A hierarchical vascularized microfluidic organ chip and its preparation method

Abstract

The present invention provides a hierarchical vascularized microfluidic organ chip and its preparation method. The chip includes: a polydimethylsiloxane module, the lower surface of which is provided with a central tissue chamber and a hierarchical microfluidic channel branching structure, the hierarchical microfluidic channel branching structure being symmetrical about the central tissue chamber; the upper surface of a glass slide is integrated with the lower surface of the polydimethylsiloxane module, and the hierarchical microfluidic channel branching structure is constructed into a large blood vessel simulating a venous artery by lining the hierarchical microfluidic channel with endothelial cells; gel perfusion holes are provided at both ends of the central tissue chamber, and liquid reservoirs and culture medium perfusion holes are provided at both ends of the hierarchical microfluidic channel branching structure. Under the action of static pressure difference, cell culture fluid with a liquid level difference flows from one side of the hierarchical microfluidic channel branching structure to the other side, stimulating the formation of a capillary network in the central tissue chamber. The present invention can provide an effective biological model for the study of vascularization-related diseases in vitro.

Description

Technical Field

[0001] The present invention relates to the field of biomedical engineering technology, and in particular to a hierarchical vascularized microfluidic organ chip and a preparation method thereof. Background Art

[0002] The human circulatory system is a highly organized system composed of blood vessels of varying diameters. It transports nutrients and oxygen, excretes metabolic products, and maintains the dynamic balance and physiological functions of various organs and tissues. Simulating angiogenesis in vitro and characterizing its functional characteristics are crucial for studying vascular mechanisms and diseases, with applications in areas such as tumor formation and metastasis, drug screening, and cancer therapy.

[0003] Organ-on-a-chips, as a novel technology, can simulate the physiological and pathological responses of different tissues and organs in vitro. Through precise fluid and mechanical control and the construction of a dynamic physiological microenvironment, microfluidic organ-on-chips can reproduce the cellular microenvironment, cell-cell and tissue-tissue interface interactions, and other characteristics of living organs. This circumvents metabolic differences between humans and animals, providing a portable, cost-effective, and novel biological model for disease modeling, drug research, and personalized medicine.

[0004] Microfluidic organ-on-a-chip (OIC) overcomes the limitations of traditional 2D (e.g., culture dishes) or static 3D cell culture models (e.g., scaffold technology), enabling the simulation of the multiscale microstructure and complex organ-specific microphysiological functions of in vivo 3D tissues. The vascular system is crucial for the interconnection between organs and serves as a semipermeable barrier to dynamically regulate molecular and cellular exchange between flowing fluids and surrounding tissues. Currently, two approaches are used to construct vascularized OICs: 1) Microfluidic channel endothelial cell lining: Human umbilical vein endothelial cells (HUVECs) are seeded within microfluidic channels to completely cover the inner surface of the channel, thereby simulating the construction of perfusable vascular networks in vitro. However, this method has difficulty constructing capillaries with complex structures and diameters less than 100 microns. 2) Endothelial cell self-assembly: By co-culturing human lung fibroblasts (NHLF) with human umbilical vein endothelial cells (HUVECs), a vascular network is formed by the proliferation, differentiation, and connection of HUVECs under the induction of vascular endothelial growth factor (VEGF), generating vascular lumens after several days of in vitro culture.

[0005] However, vascular networks in real tissues typically have a hierarchical structure, encompassing vessels of varying scales and structures. The diverse hemodynamic conditions inherent in this hierarchical structure significantly impact physiological and pathological processes, such as cell adhesion and thrombosis within the vascular network. Therefore, a hierarchical vascular network composed of vessels of varying scales is urgently needed to simulate the complex cardiovascular system within the human body. Summary of the Invention

[0006] In view of the defects in the prior art, the purpose of the present invention is to provide a hierarchical vascularized microfluidic organ chip and a preparation method thereof.

[0007] According to one aspect of the present invention, a hierarchical vascularized microfluidic organ chip is provided, comprising:

[0008] A polydimethylsiloxane module, the lower surface of which is provided with a central tissue chamber and a hierarchical microfluidic channel branch structure with different lumen diameters, wherein the hierarchical microfluidic channel branch structure is symmetrical with respect to the central tissue chamber; the hierarchical microfluidic channel branch structure is connected to the central tissue chamber via a phase guide structure;

[0009] A glass sheet, wherein the upper surface of the glass sheet is integrated with the lower surface of the polydimethylsiloxane module, and the hierarchical microfluidic channel branch structure is constructed into a large blood vessel simulating a venous artery by an endothelial cell lining method;

[0010] Gel perfusion holes are provided at both ends of the central tissue chamber, and liquid reservoirs and culture fluid perfusion holes are provided at both ends of the hierarchical microfluidic channel branch structure. The culture fluid perfusion holes are connected to the liquid reservoirs, and cell culture fluids of different heights are injected from the liquid reservoirs at both ends. Under the action of static pressure difference, the cell culture fluids with liquid level height difference flow from the hierarchical microfluidic channel branch structure on one side to the hierarchical microfluidic channel branch structure on the other side, thereby stimulating the formation of a capillary network in the central tissue chamber.

[0011] Optionally, the glass sheet and the polydimethylsiloxane module are bonded together through oxygen plasma.

[0012] Optionally, each final blood vessel of the hierarchical microfluidic channel branch structure is provided with a bubble exhaust port, and bubbles in the lumen are exhausted from the bubble exhaust port when the culture fluid is injected.

[0013] Optionally, the cross section of the hierarchical microfluidic channel branch structure is 3 / 4 circular.

[0014] Optionally, the lumen radius of the hierarchical microfluidic channel branch structure is 0.6-2.5 mm.

[0015] Optionally, the central tissue chamber is circular or rectangular in shape.

[0016] Optionally, the height of the central tissue chamber is 300-500 μm.

[0017] Optionally, the polydimethylsiloxane module has a thickness of 4-7 mm.

[0018] Optionally, the phase-guiding structure has a width of 150-250 μm and a height of 150-250 μm.

[0019] According to another aspect of the present invention, a method for preparing the hierarchical vascularized microfluidic organ chip is provided, the method comprising:

[0020] Providing a glass sheet, and using a resin material to be 3D printed into a microfluidic chip mold having a hierarchical microfluidic channel branch structure and a central tissue chamber structure;

[0021] Providing a bubble-free PDMS mold material, slowly pouring the PDMS mold material into the mold, and then curing the PDMS mold material;

[0022] Cutting the solidified PDMS mold material from the mold to obtain a polydimethylsiloxane module; punching a gel infusion hole, a culture medium infusion hole, and a bubble exhaust port on the polydimethylsiloxane module;

[0023] performing oxygen plasma bonding on the glass sheet and the polydimethylsiloxane module;

[0024] Provide a culture fluid reservoir, apply PDMS packaging material to the bottom of the culture fluid reservoir, and align it with the culture fluid perfusion hole to fix it;

[0025] After the glass sheet and the polydimethylsiloxane module are cured, they are sterilized at high temperature and high pressure to obtain a hierarchical vascularized microfluidic organ chip.

[0026] Compared with the prior art, the present invention has at least one of the following beneficial effects:

[0027] 1. The hierarchical vascularized microfluidic organ chip and its preparation method provided by the present invention include a polydimethylsiloxane module provided with a central tissue chamber and a hierarchical microfluidic channel branch structure with different lumen diameters. After bonding to a glass sheet, the microfluidic channel branches with different lumen diameters are constructed into large blood vessels simulating venous arteries through an endothelial cell lining method; the central tissue chamber is filled with fibrin gel or collagen gel mixed with endothelial cells and fibroblasts, and a three-dimensional perfusable capillary network is constructed in the central tissue chamber through the angiogenesis mechanism; the microfluidic channel branch structures on both sides are symmetrical with respect to the central tissue chamber structure, and the cell culture fluid with a liquid level difference flows from one microfluidic channel branch to the other side under the action of the static pressure difference, thereby promoting the generation of a three-dimensional capillary network in the central tissue chamber.

[0028] 2. The present invention constructs a hierarchical vascular network structure with different vascular scales by endothelializing the lumen and self-assembling capillary network, which can highly simulate the complex cardiovascular system of the human body and provide an effective biological model for the study of vascularization-related diseases in vitro. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Other features, objects and advantages of the present invention will become more apparent upon reading the detailed description of non-limiting embodiments with reference to the following drawings:

[0030] Figure 1 A schematic two-dimensional cross-sectional view of a hierarchical vascularized microfluidic organ chip according to one embodiment of the present invention;

[0031] Figure 2 Schematic top view of a hierarchical vascularized microfluidic organ chip according to one embodiment of the present invention;

[0032] Figure 3 Schematic diagram of the process of preparing a hierarchical vascularized microfluidic organ chip in one embodiment of the present invention;

[0033] Figure 4 Schematic diagram of a hierarchical vascularized microfluidic organ chip used for endothelialization of a lumen in one embodiment of the present invention;

[0034] Figure 5 Schematic diagram of the principle of endothelialized lumen of a hierarchical vascularized microfluidic organ chip in one embodiment of the present invention;

[0035] Figure 6 The fluorescence characterization results of the self-assembled capillary network of the hierarchical vascularized microfluidic organ chip at different days in one embodiment of the present invention are shown;

[0036] Figure 7 The fluorescence characterization results of the blood vessels in the central tissue chamber of the self-assembled capillary network of the hierarchical vascularized microfluidic organ chip in one embodiment of the present invention on the fifth day of culture are shown;

[0037] Figure 8 This is the 20-fold magnification of the blood vessel fluorescence characterization result of the self-assembled capillary network of the hierarchical vascularized microfluidic organ chip according to one embodiment of the present invention;

[0038] Figure 9 This is the fluorescence characterization result of the hierarchical vascular network formed by inducing endothelialization of the lumen and self-assembled capillary network in one embodiment of the present invention.

[0039] The corresponding reference numerals in the figure are: 1-polydimethylsiloxane module, 2-glass slide, 3-central tissue chamber, 4-hierarchical microfluidic channel branching structure, 5-phase guiding structure, 6-liquid reservoir, 7-cell culture fluid, 8-bubble exhaust port, 9-gel perfusion hole, 10-culture fluid perfusion hole. DETAILED DESCRIPTION

[0040] The present invention will be described in detail below with reference to specific embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those skilled in the art, several variations and improvements can be made without departing from the scope of the present invention. These all fall within the scope of protection of the present invention.

[0041] Reference Figure 1 and Figure 2 The hierarchical vascularized microfluidic organ chip provided by one embodiment of the present invention includes a polydimethylsiloxane (PDMS) module and a glass sheet 2. The lower surface of the polydimethylsiloxane module 1 is provided with a central tissue chamber 3 and a hierarchical microfluidic channel branch structure 4 with different lumen diameters. The hierarchical microfluidic channel branch structure 4 is symmetrical about the central tissue chamber 3; the hierarchical microfluidic channel branch structure 4 is connected to the central tissue chamber 3 through a phase guide structure 5; the glass sheet 2 is arranged below the polydimethylsiloxane module 1, and the upper surface of the glass sheet 2 is integrated with the lower surface of the polydimethylsiloxane module 1. The hierarchical microfluidic channel branch structure 4 is constructed into a large blood vessel simulating a venous artery by an endothelial cell lining method. Specifically, the hierarchical microfluidic channel branch structure 4 is first coated with laminin and incubated at 37°C for one hour. Then, a high concentration of endothelial cells is inoculated into the hierarchical microfluidic channel branch structure 4, and a stepper motor is used to rotate the device so that the endothelial cells are evenly covered and adhered to the lumen wall. After lining, the endothelial cells proliferate on the surface of the lumen wall, form a single layer fusion after one day of culture, and present an elongated shape along the laminar flow direction in the hierarchical microfluidic channel branch structure 4; gel perfusion holes 9 are provided at both ends of the central tissue chamber 3, and fibrin gel or collagen gel is injected into the central tissue chamber 3 through the gel perfusion holes 9; both ends of the hierarchical microfluidic channel branch structure 4 are provided with a liquid reservoir 6 and a culture fluid perfusion hole 10, and the culture fluid perfusion hole 10 is connected to the liquid reservoir 6, and cell culture fluid 7 of different heights is injected from the liquid reservoirs 6 at both ends. Under the action of the static pressure difference, the cell culture fluid 7 with a liquid level difference flows from the hierarchical microfluidic channel branch structure 4 on one side to the hierarchical microfluidic channel branch structure 4 on the other side, stimulating the formation of a capillary network in the central tissue chamber 3.

[0042] In an embodiment of the present invention, cell culture fluid 7 at different heights is injected into the liquid reservoirs 6 at both ends of the hierarchical microfluidic channel branch structure 4. Under the action of the static pressure difference, the cell culture fluid 7 with a liquid level difference flows from one side of the microfluidic channel branch to the other side, which can promote the generation of a three-dimensional capillary network in the central tissue chamber 3, and thus construct a three-dimensional vascularized microtissue.

[0043] To construct a hierarchical vascular network structure with varying vessel sizes, the central tissue chamber 3 is first filled with a fibrin gel or collagen gel mixed with endothelial cells and fibroblasts. The gel is then polymerized in a 37°C incubator for 5 minutes. Culture fluid is then injected through the culture fluid infusion holes 10 on both sides, and an initial pressure drop is established to maintain the physiological fluid level required for angiogenesis. Each terminal vessel of the hierarchical microfluidic channel branching structure 4 is equipped with a bubble vent 8, which removes bubbles from the lumen during culture fluid injection.

[0044] In some embodiments, the upper surface of the glass sheet 2 and the lower surface of the polydimethylsiloxane module 1 are bonded together by oxygen plasma, and the hierarchical microfluidic channel branch structure 4 with different lumen diameters is constructed into a large blood vessel simulating a venous artery by an endothelial cell lining method.

[0045] The middle region of the lower surface of the polydimethylsiloxane module 1 and the glass sheet 2 form a hollow central tissue chamber 3. This central tissue chamber 3 is filled with a fibrin gel or collagen gel mixed with endothelial cells and fibroblasts. This provides space for the formation of a three-dimensional capillary network. In some embodiments, the thickness of the polydimethylsiloxane module 1 is 4-7 mm, determined by the frame height of the 3D-printed mold. In other embodiments, the thickness of the polydimethylsiloxane module 1 can also be of other dimensions. The central tissue chamber 3 is circular or rectangular in shape and 300-500 μm in height. The patterned structural design of the central tissue chamber 3 confines cells within a restricted area. The height of the central tissue chamber 3 determines the height of the 3D extracellular matrix, i.e., the space within which the three-dimensional capillary network can spontaneously form. A larger central tissue chamber 3 height allows for the formation of a more complex three-dimensional capillary network.

[0046] In some embodiments, the cross-section of the hierarchical microfluidic channel branch structure 4 is 3 / 4 circular, and the lumen radius of the hierarchical microfluidic channel branch structure 4 is 0.6-2.5 mm. The hierarchical microfluidic channel branch structure 4 with this structure and size can simulate the blood vessel structures of different size ranges in the human body. At the same time, the 3 / 4 circular structural design of the branch structure has a vascular bionic structure design and is convenient for bonding with glass. Lumens of different diameters have different flow rates under the same pressure, which is more in line with the real physiological state of the human vascular system.

[0047] The hierarchical microfluidic channel branch structure 4 is symmetrical about the central tissue chamber 3. The hierarchical microfluidic channel branch structures 4 on both sides are connected to the central tissue chamber 3 through a phase guide structure 5. Specifically, the phase guide structure 5 is a groove with a width of 150-250μm and a depth of 150-250μm. When the pressure at the interface between the phase guide structure 5 and the gel is greater than the minimum pressure required for the gel to move within the central tissue chamber 3 and less than the overflow pressure, the gel will not leak into adjacent channels due to pressure accumulation during injection. According to the Yong-Laplace equation, the width and height design of the phase guide structure 5 can ensure that the pressure of the gel moving within the central tissue chamber 3 is much lower than the overflow pressure, thereby reducing the possibility of gel leakage.

[0048] In the above embodiment, fibrin gel or collagen gel is injected into the central tissue chamber 3 through the gel perfusion hole 9 by capillary force, and the phase guide structure 5 prevents the patterned fibrin gel or collagen gel from flowing into the adjacent channels; after the fibrin gel or collagen gel solidifies, cell culture fluid 7 of different heights is injected from the culture fluid perfusion holes 10 on both sides. The pressure difference generated by the cell culture fluid 7 with a liquid level difference can stimulate the growth of blood vessels of the co-cultured cells in the fibrin gel.

[0049] Another embodiment of the present invention provides a method for preparing the aforementioned hierarchical vascularized microfluidic organ chip, the method comprising:

[0050] S1. Providing a glass slide and 3D printing a hierarchical vascularized microfluidic chip mold, wherein a resin material is 3D printed to form a microfluidic chip mold having a hierarchical microfluidic channel branch structure and a central tissue chamber structure;

[0051] S2. Provide bubble-free PDMS mold material, slowly pour the PDMS mold material into the mold, and then solidify the PDMS mold material;

[0052] S3, cutting the cured PDMS mold material from the mold to obtain a polydimethylsiloxane module; punching holes for gel infusion, culture medium infusion, and bubble exhaust ports on the polydimethylsiloxane module;

[0053] S4, performing oxygen plasma bonding on the glass sheet and the polydimethylsiloxane module;

[0054] S5. Provide a culture fluid reservoir, apply PDMS packaging material to the bottom of the culture fluid reservoir, and align it with the culture fluid perfusion hole to fix it;

[0055] S6. After the glass slide and the polydimethylsiloxane module are solidified, they are sterilized at high temperature and high pressure to obtain a hierarchical vascularized microfluidic organ chip.

[0056] In one embodiment, a hierarchical vascularized microfluidic organ chip is prepared using a resin material with a printing accuracy of 0.025 mm. A microfluidic chip mold with a hierarchical microfluidic channel branching structure and a central tissue chamber structure with different lumen diameters is prepared by 3D printing. The mold is then re-molded using a highly biocompatible PDMS material to obtain a PDMS module with a microstructure. The specific steps are as follows:

[0057] Reference Figure 3 The hierarchical vascularized microfluidic organ chip can be prepared by the following method, including the following steps:

[0058] S1, such as Figure 3 As shown in (a), the glass slide was cleaned; the glass slide was ultrasonically cleaned in acetone, ethanol, and water in this order for 3 minutes, and dried at 70°C after each cleaning.

[0059] like Figure 3 As shown in (b), according to the designed hierarchical vascular organ chip size, a hierarchical vascularized microfluidic organ chip mold was prepared by 3D printing using a resin material with a printing accuracy of 0.025 mm. The microfluidic chip mold has a hierarchical microfluidic channel branch structure and a central tissue chamber structure.

[0060] Soak the printed resin mold in deionized water and perform ultrasonic cleaning; dry the cleaned resin mold and then wipe it with alcohol for disinfection;

[0061] S2, such as Figure 3 As shown in Figure (c), PDMS prepolymer and curing agent are uniformly mixed in a mass ratio of 10:1. Vacuum the mixture in a moisture-proof vacuum pan to remove air bubbles, resulting in a PDMS mold material. This bubble-free PDMS mold material is then slowly poured into a resin mold to prevent air bubbles from forming during the pouring process. The PDMS mold material is then cured in a 90°C oven for 2 hours. In practice, the PDMS prepolymer and curing agent can be mixed in other ratios to adjust the hardness of the cured mold.

[0062] S3, such as Figure 3 As shown in (d), the cured PDMS overmold was cut from the resin mold with a scalpel to obtain a PDMS module. A PDMS biopsy punch was used to punch holes for gel infusion, culture medium infusion, and a vent for bleed air at the end of the blood vessel. Specifically, the two culture medium infusion holes had a diameter of 2 mm, the gel infusion hole had a diameter of 1 mm, and the vent for bleed air at the end of the blood vessel had a diameter of 1 mm. After punching, the holes were sealed with Scotch tape.

[0063] S4, such as Figure 3As shown in (e), the ultrasonically cleaned glass slide and the punched PDMS module were then oxygen plasma cleaned. The gas was ignited at 0.7 mbar for 45 seconds. The glass slide was then removed and used as the base layer for bonding. The microstructured side of the PDMS module was placed on the glass slide, and air bubbles between the two were gently pressed out with tweezers. The bonded glass slide and PDMS module were then heated on a 120°C hotplate for 2 hours to enhance the bonding effect.

[0064] S5, such as Figure 3 As shown in (f), PDMS prepolymer and curing agent are uniformly mixed in a ratio of 10:1, and bubbles in the mixture are removed in a vacuum pump to obtain PDMS packaging material; the bottom of a 1.8 ml cryovial is cut flat as a culture medium reservoir and wiped with alcohol for disinfection; the bottom of the disinfected cryovial is coated with PDMS packaging material, aligned with the culture medium perfusion hole, and placed on a 120°C hot plate and heated for 2 hours to ensure the firmness of the cryovial.

[0065] S6. Sterilize the cured glass sheet and PDMS module under high temperature and high pressure to obtain a hierarchical vascularized microfluidic organ chip.

[0066] The actual culture effect of the hierarchical vascularized microfluidic organ chip in the above embodiment is described below to demonstrate the feasibility of in vitro culture of a hierarchical perfusable vascular system consisting of endothelialized lumens and self-assembled capillary networks and its preparation process.

[0067] Continue to refer to Figure 2 The central tissue chamber of the hierarchical vascularized microfluidic organ chip was filled with fibrin gel. The cells contained in the fibrin gel were human umbilical vein endothelial cells (HUVEC) and human lung fibroblasts (NHLF), and the initial seeding concentration of both was 7×10 6 In a branched hierarchical microfluidic channel structure on both sides, the inner wall was first coated with laminin. Human umbilical vein endothelial cells (HUVEC) were injected through the culture medium perfusion hole. After incubation for 6 hours, the non-adherent HUVECs were washed away with cell culture medium.

[0068] like Figure 4 As shown, during the endothelial cell lining process, an Arduino-controlled stepper motor rotation device was developed to ensure uniform cell adhesion to the lumen wall. A 3D-printed scaffold was used to secure the vascularized chip. The rotation device rotated at regular intervals to ensure uniform cell adhesion. In this example, the incubation period was 3-4 hours, with 90° rotations every 15 minutes.

[0069] like Figure 5The figure shows a schematic diagram of the endothelial cell lining by the rotating device as described above, in which a high-density endothelial cell suspension is injected into the lumen, and the rotating device rotates at certain time intervals during the incubation process to ensure that the endothelial cells cover the entire lumen contour.

[0070] Reference Figure 6 As shown in the figure, the hierarchical vascularized microfluidic organ chip self-assembled capillary network after inoculation and at different growth days. The size of the central tissue chamber is 10mm×3mm, and the diameter of the culture medium perfusion hole is 2mm. Every two days, the culture medium in the reservoirs at both ends of the hierarchical microfluidic channel branch structure is sucked out and replaced with new culture medium. At the same time, in order to promote the formation of capillary network in the central tissue chamber, the culture medium on both sides needs to maintain a certain liquid level difference. Figure 6 As shown, on the second day of culture after the fibrin gel mixed with cells was injected into the central tissue chamber, HUVECs formed vascular fragments and initially connected under the induction of growth factors produced by NHLF; on the fourth day of culture, a three-dimensional capillary network lumen was formed; Figure 7 Shown is a fluorescent image of self-assembled blood vessels in the central tissue chamber on the 5th day of culture, in which a relatively thick lumen has been formed.

[0071] Reference Figure 8 Shown is a 10x magnified fluorescence image of the three-dimensional capillary network in the central tissue chamber on the 7th day of vascular network culture. The lumen diameter is 25-50 μm, providing a channel for the perfusion of drug particles.

[0072] Reference Figure 9 Figure 2 shows a fluorescent image of an endothelialized lumen formed by endothelial cells lining the hierarchical microfluidic channels on both sides on day 5 of vascular culture. The microfluidic channels were first coated with laminin, and then a high concentration of endothelial cells was introduced. A stepper motor was used to rotate the device to ensure uniform cell adhesion to the lumen wall. After 6 hours of incubation, non-adherent human umbilical vein endothelial cells were rinsed away with cell culture medium. Under the action of laminar shear stress of the cell culture medium, after 24 hours, the endothelial cells lining the hierarchical microfluidic channels on both sides proliferated and formed a monolayer, constructing a lumen lined with endothelial cells that simulates the large blood vessels of the venous artery.

[0073] In the above-described embodiment of the present invention, a microfluidic chip mold with a hierarchical vascular network structure and a central tissue chamber with different lumen diameters is first prepared by 3D printing. The mold is then re-molded using the biosafe PDMS material, and then bonded to a glass sheet via oxygen plasma, thereby achieving the preparation of a hierarchical vascularized microfluidic organ chip based on the 3D printing method. The chip is equipped with a hierarchical microfluidic channel branching structure with different lumen diameters. A three-dimensional capillary network is formed in the central tissue chamber through angiogenesis mechanisms, and a hierarchical vascular network structure with different vascular scales is constructed in vitro. This can highly simulate the complex cardiovascular system of the human body and provide an effective biological model for the study of vascularization-related diseases in vitro. The lumen diameter of the hierarchical microfluidic channel branching structure can be freely designed according to the specific application, achieving personalized design. The microfluidic organ chip in the embodiment of the present invention provides innovative application value in the field of biomedical engineering, especially for the construction of in vitro vascularized tumor disease models and the screening of new anti-tumor drugs.

[0074] The above describes specific embodiments of the present invention. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various modifications or variations within the scope of the claims without affecting the essence of the present invention. The above preferred features may be used in any combination as long as they do not conflict with each other.

Claims

1. A hierarchical vascularized microfluidic organ chip, characterized in that: include: A polydimethylsiloxane module, the lower surface of which is provided with a central tissue chamber and hierarchical microfluidic channel branch structures with different lumen diameters, wherein the hierarchical microfluidic channel branch structures are symmetrical about the central tissue chamber; The hierarchical microfluidic channel branch structure is connected to the central tissue chamber via a phase guiding structure; A glass sheet, wherein the upper surface of the glass sheet is integrated with the lower surface of the polydimethylsiloxane module, and the hierarchical microfluidic channel branch structure is constructed into a large blood vessel simulating a venous artery by an endothelial cell lining method; Gel perfusion holes are provided at both ends of the central tissue chamber, and liquid reservoirs and culture fluid perfusion holes are provided at both ends of the hierarchical microfluidic channel branch structure. The culture fluid perfusion holes are connected to the liquid reservoirs, and cell culture fluids of different heights are injected from the liquid reservoirs at both ends. Under the action of static pressure difference, the cell culture fluids with liquid level height difference flow from the hierarchical microfluidic channel branch structure on one side to the hierarchical microfluidic channel branch structure on the other side, thereby stimulating the formation of a capillary network in the central tissue chamber.

2. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: The glass sheet and the polydimethylsiloxane module are bonded into one body through oxygen plasma.

3. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: Each final blood vessel of the hierarchical microfluidic channel branch structure is provided with a bubble discharge port, and bubbles in the lumen are discharged from the bubble discharge port when the culture fluid is injected.

4. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: The cross section of the hierarchical microfluidic channel branch structure is 3 / 4 circular.

5. The hierarchical vascularized microfluidic organ chip according to claim 4, characterized in that: The lumen radius of the hierarchical microfluidic channel branch structure is 0.6-2.5 mm.

6. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: The central tissue chamber is circular or rectangular in shape.

7. The hierarchical vascularized microfluidic organ chip according to claim 6, characterized in that: The height of the central tissue chamber is 300-500 μm.

8. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: The thickness of the polydimethylsiloxane module is 4-7 mm.

9. The hierarchical vascularized microfluidic organ chip according to claim 1, characterized in that: The phase-guiding structure has a width of 150-250 μm and a height of 150-250 μm.

10. A method for preparing a hierarchical vascularized microfluidic organ chip according to any one of claims 1 to 9, characterized in that: include: Providing a glass sheet, and using a resin material to be 3D printed into a microfluidic chip mold having a hierarchical microfluidic channel branch structure and a central tissue chamber structure; Providing a bubble-free PDMS mold material, slowly pouring the PDMS mold material into the mold, and then curing the PDMS mold material; Cutting the solidified PDMS mold material from the mold to obtain a polydimethylsiloxane module; Punching the gel perfusion hole, the culture medium perfusion hole, and the bubble exhaust port of the polydimethylsiloxane module; performing oxygen plasma bonding on the glass sheet and the polydimethylsiloxane module; Provide a culture fluid reservoir, apply PDMS packaging material to the bottom of the culture fluid reservoir, and align it with the culture fluid perfusion hole to fix it; After the glass sheet and the polydimethylsiloxane module are cured, they are sterilized at high temperature and high pressure to obtain a hierarchical vascularized microfluidic organ chip.

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