Skin-liver organoid microfluidic co-culture chip and culture method thereof

Abstract

The application provides a skin-liver organoid microfluidic co-culture chip, which comprises a skin culture area, a liver culture area and a plug, the skin culture area and the liver culture area are communicated through a co-culture channel, and a plug hole is arranged in the co-culture channel, the plug can be inserted into the plug hole, and the plug is a co-culture plug or a separate culture plug.The skin-liver organoid microfluidic co-culture chip can realize co-culture of liver tissue and skin tissue or separate culture of liver tissue and skin tissue by inserting the plug into the plug hole.

Description

Technical Field

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

[0002] Numerous clinical and basic studies have confirmed a close pathological link between skin diseases and liver damage. Several studies suggest that this link may be bidirectional.

[0003] In psoriasis, a study published in JAMA Dermatology showed that psoriasis patients have a significantly higher probability of developing non-alcoholic fatty liver disease than healthy individuals. Furthermore, P. Vasseur's research group found that psoriatic dermatitis is associated with the progression of liver fibrosis in an imiquimod-induced mouse model of psoriasis. Similarly, the prevalence of fatty liver disease is also significantly increased in patients with atopic dermatitis.

[0004] On the other hand, liver injury can also affect skin condition. For example, a study published in Cutaneous and Ocular Toxicology by Satoshi Yokoyama's team showed that mouse models induced with liver injury exhibited impaired skin barrier function. Dr. Shikha Bansal's clinical observation of 110 patients with chronic liver disease also found that approximately 49.1% of the patients had abnormal skin pigmentation. These studies collectively support the pathological link of bidirectional regulation between the liver and skin.

[0005] Although the pathological link between the liver and skin has been widely confirmed, the current in vitro models used to study this link still have significant limitations and are insufficient to meet the needs of mechanism analysis and drug development.

[0006] Currently available organoid co-culture chips mostly focus on single-organ function simulation systems, and mature chip systems specifically designed for liver and skin co-culture have not yet emerged. The few existing liver-skin co-culture models either simply seed both types of cells into the same culture chamber, lacking a recreation of in vivo physiological structures; or they cannot achieve long-term stable co-culture and dynamic functional monitoring of liver and skin cells, making it difficult to accurately replicate the bidirectional pathological relationship between the two, and thus failing to provide a reliable in vitro experimental platform for mechanism research, drug screening, and efficacy evaluation of related diseases. Summary of the Invention

[0007] The technical problem to be solved by this invention is how to accurately simulate the in vivo physiological microenvironment of the liver-skin microenvironment in a skin-liver organoid microfluidic co-culture chip.

[0008] The present invention solves the above-mentioned technical problems through the following technical means:

[0009] The first aspect of the present invention provides a skin-liver organoid microfluidic co-culture chip, including a skin culture area, a liver culture area and a pin, wherein the skin culture area and the liver culture area are connected through a co-culture channel and a pin hole is provided in the co-culture channel, and the pin can be inserted into the pin hole. The pin is a co-culture pin or a separate culture pin.

[0010] Beneficial effects: The skin-liver organoid microfluidic co-culture chip of the present invention can realize the co-culture of liver tissue and skin tissue or the separate culture of liver tissue and skin tissue by inserting the pin into the pin hole.

[0011] This invention, by inserting a separate culture pin into the pin hole, can prevent the culture medium in the skin culture area from mixing with the culture medium in the liver culture area. It allows for independent culture of both skin and liver tissues before they mature. Once the tissues mature or the disease is successfully induced, the culture medium in the liver tissue can be mixed with the culture medium in the skin tissue, achieving liver-skin co-culture. This enables dynamic interactive microfluidic co-culture experiments and establishes the pathological link between liver-skin and psoriasis.

[0012] Preferably, the co-culture channel is provided with a first outlet channel and a second liquid inlet, and the pin hole is located at the intersection of the co-culture channel and the first outlet channel.

[0013] Beneficial effects: This invention inserts a separate culture pin into the pin hole, preventing the culture medium in the skin culture area from mixing with the culture medium in the liver culture area. The culture medium in the skin culture area flows out through the first outlet channel, achieving separate culture of skin tissue, while the culture medium in the liver culture area flows in through the second inlet, achieving separate culture of liver tissue. This invention also inserts a co-culture pin into the pin hole, ensuring that the culture medium in the skin culture area can only flow into the liver culture area through the co-culture channel, achieving liver-skin co-culture.

[0014] Preferably, the co-cultivation pin is provided with a first guide groove, and the shape of the first guide groove is "I".

[0015] Beneficial effects: This invention uses a "I"-shaped co-culture pin to ensure that the culture medium in the skin culture area can only flow into the liver culture area through the first guide channel, thus achieving liver-skin co-culture.

[0016] Preferably, the individual cultivation pin is provided with a second guide groove, and the shape of the second guide groove is "7".

[0017] Beneficial effects: The present invention uses a figure-7 shaped individual culture pin to allow the culture medium in the skin culture area to flow out through the second guide channel along the first outlet channel, thereby achieving individual culture of skin tissue.

[0018] Preferably, the skin culture area includes a first liquid inlet, a culture area, and a gas channel. The first liquid inlet is connected to one end of the culture area, and the other end of the culture area is connected to a co-culture channel. A gas channel is provided above the culture area, and a porous membrane is provided between the culture area and the gas channel.

[0019] Beneficial effects: The skin culture area of ​​the present invention enables the gas-liquid interface culture required for skin tissue.

[0020] Preferably, the liver culture area includes a vertical diversion area and a second fluid outlet, the second fluid inlet is connected to one end of the vertical diversion area, and the other end of the vertical diversion area is connected to the second fluid outlet.

[0021] Preferably, the vertical diversion zone includes a first diversion zone, a second diversion zone, and a storage zone. The first diversion zone is provided with a plurality of first diversion holes, and the second diversion zone is provided with a plurality of second diversion holes. The first diversion zone is connected to the second diversion zone through the first diversion holes, and the second diversion zone is connected to the storage zone through the second diversion holes. The storage zone is connected to the second liquid outlet.

[0022] Preferably, the number of first diversion holes is greater than the number of second diversion holes.

[0023] Beneficial effects: The liver culture area of ​​this invention simulates liver partitions through a vertical diversion zone, thus reproducing the biomimetic liver sinusoidal culture microenvironment.

[0024] Preferably, the diameter of the first diversion hole is 20um to 100um.

[0025] Beneficial effects: The present invention sets the aperture of the first diversion hole to be 20um~100um. If the aperture is less than 20um, the aperture is too small, the flow resistance increases, and the purpose of diversion cannot be achieved; if the aperture is greater than 100um, the nutrient solution cannot be collected in the first diversion zone, and the purpose of mixing cannot be achieved.

[0026] Preferably, the diameter of the second diversion hole is 80um~200um.

[0027] Beneficial effects: The present invention sets the diameter of the second diversion hole to be larger than that of the first diversion hole in order to reduce resistance and promote the flow of liquid from the periphery to the center, forming a microflow from the periphery to the center.

[0028] Preferably, the pore size of the porous membrane is 400um~1000um.

[0029] Beneficial effects: The present invention sets the pore size of the porous membrane to be relatively large, so as to provide nutrients to the nutrient solution in the skin culture area and improve the excretion of metabolites.

[0030] Preferably, the skin-liver organoid microfluidic co-culture chip is made of PLA material and is integrally formed by 3D printing.

[0031] The second aspect of the present invention provides a method for culturing the above-mentioned skin-liver organoid microfluidic co-culture chip. First, a separate culture pin is inserted into the pin hole, and skin tissue is cultured in the skin culture area by circulating skin culture medium, and liver tissue is cultured in the liver culture area by circulating liver culture medium. Then, the separate culture pin is replaced with a co-culture pin to realize liver-skin co-culture. Attached Figure Description

[0032] Figure 1 This is an overall structural diagram of the skin culture area and liver culture area in Embodiment 1 of the present invention; Figures 2-4 This is a partial structural diagram of the skin culture area in Embodiment 1 of the present invention; Figures 5-7 This is a partial structural diagram of the liver culture area in Embodiment 1 of the present invention; Figure 8 This is a structural diagram of the pin in Embodiment 1 of the present invention; Figure 9 This is a physical image of the dermal layer of skin 3D printed according to Application Example 1 of the present invention; Figure 10 This is a microscopic image of skin tissue induced to form psoriasis, as described in Application Example 1 of this invention; Figure 11 This is a physical image of the 3D-printed liver tissue from Application Example 1 of this invention; Figure 12 This is a microscopic image of liver tissue from Application Example 1 of this invention; Figure 13 This is a bright-field image of the liver tissue scaffold in Application Example 1 of the present invention; Figure 14 This is a staining image of liver tissue from Application Example 1 of the present invention; Figure 15 This is a staining image of liver tissue from Application Example 2 of the present invention; Figure 16 This is a staining image of liver tissue from Comparative Application Example 1 of the present invention. Detailed Implementation

[0033] 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.

[0034] Unless otherwise specified, all test materials and reagents used in the following examples are commercially available.

[0035] 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.

[0036] Example 1 according to Figure 1-8 As shown, this embodiment provides a skin-liver organoid microfluidic co-culture chip, including a skin culture area 10, a liver culture area 20, and a pin 30. The skin culture area 10 and the liver culture area 20 are connected by a co-culture channel 11, which is linear. The co-culture channel 11 has a first outlet channel 16 and a second fluid inlet 21. A pin hole 12 is located at the intersection of the co-culture channel 11 and the first outlet channel 16. The pin hole 12 matches the pin 30, which is cylindrical. This embodiment provides... Two types of pins 30 are provided. The first type of pin 30 is a co-culture pin. The bottom of the co-culture pin is provided with a first guide groove 31. The first guide groove 31 is in the shape of an "I". When the co-culture pin is inserted into the pin hole 12, the nutrient solution of the skin culture area 10 can only flow into the liver culture area 20 through the co-culture channel 11 and the first guide groove 31, so as to achieve the mixing of the culture solution of liver tissue and the culture solution of skin tissue, and to achieve liver-skin co-culture, thereby carrying out dynamic interactive microfluidic co-culture experiments.

[0037] The second type of pin is a separate culture pin. The bottom of the separate culture pin is provided with a second guide groove 32. The shape of the second guide groove 32 is "7". When the separate culture pin is inserted into the pin hole 12, the flow direction of the culture medium in the skin culture area 10 can be changed, so that the culture medium in the skin culture area 10 flows out along the first outlet channel 16 through the second guide groove 32, realizing the self-circulation culture of the culture medium in the skin culture area 10, thereby realizing the separate culture of skin tissue. Since the culture medium in the skin culture area 10 cannot be mixed with the culture medium in the liver culture area 20, the culture medium in the liver culture area 20 enters the liver culture area 20 through the second liquid inlet for self-circulation culture, thereby realizing the separate culture of liver tissue.

[0038] The skin culture area 10 includes a first liquid inlet 13, a culture area 14, a gas channel 15, and a porous membrane 17. The first liquid inlet 13 is connected to one end of the culture area 14. In this embodiment, there are two culture areas 14 arranged side by side and connected to each other. The other end of the culture area 14 is connected to the co-culture channel 11. A porous membrane 17 is laid on top of the culture area 14. The pore size of the porous membrane 17 is 500 μm. A gas channel 15 is provided above the porous membrane 17, covering the culture area 14. In this embodiment, there are four gas channels 15.

[0039] In this embodiment, skin tissue is cultured in the skin culture zone 10 using a gas-liquid two-phase interface culture to simulate the physiological state of skin tissue in contact with the external environment, thus more realistically simulating the growth process of skin tissue.

[0040] The liver culture area 20 includes a vertical diversion area 22 and a second liquid outlet 23. The second liquid inlet 21 is connected to one end of the vertical diversion area 22, and the other end of the vertical diversion area 22 is connected to the second liquid outlet 23.

[0041] The vertical diversion zone 22 includes a first diversion zone 221, a second diversion zone 222, and a storage zone 223. The first diversion zone 221 is provided with a plurality of first diversion holes 221a, and the second diversion zone 222 is provided with a plurality of second diversion holes 222a. The first diversion zone 221 is connected to the second diversion zone 222 through the first diversion holes 221a, and the second diversion zone 222 is connected to the storage zone 223 through the second diversion holes 222a. The storage zone 223 is connected to the second liquid outlet 23.

[0042] The first diversion area 221 is concave circular in shape, and multiple first diversion holes 221a are arranged along the circumference of the first diversion area 221, such as... Figure 5 As shown, 12 first diversion holes 221a are evenly arranged at the circular edge of the first diversion zone 221, and 6 first diversion holes 221a are evenly arranged at the center. There are no first diversion holes 221a at the center. In this embodiment, a total of 24 first diversion holes 221a are arranged in the first diversion zone 221.

[0043] The second diversion area 222 is concave circular in shape, such as... Figure 6 As shown, a second diversion hole 222a is provided at the center of the second diversion zone 222, and then six second diversion holes 222a are evenly provided at a distance of 4cm from the center.

[0044] The first diversion zone 221, the second diversion zone 222, and the storage zone 223 are located in the same position. However, the first diversion orifice 221a and the second diversion orifice 222a are staggered in the first diversion zone 221 and the second diversion zone 222, respectively, with the number of first diversion orifices 221a exceeding the number of second diversion orifices 222a. This arrangement is intended to simulate the fluid microenvironment of the liver lobule. Under physiological conditions, the liver lobule is hexagonal, with the arteries / veins of the liver lobule located on its periphery. Blood flow in the liver is from the periphery of the liver lobule towards the center, flowing out through the central vena cava. Therefore, the first diversion orifice 221a simulates the artery / vein region of the liver lobule on its periphery, while the second diversion orifice 222a simulates the central vena cava of the liver lobule.

[0045] The first diversion orifice 221a has a pore size of 20µm to 80µm; the second diversion orifice 222a has a pore size of 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.

[0046] In this embodiment, the nutrient solution in the liver culture area 20 enters the first diversion area 221 through the second liquid inlet 21, so that the liver tissue fluid enters the second diversion area 222 through multiple first diversion holes 221a, and then enters the storage area 223 through multiple second diversion holes 222a, thereby realizing the cyclic culture of liver tissue or the dual-tissue cyclic culture of skin tissue and liver tissue.

[0047] Application Example 1 This application example uses the skin-liver organoid microfluidic co-culture chip from Example 1, and specifically includes the following steps: Preparation of S1 skin tissue: (1) Prepare a 14% (w / w) gelma hydrogel using physiological saline as the solvent. Mix the gelma hydrogel with 10 mg / ml rat tail collagen at a volume ratio of 1:1 to obtain a mixed solution. Add fibroblasts, endothelial cells, TH17 cells, and 0.5% (w / w) LAP photoinitiator to the mixed solution to make the concentration of fibroblasts 1×10⁻⁶. 6 2 × 10⁶ cells / ml, endothelial cell concentration 6 The concentration of TH17 cells was 1×10⁶ cells / ml. 5 Bio-ink for skin models was obtained by using samples per ml.

[0048] (2) The bio-ink of the skin model is printed out using an extrusion 3D printer to obtain the skin model as shown in the figure. Figure 9 As shown, the skin model has a fence-like structure, which can effectively fix the seeded cells and prevent them from shifting, falling off or collapsing during the flow of culture medium and the gas-liquid interface culture process. This ensures that the skin organoid structure is intact and the layers are clear. The fence can limit the cell growth space, so that the cells can proliferate and differentiate in an orderly manner.

[0049] (3) The dermis of the skin was irradiated with a UV lamp (504nm) for 1 min to fully cross-link and solidify it. After solidification, it was cultured in DMEM complete medium for 6 h and then inoculated with human epidermal keratinocytes (1×10⁻⁶). 7 (each cell / ml), cultured on differentiation medium for 24 hours to obtain an early skin tissue model.

[0050] S2. A separate culture pin is inserted into the pin hole 12. Then, the culture medium (i.e., the skin tissue model) in the skin culture area 10 is introduced into the culture area 14 through the first liquid inlet 13 for skin tissue culture. During culture, the gas channel 15 provides the air needed by the skin tissue. The air enters the culture area 14 through the gas channel 15 and the porous membrane 17 to promote the differentiation of the epidermal layer of the skin tissue, thereby realizing gas-liquid phase culture and simulating the real microenvironment of skin growth. The function of the porous membrane 17 is to support the skin tissue, and the skin tissue carries away metabolites through the circulating nutrient solution.

[0051] Under the action of the co-culture channel 11 and the second guide groove 32 of the individual culture pin, the culture medium in the skin culture area 10 flows out through the first outlet channel 16, and then enters the culture area 14 through the sterile tubing and the first liquid inlet 13. The sterile tubing is equipped with a peristaltic pump, and the first outlet channel 16 and the first liquid inlet 13 are connected through the sterile tubing and the peristaltic pump, so as to realize the self-circulating microfluidic culture of the culture medium in the skin culture area 10 and realize the individual culture of skin tissue.

[0052] The cultivation process is divided into four stages, as follows: Phase 1: Basic training (12 hours) The nutrient solution in the first stage is a DMEM culture medium containing 1% penicillin and streptomycin by volume, which enters the culture zone 14 through the first liquid inlet 13.

[0053] Phase 2: IL-22 induces keratinocyte proliferation (12h) The second stage of nutrient solution: 100 ng / mL of IL-22 is added to the nutrient solution of the first stage, and it enters the culture zone 14 through the first liquid inlet 13.

[0054] Phase 3: TNF-α-induced inflammatory microenvironment (6h) The nutrient solution for the third stage: The nutrient solution for the second stage is replaced with DMEM culture medium containing 10 ng / mL TNF-α, which enters the culture zone 14 through the first liquid inlet 13.

[0055] Phase 4: Differentiation and culture to consolidate pathological features (7 days) The fourth stage nutrient solution: Replace the third stage nutrient solution with 100x HKGS (human keratinocyte growth factor) and 46x 1.3mM Ca. 2+ Differentiation culture medium solution of 50 mg / mL vitamin C at 1000x.

[0056] The final cultured skin tissue, such as Figure 10As shown in the figure, after skin tissue induces psoriasis, a white film-like substance (stratum corneum) forms on the surface. At this time, the intercellular connections of the epidermal layer become loose, the epidermal layer is damaged, and the skin's water retention capacity decreases.

[0057] Preparation of S3 liver tissue: (1) Mix 5% gelatin, 1% sodium alginate, 1 mg / ml fibrinogen, 30 ug / ml laminin, human liver progenitor cells, human hepatic stellate cells, and hepatic sinusoidal endothelial cells to obtain a final concentration of 5 × 10⁻⁶ human liver progenitor cells. 6 The concentration of human hepatic stellate cells was 5 × 10⁶ cells / ml. 5 The concentration of hepatic sinusoidal endothelial cells was 1×10⁻⁶ cells / ml 6 Liver tissue bio-ink per ml.

[0058] (2) Liver tissue bio-ink was printed using an extrusion 3D printer to obtain liver tissue as shown in the image. Figure 11 As shown, liver tissue was immersed in a 2% CaCl2 solution for 5 min for cross-linking, and then rinsed three times with physiological saline to remove residual CaCl2, finally obtaining the treated liver tissue.

[0059] S4 While culturing skin tissue, the culture medium (containing the processed liver tissue) in the liver culture area 20 is introduced into the vertical diversion area 22 through the second liquid inlet 21 for culturing. The culture medium first flows into the first diversion area 221 through the second liquid inlet 21, then flows into the second diversion area 222 through multiple first diversion holes 221a, and then flows into the storage area 223 through multiple second diversion holes 222a for culturing. Finally, the culture medium flows out through the second liquid outlet 23 and then flows into the first diversion area 221 through the second liquid inlet 21, realizing the self-circulating microfluidic culture of the culture medium in the liver culture area 20.

[0060] The culture medium used during the cultivation process is DMEM medium containing 10% fetal bovine serum and 1% penicillin-antibiotics. The amount of culture medium injected is sufficient to cover the vertical shunt zone 22, ensuring that both the first shunt zone 221 and the second shunt zone 222 are in contact with the culture medium. The self-circulation frequency is maintained at 1 / 2 of the culture medium every 24 hours to maintain a balance between nutrient concentration and metabolic waste removal.

[0061] The culture time is 10 days, and the final cultured liver tissue is as follows: Figure 12 and Figure 13 As shown in the image, liver tissue forms clusters of hepatocytes. Figure 14 As can be seen, the cell survival rate is very high, with green representing live cells and red representing dead cells.

[0062] Application Example 2 This application example uses the skin-liver organoid microfluidic co-culture chip of Example 1, specifically including the following steps: Based on the culture of Example 1, skin tissue was induced to form psoriasis and liver tissue with very high cell inventory, and the pathological association between skin psoriasis and liver tissue was studied, as follows: The individual culture pin is removed and replaced with a co-culture pin, which is then inserted into the pin hole 12. At this time, the culture medium in the skin culture area 10 flows into the liver culture area 20 under the action of the co-culture channel 11 and the first guide groove 31 of the co-culture pin. The culture medium in the skin culture area 10 mixes with the culture medium in the liver culture area 20 to obtain a mixed solution. This mixed solution enters the vertical diversion area 22. The mixed solution first flows into the first diversion area 221 through the second liquid inlet 21, then flows into the second diversion area 222 through multiple first diversion holes 221a, and then flows into the storage area 223 for culture through multiple second diversion holes 222a. Finally, the mixed solution flows out through the second liquid outlet 23 and then flows back into the first diversion area 221 through the second liquid inlet 21, realizing the self-circulating microfluidic culture of the mixed solution, i.e., realizing the dynamic interactive culture of liver and skin dual tissues. The culture time is 7 days, and the final cultured liver tissue is as follows: Figure 15 As shown, after 7 days of co-culture of skin and liver tissue, lipid droplets accumulated in the liver tissue, mimicking the pathological features of psoriasis-induced fatty liver. This indicates that inflammatory factors produced by psoriasis can induce fatty liver formation in liver tissue. In the figure, red represents lipid droplets and blue represents cell nuclei.

[0063] Comparative Example 1 Comparative Example 1 provides a skin-liver organoid microfluidic co-culture chip. The difference between Comparative Example 1 and Example 1 is that the diameter of the first shunt hole is 50um~100um and the diameter of the second shunt hole is 20um~80um.

[0064] Comparative Application Example 1 This comparative application example 1 uses the skin-liver organoid microfluidic co-culture chip of Comparative Example 1. This comparative application example 1 is the same as application example 2, and the final cultured liver tissue is as follows: Figure 16 As shown, due to the change in the pore diameter of the first and second diversion holes, the culture medium is unevenly distributed, and some of the cultured tissues cannot obtain nutrients and transfer metabolic waste in time, resulting in a large number of cell deaths.

[0065] 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 skin-liver organoid microfluidic co-culture chip, characterized in that, It includes a skin culture area (10), a liver culture area (20) and a plug (30). The skin culture area (10) is connected to the liver culture area (20) through a co-culture channel (11), and a plug hole (12) is provided in the co-culture channel (11). The plug (30) can be inserted into the plug hole (12); the plug (30) is a co-culture plug or a single-culture plug.

2. The skin-liver organoid microfluidic co-culture chip according to claim 1, characterized in that, The co-culture channel (11) is provided with a first outlet channel (16) and a second liquid flow inlet (21), and the plug hole (12) is provided at the intersection of the co-culture channel (11) and the first outlet channel (16).

3. The skin-liver organoid microfluidic co-culture chip according to claim 1, characterized in that, The co-culture plug is provided with a first diversion groove (31), and the shape of the first diversion groove (31) is "one"-shaped.

4. The skin-liver organoid microfluidic co-culture chip according to claim 1, characterized in that, The single-culture plug is provided with a second diversion groove (32), and the shape of the second diversion groove (32) is "7"-shaped.

5. The skin-liver organoid microfluidic co-culture chip according to claim 1, characterized in that, The skin culture area (10) includes a first liquid flow inlet (13), a culture area (14) and a gas channel (15). The first liquid flow inlet (13) is connected to one end of the culture area (14), the other end of the culture area (14) is connected to the co-culture channel (11), the gas channel (15) is provided above the culture area (14), and a porous membrane (17) is provided between the culture area (14) and the gas channel (15).

6. The skin-liver organoid microfluidic co-culture chip according to claim 2, characterized in that, The liver culture area (20) includes a vertical diversion area (22) and a second liquid flow outlet (23). The second liquid flow inlet (21) is connected to one end of the vertical diversion area (22), and the other end of the vertical diversion area (22) is connected to the second liquid flow outlet (23).

7. The skin-liver organoid microfluidic co-culture chip according to claim 6, characterized in that, The vertical diversion area (22) includes a first diversion area (221), a second diversion area (222) and a storage area (223). The first diversion area (221) is provided with a plurality of first diversion holes (221a), the second diversion area (222) is provided with a plurality of second diversion holes (222a), the first diversion area (221) is connected to the second diversion area (222) through the first diversion holes (221a), the second diversion area (222) is connected to the storage area (223) through the second diversion holes (222a), and the storage area (223) is connected to the second liquid flow outlet (23).

8. The skin-liver organoid microfluidic co-culture chip according to claim 7, characterized in that, The number of the first diversion holes (221a) is more than that of the second diversion holes (222a).

9. The skin-liver organoid microfluidic co-culture chip according to claim 6, characterized in that, The aperture of the first diversion holes (221a) is 20um to 100um; The aperture of the second diversion holes (222a) is 80um to 200um; The aperture of the porous membrane (15) is 400um to 1000um.

10. A method for culturing a skin-liver organoid microfluidic co-culture chip as described in any one of claims 1-9, characterized in that, First, insert the single-culture plug into the plug hole, culture the skin tissue in the skin culture area (10) by circulating the skin culture solution, and culture the liver tissue in the liver culture area (20) by circulating the liver culture solution; then replace the single-culture plug with the co-culture plug to achieve liver-skin co-culture.

Images