Method for constructing multi-organ cascade chip model of cerebral lymphatic circulation return system and model

Abstract

The application belongs to the technical field of organ chip, and proposes a brain lymphatic circulation reflux system multi-organ cascade chip model construction method and model, which adjusts the size of the channel inlet and outlet of the brain lymphoid chip to be consistent with the size of the middle channel inlet of the meningeal lymphatic chip; adjusts the size of the middle channel outlet of the meningeal lymphatic tube chip to be consistent with the size of the channel inlet of the deep cervical lymph node chip; uses a catheter to connect the corresponding outlets and inlets of the brain lymphoid chip, the meningeal lymphatic tube chip and the deep cervical lymph node chip, realizes the series connection of the three chips, the flow process is consistent with the cerebrospinal fluid lymphatic circulation reflux process, fills the gap that the cerebrospinal fluid lymphatic circulation reflux process cannot be realized in vitro before; and the brain lymphoid chip, the meningeal lymphatic tube chip and the deep cervical lymph node chip are cascaded on one substrate, the actual size of the three chips is reduced, and the cascade operation of the multi-chip is more convenient.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of organ chip, and particularly relates to a brain lymphatic circulation reflux system multi-organ cascade chip model construction method and model. BACKGROUND

[0002] The cerebrospinal fluid diffuses to the parenchyma through the perivascular space of the brain by the boundary of glial cells, and is finally drained into the deep cervical lymph node through the perivascular space of the brain and the meningeal lymphatic vessels. The research on the brain lymphatic circulation reflux process of cerebrospinal fluid is still in its infancy, and the existing animal chip model has problems such as long modeling time, low research efficiency, and differences from the human body response, and cannot be applied to the research on the brain lymphatic circulation reflux process of cerebrospinal fluid.

[0003] At present, various structures of microfluidic organ chips have been developed, which have certain limitations in imaging depth, and it is difficult to realize the simulation of the complex multi-organ brain lymphoid system-meningeal lymphatic vessels-deep cervical lymph nodes. The network vascular structure chip based on gel formation is easy to damage the vascular network structure in the stable flow process of cerebrospinal fluid. The left and right structure chip separated by microchannels / microcolumns is suitable for the cascade of multi-organ chips. However, the specific size and internal structure of the existing left and right structure chip on the market are not suitable for the simulation of the more complex and larger brain lymphoid system-meningeal lymphatic vessels-deep cervical lymph nodes. At the same time, due to the differences in structure and size of different chips, it is difficult to meet the smooth flow of cerebrospinal fluid in different chips. SUMMARY

[0004] In order to solve the above problems, the application provides a brain lymphatic circulation reflux system multi-organ cascade chip model construction method and model. The size of the channel inlet and outlet of the brain lymphoid chip is adjusted to be consistent with the size of the middle channel inlet of the meningeal lymphatic chip. The size of the middle channel outlet of the meningeal lymphatic vessel chip is adjusted to be consistent with the size of the channel inlet of the deep cervical lymph node chip. The corresponding outlets and inlets of the brain lymphoid chip, the meningeal lymphatic vessel chip and the deep cervical lymph node chip are connected by a catheter to realize the series connection of the three chips. The flow process is consistent with the brain lymphatic circulation reflux process of cerebrospinal fluid, filling the gap that the brain lymphatic circulation reflux process of cerebrospinal fluid cannot be realized in vitro before. The brain lymphoid chip, the meningeal lymphatic vessel chip and the deep cervical lymph node chip are cascaded on one substrate, which reduces the actual size of the three chips and makes the cascade operation of multiple chips more convenient.

[0005] In order to achieve the above purpose, in a first aspect, the application provides a brain lymphatic circulation reflux system multi-organ cascade chip model construction method, which adopts the following technical scheme:

[0006] A brain lymphatic circulation reflux system multi-organ cascade chip model construction method, comprising:

[0007] Preparation of the brain lymphoid chip, the meningeal lymphatic chip and the deep cervical lymph node chip;

[0008] The size of the channel inlet and outlet of the brain lymphoid chip is adjusted to be consistent with the size of the intermediate channel inlet of the meningeal lymphatic chip; the size of the intermediate channel outlet of the meningeal lymphatic chip is adjusted to be consistent with the size of the channel inlet of the deep cervical lymph node chip; the corresponding outlets and inlets of the brain lymphoid chip, the meningeal lymphatic chip and the deep cervical lymph node chip are connected by a catheter;

[0009] The brain lymphoid chip, the meningeal lymphatic chip and the deep cervical lymph node chip are cascaded on a substrate.

[0010] Further, the brain lymphoid chip, the meningeal lymphatic chip and the deep cervical lymph node chip each comprise a substrate and a polydimethylsiloxane layer bonded by a silicon-oxygen bond.

[0011] Further, the brain lymphoid chip comprises an arterial blood vessel channel, an arterial perivascular space channel, a brain parenchymal channel, a venous blood vessel channel and a venous perivascular space; the meningeal lymphatic chip comprises two culture medium channels and an intermediate channel for simulating meningeal lymphatic vessels by adherent culture of lymphatic endothelial cells; the deep cervical lymph node chip is designed as an elliptical channel and comprises at least an intermediate channel for culture of lymphocytes.

[0012] Further, human umbilical arterial endothelial cells and human umbilical venous endothelial cells are injected into the blood vessel channels on both sides of the brain lymphoid part, and lymphatic endothelial cells are injected into the meningeal lymphatic part, and then cell culture is performed until the cells adhere to the top surface of the channels; human umbilical arterial endothelial cells, human umbilical venous endothelial cells and lymphatic endothelial cells are added into the corresponding channels again, the chip is turned over and cell culture is performed until the cells adhere to the top surface of the channels.

[0013] Further, brain parenchymal cell gel mixed solution and lymphocyte gel mixed solution are prepared respectively and are introduced into the intermediate brain parenchymal channel of the brain lymphoid part and the intermediate circular channel of the deep cervical lymph node part respectively, and cell culture is performed to complete gel solidification.

[0014] Further, four transparent catheters with equal lengths are taken, the two ends of the first transparent catheter are connected to a syringe pump and the inlet of the arterial perivascular space of the meningeal lymphatic part respectively, the two ends of the second transparent catheter are connected to the outlet of the venous perivascular space and the inlet of the intermediate channel of the meningeal lymphatic part respectively, the two ends of the third transparent catheter are connected to the outlet of the intermediate channel of the meningeal lymphatic part and the inlet of the intermediate circular channel of the deep cervical lymph node respectively, and the two ends of the fourth transparent catheter are connected to the outlet of the intermediate circular channel of the deep cervical lymph node and the inlet of a liquid storage pool respectively.

[0015] Further, the culture medium is pushed to flow at a flow rate of 1 μL / min, and the culture medium flows out in the order of the periarterial space, the brain parenchymal pathway, the perivenous space pathway, the meningeal lymphatic vessel pathway, and the cervical lymph node pathway, and is temporarily stored in the reservoir.

[0016] Further, when the culture medium is continuously perfused in the two arterial pathways and the venous pathway of the brain lymphoid chip, the culture medium is perfused in a cycle of 1 s at a flow rate of 1 μL / min.

[0017] To achieve the above object, in a second aspect, the present application further provides a brain lymphatic circulation reflux system multi-organ cascade chip model, which adopts the following technical scheme:

[0018] The brain lymphatic circulation reflux system multi-organ cascade chip model is obtained by the brain lymphatic circulation reflux system multi-organ cascade chip model construction method of the first aspect, and comprises a cascade of a lymphoid chip, a meningeal lymphatic vessel chip, and a cervical deep lymph node chip.

[0019] Further, the brain lymphoid chip comprises an arterial blood vessel pathway, a periarterial space pathway, a brain parenchymal pathway, a venous blood vessel pathway, and a perivenous space; the meningeal lymphatic vessel chip comprises two culture medium pathways and an intermediate pathway for simulating a meningeal lymphatic vessel by adherent culture of lymphatic endothelial cells; and the cervical deep lymph node chip is designed as an elliptical pathway and comprises at least an intermediate pathway for lymphocyte culture.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] In the present application, the size of the inlet and outlet of the pathway of the brain lymphoid chip is adjusted to be consistent with the size of the inlet of the intermediate pathway of the meningeal lymphatic vessel chip; the size of the outlet of the intermediate pathway of the meningeal lymphatic vessel chip is adjusted to be consistent with the size of the inlet of the pathway of the cervical deep lymph node chip; the corresponding outlet and inlet of the brain lymphoid chip, the meningeal lymphatic vessel chip, and the cervical deep lymph node chip are connected by a catheter, thereby realizing the series connection of the three chips, the flow process is consistent with the brain cerebrospinal fluid lymphatic circulation reflux process, and the blank of the brain cerebrospinal fluid lymphatic circulation reflux process in vitro is filled; and the brain lymphoid chip, the meningeal lymphatic vessel chip, and the cervical deep lymph node chip are cascaded on one substrate, thereby reducing the actual size of the three chips and facilitating the cascade operation of the multiple chips. BRIEF DESCRIPTION OF DRAWINGS

[0022] The drawings constituting a part of this embodiment are used to provide a further understanding of the embodiment, and the schematic embodiment and its description are used to explain the embodiment, and do not constitute an improper limitation on the embodiment.

[0023] Figure 1 It is a size chart of the first-generation microfluidic chip structure design chart of the present application.

[0024] Figure 2 Microscopic bright field image of the first generation microfluidic chip of the present application;

[0025] Figure 3 Live-dead cell imaging image of the first generation microfluidic chip of the present application;

[0026] Figure 4 Second generation integrated chip structure design and cerebrospinal fluid circulation and reflux schematic diagram and tracer flow fluorescence imaging image in the chip. DETAILED DESCRIPTION

[0027] The present application is further described below in conjunction with the accompanying drawings and examples.

[0028] It should be noted that the following detailed description is exemplary in nature and is intended to provide further description of the application. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0029] The cerebrospinal fluid (CSF) dynamics in the brain plays an important role in the maintenance of the central nervous system homeostasis. The changes of biomolecules in the CSF during the drainage process are closely related to the normal development of neural cells, hydrocephalus and cerebral ischemia, aging and related neurodegenerative diseases, etc. For example, the metabolic waste and toxic proteins in the brain can be removed through the efflux of CSF, ensuring the normal physiological activities of the central nervous system.

[0030] The traditional view generally believes that the biomolecules in the CSF are mainly removed through the arachnoid granulations into the brain meningeal sinuses, the nasal mucosa lymphatic vessels into the deep cervical lymph nodes, or the transporters and receptors at the top of the choroid plexus epithelium. However, in recent years, it has been found that the CSF can flow into the meningeal lymphatic vessels to remove the CSF and biomolecules in the CSF through the brain lymphatic pathway. In addition, with the deepening understanding of the newly discovered brain lymphoid system in recent years, the intracerebral lymphatic outflow process of the CSF has also been basically elucidated: the CSF in the perivascular space diffuses to the parenchyma through the boundary of glial cells, and is removed to the meningeal lymphatic vessels through the perivascular space, and finally flows into the deep cervical lymph nodes. However, the research on the brain lymphatic circulation and reflux process of the CSF is still in its infancy, and the existing animal models have problems such as long modeling time, low research efficiency, and differences from the human body response, etc. Therefore, it is urgent to develop more efficient and simulated animal alternative models.

[0031] At present, various structures of microfluidic organ chips have been developed, including upper and lower structures separated by porous membranes, left and right structures separated by microchannels / microcolumns, and network vascular structures. The upper and lower structure chip has certain limitations in imaging depth, and it is relatively difficult to realize the simulation of complete brain lymphoid system-meningeal lymphatic vessels-deep cervical lymph nodes. The network vascular structure chip is formed based on gel, and the stable flow of cerebrospinal fluid can easily destroy the vascular network structure. The left and right structure chip has low imaging depth, and it is easy to realize the simulation of complex multi-organ chip through the design of multiple parallel structures, and it can realize the stable driving of liquid through high-precision injection pump, which is more suitable for multi-organ chip cascade. However, the specific size and internal structure of the existing left and right structure chip on the market are not suitable for more complex and larger brain lymphatic system-meningeal lymphatic vessels-deep cervical lymph node simulation. At the same time, due to the differences in structure and size of different chips, it is difficult to meet the smooth flow of cerebrospinal fluid in different chips.

[0032] In order to solve at least one of the above problems, the present application provides a brain lymphatic circulation reflux system multi-organ cascade chip model construction method, which can be used for the cascade of multiple complex organ chips and the co-culture of different in vitro organs. First, three microfluidic chips with different structures are designed according to the real physiological structure of brain lymphoid system-meningeal lymphatic vessels-deep cervical lymph nodes, a multi-organ microfluidic chip in vitro model construction method is developed, and in vitro organ chip models of brain lymphoid system, meningeal lymphatic vessels and deep cervical lymph nodes are constructed. In order to further improve the portability and research efficiency of the product and realize the in vitro reproduction of cerebrospinal fluid circulation reflux, the product is upgraded and integrated, making the product operation more flexible and convenient, and being able to meet the cascade between different chips, which is conducive to the development of commercial products, and the integrated chip can realize the liquid flow across the chip conveniently, filling the gap of the previous inability to realize the cerebrospinal fluid lymphatic circulation reflux process in vitro.

[0033] Example 1:

[0034] As Figure 1 and Figure 2As shown, the present embodiment provides a chip model construction method for simulating a complete brain lymphoid system-meningeal lymphatic vessel-cervical deep lymph node. The multi-organ chip is a first-generation product. The multi-organ in vitro model mainly includes three parts of a brain lymphoid chip, a meningeal lymphatic vessel chip and a cervical deep lymph node chip. The three chips are all sealed by bonding a glass substrate with high light transmittance and a polydimethylsiloxane (PDMS) layer through a siloxane bond to form a complete microfluidic chip. Among them, the microfluidic channel is imprinted on the PDMS layer through soft lithography technology. Three kinds of microfluidic chips are designed according to the real physiological structure of different organs. The brain lymphoid chip is composed of five parallel straight channels, which respectively simulate the arterial blood vessels, the arterial surrounding space, the brain parenchyma, the venous blood vessels and the venous surrounding space. The meningeal lymphatic vessel is composed of three parallel straight channels, including two culture medium channels for system balance, and the middle channel for simulating the meningeal lymphatic vessel by adherent culture of lymphatic endothelial cells. The cervical deep lymph node channel is designed as an elliptical channel according to the shape and cell composition of the real lymph node. The middle channel is used for lymphocyte culture, and the lymphocyte response can be investigated in the later stage to evaluate the function of lymphatic circulation. In order to restore the mechanical stimulation in the above organs, the blood flow stimulation simulation is realized by a high-precision syringe pump in the arterial and surrounding channels of the brain lymphoid chip, and the cerebrospinal fluid flow simulation is realized by the same way in the lymphatic vessel channel of the meningeal lymphatic vessel chip.

[0035] The specific method and steps are as follows:

[0036] S101, brain lymphoid chip:

[0037] S1011, chip pretreatment.

[0038] First, inject deionized water into all channels of the chip with a syringe, rinse three times, rinse with anhydrous ethanol three times, and then place the chip under ultraviolet light overnight for disinfection and sterilization after observing that there is no impurity in the channel through a microscope. Before injecting cells, rinse the microfluidic channel with PBS buffer solution and pass 100 μg / mL of a mixture of fibronectin and collagen IV into a 37°C cell incubator to stand overnight to complete the chip pretreatment.

[0039] S1012, cell culture.

[0040] First, rinse all channels of the chip with cell culture medium, then pass 1×10 6 6 ​Human umbilical artery endothelial cells and human umbilical vein endothelial cells were collected at a density of 100 cells / mL. The chip was inverted and placed in a cell culture incubator for 6 hours until the cells adhered to the top surface of the channels. Fresh culture medium was injected into the two channels every 6 hours using a syringe pump. After the four inner walls of the channels were confluent with cells, the brain parenchymal cells were injected into the middle channel. First, under ice bath conditions, 100 μL of 10×PBS buffer was added to 200 μL of 0.1mol / L NaOH solution and mixed thoroughly. Then, 500 μL of 10 mg / mL rat tail type I collagen was added and quickly mixed. The solution was then stored at 4°C. Astrocytes, neurons, and microglia were digested with trypsin and resuspended in EP tubes and mixed thoroughly. The cell density was adjusted to 1×10⁻⁶ cells / mL. 5 After achieving a cell-to-mL ratio, 200 μL of cell suspension was added to the pre-prepared rat tail type I collagen mixture and mixed thoroughly. Next, the cell-gel mixture was injected into the brain parenchyma channel using a syringe. The chip was then placed in a 37°C cell culture incubator and allowed to stand for 1 hour. The cells then grew in three dimensions on the solidified gel scaffold.

[0041] S1013, Multi-cell co-culture under flow stimulation conditions.

[0042] The culture medium was continuously perfused into the arterial and venous channels on both sides using a high-precision syringe pump at a flow rate of 1 μL / min, creating a mechanical stimulation similar to blood flow. The growth status of all cells in the chip was observed to confirm the cell growth status in the brain parenchyma and vascular channels until a complete monolayer of endothelial cells and a relatively complete in vitro lymphoid model were formed.

[0043] In some embodiments of the brain-like lymphatic system, the blood flow velocity in the arteries is not uniform but rather fluctuates periodically due to the contraction and relaxation of the heart, generating pulsatile blood flow. Simultaneously, this pulsatile fluctuation pumps the solution in the periarterial spaces to the brain tissue and perivenous spaces, propelling the overall fluid flow and completing the drainage of cerebrospinal fluid and brain parenchymal metabolic products. Therefore, due to the unique characteristics of fluid flow in the brain-like lymphatic system, arterial fluid flow cannot be simulated solely by a single flow velocity. During perfusion, by adjusting the flow rate of the infusion pump, the fluid in the arterial channel is perfused at a rate of 1 μL / min to achieve a volume of 60 nL, with the process repeated every 1 second, thus achieving intermittent fluid flow similar to arterial fluctuations.

[0044] S102, Meningeal Lymphatic Vessel Chip:

[0045] After completing the chip preprocessing process using the same method, 1×10⁻⁶ ohms is introduced into the intermediate channel. 6Cells / mL of human lymphoendothelial cells were added to the microfluidic chip, and the chip was incubated for 6 hours to allow the cells to adhere to the bottom of the chip. Then, 1×10⁶ cells / mL were added to the aforementioned channels. 6 The chip was inverted and placed in a cell culture incubator for 6 hours until the cells adhered to the top surface of the channel. The culture medium was continuously perfused into the middle of the channel using a high-precision syringe pump at a flow rate of 1 μL / min. Microscopic observation was performed until all four inner walls of the channel were fully covered with cells, thus completing the in vitro construction of the meningeal lymphatic vessel model. Similarly, in some embodiments, by adjusting the syringe pump flow rate, the fluid in the arterial channel was perfused at a flow rate of 1 μL / min to achieve a volume of 60 nL, with the process repeated every 1 second, achieving intermittent fluid flow similar to arterial pulsations.

[0046] S103, Deep Cervical Lymph Node Chip:

[0047] Culture medium was added to both channels of a three-channel microfluidic chip with circular channels to equilibrate the entire system. Peripheral circulating mononuclear cells (PBMCs) were separated by density centrifugation. B cells and T cells were sorted using antibody-labeled magnetic beads, and the cell density was adjusted to 1 × 10⁻⁶ cells / cells. 5 After passing through mL, 200 μL of 1×10⁻⁶ cells / mL was added under ice bath conditions. 5 A mixture of B and T cell suspensions (number of cells / mL) and rat tail type I collagen (100 μL 10×PBS buffer, 200 μL 0.1 mol / L NaOH solution, 500 μL 10 mg / mL rat tail type I collagen) was thoroughly mixed. The mixture was then injected into the central circular channel using a syringe. The chip was placed in a 37°C cell culture incubator and incubated for 1 hour. Cells then grew three-dimensionally on the solidified gel scaffold. Fresh culture medium was subsequently injected into the chip every 6 hours, and the growth status of all cells was observed until a complete in vitro deep cervical lymph node model was formed.

[0048] Example 2:

[0049] Based on Example 1, this example provides a method for constructing a multi-organ cascade chip model of the cerebral lymphatic circulation reflux system, realizing in vitro simulation of the cerebrospinal fluid circulation reflux process. This device is a second-generation product.

[0050] The simulation of the brain lymphoid system, meningeal lymphatic vessels and deep cervical lymph nodes was realized in the first product of Example 1, but the in-vitro reproduction of the circulation and reflux of cerebrospinal fluid needs to be realized through the cascade of three chips. In the brain, cerebrospinal fluid passes through the perivascular space, brain parenchyma, perivascular space, meningeal lymphatic vessels and finally flows into the cervical lymph nodes. The size of the two blood vessel perivascular space channels of the brain lymphoid chip in the first product is 2 mm, which is difficult to match the size of the outlet and inlet of the meningeal lymphatic vessel chip. Therefore, first, the outlet and inlet of the brain lymphoid chip channel in the first product are adjusted to meet the cascade between different chips and realize the penetration of cerebrospinal fluid in different chips. The outlet and inlet of the brain lymphoid chip are adjusted to 800 μm, which is consistent with the inlet of the intermediate endothelial channel of the meningeal lymphatic vessel, and at the same time, the outlet size of the intermediate channel of the meningeal lymphatic vessel chip is consistent with the inlet size of the downstream deep cervical lymph node chip, so that the cascade of the three chips can be realized through the corresponding size of the catheter. In addition, in order to further improve the portability and operation convenience of the chip, the three chips of the second product are cascaded on a glass substrate, which greatly reduces the actual size of the three chips and makes the cascade operation of multiple chips more convenient.

[0051] The specific method and steps of the second product are as follows:

[0052] S201, in-vitro model construction of brain lymphoid system-meningeal lymphatic vessels-deep cervical lymph nodes: the three parts of the integrated model can be constructed at the same time, which shortens the construction time of the three independent chips in the first product and improves the experimental efficiency.

[0053] S2011, first, 1×10 6 human umbilical artery endothelial cells and human umbilical vein endothelial cells are injected into the blood vessel channels on both sides of the brain lymphoid part, and 1×10 6 lymphatic endothelial cells are injected into the meningeal lymphatic vessel part, and the chip is placed in a cell incubator for 6 hours until the cells adhere to the top surface of the channel. Human umbilical artery endothelial cells, human umbilical vein endothelial cells and lymphatic endothelial cells are added to the above channels again, the chip is turned over and placed in a cell incubator for 6 hours until the cells adhere to the top surface of the channel.

[0054] S2012, under ice bath conditions, brain parenchyma cell gel mixed solution (100 μL 10×PBS buffer solution, 200 μL 0.1 mol / L NaOH solution, 500 μL 10 mg / mL rat tail type I collagen, 200 μL 1×10 5100 μL 10×PBS buffer solution, 200 μL 0.1 mol / L NaOH solution, 500 μL 10 mg / mL mouse tail type I collagen, 200 μL 1×10 5 100 μL 10×PBS buffer solution, 200 μL 0.1 mol / L NaOH solution, 500 μL 10 mg / mL mouse tail type I collagen, 200 μL 1×10

[0055] S202, cerebrospinal fluid circulation backflow process in vitro simulation: four equal length of translucent catheter, the first section of the transparent catheter two ends are connected with high precision syringe pump and brain lymphatic part of the arterial surrounding gap entrance, the second section of the transparent catheter two ends are connected with brain lymphatic part of the venous surrounding gap exit and meningeal lymphatic vessel part of the intermediate channel entrance, the third section of the transparent catheter two ends are connected with meningeal lymphatic vessel part of the intermediate channel exit and cervical deep lymph node intermediate circular channel entrance, the fourth section of the transparent catheter two ends are connected with cervical deep lymph node intermediate circular channel exit and storage pool entrance. With high precision syringe pump to push the culture medium flow at the flow rate of 1 μL / min, the culture medium flows out in the order of arterial surrounding gap, brain parenchymal channel, venous surrounding gap channel, meningeal lymphatic vessel channel and cervical lymph node channel, and is temporarily stored in the storage pool. Subsequently, the culture medium in the storage pool can be directly used for real-time analysis of protein, cytokine, metal ion and other substances. More importantly, the flow process is consistent with the cerebrospinal fluid lymphatic circulation backflow process, filling the gap that the cerebrospinal fluid lymphatic circulation backflow process cannot be realized in vitro before.

[0056] In summary, as shown in Figure 3 and Figure 4 The organ chip disclosed by the application first realizes the complete simulation of the complex brain lymphatic-meningeal lymphatic vessel-cervical deep lymph node brain lymphatic drainage system through three different structures of the chip design, and then realizes the simulation of the lymphatic circulation backflow process by adjusting the sizes of the outlets and inlets of the three organ chips and cascading the three chips on a glass substrate to construct an integrated brain lymphatic organ chip. Compared with the existing organ chip, the organ chip can solve the problem that the existing microfluidic chip is not suitable for the construction of the more complex brain lymphatic system, and the integrated chip design improves the convenience of operating the organ chip, shortens the construction time of the in vitro model, and improves the experimental efficiency. In addition, the integrated organ chip provides a new method for the in vitro simulation of other organs containing liquid flow process.

[0057] The above merely provides preferred embodiments of the present embodiment, but is not intended to limit the present embodiment. For those skilled in the art, the present embodiment can have various modifications and variations. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present embodiment shall fall into the scope of protection of the present embodiment.

Claims

1. A method for constructing a multi-organ cascade chip model of brain lymphatic circulation backflow system, characterized in that, The method comprises the following steps: Preparation of brain lymphoid chip, meningeal lymphatic vessel chip and deep cervical lymph node chip; Adjusting the size of the channel entrance and exit of the brain lymphoid chip to be consistent with the size of the middle channel entrance of the meningeal lymphatic vessel chip; adjusting the size of the middle channel exit of the meningeal lymphatic vessel chip to be consistent with the size of the channel entrance of the deep cervical lymph node chip; Connecting the corresponding exit and entrance of the brain lymphoid chip, meningeal lymphatic vessel chip and deep cervical lymph node chip by using a catheter; Cascading the brain lymphoid chip, meningeal lymphatic vessel chip and deep cervical lymph node chip on a substrate; Taking four equal-length transparent catheters, connecting the two ends of the first transparent catheter to an injection pump and the entrance of the brain lymphoid part of the arterial surrounding space respectively, connecting the two ends of the second transparent catheter to the exit of the venous surrounding space and the entrance of the meningeal lymphatic vessel part of the middle channel respectively, connecting the two ends of the third transparent catheter to the exit of the meningeal lymphatic vessel part of the middle channel and the entrance of the deep cervical lymph node part of the middle circular channel respectively, and connecting the two ends of the fourth transparent catheter to the exit of the deep cervical lymph node part of the middle circular channel and the entrance of the liquid storage pool respectively; pushing the culture medium to flow at a flow rate of 1 μL / min, and the culture medium flows out in the order of the arterial surrounding space, the brain parenchymal channel, the venous surrounding space channel, the meningeal lymphatic vessel channel and the cervical lymph node channel, and is temporarily stored in the liquid storage pool; When the culture medium is continuously perfused in the arterial channel and the venous channel of the brain lymphoid chip, the culture medium is circulated and perfused at an interval of 1 s at a flow rate of 1 μL / min.

2. The method of claim 1, wherein the brain lymphatic circulation backflow system multi-organ cascade chip model is constructed by using a microfluidic chip. The brain lymphoid chip, meningeal lymphatic vessel chip and deep cervical lymph node chip each comprise a substrate and a polydimethylsiloxane layer which are bonded and sealed by a siloxane bond.

3. The method for constructing a multi-organ cascade chip model of the brain lymphatic circulation return system as described in claim 1, characterized in that, The brain lymphoid chip comprises an arterial blood vessel channel, an arterial surrounding space channel, a brain parenchymal channel, a venous blood vessel channel and a venous surrounding space; the meningeal lymphatic vessel chip comprises two culture medium channels and a middle channel for simulating the meningeal lymphatic vessel by adherent culture of lymphatic endothelial cells; the deep cervical lymph node chip is designed as an elliptical channel and comprises at least a middle channel for culture of lymphocytes.

4. The method for constructing a multi-organ cascade chip model of the brain lymphatic circulation reflux system as described in claim 3, characterized in that, Human umbilical artery endothelial cells and human umbilical vein endothelial cells are respectively injected into the two blood vessel channels of the brain lymphoid part, and lymphatic endothelial cells are injected into the meningeal lymphatic vessel part, and then cell culture is performed until the cells adhere to the top surface of the channel; human umbilical artery endothelial cells, human umbilical vein endothelial cells and lymphatic endothelial cells are respectively added to the corresponding channels again, the chip is turned over and cell culture is performed until the cells adhere to the top surface of the channel.

5. The method for constructing a multi-organ cascade chip model of the brain lymphatic circulation reflux system as described in claim 4, characterized in that, Brain parenchymal cell gel mixture and lymphocyte gel mixture are respectively prepared and introduced into the middle brain parenchymal channel of the brain lymphoid part and the middle circular channel of the deep cervical lymph node part, and cell culture is performed to complete gel solidification.

6. A multi-organ cascade chip model of the cerebral lymphatic circulation return system, characterized in that, The brain lymphoid chip, meningeal lymphatic vessel chip and deep cervical lymph node chip obtained by the method for constructing a multi-organ cascading chip model of a brain lymphatic circulation reflux system according to any one of claims 1-5.

7. The cerebral lymphatic circulation backflow system multi-organ cascade-chip model according to claim 6, wherein, The brain lymphoid core chip comprises an arterial blood vessel channel, an arterial perivascular space channel, a brain parenchymal channel, a venous blood vessel channel and a venous perivascular space; the meningeal lymphatic vessel chip comprises two culture medium channels and an intermediate channel for simulating meningeal lymphatic vessels by adherent culture of lymphatic endothelial cells; and the deep cervical lymph node chip is designed as an elliptical channel and comprises at least an intermediate channel for lymphocyte culture.

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