Arterial organ-on-chip based on multi-material suspended biological 3D printing and preparation method

Abstract

The application provides an arterial organ chip based on multi-material suspended biological 3D printing and a preparation method, wherein a biological ink material is derived from a decellularized matrix of mammalian soft tissue, and the application takes pig skin decellularized matrix dECM as an example, combines with triple pyridine ruthenium chloride Ru and sodium persulfate SPS, and prepares biological ink material VLC-dECM with light irradiation crosslinking characteristics, and PF-127 which is easily soluble in water is used as a sacrificial ink material; the VLC-dECM material encapsulating human aortic smooth muscle cells is suspended printed in the support bath material VLC-dECM in sequence to form a first layer, and the second layer of the sacrificial material PF-127 is suspended printed; after visible light crosslinking, the sacrificial material PF-127 is dissolved and removed by using a phosphate buffer PBS, and a blood vessel channel is formed. The application saves the manufacturing time; the manufactured arterial organ chip can realize pumpless perfusion under the guidance of gravity, simplifies the overall structure of the arterial organ chip, and has a good multilayer arterial biomimetic structure.

Description

TECHNICAL FIELD

[0001] The application provides an arterial organ chip based on multi-material suspended biological 3D printing and a preparation method, and belongs to the technical field of biological tissue engineering. BACKGROUND

[0002] The arterial system of the human body plays an extremely important role in blood circulation, responsible for transporting blood containing oxygen and nutrients from the heart to the whole body. When any link of the arterial system is disturbed, it can cause various fatal diseases and complications. Abnormal symptoms related to arteries can cause ischemic diseases such as myocardial infarction, cerebral infarction, etc. These diseases are the main cause of death of millions of people worldwide every year, usually triggered by arterial-related diseases such as atherosclerosis, thrombosis, and aneurysm. Although clinical treatment methods can save some patients with arterial-related diseases, the underlying pathology of these arterial abnormalities is not fully understood.

[0003] A large number of vascular disease models have been established based on experimental animals or planar cell culture, however, both types of models face major limitations that limit their application. First, interspecies differences between animals and humans inevitably lead to pathological differences and unpredictable drug response changes. Results obtained from experimental animals can provide limited or even ineffective reference for clinical application. On the other hand, two-dimensional cell culture mostly lacks the three-dimensional microenvironment present in natural tissues, and therefore cannot accurately reflect human physiological and pathological mechanisms, for example, planar substrates not only inhibit cell polarization and arrangement, but also limit the interaction between multiple types of cells.

[0004] In order to overcome these limitations, to study the pathological mechanisms of arterial blood vessels and the effects of various factors on diseased arteries, three-dimensional arterial in vitro chips can simulate biological environments at multiple levels and scales, which is conducive to understanding and studying the pathological mechanisms of arteries. At present, methods for constructing three-dimensional arterial blood vessel chips include micro-needle template method, polydimethylsiloxane (PDMS) soft lithography method and coaxial biological 3D printing method. The micro-needle template method and the polydimethylsiloxane (PDMS) soft lithography method cannot prepare a double-layer blood vessel in vitro model, even if the coaxial biological 3D printing method, due to the limitations of biological ink materials and manufacturing processes, still cannot effectively realize the manufacturing of multi-layer blood vessel chips. Therefore, there is an urgent need for an arterial disease model that can accurately reflect the physiological and pathological mechanisms of human arteries, as a diagnostic, detection and screening platform for exploring disease mechanisms and developing specific drugs.

[0005] In summary, the present application aims at the technical problems of existing biological 3D printing for manufacturing multi-layered arterial organ chip, and the model demand of multi-layered arterial organ chip in biomedical field, and proposes a human arterial organ chip manufacturing method based on multi-material suspension biological 3D printing based on suspension biological 3D printing technology. The method involves the preparation of a new type of biological ink, and the biological ink material has the characteristics of light irradiation crosslinking, good biocompatibility, rheological property and printability. SUMMARY

[0006] Based on the existing biological manufacturing method, it is difficult to prepare a multi-layered arterial organ chip with complex structure, size controllable shape, and cannot guarantee the structure shape fidelity of the arterial organ chip, and the existing arterial organ chip cannot accurately reflect the physiological and pathological mechanism of human blood vessels, and the complex program of pump control perfusion, the present application based on biological 3D printing technology, proposes a human arterial organ chip manufacturing method based on multi-material suspension biological 3D printing, which constructs a multi-layered structure pipeline by using the special properties of various biological ink materials, and can bionic manufacturing of complex blood vessel structure such as bending and branching, and complex arterial organ chip model with different inner diameter and wall thickness, at the same time, it can realize pump-free perfusion depending on gravity, the multi-layered arterial organ chip involved in the present application can become a useful tool for studying the relationship between hemodynamics and arterial diseases, and can be used for drug screening, pathological mechanism research and other biomedical fields.

[0007] The specific technical scheme is:

[0008] The arterial organ chip manufacturing method proposed by the present application involves decellularized matrix material derived from pig, cow, human and other mammalian soft tissues, preferably, the present application takes fresh pig skin decellularized matrix (decellularized extracellular matrix, dECM) as an example, and combines with light initiator trispyridine ruthenium chloride Ru and sodium persulfate SPS to prepare biological ink material with light irradiation crosslinking property (visible light curable dECM, VLC-dECM). PF-127 which is easy to dissolve in water is used as a sacrificial ink material, and the VLC-dECM material encapsulating human aortic smooth muscle cells (HAoSMCs) is first printed in the support bath material VLC-dECM, and then the second layer of sacrificial material PF-127 is printed, after visible light irradiation crosslinking, the sacrificial material PF-127 is dissolved and removed by using phosphate buffer PBS, forming a blood vessel channel, and achieving the purpose of manufacturing a multi-layered arterial organ chip. In addition, by controlling the printing process parameters such as printing path, printing speed and extrusion air pressure, the complex arterial organ chip model with controllable structure and size can be accurately realized.

[0009] The specific steps of the method include:

[0010] Step one, prepare support bath ink;

[0011] Mix 2% dECM with 10x DMEM solution at a volume ratio of 9:1, add sodium hydroxide solution, neutralize the acidity of the material, and adjust the pH of the dECM material to 7. Mix the dECM material with pH 7 and PBS buffer at a volume ratio of 1:0.6, then put it in a biological incubator (37℃, 5.0% CO2 by volume) for 20-30 minutes for pre-crosslinking to form a gel. Then take it out, use a cell crusher to crush the dECM gel for 3 minutes to form dECM microgel particles. Centrifuge the dECM microgel particles in a high-speed centrifuge (10000r / min) for 5 minutes, and aspirate the supernatant after centrifugation.

[0012] Mix the dECM microgel particles with trispyridine ruthenium chloride Ru (concentration 50mM / L solution) at a volume ratio of 1:100, and then mix the dECM microgel particles with sodium persulfate SPS (concentration 50mM / L solution) at a volume ratio of 1:10. Since the dECM gel mixed with trispyridine ruthenium chloride Ru and sodium persulfate SPS is a temperature-sensitive and light-sensitive material, this operation needs to be carried out in a light-proof and temperature less than 15℃ environment. The obtained mixed biological ink VLC-dECM is used as a support bath material, hereinafter referred to as 0.5VLC-dECM, which has shear recovery and light irradiation crosslinking properties. This method improves the self-healing ability of VLC-dECM, which is beneficial to realize embedded suspension printing manufacturing. The 0.5VLC-dECM ink is extruded into the first nozzle barrel of the biological 3D printer, and the printing temperature is set to 15℃.

[0013] Step two, prepare printing biological ink;

[0014] Mix 2% dECM with 10x DMEM solution at a volume ratio of 9:1, add sodium hydroxide solution, neutralize the acidity of the material, and adjust the pH of the dECM material to 7. Mix the dECM material with pH 7 and PBS buffer at a volume ratio of 1:0.7. Mix trispyridine ruthenium chloride Ru (concentration 50mM / L solution) and sodium persulfate SPS (concentration 50mM / L solution) into the dECM gel in turn at a ratio of 1:200 and 1:20 respectively, since the dECM gel mixed with trispyridine ruthenium chloride Ru and sodium persulfate SPS is a temperature-sensitive and light-sensitive material, this operation needs to be carried out in a light-proof and temperature less than 15℃ environment. Prepare the mixed biological ink VLC-dECM for biological 3D printing, hereinafter referred to as 0.25VLC-dECM, which has shear recovery and light irradiation crosslinking properties. Use a pipette to extrude 0.25VLC-dECM into the first nozzle barrel of the biological 3D printer, and set the printing temperature to 15℃. 60.25VLC-dECM mixed with 1000 cells / mL human aortic smooth muscle cells (HAoSMCs) were extruded into the second nozzle cartridge of the bioprinter, and the printing temperature was set to 15℃.

[0015] Step three, the receiving platform temperature of the bioprinter was set to 15℃, and the bioprinting was started in a light-proof environment. First, the first nozzle (needle diameter 0.8mm) was used to print 1-2mm thick 0.5VLC-dECM material as the support bath material of the arterial organ chip, the printing speed was 150mm / min, and the printing air pressure was 8kPa. The chip frame was made by bioprinting at a printing temperature of 120℃ and an extrusion air pressure of 330kPa using a biopolymer material polyethylene. Its structure is similar to a hollow "H" shape, and it is designed with a perfusion drainage port to guide the cell culture solution into the arterial organ chip pipeline, so that the chip can realize pump-free perfusion depending on gravity. The material can be replaced by other biopolymer materials.

[0016] Step four, the second nozzle (needle diameter 0.8mm) was converted to extrude 0.25VLC-dECM encapsulating HAoSMCs cells, the printing speed was 250mm / min, the printing air pressure was 8kPa, and the printing path was completed according to the G code instructions to form the first layer structure of the arterial organ chip.

[0017] Step five, the third nozzle (needle diameter 0.8mm) was converted to extrude sacrificial material PF-127 (40% w / v), and the printing was completed according to the same printing path as step four at a printing temperature of 25℃, a printing speed of 350mm / min, and a printing air pressure of 170kPa.

[0018] Step six, the arterial organ chip was irradiated with visible light with an intensity of 25mW / cm 2 , wavelength 405nm for 20s to cause photo-radiation crosslinking;

[0019] Step seven, the sacrificial ink material PF-127 was removed using phosphate buffer PBS to form the microchannels of the arterial organ chip, and the structure of the arterial organ chip model was completed at this time.

[0020] Step eight, an appropriate amount of DMEM culture solution was added to the arterial organ chip model, and the model was placed in a biological incubator at a temperature of 37℃ and a volume ratio of 5% CO2 for 30 minutes. The dECM contained in the VLC-dECM material was subjected to temperature-sensitive crosslinking to form an interpenetrating network with the photo-radiation crosslinked gel, thereby improving the material strength.

[0021] Step nine, the arterial organ chip model was taken out, the DMEM culture solution was aspirated, and the model pipeline was seeded with 2*106 cells / mL of human umbilical vein endothelial cells (HUVECs), and then adding culture solution again and placing in a biological incubator at 37 DEG C and 5% CO2 by volume;

[0022] Step ten, after culturing for 2 hours, observing the state of the cells, replacing new DMEM culture solution, and placing the arterial organ chip in a shaker to assist in pumpless perfusion of the culture solution under the guidance of gravity, setting the shaker to oscillate up and down with a maximum oscillation angle of 15 DEG with respect to the horizontal plane and an oscillation speed of 30 r / min, so that the culture solution can flow into the chip pipeline fully, and placing in a biological incubator at 37 DEG C and 5% CO2 by volume to continue culturing until the vascular model pipeline is full of cells to form an endothelial layer and HAoSMCs cells form a medial layer, at which time the arterial organ chip model has biological functions and can be used for predicting vascular diseases and simulating drug delivery.

[0023] The technical scheme of the present application has the following technical effects:

[0024] 1. The biological ink material VLC-dECM prepared by the present application is related to a decellularized matrix derived from mammalian soft tissue, which can provide a necessary physiological microenvironment for cell proliferation and growth;

[0025] 2. The biological ink material VLC-dECM prepared by the present application is a biological material with light irradiation crosslinking characteristics, and also has temperature-sensitive crosslinking characteristics, and the two crosslinking methods form an interpenetrating network, which improves the material strength and improves the forming quality of the arterial organ chip;

[0026] 3. The present application provides a human arterial organ chip manufacturing method based on multi-material suspension biological 3D printing, which only needs 120s to manufacture an arterial organ chip model, saves manufacturing time, and improves the biological manufacturing efficiency;

[0027] 4. The human arterial organ chip manufactured by suspension biological 3D printing in the present application can only rely on pumpless perfusion guided by gravity in the post-processing perfusion process, which simplifies the chip structure and is easy to operate;

[0028] 5. The biological ink material involved in the present application has the characteristics of vascular tissue specificity, high biological activity and light irradiation crosslinking, and the proportion of the materials contained in the biological ink material is verified by experiments, which can ensure the crosslinking of the arterial organ chip under visible light and also ensure the cell activity and improve the biological function of the chip;

[0029] 6. The arterial organ chip manufactured by the present application has a good multi-layer arterial biomimetic structure, can realize straight, curved, bifurcated and other configurations, and the size is controllable, which can be used in the biomedical field such as drug screening and pathological exploration. BRIEF DESCRIPTION OF DRAWINGS

[0030] Figure 1Manufacturing process of the double-layer vascular in-vitro model of the application;

[0031] Figure 2a Process diagram of the preparation of the double-layer arterial organ chip in-vitro model by the embedded suspended biological 3D printing of the application;

[0032] Figure 2b Structure of the double-layer arterial organ chip in-vitro model of the application and pump-free perfusion arterial organ chip frame structure;

[0033] Figure 3 Double-layer arterial organ chip model of the embodiment. DETAILED DESCRIPTION

[0034] The specific technical solutions of the application are described in combination with the embodiments.

[0035] The preparation method of the arterial organ chip based on multi-material suspended biological 3D printing proposed by the application can prepare a multi-layer arterial organ chip model with high fidelity of structural shape and controllable structure and size, and the specific implementation scheme is as follows: Figure 1

[0036] Step one, preparation of support bath ink. Optimized, for example, with fresh pig skin decellularized matrix dECM, mix 2% dECM with 10x DMEM solution at a volume ratio of 9:1, add sodium hydroxide solution, neutralize the acidity of the material, and adjust the pH of the dECM material to 7. Mix the dECM with pH 7 and phosphate buffer PBS at a volume ratio of 1:0.6, then put it in a biological incubator (37℃, volume ratio 5.0% CO2) for 20-30 minutes for pre-crosslinking to form a gel, then take it out, use a cell disruptor to break the dECM gel for 3 minutes to form dECM microgel particles. Centrifuge the dECM microgel particles in a high-speed centrifuge (10000r / min) for 5 minutes, and aspirate the supernatant after centrifugation. Finally, mix the dECM microgel particles with trispyridine ruthenium chloride Ru and sodium persulfate SPS (concentration 50mM / L solution) at a ratio of 1:100 and 1:10 respectively. Since the dECM gel mixed with trispyridine ruthenium chloride Ru and sodium persulfate SPS is a temperature-sensitive and light-sensitive material, this operation needs to be carried out in a light-proof and temperature less than 15℃ environment. The final preparation of mixed biological ink VLC-dECM is used as a support bath material, hereinafter referred to as 0.5VLC-dECM, which has shear recovery and light irradiation crosslinking properties. This method improves the self-healing ability of VLC-dECM, which is beneficial to the realization of embedded suspended printing manufacturing. The 0.5VLC-dECM ink is extruded into the first nozzle (1) of the biological 3D printer, and the printing temperature is set to 15℃.

[0037] ​Step 2: Prepare the printing bio-ink. Preferably, using fresh decellularized pig skin matrix as an example, mix 2% (w / w) dECM with 10×DMEM solution at a volume ratio of 9:1. Add sodium hydroxide solution to neutralize the acidity of the material, adjusting the pH of the dECM material to 7. Mix the pH 7 dECM material with phosphate-buffered saline (PBS) at a volume ratio of 1:0.7. Then, sequentially mix ruthenium tripyridine chloride (Ru) and sodium persulfate (SPS) (50 mM / L solution) into the dECM gel at ratios of 1:200 and 1:20, respectively. Since the dECM gel containing Ruthenium tripyridine chloride (Ru) and sodium persulfate (SPS) is a thermosensitive and photosensitive material, this operation needs to be performed in a light-protected environment at a temperature below 15°C. Prepare a hybrid bio-ink VLC-dECM for bio-3D printing, hereinafter referred to as 0.25VLC-dECM, which has shear recovery and photo-irradiation crosslinking properties. Use a pipette to mix a 2*10... 6 Human aortic vascular smooth muscle cells (HAoSMCs) were uniformly mixed into 0.25 VLC-dECM and squeezed into the second nozzle (2) of the bio-3D printer. The printing temperature was set to 15℃.

[0038] Step 3: Set the receiving platform temperature of the bio-3D printer to 15℃ and begin bio-3D printing in a light-protected environment. First, use the first nozzle (1) (needle diameter 0.8mm) to print a 1-2mm thick layer of 0.5VLC-dECM material into the pre-prepared chip framework as the support bath material for the arterial organ chip. The printing speed is 150mm / min, and the printing pressure is 8kPa. Figure 2a As shown in (i) above, the chip frame is manufactured using a biopolymer material, polyethylene, at a printing temperature of 120°C and an extrusion pressure of 330 kPa via bio-3D printing. Its structure resembles a hollow "I" shape, as shown below. Figure 2b As shown, the structure is designed with a pumpless perfusion drainage port, which assists the arterial organ-on-a-chip in relying on gravity to induce the culture medium to flow into the chip channels. Its material can be replaced by other biopolymer materials.

[0039] Step 4: Switch to the second nozzle (2) (needle diameter 0.8mm) to extrude 0.25VLC-dECM encapsulating HAoSMCs cells. Print speed 250mm / min, printing pressure 8kPa, and follow the G-code instructions to complete the printing path, forming the first layer structure of the arterial organ-on-a-chip, such as... Figure 2a As shown in (ii);

[0040] Step five, the third nozzle (3) (needle diameter 0.8mm) is used to extrude the sacrificial material PF-127 (40% w / v), and the printing is completed according to the same printing path as step four, with the printing temperature of 25℃, the printing speed of 350mm / min, and the printing air pressure of 170kPa, as shown in (iii) of Figure 2a ;

[0041] Step six, the artery organ chip is irradiated with visible light with the light intensity of 25mW / cm 2 and the wavelength of 405nm for 20s, so that the light irradiation crosslinking is performed, as shown in (iv) of Figure 2a ;

[0042] Step seven, the sacrificial ink material PF-127 is removed by using the phosphate buffer PBS, as shown in (v) of Figure 2a , so that the artery organ chip microchannel is formed, and at this time, the artery organ chip model structure manufacturing is completed;

[0043] Step eight, the appropriate DMEM culture solution is added to the artery organ chip model, and the model is placed in a biological incubator with the temperature of 37℃ and the CO2 concentration of 5% for 30 minutes, so that the dECM contained in the VLC-dECM material is subjected to the temperature-sensitive crosslinking, and the light irradiation crosslinking gel forms the interpenetrating network, so as to improve the material strength;

[0044] Step nine, the artery organ chip model is taken out, the DMEM culture solution is absorbed, and the human umbilical vein endothelial cells HUVECs with the cell density of 2*10 6 cells / mL are planted in the model pipeline, the culture solution is added again, and the biological incubator with the temperature of 37℃ and the CO2 concentration of 5% is placed;

[0045] Step ten, after 2 hours of culture, the cell state is observed, the new DMEM culture solution is replaced, and the artery organ chip is placed on the shaker to assist the culture solution to flow into the chip pipeline under the guidance of gravity, the shaker is set to swing up and down, the maximum swing angle with the horizontal plane is set to 15°, and the swing speed is 30r / min, so that the culture solution can fully flow into the chip pipeline, and the biological incubator with the temperature of 37℃ and the CO2 concentration of 5% is placed for continuous culture, until the vascular model pipeline is full of cells to form the endothelial layer and the HAoSMCs cells form the media layer, at this time, the artery organ chip model has the biological function, and can be used for predicting vascular diseases and simulating drug delivery. According to the requirements, the artery organ chip model can be of various structures, as shown in Figure 2b .

[0046] In order to clearly show the human artery organ chip manufacturing method based on multi-material suspended biological 3D printing provided in the application, red and blue fluorescent particles are uniformly mixed in 0.25VLC-dECM material and PF-127 material respectively, and an inverted fluorescence microscope is used to observe the forming condition of the artery organ chip, as shown in Figure 3 The double-layer artery organ chip model obtained for the embodiment is placed on a shaking table, under the swing action of the shaking table, the cell culture solution flows into the inside of the artery organ chip pipeline through the drainage port by gravity, and reciprocating circulation flow is accompanied by the shaking table.

[0047] The human artery organ chip manufacturing method based on multi-material suspended biological 3D printing provided in the application has wide application prospects, can plant human umbilical vein endothelial cells in the pipeline of the printed artery organ chip model sample to simulate blood perfusion, and can make the vascular in-vitro model of the bifurcation structure appear endothelial layer damage at the bifurcation position, so that the bifurcation structure position of the human body is prone to vascular diseases, such as atherosclerosis induced by endothelial layer damage.

Claims

1. A method for the fabrication of an arterial organ-on-chip based on multi- material suspended biological 3D printing, characterized in that, Comprising the following steps: Step one, prepare support bath ink; Mix 2% dECM with 10×DMEM solution at a volume ratio of 9:1, add sodium hydroxide solution, neutralize the material acidity, and adjust the pH of the dECM material to 7; Mix the dECM material with the phosphate buffer PBS at a volume ratio of 1:0.7; In the dark and temperature less than 15℃ environment, mix the dECM gel with tris (2-pyridyl) ruthenium chloride at a volume ratio of 1:200 to prepare a solution with a concentration of 50mM / L, and continue to mix with sodium persulfate SPS at a volume ratio of 1:20 to prepare a solution with a concentration of 50mM / L; The mixed biological ink VLC-dECM for biological 3D printing is prepared, which is referred to as 0.25 VLC-dECM hereinafter, and has the properties of shear recovery and light irradiation crosslinking; Step three, set the receiving platform temperature of the biological 3D printer to 15℃, and start the biological 3D printing in a dark environment; First, use the first nozzle (1) with a needle diameter of 0.8 mm to print 1-2 mm thick 0.5 VLC-dECM material as the support bath material of the arterial organ chip in the pre-prepared chip frame, the printing speed is 150 mm / min, and the printing air pressure is 8 kPa, wherein the chip frame is made of biopolymer material polyethylene by biological 3D printing under the conditions of printing temperature 120℃ and extrusion air pressure 330 kPa, and its structure is hollow "H" type, and is designed with perfusion drainage port for guiding the cell culture solution into the pipeline of the arterial organ chip to assist pump-free perfusion; ​ ​ ​ Using a pipette gun, 2 x 10 6 cells / mL human aortic vascular smooth muscle cells HAoSMCs were uniformly mixed into 0.25 VLC-dECM and extruded into the second nozzle (2) cartridge of the bioprinter, with a printing temperature of 15°C; ​ Step four, the second nozzle (2) with a needle diameter of 0.8 mm is used to extrude 0.25 VLC-dECM encapsulating HAoSMCs, the printing speed is 250 mm / min, the printing air pressure is 8 kPa, and the printing path is completed according to the G code instructions, thereby forming the first layer structure of the arterial organ chip; Step five, the third nozzle (3) with a needle diameter of 0.8 mm is used to extrude the sacrificial material PF-127, which is a 40% w / v solution, and the printing is completed by moving according to the same printing path as step four under the printing temperature of 25℃, the printing speed of 350 mm / min, and the printing air pressure of 170 kPa; Step six, using light intensity 25 mW / cm 2 The arterial organ chip 20s is irradiated with visible light of wavelength 405 nm to cause photo irradiation crosslinking. Step seven, the sacrificial ink material PF-127 is removed by using PBS, thereby forming the microchannel of the arterial organ chip, and at this time, the manufacturing of the arterial organ chip model structure is completed; Step eight, an appropriate amount of DMEM culture solution is added to the arterial organ chip model, and the model is placed in a biological incubator with a temperature of 37℃ and a volume ratio of 5% CO2 for 30 minutes, so that the dECM contained in the VLC-dECM material is temperature-sensitive cross-linked, and the interpenetrating network is formed with the photo-cross-linked gel; Step nine, take out the arterial organ chip model, suck away DMEM culture solution, plant density 2×10 6 cells / mL human umbilical vein endothelial cells HUVECs into the model pipeline, add culture solution again, and place in a biological incubator with a temperature of 37℃ and a volume ratio of 5% CO2; Step ten, after 2 hours of culture, the cell state is observed, new DMEM culture solution is replaced, and the arterial organ chip is placed on a shaker to assist the pump-free perfusion of the culture solution under the guidance of gravity, the shaker is set to oscillate up and down, the maximum oscillation angle with the horizontal plane is set to 15°, and the oscillation speed is 30 r / min, so that the culture solution can fully perfuse the chip pipeline, and the culture is continued in a biological incubator with a temperature of 37℃ and a volume ratio of 5% CO2 until the vascular model pipeline is fully covered with cells to form an endothelial layer, and the HAoSMCs form a media layer.

2. The method for preparing an arterial organ-on-chip based on multi- material suspension biological 3D printing according to claim 1, characterized in that, The decellularized matrix is derived from soft tissue of a mammal; the soft tissue includes one or more of aortic, dermis, skeletal muscle, and gastric mucosa.

3. The method for preparing an arterial organ-on-chip based on multi- material suspension biological 3D printing according to claim 1, characterized in that, The decellularized matrix is derived from pig skin decellularized matrix dECM.

4. The method for preparing an arterial organ-on-chip based on multi- material suspension biological 3D printing according to claim 1, characterized in that, The chip frame realizes pump-free perfusion by gravity.

5. An arterial organ-on-a-chip based on multi-material suspended biological 3D printing, characterized in that, The preparation method according to any one of claims 1-4. The preparation method according to any one of claims 1-4.

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