Peritoneal chip and peritoneal in-vitro model construction method

Abstract

The invention discloses a peritoneal chip and a peritoneal in-vitro model construction method, the peritoneal chip comprises a first chip, a second chip and a third chip, the second chip is arranged between the first chip and the third chip, the first chip is provided with a peritoneal surface mesothelial tissue simulation module, the peritoneal surface mesothelial tissue simulation module is used for culturing peritoneal mesothelial cells, and the third chip is provided with a peritoneal surface mesothelial tissue simulation module. The third chip is provided with a peritoneal connective tissue simulation module, the peritoneal connective tissue simulation module is used for culturing vascular endothelial cells and fat cells, and the second chip is communicated with the peritoneal surface mesothelial tissue simulation module and the peritoneal connective tissue simulation module. According to the peritoneal chip disclosed by the invention, peritoneal mesothelial cells are cultured on the first chip to simulate peritoneal surface mesothelial tissues, vascular endothelial cells and fat cells are cultured on the third chip to simulate peritoneal connective tissues, and the peritoneal surface mesothelial tissues and the peritoneal connective tissues are communicated under the assistance of the second chip; the reconstruction of an extracorporeal peritoneal tissue interface and the reproduction of tissue functions are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of organ chips, and more specifically, to a peritoneum chip and a method for constructing an in vitro peritoneum model. Background Art

[0002] The peritoneum is a layer of serous membrane in the human abdominal cavity, usually composed of peritoneal mesothelial cells, fat cells, blood vessels and other structures. Its surface is mesothelial cells, and under the membrane is tissue containing capillaries. The peritoneum wraps the internal organs in the abdominal cavity, such as the stomach, spleen and intestines, which can reduce the damage to the organs in the abdominal cavity caused by external impact, and at the same time reduce the lateral displacement or dislocation of the organs. The mesothelial cells on the surface of the peritoneum have phagocytic ability, and the abundant capillaries in the peritoneum can transport blood to the organs in the abdominal cavity to provide them with nutrients and oxygen. The peritoneal cavity usually contains a small amount of serous fluid, which can lubricate the surface of the peritoneum and reduce friction during the movement of internal organs.

[0003] With the development of microfluidics and tissue engineering technology, organ chips that reconstruct tissues and organs on microfluidic chips have been widely reported. Organ chips are usually made of transparent materials such as polydimethylsiloxane or plexiglass. Organ chips are equipped with structures such as microchannels, microchambers or microvalves. By dynamically culturing different types of cells on organ chips, the basic structure and function of tissues and organs can be reconstructed in vitro. In addition, organ chips also have the advantages of visualization, easy integration, and customization. They are a novel platform for current biomedical research.

[0004] Although organ chips have made great progress in recent years, the production of peritoneal chips is still very difficult due to the special structure of the peritoneum, which has the mesothelial layer and submembranous connective tissue.

[0005] Therefore, there is an urgent need for a peritoneum chip to achieve peritoneal tissue interface reconstruction and tissue function reproduction. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a peritoneum chip to achieve peritoneal tissue interface reconstruction and tissue function reproduction.

[0007] Another core of the present invention is to disclose a method for constructing an in vitro peritoneal model.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] A peritoneal chip comprises a first chip, a second chip and a third chip, wherein the second chip is arranged between the first chip and the third chip;

[0010] The first chip is provided with a peritoneal surface mesothelial tissue simulation module, which is used to culture peritoneal mesothelial cells; the third chip is provided with a peritoneal connective tissue simulation module, which is used to culture vascular endothelial cells and fat cells;

[0011] The second chip is connected to the peritoneal surface mesothelial tissue simulation module and the peritoneal connective tissue simulation module.

[0012] Optionally, in the above-mentioned peritoneal chip, the peritoneal surface mesothelial tissue simulation module includes a mesothelial cell entrance, a peritoneal mesothelial tissue area and a mesothelial cell exit, and the peritoneal mesothelial tissue area is connected to the mesothelial cell entrance and the mesothelial cell exit, respectively.

[0013] Optionally, in the above peritoneal chip, the peritoneal connective tissue simulation module includes a first connective tissue area inlet, a connective tissue area and a first connective tissue area outlet, and the connective tissue area is connected to the first connective tissue area inlet and the first connective tissue area outlet respectively;

[0014] The second chip connects the peritoneal mesothelial tissue region and the connective tissue region.

[0015] Optionally, in the above-mentioned peritoneal chip, a second connective tissue area inlet and a second connective tissue area outlet are provided on the first chip, the second connective tissue area inlet is connected to the first connective tissue area inlet, and the second connective tissue area outlet is connected to the first connective tissue area outlet.

[0016] Optionally, in the above peritoneal chip, the third chip is provided with a first connective tissue region culture medium inlet, a first connective tissue region culture medium outlet, a second connective tissue region culture medium inlet and a second connective tissue region culture medium outlet;

[0017] The first connective tissue region culture medium inlet and the first connective tissue region culture medium outlet are connected through a first culture medium channel;

[0018] The second connective tissue region culture medium inlet and the second connective tissue region culture medium outlet are connected through a second culture medium channel;

[0019] The first culture medium channel and the second culture medium channel are both in communication with the connective tissue region.

[0020] Optionally, in the above peritoneal chip, a cell activity limiting component is provided in the connective tissue region to limit the infiltration of cells from the connective tissue region to the first culture medium channel and the second culture medium channel.

[0021] Optionally, in the above peritoneal chip, the cell activity limiting component includes a plurality of trapezoidal protrusions arranged in sequence to form a dam structure.

[0022] Optionally, in the above peritoneal chip, the first chip is provided with a third connective tissue region culture medium inlet, a third connective tissue region culture medium outlet, a fourth connective tissue region culture medium inlet and a fourth connective tissue region culture medium outlet;

[0023] The culture medium inlet of the third connective tissue region is communicated with the culture medium inlet of the first connective tissue region, and the culture medium outlet of the third connective tissue region is communicated with the culture medium outlet of the first connective tissue region;

[0024] The culture medium inlet of the fourth connective tissue zone is connected with the culture medium inlet of the second connective tissue zone, and the culture medium outlet of the fourth connective tissue zone is connected with the culture medium outlet of the second connective tissue zone.

[0025] Optionally, in the above peritoneal chip, a plurality of through holes are provided on the second chip.

[0026] A method for constructing an in vitro peritoneal model, using the above-mentioned peritoneal chip, comprises the following steps:

[0027] Assembling the peritoneal chip, assembling the first chip, the second chip and the third chip to form a peritoneal chip;

[0028] Constructing peritoneal connective tissue, and sequentially injecting the mixed solution and vascular endothelial cell culture medium into the peritoneal connective tissue simulation module;

[0029] Construct the peritoneal surface mesothelial tissue, and inject the mesothelial cell suspension and mesothelial cell culture medium into the peritoneal surface mesothelial tissue simulation module in sequence.

[0030] Optionally, in the above-mentioned peritoneal in vitro model construction method, the step of constructing peritoneal connective tissue specifically includes:

[0031] preparing a mixed solution, mixing a vascular endothelial cell suspension of a first preset concentration and a fat cell suspension of a second preset concentration with a bovine fibrinogen solution to obtain a multicellular suspension, and mixing the multicellular suspension with a thrombin solution to obtain a mixed solution;

[0032] injecting the mixed solution into the connective tissue area of ​​the peritoneal connective tissue simulation module;

[0033] Placing the peritoneal chip in a cell culture incubator and leaving it to stand for a first preset time to allow the mixed solution to solidify into a hydrogel;

[0034] The peritoneal chip was taken out from the cell culture incubator, and vascular endothelial cell culture medium was injected into the peritoneal connective tissue simulation module.

[0035] Optionally, in the above-mentioned peritoneal in vitro model construction method, the step of constructing peritoneal surface mesothelial tissue specifically includes:

[0036] injecting a mesothelial cell suspension of a third preset concentration into the peritoneal surface mesothelial tissue simulation module;

[0037] placing the peritoneal chip in a cell culture incubator and letting it stand for a second preset time;

[0038] The peritoneal chip was taken out from the incubator and the mesothelial cell culture medium was injected into the mesothelial tissue simulation module on the peritoneal surface.

[0039] From the above scheme, it can be seen that the peritoneal chip disclosed in the present invention cultures peritoneal mesothelial cells on the first chip to simulate peritoneal surface mesothelial tissue, cultures vascular endothelial cells and fat cells on the third chip to simulate peritoneal connective tissue, and the peritoneal surface mesothelial tissue and peritoneal connective tissue are connected and interacted with the assistance of the second chip to achieve in vitro peritoneal tissue interface reconstruction and tissue function reproduction. The peritoneal chip can be used to study the physiological changes of the peritoneum and the changes in the peritoneal tissue structure and function caused by tumor peritoneal metastasis.

[0040] The method for constructing an in vitro peritoneal model disclosed in the present invention has the same technical effect as the peritoneal chip and will not be described in detail. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0042] Figure 1 An exploded view of the peritoneal chip disclosed in an embodiment of the present invention;

[0043] Figure 2 A top view of a first chip disclosed in an embodiment of the present invention;

[0044] Figure 3 An enlarged view of the second chip disclosed in an embodiment of the present invention;

[0045] Figure 4 This is a top view of the third chip disclosed in the embodiment of the present invention.

[0046] Among them, 10, first chip, 101, mesothelial cell entrance, 102, peritoneal mesothelial tissue area, 103, mesothelial cell exit, 104, second connective tissue area entrance, 105, second connective tissue area exit, 106, third connective tissue area culture medium entrance, 107, third connective tissue area culture medium exit, 108, fourth connective tissue area culture medium entrance, 109, fourth connective tissue area culture medium exit;

[0047] 20. Second chip;

[0048] 30. The third chip, 301. The inlet of the first connective tissue area, 302. The connective tissue area, 303. The outlet of the first connective tissue area, 304. The culture medium inlet of the first connective tissue area, 305. The culture medium outlet of the first connective tissue area, 306. The first culture medium channel, 307. The culture medium inlet of the second connective tissue area, 308. The culture medium outlet of the second connective tissue area, 309. The second culture medium channel, 310. The cell activity limiting component. DETAILED DESCRIPTION

[0049] The core of the present invention is to disclose a peritoneum chip to achieve peritoneal tissue interface reconstruction and tissue function reproduction.

[0050] Another core of the present invention is to disclose a method for constructing an in vitro peritoneal model.

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] It should be noted that the various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0053] like Figure 1 As shown, an embodiment of the present invention discloses a peritoneal chip, including a first chip 10 , a second chip 20 and a third chip 30 .

[0054] The first chip 10, the second chip 20 and the third chip 30 are stacked, and the second chip 20 is arranged between the first chip 10 and the third chip 30. The first chip 10 is provided with a peritoneal surface mesothelial tissue simulation module, which is used to culture peritoneal mesothelial cells in two dimensions to simulate peritoneal surface mesothelial tissue. The third chip 30 is provided with a peritoneal connective tissue simulation module, which is used to culture vascular endothelial cells and adipocytes in three dimensions to simulate peritoneal connective tissue. The second chip 20 is connected to the peritoneal surface mesothelial tissue simulation module and the peritoneal connective tissue simulation module.

[0055] The peritoneal chip disclosed in the embodiment of the present invention cultures peritoneal mesothelial cells on the first chip 10 to simulate peritoneal surface mesothelial tissue, cultures vascular endothelial cells and adipocytes on the third chip 30 to simulate peritoneal connective tissue, and the peritoneal surface mesothelial tissue and peritoneal connective tissue are interconnected and interacted with the assistance of the second chip 20, thereby achieving in vitro peritoneal tissue interface reconstruction and tissue function reproduction. The peritoneal chip can be used to study the physiological changes of the peritoneum and the changes in the peritoneal tissue structure and function caused by tumor peritoneal metastasis.

[0056] It should be noted that the shapes of the first chip 10 and the third chip 30 can be circular, elliptical or polygonal, or chamfered on the basis of the polygon. The specific shapes can be determined according to actual needs. Figure 1 The first chip 10 and the third chip 30 are both in the shape of a rounded rectangle. The first chip 10 and the third chip 30 can have the same shape or different shapes. Preferably, the lower surface of the first chip 10 and the upper surface of the third chip 30 have the same area.

[0057] Specifically, Figure 2 As shown, the peritoneal surface mesothelial tissue simulation module includes a mesothelial cell inlet 101, a peritoneal mesothelial tissue area 102 and a mesothelial cell outlet 103. The peritoneal mesothelial tissue area 102 is connected to the mesothelial cell inlet 101 and the mesothelial cell outlet 103 through a fluid channel. The mesothelial cell suspension and the mesothelial cell culture fluid can be injected from the mesothelial cell inlet 101, flow through the peritoneal mesothelial tissue area 102, and flow out from the mesothelial cell outlet 103.

[0058] In order to ensure that the mesothelial cell inlet 101, the peritoneal mesothelial tissue area 102 and the mesothelial cell outlet 103 can store the cell culture fluid, the thickness of the first chip 10 is greater than 8 mm, preferably 10 mm.

[0059] Furthermore, if Figure 3 As shown, the peritoneal connective tissue simulation module includes a first connective tissue area inlet 301, a connective tissue area 302 and a first connective tissue area outlet 303, and the connective tissue area 302 is respectively connected to the first connective tissue area inlet 301 and the first connective tissue area outlet 303. The second chip 20 is connected to the peritoneal mesothelial tissue area 102 and the connective tissue area 302. Preferably, the peritoneal mesothelial tissue area 102 and the connective tissue area 302 have the same size, and the size of the second chip 20 is preferably greater than or equal to the size of the peritoneal mesothelial tissue area 102.

[0060] Specifically, the first connective tissue region entrance 301, the connective tissue region 302 and the first connective tissue region exit 303 are all grooves provided on the third chip 30, and the shapes are not specifically limited. The connective tissue region 302 may be a rectangular groove, the cross-sectional area may be 2mm²-8mm², preferably 5mm², and the depth may be 80μm-200μm, preferably 150μm.

[0061] In order to ensure that the first connective tissue area inlet 301, the connective tissue area 302 and the first connective tissue area outlet 303 can store the cell culture fluid while reducing the influence on the microscopic imaging effect observation, the thickness of the third chip 3 can be 1mm-3mm, preferably 2mm.

[0062] Based on the above embodiments, Figure 2 As shown, the first chip 10 is provided with a second connective tissue region inlet 104 and a second connective tissue region outlet 105, the second connective tissue region inlet 104 is connected to the first connective tissue region inlet 301, and the second connective tissue region outlet 105 is connected to the first connective tissue region outlet 303. The cell fluid and cell culture fluid for culturing vascular endothelial cells and adipocytes are injected into the first connective tissue region inlet 301 through the second connective tissue region inlet 104, and the injection is stopped when the fluid flows out from the second connective tissue region outlet 105.

[0063] In other specific embodiments, the size of the first chip 10 is smaller than that of the third chip 30, and the positions of the first connective tissue area inlet 301 and the first connective tissue area outlet 303 on the third chip 30 corresponding to the first chip 10 are exposed to the first chip 10, so that the cell fluid and cell culture fluid for culturing vascular endothelial cells and adipocytes are injected from the first connective tissue area inlet 301 until the cell fluid or cell culture fluid for culturing vascular endothelial cells and adipocytes flows out from the first connective tissue area outlet 303 and the injection is stopped.

[0064] Furthermore, if Figure 4 As shown, the third chip 30 is provided with a first connective tissue region culture medium inlet 304, a first connective tissue region culture medium outlet 305, a second connective tissue region culture medium inlet 307 and a second connective tissue region culture medium outlet 308, the first connective tissue region culture medium inlet 304 and the first connective tissue region culture medium outlet 305 are connected through a first culture medium channel 306, the second connective tissue region culture medium inlet 307 and the second connective tissue region culture medium outlet 308 are connected through a second culture medium channel 309, and the first culture medium channel 306 and the second culture medium channel 309 are both connected to the connective tissue region 302. Preferably, the first culture medium channel 306 and the second culture medium channel 309 are respectively located on both sides of the connective tissue region 302 in the length direction.

[0065] The first culture medium channel 306 and the second culture medium channel 309 can provide the connective tissue region 302 with cell fluid for culturing vascular endothelial cells and adipocytes. The two culture medium channels can provide a pressure difference so that the culture fluid can flow smoothly to the connective tissue region 302 .

[0066] In order to limit the cells in the connective tissue region 302 from infiltrating and transferring to the first culture medium channel 306 and the second culture medium channel 309 , a cell movement limiting component 310 is provided at the connection points between the connective tissue region 302 and the first culture medium channel 306 and the second culture medium channel 309 .

[0067] In some specific embodiments, the cell activity limiting component 310 includes a plurality of trapezoidal protrusions arranged in sequence to form a dam structure. The spacing between any two adjacent trapezoidal protrusions can be the same or different, as long as the cells can be limited from oozing out of the connective tissue region 302 to the first culture medium channel 306 and the second culture medium channel 309, and the culture fluid can flow into the connective tissue region 302 through the first culture medium channel 306 and the second culture medium channel 309. The depth of the trapezoidal protrusion is the same as the depth of the connective tissue region 302, and the distance between two adjacent trapezoidal protrusions can be 50μm-100μm, preferably 100μm.

[0068] like Figure 1 and Figure 2 As shown, in some specific embodiments, the first chip 10 is provided with a third connective tissue region culture medium inlet 106, a third connective tissue region culture medium outlet 107, a fourth connective tissue region culture medium inlet 108 and a fourth connective tissue region culture medium outlet 109. The third connective tissue region culture medium inlet 106 is connected to the first connective tissue region culture medium inlet 304, the third connective tissue region culture medium outlet 107 is connected to the first connective tissue region culture medium outlet 305; the fourth connective tissue region culture medium inlet 108 is connected to the second connective tissue region culture medium inlet 307, and the fourth connective tissue region culture medium outlet 109 is connected to the second connective tissue region culture medium outlet 308.

[0069] When in use, culture fluid can be injected through the third connective tissue zone culture fluid inlet 106 and the fourth connective tissue zone culture fluid inlet 108 respectively, and the injection is stopped when the culture fluid is observed to flow out from the third connective tissue zone culture fluid outlet 107 and the fourth connective tissue zone culture fluid outlet 109.

[0070] In other specific embodiments, the areas of the first chip 10 corresponding to the first connective tissue area culture medium inlet 304, the first connective tissue area culture medium outlet 305, the second connective tissue area culture medium inlet 307 and the second connective tissue area culture medium outlet 308 on the third chip 30 can be set as hollow areas. Alternatively, the size of the first chip 1 is smaller than that of the third chip 30, so that the first connective tissue area culture medium inlet 304, the first connective tissue area culture medium outlet 305, the second connective tissue area culture medium inlet 307 and the second connective tissue area culture medium outlet 308 on the third chip 30 are all exposed to the first chip 10, so as to facilitate the injection of culture solution.

[0071] like Figure 3 As shown, in the peritoneal chip disclosed in the embodiment of the present invention, a plurality of through holes are provided on the second chip 20, and the shape of the through holes may be one of circular, elliptical or polygonal. The plurality of through holes may be arranged evenly or unevenly, and the diameter or width of each through hole may be the same or different. The porosity of the second chip 20 may be 80%-90%, preferably 90%, and the diameter or width of the through hole may be 5μm-8μm, preferably 8μm. The second chip 20 preferably has a porous membrane structure. In order to reduce the breakage of the second chip 20 due to its thin thickness, the thickness of the second chip 2 is preferably 20μm-200μm.

[0072] In the peritoneal chip disclosed in the embodiment of the present invention, the first chip 10 and the third chip 30 are made of organic glass or polydimethylsiloxane.

[0073] In addition, an embodiment of the present invention also discloses a method for constructing an in vitro peritoneal model, using the above-mentioned peritoneal chip, comprising the following steps:

[0074] S1, assembling the peritoneal chip;

[0075] The first chip 10, the second chip 20 and the third chip 30 are bonded and assembled to form a peritoneal chip. Specifically, the bonding and assembly of the first chip 10, the second chip 20 and the third chip 30 can be achieved by a plasma bonding machine.

[0076] Specifically, the axis of the peritoneal mesothelial tissue zone 102 is collinear with the axis of the second chip 20 and the axis of the connective tissue zone 302, the axis of the second connective tissue zone inlet 104 is collinear with the axis of the first connective tissue zone inlet 301, the axis of the second connective tissue zone outlet 105 is collinear with the axis of the first connective tissue zone outlet 303, the axis of the third connective tissue zone culture medium inlet 106 is collinear with the axis of the first connective tissue zone culture medium inlet 304, the axis of the third connective tissue zone culture medium outlet 107 is collinear with the axis of the first connective tissue zone culture medium outlet 305, the axis of the fourth connective tissue zone culture medium inlet 108 is collinear with the axis of the second connective tissue zone culture medium inlet 307, the axis of the fourth connective tissue zone culture medium outlet 109 is collinear with the axis of the second connective tissue zone culture medium outlet 308, and then the first chip 10, the second chip 20 and the third chip 30 are assembled and bonded to form a peritoneal chip.

[0077] S2, build peritoneal connective tissue;

[0078] The mixed solution and the vascular endothelial cell culture medium are sequentially injected into the peritoneal connective tissue simulation module, specifically comprising the following steps:

[0079] S2-1, preparing a mixed solution;

[0080] The vascular endothelial cell suspension of the first preset concentration and the adipocyte suspension of the second preset concentration are mixed with the bovine fibrinogen solution to obtain a multicellular suspension, and the multicellular suspension is mixed with the thrombin solution to obtain a mixed solution. Specifically, the first preset concentration is preferably 3×10 6 The second preset concentration is preferably 1×10 5 / mL, the concentration of bovine fibrinogen in the multi-cell suspension is 2.5mg / mL, and the concentration of thrombin solution in the mixed solution is 3U / mL.

[0081] S2-2, injecting the mixed solution into the connective tissue area 302 of the peritoneal connective tissue simulation module;

[0082] The mixed solution is injected from the second connective tissue area inlet 104 by a pipette so that the mixed solution flows to the connective tissue area 302 . When the mixed solution flows out from the second connective tissue area outlet 105 , the injection is stopped.

[0083] S2-3, placing the peritoneal chip in a cell culture incubator and letting it stand for a first preset time;

[0084] Specifically, the temperature in the cell culture incubator is set to 37° C., and 5% carbon dioxide is filled into the cell culture incubator. The first preset time is preferably 20 minutes, so that the mixed liquid in the connective tissue area 302 solidifies into a hydrogel, providing a three-dimensional extracellular matrix for the vascular endothelial cells and adipocytes therein.

[0085] S2-4, taking out the peritoneal chip from the cell culture incubator, and injecting vascular endothelial cell culture medium into the peritoneal connective tissue simulation module;

[0086] The peritoneal chip is taken out of the cell culture incubator, and endothelial cell culture fluid is injected from the third connective tissue zone culture medium inlet 106 and the fourth connective tissue zone culture medium inlet 108 respectively. The endothelial cell culture fluid flows through the first culture medium channel 306 and the second culture medium channel 309 respectively until the endothelial cell culture fluid flows out from the third connective tissue zone culture medium outlet 107 and the fourth connective tissue zone culture medium outlet 109. The injection is stopped, and air can be discharged while the endothelial cell culture fluid is injected.

[0087] S3, build mesothelial tissue on the peritoneal surface;

[0088] The mesothelial cell suspension and mesothelial cell culture medium are injected into the peritoneal surface mesothelial tissue simulation module in sequence. The specific steps include:

[0089] S3-1, injecting a mesothelial cell suspension of a third preset concentration into the peritoneal surface mesothelial tissue simulation module;

[0090] Specifically, a mesothelial cell suspension of a third preset concentration is injected through the mesothelial cell inlet 101, and the mesothelial cell suspension flows into the peritoneal mesothelial tissue area 102 until it flows out from the mesothelial cell outlet 103, and the injection is stopped. The third preset concentration is preferably 3×10 6 Pieces / mL.

[0091] S3-2, placing the peritoneal chip in a cell culture incubator and letting it stand for a second preset time;

[0092] The second preset time is preferably 120 minutes, so that the mesothelial cells adhere to the bottom of the fluid channel, that is, the surface of the second chip 20 .

[0093] S3-3. Take out the peritoneal chip from the incubator and inject mesothelial cell culture medium into the mesothelial tissue simulation module on the peritoneal surface.

[0094] Specifically, the mesothelial cell culture fluid is injected through the mesothelial cell inlet 101 , and the mesothelial cell culture fluid flows into the peritoneal mesothelial tissue region 102 , and the injection is stopped until the fluid flows out from the mesothelial cell outlet 103 .

[0095] As shown in this application and claims, unless the context clearly indicates an exception, the words "a", "an", "a kind" and / or "the" do not refer to the singular, but also include the plural. Generally speaking, the terms "include" and "comprise" only indicate the inclusion of clearly identified steps and elements, and these steps and elements do not constitute an exclusive list. The method or device may also include other steps or elements. The elements defined by the sentence "includes a..." do not exclude the existence of other identical elements in the process, method, commodity or device that includes the elements.

[0096] In the following, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features.

[0097] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principles of the present invention, the present invention can also be improved and modified, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. A peritoneal chip, characterized in that: Comprising a first chip (10), a second chip (20) and a third chip (30), wherein the second chip (20) is arranged between the first chip (10) and the third chip (30); The first chip (10) is provided with a peritoneal surface mesothelial tissue simulation module, and the peritoneal surface mesothelial tissue simulation module is used to culture peritoneal mesothelial cells. The third chip (30) is provided with a peritoneal connective tissue simulation module, and the peritoneal connective tissue simulation module is used to culture vascular endothelial cells and fat cells. The second chip (20) is connected to the peritoneal surface mesothelial tissue simulation module and the peritoneal connective tissue simulation module.

2. The peritoneal chip according to claim 1, characterized in that The peritoneal surface mesothelial tissue simulation module comprises a mesothelial cell entrance (101), a peritoneal mesothelial tissue region (102) and a mesothelial cell exit (103), and the peritoneal mesothelial tissue region (102) is connected to the mesothelial cell entrance (101) and the mesothelial cell exit (103), respectively.

3. The peritoneal chip according to claim 2, characterized in that: The peritoneal connective tissue simulation module comprises a first connective tissue area inlet (301), a connective tissue area (302) and a first connective tissue area outlet (303), wherein the connective tissue area (302) is connected to the first connective tissue area inlet (301) and the first connective tissue area outlet (303) respectively; The second chip (20) is connected to the peritoneal mesothelial tissue area (102) and the connective tissue area (302).

4. The peritoneal chip according to claim 3, characterized in that The first chip (10) is provided with a second connective tissue area inlet (104) and a second connective tissue area outlet (105); the second connective tissue area inlet (104) is connected to the first connective tissue area inlet (301), and the second connective tissue area outlet (105) is connected to the first connective tissue area outlet (303).

5. The peritoneal chip according to claim 3, characterized in that: The third chip (30) is provided with a first connective tissue region culture medium inlet (304), a first connective tissue region culture medium outlet (305), a second connective tissue region culture medium inlet (307) and a second connective tissue region culture medium outlet (308); The first connective tissue region culture medium inlet (304) and the first connective tissue region culture medium outlet (305) are connected via a first culture medium channel (306); The second connective tissue region culture medium inlet (307) and the second connective tissue region culture medium outlet (308) are connected via a second culture medium channel (309); The first culture medium channel (306) and the second culture medium channel (309) are both connected to the connective tissue region (302).

6. The peritoneal chip according to claim 5, characterized in that The connective tissue region (302) is provided with a cell activity limiting component (310) to limit the infiltration of cells from the connective tissue region (302) to the first culture medium channel (306) and the second culture medium channel (309).

7. The peritoneal chip according to claim 6, characterized in that The cell activity limiting component (310) comprises a plurality of trapezoidal protrusions arranged in sequence to form a dam structure.

8. The peritoneal chip according to claim 5, characterized in that The first chip (10) is provided with a third connective tissue region culture medium inlet (106), a third connective tissue region culture medium outlet (107), a fourth connective tissue region culture medium inlet (108) and a fourth connective tissue region culture medium outlet (109); The third connective tissue region culture medium inlet (106) is connected to the first connective tissue region culture medium inlet (304), and the third connective tissue region culture medium outlet (107) is connected to the first connective tissue region culture medium outlet (305); The fourth connective tissue region culture medium inlet (108) is connected to the second connective tissue region culture medium inlet (307), and the fourth connective tissue region culture medium outlet (109) is connected to the second connective tissue region culture medium outlet (308).

9. The peritoneal chip according to any one of claims 1 to 8, characterized in that: The second chip (20) is provided with a plurality of through holes.

10. A method for constructing an in vitro peritoneal model, using the peritoneal chip according to any one of claims 1 to 9, characterized in that: The steps include: Assembling a peritoneal chip, assembling the first chip (10), the second chip (20) and the third chip (30) to form a peritoneal chip; Constructing peritoneal connective tissue, and sequentially injecting the mixed solution and vascular endothelial cell culture medium into the peritoneal connective tissue simulation module; Construct the peritoneal surface mesothelial tissue, and inject the mesothelial cell suspension and mesothelial cell culture medium into the peritoneal surface mesothelial tissue simulation module in sequence.

11. The method for constructing an in vitro peritoneal model according to claim 10, characterized in that: The step of constructing peritoneal connective tissue specifically includes: preparing a mixed solution, mixing a vascular endothelial cell suspension of a first preset concentration and a fat cell suspension of a second preset concentration with a bovine fibrinogen solution to obtain a multicellular suspension, and mixing the multicellular suspension with a thrombin solution to obtain a mixed solution; injecting the mixed solution into the connective tissue area (302) of the peritoneal connective tissue simulation module; Placing the peritoneal chip in a cell culture incubator and leaving it to stand for a first preset time to allow the mixed solution to solidify into a hydrogel; The peritoneal chip was taken out from the cell culture incubator, and vascular endothelial cell culture medium was injected into the peritoneal connective tissue simulation module.

12. The method for constructing an in vitro peritoneal model according to claim 10, characterized in that: The step of constructing peritoneal surface mesothelial tissue specifically includes: injecting a mesothelial cell suspension of a third preset concentration into the peritoneal surface mesothelial tissue simulation module; placing the peritoneal chip in a cell culture incubator and letting it stand for a second preset time; The peritoneal chip was taken out from the incubator and the mesothelial cell culture medium was injected into the mesothelial tissue simulation module on the peritoneal surface.

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