Micro-fluidic blood vessel chip for diagnosing incomplete Kawasaki disease as well as preparation method and application of micro-fluidic blood vessel chip

Abstract

The invention discloses a micro-fluidic blood vessel chip for diagnosing incomplete Kawasaki disease as well as a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the following steps: step 1, preparing a micro-fluidic chip containing a collagen channel; step 2, planting the HCASMCs in the collagen channel, planting the HUVECs after the HCASMCs converge into a single layer to cover the collagen channel, enabling the HUVECs to converge into a single layer to cover the surface of the HCASMCs, and then dynamically pouring a culture medium to obtain the micro-fluidic blood vessel chip. According to the invention, the tissue structure of the blood vessel can be effectively simulated, and the blood flow of the blood vessel can be simulated through continuous dynamic perfusion culture of cells. In practical application, serum to be diagnosed is injected into the microfluidic blood vessel chip, and the physical change of a collagen channel and the change of a cell inflammatory reaction antibody are observed, so that the incomplete Kawasaki disease can be diagnosed. The invention provides a brand new thought and technical platform for diagnosis and treatment of Kawasaki disease, and screening of incomplete Kawasaki disease can be carried out in a short time.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials and relates to a microfluidic vascular chip, and in particular to a microfluidic vascular chip for diagnosing incomplete Kawasaki disease and a preparation method and application thereof. Background Art

[0002] Kawasaki disease (KD), also known as mucocutaneous lymph node syndrome, is an acute febrile rash disease with systemic vasculitis as the main lesion. Coronary artery disease is the main complication of Kawasaki disease and a key factor affecting its prognosis. However, the clinical manifestations of Kawasaki disease in some children are often atypical and do not meet the diagnostic criteria for complete Kawasaki disease, which often makes it difficult for doctors to make a diagnosis. Therefore, missed diagnoses often occur, resulting in delayed treatment and a significant increase in coronary complications. Therefore, how to identify incomplete Kawasaki disease as early as possible and reduce missed diagnoses or even misdiagnoses in children is one of the current clinical problems that need to be solved.

[0003] At present, there is no accurate in vitro model dedicated to Kawasaki disease research in the field of biomedical research. The existing Kawasaki disease models rely only on animal models and planar cell culture. As we all know, animal models are subject to great ethical controversy and species differences. Due to different genes and genetic backgrounds, many pathogenic mechanisms or effective drugs confirmed in animal experiments are ineffective or have little effect after clinical application; and traditional planar cell culture has been proven to be unable to simulate the complex internal environment of the human body, especially unable to reproduce the characteristics of vascular fluid mechanics. Summary of the invention

[0004] The present invention provides a microfluidic blood vessel chip for incomplete Kawasaki disease diagnosis and a preparation method and application thereof, so as to overcome the defects of the prior art.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for preparing a microfluidic vascular chip for diagnosing incomplete Kawasaki disease, comprising the following steps: step 1, preparing a microfluidic chip containing a collagen channel; step 2, planting HCASMCs (primary human coronary artery smooth muscle cells) in the collagen channel, and after the HCASMCs merge into a single layer covering the collagen channel, planting HUVECs (primary human umbilical vein endothelial cells), allowing the HUVECs to merge into a single layer covering the surface of the HCASMCs, and then dynamically perfusing (i.e., continuously perfusing) culture medium to obtain the microfluidic vascular chip.

[0007] Furthermore, in step one, the preparation method of the microfluidic chip is as follows: a PMMA (polymethyl methacrylate) template is obtained by etching, and a PDMS (polydimethylsiloxane) chip is obtained by a reverse molding method; the mass ratio of PDMS to curing agent is 10:1, the curing temperature is 70°C, and the curing time is 3 hours; the PDMS chip has a collagen groove with an opening facing upward, and both sides of the collagen groove have punched grooves with openings facing the collagen groove; a needle is inserted from the side of the PDMS chip so that it is placed in the collagen groove between the two punched grooves, and the bottom of the punched groove is lower than the bottom of the collagen groove, so that the needle placed in the punched groove is in a suspended state in the collagen groove; PDMS is poured into the punched groove, and the needle is pulled out after it solidifies to form a channel; a hole is drilled above the channel to form an inlet and an outlet connected to the two channels, and two liquid reservoirs are fixed at the inlet and outlet; the needle is inserted into the collagen groove from the side of the PDMS chip through the channel again, and then the collagen solution is poured in, and the microfluidic chip containing the collagen channel is obtained after curing.

[0008] Furthermore, in step one, the collagen channel is formed by solidifying a collagen solution; the collagen solution is a rat tail type I collagen solution, with a concentration of 6 to 8 mg / mL, a solidification temperature of 35 to 40°C, and a solidification time of 30 to 60 min; and the diameter of the collagen channel is 200 to 500 μm.

[0009] Furthermore, in step 2, the HCASMCs and HUVECs are planted by the flipping planting method; the specific method is: after injecting the cells, they are cultured in an incubator for 30 minutes, and then the same cells are injected again, and the chip is flipped 180 degrees and continued to be cultured for 30 minutes. The time for HCASMCs to merge into a monolayer is 24 hours, and the time for HUVECs to merge into a monolayer is 48 hours; the planting concentration of HCASMCs is 150,000 to 250,000 / mL, and the planting concentration of HUVECs is 250,000 to 350,000 / mL.

[0010] Furthermore, in step 2, the culture medium includes HCASMCs complete culture medium and HUVECs complete culture medium; the volume ratio of HCASMCs complete culture medium to HUVECs complete culture medium is 1:1; the culture medium is dynamically perfused by a pump and through a reservoir at the inlet of the microfluidic chip; the perfusion rate of the culture medium is 1 to 3 μL / min, and the perfusion time is more than 48 hours.

[0011] In a second aspect, the present invention provides a microfluidic vascular chip for diagnosing incomplete Kawasaki disease, which is prepared by the above-mentioned preparation method.

[0012] In a third aspect, the present invention provides the use of the above-mentioned microfluidic vascular chip in the preparation of a diagnostic product for incomplete Kawasaki disease.

[0013] In a fourth aspect, the present invention provides the use of the above-mentioned microfluidic vascular chip in constructing an incomplete Kawasaki disease pathological model.

[0014] In a fifth aspect, the present invention provides a method for constructing an incomplete Kawasaki disease pathological model, in which a culture medium containing serum from patients with incomplete Kawasaki disease is dynamically perfused into the above-mentioned microfluidic vascular chip; the culture medium containing serum from patients with incomplete Kawasaki disease is a culture medium in which the serum in HCASMCs complete culture medium and HUVECs complete culture medium is replaced with serum from patients with incomplete Kawasaki disease; the perfusion speed is 40 to 60 μL / min, and the perfusion time is more than 4 hours.

[0015] In a fifth aspect, the present invention provides the use of the above-mentioned microfluidic vascular chip in screening drugs for treating incomplete Kawasaki disease.

[0016] The beneficial effects of the present invention are:

[0017] 1. The microfluidic vascular chip for incomplete Kawasaki disease diagnosis provided by the present invention is a "straight-line" microfluidic chip, which has a simple method and is easy to prepare. A tubular structure is used as the matrix structure for building a three-dimensional co-culture system of HCASMCs and HUVECs, and a circulating micropump system (a pump device for dynamic perfusion of culture medium) is introduced to achieve dynamic perfusion culture of cells, which more realistically simulates the structure and physiological characteristics of human blood vessels, avoiding the differences caused by different species and the deficiency that flat static culture cannot well simulate the in vivo environment.

[0018] 2. The microfluidic vascular chip for the diagnosis of incomplete Kawasaki disease provided by the present invention has a tubular channel prepared from rat tail type I collagen, which provides an extracellular matrix environment close to the in vivo for the construction of bionic vascular tissue. Rat tail type I collagen has a wide elastic range and can well simulate the elastic modulus of blood vessels in the body, and more accurately reflect the physical changes of coronary artery vascular lesions in Kawasaki disease.

[0019] 3. The microfluidic vascular chip for the diagnosis of incomplete Kawasaki disease provided by the present invention can be used to construct an in vitro disease model of Kawasaki disease, as well as to perform in vitro diagnosis and drug screening for incomplete Kawasaki disease, providing a reliable tool for clinical scientific research on incomplete Kawasaki disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the structure of the microfluidic chip of the present invention, wherein a is a schematic diagram of the vascular chip, and b is a physical diagram of the vascular chip;

[0021] Figure 2 It is a schematic diagram of the preparation process of the microfluidic chip of the present invention;

[0022] Figure 3Schematic diagram of the structure of the microfluidic chip for constructing a three-dimensional co-culture system of HCASMCs and HUVECs of the present invention, wherein a is a schematic diagram of the co-culture system, and b is a 3D image of calcein staining taken by confocal microscopy of the co-culture system;

[0023] Figure 4 It is the characterization of the vascular tissue constructed by the microfluidic vascular chip of the present invention, wherein HCASMCs and HUVECs are respectively labeled with α-smooth muscle actin (α-SMA) and platelet endothelial cell adhesion molecule (CD31), and the cell nucleus is labeled with 4',6-diamidino-2-phenylindole (DAPI);

[0024] Figure 5 It is an immunofluorescence staining diagram of inflammatory markers (ICAM-1, VCAM, E-Selectin, NLRP3, CXCL10) after adding serum samples of children to the microfluidic vascular chip of the present invention and its corresponding statistical diagram. DETAILED DESCRIPTION

[0025] The present invention is further described below in conjunction with specific embodiments and drawings.

[0026] Example 1

[0027] This embodiment provides a microfluidic vascular chip for incomplete Kawasaki disease diagnosis, and the preparation method thereof comprises the following steps:

[0028] Step 1: Figure 1 and 2 As shown, a microfluidic chip containing collagen channels was prepared.

[0029] Preparation of PDMS chip:

[0030] The PMMA template was designed using AutoCAD software. The chip contains collagen grooves (0.8*0.4*0.3cm), channels (0.9*0.5*0.3cm), inlets and outlets (D=0.1cm), and two reservoirs located at the inlets and outlets. The reservoirs are made of 1cm 3 The PDMS block was punched with holes (D = 0.5 cm).

[0031] The PDMS solution and curing agent were mixed at a mass ratio of 10:1, placed in a vacuum drying oven to remove bubbles in the solution, and then the solution was poured into the PMMA template and vacuumed again to remove bubbles. After curing in a 70°C oven for 3 hours, the cured PDMS chip was separated with a scalpel.

[0032] A tattoo needle with a diameter of 350 μm was fixed in the punched groove of PDMS with liquid PDMS. After the PDMS solidified, the needle was pulled out, and a hole with a diameter of 1 mm was punched in the punched groove using a puncher. The PDMS chip and glass sheet were combined after plasma treatment, and then two liquid reservoirs were fixed at the inlet and outlet of the chip with liquid PDMS.

[0033] After the PDMS chip was made, in order to promote a better fit between PDMS and collagen, it was coated with 1% polyethyleneimine (PEI) for 10 min, 0.1% glutaraldehyde (GTA) for 20 min, and then coated with 70% ethanol overnight, followed by treatment with a plasma processor for 45 s, and then the PDMS chip was irradiated with ultraviolet light for more than 20 min.

[0034] Constructing collagen channels:

[0035] Re-insert the sterilized tattoo needle into the PDMS chip, add the collagen solution into the groove of the PDMS, wait for 30 to 60 minutes for the collagen to solidify, carefully pull out the tattoo needle, fill the needle hole with vacuum grease, and you can get a closed collagen channel with a diameter of 350μm.

[0036] The reagents required for preparing the collagen solution are: rat tail type I collagen (10 mg / mL), 1N sodium hydroxide (NAOH), 10X phosphate buffered saline (PBS), sterile ultrapure water. The concentration of the collagen solution is 6 mg / mL.

[0037] The preparation method of collagen solution is:

[0038] V 鼠尾I型胶原 =V 胶原溶液 *C 所需浓度 / 鼠尾I型胶原原浓度

[0039] V 1N NAOH =V 鼠尾I型胶原 *0.025

[0040] V 10X PBS =V 胶原溶液 / 10

[0041] V 超纯水 =V 胶原溶液 -V 鼠尾I型胶原 -V 1N NAOH -V 10X PBS

[0042] The preparation of collagen solution should be done on ice or in a 4℃ refrigerator. When preparing collagen solution, first prepare alkaline neutralization solution, then add rat tail type I collagen to prevent the collagen from solidifying due to local overtemperature or local acid-base neutralization. Be careful during the stirring process to avoid bubbles. If bubbles are generated, centrifuge it.

[0043] Step 2: Figure 3 As shown, a three-dimensional cell co-culture system was constructed.

[0044] Cell planting: According to the microfluidic chip obtained in step 1, use a pipette to suck 10 μL of 200,000 / mL HCASMCs suspension and slowly inject it into the chip. After culturing in a carbon dioxide incubator for 30 minutes, inject 200,000 / mL HCASMCs suspension again, flip the microfluidic chip 180° and continue to culture in the incubator for 30 minutes, then flip the microfluidic chip 180° again and continue to culture for 24 hours. After 24 hours, use a pipette to suck 10 μL of 300,000 / mL HUVECs suspension and plant HUVECs in the microfluidic chip according to the HCASMCs planting method, and continue to culture for 48 hours. HCASMCs are attached to the surface of the collagen channel, and HUVECs are attached to the surface of HCASMCs to form bionic vascular tissue.

[0045] Dynamic perfusion culture:

[0046] The microfluidic chip after cell culture was connected to a circulating micropump, and the culture medium was dynamically perfused through the circulating micropump and the reservoir at the entrance of the microfluidic chip. The pump flow rate was adjusted to perfuse and culture for 48 hours to obtain a microfluidic vascular chip. The culture medium was a mixture of HCASMCs complete culture medium and HUVECs complete culture medium in a volume ratio of 1:1. The flow rate of 2μL / min simulated the blood flow rate under physiological conditions. The characterization results after culture are shown in Figure 2. Figure 4 shown.

[0047] Example 2

[0048] This embodiment provides the use of a microfluidic vascular chip in preparing diagnostic products for incomplete Kawasaki disease, constructing a pathological model of incomplete Kawasaki disease, and screening drugs for treating incomplete Kawasaki disease.

[0049] In actual applications, by injecting the serum to be diagnosed into the microfluidic vascular chip and observing the physical changes in the collagen channel and the changes in the inflammatory response antibodies of the cells, incomplete Kawasaki disease can be diagnosed. The following is an effect experiment.

[0050] Serum from patients was added to the microfluidic vascular chip prepared in Example 1. The adding method was as follows: serum in the complete medium of HCASMCs and HUVECs was replaced with serum from patients with incomplete Kawasaki disease, and the serum was dynamically perfused into the microfluidic vascular chip. The flow rate of 50 μL / min simulated the blood flow rate under pathological conditions.

[0051] After adding the patient's serum and culturing for 4 hours, the inflammatory characteristics of the cells in the microfluidic vascular chip can be observed under a confocal microscope. Figure 5 shown.

[0052] From the results of inflammation characterization, it can be seen that after adding serum, the microfluidic vascular chip of the healthy control group was negative for all five inflammatory markers (the number of positive endothelial cells was <1%), and the microfluidic vascular chip of the common fever group only showed a weak positive for ICAM-1 (about 3%). Taking purpura as an example, the microfluidic vascular chip of other vasculitis groups showed strong positivity for ICAM-1, VCAM, and E-Selectin (the number of positive endothelial cells was >50%), but there was no obvious response to NLRP3 and CXCL10. Among the 20 typical Kawasaki disease microfluidic vascular chips, 17 showed strong positive responses to the above five markers; among the 20 incomplete Kawasaki disease cases, 16 responded. These results show that the microfluidic vascular chip for the diagnosis of incomplete Kawasaki disease can make a diagnosis of incomplete Kawasaki disease.

[0053] Similarly, the above method can be used to construct an incomplete Kawasaki disease pathology model, and the incomplete Kawasaki disease pathology model can be further used to screen drugs for treating incomplete Kawasaki disease.

[0054] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the reagents, materials and operating procedures used herein are reagents, materials and conventional procedures widely used in the corresponding fields.

[0055] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a microfluidic vascular chip for incomplete Kawasaki disease diagnosis, characterized in that: The following steps are involved: Step 1, preparing a microfluidic chip containing a collagen channel; Step 2: Plant HCASMCs in the collagen channel, wait for the HCASMCs to merge into a single layer covering the collagen channel, then plant HUVECs, allow the HUVECs to merge into a single layer covering the surface of the HCASMCs, and then dynamically perfuse the culture medium to obtain the microfluidic vascular chip.

2. The method for preparing a microfluidic vascular chip for incomplete Kawasaki disease diagnosis according to claim 1, characterized in that: In step one, the preparation method of the microfluidic chip is: a PMMA template is obtained by etching, and a PDMS chip is obtained by a reverse molding method; the PDMS chip has a collagen groove; a needle is inserted into the collagen groove, and then a collagen solution is poured in, and after curing, the microfluidic chip containing the collagen channel is obtained.

3. The method for preparing a microfluidic vascular chip for incomplete Kawasaki disease diagnosis according to claim 1, characterized in that: In step 1, the collagen channel is formed by solidifying a collagen solution; the collagen solution is a rat tail type I collagen solution, with a concentration of 6 to 8 mg / mL, a solidification temperature of 35 to 40° C., and a solidification time of 30 to 60 min; and the diameter of the collagen channel is 200 to 500 μm.

4. The method for preparing a microfluidic vascular chip for incomplete Kawasaki disease diagnosis according to claim 1, characterized in that: In step 2, the HCASMCs and HUVECs are planted by a flip planting method; The seeding concentration of HCASMCs was 150,000 to 250,000 / mL, and the seeding concentration of HUVECs was 250,000 to 350,000 / mL.

5. The method for preparing a microfluidic vascular chip for incomplete Kawasaki disease diagnosis according to claim 1, characterized in that: In step 2, the culture medium includes HCASMCs complete culture medium and HUVECs complete culture medium; The volume ratio of HCASMCs complete medium and HUVECs complete medium was 1:1; The perfusion rate of the culture medium is 1-3 μL / min, and the perfusion time is more than 48 hours.

6. A microfluidic vascular chip for the diagnosis of incomplete Kawasaki disease obtained by the preparation method according to any one of claims 1 to 5.

7. Use of the microfluidic vascular chip as described in claim 6 in preparing a diagnostic product for incomplete Kawasaki disease.

8. Use of the microfluidic vascular chip as described in claim 6 in constructing an incomplete Kawasaki disease pathological model.

9. A method for constructing an incomplete Kawasaki disease pathological model, characterized in that: Dynamically perfusing a culture medium containing serum from a patient with incomplete Kawasaki disease into the microfluidic vascular chip as claimed in claim 6; The culture medium containing incomplete Kawasaki disease patient serum is a culture medium in which the serum in the HCASMCs complete culture medium and the HUVECs complete culture medium is replaced with the serum of the incomplete Kawasaki disease patient; the perfusion speed is 40 to 60 μL / min, and the perfusion time is more than 4 hours.

10. Use of the microfluidic vascular chip as claimed in claim 6 in screening drugs for treating incomplete Kawasaki disease.

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