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CN-119752624-B - Microfluidic intestinal organ chip, construction method and application thereof

CN119752624BCN 119752624 BCN119752624 BCN 119752624BCN-119752624-B

Abstract

The invention relates to the technical field of microfluidic intestinal organ chips, and particularly discloses a microfluidic intestinal organ chip, a construction method and application thereof; the chip body comprises an upper chip, a porous film and a lower chip, wherein the porous film is arranged between the venous unit and the arterial unit, and the construction method comprises the steps of (1) adding collagen into the intestinal culture unit to induce gel formation, (2) inoculating endothelial cells to the lower layer of the porous film, and (3) inoculating intestinal epithelial cells. The invention can be applied to simulation of the influence of intestinal environment on organisms, simulation of nutrient and toxin absorption by intestinal tracts, simulation of the influence of brain on intestinal tracts, simulation of the influence of environmental changes (such as chronic pressure and depression) in the organisms on intestinal tracts, simulation of intestinal structures and barrier functions by hormones, neurotransmitters, inflammatory factors or other bioactive substances in blood circulation, in-vivo simulation of immunity and inflammatory reactions absorbed after digestion, and substitution of experimental animals in the drug screening process.

Inventors

  • YUE FENG
  • LIU MINGHUI

Assignees

  • 海南大学

Dates

Publication Date
20260508
Application Date
20241231

Claims (8)

  1. 1. A microfluidic intestinal organ chip is characterized in that a chip body comprises an upper chip, a porous film and a lower chip, wherein 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 device comprises an intestinal culture unit, a venous unit, an arterial unit, a venous channel inlet shallow slot, a venous channel outlet shallow slot, a connecting channel, a venous channel inlet shallow slot, a venous channel outlet shallow slot, a venous channel inlet shallow slot and a venous channel outlet shallow slot, wherein the venous unit is arranged below the intestinal culture unit; The upper layer of the porous film is filled with a porous micro-fluid medium and is set to be a capillary network and intestinal submucosa structure, and the upper surface of the porous micro-fluid medium is inoculated with intestinal epithelial cells to be used as an intestinal cavity structure; The venous channel is located at the bottom of the porous microfluidic medium, the two are separated by a layer of porous film; substances in the intestinal lumen enter the porous microfluidic medium after penetrating through intestinal epithelial cells and finally flow into the venous channel to simulate the absorption process of food, digestive tract substances or toxins from the intestinal lumen into blood; In addition, the fluid from the arterial region only flows in the porous microfluidic medium and flows towards the endothelial cells of the vein, but cannot overflow to the surface of intestinal epithelial cells, and the fluid from the arterial region is gathered in a venous channel after passing through the porous microfluidic medium, so that the simulation of the intestinal blood circulation of the artery-capillary-vein is realized.
  2. 2. The microfluidic intestinal organ chip according to claim 1, wherein the porous membrane is PC or PET.
  3. 3. The microfluidic intestinal organ chip according to claim 1, wherein the chip body is made of PDMS, PMMA, PC or COC.
  4. 4. The microfluidic intestinal organ chip according to claim 1, wherein the final concentration of gel used in the porous microfluidic medium is 3-6mg/mL.
  5. 5. A microfluidic intestinal organ chip according to claim 1, wherein the height of the upper chip is set to 6-8mm, the narrowest distance of the connecting channel is set to 1-2mm, the vertical distance is set to 2-4mm, 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 inlet through hole and the venous channel outlet through hole are set to 3-4mm, the characteristic depths in the lower chip are set to 0.4-0.6mm, the diameters of the venous channel inlet shallow groove and the venous channel outlet shallow groove are set to 3-4mm, the diameters of the venous unit are set to 6-8mm, the widths of the venous inlet connecting channel and the venous outlet connecting channel are set to 1-2mm, and the pore diameter of the porous membrane is 3-8 μm.
  6. 6. The method for constructing the microfluidic intestinal organ chip is characterized in that the microfluidic intestinal organ chip according to any one of claims 1-5 is constructed, and the intestinal organ chip is made of PDMS or thermoplastic; the PDMS chip manufacturing process comprises the following steps: s1 pretreatment Mixing PDMS prepolymer and curing agent according to the mass ratio of 10:1, stirring to fully mix, removing bubbles from the mixed PDMS prepolymer by using a vacuumizing mode, pouring the bubble-removed PDMS prepolymer on a mould to ensure that the PDMS prepolymer does not exist, transferring the PDMS prepolymer into an oven, and heating and curing; s2 punching Punching the PDMS upper chip at a vertical angle at a corresponding position by using a puncher with a corresponding size; S3 chip package Because PDMS and PET film can not be directly bonded through oxygen plasma treatment, bonding is realized through amino silanization of the PET film; The amino silanization process is as follows: (1) Firstly, preparing a 5% 3-aminopropyl triethoxysilane solution by using water, preheating to 80 ℃, cleaning a PDMS chip by using isopropanol, drying by using nitrogen or compressed air, and cleaning two sides of the PDMS chip to remove any obvious scraps; (2) The PET film was treated with oxygen plasma for 30 seconds and transferred to a preheated 5% aptes solution for 30 minutes; (3) Washing the PET film with deionized water and drying at room temperature; (4) Simultaneously treating the amino silanized PET film and the PDMS chip with oxygen plasma for 30 seconds and bonding; (5) Putting the assembled PDMS chip into an oven, and heating for 24 hours at 60 ℃; s4 intestinal organ chip construction Sterilizing the assembled PDMS chip using a 70% ethanol washing channel, and irradiating the assembled PDMS chip with ultraviolet light in a biosafety cabinet for at least 15 minutes before using the PDMS chip; (1) Mixing type I collagen, 5 XPBS, 1 XPBS and 0.5N NaOH to prepare the final concentration of the collagen is 6mg/ml, then adding the collagen into an upper chip to form a collagen layer with the thickness of 1.5mm in an intestinal culture unit, a connecting channel and an arterial unit, and incubating the chip in a 37 ℃ incubator for 1 hour to induce gel formation; (2) Inoculating endothelial cells to the lower layer of a porous film, firstly introducing 50 mug/ml of fibronectin solution into a lower layer channel, inversely placing the solution for at least 2 hours in a 37 ℃ incubator, washing the channel with endothelial cell culture medium, then introducing the endothelial cells with the final cell density of 1X 10 5 per cm 2 into a chip, inversely placing the chip so that the endothelial cells can be attached to the lower side of the film, flushing the unattached endothelial cells after the endothelial cells are attached for 1-2 hours, and then filling the endothelial cell culture medium; (3) Intestinal epithelial cells are inoculated, the intestinal epithelial cells with the final cell concentration of 5 multiplied by 10 5 /cm 2 are introduced to the top of the porous micro-fluid medium, the intestinal epithelial cells are placed for 2 to 4 hours after being transferred to an incubator, and then unattached intestinal epithelial cells are washed away.
  7. 7. The method of claim 6, wherein in the step (1) of constructing the S4 intestinal organ chip, the simulation of constructing the enteric nerve and the intestinal immunity comprises embedding enteric nerve cells and immune cells in collagen.
  8. 8. The microfluidic intestinal organ chip is characterized by being applied to the simulation of the influence of the internal and external environments of an organism on intestinal tracts, the influence on health and the influence of the intestinal tract environment on the organism according to any one of claims 1-5; Or applied to the simulation of the digestion and absorption of food by the intestinal tract; Or applied to the simulation of the effect of brain on intestinal tract, the effect of environmental change in the organism on intestinal tract, and the simulation of the effect of hormone, neurotransmitter, inflammatory factor or other bioactive substances in blood circulation on intestinal tract structure and barrier function; Or applied to the simulation of immune and inflammatory reactions caused by the absorption of substances and toxins absorbed after digestion; or to replace the use of experimental animals in drug screening processes.

Description

Microfluidic intestinal organ chip, construction method and application thereof Technical Field The invention relates to the technical field of microfluidic intestinal organ chips, in particular to a microfluidic intestinal organ chip, a construction method and application thereof. Background The surface of the small intestine facing the intestinal lumen is called the mucosa, which together with the underlying submucosa, lateral myolayer and serosa constitutes the intestinal tissue. The mucosal surface is composed of simple columnar epithelial cells, and forms an intestinal barrier by the connection between epithelial cells and cells, which are sequentially arranged from the top of the intestinal epithelial cells to the position of the basal membrane through tight connection, adhesive connection, desmosome connection and gap connection, seals the top and the basal membrane and limits paracellular permeability, which is regulated by a variety of factors including chronic stress, diet, intestinal flora, cytokines and the like. The natural layer of the substrate has rich capillaries. Arterial blood vessels of the intestinal tract are governed by an arterial arch of an upper mesenteric artery extending radially in the mesenteric to the intestinal wall, gradually form a capillary network, then collect into a capillary venous network and an intestinal vein, and finally collect into hepatic portal veins. The walls of capillaries are composed of a single, thin layer of endothelial cells and a basement membrane that surrounds it. The distance between the intestinal epithelial cell basement membrane and the capillaries is not more than a few microns. These vessels are not only close to the epithelial layer, but also fenestrations in endothelial cells covered only by the basement membrane. Thus, when food is subjected to mechanical and chemical digestion, nutrients can enter capillaries located in the lamina propria through the connection between epithelial cells, and the substances are transferred from the lumen to the vascular system, completing the absorption of the nutrients. In addition, the gut acts as the largest and most important barrier against the external environment, allowing the passage of selective nutrients like amino acids, carbohydrates, electrolytes, lipids and water, while hindering the entry of toxins and bacterial endoluminal toxins, antigens and gut flora to maintain a balanced steady state. Disruption of the intestinal barrier increases permeability, which may be detrimental to the host, as it may allow intestinal lumen antigens and toxins to migrate through the intestinal wall into the subcutaneous tissue and blood. In turn, such translocation may induce local and systemic immune responses, possibly leading to the development of pathology. In addition, increased intestinal permeability is associated with a variety of autoimmune and gastrointestinal disorders. The digestion and absorption processes of the intestinal tract are very complex, and the internal and external environments have various profound effects on the organism. For example, external dietary intake, microbial environment, internal hormonal regulation, neurotransmitter action, etc. affect intestinal function. At present, animal models are a common way to study intestinal pathophysiology. Due to species differences, animal models do not accurately reflect human responses to pathogens, diseases and drugs. For example, in animal experimental studies of chronic stress, the stress is generally induced for days ranging from 7 to 14 days. On the other hand, due to ethical problems associated with clinical studies, observation studies can only be performed under natural stress conditions. Because of the large differences in pressure source type and duration and intestinal permeability measurements between animals and clinical studies, it is difficult to compare animal pressure studies with human pressure studies. In addition, conventional cellular models also fail to mimic the characteristics of the intestinal microenvironment, such as fluid flow, villus structure, and peristalsis. Therefore, it is difficult to simulate the comprehensive functions of human intestinal tracts and realize real-time observation of interactive dynamics. At present, 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, organoids are stationary and do not have fluid flow and peristalsis found in the human intestinal tract. Although the intestinal organoids can mimic intestinal physiology, it is difficult to deliver microorganisms or foods, etc. into the interior of the intestinal organoids due to the occlusion of the lumen of the intestinal organoids. The organ chip is a micro cell culture device with a micro fluid channel and living cells by taking a micro fluid technology as a core, can simulat