Micro-fluidic chip for multi-cell co-culture of liver

Abstract

The utility model discloses a micro-fluidic chip for liver multi-cell co-culture, which belongs to the technical field of biomedical engineering and comprises a perfusion layer and a culture layer, and the perfusion layer is provided with a first culture solution inlet and a culture medium outlet; the culture layer is provided with a micro-perfusion liver chip; bonding and forming the perfusion layer and the culture layer; two groups of micro-perfusion liver chips are arranged, and each group of micro-perfusion liver chips comprises a set of micro-perfusion network system; the micro-perfusion network system comprises a micro-perfusion channel, a multifunctional cell co-culture area and a liquid storage tank, the micro-perfusion channel, the multifunctional cell co-culture area and the liquid storage tank are communicated, and the micro-perfusion network system is communicated with the first culture solution inlet and the culture medium outlet; the multifunctional cell co-culture area comprises four cell culture areas. The problems that in the prior art, the liver structure is complex, and experimental observation and operation are difficult are solved. The device has the advantages that the in-vivo environment and interaction of liver organ diseases are fully simulated, and observation is convenient.

Description

Technical Field

[0001] The utility model relates to the technical field of biomedical engineering, and particularly relates to a microfluidic chip for co-culturing multiple liver cells. Background Art

[0002] In clinical practice, drug-induced liver injury has become a major challenge in the public health field due to its high incidence and mortality. Therefore, drug hepatotoxicity evaluation is very important, and the toxic reactions in the liver involve the interaction of multiple cells in the liver.

[0003] However, this process faces significant obstacles: Although traditional animal models are widely used, they are limited by species differences and animal ethics disputes; while in vitro 2D static cell models are unable to fully simulate complex cell-cell interactions and physiological metabolic processes, resulting in their inability to effectively observe long-term changes in hepatocyte function. The maintenance of liver physiological functions is a process of multi-cell cooperation, and the interaction between hepatocytes and liver sinusoidal endothelial cells, stellate cells, Kupffer cells, etc. is particularly crucial. In view of this, multi-cell co-culture models, as research means closer to the actual in vivo situation, have increasingly received the favor of the scientific research community.

[0004] In recent years, in order to more accurately reflect the key elements of drug safety and efficacy evaluation, a series of in vitro three-dimensional hepatocyte models have emerged, aiming to reproduce the complex structure and functions of the liver. The development of these models has greatly benefited from the latest progress in microfluidic technology and tissue engineering, especially the rise of liver organ-on-a-chip technology, which has brought innovation to the drug evaluation system. By precisely simulating the in vivo liver microenvironment, liver organ-on-a-chip provides a highly simulated experimental platform for researchers, greatly improving the accuracy and efficiency of drug screening and toxicity testing. Despite the bright prospects, the current liver organ-on-a-chip technology still faces several limitations. Most studies tend to select a few key hepatocyte types for co-culture, ignoring the complexity of the liver as a whole. In addition, although the use of materials such as hydrogels for cell encapsulation can promote cell-cell interaction to a certain extent, it also brings additional technical difficulties to experimental observation and operation. Therefore, developing a new type of liver organ-on-a-chip model that can comprehensively reflect liver physiological characteristics and is convenient for operation and observation has become the key to solving the above problems and promoting the progress of the field of drug hepatotoxicity evaluation.

[0005] Therefore, there is an urgent need for a microfluidic chip for co-culturing multiple liver cells to solve the above problems. Summary of the Utility Model

[0006] For this reason, the utility model provides a microfluidic chip for co-culturing multiple liver cells to solve the problems of complex liver structure and difficult experimental observation and operation in the prior art.

[0007] To achieve the above object, the present utility model provides the following technical solutions:

[0008] According to a first aspect of the present utility model, there is provided a microfluidic chip for multi - cell co - culture of the liver, comprising:

[0009] A perfusion layer provided with a first culture medium inlet and a culture medium outlet; and

[0010] A culture layer provided with a micro - perfusion liver chip;

[0011] The perfusion layer and the culture layer are bonded and formed.

[0012] Further, there are 2 sets of the micro - perfusion liver chips, and each set of micro - perfusion liver chips includes 1 set of micro - perfusion network systems.

[0013] Further, the micro - perfusion network system includes a micro - perfusion channel, a multi - functional cell co - culture area, and a liquid storage pool. The micro - perfusion channel, the multi - functional cell co - culture area, and the liquid storage pool are connected and communicated, and the micro - perfusion network system is connected and communicated with the first culture medium inlet and the culture medium outlet.

[0014] Further, the multi - functional cell co - culture area includes: an LX - 2 culture area, an HUVEC culture area, a HepaRG culture area, and a THP - 1 culture area, and the areas of the 4 cell culture areas correspond to the quantity ratio of hepatic stellate cells, hepatic sinusoidal endothelial cells, hepatocytes, and Kupffer cells in the liver.

[0015] Further, the micro - perfusion channel includes a second culture medium inlet and a perfusion channel. The second culture medium inlet is vertically connected to the end of the perfusion channel, and the second culture medium inlet is connected and communicated with the first culture medium inlet.

[0016] Further, the perfusion channel includes:

[0017] A perfusion inlet, with 1 provided. One end of the perfusion inlet is connected and communicated with the second culture medium inlet, and the other end of the perfusion inlet is connected and communicated with a perfusion buffer area;

[0018] A perfusion buffer area surrounding the outside of the 4 cell culture areas; and

[0019] Four perfusion outlets, with one end of each perfusion outlet connected and communicated with the perfusion buffer area, and the other end of each perfusion outlet respectively connected and communicated with the inner cavities of the 4 cell culture areas.

[0020] Further, it further includes a fence. The fence is arranged between the 4 cell culture areas, and the fence is connected to the side walls of the cell culture areas to enclose a closed area.

[0021] Further, the fence divides the side wall of the multi-functional cell co-culture area into an outer wall and an inner wall. The outer wall is of the same height as the fence, and the inner wall is lower than the fence.

[0022] Further, the inner walls of the 4 cell culture areas and the fences on both sides of the cell culture area enclose a liquid storage pool. The cell culture area is communicated with the liquid storage pool, and the liquid storage pool is communicated with the culture medium outlet of the perfusion layer.

[0023] Further, different cell culture areas are communicated with each other through perfusion channels.

[0024] The utility model has the following advantages:

[0025] 1. The microfluidic chip proposed in this application optimizes the area design of the cell culture area by accurately simulating the types and distribution ratios of cells in the liver, ensuring that the main liver function cells such as hepatocytes, Kupffer cells, hepatic sinusoidal endothelial cells, and hepatic stellate cells can grow in an environment matching their in-vivo ratios, thereby maintaining the specific functions and phenotypic expressions of these cells. It guarantees the nutrition and microenvironment of each main liver function cell, which helps to maintain its specific phenotype. At the same time, it can simulate the blood flow in the body, conduct signal communication between liver organs, and fully simulate the in-vivo environment and interactions of liver organ diseases.

[0026] 2. The liver multi-cell co-culture microfluidic chip of this application customizes the sizes of each culture chamber according to the actual ratio of parenchymal cells to non-parenchymal cells in the liver. This not only promotes more physiologically relevant interactions between different cell types but also facilitates the direct observation and analysis of the interaction status and functional performance between parenchymal cells and non-parenchymal cells. The chip of this application not only strengthens the understanding of the interaction mechanism of liver cells but also provides a more accurate and intuitive experimental platform for researching the pathogenesis of liver diseases, drug screening, and efficacy evaluation. Description of the Drawings

[0027] In order to more clearly illustrate the embodiments of the present utility model or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings described below are only exemplary, and for those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained based on the provided drawings.

[0028] The structures, proportions, sizes, etc. shown in this specification are only used to match the content disclosed in the specification for those familiar with this technology to understand and read, and are not used to limit the implementation conditions of the present utility model. Therefore, they do not have substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size, without affecting the efficacy that the present utility model can produce and the purpose that can be achieved, should still fall within the scope covered by the technical content disclosed by the present utility model.

[0029] Figure 1 It is a schematic diagram of the overall structure of a microfluidic chip for multi - cell co - culture of the liver provided by the present utility model;

[0030] Figure 2 It is a schematic diagram of the perfusion layer structure provided by the present utility model;

[0031] Figure 3 It is a schematic diagram of the micro - perfusion liver chip structure provided by the present utility model;

[0032] In the figure: 1. Perfusion layer; 11. First culture medium inlet; 12. Culture medium outlet; 2. Culture layer; 21. Micro - perfusion liver chip; 211. Micro - perfusion network system; 212. Micro - perfusion channel; 2121. Second culture medium inlet; 2122. Perfusion channel; 21221. Perfusion inlet; 21222. Perfusion buffer zone; 21223. Perfusion outlet; 213. Multi - functional cell co - culture area; 2131. LX - 2 culture area; 2132. HUVEC culture area; 2133. HepaRG culture area; 2134. THP - 1 culture area; 214. Fence; 215. Liquid storage pool. Specific embodiments

[0033] The following specific embodiments illustrate the implementation manners of the present utility model. Those familiar with this technology can easily understand other advantages and effects of the present utility model from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present utility model, rather than all of them. Based on the embodiments in the present utility model, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present utility model.

[0034] This application provides a microfluidic chip for liver multicellular co - culture, including a perfusion layer 1 and a culture layer 2. The perfusion layer 1 is successively provided with a first culture medium inlet 11 and a culture medium outlet 12, and the diameters of both the first culture medium inlet 11 and the culture medium outlet 12 are 1.58 mm. The first culture medium inlet 11 is the inlet for fresh culture medium required for cell culture, while the culture medium outlet 12 is responsible for removing metabolic wastes to maintain the freshness and stability of the culture environment. The culture layer 2 is provided with a micro - perfusion liver chip 21, which is the core component of this system. It not only contains carefully designed micro - perfusion channels 212 to support multicellular co - culture, but also precisely replicates the micro - environmental structure inside the liver, providing an in vitro model that highly simulates real liver tissue. The perfusion layer 1 and the culture layer 2 are bonded and formed into a stable and functionally clear double - layer structure, ensuring the sealing of the entire microfluidic system and the convenience of operation.

[0035] The overall size of the microfluidic chip is 125 ± 0.5 mm in length, 85 ± 0.25 mm in width, and 7 mm in height (perfusion layer 1: 2 ± 0.5 mm, culture layer 2: 5 ± 0.5 mm). And the materials of both the perfusion layer 1 and the culture layer 2 of the microfluidic chip are polymers of transparent and breathable biocompatible material polydimethylsiloxane. Its transparency is convenient for directly observing the growth state and behavior of cells, and its breathability is conducive to gas exchange to maintain an appropriate oxygen level for cell culture. The polymer of polydimethylsiloxane can be sterilized by high - temperature and high - pressure treatment, ensuring the aseptic state of the microfluidic chip. Before use, it can be washed several times with culture medium and soaked overnight for standby to further remove harmful substances or impurities that may remain on the material surface, making the microfluidic chip reach the best preparation state before formal use.

[0036] Among them, as Figure 1 shown, the micro - perfusion liver chip 21 is provided with 2 groups of the same size, and each group of micro - perfusion liver chips 21 contains 1 set of micro - perfusion network system 211. Specifically, the micro - perfusion network system 211 includes micro - perfusion channels 212, a multi - functional cell co - culture area 214, and a liquid storage pool 215, and the micro - perfusion channels 212, the multi - functional cell co - culture area 214, and the liquid storage pool 215 are connected and communicated. The design of each group of micro - perfusion network systems 211 follows the proportion of cell types in the liver, ensuring the physiological relevance of the cell culture environment; as Figure 2 and Figure 3As shown, the multi-functional cell co-culture area 214 includes 4 cell culture areas, and the areas of the 4 cell culture areas correspond to the quantity ratio of hepatocytes, Kupffer cells, hepatic sinusoidal endothelial cells, and hepatic stellate cells in the liver (i.e., 60:15:20:5), and are respectively the HepaRG (human hepatocyte cell line) culture area 2133, the THP-1 (human acute monocytic leukemia cell line) culture area 2134, the HUVEC (human umbilical vein endothelial cell) culture area 2132, and the LX-2 (human hepatic stellate cell) culture area 2131. The diameter of the HepaRG culture area 2133 is 26 mm, corresponding to the dominant position of hepatocytes in the liver; the diameter of the THP-1 culture area 2134 is 13 mm, corresponding to immune cells such as Kupffer cells; the diameter of the LX-2 culture area 2131 is 9 mm, corresponding to hepatic stellate cells; the diameter of the HUVEC culture area 2132 is 17 mm, corresponding to hepatic sinusoidal endothelial cells.

[0037] By arranging these different types of cell culture areas in a specific ratio and position, the micro-perfusion liver chip 21 can not only promote direct cell-cell interactions, but also simulate blood flow through the micro-perfusion channels 212 to achieve effective exchange of nutrients and metabolites, as well as precise delivery and effect evaluation of drugs or toxins. The integration of the reservoir 215 further enhances the system's ability to regulate the culture environment, ensuring long-term stability of cell culture and repeatability of experiments. In the design of this application, the two groups of micro-perfusion liver chips 21 are arranged in parallel, which not only provides the possibility of a control group for experiments, enhances the reliability and persuasiveness of experimental data, but also facilitates large-scale screening experiments or parallel comparison studies, making it convenient to conduct experiments under multiple conditions simultaneously, accelerating the research process, and improving research efficiency.

[0038] Specifically, the micro-perfusion channel 212 includes a second culture medium inlet 2121 and a perfusion channel 2122. The second culture medium inlet 2121 is vertically connected to the end of the perfusion channel 2122, and the second culture medium inlet 2121 corresponds to the first culture medium inlet 11. When the perfusion layer 1 and the culture layer 2 are bonded together, the second culture medium inlet 2121 and the first culture medium inlet 11 are in a connected state. In this state, the culture medium can be injected into the micro-perfusion channel 212 through the first culture medium inlet 11 of the perfusion layer 1 and the second culture medium inlet 2121 of the culture layer 2. As the core flow path of the entire system, the micro-perfusion channel 212 is not only responsible for transporting nutrient-rich culture medium, but also bears the responsibility of regulating the hydrodynamic environment, simulating the flow patterns of blood and lymph in the liver.

[0039] Among them, the perfusion channel 2122 includes: a perfusion inlet 21221, with at least 1 being provided. Preferably, in this application, 1 perfusion inlet 21221 is provided in 1 set of micro-perfusion network system 211. The length of the perfusion inlet 21221 is 2.37 mm, the width is 0.5 mm, and the height is 1 mm. This design achieves minimizing the physical interference to the cell culture system while ensuring sufficient fluid flux to quickly and evenly deliver the culture medium into the system.

[0040] One end of the perfusion inlet 21221 is connected to the second culture medium inlet 2121, and the other end is connected to the perfusion buffer zone 21222. This design facilitates liquid perfusion distribution to control the liquid perfusion of the culture chamber and realizes high-throughput culture of cells or microorganisms. The perfusion buffer zone 21222 is in a channel shape and surrounds the outside of the multi-functional cell co-culture area 214. Its outer diameter is 52 mm, the inner diameter is 50 mm, and the height is 1 mm. This design ensures sufficient volume to store and regulate the culture medium flow rate and also ensures that it is adjacent to the multi-functional cell co-culture area 214, enabling it to quickly respond to changes in the metabolic requirements of cells.

[0041] As Figure 3 shown, 4 perfusion outlets 21223 are provided. One end of the perfusion outlet 21223 is connected to the perfusion buffer zone 21222, and the other end of the perfusion outlet 21223 is respectively connected to the inner cavity of the multi-functional cell co-culture area 214. As a bridge connecting the perfusion inlet 21221 and multiple perfusion outlets 21223, the perfusion buffer zone 21222 not only helps maintain the stability of fluid dynamics but also can serve as a temporary liquid storage space when necessary to adjust the perfusion speed and avoid causing instantaneous pressure fluctuations directly to the multi-functional cell co-culture area 214.

[0042] This design realizes the uniform distribution of the culture medium in the multi-functional cell co-culture area 214, ensuring that all cells can obtain consistent nutrient supply and metabolite excretion opportunities. By dispersing the outlets, the problem of excessive or too low local fluid shear force can be effectively reduced, simulating the diversity of the in-vivo microenvironment. This design not only improves the efficiency and quality of cell culture but also provides a high degree of flexibility and control precision.

[0043] Among them, it also includes a fence 214, as Figure 2, the fence 214 is arranged between the 4 multi-functional cell co-culture areas 214, and the fence 214 is connected to the side wall of the multi-functional cell co-culture area 214. The inner wall and the outer wall of the multi-functional cell co-culture area 214 are isolated by its height difference, forming a differential space to enclose a closed area. Specifically, the outer wall is at the same height as the fence 214, with a height of 5 mm; the inner wall is lower than the fence 214, with a height of 1 mm. The inner walls of the 4 multi-functional cell co-culture areas 214 and the fences 214 on both sides of the multi-functional cell co-culture area 214 enclose a liquid storage pool 215, and the multi-functional cell co-culture area 214 is communicated with the liquid storage pool 215.

[0044] When performing cell perfusion culture, the liquid storage pool 215 serves as a reserve space for the culture medium, which can continuously provide fresh nutrients for the multi-functional cell co-culture area 214, while receiving and diluting the metabolic wastes flowing out from the multi-functional cell co-culture area 214. Through the connection with the multi-functional cell co-culture area 214, the circulation and update of the culture medium are realized, maintaining the ideal environment required for cell growth. And the connection design between the multi-functional cell co-culture area 214 and the liquid storage pool 215 promotes the flow of the culture medium, and this flow can generate a gentle fluid shear force, simulating the blood flow or other body fluid flow conditions in the physiological environment.

[0045] The relatively large volume of the liquid storage pool 215 also helps to stabilize the temperature, pH value and osmotic pressure of the culture medium, reducing the impact of environmental fluctuations on cells. Especially when external conditions change, the liquid storage pool 215 can serve as a buffer system to maintain the relative constancy of the environment in the multi-functional cell co-culture area 214. By adding, replacing or adjusting the liquid through the liquid storage pool 215, compared with directly operating on the multi-functional cell co-culture area 214, it can reduce the direct interference with cells and improve the controllability and repeatability of the experiment. Due to the height difference between the inner and outer side walls, different microenvironments can be created. For example, the shallower area formed at the lower part of the inner wall may be more suitable for cell adhesion and observation, while the deep liquid storage pool 215 focuses on maintaining liquid exchange and environmental stability. This differential design increases the flexibility and complexity of the microfluidic chip.

[0046] Among them, the liquid storage pool 215 corresponds to the culture medium outlet 12 of the perfusion layer 1. This design can introduce the culture medium containing drugs or reagents into the multi-functional cell co-culture area 214 through the first culture medium inlet 11 of the perfusion layer 1 when performing drug screening or specific treatment, and the liquid storage pool 215 can serve as a place to recover or dilute these substances, facilitating subsequent analysis and treatment; it is an important part to realize the nutrient supply, metabolite clearance, environmental control and experimental operation facilitation in the cell perfusion culture system.

[0047] The microperfusion network system 211, through its precise pipeline design, ensures that after the culture medium enters from the second culture medium inlet 2121, it can be evenly and effectively distributed to the multi-functional cell co-culture area 214, and then flows back to the storage pool 215 through the perfusion channel 2122, forming a closed and self-circulating culture system. The storage pool 215, as the "heart" of the entire system, not only stores the culture medium, but also provides stable environmental parameters through its large volume, ensuring the long-term stability of cell culture and the repeatability of experiments.

[0048] Steps for using the microfluidic chip for liver multi-cell co-culture:

[0049] 1. Sterilization and pretreatment: The microfluidic chip is a polymer of the transparent and breathable biocompatible material polydimethylsiloxane, which can be sterilized by high-temperature and high-pressure treatment to ensure a sterile state. Subsequently, it is washed and soaked overnight with the culture medium multiple times to remove any potential contaminants and prepare for cell culture.

[0050] 2. Assembly structure: The perfusion layer 1 is bonded to the culture layer 2 to form a double-layer structure, ensuring tightness.

[0051] 3. Perfusion channel connection: The perfusion layer 1 is provided with a first culture medium inlet 11, a culture medium outlet 12, and the microperfusion channel 212 network of the culture layer 2, including a perfusion inlet 21221, a perfusion buffer area 21222, a perfusion outlet 21223, and a storage pool 215, forming a closed and self-circulating microfluidic system.

[0052] The second culture medium inlet 2121 is vertically connected to the end of the perfusion channel 2122 and is connected to the first culture medium inlet 11, ensuring that the culture medium can enter the microperfusion channel 212 from the first culture medium inlet 11 and the second culture medium inlet 2121 and flow to the multi-functional cell co-culture area 214.

[0053] 4. Cell seeding: According to the ratio of cell types in the liver (hepatocytes: Kupffer cells: hepatic sinusoidal endothelial cells: hepatic stellate cells = 60:15:20:5), HepaRG, THP-1, HUVEC, and LX-2 cells are seeded into the corresponding cell culture areas respectively.

[0054] 5. Perfusion culture: Start the culture medium circulation. Fresh culture medium enters through the first culture medium inlet 11 and the second culture medium inlet 2121, is evenly distributed to each cell culture area through the microperfusion channel 212 to provide nutrients, and at the same time, the waste products generated by cell metabolism return to the storage pool 215 through the perfusion outlet 21223 to achieve cyclic renewal.

[0055] 6. Environmental control: The liquid storage tank 215 serves as a buffer to regulate the volume, temperature, pH value, and osmotic pressure of the culture medium, maintaining the stability of the cell culture environment; through the design of the perfusion channel 2122, the hydrodynamic is controlled to simulate the in-vivo blood flow.

[0056] Although the present utility model has been described in detail with general descriptions and specific embodiments above, based on the present utility model, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present utility model all fall within the scope of protection required by the present utility model.

Claims

1. A microfluidic chip for multi-cell co-culture of liver, characterized in that: include: The perfusion layer (1) is provided with a first culture solution inlet (11) and a culture medium outlet (12); and A culture layer (2) provided with a micro-perfusion liver chip (21); The perfusion layer (1) and the culture layer (2) are bonded and formed.

2. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 1, characterized in that: The micro-perfusion liver chip (21) is provided in two groups, and each group of micro-perfusion liver chips (21) comprises a set of micro-perfusion network system (211).

3. The microfluidic chip for liver multi-cell co-culture as claimed in claim 2, characterized in that: The micro-perfusion network system (211) comprises a micro-perfusion channel (212), a multifunctional cell co-culture area (213) and a liquid reservoir (215); the micro-perfusion channel (212), the multifunctional cell co-culture area (213) and the liquid reservoir (215) are connected, and the micro-perfusion network system (211) is connected to the first culture fluid inlet (11) and the culture medium outlet (12).

4. The microfluidic chip for liver multi-cell co-culture as claimed in claim 3, characterized in that: The multifunctional cell co-culture area (213) includes: an LX-2 culture area (2131), a HUVEC culture area (2132), a HepaRG culture area (2133) and a THP-1 culture area (2134), and the areas of the four cell culture areas correspond to the number ratio of hepatic stellate cells, hepatic sinusoidal endothelial cells, hepatic parenchymal cells and Kupffer cells in the liver.

5. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 4, characterized in that: The micro-perfusion channel (212) comprises a second culture fluid inlet (2121) and a perfusion channel (2122), wherein the second culture fluid inlet (2121) is vertically connected to the end of the perfusion channel (2122), and the second culture fluid inlet (2121) is connected to the first culture fluid inlet (11).

6. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 5, characterized in that: The perfusion channel (2122) comprises: A perfusion inlet (21221) is provided, one end of the perfusion inlet (21221) is connected to the second culture fluid inlet (2121), and the other end of the perfusion inlet (21221) is connected to the perfusion buffer (21222); a perfusion buffer (21222), surrounding the outside of the four cell culture areas; and There are four perfusion outlets (21223), one end of each of which is connected to the perfusion buffer zone (21222), and the other end of each of which is connected to the inner cavities of four cell culture zones.

7. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 4, characterized in that: It also includes a fence (214), which is arranged between the four cell culture areas, and the fence (214) is connected to the side walls of the cell culture area to form a closed area.

8. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 7, characterized in that: The fence (214) divides the side wall of the multifunctional cell co-culture area (213) into an outer wall and an inner wall. The outer wall is at the same height as the fence (214), and the inner wall is lower than the fence (214).

9. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 8, characterized in that: The inner walls of the four cell culture areas and the fences (214) on both sides of the cell culture areas together form a liquid reservoir (215); the cell culture areas are connected to the liquid reservoir (215), and the liquid reservoir (215) is connected to the culture medium outlet (12) of the perfusion layer (1).

10. The microfluidic chip for multi-cell co-culture of liver as claimed in claim 9, characterized in that: Different cell culture areas are connected via perfusion channels (2122).

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