Microfluidic intestinal organ chip, construction method and application thereof

Abstract

The present application relates to the technical field of microfluidic intestinal organ chip, and specifically discloses a microfluidic intestinal organ chip, a construction method and application thereof; the chip body comprises an upper layer chip, a porous membrane and a lower layer chip; the porous membrane is arranged between a vein unit and an artery unit; the construction method comprises the following steps: (1) collagen is added into an intestinal culture unit to induce gel formation; (2) endothelial cells are inoculated under the lower layer of the porous membrane; and (3) intestinal epithelial cells are inoculated. The present application can be applied to simulation of the influence of intestinal environment on the body, simulation of the absorption of nutrients and toxins by the intestinal tract, simulation of the influence of the brain on the intestinal tract, simulation of the influence of the internal environment of the body (such as chronic stress and depression) on the intestinal tract through hormones, neurotransmitters, inflammatory factors or other bioactive substances in the blood circulation on the structure and barrier function of the intestinal tract, simulation of the immune and inflammatory reactions in the body after digestion and absorption, and replacement of the use of experimental animals in the drug screening process.

Description

Technical Field

[0001] This invention relates to the field of microfluidic intestinal organ-on-a-chip technology, specifically to a microfluidic intestinal organ-on-a-chip, its construction method, and its applications. Background Technology

[0002] The surface of the small intestine facing the intestinal lumen is called the mucosa, which, together with the underlying submucosa, lateral muscularis propria, and serosa, constitutes the intestinal tissue. The mucosal surface is composed of simple columnar epithelial cells, and the junctions between these cells form the intestinal barrier. Tight junctions, adhesive junctions, desmosome junctions, and gap junctions are arranged sequentially from the apex of the intestinal epithelial cells to the basement membrane, sealing the apex and limiting permeability. This permeability is regulated by various factors, including chronic stress, diet, gut microbiota, and cytokines. The lamina propria at the base of the mucosa is richly supplied with capillaries. The arteries of the intestine are innervated by the superior mesenteric artery, which extends radially within the mesentery to the intestinal wall via arterial arches, gradually forming a capillary network. These capillaries then converge into a capillary venous network and intestinal veins, ultimately draining into the portal vein. The capillary walls consist of a single, thin layer of endothelial cells and a basement membrane surrounding them. The distance between the intestinal epithelial cell basement membrane and the capillaries is no more than a few micrometers. These blood vessels are not only located close to the epithelial layer, but also have fenestrations in the endothelial cells, which are covered only by the basement membrane. Therefore, after food undergoes mechanical and chemical digestion, nutrients can pass through the connections between epithelial cells into the capillaries located in the lamina propria, where substances are transferred from the lumen to the vascular system, completing the absorption of nutrients.

[0003] Furthermore, the gut, as the largest and most important barrier against the external environment, allows the passage of selective nutrients such as amino acids, carbohydrates, electrolytes, lipids, and water, while hindering the entry of toxins, bacterial luminal toxins, antigens, and gut microbiota to maintain homeostasis. Disruption of the intestinal barrier increases permeability, which can be detrimental to the host because it may allow intestinal antigens and toxins to translocate through the intestinal wall into the subepithelial tissue and bloodstream. In turn, this translocation may induce local and systemic immune responses, potentially leading to pathological development. Moreover, increased intestinal permeability is associated with a variety of autoimmune diseases and gastrointestinal disorders.

[0004] The digestive and absorptive processes in the gut are extremely complex, and the internal and external environments have a profound and multifaceted impact on the body. For example, external factors such as dietary intake and the microbial environment, as well as internal factors such as hormonal regulation and neurotransmitter activity, all affect gut function.

[0005] Animal models are currently a common method for studying intestinal pathophysiology. However, due to species differences, animal models cannot accurately reflect the human response to pathogens, diseases, and drugs. For example, in animal studies of chronic stress, stress typically needs to be induced for several days, ranging from 7 to 14 days. On the other hand, due to ethical concerns associated with clinical research, observational studies can only be conducted under natural stress conditions. Because animal and clinical studies differ significantly in the type and duration of stressors and intestinal permeability measurements, it is difficult to compare animal stress studies with human stress studies.

[0006] Furthermore, traditional cell models cannot simulate the characteristics of the intestinal microenvironment, such as fluid flow, villus structure, and peristalsis. Therefore, it is difficult to simulate the comprehensive functions of the human gut and achieve real-time observation of interactive dynamics. Currently, organoid culture using induced pluripotent stem cells or primary intestinal cells is an emerging method to regenerate three-dimensional intestinal structures from fully differentiated intestinal epithelial cells. However, organoid culture is static and lacks the fluid flow and peristalsis present in the human gut. Although intestinal organoids can mimic intestinal physiology, the closed lumen of intestinal organoids makes it difficult to deliver microorganisms or food into their interior.

[0007] Organ-on-a-chip (OA-chip) is a miniature cell culture device containing microfluidic channels and living cells, based on microfluidic technology. It can physiologically simulate key structures and functional units of human tissues and organs in vitro, to some extent overcoming the limitations of animal and static models. Currently, there are three main types of intestinal OA-chip models: 1) Vertical clip-on design. This design consists of upper and lower channels interconnected by a suspended porous membrane. Typically, intestinal epithelial cells and vascular endothelial cells are seeded in the upper and lower channels respectively. Fluid perfusion within the channels generates the shear forces experienced by the cells and transports substances, simulating the vascular and luminal environment. 2) Planar parallel design. This design incorporates a central channel filled with collagen gel in addition to the left and right microchannels. Micropillars or phase guidance are used to fill the central channel with gel, and intestinal epithelial cells and vascular endothelial cells are seeded in the left and right microchannels. 3) 3D gel structure. In this design, the chip channel is filled with gel and ablated with a laser to create multiple side indentations shaped like crypts, thereby polarizing basal stem cells. Alternatively, a hollow cylindrical channel can be formed within the 3D gel using tiny sticks or adhesive fingers, and cells can then be cultured along the surface of the central cavity.

[0008] Most existing intestinal organ-on-a-chip systems can only simulate single vascular structures, such as the intestinal barrier structure (intestinal epithelial cells). Compared with the anatomy and physiology of the human intestine, they cannot accurately simulate the submucosal structure and function. In particular, they lack the simulation of the perfusion arteries that innervate the intestinal wall and even the intestinal capillary network. They cannot simulate the blood circulation between arteries and veins, as well as the material exchange between blood and tissues. They also cannot simulate the influence of the internal environment on the intestine, such as the effects of chronic stress, neuroendocrine disorders, and brain diseases on the intestine through the circulation of neurotransmitters. In other words, they cannot achieve a comprehensive simulation of intestinal structure and function. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a microfluidic intestinal organ chip, its construction method, and its applications, thereby achieving comprehensive simulation of intestinal structure and function.

[0010] To achieve the above objectives, the present invention adopts the following technical solution:

[0011] A microfluidic intestinal organ-on-a-chip includes an upper chip, a porous membrane, and a lower chip. The upper chip has a venous channel inlet and outlet through-hole, an intestinal culture unit, an arterial unit, and a connecting channel. The lower chip has a venous channel inlet shallow groove, a venous channel outlet shallow groove, a venous unit, a venous inlet connecting channel, and a venous outlet connecting channel. The venous unit is located below the intestinal culture unit, and a porous membrane is disposed between the venous unit and the arterial unit. The position of the venous channel inlet through-hole corresponds vertically to the position of the venous channel inlet shallow groove, and the position of the venous channel outlet through-hole corresponds vertically to the position of the venous channel outlet shallow groove. The intestinal culture unit and the arterial unit are cylindrical. The arterial unit is located to the right of the intestinal culture unit and is connected via a connecting channel. The venous channel inlet shallow groove is connected to the venous unit via the venous inlet connecting channel, and the venous channel outlet shallow groove is connected to the venous unit via the venous outlet connecting channel.

[0012] Optionally, the porous film is made of PC or PET material.

[0013] Optionally, the material used for the chip body may be PDMS, PMMA, PC, or COC.

[0014] Optionally, the lower layer of the porous membrane is seeded with vascular endothelial cells.

[0015] Optionally, the upper layer of the porous membrane is filled with a porous microfluidic medium, which is configured as a capillary network and a submucosal structure of the intestine; intestinal epithelial cells are seeded on the upper surface of the porous microfluidic medium as an intestinal lumen structure; the final concentration of the gel used in the porous microfluidic medium is 3-6 mg / mL.

[0016] Optionally, the right side of the upper chip is an arterial structure; within the arterial structure, liquid can flow to and fill the porous microfluidic medium without overflowing onto the surface of the intestinal epithelial cells.

[0017] Optionally, the height of the upper chip is set to 6-8 mm; the narrowest distance of the connecting channels is set to 1-2 mm, the vertical distance is set to 2-4 mm, and the height is set to 1-2 mm; the diameter of the intestinal culture unit and the arterial unit is set to 4-6 mm; the diameter of the venous channel inlet and outlet through-holes is set to 3-4 mm; the feature depth within the lower chip is set to 0.4-0.6 mm; the diameter of the venous channel inlet and outlet shallow grooves is set to 3-4 mm; the diameter of the venous unit is set to 6-8 mm; the width of the venous inlet connecting channel and the venous outlet connecting channel is set to 1-2 mm; and the pore size of the porous membrane is 3-8 μm.

[0018] A microfluidic intestinal organ-on-a-chip fabrication method, wherein a microfluidic intestinal organ-on-a-chip is fabricated as described above, and the intestinal organ-on-a-chip is made using PDMS or thermoplastic plastic;

[0019] The PDMS chip manufacturing process includes:

[0020] S1 Pre-processing

[0021] Mix the PDMS prepolymer and curing agent at a mass ratio of 10:1 and stir until fully mixed; remove air bubbles from the mixed PDMS prepolymer using a vacuum method; pour the defoamed PDMS prepolymer onto a mold, ensuring that there are no air bubbles present; then transfer it to an oven for heating and curing.

[0022] S2 drilling

[0023] Drill holes at the corresponding positions and vertical angles using a punch of the appropriate size on the upper-layer chip of the PDMS.

[0024] S3 chip package

[0025] Since PDMS and PET film cannot be directly bonded by oxygen plasma treatment, bonding is achieved by aminosilanizing the PET film.

[0026] The aminosilanization process is as follows:

[0027] (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water, preheat it to 80°C, clean the PDMS chip with isopropanol and dry it with nitrogen or compressed air, and clean both sides of the PDMS chip to remove any obvious debris.

[0028] (2) Treat the PET film with oxygen plasma for 30 seconds and then transfer it to a preheated 5% APTES solution for 30 minutes;

[0029] (3) Wash the PET film with deionized water and dry it at room temperature;

[0030] (4) Simultaneously treat the aminosilanized PET film and the PDMS chip with oxygen plasma for 30 seconds and then bond them together;

[0031] (5) Place the assembled PDMS chip into an oven and heat it at 60°C for 24 hours;

[0032] S4 Intestinal Organ Microarray Construction

[0033] The assembled PDMS chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use.

[0034] (1) Type I collagen, 5×PBS, 1×PBS and 0.5N NaOH were mixed to prepare a final collagen concentration of 6 mg / ml; then, the collagen was added to the upper chip to form a collagen layer with a thickness of 1.5 mm in the intestinal culture unit, connecting channel and arterial unit; after incubating the chip in a 37℃ incubator for 1 hour, gel formation was induced.

[0035] (2) Endothelial cells were seeded into the lower layer of a porous membrane. First, a 50 μg / ml fibronectin solution was introduced into the lower channel, and the cell culture was inverted at 37°C for at least 2 hours. The channel was then washed with endothelial cell culture medium. Subsequently, the final cell density was increased to 1×10⁻⁶ cells / mL. 5 pcs / cm 2 Endothelial cells were introduced into the chip and the chip was inverted so that the endothelial cells could attach to the underside of the membrane. After the endothelial cells had attached for 1 to 2 hours, the unattached endothelial cells were rinsed off, and then the chip was filled with endothelial cell culture medium.

[0036] (3) Inoculate intestinal epithelial cells to a final cell concentration of 5 × 10⁻⁶. 5 pcs / cm 2 Intestinal epithelial cells were introduced onto the top of a porous microfluidic medium, transferred to an incubator, and placed for 2–4 hours. Subsequently, unattached intestinal epithelial cells were washed away.

[0037] Optionally, in step (1) of S4 intestinal organ-on-a-chip construction, for the construction of a simulation of the enteric nervous system and the intestinal immune system, the enteric nerve cells and immune cells are embedded in collagen.

[0038] Applications of microfluidic gut organ-on-a-chip, as described above, include: the impact of the internal and external environment on the gut and thus on health, and the simulation of the impact of the gut environment (such as gut microbiota) on the body;

[0039] Or it can be applied to: simulation of food digestion, nutrient and toxin absorption (nutrients and toxins);

[0040] Or it can be applied to: simulating the brain's influence on the gut, the effects of changes in the body's internal environment (such as chronic stress and depression) on the gut, and simulating the effects of hormones, neurotransmitters, inflammatory factors, or other bioactive substances in the bloodstream on the gut's structure and barrier function;

[0041] Or it can be applied to: the simulation of immune and inflammatory responses caused by the absorption of substances and toxins after food digestion;

[0042] Or it can be used to replace the use of laboratory animals in the drug screening process.

[0043] 1. This intestinal organ-on-a-chip features an intestinal barrier structure formed by arteries, the intestinal lumen (including simulated mucosa and submucosa), and the venous system, simulating the absorption process. It also incorporates perfusion arteries implanted into the intestine, achieving blood supply to the intestinal mucosa and submucosa (e.g., perfusion of neurotransmitters or neuroendocrine factors to simulate the effects of changes in the in vivo environment on the intestine). Furthermore, it utilizes a porous microfluidic medium to create a capillary system from arteries to veins, a biomimetic intestinal organ-on-a-chip system. Another unique feature is the use of porous microfluidic media as the submucosal structure. Based on this comprehensive simulation of intestinal structure and function, the intestinal organ-on-a-chip boasts a reliable structural design, facilitating smooth control of subsequent fluid flow and maximizing chip performance. Simultaneously, it facilitates standardized and simple chip fabrication, ensuring controllable fabrication quality and significantly improving the chip's yield rate.

[0044] 2. This chip structure provides support for intestinal epithelial cells. Its microporous structure allows fluid to flow internally, simulating capillary exchange. It can potentially simulate the submucosal muscle layer and enteric nerves, and can be inoculated with immune cells to simulate intestinal immunity. In this chip, human intestinal epithelial cells are seeded on top of a porous microfluidic medium. The arterial region is unidirectionally connected to the porous microfluidic medium; fluid from the arterial region flows only unidirectionally into the medium and cannot flow back. Furthermore, fluid from the arterial region is confined to the porous microfluidic medium and flows towards the venous endothelial cells, preventing leakage onto the surface of the intestinal epithelial cells. The venous channel is located at the bottom of the porous microfluidic medium, separated from the intestinal epithelial cells by a porous membrane. Substances from the intestinal lumen pass through the intestinal epithelial cells into the porous microfluidic medium and finally flow into the venous channel, simulating the absorption process of food, digestive substances, or toxins from the intestinal lumen into the bloodstream. Simultaneously, fluid from the arterial region is collected in the venous channel after passing through a porous microfluidic medium, simulating the intestinal blood circulation of arteries-capillaries-venules. This intestinal organ-on-a-chip provides a simulated in vitro platform for intestinal-related digestion, absorption, the influence of internal and external environments on the body, the pathophysiological mechanisms of intestinal-related diseases, and drug screening research. Attached Figure Description

[0045] Figure 1 This is an exploded structural diagram of the intestinal organ chip of the present invention;

[0046] Figure 2 This is a cross-sectional view of the intestinal organ chip of the present invention;

[0047] Figure 3 This is a schematic diagram of the structure of the first embodiment of the toothed rolling roller device of the present invention;

[0048] Figure 4 yes Figure 3 A diagram illustrating the usage status;

[0049] Figure 5 This is a schematic diagram of the structure of a second embodiment of the toothed rolling roller device of the present invention;

[0050] Figure 6 This is a cross-sectional view of the rolling roller of the present invention;

[0051] Figure 7 This is a schematic diagram of the usage state of the dual negative pressure defoaming device of the present invention;

[0052] Figure 8 This is a cross-sectional view of the dual negative pressure defoaming device of the present invention;

[0053] Figure 9 yes Figure 8 Enlarged diagram of area A in the middle. Detailed Implementation

[0054] The above description is a detailed explanation of this patent in conjunction with specific embodiments, and it should not be construed that the specific embodiments of this patent are limited to the above description. For those skilled in the art, any substitutions or modifications made to the described embodiments without departing from the concept of this patent should be considered within the scope of protection of this patent. In this specification, specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification and the features of different embodiments or examples. Although the embodiments of the present invention and their advantages have been described in detail, it should be understood that various changes, substitutions, and modifications can be made herein without departing from the scope of protection of the patent application.

[0055] A microfluidic intestinal organ-on-a-chip, such as Figures 1-2 As shown, the chip body includes an upper chip 1, a porous thin film 2, and a lower chip 3. The upper chip includes a venous channel inlet hole 11, a venous channel outlet hole 12, an intestinal culture unit 13, an arterial unit 14, and a connecting channel 15. The lower chip includes a venous channel inlet shallow groove 31, a venous channel outlet shallow groove 32, a venous unit 33, a venous inlet connecting channel 34, and a venous outlet connecting channel 35. The venous unit 33 is located below the intestinal culture unit 13, and a porous membrane 2 is disposed between the venous unit 33 and the arterial unit 13. The position of the venous channel inlet hole 11 corresponds vertically to the position of the venous channel inlet shallow groove 31, and the position of the venous channel outlet hole 12 corresponds vertically to the position of the venous channel outlet shallow groove 32. The intestinal culture unit 13 and the arterial unit 14 are cylindrical. The arterial unit 14 is located to the right of the intestinal culture unit 13 and is connected by the connecting channel 15. The venous channel inlet shallow groove 31 is connected to the venous unit 33 through the venous inlet connecting channel 34, and the venous channel outlet shallow groove 32 is connected to the venous unit 33 through the venous outlet connecting channel 35.

[0056] The porous film 2 is made of PC or PET material.

[0057] The height of the upper chip is set to 6-8 mm; the narrowest distance of the connecting channel 15 is set to 1-2 mm, the vertical distance is set to 2-4 mm, and the height is set to 1-2 mm; the diameter of the intestinal culture unit 13 and the arterial unit 14 is set to 4-6 mm; the diameter of the venous channel inlet through-hole 11 and the venous channel outlet through-hole 12 is set to 3-4 mm; the feature depth within the lower chip is set to 0.4-0.6 mm; the diameter of the venous channel inlet shallow groove 31 and the venous channel outlet shallow groove 32 is set to 3-4 mm; the diameter of the venous unit 33 is set to 6-8 mm; the width of the venous inlet connecting channel 34 and the venous outlet connecting channel 35 is set to 1-2 mm; and the pore size of the porous membrane 2 is 3-8 μm.

[0058] Considering the overall experimental design and subsequent application requirements, the chip can be made of PDMS material, which has good air permeability, high transparency and is easy to process. Alternatively, depending on the experimental conditions, materials with good biocompatibility and thermoplastic mechanical processing, such as PMMA, PC, and COC, can be selected.

[0059] During implementation, vascular endothelial cells are seeded in the lower layer of the porous membrane 2. The upper layer of the porous membrane is filled with a porous microfluidic medium, designed as a capillary network and submucosal structure of the intestinal tract. Intestinal epithelial cells are seeded on the upper surface of the porous microfluidic medium as an intestinal lumen structure. Materials for the porous microfluidic medium can include fibrinogen, gelatin, laminin, and collagen. In practical applications, collagen is preferred as the porous microfluidic medium. The final gel concentration used in the porous microfluidic medium is 3–6 mg / mL. An arterial structure is located on the right side of the upper chip. Within the arterial structure, liquid can flow into and fill the porous microfluidic medium without overflowing onto the surface of the intestinal epithelial cells.

[0060] This structure provides support for intestinal epithelial cells, and its microporous structure allows fluid to flow internally. It can be used to simulate capillary exchange, potentially mimicking the submucosal muscle layer and enteric nerves, and can be seeded with immune cells to simulate intestinal immunity. In this chip, human intestinal epithelial cells are seeded on top of a porous microfluidic medium. Arterial regions are unidirectionally connected to the porous microfluidic medium; fluid flows only unidirectionally into the medium from the arterial region and cannot flow back. Furthermore, fluid from the arterial region is confined to the porous microfluidic medium and flows towards the venous endothelial cells, preventing leakage onto the surface of the intestinal epithelial cells. Venous channels are located at the bottom of the porous microfluidic medium, separated from the intestinal epithelial cells by a porous membrane. Substances within the intestinal lumen pass through the intestinal epithelial cells into the porous microfluidic medium and finally flow into the venous channels, simulating the absorption process of food, digestive substances, or toxins from the intestinal lumen into the bloodstream. Meanwhile, the fluid from the arterial region is collected in the venous channel after passing through a porous microfluidic medium, thus simulating the intestinal blood circulation of arteries-capillaries-venules.

[0061] Microfluidic intestinal organ-on-a-chip construction method

[0062] The chip design is created using SolidWorks software. Chips can be fabricated using PDMS and thermoplastics. For thermoplastic chips, chip modules can be obtained through injection molding or CNC machining, and then assembled using thermoforming bonding. For PDMS chips, molds can be obtained by CNC machining of PMMA, or through 3D printing. The PDMS chip fabrication process is as follows:

[0063] The PDMS chip manufacturing process includes:

[0064] S1 Pre-processing

[0065] The PDMS prepolymer and curing agent are mixed at a mass ratio of 10:1. The mixture is stirred using an existing stirring device (such as a stirring rod or fork) or the toothed roller device of this invention until fine bubbles are generated to ensure thorough mixing (the PDMS prepolymer and curing agent must be thoroughly mixed, otherwise chip quality will be affected). Because stirring the PDMS prepolymer and curing agent naturally generates bubbles, using existing stirring devices such as stirring rods not only introduces excessive air into the liquid but also results in low mixing efficiency (PDMS prepolymer is a viscous liquid). To reduce the amount of air introduced during stirring, improve the efficiency of subsequent vacuum degassing operations, and enhance the mixing efficiency, the toothed roller device of this invention is preferred in this embodiment to achieve the above-mentioned technical effects. The first embodiment of the toothed roller device 4 is as follows... Figures 3-4 As shown, the device includes a left support and a right support 41, connected by a crossbar 49. Three or more rolling rollers are arranged between the left and right supports 41, and the outer walls of the rolling rollers have toothed grooves. The three rolling rollers are arranged in a triangular formation, with the first rolling roller 42 and the second rolling roller 48 spaced apart at the bottom along the same horizontal line. A third rolling roller 43 is positioned between the first and second rolling rollers, above them, and its toothed grooves engage with those of the first rolling roller 42 and the second rolling roller 48. This arrangement serves to: by pushing the first and second rolling rollers 42 and 48 to roll, the third rolling roller 43 is driven, and through the engagement and rotation of the toothed grooves of the three rolling rollers, the PDMS prepolymer and curing agent are agitated and mixed. Figure 4As shown, in use, three rolling rollers are submerged below the liquid surface to achieve mixing, effectively preventing excessive air from entering the liquid. Agitation is achieved through the toothed grooves of the three rotating rollers, significantly improving the mixing efficiency of the two components. For ease of operation, in this embodiment, as... Figure 3 As shown, it also includes a left connecting rod and a right connecting rod 45. The left connecting rod and the right connecting rod 45 are rotatably connected to the left bracket and the right bracket 41 respectively via a rotating shaft 44. The upper parts of the left connecting rod and the right connecting rod 45 are connected by a connecting rod. An operating rod 46 is connected to the middle of the connecting rod. A handle 47 is provided on the top of the operating rod 46 to facilitate the above-mentioned pushing operation.

[0066] like Figure 5 As shown, in a second embodiment of the toothed roller device 4, the height of the three rollers can be adjusted according to the depth of the liquid to ensure that the height of the three rollers is adjusted with the depth of the target liquid, ensuring that all three rollers are submerged in the liquid. The difference between this embodiment and the first embodiment lies only in that: a first adjusting elongated hole 413, a second adjusting elongated hole 416, and a third adjusting elongated hole 411 are respectively provided on the left support and the right support 41; the first adjusting elongated hole 413 and the second adjusting elongated hole 416 are arranged horizontally, while the third adjusting elongated hole 411 is arranged vertically; as shown... Figure 6 As shown, taking the first adjusting elongated hole 413 as an example, a T-shaped bushing 415 is movably disposed within the first adjusting elongated hole 413, and a thread is provided on the outside of the T-shaped bushing 415. An upper nut 414 is provided on the outside to lock and fix the T-shaped bushing 415. The rolling shaft of the first rolling roller 42 can be rotatably inserted into the T-shaped bushing 415, that is, the position of the first rolling roller 42 is adjusted by adjusting the positions of the T-shaped bushings 415 on both sides. In some embodiments, the first adjusting elongated hole 41... 3. The second and third adjustment elongated holes 416 and 411 are also provided with scales 412 to improve the accuracy of adjustment. In use, if the liquid level is shallow, the gap between the first rolling roller 42 and / or the second rolling roller 48 is widened and the position of the third rolling roller 43 is lowered, so that the third rolling roller 43 engages with the first rolling roller 42 and the second rolling roller 48 respectively, thereby reducing the height of the three rolling rollers and ensuring that the three rolling rollers can always be submerged below the liquid level for stirring.

[0067] The mixed PDMS prepolymer can be placed in a vacuum desiccator for bubble removal, but using this traditional method, bubble removal generally requires waiting for more than 1 hour (bubbles must be completely removed, otherwise defects such as bubble holes will appear after curing, leading to chip defects). In this embodiment, the dual negative pressure debubbling device of the present invention is preferred, which can significantly improve the bubble removal speed and effect, and greatly improve work efficiency. Figures 7-9As shown, the dual negative pressure defoaming device 6 includes a base 63, a separation disc 62, and a top cover 61. The separation disc 62 is detachably disposed between the base 63 and the top cover 61, and its contact surfaces can be provided with silicone rings to improve sealing. The bottom surface 621 of the separation disc 62 is provided with a plurality of extrusion holes 622. The extrusion holes 622 have a conical structure, and the diameter d1 of the upper opening is larger than the diameter d2 of the lower opening. The purpose of this design is to enable the mixed PDMS prepolymer to flow downwards and defoam through the extrusion holes. The structure of 622 pushes out small air bubbles uniformly mixed in the liquid upwards, significantly improving the efficiency of eliminating small air bubbles in the liquid; a first suction pipe 67 and a second suction pipe 66 are respectively provided on one side of the chassis 63 and the separation plate 62, and a first valve 65 and a second valve 64 are respectively provided on the first suction pipe 67 and the second suction pipe 66; a first pressure gauge 611, an exhaust valve (not shown), a safety valve (not shown), etc. are provided on the upper cover 61; a second pressure gauge 651 is provided on the first suction pipe 67. In use, the mixed liquid to be degassed is poured onto the bottom surface 621. Then, the air pump 7 is used to evacuate air from above the separation plate 62 and the base plate 63. Gravity can be used to force the mixed liquid to flow downwards from the degassing hole 622. Alternatively, the following control method can be selected: the pressure on the upper and lower sides of the liquid is checked by the first pressure gauge 611 and the second pressure gauge 651. Then, the pressure difference between the upper and lower sides is controlled by the first valve 65 and the second valve 64, that is, the air pressure on the lower side of the liquid is controlled to be lower than the air pressure on the upper side, thereby increasing the downward flow speed of the liquid. The gas in the liquid is squeezed upwards and then pumped to the upper surface of the liquid. The downward flowing liquid is also subject to the vacuum of the base plate 63 to continue to degas the bubbles. Through the above principle, the bubble degassing speed and degassing effect in this step can be significantly improved, greatly improving the quality and efficiency of chip fabrication, and with good repeatability.

[0068] Pour the defoamed PDMS prepolymer onto the mold and perform vacuum defoaming again until no bubbles are present in the PDMS prepolymer. Transfer it to an 80℃ oven and heat to cure for 1 hour. Finally, carefully peel the cured PDMS off the mold and cut it into PDMS blocks of appropriate size for subsequent use.

[0069] S2 drilling

[0070] Use a punch of the appropriate size to drill holes at the corresponding positions and at a vertical angle on the upper-layer chip of the PDMS.

[0071] S3 chip package

[0072] Since PDMS and PET films cannot be directly bonded via oxygen plasma treatment, aminosilanization of the PET film is required for bonding. In the aminosilanization process, a 5% 3-aminopropyltriethoxysilane (APTES) solution is first prepared with water and preheated to 80°C using a hot plate. The PDMS chip is then cleaned with isopropanol and dried with nitrogen or compressed air. Both sides of the PDMS chip are cleaned with Scotch tape to remove any visible debris. The PET film is then treated with oxygen plasma for 30 seconds (80W) and transferred to the preheated 5% APTES solution for 30 minutes. Next, the PET film is rinsed with deionized water and dried at room temperature. The aminosilanized PET film and the PDMS chip are simultaneously treated with oxygen plasma for 30 seconds (80W) and then bonded. Finally, the assembled PDMS chip is placed in an oven and heated at 60°C for 24 hours.

[0073] Intestinal organ-on-a-chip construction

[0074] The assembled intestinal organ-on-a-chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use.

[0075] Type I collagen was prepared using 5×PBS, 1×PBS, and 0.5N NaOH to achieve a final collagen concentration of 6 mg / ml. The collagen was then added to the upper chip layer, forming a 1.5 mm thick collagen layer within the intestinal culture unit, connecting channels, and arterial units. The chip was incubated at 37°C for 1 hour to induce gel formation.

[0076] To mimic the enteric nervous system and intestinal immunity, enteric nerve cells and immune cells can be embedded in collagen.

[0077] For seeding endothelial cells into the lower layer of a porous membrane, a 50 μg / ml fibronectin solution was first introduced into the lower channel, and the cells were incubated upside down at 37°C for at least 2 hours. The channels were then washed three times with endothelial cell culture medium. Subsequently, the final cell density was increased to 1 × 10⁻⁶ cells / mL. 5 pcs / cm 2 Endothelial cells were introduced into the chip, and the chip was inverted so that the endothelial cells could attach to the underside of the membrane. After the endothelial cells had attached for 1–2 hours, the unattached endothelial cells were rinsed with endothelial cell culture medium, and then the chip was filled with endothelial cell culture medium.

[0078] For intestinal epithelial cell inoculation, the final cell concentration will be 5 × 10⁻⁶. 5 pcs / cm 2 Intestinal epithelial cells were introduced onto the top of a porous microfluidic medium, transferred to an incubator, and placed for 2–4 hours. Subsequently, unattached intestinal epithelial cells were washed away with culture medium.

[0079] The volume of culture medium added can be calculated and set appropriately based on the actual experimental conditions to ensure that the fluid in the artery flows into and fills the porous microfluidic medium without overflowing onto the surface of the intestinal epithelial cells. Simultaneously, fluid from the intestinal lumen can also enter the porous microfluidic medium without flowing into the artery, ultimately allowing the fluid from both the artery and the intestinal lumen to converge in the underlying venous channels, realistically simulating intestinal physiological function. A pressure control device can also be connected to the port to control the pressure difference, achieving the aforementioned control of fluid flow.

[0080] Applications of microfluidic intestinal organ-on-a-chip

[0081] The intestinal organ-on-a-chip proposed in this study has a wide range of applications. Because the chip simulates the intestinal lumen, intestinal barrier, intestinal mucosa and submucosal structures, as well as the arterial-capillary-venous blood supply system, it can be used to study the effects of the intestinal environment on the body (such as the effects of food and gut microbiota on the body, establishing gut-microbiota interactions to elucidate the early pathogenesis of intestinal diseases), and to simulate the processes of food digestion, nutrient absorption, and toxin absorption. The structural simulation of the intestinal blood supply system makes it possible to study the effects of the brain and internal environment (such as chronic stress and depression) on the intestine, such as the effects of hormones or other bioactive substances in the circulatory system on the function of the intestinal structural barrier, and the effects of inflammatory factors on the intestine; or to simulate the immune and inflammatory responses caused by the absorption of substances and toxins after food digestion; it can also replace the use of experimental animals in drug screening to avoid the ethical issues arising from animal testing, improving efficiency and accuracy while saving costs.

Claims

1. A microfluidic intestinal organ-on-a-chip, characterized in that: The chip body includes an upper chip, a porous film, and a lower chip; the upper chip is provided with a venous channel inlet through-hole, a venous channel outlet through-hole, an intestinal culture unit, an arterial unit, and a connection channel; the lower chip is provided with a venous channel inlet shallow groove, a venous channel outlet shallow groove, a venous unit, a venous inlet connection channel, and a venous outlet connection channel. The venous unit is positioned below the intestinal culture unit, with a porous membrane separating them. The position of the venous channel inlet orifice corresponds vertically to the position of the venous channel inlet groove, and the position of the venous channel outlet orifice corresponds vertically to the position of the venous channel outlet groove. The intestinal and arterial units are cylindrical. The arterial unit is positioned to the right of the intestinal culture unit, and the two units are connected via a connecting channel. The venous channel inlet groove is connected to the venous unit via a venous inlet connecting channel, and the venous channel outlet groove is connected to the venous unit via a venous outlet connecting channel. Vascular endothelial cells are seeded in the lower layer of a porous membrane; the upper layer of the porous membrane is filled with a porous microfluidic medium, which is configured as a capillary network and intestinal submucosa structure; intestinal epithelial cells are seeded on the upper surface of the porous microfluidic medium as an intestinal lumen structure. The venous access is located at the bottom of the porous microfluidic medium, and the two are separated by a porous membrane. Substances in the intestinal lumen pass through the intestinal epithelial cells and enter the porous microfluidic medium, eventually flowing into the venous channel to simulate the absorption process of food, digestive substances, or toxins from the intestinal lumen into the bloodstream. Within the arterial region, fluid flows into the porous microfluidic medium in only one direction and cannot flow back; furthermore, fluid from the arterial region is confined to flowing within the porous microfluidic medium and towards the venous endothelial cells, but cannot overflow onto the surface of the intestinal epithelial cells; after passing through the porous microfluidic medium, the liquid from the arterial region is collected in the venous channel, thus simulating the intestinal blood circulation of artery-capillary-venous.

2. The microfluidic intestinal organ chip according to claim 1, characterized in that: The porous film is made of PC or PET material.

3. The microfluidic intestinal organ chip according to claim 1, characterized in that: The chip body is made of PDMS, PMMA, PC, or COC.

4. The microfluidic intestinal organ chip according to claim 1, characterized in that: The final gel concentration used in porous microfluidic media is 3-6 mg / mL.

5. A microfluidic intestinal organ chip according to claim 1, characterized in that: The height of the upper chip is set to 6-8 mm; the narrowest distance of the connecting channels is set to 1-2 mm, the vertical distance is set to 2-4 mm, and the height is set to 1-2 mm; the diameter of the intestinal culture unit and the arterial unit is set to 4-6 mm; the diameter of the venous channel inlet and outlet through-holes is set to 3-4 mm; the feature depth within the lower chip is set to 0.4-0.6 mm; the diameter of the venous channel inlet and outlet shallow grooves is set to 3-4 mm; the diameter of the venous unit is set to 6-8 mm; the width of the venous inlet connecting channel and the venous outlet connecting channel is set to 1-2 mm; and the pore size of the porous membrane is 3-8 μm.

6. A method for constructing a microfluidic intestinal organ-on-a-chip, characterized in that: Construct a microfluidic intestinal organ chip as described in any one of claims 1 to 5, wherein the intestinal organ chip is fabricated using PDMS or thermoplastic plastic; The PDMS chip manufacturing process includes: S1 Pre-processing Mix the PDMS prepolymer and curing agent at a mass ratio of 10:1 and stir until fully mixed; The mixed PDMS prepolymer is degassed using a vacuum method; the degassed PDMS prepolymer is poured onto a mold, ensuring that there are no air bubbles present; then it is transferred to an oven for heating and curing. S2 drilling Drill holes at the corresponding positions and vertical angles using a punch of the appropriate size on the upper-layer chip of the PDMS. S3 chip package Since PDMS and PET film cannot be directly bonded by oxygen plasma treatment, bonding is achieved by aminosilanizing the PET film. The aminosilanization process is as follows: (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water, preheat it to 80°C, clean the PDMS chip with isopropanol and dry it with nitrogen or compressed air, and clean both sides of the PDMS chip to remove any obvious debris. (2) Treat the PET film with oxygen plasma for 30 seconds and then transfer it to a preheated 5% APTES solution for 30 minutes; (3) Wash the PET film with deionized water and dry it at room temperature; (4) The aminosilanized PET film and the PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and then bonded together; (5) Place the assembled PDMS chip into an oven and heat it at 60°C for 24 hours; S4 Intestinal Organ Microarray Construction The assembled PDMS chip was sterilized by washing the channel with 70% ethanol and then irradiated with ultraviolet light in a biosafety cabinet for at least 15 minutes before use. (1) Type I collagen, 5×PBS, 1×PBS and 0.5N NaOH were mixed to prepare a final collagen concentration of 6 mg / ml; then, the collagen was added to the upper chip to form a collagen layer with a thickness of 1.5 mm in the intestinal culture unit, connecting channel and arterial unit; after incubating the chip in a 37°C incubator for 1 hour, gel formation was induced. (2) Endothelial cells were seeded into the lower layer of the porous membrane. First, a 50 μg / ml fibronectin solution was introduced into the lower channel. The cell culture was inverted at 37°C for at least 2 hours. The channel was then washed with endothelial cell culture medium. Subsequently, the final cell density was increased to 1×10⁻⁶ cells / mL. 5 pcs / cm 2 Endothelial cells were introduced into the chip, and the chip was inverted so that the endothelial cells could attach to the underside of the membrane. After the endothelial cells had attached for 1-2 hours, the unattached endothelial cells were rinsed off, and then the chip was filled with endothelial cell culture medium. (3) Inoculate intestinal epithelial cells to a final cell concentration of 5 × 10⁻⁶. 5 pcs / cm 2 Intestinal epithelial cells were introduced onto the top of a porous microfluidic medium, transferred to an incubator, and placed for 2-4 hours. Then, unattached intestinal epithelial cells were washed away.

7. The microfluidic intestinal organ-on-a-chip construction method according to claim 6, characterized in that: In step (1) of the S4 intestinal organ-on-a-chip construction, the construction of the intestinal nerve and intestinal immune simulation includes embedding intestinal nerve cells and immune cells in collagen.

8. The application of microfluidic intestinal organ-on-a-chip, characterized by: The microfluidic intestinal organ chip as described in any one of claims 1 to 5 is applied to: the influence of the internal and external environment of the body on the intestine, and thus on health, and the simulation of the influence of the intestinal environment on the body; Or it can be applied to: simulating the digestion and absorption of food in the intestines; Or it can be applied to: simulating the effects of the brain on the gut, the effects of changes in the body's internal environment on the gut, and simulating the effects of hormones, neurotransmitters, inflammatory factors or other bioactive substances in the bloodstream on the gut structure and barrier function; Or it can be applied to: the simulation of immune and inflammatory responses caused by the absorption of substances and toxins after digestion; Or it can be used to replace the use of laboratory animals in the drug screening process.

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