc.), NO synthase, etc.

[0107] In this embodiment, the culture medium flow rate control unit 52 controls the flow rate of the culture medium to perfuse the artificial liver 10 based on the flow rate measurement value measured by the culture medium flow rate measuring unit 53. This makes it possible to control the flow rate of the culture medium to perfuse the artificial liver 10 based on the actual flow rate of the culture medium perfusing the artificial liver 10. Therefore, the flow rate of the culture medium to perfuse the artificial liver 10 can be controlled with high precision.

[0108] In this embodiment, the oxygen supply control unit 54 controls the oxygen supply amount based on a preset algorithm corresponding to the artificial liver 10. This optimizes the timing and amount of oxygen supply to the artificial liver 10, and automatically adjusts the oxygen concentration inside the artificial liver 10 to a level suitable for culturing liver organoids. Therefore, liver organoids can be cultured effectively. [Explanation of symbols]

[0109] 1. Artificial liver culture system 10 Artificial liver 11. Inlet of the first channel 12. First channel outlet 13 Second channel, first opening 14 Second channel, second opening 15 Third channel entrance 16 Third channel outlet 20 Oxygen column 30 Liver circulation lines 31 Oxygen line 32. Biliary circulation line 50 Control Unit 51 Metabolite Measurement Section 52 Culture medium flow control unit 53 Culture solution flow rate measuring section 54 Oxygen supply control unit 61 HSEC liver organoids (coated liver organoids) 62 Liver sinusoidal endothelial cells 63 Hepatocytes 64 Hepatic stellate cells 65 Kupffer cells 71 HSEC bile duct organoids (coated bile duct organoids) 72 Bile duct organoids 80,80' End Cap 81 O-ring 82 Opening 83 Expanded diameter part 100-layer structure 110 1st-1st layer (1st layer) 111 Small hole 112 Wall 113 Peripheral wall 114 First channel 115 Diffusion section 116 Convergence section 117 High-voltage area 118 Medium pressure range 119 Low-pressure area 120 1st-2nd layer (2nd layer) 121 Oxygen permeable membrane 124 Second channel 125 Pipe section 130 1st-3rd layer (3rd layer) 131 Small hole 132 Wall 133 Peripheral wall 134 Third channel 135 Pipe section 210 2nd-1st layer (1st layer) 220 2nd-2nd layer (2nd layer) 230 2nd-3rd layer (3rd layer) 301 Liver circulation line valve 302 Liver circulation line pump 312 Oxygen line pump 321 Biliary circulation line valve 322 Biliary circulation line pump

Claims

1. A first layer made of porous ceramic, in which a first channel through which culture medium or blood flow perfuses is formed along one direction, A second layer is laminated on the first layer, and a second channel through which perfluorocarbons flow or bile is discharged is formed along the one direction, A third layer made of porous ceramic is laminated on the second layer, and a third channel through which culture medium or blood flow perfuses is formed along the one direction. Equipped with, The first channel is filled with liver organoids or liver spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells. An artificial liver in which the third channel is filled with bile duct organoids or bile duct spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells.

2. The artificial liver according to claim 1, wherein a three-layer structure consisting of a first layer, a second layer, and a third layer is repeatedly stacked in this order multiple times.

3. The artificial liver according to claim 1, wherein the first layer, the second layer and the third layer are composed of a plurality of members divided in the stacking direction, and the plurality of members are configured to be slidable from one another in a direction opposite to the one direction.

4. A plurality of units formed by attaching a pair of end caps from both ends of the laminate of the first layer, the second layer and the third layer, The artificial liver according to claim 1, wherein the plurality of units are connected via the end caps.

5. An artificial liver according to any one of claims 1 to 4, A metabolite measurement unit for measuring metabolites produced from the artificial liver, A culture medium flow control unit controls the flow rate of the culture medium that perfuses the artificial liver, Equipped with, The culture medium flow rate control unit is an artificial liver culture system that provides feedback control of the flow rate of the culture medium based on the measurement results measured by the metabolite measurement unit.

6. The artificial liver is perfused with a culture medium flow rate measuring unit, which measures the flow rate of the culture medium. The artificial liver culture system according to claim 5, wherein the culture medium flow rate control unit controls the flow rate of the culture medium based on the flow rate measurement value measured by the culture medium flow rate measuring unit.

7. The system includes an oxygen supply control unit that controls the amount of oxygen supplied to the artificial liver, The artificial liver culture system according to claim 5, wherein the oxygen supply control unit controls the oxygen supply amount based on an algorithm set in advance in correspondence with the artificial liver.

8. A first layer formation step in which a first channel through which culture medium or blood flow perfuses is formed along one direction is formed on a porous ceramic first layer, The first layer is filled with liver organoids or liver spheroids whose surfaces are coated with hepatic sinusoidal endothelial cells, A second layer formation step is to form a second layer which is laminated on the first layer and in which a second channel is formed along the one direction through which perfluorocarbons flow or bile is discharged, A third layer formation step is to form a third layer made of porous ceramic, which is laminated on the second layer and has a third channel formed along the one direction through which a culture medium or blood flow perfuses, The third layer is filled with a third layer, which consists of a bile duct organoid or bile duct spheroid whose surface is coated with hepatic sinusoidal endothelial cells. A method for manufacturing an artificial liver having [a certain characteristic].

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