Analog intestinal-liver axis organoid microfluidic chip and a culture method thereof

Abstract

The application provides a simulated enterohepatic axis organoid microfluidic chip and a culture method thereof, which comprises a liver culture area and an intestinal culture area, the liver culture area and the intestinal culture area exchange substances through a chitosan porous membrane, and the pore size of the chitosan porous membrane is 0.4 um to 10 um. The simulated enterohepatic axis organoid microfluidic chip simulates multiple liver lobule structures to simulate liver subareas through the liver culture area, and simulates the hollow structure of the intestinal tract to simulate the physiological structure of the intestinal tract through the intestinal culture area. The liver culture area and the intestinal culture area exchange substances through the chitosan porous membrane, the chitosan porous membrane allows small-molecule nutrients, metabolites and signal factors to pass through, and blocks organoid fragments or macromolecular impurities, thereby guaranteeing the specificity and safety of the exchange.

Description

Technical Field

[0001] This invention belongs to the field of biomedical engineering technology, specifically relating to a microfluidic chip simulating the gut-liver axis organoid and its culture method. Background Technology

[0002] The human body is an organic whole closely interconnected through the circulatory, nervous, and endocrine systems. Among these, the "gut-hepatic axis," formed by the portal vein, biliary system, and systemic circulation, is a core hub for maintaining metabolic homeostasis, immune balance, and toxin defense. Under physiological conditions, nutrients absorbed by the intestines, microbial metabolites, and potential endogenous and exogenous toxins are directly transported to the liver via the portal vein for first-pass metabolism, detoxification, and biotransformation. Simultaneously, bile acids synthesized by the liver are secreted into the intestines, participating in lipid digestion and regulating the gut microbiota. This bidirectional, dynamic communication network is crucial for bodily health, and its dysfunction has been proven to be closely related to the pathogenesis of various complex diseases, including fatty liver, cirrhosis, inflammatory bowel disease, sepsis, and even neurodegenerative diseases. Given the central role of the gut-hepatic axis, establishing an in vitro model that faithfully simulates its physiological and pathological processes has become a critical requirement for accelerating new drug discovery and reducing the risk of clinical failure in drug development and disease research. However, current mainstream research models have fundamental deficiencies in simulating the interactions between these complex organs, constituting a major bottleneck in technological development.

[0003] Traditional two-dimensional cell culture models involve culturing a single layer of cells (such as the Caco-2 intestinal epithelial cell line or the HepG2 hepatocellular carcinoma cell line) on the surface of a plastic culture dish. While this method offers advantages such as low cost, ease of operation, and suitability for high-throughput screening, its limitations are fundamental. The two-dimensional culture environment cannot simulate the complex three-dimensional structure of human organs, the dynamic interactions between cells and the extracellular matrix, and inherent cell polarity. In this non-physiological environment, cells rapidly lose their key phenotypes and functions; for example, the activity of drug-metabolizing enzymes (such as cytochrome P450 enzymes) in liver cells decreases sharply. More importantly, the two-dimensional model is static and isolated, completely severing the continuous communication between the intestine and liver through fluid flow, hormonal signals, and immune factors. Therefore, it cannot be used to study any scientific questions related to organ-to-organ interactions, such as the hepatic metabolism of orally administered drugs after intestinal absorption or drug-induced enterogenic liver injury.

[0004] Animal models (such as mice, rats, dogs, and non-human primates) serve as the "gold standard" bridging in vitro experiments and human clinical trials, providing a complete in vivo physiological environment encompassing multi-organ interactions, neuroendocrine regulation, and immune system responses. However, there is an insurmountable "species difference" barrier to using animal models to predict human responses. Different species exhibit fundamental differences in gene expression profiles, drug-metabolizing enzyme activity, gut microbiota composition, and immune system function, leading to the failure of many candidate drugs that are safe and effective in animal models in human clinical trials, and vice versa.

[0005] Statistics show that over 90% of drugs that enter Phase I clinical trials ultimately fail to gain market approval, with the inability of preclinical animal models to accurately predict human toxicity or efficacy being a significant contributing factor. Furthermore, animal testing faces high costs, lengthy cycles, complex procedures, and increasing ethical scrutiny, making it difficult to meet the high-throughput and rapid-iteration demands of modern drug development. Summary of the Invention

[0006] The technical problem to be solved by this invention is how to provide an integrated and functional liver-intestinal organoid microfluidic chip.

[0007] The present invention solves the above-mentioned technical problems through the following technical means:

[0008] The first aspect of the present invention provides a microfluidic chip for simulating the gut-liver axis organoid, including a liver culture region and an intestinal culture region, wherein the liver culture region and the intestinal culture region exchange substances through a chitosan porous membrane with a pore size of 0.4 μm to 10 μm.

[0009] The preparation method of the chitosan porous membrane is as follows: chitosan solution is added to glacial acetic acid, mixed and stirred, and placed in an oven until the chitosan dissolves to obtain a mixed solution. 2xHEPES buffer solution is added to the mixed solution and stirred. Then gelatin is added and placed in an oven until the gelatin dissolves to obtain a mixed solution. Finally, the mixed solution is printed using a 3D bioprinter to obtain a chitosan porous membrane.

[0010] Beneficial effects: Compared with traditional porous membranes, the chitosan porous membrane of the present invention is non-toxic, can slightly adhere to organoids to prevent them from falling off, slowly degrades to provide micronutrients, and improves the survival rate and maturity of organoids; it has both mechanical strength and flexibility, and its interconnected micropores are permeable and have good hydrophilicity, ensuring smooth and stable convection exchange.

[0011] The microfluidic chip for simulating the gut-liver axis organoids of this invention simulates multiple liver lobule structures to represent liver regions through the liver culture region, and simulates the hollow structure of the intestine through the intestinal culture region to represent the physiological structure of the intestine. This invention facilitates material exchange between the liver culture region and the intestinal culture region through a chitosan porous membrane. The chitosan porous membrane allows small molecule nutrients, metabolites, and signaling factors to pass through, while blocking organoid fragments or large molecular impurities, ensuring the specificity and safety of the exchange.

[0012] Preferably, the liver culture area includes a first liquid inlet, a vertical diversion area, and a first liquid outlet. The first liquid inlet is connected to the vertical diversion area, and the vertical diversion area is connected to the second liquid outlet.

[0013] Preferably, the vertical diversion area includes a first collection area, a first cultivation area, and a second collection area, wherein the first collection area is connected to the first cultivation area, and the first cultivation area is connected to the second collection area.

[0014] Preferably, the first collection zone is provided with a plurality of first diversion holes, the first culture zone is provided with a plurality of second diversion holes, the first collection zone is connected to the first culture zone through the first diversion holes, and the first culture zone is connected to the second collection zone through the second diversion holes.

[0015] Preferably, the number of first diversion holes is greater than the number of second diversion holes.

[0016] Preferably, the diameter of the first diversion hole is 20um to 100um; the diameter of the second diversion hole is 80um to 200um.

[0017] Preferably, the second collection zone has a through hole at its center, and the through hole is connected to the first liquid outlet through the first exchange zone.

[0018] Preferably, the intestinal culture zone includes a second liquid inlet, a second culture zone, a second exchange zone, and a second liquid outlet. The first liquid inlet is connected to the second culture zone, the second culture zone is connected to the second exchange zone, and the second exchange zone is connected to the second liquid outlet.

[0019] Preferably, a chitosan porous membrane is disposed between the first exchange region and the second exchange region.

[0020] Preferably, both the first and second exchange zones are intestinal-like tubules.

[0021] Preferably, the liquid flow direction in the first exchange zone is opposite to the liquid flow direction in the second exchange zone.

[0022] The second aspect of the present invention provides a method for culturing the above-mentioned microfluidic chip simulating the intestinal-liver axis organoids. Liver organoids are printed onto the liver culture area by inkjet printing, and liver culture medium is circulated into the liver culture area. At the same time, intestinal organoids are printed onto the intestinal culture area by inkjet printing, and intestinal nutrient solution is circulated into the intestinal culture area. During the culture process, the liquid in the liver culture area and the liquid in the intestinal culture area exchange substances through a chitosan porous membrane, and the flow directions of the liquid in the liver culture area and the liquid in the intestinal culture area are opposite.

[0023] Beneficial effects: This invention achieves transmembrane convection on both sides of the chitosan porous membrane by having the liquid in the liver culture zone and the liquid in the intestinal culture zone flow in opposite directions, driving bidirectional diffusion and exchange of nutrients and metabolites between the liver and intestinal sides, thus simulating the material transport mechanism of the gut-liver axis. Attached Figure Description

[0024] Figure 1 This is an exploded view of the microfluidic chip simulating the gut-liver axis organoid in Embodiment 1 of the present invention; Figure 2 This is an overall diagram of the microfluidic chip simulating the gut-liver axis organoid in Embodiment 1 of the present invention; Figure 3 This is a schematic diagram of the structure of the first collection area in Embodiment 1 of the present invention; Figure 4 This is a schematic diagram of the structure of the first culture region in Embodiment 1 of the present invention; Figure 5 This is a diagram of the mature liver organoid in Application Example 1 of this invention; Figure 6 This is a diagram of intestinal organoid maturation in Application Example 1 of this invention; Figure 7 This is a diagram of the growth state of intestinal organoids in Comparative Application Example 1 of the present invention; Figure 8 This is a diagram of the growth state of liver organoids in Comparative Application Example 1 of this invention; Figure 9 This is a diagram showing the growth state of intestinal organoids in Comparative Application Example 2 of the present invention; Figure 10 This is a diagram of the growth state of liver organoids in Comparative Application Example 2 of the present invention; Figure 11 This is a diagram of the liver organoid growth state in Comparative Application Example 3 of the present invention; Figure 12 This is a diagram showing the growth state of intestinal organoids in Comparative Application Example 3 of the present invention. Detailed Implementation

[0025] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0027] Unless otherwise specified in the embodiments, the techniques or conditions described in the literature in this field or in accordance with the product manual may be followed.

[0028] Example 1 according to Figure 1-2 As shown, this embodiment provides a microfluidic chip simulating the gut-liver axis organoid. The chip includes a liver culture region, an intestinal culture region, and a chitosan porous membrane 30. The liver culture region and the intestinal culture region exchange substances through the chitosan porous membrane, thereby constructing a physiological structure simulating the gut-liver axis. The pore size of the chitosan porous membrane is 0.4µm~10µm. The chitosan porous membrane can accurately sieve molecules, protect the culture system, adapt to low-flow-rate microfluidics, ensure stable organoid growth, and also take into account the simulation of intestinal microecology, restoring the physiological interaction characteristics of the gut-liver axis. The chitosan porous membrane allows small molecule nutrients (such as glucose and amino acids), metabolites (such as bile acids and intestinal microbial derivatives), and signaling factors to pass through, while blocking organoid debris or large molecular impurities, ensuring the specificity and safety of the exchange; at the same time, convection is formed on both sides of the chitosan porous membrane, driving bidirectional diffusion exchange of nutrients and metabolites between the liver side and the intestinal side, simulating the material transport mechanism of the gut-liver axis.

[0029] The chitosan porous membrane is prepared as follows: 50 ml of a 6% (w / w) chitosan solution was added to 1% (w / w) glacial acetic acid and mixed. The mixture was then placed in a 60°C oven until the chitosan dissolved, resulting in a mixed solution. 5 ml of the mixed solution was taken and 5 ml of 2xHEPES buffer was added and stirred until the pH of the mixed solution was between 5 and 6. 5% (w / w) gelatin was then added and placed in a 60°C oven until the gelatin dissolved, resulting in a mixed solution. The mixed solution was poured into the printing syringe of a 3D bioprinter, cooled to room temperature, and finally printed using the 3D bioprinter to obtain a chitosan porous membrane.

[0030] Compared to traditional porous membranes, chitosan porous membranes are non-toxic, can slightly adhere to organoids to prevent them from falling off, slowly degrade to provide micronutrients, and improve the survival rate and maturity of organoids. They also have the advantages of combining mechanical strength and flexibility, with interconnected micropores that are permeable and hydrophilic, ensuring smooth and stable convection exchange.

[0031] The liver culture area includes a first liquid inlet 11, a vertical diversion area 12, and a first liquid outlet 13. The first liquid inlet 11 is connected to the vertical diversion area 12, and the vertical diversion area 12 is connected to the first liquid outlet 13.

[0032] The vertical diversion zone 12 includes a first collection zone 121, a first culture zone 122, and a second collection zone 123. The first collection zone 121, the first culture zone 122, and the second collection zone 123 are divided into three layers in the vertical direction. Figure 3 As shown, the first collection area 121 is provided with a plurality of first diversion holes 121a, according to Figure 4 As shown, the first culture zone 122 is provided with multiple second diversion holes 122a, and the number of first diversion holes 121a is greater than the number of second diversion holes 122a. The first collection zone 121 is connected to the first culture zone 122 through the first diversion holes 121a, and the first culture zone 122 is connected to the second collection zone 123 through the second diversion holes 122a. This arrangement is intended to simulate the fluid microenvironment of the liver lobules. Under physiological conditions, the liver lobules are hexagonal, and the arteries / veins of the liver lobules are located on the periphery of the liver lobules. The blood flow in the liver flows from the periphery of the liver lobules towards the center, flowing towards the central vena cava, and then out. Therefore, the first diversion hole 121a simulates the artery / vein region of the liver lobules on the periphery, while the second diversion hole 122a simulates the central vena cava of the liver lobules.

[0033] A through hole (not shown in the figure) is provided at the center of the second collection area 123. The through hole is connected to the first liquid outlet through the first exchange area 14. The first exchange area 14 is an intestinal-like tube, which is formed by bending and stacking.

[0034] The diameter of the first diversion orifice 121a is 20µm to 80µm; the diameter of the second diversion orifice 122a is 50µm to 100µm. This configuration in this embodiment is designed to meet the requirements of biomimetic liver lobule culture and the characteristics of microfluidic fluid, and it works synergistically with the ratio of the number of diversion orifices, which is beneficial for liver culture.

[0035] The intestinal culture zone includes a second liquid inlet 21, a second culture zone 22, a second exchange zone 23, and a second liquid outlet 24. The second liquid inlet 21 is connected to the second culture zone 22, the second culture zone 22 is connected to the second exchange zone 23, and the second exchange zone 23 is connected to the second liquid outlet 24. The second liquid inlet 21, the second culture zone 22, the second exchange zone 23, and the second liquid outlet 24 are all on the same layer. The second exchange zone 23 is an intestinal-like tube, which is formed by bending and stacking.

[0036] In this embodiment, a chitosan porous membrane is provided between the first exchange zone 14 and the second exchange zone 23. The first exchange zone 14 and the second exchange zone 23 overlap in the vertical direction. Convection is formed between the first exchange zone 14 and the second exchange zone 23, generating microflow and allowing nutrients to be exchanged.

[0037] Meanwhile, the liquid flow direction in the first exchange zone 14 is opposite to the liquid flow direction in the second exchange zone 23.

[0038] Application Example 1 This application example uses the microfluidic chip simulating the hepatic-gut axis organoid from Example 1. It utilizes a chitosan porous membrane for convective material exchange. The chitosan porous membrane allows small molecule nutrients, metabolites, and signaling factors to pass through while blocking organoid debris or large molecular impurities, ensuring the specificity and safety of the exchange. Convection is formed on both sides of the chitosan porous membrane, driving bidirectional diffusion exchange of nutrients and metabolites between the liver and intestine sides, simulating the material transport mechanism of the gut-liver axis.

[0039] The specific cultivation methods are as follows: Liver organoids were printed in the first culture zone 122 using inkjet printing technology, following the natural arrangement of liver lobules. The first fluid outlet 13 is connected to the liver culture medium via a sterile tubing-connected peristaltic pump, and the liver culture medium is connected to the first fluid inlet 11, enabling microfluidic culture of the liver organoids. The liver culture medium is DMEM. The mature liver organoids were cultured on day 16 as shown in the image. Figure 5 As shown in the figure, the liver organoids have formed a large number of well-defined and structurally complete 3D spherical structures, which are consistent with the typical morphology of mature liver organoids; the size distribution gradient of the spheres is reasonable (tens to hundreds of micrometers), and the growth synchronization is good; the overall survival rate is high, and the small amount of debris in the background indicates that the culture status is stable and suitable for subsequent functional experiments.

[0040] The preparation of the bioprinting ink for the liver organoid is as follows: Mix 10 mg / mL matrix gel with DMEM culture medium at a volume ratio of 8:2, then mix with hepatocytes. At this point, the density of hepatocytes is 3 × 10⁻⁶. 3 per ml.

[0041] Intestinal organoids were printed onto the inner walls of both sides of the second culture zone 122 using inkjet printing technology to prevent detachment due to fluid erosion. One side of the intestinal cells was in contact with the flowing enteric nutrient solution, while the other side adhered to the lumen wall, mimicking the physiological structure of the intestine. The second fluid outlet 24 was connected to the intestinal culture medium via a sterile tubing connected to an independent peristaltic pump. The intestinal culture medium was connected to the second fluid inlet 21, enabling microfluidic culture of the intestinal organoids. The enteric nutrient solution was colon organoid amplification culture medium (without the Y-27632 inhibitor). The mature intestinal organoids were observed on day 16. Figure 6 As shown in the figure, the intestinal organoids have formed typical cystic structures with clear boundaries and thick walls, which are the core morphological markers of maturity; the size gradient of the intestinal organoids is reasonable, and the dense cyst walls indicate that the intestinal epithelial cells have differentiated and have the potential for intestinal physiological functions; the overall condition is stable, with only a small amount of damage and fragmentation indicating mild culture stress, and it can be used for subsequent experiments.

[0042] The preparation of the bioprinting ink for the intestinal organoids is as follows: 10 mg / mL of matrix gel was mixed with colon organoid amplification medium (without Y-27632 inhibitor) at a volume ratio of 8:2, and then mixed with the cultured mature intestinal organoids. At this point, the density of the intestinal organoids was 2 × 10⁻⁶. 3 per mL.

[0043] Comparative Example 1 This comparative example provides a microfluidic chip that simulates the intestinal-hepatic axis organoid. The difference between this comparative example and Example 1 is that the pore size of the chitosan porous membrane is different, ranging from 12µm to 15µm. All other aspects are the same as in Example 1.

[0044] Comparative Application Example 1 In this comparative application example 1, liver organoids and intestinal organoids were cultured using the microfluidic chip of the simulated intestinal-liver axis organoids in Comparative Example 1. The culture method was exactly the same as in application example 1.

[0045] On day 16 of culture, the growth status of the intestinal organoids was observed, and the results were as follows: Figure 7 As shown, after staining for dead and live cells, fluorescence imaging of intestinal organoids showed that red fluorescence (dead cells) signal was significantly more than green fluorescence (live cells) signal, indicating that the overall cell viability of the organoids was low and the culture status was poor.

[0046] On day 16 of culture, the liver organoids were in the following condition: Figure 8 As shown, the presence of more red fluorescence (dead cells) signals than green fluorescence (live cells) signals indicates that the liver organoids are in poor condition.

[0047] Comparative Application Example 2 This comparative application example uses the microfluidic chip for simulating the liver-gut axis organoids of Example 1. The difference between this comparative application example and Example 1 is that after adding intestinal culture medium to the second liquid inlet 21, the second liquid inlet 21 is directly blocked, and the second liquid outlet 24 is blocked at the same time. The intestinal organoids are then statically cultured in the second culture zone 122.

[0048] The final results of the cultured intestinal organoids are as follows Figure 9 As shown, the weak and sparse live signal (green fluorescence) of intestinal organoid cells cultured in a quiescent state indicates a low cell number and low overall viability.

[0049] The final cultured liver organoids, such as Figure 10 As shown in the image, the liver organoids are stained with ALB immunofluorescence (blue represents DAPI nuclear staining, and red represents ALB). It can be seen that the liver organoids form a structurally complete 3D cystic structure, and the cells in the cyst wall highly express ALB, proving that there are a large number of functionally mature hepatocytes in them.

[0050] Comparative Application Example 3 This comparative application example uses the microfluidic chip for simulating the liver-gut axis organoids of Example 1. The difference between this comparative application example and Example 1 is that after adding liver culture medium to the first liquid inlet 11, the first liquid inlet 11 is directly blocked, and the first liquid outlet 13 is blocked at the same time. The liver organoids are then statically cultured in the first culture area 122.

[0051] The final results of the cultured liver organoids are as follows: Figure 11 As shown, dead / non-living cells (red) are clearly visible in liver organoids cultured in a quiescent state: significant red fluorescent signals are present in the image, and are widely distributed, even overlapping with green signals. This indicates a high proportion of dead cells, apoptotic cells, or severely damaged living cells.

[0052] The final cultured intestinal organoids, such as Figure 12 As shown in the microscopic image of the intestinal organoids, numerous spherical structures are visible, proving that early cystic structures have been formed, which is consistent with the typical morphology of 3D intestinal organoid culture, indicating that preliminary self-assembly has been completed.

[0053] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A microfluidic chip simulating the hepato-gut axis organoid, characterized in that, It includes a liver culture zone and an intestinal culture zone, and the liver culture zone and the intestinal culture zone exchange substances through a chitosan porous membrane; the pore size of the chitosan porous membrane is 0.4um~10um; The preparation method of the chitosan porous membrane is as follows: chitosan solution is added to glacial acetic acid, mixed and stirred, and placed in an oven until the chitosan dissolves to obtain a mixed solution. 2xHEPES buffer solution is added to the mixed solution and stirred. Then gelatin is added and placed in an oven until the gelatin dissolves to obtain a mixed solution. Finally, the mixed solution is printed using a 3D bioprinter to obtain a chitosan porous membrane.

2. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 1, characterized in that, The liver culture area includes a first fluid inlet, a vertical diversion area, and a first fluid outlet. The first fluid inlet is connected to the vertical diversion area, and the vertical diversion area is connected to the second fluid outlet.

3. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 2, characterized in that, The vertical diversion area includes a first collection area, a first cultivation area, and a second collection area. The first collection area is connected to the first cultivation area, and the first cultivation area is connected to the second collection area.

4. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 3, characterized in that, The first collection zone is provided with multiple first diversion holes, and the first culture zone is provided with multiple second diversion holes. The first collection zone is connected to the first culture zone through the first diversion holes, and the first culture zone is connected to the second collection zone through the second diversion holes.

5. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 4, characterized in that, The number of first diversion orifices is greater than that of second diversion orifices.

6. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 4, characterized in that, The diameter of the first diversion orifice is 20um~100um; the diameter of the second diversion orifice is 80um~200um.

7. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 4, characterized in that, The second collection zone has a through hole at its center, which is connected to the first liquid outlet through the first exchange zone.

8. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 7, characterized in that, The intestinal culture zone includes a second liquid inlet, a second culture zone, a second exchange zone, and a second liquid outlet. The first liquid inlet is connected to the second culture zone, the second culture zone is connected to the second exchange zone, and the second exchange zone is connected to the second liquid outlet.

9. The microfluidic chip for simulating the hepato-gut axis organoid according to claim 8, characterized in that, A chitosan porous membrane is placed between the first and second exchange zones. Both the first and second exchange zones are intestinal-like tubules. The liquid flow direction in the first exchange zone is opposite to that in the second exchange zone.

10. A method for culturing a microfluidic chip simulating the hepato-gut axis organoid as described in any one of claims 1-9, characterized in that, Liver organoids are printed onto the liver culture area using inkjet printing, and liver culture medium is circulated into the liver culture area. At the same time, intestinal organoids are printed onto the intestinal culture area using inkjet printing, and intestinal nutrient solution is circulated into the intestinal culture area. During the culture process, the liquids in the liver culture area and the intestinal culture area exchange substances through a chitosan porous membrane, and the liquids in the liver culture area and the intestinal culture area flow in opposite directions.

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