Uterine rhythmic contraction organ chip simulating primary dysmenorrhea and preparation method thereof

Abstract

The application discloses a uterus rhythmic contraction organ chip simulating primary dysmenorrhea and a preparation method thereof, and belongs to the technical field of organ chips. The uterus rhythmic contraction organ chip comprises, from top to bottom, an upper chamber layer, a middle porous membrane and a lower chamber layer. The upper chamber layer is internally provided with five parallel microchannels, including a first channel, a second channel and a third channel arranged in sequence in the middle, and a fourth channel and a fifth channel arranged on both sides. The adjacent microchannels are separated by microcolumns arranged at intervals, and the gap between the adjacent microcolumns constitutes a microenvironment interaction path between the microchannels. The bottom of the third channel penetrates through the upper chamber layer to form an open bottom end. The lower chamber layer is internally provided with an airway chamber located directly below the third channel. The middle porous membrane blocks the open bottom end of the third channel and physically isolates the airway chamber from the third channel.

Description

Technical Field

[0001] This invention belongs to the field of organ-on-a-chip technology, specifically relating to an organ-on-a-chip that simulates the rhythmic contractions of the uterus in primary dysmenorrhea and its preparation method. Background Technology

[0002] With the continuous development of medical technology, the shortcomings of traditional drug efficacy evaluation models have gradually become apparent. While two-dimensional cell culture systems are simple to operate and inexpensive, their overly simplistic culture and modeling systems lack three-dimensional tissue structure support, making it impossible to simulate in vivo cell-cell interactions, reproduce in vivo tissue conditions and microenvironments, and replicate highly differentiated human tissue structures and functions, thus limiting their predictive accuracy for disease treatment. Animal models, to a certain extent, preserve the complete in vivo physiological environment and can reflect systemic hormonal regulation, neuroendocrine feedback, and pathological changes at the tissue level. However, animal experiments suffer from significant species differences and are subject to problems such as long experimental cycles, high costs, and strict ethical reviews.

[0003] In recent years, organ-on-a-chip technology, as an important supplement between two-dimensional culture and animal models, has rapidly developed and been applied to the in vitro simulation of various organs such as the liver, lungs, intestines, and kidneys. Organ-on-a-chip technology partially reproduces the microphysiological environment of organs by constructing three-dimensional cell culture microchambers on a microfluidic platform and introducing functional modules such as fluid perfusion, mechanical stress, and cell co-culture.

[0004] As the core organ of the female reproductive system, the uterus's physiological functions are not only involved in the menstrual cycle, embryo implantation, and pregnancy maintenance, but also closely related to the pathogenesis of various gynecological diseases, such as primary dysmenorrhea, endometriosis, and adenomyosis. In uterine-related research, a few studies have attempted to construct endometrial organoid microarrays and vascularized endometrial composite systems. However, these models are often structurally singular, focusing only on a single functional layer, lacking integration of the "vascular-endometrial-myomyelin" physiological structure, and failing to simulate the biomechanical characteristic of uterine rhythmic contractions. Furthermore, research on uterine organoid microarrays has largely focused on embryonic development and infertility treatment, with relatively little research on diseases closely related to abnormal uterine contractions, local ischemia, and inflammatory responses, such as primary dysmenorrhea.

[0005] Therefore, there is an urgent need in this field for a dedicated organ-on-a-chip system that can highly simulate the anatomical structure, cellular composition, mechanical function, and microenvironment interaction of the human uterus. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide an organ-on-a-chip that simulates the rhythmic contractions of the uterus in simulating primary dysmenorrhea and its preparation method, thereby solving the problems in the prior art.

[0007] The objective of this invention can be achieved through the following technical solutions: An organ-on-a-chip simulating the rhythmic contractions of the uterus in primary dysmenorrhea comprises, from top to bottom, an upper chamber layer, a middle porous membrane, and a lower chamber layer. The upper chamber layer has five parallel microchannels inside, including: a first channel, a second channel, and a third channel arranged in sequence in the middle, and a fourth channel and a fifth channel located on both sides respectively; adjacent microchannels are separated by spaced micropillars, and the gaps between adjacent micropillars constitute the microenvironmental interaction path between microchannels; The bottom of the third channel extends downward through the upper chamber layer to form a bottom opening; the lower chamber layer contains an airway chamber located directly below the third channel; the intermediate porous membrane seals the bottom opening of the third channel and physically isolates the airway chamber from the third channel.

[0008] Furthermore, the micropillars are cuboid in shape; the spacing between adjacent micropillars is 100 µm.

[0009] Furthermore, the intermediate porous film is made of polyethylene terephthalate with a pore size range of 0.4~1.0 µm.

[0010] Furthermore, each of the five microchannels in the upper chamber layer is provided with an independent fluid inlet and outlet, which are configured to be connected to an external circulation perfusion system; The lower chamber layer is provided with an independent pneumatic inlet / outlet, which is connected to the airway chamber and configured to connect to an external mechanical stimulation device.

[0011] The above-mentioned method for preparing the organ-on-a-chip simulating the rhythmic contractions of the uterus in primary dysmenorrhea includes the following steps: Based on the chip size and thickness, a mold is prepared using SU-8 photoresist. The base adhesive and the curing adhesive are mixed, cross-linked, and cured to obtain an upper chamber layer with the microchannels and micropillars, and a lower chamber layer with the airway chambers. After the porous membrane is cut, it is then subjected to anhydrous ethanol soaking to remove oil stains, deionized water rinsing and drying treatment in sequence. The upper chamber layer, the treated intermediate porous film, and the lower chamber layer are subjected to surface activation treatment. The upper chamber layer, the middle porous film, and the lower chamber layer are stacked sequentially and bonded by applying pressure.

[0012] Furthermore, the base adhesive and curing adhesive are Dow Corning Sylgard 184 base adhesive and curing adhesive, which are mixed in a mass ratio of 10:1 and cured at 65°C.

[0013] The above-mentioned organ-on-a-chip model of uterine rhythmic contractions is used in the in vitro simulation of primary dysmenorrhea.

[0014] A method for constructing an in vitro primary dysmenorrhea model using the above-mentioned organ-on-a-chip includes the following steps: Human umbilical vein endothelial cells, endometrial organoids, and uterine smooth muscle cells were seeded into the microchannels corresponding to the upper chamber layer and cultured statically. Cell culture was performed by circulating and perfusing culture medium containing prostaglandins or dysmenorrhea-related inflammatory factors into the microchannels. Periodic positive and negative pressure changes are applied to the airway chamber to drive the deformation of the intermediate porous membrane that is sealed at the bottom opening of the third channel, thereby applying periodic mechanical stimulation to the uterine smooth muscle cells inoculated thereon.

[0015] Furthermore, the human umbilical vein endothelial cells are seeded in the first channel; the endometrial organoids are seeded in the second channel; and the uterine smooth muscle cells are seeded in the third channel and attached to the intermediate porous membrane.

[0016] Furthermore, the culture medium is perfused through the fourth and fifth channels.

[0017] The beneficial effects of this invention are: 1. This invention employs an upper chamber containing five parallel channels, separated by cuboid micropillars, into which human umbilical vein endothelial cells, endometrial organoids, and uterine smooth muscle cells are seeded. This arrangement not only physically reconstructs the complete "blood vessel-endometrium-myophyll" tissue anatomy, but also allows for free biochemical communication and exchange of key factors such as prostaglandins between different tissue layers through the gaps between the micropillars. This overcomes the limitations of existing technologies in simulating complex inter-tissue interactions, providing a highly biomimetic in vitro microenvironment.

[0018] 2. This invention designs a differentiated through-structure at the bottom, ensuring that the flexible porous membrane in the middle only blocks the lower opening of channel 3 (uterine smooth muscle cell culture chamber), and precisely sets a lower airway chamber directly below it. A mechanical stimulation device (pneumatically driven) applies positive and negative pressure changes to the lower chamber, driving the flexible porous membrane to deform. This locally targeted mechanical stimulation method can directly drive the uterine smooth muscle cells attached to it to contract, thereby highly replicating the rhythmic physiological contractions of the uterus and the abnormal spasms in the pathological state of dysmenorrhea in vitro. For the first time, this provides a controllable and observable dynamic biomechanical model for exploring the mechanism of primary dysmenorrhea. 3. This invention provides independent micro-inlet / outlet pumps for each channel in the upper chamber, particularly utilizing channels 4 and 5 as bypass channels for independent perfusion of culture media and drugs. This hydrodynamic design establishes a stable concentration gradient within the chip, highly simulating the real dynamic process of drugs and other nutrients gradually permeating and diffusing into the uterine tissue after being introduced via blood vessels to exert their effects. This enhances the flexibility of regulating different cellular microenvironments and provides a reliable validation platform for high-throughput screening of dysmenorrhea drugs and the development of treatment strategies.

[0019] 4. The organ-on-a-chip of this invention employs mature SU-8 photoresist molding and PDMS casting processes, and permanently bonds the upper PDMS chamber, PET porous film, and lower PDMS chamber using oxygen plasma. This standardized soft lithography and bonding process not only ensures the dual-chamber chip possesses excellent sealing properties to withstand continuous aerodynamic stress at the bottom without cracking, but also guarantees a high degree of consistency in the chip structure. This provides extremely high experimental reproducibility for scientific research, lays a solid foundation for reliable data, and significantly reduces R&D costs compared to animal experiments. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 This is a physical image of the uterine organ microarray of the present invention; Figure 2 This is a schematic diagram of the three-dimensional structure of the uterine organ-on-a-chip of the present invention; Figure 3 This is a schematic diagram of the upper channel structure of the uterine organ chip of the present invention; Figure 4 This is a schematic diagram of the lower airway structure of the uterine organ-on-a-chip according to the present invention; Figure 5 This is a schematic diagram of the fluid dynamics simulation within the channel of the present invention; Figure 6 This is a schematic diagram simulating the concentration diffusion after channel-based drug delivery according to the present invention.

[0022] Wherein: 1-first channel, 2-second channel, 3-third channel, 4-fourth channel, 5-fifth channel. Detailed Implementation

[0023] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and 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.

[0024] Example 1 In this embodiment, the structure, preparation method, and application of the uterine rhythmic contraction organ-on-a-chip of the present invention are summarized.

[0025] like Figure 2 As shown, the organ-on-a-chip simulating the rhythmic contractions of the uterus in primary dysmenorrhea includes: an upper chamber layer, a middle porous membrane, and a lower chamber layer stacked from top to bottom; like Figure 3 As shown, the upper chamber layer contains five parallel microchannels: a first channel 1, a second channel 2, and a third channel 3 arranged sequentially in the middle, and a fourth channel 4 and a fifth channel 5 located on either side. During chip use, the first channel 1 is a cell culture chamber for human umbilical vein endothelial cell culture; the second channel 2 is an organoid culture chamber for endometrial organoid culture; the third channel 3 is a cell culture chamber for uterine smooth muscle cell culture; and the fourth and fifth channels 4 and 5 are channels for perfusion of culture medium and drugs, simulating the process by which drugs and other nutrients diffuse through blood vessels to the uterine tissue to exert their effects.

[0026] Adjacent microchannels are separated by spaced micropillars, and the gaps between adjacent micropillars constitute the microenvironmental interaction path between microchannels, thus enabling microenvironmental interaction between cells while ensuring independent cell culture in different channels.

[0027] The bottoms of the first channel 1, the second channel 2, the fourth channel 4, and the fifth channel 5 are not through-holes. The bottom of the third channel 3 penetrates downward through the upper chamber layer to form a bottom opening. The intermediate porous membrane is sandwiched and bonded between the upper chamber layer and the lower chamber layer, which can seal the bottom opening of the third channel 3. Furthermore, due to the small pores on the intermediate porous membrane, the culture medium in the third channel 3 will not leak.

[0028] like Figure 4As shown, an airway chamber is provided inside the lower chamber layer. The airway chamber is located directly below the third channel 3 and is connected to a mechanical stimulation device. The mechanical stimulation device is pneumatically driven and can apply periodic mechanical stimulation to the airway chamber in the lower chamber layer by changing positive and negative pressure, causing the intermediate porous membrane (flexible) to deform, thereby applying periodic mechanical stimulation to the uterine smooth muscle cells inoculated on the intermediate porous membrane.

[0029] The upper chamber layer has five microchannels, each with an independent fluid inlet / outlet, configured to connect to an external circulation perfusion system. The lower chamber layer has an independent pneumatic inlet / outlet, connected to the airway chamber and configured to connect to an external mechanical stimulation device. The upper and lower chamber layers are both made of PDMS with a thickness of 3-5 mm. The middle porous film is made of polyethylene terephthalate (PET) with a thickness of 14-15 μm and a pore size range of 0.4-1.0 μm.

[0030] In the upper chamber layer, the micropillars between the microchannels are cuboid in shape (300 μm long, 200 μm wide, and 100 μm high), with a spacing of 100 μm between adjacent micropillars. After the cells are mixed with the matrix gel and injected into the channels, the cells cannot pass through the gaps due to surface tension.

[0031] In the upper chamber layer, the width of each of the five microchannels is 3mm; the depth of the first channel 1, the second channel 2, the fourth channel 4 and the fifth channel 5 is 100μm.

[0032] In the lower chamber layer, the airway chamber is 3 mm wide and 100 μm deep.

[0033] The method for preparing the above-mentioned uterine rhythmic contraction organ-on-a-chip includes the following steps: S1: Determine the chip size and thickness, and prepare the corresponding amount of SU-8 photoresist and Sylgard 184 base adhesive according to the design size of the dual-chamber uterus chip; mix the base adhesive and curing adhesive together in a certain proportion, crosslink and cure at a certain temperature to ensure that the two are evenly dispersed, and then apply them to the upper and lower PDMS chambers.

[0034] S2: Select a PET film with a suitable pore size. Before use, cut the PET film to the appropriate size according to the chip design. Soak it in anhydrous ethanol to remove surface oil before use, then rinse it with deionized water and dry it in a vacuum drying oven.

[0035] S3: The upper PDMS chamber, the treated PET porous membrane, and the lower PDMS chamber are placed together in an oxygen plasma treatment machine for surface activation at specific power and processing time. After treatment, pressure is applied to permanently bond the three-layer structure to form the desired chip.

[0036] After the uterine rhythmic contraction organ-on-a-chip is prepared, the process of constructing an in vitro primary dysmenorrhea model using the uterine rhythmic contraction organ-on-a-chip specifically includes: Human umbilical vein endothelial cells were seeded into the first channel 1 of the chip, endometrial organoids into the second channel 2, and uterine smooth muscle cells into the third channel 3. After 6 hours of incubation, the chip was connected to a circulating perfusion system and perfused with culture medium (containing 0.1 μM or 1 μM estradiol) at a flow rate of 1 μL / min through the fourth and fifth channels. After 24 hours of stable culture, the lower airway chamber was connected to a mechanical stimulation device to provide periodic mechanical stimulation to the uterine smooth muscle cell culture chamber, simulating its rhythmic contractions in the human body. Subsequently, the contraction frequency and amplitude of the uterine smooth muscle cells and the release of related inflammatory factors in the chip were monitored to evaluate the relieving effect of drugs or interventions on the dysmenorrhea model (the drugs were dissolved in the culture medium at a certain concentration, in the same manner as the above-mentioned culture medium perfusion).

[0037] The lower airway chamber of the chip is connected to an external mechanical stimulation device. This device applies mechanical stimulation to the lower chamber periodically through program control, thereby enabling the uterine smooth muscle cells to mimic the rhythmic physiological contractions of the uterus.

[0038] The first channel 1 to the fifth channel 5 of the upper chamber are connected to five independent flow pumps of the circulating perfusion pump to achieve independent perfusion, which is used to deliver nutrients, hormones or drugs to simulate the in vivo microenvironment.

[0039] Example 2 This embodiment describes the specific chip structure design process; Chip Structure Design and Fluid Dynamics Simulation Verification: Before chip fabrication, the multi-channel perfusion system of the cell culture chamber on the upper layer of the chip was simulated using the Comsol Multiphysics 6.4 simulation software. The perfusion flow rate was set to 1 μL / min, simulating the flow of culture medium in channels 1-5, and evaluating the flow rate, pressure distribution, and concentration diffusion under drug administration conditions within the channels. Detailed fluid dynamics simulation results can be found in [link to simulation]. Figure 5 For a detailed simulation of concentration diffusion, please refer to [link / reference]. Figure 6 ; from Figure 5 It can be seen from this that the reciprocal of the step size is 10. -2 The graph shows a downward trend. Figure 5The 'a' in the model indicates that the step size increases with time, and the established model has good convergence and can be used to simulate fluid flow in a channel. Figure 5 The bd fields in the microarray reflect the concentration field, velocity field, and pressure field, respectively, which can effectively reflect the instantaneous concentration, velocity, and pressure conditions within the organ-on-a-chip.

[0040] Figure 6 As can be seen from the af data, the distribution of drug concentration within the chip at 1, 5, 10, 15, 20, and 25 minutes shows that the drug-containing culture medium injected from channels four and five gradually fills channels one, two, and three, respectively, and at 25 minutes (…). Figure 6 f) The drug concentration within the chip stabilizes (uniform color in the figure), which can be used to simulate concentration diffusion during drug administration.

[0041] The final chip dimensions are as follows: Both the upper and lower chamber layers are made of PDMS and have a thickness of 5 mm.

[0042] In the upper chamber layer, the micropillars between the microchannels are cuboid in shape (300 μm long, 200 μm wide, and 100 μm high), with a spacing of 100 μm between adjacent micropillars.

[0043] In the upper chamber layer, the width of each of the five microchannels is 3 mm; the depth of the first channel 1, the second channel 2, the fourth channel 4 and the fifth channel 5 is 100 μm.

[0044] In the lower chamber layer, the airway chamber is 3 mm wide and 100 μm deep.

[0045] Example 3 In this embodiment, based on the structural design of Embodiment 2, the fabrication process of the uterine rhythmic contraction organ-on-a-chip is introduced through a specific fabrication case. S1. After determining the chip size and thickness, prepare 4 g of Zhongxin Hengqi negative SU-8 photoresist according to the design dimensions of the dual-chamber uterus chip. Spin-coat the photoresist evenly onto a 4-inch silicon wafer, and fix the channels onto the silicon wafer using ultraviolet lithography to form an organ-on-a-chip mold. Prepare an appropriate amount of Dow Corning Sylgard 184 base adhesive and curing agent. Mix the base adhesive and curing agent together at a mass ratio of 10:1, stir evenly, remove air bubbles, pour into the mold, and place in an oven at 65℃ for 2 hours for complete curing.

[0046] S2. Select a porous PET film with a thickness of 14-15 μm and a pore size in the range of 0.4-1 μm. Before use, cut the PET film into rectangles of 3 mm × 20 mm. Soak it in anhydrous ethanol to remove surface oil before use, then rinse it with deionized water, and finally dry it in a vacuum drying oven at 40°C for 30 min.

[0047] S3, the upper PDMS chamber, the treated PET porous membrane, and the lower PDMS chamber are placed together in an oxygen plasma treatment machine for surface activation at a specific power and time of 600W for 1 minute. After treatment, pressure is applied, and the mixture is placed in a 65℃ oven for 3 hours to permanently bond the three-layer structure, forming the desired chip (e.g., ...). Figure 1 (As shown).

[0048] Example 4 In this embodiment, the organ-on-a-chip prepared in Example 3 is used to construct an in vitro primary dysmenorrhea model, specifically including: Step 1: Cultured and stable human umbilical vein endothelial cells, endometrial organoids, and uterine smooth muscle cells were mixed with an appropriate amount of matrix gel (VivoMatter, VMO02-PRF-10) at a volume ratio of 50:1 and injected into the first channel 1, the second channel 2, and the third channel 3 of the upper chamber of the chip. After culturing in an incubator at 37°C and 5% CO2 for 6 h, the upper chamber channels 1-5 were independently connected to a circulating perfusion system and the culture medium (containing 0.1 μM estradiol) was stably perfused at a flow rate of 1 μL / min.

[0049] Step 2: After 24 hours of cyclic perfusion culture, the lower airway chamber is connected to a mechanical stimulation device. This device applies mechanical stimulation to the lower chamber periodically through program control, thereby enabling the uterine smooth muscle cells to mimic the rhythmic physiological contractions of the uterus.

[0050] In the description of this specification, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0051] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. An organ-on-a-chip that simulates the rhythmic contractions of the uterus in primary dysmenorrhea, characterized in that, It consists of, from top to bottom, the following layers stacked sequentially: an upper chamber layer, a middle porous membrane, and a lower chamber layer; The upper chamber layer has five parallel microchannels inside, including: a first channel, a second channel, and a third channel arranged in sequence in the middle, and a fourth channel and a fifth channel located on both sides respectively; adjacent microchannels are separated by spaced micropillars, and the gaps between adjacent micropillars constitute the microenvironmental interaction path between microchannels; The bottom of the third channel extends downward through the upper chamber layer to form a bottom opening; the lower chamber layer contains an airway chamber located directly below the third channel; the intermediate porous membrane seals the bottom opening of the third channel and physically isolates the airway chamber from the third channel.

2. The organ-on-a-chip for simulating primary dysmenorrhea with rhythmic uterine contractions according to claim 1, characterized in that, The micropillars are cuboid in shape; the spacing between adjacent micropillars is 100 µm.

3. The organ-on-a-chip for simulating primary dysmenorrhea with rhythmic uterine contractions according to claim 1, characterized in that, The intermediate porous film is made of polyethylene terephthalate with a pore size range of 0.4~1.0 µm.

4. The organ-on-a-chip for simulating primary dysmenorrhea with rhythmic uterine contractions according to claim 1, characterized in that, The five microchannels in the upper chamber layer are each equipped with an independent fluid inlet and outlet, which are configured to connect to an external circulation perfusion system. The lower chamber layer is provided with an independent pneumatic inlet / outlet, which is connected to the airway chamber and configured to connect to an external mechanical stimulation device.

5. The method for preparing the uterine rhythmic contraction organ-on-a-chip simulating primary dysmenorrhea according to any one of claims 1-4, characterized in that, Includes the following steps: Based on the chip size and thickness, a mold is prepared using SU-8 photoresist. The base adhesive and the curing adhesive are mixed, cross-linked, and cured to obtain an upper chamber layer with the microchannels and micropillars, and a lower chamber layer with the airway chambers. After the porous membrane is cut, it is then subjected to anhydrous ethanol soaking to remove oil stains, deionized water rinsing and drying treatment in sequence. The upper chamber layer, the treated intermediate porous film, and the lower chamber layer are subjected to surface activation treatment. The upper chamber layer, the middle porous film, and the lower chamber layer are stacked sequentially and bonded by applying pressure.

6. The method for preparing the organ-on-a-chip simulating primary dysmenorrhea with rhythmic uterine contractions according to claim 5, characterized in that, The base adhesive and curing adhesive are Dow Corning Sylgard 184 base adhesive and curing adhesive, which are mixed in a mass ratio of 10:1 and cured at 65°C.

7. The application of the uterine rhythmic contraction organ-on-a-chip according to any one of claims 1-4 in in vitro simulation of primary dysmenorrhea.

8. A method for constructing an in vitro primary dysmenorrhea model using organ-on-a-chip according to any one of claims 1 to 4, characterized in that, Includes the following steps: Human umbilical vein endothelial cells, endometrial organoids, and uterine smooth muscle cells were seeded into the microchannels corresponding to the upper chamber layer and cultured statically. Cell culture was performed by circulating and perfusing culture medium containing prostaglandins or dysmenorrhea-related inflammatory factors into the microchannels. Periodic positive and negative pressure changes are applied to the airway chamber to drive the deformation of the intermediate porous membrane that is sealed at the bottom opening of the third channel, thereby applying periodic mechanical stimulation to the uterine smooth muscle cells inoculated thereon.

9. The method for constructing an in vitro primary dysmenorrhea model according to claim 8, characterized in that, The human umbilical vein endothelial cells are seeded in the first channel; the endometrial organoids are seeded in the second channel; and the uterine smooth muscle cells are seeded in the third channel and attached to the intermediate porous membrane.

10. The method for constructing an in vitro primary dysmenorrhea model according to claim 8, characterized in that, The culture medium is perfused through the fourth and fifth channels.

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