Microorganism-intestine-joint axis multi-organ chip and preparation method thereof

Abstract

The invention discloses a microorganism-intestine-joint axis multi-organ chip and a preparation method thereof, and belongs to the field of biomedical engineering.The preparation method comprises the following specific steps that firstly, an upper-layer chip male mold, a middle-layer chip male mold and a lower-layer chip male mold of the multi-organ chip are prepared through the high-precision 3D printing technology; iI, processing the silicon wafer with the cylindrical microarray through photoetching to prepare a porous membrane; iII, preparing an upper-layer chip, a middle-layer chip and a lower-layer chip; iV, assembling each group of prepared chips; according to the invention, human cells or organoids can be used for experiments, the experiment result has good correlation with human beings, and there is no species difference between animal experiments and human beings; the experiment process can be obviously accelerated, and the experiment time is shortened; according to the method, the experimental cost can be greatly saved, and the dispute of experimental ethics does not exist, so that the method can be expanded to large-scale standardized processing production so as to carry out high-throughput and large-scale experimental research.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering, and particularly to a multi-organ chip of the microbiota-gut-joint axis and a preparation method thereof. Background Art

[0002] Osteoarthritis (OA) is one of the most common types of arthritis, a chronic degenerative and disabling disease that causes significant health, economic, and social problems, but the research work to date has not been able to identify its exact cause. The corresponding treatment strategies for OA are relatively limited, and most are symptomatic treatments, including moderate exercise and rehabilitation strategies, drug interventions, and joint replacement surgeries for advanced OA. There is currently no treatment method that can satisfactorily stop or delay the progression of osteoarthritis or provide effective and lasting symptom relief.

[0003] The gut microbiota (GM) is a key environmental factor that widely participates in various physiological processes of the growth, development, and inflammatory lesions of bones and joints. The relationship between the gut microbiota and joints is called the "microbiota-gut-joint axis", which contains complex pathways and mechanisms of the interaction between the gut microbiota and joints. Studying the microbiota-gut-joint axis helps to discover potential targets for treating arthritis and is expected to improve joint system diseases by regulating the gut microbiota.

[0004] Currently, the mechanism of the impact of the gut microbiota on arthritis is still unclear, mainly because of the lack of effective in vitro models. The widely used animal models have obvious limitations and defects. In addition to experimental ethics and the naturally existing genetic barriers, there are also significant differences in the composition of the gut microbiota between animals and humans. The gut microbiota of simple model animals is mostly aerobic, while obligate anaerobic bacteria dominate in the human gut. The gut microbiota of animal models cannot generalize the characteristics of the human gut microbiota in terms of quantity, variety, and complexity; secondly, there are significant differences in the structure, composition, function, response, stress, etc. between animal and human joints, making it difficult to translate the experimental results of animal models to human patients. In addition, there are also huge differences in aspects such as the age, gender, physiological conditions, disease severity, gut microbiota composition, diet, and supplements of the subjects in clinical studies. And the clinical samples have high heterogeneity and the research time span is generally long. The research on human patients' joints can only rely on limited means, and the number of biopsy samples is limited and it is difficult to conduct subsequent research. Therefore, constructing an effective microbiota-gut-joint axis model in vitro is the key to studying the impact of the gut microbiota on joint development, degeneration, inflammation, etc. Summary of the Invention

[0005] The purpose of the present invention is to solve the defects existing in the prior art, and to propose a multi-organ chip of the microbiota-gut-joint axis and a preparation method thereof.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A preparation method of a multi-organ chip of the microbiota-gut-joint axis, and the specific steps of the preparation method are as follows:

[0008] Ⅰ. Use high-precision 3D printing technology to prepare the upper chip male mold, the middle layer chip male mold and the lower layer chip male mold of the multi-organ chip respectively;

[0009] Ⅱ. Prepare a porous membrane by lithographically processing a silicon wafer with a cylindrical microarray;

[0010] Ⅲ. Prepare the upper chip, the middle layer chip and the lower layer chip;

[0011] Ⅳ. Assemble the prepared groups of chips.

[0012] As a further solution of the present invention, the upper chip male mold, the middle layer chip male mold and the lower layer chip male mold described in step Ⅰ are respectively based on AutoCAD files and generate 3D files with Solidwork;

[0013] Among them, the thickness of the upper chip is 2 mm, the thickness of the middle layer chip is 0.2 mm, and the thickness of the lower layer chip is 8 mm;

[0014] The upper chip includes the microbiota-gut symbiotic module Ⅰ; the middle layer chip includes the vascular microchannel module Ⅱ and the synovial organoid module Ⅲ; the lower layer chip includes the cartilage organoid module Ⅳ.

[0015] As a further solution of the present invention, the specific steps of preparing the upper chip, the middle layer chip and the lower layer chip described in step Ⅲ are as follows:

[0016] S1.1: Pour the PDMS prepolymer into the upper chip male mold, the middle layer chip male mold and the lower layer chip male mold respectively;

[0017] S1.2: After pouring, degas at -80 kPa until all bubbles are removed;

[0018] S1.3: After degassing is completed, cure the upper chip and the middle layer chip at 60 °C for more than 6 h, and cure the lower layer chip at 60 °C for more than 10 h.

[0019] As a further solution of the present invention, the specific steps of assembling the prepared groups of chips described in step Ⅳ are as follows:

[0020] S2.1: Demold the upper chip, punch the punching positions listed on the upper chip with a punch, then perform plasma treatment on the lower surface of the upper chip, i.e., the structural opening surface, together with the porous membrane, and then irreversibly bond the porous membrane to the curved channel part of the microorganism-intestine symbiotic module I;

[0021] S2.2: After performing plasma treatment on the lower surface of the upper chip bonded with the porous membrane and the uncured but undemolded intermediate chip together, align and bond the two surfaces. When bonding, the curved channel parts of the microorganism-intestine symbiotic module I and the vascular microchannel module II overlap and are separated by the porous membrane. After bonding is completed, demold the intermediate layer from the male mold;

[0022] S2.3: Punch the punching positions listed on the bonded body of the upper and intermediate chips with a punch. The punching positions correspond to the inlet and outlet positions on the lower chip. After performing plasma treatment on the lower surfaces of the bonded upper and intermediate chips and the upper surface structural opening surface of the lower chip together, align and bond the two surfaces to obtain the microorganism-intestine-joint axis multi-organ chip. At this time, the synovial organoid module III in the intermediate layer overlaps with the cartilage organoid module IV in the lower layer, and the circular structures of the synovial organoid module III and the cartilage organoid module IV are concentric circles.

[0023] As a further solution of the present invention, the punching positions listed on the upper chip in S2.1 include: the inlet ① and outlet ② of the microorganism-intestine symbiotic module I correspond to the opening positions on the upper chip; the ventilation ports ③ and ④ of the vacuum chambers on both sides of the microorganism-intestine symbiotic module I correspond to the opening positions on the upper chip; the inlet ⑤ and outlet ⑥ of the vascular microchannel module II in the intermediate chip correspond to the opening positions on the upper chip; the inlets ⑦, ⑨, and outlets ⑧, ⑩, of the synovial organoid module III in the intermediate chip correspond to the opening positions on the upper chip;

[0024] The punching positions listed on the bonded body of the upper and intermediate chips in S2.3 include the inlet and outlet of the cartilage organoid module IV in the lower chip, which are located at the opening positions in the intermediate chip.

[0025] A microorganism-intestine-joint axis multi-organ chip, comprising an upper chip, a porous membrane, an intermediate chip and a lower chip;

[0026] The upper chip includes a microorganism-intestine symbiotic module I, and the thickness of the upper chip is 2 mm;

[0027] The porous membrane is 6 mm wide, 10 mm long, 30 μm thick, with a pore diameter of 8 μm and a center-to-center distance of 30 μm between the round holes;

[0028] The middle layer chip includes a vascular microchannel module II and a synovial organoid module III, and the middle layer chip is 200 μm thick. The structural thickness of the synovial organoid module III is 200 μm;

[0029] The lower layer chip includes a cartilage organoid module IV, and the lower layer chip is 8 mm thick.

[0030] As a further aspect of the present invention, the curved channel portions of the microorganism-intestinal symbiosis module I and the vascular microchannel module II overlap in structure and are separated by the porous membrane;

[0031] The synovial organoid module III and the cartilage organoid module IV overlap in structure, and their circular structures are concentric circles. The diameter R1 of the circular structure of the synovial organoid module III is greater than the diameter R2 of the circular structure of the cartilage organoid module IV.

[0032] As a further aspect of the present invention, the microorganism-intestinal symbiosis module I includes a curved upper microchannel a and two curved upper vacuum chambers b and c located on both sides of the upper microchannel a respectively;

[0033] Both ends of the upper microchannel a are respectively connected to the inlet ① and the outlet ② of the upper microchannel. The two upper vacuum chambers b and c are respectively connected to the connection ports ③ and ④;

[0034] The upper microchannel a is 750 μm wide and 500 μm thick; the upper vacuum chambers b and c are 750 μm wide and 500 μm thick.

[0035] As a further aspect of the present invention, the vascular microchannel module II includes a curved middle layer curved microchannel d, which is respectively connected to the inlet ⑤ and the outlet ⑥. The middle layer curved microchannel d is 750 μm wide and 200 μm thick;

[0036] The synovial organoid module III includes a total of 4 array structures e-1, e-2, e-3, and e-4. Among them, e-1 and e-4 have 4 structural units, and e-2 and e-3 have 5 structural units;

[0037] Both ends of the e-1 include the inlet ⑦ and the outlet ⑧; both ends of the e-2 include the inlet ⑨ and the outlet ⑩; both ends of the e-3 include the inlet and the outlet Both ends of the e-4 include the inlet and the outlet

[0038] The inner diameter R1 of the single structural unit is 5 mm, the outer diameter R3 is 7 mm, and there are two sets of trapezoidal arrays g-1 and g-2 between the inner circle and the outer circle of the single structural unit;

[0039] The trapezoidal arrays g-1 and g-2 respectively contain two columns of staggered trapezoids. A channel structure h-1 is formed between the trapezoidal arrays g-1 and g-2, and a channel structure h-2 is formed between the trapezoidal array g-2 and the outer circle. The widths of the channel structure h-1 and the channel structure h-2 are both 300 μm;

[0040] For the trapezoids of the trapezoidal arrays g-1 and g-2, the short side length D1 is 50 μm, the long side length D2 is 100 μm, the height H1 is 100 μm, the spacing D3 between the short sides is 30 μm, the distance D4 between the long sides on the inner circle side is 50 μm, and the distance D5 between the long sides on the outer circle side is 60 μm;

[0041] Both ends of the synovial organoid module Ⅲ are respectively connected to the inlet and the outlet

[0042] As a further solution of the present invention, the cartilage organoid module Ⅳ includes 18 conical culture units i with spherical bottoms and channels j connecting the culture units;

[0043] The channel j includes an inlet and an outlet

[0044] The conical depth H2 of the conical culture unit i is 6 mm, the maximum diameter R2 is 48 mm, and the spherical diameter of the spherical bottom is 3 mm.

[0045] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0046] The present invention is composed of an upper layer chip, a porous membrane structure, an intermediate layer chip and a lower layer chip, and is used for in vitro modeling and physiological and pathological research of the microbiota-gut-joint axis. This multi-organ chip of the microbiota-gut-joint axis can be used for experiments with human cells or organoids, and the experimental results have good correlation with humans, and there are no inter-species differences between animal experiments and humans; at the same time, this multi-organ chip of the microbiota-gut-joint axis adopts microfluidic chip technology, and continuously perfuses fluid in the microchannels and chambers to culture cells and tissues, which can significantly accelerate the experimental process and shorten the experimental time; in addition, the multi-organ chip of the microbiota-gut-joint axis of the present invention combines microfluidic technology and hydrodynamics, and uses a multi-organ chip processed by PDMS instead of animal experiments, which can greatly save experimental costs and there is no controversy over experimental ethics, so that it can be extended to large-scale standardized processing and production for high-throughput and large-scale experimental research. Description of the Drawings

[0047] The accompanying drawings are used to provide a further understanding of the present invention and form a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention.

[0048] Figure 1 It is a flowchart of a method for preparing a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0049] Figure 2 It is a schematic diagram of the chip structure of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0050] Figure 3 It is a schematic diagram of the structure of the microorganism-intestine symbiotic module I of the upper chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0051] Figure 4 It is a schematic diagram of the channels of the microorganism-intestine symbiotic module I of the upper chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0052] Figure 5 It is a schematic diagram of the inlet and outlet of the microorganism-intestine symbiotic module I of the upper chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0053] Figure 6 It is a schematic diagram of the structure of the blood vessel microchannel module II and the synovial organoid module III of the middle chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0054] Figure 7 It is a schematic diagram of the channels and inlets and outlets of the blood vessel microchannel module II of the middle chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0055] Figure 8 It is a schematic diagram of the array structure of the synovial organoid module III of the middle chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0056] Figure 9 It is a schematic diagram of the inlets and outlets of the synovial organoid module III of the middle chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0057] Figure 10 It is a schematic diagram of the structural unit of the synovial organoid module III of the middle chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0058] Figure 11Schematic diagram of the trapezoidal array of the synovial organoid module Ⅲ of the middle layer chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0059] Figure 12 Schematic diagram of the structure of the cartilage organoid module Ⅳ of the lower layer chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0060] Figure 13 Schematic diagram of the inlet / outlet and culture chamber structure of the cartilage organoid module Ⅳ of the lower layer chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0061] Figure 14 Schematic diagram of a single culture unit in the cartilage organoid module Ⅳ of the lower layer chip of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention;

[0062] Figure 15 Schematic diagram of the structure of the porous membrane of a multi-organ chip of the microorganism-intestine-joint axis proposed by the present invention. Detailed implementation manners

[0063] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments.

[0064] Embodiment 1

[0065] Refer to Figures 1-15 , this embodiment discloses a preparation method of a multi-organ chip of the microorganism-intestine-joint axis. The specific steps of the preparation method are as follows:

[0066] Use high-precision 3D printing technology to prepare the upper layer chip male mold, the middle layer chip male mold and the lower layer chip male mold of the multi-organ chip respectively.

[0067] It should be noted that the upper layer chip male mold, the middle layer chip male mold and the lower layer chip male mold are respectively based on AutoCAD files as standards, and 3D files are generated by Solidwork; among them, the thickness of the upper layer chip is 2 mm, the middle layer thickness is 0.2 mm, and the thickness of the lower layer chip is 8 mm; the upper layer chip includes the microorganism-intestine symbiotic module Ⅰ; the middle layer chip includes the vascular microchannel module Ⅱ and the synovial organoid module Ⅲ; the lower layer chip includes the cartilage organoid module Ⅳ.

[0068] Prepare the porous membrane by lithographically processing a silicon wafer with a cylindrical microarray.

[0069] Prepare the upper layer chip, the middle layer chip and the lower layer chip.

[0070] Specifically, pour the PDMS prepolymer into the upper-layer chip male mold, the middle-layer chip male mold, and the lower-layer chip male mold respectively. After pouring, degas at -80 kPa until all bubbles are removed. After degassing is completed, cure the upper-layer chip and the middle-layer chip at 60 °C for more than 6 h, and cure the lower-layer chip at 60 °C for more than 10 h.

[0071] Assemble the prepared groups of chips.

[0072] Specifically, referring to Figure 2 it can be seen that the upper-layer chip is demolded, then punch holes at the punching positions listed on the upper-layer chip with a punch. Then, the lower surface of the upper-layer chip, i.e., the structural opening surface, and the porous membrane are subjected to plasma treatment together. After that, the porous membrane is irreversibly bonded to the curved channel part of the microorganism-intestine symbiotic module I. After the lower surface of the upper-layer chip bonded with the porous membrane and the middle-layer chip that has been cured but not demolded are subjected to plasma treatment together, align the two surfaces and bond them. When bonding, the curved channel parts of the microorganism-intestine symbiotic module I and the vascular microchannel module II overlap and are separated by the porous membrane. After bonding is completed, demold the middle layer from the male mold, punch holes at the punching positions listed on the bonded body of the upper-layer and middle-layer chips with a punch, and the punching positions correspond to the inlet and outlet positions on the lower-layer chip. After the lower surfaces of the bonded upper-layer and middle-layer chips and the upper surface structural opening surface of the lower-layer chip are subjected to plasma treatment together, align the two surfaces and bond them to obtain the multi-organ chip of the microorganism-intestine-joint axis. At this time, the synovial organ module III in the middle layer overlaps with the cartilage organ module IV in the lower layer, and the circular structures of the synovial organ module III and the cartilage organ module IV are concentric circles.

[0073] In this embodiment, referring to Figures 3-14 it can be seen that the punching positions listed on the upper-layer chip include: the inlet ① and outlet ② of the microorganism-intestine symbiotic module I correspond to the opening positions on the upper-layer chip; the vent holes ③ and ④ of the vacuum chambers on both sides of the microorganism-intestine symbiotic module I correspond to the opening positions on the upper-layer chip; the inlet ⑤ and outlet ⑥ of the vascular microchannel module II in the middle-layer chip correspond to the opening positions on the upper-layer chip; the inlets ⑦, ⑨, and outlets ⑧, ⑩, of the synovial organ module III in the middle-layer chip correspond to the opening positions on the upper-layer chip;

[0074] The punching positions listed on the bonded body of the upper-layer and middle-layer chips include the inlet and outlet of the cartilage organ module IV in the lower-layer chip, which are located at the opening positions in the middle-layer chip.

[0075] In addition, after the multi-organ chip of the microbiota-gut-joint axis in this embodiment, the chip is inverted, and a mixture of 1% Matrigel and rat tail collagen type I with a ratio of 1:1 is injected into inlet ⑤ of the middle-layer chip to fill the curved microchannel d of the entire vascular microchannel module II. After placing it in an incubator at 37 °C for 1 h, the Matrigel and collagen mixture is slowly aspirated, and human umbilical vein endothelial cells (HUVECs) are digested and resuspended at a density of 1.5×10 5 cells / cm 2 . After that, it is injected into the microchannel d and placed in an incubator at 37 °C. After standing for 12 h, the chip is flipped. At this time, the HUVECs adhere to the lower surface of the porous membrane V. The endothelial cell medium is introduced into inlet ⑤ of the vascular microchannel module II at a flow rate of 50 μL / h;

[0076] A mixture of Matrigel and rat tail collagen type I with a ratio of 1:1 is injected into inlet ① of the upper-layer chip to fill the curved microchannel a of the entire microbiota-gut module I. After placing it in an incubator at 37 °C for 1 h, the Matrigel and collagen mixture is slowly aspirated, and human intestinal epithelial cells (Caco-2) are digested and resuspended at a density of 1.5×10 5 cells / cm 2 . After that, it is injected into the microchannel a and left standing in an incubator at 37 °C for 12 h. At this time, the Caco-2 cells adhere to the upper surface of the porous membrane V. The intestinal epithelial cell medium is introduced into inlet ① of the microbiota-gut module I at a flow rate of 50 μL / h; meanwhile, interfaces ③ and ④ are connected to a pulsating vacuum pump, and a vacuum stimulation is applied to channels b and c at a frequency of 1 Hz;

[0077] Six days after injecting Caco-2 into the upper microchannel, a medium without antibiotics and antifungal drugs is perfused in the microchannel a of the microbiota-gut module I and the microchannel d of the vascular microchannel module II for 24 h. If the microorganisms to be co-cultured include anaerobic microorganisms, the medium without antibiotics and antifungal drugs perfused in the microchannel a of the microbiota-gut module I for 24 h needs to be deoxygenated. However, the medium perfused in the microchannel d of the vascular microchannel module II does not need to be deoxygenated;

[0078] The microbial mixture (including anaerobic bacteria, facultative anaerobic bacteria, and aerobic bacteria) is added at a concentration of 1×10 7Inoculate at a density of CFU / mL in microchannel a. After static culture for 12 h, the microorganisms will adhere to the surface of the intestinal epithelial monolayer formed by Caco-2 cells. Subsequently, continue to perfuse and culture the upper microchannel with Caco-2 medium without antibiotics and antifungal drugs (select deoxygenated / non-deoxygenated treatment according to whether anaerobic microorganisms are included), and the flow rate is 50 μL / h. Then, the microorganisms that do not adhere to the intestinal epithelial surface will be washed away by the flowing medium. At this time, the medium in microchannel a of the microorganism-intestine module I flows out of the chip through outlet ②, and the medium in microchannel d of the vascular microchannel module II flows out of the chip through outlet ⑥;

[0079] After resuspending the digested human bone marrow mesenchymal stem cells (BMSCs) at a density of 1×10 6 cells / mL, inject 1.5 mL of the cell suspension into the cartilage organoid module IV from the inlet, and let the chip stand in an incubator at 37 °C for 24 h. Due to the action of gravity, the cells spontaneously aggregate at the spherical bottom of the conical culture unit i. Since the PDMS material is hydrophobic, the cells aggregate into spheres and do not adhere to the wall of the conical culture unit i. After 24 h, construct synovial organoids on the chip. After the successful construction of the synovial organoids, perfuse cartilage medium from the inlet of channel j connecting the cartilage organoid culture unit to induce the in-situ formation of cartilage organoids, and the discarded medium flows out from the outlet ;

[0080] Prepare a Gelma hydrogel solution with a concentration of 15%, and the LAP concentration is 0.15%. Resuspend the digested synovial cells in the Gelma hydrogel at a density of 4×10 6 cells / mL. After the BMSCs are statically cultured in the cartilage organoid module IV for 24 h, slowly inject the cell and hydrogel mixture from inlets ⑦, ⑨, and in the middle-layer synovial organoid module III until the mixture fills the synovial organoid culture chambers of the 4 array structures e-1, e-2, e-3, and e-4. The trapezoidal array structures on both sides of the culture chamber can effectively restrict the flow of the hydrogel mixture into the channels or cartilage organoid culture chambers, and irradiate the chip with a UV lamp with a power of 25 W and a wavelength of 365 nm for 2 min to solidify the hydrogel; then, flow the synovial cell medium into inlets and of the synovial organoid module III at a flow rate of 50 μL / h, and the discarded medium flows out from the outlet ;

[0081] At a specific time point (3 days after the formation of cartilage organoids), connect the outlet ⑥ of microchannel d in the vascular microchannel module II to the inlet of the synovial organoid module III They are connected. At this time, after the metabolites of intestinal microorganisms pass through the intestinal epithelial barrier and the vascular endothelial barrier through selective passage, they enter the synovial organ module III and further affect the cartilage organ.

[0082] Example 2

[0083] Refer to Figures 2-15 , this example discloses a multi-organ chip of the microbiota-gut-joint axis, including an upper chip, a porous membrane, a middle chip and a lower chip;

[0084] The upper chip includes the microbiota-gut symbiotic module I, and the thickness of the upper chip is 2 mm.

[0085] Specifically, referring to Figures 3-5 , the curved channel parts of the microbiota-gut symbiotic module I and the vascular microchannel module II overlap in structure and are separated by a porous membrane. The synovial organ module III and the cartilage organ module IV overlap in structure, and their circular structures are concentric circles. The diameter R1 of the circular structure of the synovial organ module III is greater than the diameter R2 of the circular structure of the cartilage organ module IV.

[0086] It should be further noted that referring to Figures 4-5 , the microbiota-gut symbiotic module I includes a curved upper microchannel a and two curved upper vacuum chambers b and c located on both sides of the upper microchannel a respectively. The two ends of the upper microchannel a are respectively connected with the inlet ① and the outlet ② of the upper microchannel. The two upper vacuum chambers b and c are respectively connected with the connection ports ③ and ④. The width of the upper microchannel a is 750 μm and the thickness is 500 μm; the width of the upper vacuum chambers b and c is 750 μm and the thickness is 500 μm.

[0087] Referring to Figure 15 , the porous membrane is 6 mm wide, 10 mm long, 30 μm thick, the pore diameter is 8 μm, and the center distance of the round holes is 30 μm.

[0088] The middle chip includes the vascular microchannel module II and the synovial organ module III, and the thickness of the middle chip is 200 μm, and the structural thickness of the synovial organ module III is 200 μm.

[0089] Specifically, referring to Figures 6-10 , the vascular microchannel module II includes a curved middle-layer curved microchannel d, which are respectively connected with the inlet ⑤ and the outlet ⑥. The width of the middle-layer curved microchannel d is 750 μm and the thickness is 200 μm. The synovial organ module III includes a total of 4 array structures of e-1, e-2, e-3 and e-4. Among them, e-1 and e-4 have 4 structural units, e-2 and e-3 have 5 structural units. The two ends of e-1 include the inlet ⑦ and the outlet ⑧; the two ends of e-2 include the inlet ⑨ and the outlet ⑩; the two ends of e-3 include the inlet and the outlet Both ends of e-4 include the inlet and the outlet The inner diameter R1 of a single structural unit is 5 mm, and the outer diameter R3 is 7 mm. There are two sets of trapezoidal arrays g-1 and g-2 between the inner circle and the outer circle of a single structural unit. The trapezoidal arrays g-1 and g-2 respectively contain two columns of staggered trapezoids. A channel structure h-1 is formed between the trapezoidal arrays g-1 and g-2, and a channel structure h-2 is formed between the trapezoidal array g-2 and the outer circle. The widths of the channel structure h-1 and the channel structure h-2 are both 300 μm. For the trapezoids of the trapezoidal arrays g-1 and g-2, the short side length D1 is 50 μm, the long side length D2 is 100 μm, the height H1 is 100 μm, the spacing D3 between the short sides is 30 μm, the distance D4 between the long sides on the inner circle side is 50 μm, and the distance D5 between the long sides on the outer circle side is 60 μm. Both ends of the synovial organoid module Ⅲ are respectively connected to the inlet and the outlet

[0090] The lower layer chip includes the cartilage organoid module Ⅳ, and the thickness of the lower layer chip is 8 mm.

[0091] Specifically, referring to Figures 12-14 , the cartilage organoid module Ⅳ contains 18 conical culture units i with spherical bottoms and channels j connecting the culture units. The channels j include the inlet and the outlet The conical depth H2 of the conical culture unit i is 6 mm, the maximum diameter R2 is 48 mm, and the spherical diameter of the spherical bottom is 3 mm.

Claims

1. A method for preparing a microorganism-intestine-joint axis multi-organ chip, characterized in that: The specific steps of the preparation method are as follows: Ⅰ. Use high-precision 3D printing technology to prepare the upper chip positive mold, the middle chip positive mold and the lower chip positive mold of the multi-organ chip respectively; II. Preparation of porous membranes by photolithographic processing of silicon wafers with cylindrical microarrays; III. Prepare upper chip, middle chip and lower chip; IV. Assemble the prepared chips.

2. The method for preparing a microorganism-intestine-joint axis multi-organ chip according to claim 1, characterized in that: In step I, the upper chip positive mold, the middle chip positive mold and the lower chip positive mold are respectively based on AutoCAD files and 3D files are generated by Solidwork; Among them, the thickness of the upper chip is 2mm, the thickness of the middle chip is 0.2mm, and the thickness of the lower chip is 8mm; The upper chip includes a microorganism-intestinal symbiosis module I; the middle chip includes a vascular microchannel module II and a synovial organoid module III; and the lower chip includes a cartilage organoid module IV.

3. The method for preparing a microorganism-intestine-joint axis multi-organ chip according to claim 2, characterized in that: The specific steps of preparing the upper chip, the middle chip and the lower chip in step III are as follows: S1.1: pouring PDMS prepolymer into the upper chip positive mold, the middle chip positive mold and the lower chip positive mold respectively; S1.2: After pouring, degas at -80 kPa until all bubbles are removed; S1.3: After the bubble removal is completed, the upper chip and the middle chip are cured at 60°C for more than 6 hours, and the lower chip is cured at 60°C for more than 10 hours.

4. The method for preparing a microorganism-intestine-joint axis multi-organ chip according to claim 3, characterized in that: The specific steps of assembling the prepared chips in step IV are as follows: S2.1: demold the upper chip, punch holes at the punching positions listed on the upper chip, and then treat the lower surface of the upper chip, i.e., the structural opening surface, with the porous membrane by plasma, and then irreversibly bond the porous membrane to the curved channel part of the microorganism-intestinal symbiosis module I; S2.2: After plasma treatment of the lower surface of the upper chip bonded with the porous membrane and the cured but undemolded middle chip, the two surfaces are aligned and bonded. During bonding, the curved channels of the microorganism-intestinal symbiosis module I and the vascular microchannel module II overlap and are separated by the porous membrane. When bonding is completed, the middle layer is demolded from the positive mold; S2.3: Punch the holes listed on the upper and middle chip bonding bodies with a punch, and the punching positions correspond to the entrance and exit positions on the lower chip. After the lower surfaces of the bonded upper and middle chip are plasma treated together with the opening surface of the upper surface structure of the lower chip, the two surfaces are aligned and bonded to obtain the microbiome-intestine-joint axis multi-organ chip. At this time, the synovial organoid module III of the middle layer overlaps with the cartilage organoid module IV of the lower layer, and the circular structure of the synovial organoid module III is concentric with the circular structure of the cartilage organoid module IV.

5. The method for preparing a microorganism-intestine-joint axis multi-organ chip according to claim 4, characterized in that: S2.1 The punching positions listed in the upper chip include: the inlet ① and outlet ② of the microorganism-intestinal symbiosis module I correspond to the opening positions of the upper chip; the vents ③ and ④ of the vacuum chambers on both sides of the microorganism-intestinal symbiosis module I correspond to the opening positions of the upper chip; the inlet ⑤ and outlet ⑥ of the vascular microchannel module II of the middle chip correspond to the opening positions of the upper chip; the inlet ⑦, ⑨, and exits ⑧, ⑩, Corresponding to the opening position of the upper chip; The punching positions listed on the upper and middle chip bonding bodies described in S2.3 include the inlet of the cartilage organoid module IV of the lower chip and export Located at the opening position of the middle layer chip.

6. A microorganism-intestine-articular axis multi-organ chip, used to implement the microorganism-intestine-articular axis multi-organ chip preparation method according to any one of claims 1 to 5, characterized in that: It includes an upper chip, a porous membrane, a middle chip and a lower chip; The upper chip includes a microorganism-intestinal symbiosis module I, and the thickness of the upper chip is 2 mm; The porous membrane has a width of 6 mm, a length of 10 mm, a thickness of 30 μm, a pore diameter of 8 μm, and a center-to-center distance of 30 μm; The intermediate layer chip includes a vascular microchannel module II and a synovial organoid module III, and the thickness of the intermediate layer chip is 200 μm, and the structural thickness of the synovial organoid module III is 200 μm; The lower chip includes a cartilage organoid module IV, and the thickness of the lower chip is 8 mm.

7. The microorganism-intestine-joint axis multi-organ chip according to claim 6, characterized in that: The curved channel parts of the microorganism-intestinal symbiosis module I and the vascular microchannel module II are structurally overlapped and separated by the porous membrane; The synovial organoid module III and the cartilage organoid module IV overlap in structure, and the circular structures of the two are concentric circles. The diameter R1 of the circular structure of the synovial organoid module III is greater than the diameter R2 of the circular structure of the cartilage organoid module IV.

8. The microorganism-intestine-joint axis multi-organ chip according to claim 6, characterized in that: The microorganism-intestinal symbiosis module I comprises a curved upper microchannel a and two curved upper vacuum chambers b and c respectively located on both sides of the upper microchannel a; The two ends of the upper microchannel a are respectively connected to the inlet ① and the outlet ② of the upper microchannel, and the two upper vacuum chambers b and c are respectively connected to the connection ports ③ and ④; The upper microchannel a has a width of 750 μm and a thickness of 500 μm; the upper vacuum chambers b and c have a width of 750 μm and a thickness of 500 μm.

9. The microorganism-intestine-joint axis multi-organ chip according to claim 6, characterized in that: The blood vessel microchannel module II comprises a curved middle layer curved microchannel d, which is respectively connected with an inlet ⑤ and an outlet ⑥, and the middle layer curved microchannel d has a width of 750 μm and a thickness of 200 μm; The synovial organoid module III includes 4 array structures, namely e-1, e-2, e-3 and e-4, wherein e-1 and e-4 have 4 structural units, and e-2 and e-3 have 5 structural units; The two ends of e-1 include an inlet ⑦ and an outlet ⑧; the two ends of e-2 include an inlet ⑨ and an outlet ⑩; the two ends of e-3 include an inlet and export e-4 contains imports at both ends and export The inner diameter R1 of the single structural unit is 5 mm, the outer diameter R3 is 7 mm, and the single structural unit has two sets of trapezoidal arrays g-1 and g-2 from the inner circle to the outer circle; The trapezoidal arrays g-1 and g-2 respectively include two staggered trapezoids, a channel structure h-1 is formed between the trapezoidal arrays g-1 and g-2, and a channel structure h-2 is formed between the trapezoidal array g-2 and the outer circle, and the width of the channel structure h-1 and the channel structure h-2 are both 300 μm; The trapezoids of the trapezoidal arrays g-1 and g-2 have a short side length D1 of 50 μm, a long side length D2 of 100 μm, a height H1 of 100 μm, a spacing D3 between the short sides of 30 μm, a distance D4 between the long sides on the inner circle side of 50 μm, and a distance D5 between the long sides on the outer circle side of 60 μm; The two ends of the synovial organoid module III are connected to the import and export 10. The microbe-intestine-joint axis multi-organ chip according to claim 6, characterized in that: The cartilage organoid module IV comprises 18 conical culture units i with spherical bottoms and channels j connecting the culture units; The channel j contains the inlet and export The conical depth H2 of the conical culture unit i is 6 mm, the maximum diameter R2 is 48 mm, and the spherical diameter of the spherical bottom is 3 mm.

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