Microfluidic intestinal organ chip as well as construction method and application thereof

Abstract

The invention relates to the technical field of micro-fluidic intestinal organ chips, and particularly discloses a micro-fluidic intestinal organ chip as well as a construction method and application thereof. The chip body comprises an upper-layer chip, a porous film and a lower-layer chip; a porous film is arranged between the vein unit and the artery unit; the construction method comprises the following steps: (1) adding collagen into the intestinal culture unit, and inducing gel formation; (2) inoculating endothelial cells to the lower layer of the porous film; and (3) inoculating intestinal epithelial cells. The method can be applied to the simulation of the influence of the intestinal environment on the organism; the absorption of intestinal tracts on nutrient substances and toxins is simulated; the influence of the brain on the intestinal tract is simulated, and the influence of environmental changes (such as chronic pressure and depression) in the body on the intestinal tract is simulated through hormones, neurotransmitters, inflammatory factors or other bioactive substances in blood circulation on the structure and the barrier function of the intestinal tract; in-vivo simulation of immune and inflammatory reactions absorbed after digestion; and the kit can be used for replacing experimental animals in the drug screening process.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic intestinal organ chips, and in particular to a microfluidic intestinal organ chip, a construction method and applications thereof. Background Art

[0002] The surface of the small intestine facing the intestinal cavity is called the mucosa, which together with the submucosal layer, outer muscle layer and serosa below constitutes the intestinal tissue. The mucosal surface is composed of simple columnar epithelial cells, and the intestinal barrier is formed by the junctions between epithelial cells and cells. Tight junctions, adhesion junctions, desmosome junctions and gap junctions are arranged in sequence from the top of the intestinal epithelial cells to the basement membrane, sealing the top with the basement membrane and limiting paracellular permeability. Its permeability is regulated by many factors, including chronic stress, diet, intestinal flora and cytokines. The basal lamina propria has abundant capillaries. The arterial blood vessels of the intestine are innervated by the arterial arches that extend radially from the superior mesenteric artery in the mesentery to the intestinal wall, gradually forming a capillary network, and then converging to the capillary venous network and intestinal veins, and finally converging to the portal vein. The wall of the capillary is composed of a single, thin endothelial cell layer and the basement membrane surrounding it. The distance between the basement membrane of the intestinal epithelial cells and the capillaries does not exceed a few microns. These blood vessels are not only close to the epithelium, but also have fenestrations in the endothelial cells covered only by the basement membrane. Therefore, after the food is mechanically and chemically digested, the nutrients can pass through the junctions between the epithelial cells and enter the capillaries located in the lamina propria. The substances are transferred from the lumen to the vascular system to complete the absorption of nutrients.

[0003] Furthermore, the intestine, 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 blocking the entry of toxins and bacteria into the luminal toxins, antigens, and intestinal flora to maintain a balanced homeostasis. Disruption of the intestinal barrier increases permeability, which can be detrimental to the host because it may allow the translocation of luminal antigens and toxins through the intestinal wall into the subepithelial tissue and blood. In turn, this translocation may induce local and systemic immune responses, potentially leading to the development of pathology. In addition, increased intestinal permeability has been associated with a variety of autoimmune and gastrointestinal diseases.

[0004] The digestion and absorption process of the intestine is extremely complex, and the internal and external environment has a profound impact on the body in many aspects. For example, external dietary intake, microbial environment, internal hormone regulation and neurotransmitter action all affect the function of the intestine.

[0005] Animal models are currently a common way to study intestinal pathophysiology. Due to species differences, animal models cannot accurately reflect the human body's response to pathogens, diseases, and drugs. For example, in animal experimental studies of chronic stress, stress usually needs to be induced for several days, ranging from 7 to 14 days. On the other hand, due to ethical issues related to clinical research, observational studies can only be performed under natural stress conditions. Because animal and clinical studies differ greatly in both the type and duration of stressors and the measurement of intestinal permeability, it is difficult to compare animal stress studies with human stress studies.

[0006] In addition, traditional cell models are also unable to 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 intestine 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 with fully differentiated intestinal epithelial cells. However, organoid culture is static and does not have the fluid flow and peristalsis that exist in the human intestine. Although intestinal organoids can simulate intestinal physiology, it is difficult to deliver microorganisms or food to the interior of the intestinal organoids due to the closed lumen of the intestinal organoids.

[0007] Organ chips are miniature cell culture devices with microfluidic channels and living cells based on microfluidic technology. They can simulate the key structures and functional units of human tissues and organs in vitro in an approximately physiological manner, making up for the limitations of animal models and static models to a certain extent. At present, there are three main types of intestinal organ chip models: 1) Vertical sandwich design. The vertical sandwich design intestinal organ chip model is composed of upper and lower channels, and the upper and lower channels are interconnected by suspended porous films. Usually, intestinal epithelial cells and vascular endothelial cells are inoculated in the upper and lower channels respectively, and the shear force experienced by the cells is generated by fluid perfusion in the channel and the substances are transported to simulate the vascular and luminal environment. 2) Plane parallel design. In addition to the left and right microchannels, the chip of this design also adds a middle channel filled with collagen gel. The gel is filled into the middle channel using microcolumns or phase guidance, and intestinal epithelial cells and vascular endothelial cells are inoculated in the left and right microchannels. 3) 3D gel structure. In this design, the chip channels are filled with gel and laser-ablated to have multiple side indentations in the shape of crypts to polarize basal stem cells, or hollow cylindrical channels are formed within the 3D gel using tiny sticks or sticky fingers, and cells are then cultured along the surface of the central cavity.

[0008] Most of the existing intestinal organ chips can only simulate a single vascular structure, such as only simulating the intestinal barrier structure (enteroepithelial cells), etc., and compared with the human intestinal anatomy and physiology, they cannot accurately simulate the structure and function of the submucosal membrane, especially lack the simulation of the perfusion arteries that control the intestinal wall and even the intestinal capillary network. They cannot simulate the blood circulation between arteries and veins and the material exchange between blood and tissues, nor can they simulate the impact of the internal environment on the intestine, such as the impact of chronic stress, neuroendocrine disorders, brain diseases, etc. on the intestine through neurotransmitters circulating in the blood, etc., that is, they cannot achieve a full range of simulation of intestinal structure and function. Summary of the invention

[0009] In response to the above technical problems, the present invention provides a microfluidic intestinal organ chip, a construction method and its application, so as to achieve all-round simulation of intestinal structure and function.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] A microfluidic intestinal organ chip, the chip body comprises 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 connecting 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 connecting channel, and a venous outlet connecting channel; the venous unit is arranged below the intestinal culture unit, and a porous film is arranged between the venous unit and the arterial unit; the position of the venous channel inlet through hole corresponds to the position of the venous channel inlet shallow groove in the vertical direction, and the position of the venous channel outlet through hole corresponds to the position of the venous channel outlet shallow groove in the vertical direction; the intestinal culture unit and the arterial unit are arranged in a cylindrical shape; the arterial unit is arranged on the right side of the intestinal culture unit and connected through a connecting channel; the venous channel inlet shallow groove is connected to the venous unit through the venous inlet connecting channel, and the venous channel outlet shallow groove is connected to the venous unit through the venous outlet connecting channel.

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

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

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

[0015] Optionally, the upper layer of the porous film is filled with a porous microfluidic medium, which is set as a capillary network and an intestinal submucosal structure; the upper surface of the porous microfluidic medium is inoculated with intestinal epithelial cells as an intestinal cavity structure; and the final concentration of the gel used in the porous microfluidic medium is 3 to 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 6-8mm; the narrowest distance of the connecting channel is set to 1-2mm, the upper and lower distances are set to 2-4mm, and the height is set to 1-2mm; the diameters of the intestinal culture unit and the arterial unit are set to 4-6mm; the diameters of the venous channel entrance through hole and the venous channel outlet through hole are set to 3-4mm; the characteristic depths in the lower chip are all set to 0.4-0.6mm; the diameters of the venous channel entrance shallow groove and the venous channel outlet shallow groove are set to 3-4mm; the diameter of the venous unit is set to 6-8mm; the widths of the venous entrance connecting channel and the venous outlet connecting channel are set to 1-2mm; and the pore size of the porous film is 3-8μm.

[0018] A method for constructing a microfluidic intestinal organ chip, constructing the microfluidic intestinal organ chip as described above, wherein the intestinal organ chip is made of PDMS or thermoplastic;

[0019] The PDMS chip production process includes:

[0020] S1 Pre-processing

[0021] The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1 and stirred until fully mixed; the mixed PDMS prepolymer is vacuumed to eliminate bubbles; the debubbled PDMS prepolymer is poured onto the mold to ensure that there are no bubbles in the PDMS prepolymer; then it is transferred to an oven and heated to cure;

[0022] S2 Punch Hole

[0023] Use a puncher of corresponding size to punch holes at a vertical angle at the corresponding position on the PDMS upper chip;

[0024] S3 chip package

[0025] Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, bonding was achieved by aminosilanization of the PET film;

[0026] The aminosilanization process is:

[0027] (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water and preheat it to 80°C. Clean the PDMS chip with isopropanol and blow dry it with nitrogen or compressed air. 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 transfer it to a preheated 5% APTES solution for 30 minutes;

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

[0030] (4) The aminosilanized PET film and the PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and bonded;

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

[0032] S4 Intestinal Organ Chip Construction

[0033] The assembled PDMS chip was sterilized by washing the channels with 70% ethanol and irradiated with UV 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; subsequently, collagen was added to the upper chip to form a 1.5 mm thick collagen layer in the intestinal culture unit, the connecting channel, and the arterial unit; the chip was incubated in a 37°C incubator for 1 hour to induce gel formation;

[0035] (2) Endothelial cells were seeded into the lower layer of the porous film. First, a 50 μg / ml fibronectin solution was introduced into the lower channel, which was then placed upside down in a 37°C incubator for at least 2 h. The channel was then washed with endothelial cell culture medium. Subsequently, the final cell density was 1×10 5 Pieces / cm 2 The endothelial cells are introduced into the chip, and the chip is inverted so that the endothelial cells can attach to the lower side of the membrane; after the endothelial cells attach for 1 to 2 hours, the unattached endothelial cells are rinsed and then filled with endothelial cell culture medium;

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

[0037] Optionally, in step (1) of constructing the S4 intestinal organ chip, the construction of intestinal nerves and intestinal immune simulations includes embedding intestinal nerve cells and immune cells in collagen.

[0038] Applications of microfluidic intestinal organ chips: The microfluidic intestinal organ chips described above are used to: simulate the effects of the internal and external environment of the body on the intestine and thus on health, and the effects of the intestinal environment (such as intestinal flora) on the body;

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

[0040] Or applied to: simulation of the effect of the brain on the intestine, the effect of changes in the body's internal environment (such as chronic stress, depression) on the intestine, simulation of the intestinal structure and barrier function through hormones, neurotransmitters, inflammatory factors or other bioactive substances in the blood circulation;

[0041] Or applied to: simulation of immune and inflammatory responses caused by the absorption of digested food substances and toxins;

[0042] Or applied to: replacing the use of experimental animals in the drug screening process.

[0043] 1. This intestinal organ chip has an intestinal barrier structure formed by arteries, intestinal cavities (including simulation of mucosa and submucosal structures), and venous systems, which can simulate the absorption process. At the same time, the intestinal perfusion artery features implanted in the intestine can realize blood supply to the intestinal mucosa and submucosal layer (such as perfusion of neurotransmitters or neuroendocrine factors to simulate the impact of changes in the in vivo environment on the intestine) and the capillary system from arteries to veins with a porous microfluidic medium structure. Another unique feature of the bionic intestinal organ chip system is the use of porous microfluidic media as the intestinal submucosal structure. Based on the realization of the above-mentioned all-round simulation of intestinal structure and function, the structural design of this intestinal organ chip is reliable, which is conducive to the smoothness of the subsequent related liquid flow control and maximizes the relevant performance of the chip; at the same time, it is conducive to the standard preparation of the chip in the later stage and the preparation is simple, which makes the preparation quality controllable and significantly improves the qualified rate of chip preparation.

[0044] 2. The chip structure can provide support for intestinal epithelial cells. Its tiny porous structure allows liquid to flow inside, which can be used to simulate capillaries for material exchange, potentially simulate the submucosal muscle layer and intestinal nerves, and can be inoculated with immune cells to simulate intestinal immunity. In this chip, human intestinal epithelial cells are inoculated on the top of the porous microfluidic medium, and the arterial characteristic area is unidirectionally connected to the porous microfluidic medium, that is, the fluid in the arterial area only flows into the porous microfluidic medium in one direction, and vice versa. It cannot flow back. In addition, the fluid from the arterial area is limited to flowing in the porous microfluidic medium and flows toward the venous endothelial cells, but cannot overflow to the surface of the intestinal epithelial cells. The venous channel is located at the bottom of the porous microfluidic medium, and the two are separated by a layer of porous film. The substances in the intestinal cavity pass through the intestinal epithelial cells and enter the porous microfluidic medium, and finally flow into the venous channel to simulate the absorption process of food, digestive tract substances or toxins from the intestinal cavity into the blood. At the same time, the liquid from the arterial area is collected in the venous channel after passing through the porous microfluidic medium, realizing the simulation of the intestinal blood circulation of artery-capillary-venous. This intestinal organ chip can provide a simulated in vitro platform for intestinal digestion, absorption, the impact of internal and external environment on the body, the pathological and physiological mechanism of intestinal diseases, and drug screening research. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is a schematic diagram of the exploded structure of the intestinal organ chip of the present invention;

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

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

[0048] Figure 4 yes Figure 3 Schematic diagram of the usage status;

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

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

[0051] Figure 7 It is a schematic diagram of the use state of the double negative pressure defoaming device of the present invention;

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

[0053] Fig. 9 yes Figure 8 A magnified schematic diagram of area A in the middle. DETAILED DESCRIPTION

[0054] The above content is a detailed description of this patent in combination with specific implementation methods, and it cannot be determined that the specific implementation methods of this patent are limited to the above description. For ordinary technicians in the technical field to which this patent belongs, without departing from the concept of this patent, several substitutions or modifications made to the above-described implementation methods should be deemed to belong to the protection scope of this patent. In the description of this specification, in this specification, the specific features, structures, materials or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In the absence of contradictions, those skilled in the art can combine and combine 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 chip, such as Figures 1-2 As shown, the chip body includes an upper chip 1, a porous film 2 and a lower chip 3. Among them, the upper chip is provided with a venous channel inlet through hole 11, a venous channel outlet through hole 12, an intestinal culture unit 13, an arterial unit 14, and a connecting channel 15; the lower chip is provided with 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 arranged below the intestinal culture unit 13, and the venous unit 33 is arranged between the arterial unit 13 and a porous film 2 is arranged; the position of the venous channel inlet through hole 11 corresponds to the position of the venous channel inlet shallow groove 31 in the vertical direction, and the position of the venous channel outlet through hole 12 corresponds to the position of the venous channel outlet shallow groove 32 in the vertical direction; the intestinal culture unit 13 and the arterial unit 14 are arranged in a cylindrical shape; the arterial unit 14 is arranged on the right side of the intestinal culture unit 13 and is connected through a 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.

[0057] The height of the upper chip is set to 6-8mm; the narrowest distance of the connecting channel 15 is set to 1-2mm, the upper and lower distances are set to 2-4mm, and the height is set to 1-2mm; the diameters of the intestinal culture unit 13 and the arterial unit 14 are set to 4-6mm; the diameters of the venous channel entrance through hole 11 and the venous channel outlet through hole 12 are set to 3-4mm; the characteristic depths in the lower chip are all set to 0.4-0.6mm; the diameters of the venous channel entrance shallow groove 31 and the venous channel outlet shallow groove 32 are set to 3-4mm; the diameter of the venous unit 33 is set to 6-8mm; the widths of the venous entrance connecting channel 34 and the venous outlet connecting channel 35 are set to 1-2mm; the pore size of the porous film 2 is 3-8μm.

[0058] Taking into account the overall experimental design and subsequent application requirements, the chip can use PDMS material with good air permeability, high transparency and easy processing, or it can choose materials with good biocompatibility and thermoplastic mechanical processing, including PMMA, PC, COC and other materials according to the experimental conditions.

[0059] During the implementation process, the lower layer of the porous film 2 is inoculated with vascular endothelial cells. The upper layer of the porous film is filled with porous microfluidic media, which is set to a capillary network and intestinal submucosal structure. Intestinal epithelial cells are inoculated on the upper surface of the porous microfluidic medium. As the intestinal cavity structure, fibrinogen, gelatin, laminin, collagen, etc. can be selected as the material for the porous microfluidic medium. In practical applications, collagen is preferred for porous microfluidic media. The final concentration of the gel used in its porous microfluidic medium is 3 to 6 mg / mL. On the right side of the upper chip is the arterial structure. In the arterial structure, the liquid can flow to and fill the porous microfluidic medium without overflowing onto the surface of the intestinal epithelial cells.

[0060] This structure can provide support for intestinal epithelial cells. Its tiny porous structure allows liquid to flow inside, which can be used to simulate capillaries for material exchange. It can potentially simulate the submucosal muscle layer and intestinal nerves, and can be inoculated with immune cells to simulate intestinal immunity. In this chip, human intestinal epithelial cells are inoculated on the top of the porous microfluidic medium, and the arterial characteristic area is unidirectionally connected to the porous microfluidic medium, that is, the fluid in the arterial area only flows into the porous microfluidic medium in one direction, and cannot flow back in the opposite direction. In addition, the fluid from the arterial area is limited to flowing in the porous microfluidic medium and flows toward the venous endothelial cells, but cannot overflow to the surface of the intestinal epithelial cells. The venous channel is located at the bottom of the porous microfluidic medium, and the two are separated by a layer of porous film. The substances in the intestinal cavity pass through the intestinal epithelial cells and enter the porous microfluidic medium, and finally flow into the venous channel to simulate the absorption process of food, digestive tract substances or toxins from the intestinal cavity into the blood. At the same time, the fluid from the arterial area passes through the porous microfluidic medium and then gathers in the venous channel, thus simulating the intestinal blood circulation of artery-capillary-venous.

[0061] Microfluidic Intestinal Organ Chip Construction Method

[0062] Solidworks software is used to draw the design of the chip. The chip can be made of PDMS and thermoplastics. For chips made of thermoplastics, the chip module can be obtained by injection molding or CNC processing, and then the chip is assembled by hot pressing bonding. For PDMS chip production, PMMA is processed by CNC processing to obtain a mold, or the mold can be obtained by 3D printing. The PDMS chip production process is as follows:

[0063] The PDMS chip production process includes:

[0064] S1 Pre-processing

[0065] The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1, and stirred using an existing stirring device (such as a stirring rod or a fork) or the toothed rolling roller device of the present invention until fine bubbles are generated to allow for full mixing (the PDMS prepolymer and the curing agent must be fully mixed, otherwise the chip quality will be affected); because bubbles will naturally be generated when the PDMS prepolymer and the curing agent are stirred, the use of existing stirring devices such as a stirring rod will not only cause excessive air to be stirred into the liquid, but also the stirring and mixing efficiency is low (the PDMS prepolymer is a viscous liquid); in order to reduce the amount of air mixed into the stirring process, improve the efficiency of the subsequent vacuum degassing operation, and improve the stirring and mixing efficiency, in this embodiment, the toothed rolling roller device of the present invention may be preferably used to achieve the above-mentioned technical effects. The first embodiment of the toothed rolling roller device 4, such as Figures 3-4 As shown, it includes a left bracket and a right bracket 41, and the left bracket and the right bracket are connected by a cross bar 49; more than three rolling rollers are arranged between the left bracket and the right bracket 41, and the outer walls of the rolling rollers are provided with tooth marks; the three rolling rollers are arranged in a triangle to form a group, wherein the first rolling roller 42 and the second rolling roller 48 are arranged at the same horizontal line at the lower part, and the third rolling roller 43 is arranged between the first rolling roller 42 and the second rolling roller 48, and the third rolling roller 43 is arranged above the first rolling roller and the second rolling roller, and the tooth marks of the third rolling roller 43 are respectively meshed with the tooth marks of the first rolling roller 42 and the tooth marks of the second rolling roller 48; the effect of such arrangement is: when using, by pushing the first rolling roller 42 and the second rolling roller 48 to roll, the third rolling roller 43 is driven, and the PDMS prepolymer and the curing agent are stirred and mixed through the meshing and rotation of the tooth marks of the three rolling rollers, and at the same time as Figure 4As shown, when in use, the three rollers are immersed below the liquid surface to achieve mixing, effectively preventing excessive air from mixing into the liquid, and stirring is achieved by relying on the tooth marks of the three rotating rollers, which significantly improves 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 side rod and a right connecting side rod 45, which are rotatably connected to the left bracket and the right bracket 41 respectively through a rotating shaft 44, and the upper parts of the left connecting side rod and the right connecting side rod 45 are connected by a connecting rod, and an operating rod 46 is connected to the middle part of the connecting rod, and 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, the second embodiment of the toothed rolling roller device 4 can adjust the height of the three rolling rollers according to the depth of the liquid, so as to ensure that the height of the three rolling rollers is adjusted along with the depth of the target liquid, and ensure that the three rolling rollers are immersed in the liquid. The only difference between the second embodiment and the first embodiment is that the first adjustment long hole 413, the second adjustment long hole 416 and the third adjustment long hole 411 are respectively provided on the left bracket and the right bracket 41; the first adjustment long hole 413 and the second adjustment long hole 416 are provided in the horizontal direction, and the third adjustment long hole 411 is provided in the vertical direction; as shown in FIG. Figure 6 As shown, taking the first adjustment slot 413 as an example, a T-shaped sleeve 415 is movably provided in the first adjustment slot 413, and then a thread is provided on the outside of the T-shaped sleeve 415, and the T-shaped sleeve 415 is locked and fixed by providing an upward nut 414 on the outside, and the rolling shaft of the first rolling roller 42 can be rotatably inserted into the T-shaped sleeve 415, that is, the position of the first rolling roller 42 is adjusted by adjusting the positions of the T-shaped sleeves 415 on both sides; in some embodiments, the first adjustment slot 413 is provided with a T-shaped sleeve 415, and the T-shaped sleeve 415 is provided with a thread on the outside ... provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 415 is provided with a thread on the outside, and the T-shaped sleeve 4 3. The second adjustment long hole 416 and the third adjustment long hole 411 are also respectively provided with scales 412 to increase the accuracy of the adjustment; when 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 is engaged with the first rolling roller 42 and the second rolling roller 48 respectively, so as to reduce the height of the three rolling rollers, thereby ensuring that the three rolling rollers can be always immersed below the liquid surface for stirring operation.

[0067] The mixed PDMS prepolymer can be placed in a vacuum drying dish to eliminate bubbles, but using this traditional method, it generally takes more than 1 hour to eliminate bubbles (the bubbles must be fully eliminated, otherwise there will be defects such as bubbles and holes after curing, resulting in unqualified chips); in this embodiment, the double negative pressure degassing device of the present invention can be preferably used, which can significantly improve the bubble elimination speed and elimination effect, and greatly improve the work efficiency; Figures 7 to 9As shown, the double negative pressure defoaming device 6 includes a bottom plate 63, a separation plate 62 and an upper cover 61; the separation plate 62 is detachably arranged between the bottom plate 63 and the upper cover 61, and its contact surface can be provided with a silicone ring to improve the sealing performance; the bottom surface 621 of the separation plate 62 is provided with a plurality of squeezing holes 622; the squeezing holes 622 are in a conical structure, and the aperture d1 of the upper opening is larger than the aperture d2 of the lower opening. The purpose of this design is to promote the mixed PDMS prepolymer to flow downward by relying on the squeezing holes The structure of 622 squeezes small bubbles uniformly mixed in the liquid upward, significantly improving the efficiency of eliminating small bubbles in the liquid; the first exhaust pipe 67 and the second exhaust pipe 66 are respectively provided on one side of the bottom plate 63 and the separation plate 62, and the first exhaust pipe 67 and the second exhaust pipe 66 are respectively provided with a first valve 65 and a second valve 64; a first pressure gauge 611 and 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 exhaust pipe 67. When in use, pour the mixed liquid from which bubbles are to be eliminated onto the bottom surface 621, and then use the air pump 7 to evacuate the top of the separation plate 62 and the bottom plate 63. Gravity can be used to cause the mixed liquid to flow downward from the bubble squeezing hole 622, or the following control method can be selected: the pressures on the upper and lower sides of the liquid can be checked by the first pressure gauge 611 and the second pressure gauge 651, and then the upper and lower pressure differences can be 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 speed at which the liquid flows downward, and the gas in the liquid is squeezed upward and then drawn above the liquid surface, and the liquid flowing downward is further subjected to the vacuum degree of the bottom plate 63 to continue to eliminate bubbles. Through the above principle, the bubble elimination speed and elimination effect in this step can be significantly improved, thereby greatly improving the quality and work efficiency of chip preparation, and having good repeatability.

[0068] Pour the debubbled PDMS prepolymer onto the mold and perform vacuum debubbling again until there are no bubbles in the PDMS prepolymer; transfer it to an 80°C oven and heat-cure it for 1 hour; finally, carefully peel off the cured PDMS from the mold and cut it into PDMS blocks of appropriate sizes for subsequent use.

[0069] S2 Punch Hole

[0070] Use a puncher of corresponding size to punch holes at a vertical angle at the corresponding positions on the PDMS upper chip.

[0071] S3 chip package

[0072] Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, the PET film needs to be aminosilanized to achieve bonding. During the aminosilanization process, a 5% 3-aminopropyltriethoxysilane (APTES) solution is first prepared with water, preheated to 80°C with a hot plate, the PDMS chip is cleaned with isopropanol and blown dry with nitrogen or compressed air, and both sides of the PDMS chip are cleaned with Scotch tape to remove any obvious debris. The PET film is treated with oxygen plasma for 30 seconds (80W) and transferred to the preheated 5% APTES solution for 30 minutes. Then, the PET film is washed with deionized water and dried at room temperature. The aminosilanized PET film and PDMS chip are simultaneously treated with oxygen plasma for 30 seconds (80W) and 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 chip was sterilized by washing the channels with 70% ethanol and irradiated with UV light in a biosafety cabinet for at least 15 minutes before use.

[0075] Type I collagen, 5×PBS, 1×PBS, and 0.5NNaOH were prepared to a final concentration of 6 mg / ml. Subsequently, collagen was added to the upper chip to form a 1.5 mm thick collagen layer in the intestinal culture unit, the connecting channel, and the arterial unit. The chip was incubated in a 37°C incubator for 1 hour to induce gel formation.

[0076] To simulate intestinal nerves and intestinal immunity, intestinal nerve cells and immune cells can be embedded in collagen.

[0077] For endothelial cells seeded into the lower layer of the porous membrane, a 50 μg / ml fibronectin solution was first introduced into the lower channel, which was then placed upside down in a 37°C incubator for at least 2 h, and the channel was washed three times with endothelial cell culture medium. 5 Pieces / cm 2 Introduce the endothelial cells into the chip and invert the chip so that the endothelial cells can attach to the underside of the membrane. After the endothelial cells attach for 1 to 2 hours, rinse the unattached endothelial cells with endothelial cell culture medium and then fill it with endothelial cell culture medium.

[0078] For intestinal epithelial cell inoculation, the final cell concentration was 5 × 10 5 Pieces / cm 2 The intestinal epithelial cells were introduced onto the top of the porous microfluidic medium, transferred to an incubator and placed for 2 to 4 hours, and then the unattached intestinal epithelial cells were rinsed off with culture medium.

[0079] For the volume of culture medium added, the appropriate liquid height can be calculated and set according to the actual experimental situation, so that the liquid in the artery can flow into and fill the porous microfluidic medium without overflowing onto the surface of the intestinal epithelial cells. At the same time, the liquid in the intestinal cavity can also enter the porous microfluidic medium and not flow into the artery, ultimately achieving the collection of the liquid in the artery and the liquid in the intestinal cavity in the lower venous channel, truly simulating the physiological function of the intestine. The pressure control device can also be connected to the port to control the pressure difference, thereby realizing the above-mentioned control process of liquid flow.

[0080] Applications of Microfluidic Intestinal Organ Chips

[0081] The intestinal organ chip of this proposal has a wide range of applications. Since the intestinal cavity, intestinal barrier, intestinal mucosa and submucosal structure, and arterial-capillary-venous blood supply system are simulated in the chip, it can be used to study the impact of the intestinal environment on the body (such as the impact of food and intestinal microorganisms on the body, establish the relationship between the intestine and the microbiome, and clarify the early pathogenesis of intestinal diseases), as well as the simulation of food digestion, nutrients, and toxin absorption processes; the structural simulation of the intestinal blood supply system makes it possible to study the impact of the brain and the internal environment (such as chronic stress and depression) on the intestine, such as the impact of hormones or other bioactive substances in the circulatory system on the intestinal structural barrier function, and the impact of inflammatory factors on the intestine; or it can be used to simulate 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 the drug screening process to avoid ethical issues caused by animal experiments, and improve efficiency and accuracy while saving costs.

Claims

1. A microfluidic intestinal organ 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 connecting 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 connecting channel, and a venous outlet connecting channel; the venous unit is arranged below the intestinal culture unit, and a porous film is arranged between the venous unit and the arterial unit; the position of the venous channel inlet through hole corresponds to the position of the venous channel inlet shallow groove in the vertical direction, and the position of the venous channel outlet through hole corresponds to the position of the venous channel outlet shallow groove in the vertical direction; the intestinal culture unit and the arterial unit are arranged in a cylindrical shape; the arterial unit is arranged on the right side of the intestinal culture unit and is connected through a connecting channel; the venous channel inlet shallow groove is connected to the venous unit through the venous inlet connecting channel, and the venous channel outlet shallow groove is connected to the venous unit through the venous outlet connecting channel.

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

3. The microfluidic intestinal organ chip according to claim 1, characterized in that: The material used for the chip body is PDMS or PMMA or PC or COC.

4. The microfluidic intestinal organ chip according to claim 1, characterized in that: The lower layer of the porous film is seeded with vascular endothelial cells.

5. The microfluidic intestinal organ chip according to claim 1, characterized in that: The upper layer of the porous film is filled with a porous microfluidic medium, which is set as a capillary network and an intestinal submucosal structure; intestinal epithelial cells are inoculated on the upper surface of the porous microfluidic medium as an intestinal cavity structure; the final concentration of the gel used in the porous microfluidic medium is 3-6 mg / mL.

6. The microfluidic intestinal organ chip according to claim 5, characterized in that: 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.

7. The microfluidic intestinal organ chip according to claim 1, characterized in that: The height of the upper chip is set to 6-8mm; the narrowest distance of the connecting channel is set to 1-2mm, the upper and lower distances are set to 2-4mm, and the height is set to 1-2mm; the diameters of the intestinal culture unit and the arterial unit are set to 4-6mm; the diameters of the venous channel entrance through hole and the venous channel outlet through hole are set to 3-4mm; the characteristic depths in the lower chip are all set to 0.4-0.6mm; the diameters of the venous channel entrance shallow groove and the venous channel outlet shallow groove are set to 3-4mm; the diameter of the venous unit is set to 6-8mm; the widths of the venous entrance connecting channel and the venous outlet connecting channel are set to 1-2mm; and the pore size of the porous film is 3-8μm.

8. A method for constructing a microfluidic intestinal organ chip, characterized in that: Constructing a microfluidic intestinal organ chip as described in any one of claims 1 to 7, wherein the intestinal organ chip is made of PDMS or thermoplastic; The PDMS chip production process includes: S1 Pre-processing The PDMS prepolymer and the curing agent are mixed in a mass ratio of 10:1 and stirred until fully mixed; the mixed PDMS prepolymer is vacuumed to eliminate bubbles; the debubbled PDMS prepolymer is poured onto the mold to ensure that there are no bubbles in the PDMS prepolymer; then it is transferred to an oven and heated to cure; S2 Punch Hole Use a puncher of corresponding size to punch holes in the PDMS upper chip at a vertical angle at the corresponding position; S3 chip packaging Since PDMS and PET film cannot be bonded directly by oxygen plasma treatment, bonding was achieved by aminosilanization of the PET film; The aminosilanization process is: (1) First, prepare a 5% 3-aminopropyltriethoxysilane solution with water and preheat it to 80°C. Clean the PDMS chip with isopropanol and blow dry it with nitrogen or compressed air. Clean both sides of the PDMS chip to remove any obvious debris. (2) Treat the PET film with oxygen plasma for 30 seconds and transfer it to a preheated 5% APTES solution for 30 minutes; (3) washing the PET film with deionized water and drying it at room temperature; (4) The aminosilanized PET film and the PDMS chip were simultaneously treated with oxygen plasma for 30 seconds and bonded; (5) Place the assembled PDMS chip in an oven and heat at 60 °C for 24 h; S4 Intestinal Organ Chip Construction The assembled PDMS chip was sterilized by washing the channels with 70% ethanol and irradiated with UV 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; subsequently, collagen was added to the upper chip to form a 1.5 mm thick collagen layer in the intestinal culture unit, the connecting channel, and the arterial unit; the chip was incubated in a 37°C incubator for 1 hour to induce gel formation; (2) Endothelial cells were seeded into the lower layer of the porous film. First, a 50 μg / ml fibronectin solution was introduced into the lower channel, which was then placed upside down in a 37°C incubator for at least 2 h. The channel was then washed with endothelial cell culture medium. Subsequently, the final cell density was 1×10 5 Pieces / cm 2 The endothelial cells are introduced into the chip, and the chip is inverted so that the endothelial cells can attach to the lower side of the membrane; after the endothelial cells attach for 1 to 2 hours, the unattached endothelial cells are rinsed and then filled with endothelial cell culture medium; (3) Inoculate intestinal epithelial cells to a final cell concentration of 5×10 5 Pieces / cm 2 The intestinal epithelial cells were introduced onto the top of the porous microfluidic medium, transferred to an incubator and placed for 2 to 4 hours, and then the unattached intestinal epithelial cells were washed away.

9. The method for constructing a microfluidic intestinal organ chip according to claim 8, characterized in that: In step (1) of constructing the S4 intestinal organ chip, the construction of intestinal nerves and intestinal immune simulations includes embedding intestinal nerve cells and immune cells in collagen.

10. Application of microfluidic intestinal organ chip, characterized by: The microfluidic intestinal organ chip as claimed in any one of claims 1 to 7 is used to: simulate the impact of the internal and external environment of the body on the intestine, and the impact on health, and the impact of the intestinal environment on the body; Or applied to: simulation of food digestion and absorption by the intestine; Or applied to: simulation of the effect of the brain on the intestine, the effect of changes in the body's internal environment on the intestine, simulation of the intestinal structure and barrier function through hormones, neurotransmitters, inflammatory factors or other bioactive substances in the blood circulation; Or applied to: simulation of immune and inflammatory responses caused by substances absorbed after digestion or toxin absorption; or applied to: replacing the use of experimental animals in the drug screening process.

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