In-vitro brain lymphatic system chip construction method and brain lymphatic system chip

Abstract

The invention belongs to the technical field of organ chips, provides an in-vitro brain lymphatic system chip construction method and a brain lymphatic system chip, and makes up the blank of brain lymphatic system in-vitro model construction. The method comprises the following steps: firstly, carrying out flushing treatment, disinfection and sterilization treatment, rinsing treatment and cell culture on a micro-fluidic channel in the chip; then, in the treated micro-fluidic channel, blood vessel channel cell inoculation and brain parenchyma channel cell inoculation are carried out; and finally, in the blood vessel channel, perfusion is carried out once at a preset flow speed every preset duration, shear force stimulation simulating pulse type blood flow is provided, basic formation of in-vitro cerebral vessels is achieved after continuous perfusion, perfusion is carried out once at a preset flow speed every preset duration, shear force stimulation simulating pulse type blood flow is provided, and in-vitro cerebral vessels are formed. Intermittent liquid flow simulating arterial fluctuation is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of organ chips, and in particular relates to a method for constructing an in vitro brain lymphatic system chip and a brain lymphatic system chip. Background Art

[0002] A variety of brain organ-on-chips have been developed, including blood-brain barrier chips, neurovascular unit organ-on-chips, and neurodegenerative disease chips, for applications in drug evaluation, metabolomics research, viral invasion, and bacterial infection studies. However, methods for constructing in vitro models of the recently discovered brain lymphatic system remain elusive.

[0003] In the brain lymphatic system, the blood flow velocity in the arteries is not uniform, but is affected by the contraction and relaxation of the heart, showing periodic fluctuations and generating pulsatile blood flow. At the same time, this pulsatile fluctuation pumps the solution in the periarterial space to the brain tissue and the perivenous space, promoting the overall flow of the fluid and completing the drainage of cerebrospinal fluid and brain parenchymal metabolic products. Therefore, due to the special characteristics of the fluid flow in the brain lymphatic system, the construction of an in vitro model of the brain lymphatic system is restricted. In addition, the problem of cell migration crosstalk between different channels in the chip also restricts the construction of an in vitro model of the brain lymphatic system. Summary of the Invention

[0004] In order to solve the above problems, the present invention proposes an in vitro brain lymphatic system chip construction method and a brain lymphatic system chip, which fills the gap in the construction of an in vitro brain lymphatic system model; during the continuous perfusion of the vascular channel, the culture medium of the brain parenchymal channel is replaced at intervals; through the co-culture of multiple cells in the vascular channel and the brain parenchymal channel, an in vitro lymphatic model is formed through direct contact between different cells or the transmission of signal molecules; perfusion is performed at a preset flow rate and a preset time interval, providing shear force stimulation that simulates pulsatile blood flow, thereby realizing intermittent liquid flow that simulates arterial fluctuations.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing an in vitro brain lymphatic system chip, which adopts the following technical solutions: A method for constructing an in vitro brain lymphatic system chip, comprising: The microfluidic channels in the chip are flushed, sterilized, rinsed and cultured with cells; In the treated microfluidic channels, vascular channel cell seeding and brain parenchymal channel cell seeding were performed; In the vascular channel, perfusion is performed at a preset flow rate and at intervals of preset duration, providing shear force stimulation that simulates pulsatile blood flow. After continuous perfusion, the formation of in vitro cerebral blood vessels is achieved. During the continuous perfusion of the vascular channel, the culture medium of the brain parenchyma channel is replaced at intervals. By co-culturing multiple cells in vascular channels and brain parenchymal channels, an in vitro lymphoid model is formed through direct contact between different cells or the transmission of signal molecules.

[0006] Furthermore, the microfluidic channels in the chip were rinsed with deionized water and ethanol respectively. After there were no impurities in the microfluidic channels, the chip was disinfected and sterilized by ultraviolet light. After the preset disinfection and sterilization time, the microfluidic channels were rinsed with PBS solution, and then a mixture of fibronectin and collagen was introduced into all microfluidic channels, and the chip was placed in a preset temperature environment for cell culture. PBS solution was added to rinse all microfluidic channels to remove excess fibronectin and collagen mixture.

[0007] Furthermore, the cell inoculation includes: digesting the pre-cultured human brain microvascular endothelial cells with trypsin to adjust the cell density; fully rinsing the pre-treated microfluidic channel with cell culture medium, and then introducing the digested endothelial cells into the arterial channel and the venous channel respectively; placing the chip upright in a cell culture incubator for a preset time, and then re-introducing cells into the arterial channel and the venous channel, and then inverting the chip in the cell culture incubator for a preset time, introducing fresh culture medium, and removing cells that are not attached to the wall.

[0008] Furthermore, cell inoculation in the brain parenchymal channel includes: digesting astrocytes and neuronal cells, adjusting the cell density, and thoroughly mixing and pre-cooling the cells; adding 100 μL of type I collagen to 6 μL of 0.1 mol / L sodium hydroxide solution and mixing; adding 12 μL of 10×PBS solution, and then thoroughly mixing 380 μL of the cell mixture solution to obtain a cell-gel mixture solution; passing the gel mixture solution into the brain parenchymal channel to culture cells on the chip.

[0009] Furthermore, in the vascular channel, 500 nL of volume was perfused every 1 second at a flow rate of 1 μL / min to provide shear force stimulation that simulated pulsatile blood flow; after 24 hours of continuous perfusion, the growth status of endothelial cells was observed to confirm the basic formation of cerebral blood vessels in vitro.

[0010] Furthermore, during the vascular channel process, the culture medium of the brain parenchymal channel is replaced every 6 hours to ensure the supply of nutrients required for normal cell growth and the removal of metabolic waste.

[0011] Furthermore, the length×width×height dimensions of the gap channel used to connect different vascular channels, and the vascular channel and the brain parenchymal channel are 40 μm×5 μm×3 μm.

[0012] In order to achieve the above objectives, in a second aspect, the present invention further provides an in vitro brain lymphatic system chip, which adopts the following technical solutions: An in vitro brain lymphatic system chip is obtained by the in vitro brain lymphatic system chip construction method as described in the first aspect, comprising a substrate and an upper layer of polydimethylsiloxane, wherein the substrate and the upper layer of polydimethylsiloxane are connected by silicon-oxygen bonds.

[0013] Furthermore, the channels in the chip include vascular channels on both sides, perivascular gap channels and brain parenchyma channels, and the channels are connected by vertical gap channels. The length × width × height dimensions of the gap channels are 40 μm × 8 μm × 3.

[0014] Furthermore, the length × width × height dimensions of the gap channel were adjusted to 40 μm × 5 μm × 3 μm.

[0015] Compared with the prior art, the present invention has the following beneficial effects: 1. The present invention fills the gap in the construction of an in vitro model of the brain lymphatic system; first, the microfluidic channel in the chip is flushed, disinfected and sterilized, rinsed and cell cultured; then, in the treated microfluidic channel, vascular channel cell inoculation and brain parenchymal channel cell inoculation are carried out; finally, in the vascular channel, perfusion is performed at a preset flow rate and at intervals of a preset duration to provide shear force stimulation that simulates pulsatile blood flow, and after continuous perfusion, the basic formation of in vitro cerebral blood vessels is achieved; during the continuous perfusion of the vascular channel, the culture medium of the brain parenchymal channel is replaced at intervals; after co-culturing of multiple cells in the vascular channel and the brain parenchymal channel, an in vitro lymphatic model is formed through direct contact between different cells or the transmission of signal molecules; at a preset flow rate and at intervals of a preset duration, perfusion is performed to provide shear force stimulation that simulates pulsatile blood flow, thereby achieving intermittent liquid flow that simulates arterial fluctuations.

[0016] 2. The present invention adjusts the syringe pump flow rate to perform perfusion at a preset flow rate and at preset intervals for a preset duration, providing shear force stimulation that mimics pulsatile blood flow and achieving intermittent liquid flow that mimics arterial fluctuations. However, the improvement in flow stimulation leads to significant crosstalk between channels in cell distribution. Therefore, the present invention improves the chip structure based on the flow rate and cell distribution, adjusting the gap channel size to 40 μm × 5 μm × 3 μm, which can effectively improve the crosstalk problem between different cells in the main channels. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The drawings constituting a part of the specification of this embodiment are used to provide a further understanding of this embodiment. The schematic embodiments and descriptions of this embodiment are used to explain this embodiment and do not constitute an improper limitation on this embodiment.

[0018] Figure 1 This is a flow chart of Example 1 of the present invention; Figure 2This is a structural design diagram of the microfluidic chip according to Example 1 of the present invention; Figure 3 This is a bright field image of the microchannel in the first-generation microfluidic chip of Example 1 of the present invention; Figure 4 This is a partial enlarged view of the small gaps between the main channels of the first-generation chip of Example 1 of the present invention; Figure 5 This is a partial enlarged view of the small gap between the main channels of the second-generation chip of Example 1 of the present invention; Figure 6 This is a flowchart for constructing an in vitro lymphoid organ-on-a-chip model according to Example 1 of the present invention; Figure 7 The flow velocity changes over time under the uniform flow stimulation condition of Example 1 of the present invention; Figure 8 The flow rate changes over time under the intermittent cyclic flow stimulation condition of Example 1 of the present invention; Figure 9 This is a bright field image of cells on the first-generation chip of Example 1 of the present invention; Figure 10 This is a bright field image of cells on the second-generation chip of Example 1 of the present invention; Figure 11 This is a confocal fluorescence image of live and dead cell staining of the first-generation chip in Example 1 of the present invention; green: live cells; red: dead cells; Figure 12 This is a confocal fluorescence image of live and dead cell staining of the second-generation chip in Example 1 of the present invention; green: live cells; red: dead cells; Figure 13 This is a bar graph of cell viability of the first generation chip in Example 1 of the present invention; Figure 14 This is a bar graph of cell viability of the second-generation chip in Example 1 of the present invention; Figure 15 This is a whole-body confocal fluorescence image of the second-generation brain lymphoid organ chip model in Example 1 of the present invention; green: living cells; red: dead cells; Figure 16 This is a partially enlarged fluorescence imaging image of the second-generation brain lymphoid organ core according to Example 1 of the present invention; Figure 17 This is a three-dimensional reconstruction of the blood vessel channel according to Example 1 of the present invention; green: living cells; red: dead cells; Figure 18 This is a fluorescence image of the vascular channel from the main perspective of Example 1 of the present invention; green: living cells; red: dead cells; Figure 19 This is a fluorescence image of the vascular channel from the side view of Example 1 of the present invention; green: living cells; red: dead cells; Figure 20 This is a three-dimensional reconstruction of the brain parenchyma channel in Example 1 of the present invention; green: living cells; red: dead cells. DETAILED DESCRIPTION

[0019] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0020] It should be noted that the following detailed descriptions are exemplary and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present application belongs.

[0021] Previously, the academic community generally believed that the central nervous system lacked a lymphatic system. However, recent research has revealed for the first time in the mouse brain that interstitial structures formed between astrocyte end feet and blood vessel walls possess functions similar to those of the lymphatic system in clearing metabolic waste. This has revealed the existence of a novel clearance pathway in the central nervous system and has been named the glymphatic system. Current research has confirmed that the glymphatic system effectively clears soluble proteins and metabolic waste. Furthermore, researchers have discovered that the glymphatic system also facilitates the drainage of certain cells. For example, enhancing the drainage function of the dural lymphatic system accelerates the clearance of red blood cells, facilitating hematoma absorption and brain function recovery. Despite recent progress in the study of the brain's glymphatic system, many unexplored questions remain, such as the impact of central nervous system diseases on the glymphatic system, the mechanisms of its formation and development, and the glymphatic metabolism of drugs. Therefore, a reliable model of the glymphatic system is needed to further investigate these issues.

[0022] Currently, research on the brain's lymphatic system still primarily relies on animal models. However, animal models have numerous limitations. First, animal models are expensive, require long modeling times, and pose ethical challenges. Furthermore, animal models differ from human physiology in many ways, potentially producing physiological responses that differ significantly from those in humans, impacting the accuracy of research. Second, animal models themselves are complex physiological systems, and the generation of certain key signals may be masked by the synergistic effects of other organs or systems, hindering the precise study of key mechanisms of the brain's lymphatic system. More importantly, due to the limitations of the luminescence intensity of commercial fluorescent probes and the imaging depth of existing imaging technologies, in situ, real-time observation of exosome brain drainage and key molecules in the brain's lymphatic system is difficult in animal models. Therefore, we need to develop a more realistic in vitro brain lymphatic system model to replace animal models for related research.

[0023] In recent years, the emergence of organ chips has provided a very promising tool for in vitro simulation of human organs. This in vitro model based on microfluidic chips can reproduce the physiological environment and function of human organs on the chip by simulating key tissue structures and functions, extracellular matrix, biochemical factors and physicochemical parameters, and there are no ethical issues. Secondly, organ chips are repeatable and can be mass-produced, which can narrow the differences between different models and achieve efficient and high-throughput simultaneous construction of multiple models, greatly shortening the long modeling time of animal models and improving research efficiency. In addition, in the study of the clearance process of the brain lymphatic system, the highly transparent organ chip can achieve high-resolution in situ real-time monitoring of exosomes and key cellular molecules, which is conducive to the subsequent rapid analysis of different proteins and nucleic acids, and provides important information for the study of the brain lymphatic system.

[0024] A variety of brain organ-on-chips have been developed, including blood-brain barrier chips, neurovascular unit organ-on-chips, and neurodegenerative disease chips, for applications in drug evaluation, metabolomics research, viral invasion, and bacterial infection studies. However, methods for constructing in vitro models of the recently discovered brain lymphatic system remain elusive.

[0025] In order to solve at least one of the above problems, the present invention provides a method for constructing an in vitro brain lymphatic system chip, and provides a brain lymphatic system chip, which reproduces the blood flow stimulation and lymphatic system drainage function, and restores the real physiological structure of the brain lymphatic system in terms of molecular expression, spatial distribution, etc., providing a reliable tool for subsequent in-depth exploration of issues such as drug response, disease impact and developmental mechanism of the lymphatic system.

[0026] A method for constructing an in vitro brain lymphatic system chip specifically targets dynamic flow stimulation, three-dimensional co-culture of multiple cells, and in vitro restoration of basic spatial structures of the brain lymphatic system. The in vitro model constructed based on this method can, to a certain extent, reproduce the actual conditions of the brain lymphatic system, providing a powerful tool for related research. The method steps are as follows: S1. Chip pretreatment.

[0027] First, the five microfluidic channels in the chip were rinsed with deionized water and then ethanol. After microscopic inspection to ensure the channels were free of impurities, the chip was sterilized under UV light. After 24 hours, the microfluidic channels were rinsed with PBS solution. A 100 μg / mL mixture of fibronectin and type IV collagen was then introduced into all microfluidic channels. The microfluidic chip was then placed in a cell culture incubator at 37°C overnight. PBS solution was then added to rinse all microfluidic channels to remove excess fibronectin and collagen mixture. This step facilitates the adherent growth of subsequently seeded cells within the chip.

[0028] S2. Cell seeding in vascular channels.

[0029] Human brain microvascular endothelial cells grown in culture flasks were digested with trypsin and the cell density was adjusted to 5×10 5 / mL. The pretreated microfluidic chip channel was thoroughly rinsed with cell culture medium, and then the digested endothelial cells were introduced into the arterial channel and venous channel respectively. After the microfluidic chip was placed upright in a cell culture incubator for 1 hour, cells were reintroduced into both channels and the microfluidic chip was inverted in the cell culture incubator for 1 hour. Fresh culture medium was introduced to remove cells that were not attached to the wall. The microfluidic chip was then placed upright in the incubator, and the cell growth status was observed and the culture medium in the channel was replaced every 6 hours. After the four inner walls of the channel were covered with cells, the next step was carried out.

[0030] S3. Brain parenchymal channel cell inoculation.

[0031] First, pre-cool all the tools needed for the subsequent steps at 4°C, including syringes, catheters, and pipette tips. Digest astrocytes and neurons and resuspend them in EP tubes, adjusting the cell density to 1×10 5 The three cell types were thoroughly mixed and pre-cooled at 4°C for half an hour. Subsequently, 100 μL of type I collagen was added to 6 μL of 0.1 mol / L NaOH solution in an ice bath. The solution was quickly mixed using a pre-cooled pipette tip. 12 μL of 10× PBS buffer was added, and finally, 380 μL of the three cell mixture was thoroughly mixed to form a cell-gel mixture. This gel mixture was introduced into the brain parenchymal channel, and the chip was placed in a 37°C cell culture incubator for 1 hour. The gel mixture completely polymerized into a semisolid gel, and the two cell types were evenly dispersed on the gel scaffold, forming a three-dimensional spatial distribution.

[0032] S4. Flow stimulation simulation and multi-cell co-culture.

[0033] In the vascular channel, a high-precision syringe pump was used to perfuse 500 nL of volume every 1 second at a flow rate of 1 μL / min, providing shear stimulation that mimics pulsatile blood flow. After 24 hours of continuous perfusion, endothelial cell growth was observed to confirm the basic formation of in vitro cerebral vasculature. During this continuous perfusion, the culture medium in the parenchymal channel was replaced every 6 hours to ensure the supply of nutrients required for normal cell growth and the removal of metabolic waste. Through 24 hours of co-culture of multiple cells in the vascular and parenchymal channels, a relatively complete in vitro lymphoid model was established through direct contact between different cells and the transmission of signaling molecules.

[0034] S5. Characterization of the basic structure of the brain lymphatic system.

[0035] After the above-mentioned cell inoculation and co-culture process is completed, it is necessary to verify that the constructed in vitro lymphoid model restores the real in vivo lymphoid system to a certain extent through cell activity and basic structure. First, the live-dead cell staining kit Calcein-AM / PI is combined with confocal fluorescence imaging technology to analyze the cell survival in the cell nucleus model construction method and calculate the cell survival rate. The spatial distribution of cells in different channels of the constructed model is observed through the three-dimensional reconstruction technology of the fluorescence imaging image to confirm that the basic physiological structure of the in vivo lymphoid system is reproduced in vitro.

[0036] The present invention provides a brain lymphatic system chip constructed using the aforementioned method. This chip is specifically designed for simulating three-dimensional multicellular brain lymphatic structures with fluid flow stimulation and drainage capabilities. The chip also addresses the issue of cell migration crosstalk between different channels within the chip. By optimizing the fluid flow and internal structure within the channels, a more realistic second-generation lymphatic organ-on-a-chip has been developed. The chip primarily comprises a lower, highly transparent glass substrate and an upper polydimethylsiloxane (PDMS) layer, which are securely bonded together via silicon-oxygen bonds. The glass substrate's high light transmittance enables the acquisition of high-resolution fluorescence images, facilitating real-time monitoring of molecular dynamics.

[0037] The first-generation lymphatic organ-on-a-chip primarily consists of five rectangular channels: a bilateral vascular channel (length × width × height: 10,000 μm × 1,000 μm × 150 μm), a perivascular interstitial channel (10,000 μm × 200 μm × 150 μm), and a brain parenchymal channel (10,000 μm × 2,000 μm × 150 μm). The five main channels are connected by vertical interstitial channels measuring 40 μm × 8 μm × 3 μm, with a spacing of 100 μm between each channel. This microchannel structure enables direct contact between cells in different channels, recreating the interactions between cells in a real brain lymphatic system. In addition, in previous organ-on-a-chip studies, culture medium was often continuously perfused through the channels at a uniform flow rate using a high-precision syringe pump to provide shear stimulation that mimics blood flow.

[0038] However, in the brain's lymphatic system, blood flow in arteries is not uniform but rather fluctuates periodically due to the effects of cardiac contraction and relaxation, generating pulsatile blood flow. Simultaneously, these pulsatile fluctuations pump fluid from the periarterial spaces into the brain tissue and perivenous spaces, driving overall fluid flow and draining cerebrospinal fluid and brain parenchymal metabolic products. Therefore, due to the specific nature of fluid flow in the brain's lymphatic system, fluid flow in arteries cannot be simulated using a single flow rate alone. In this organ-on-a-chip, by adjusting the syringe pump flow rate to perfuse a 60 nL volume in the arterial channel at a rate of 1 μL / min, with a cycle interval of 1 second, intermittent fluid flow similar to arterial fluctuations was achieved. However, this improved flow stimulation led to significant crosstalk between channels in cell distribution. Therefore, the present invention modified the chip structure to address this flow rate and cell distribution, adjusting the dimensions of the small interstitial channels to 40 μm × 5 μm × 3 μm, effectively reducing crosstalk between different cells in the main channels.

[0039] The in vitro lymphoid organ chip constructed by the present invention fills the gap in lymphoid organ chips. Targeting the characteristics of fluid flow in brain lymphoids, it improves the uniform fluid flow rate in blood flow stimulation in other organ chips, achieving cyclic pulsed fluid flow stimulation. In addition, by optimizing the internal dimensions of the first-generation organ chip, a second-generation lymphoid organ chip model was developed, solving the problem of cell crosstalk between different channels under intermittent flow stimulation. On the other hand, the construction method of this model is relatively simple and the construction process is convenient and fast, which can effectively shorten the model construction time and improve research efficiency. At the same time, this construction method has certain universality and can be extended to the in vitro simulation of other human organs by changing the cell type and culture method, and has a very broad application prospect. In addition, the in vitro lymphoid organ chip has high flexibility and can be cascaded with a variety of analytical systems, including confocal fluorescence imaging, mass spectrometry analysis, and PCR sequencing detection, providing a convenient tool for the analysis of various biomolecules such as reactive oxygen species, various ions, proteins, and genes.

[0040] Example 1: like Figure 1 As shown, this embodiment provides a method for constructing an in vitro brain lymphatic system chip, which can be used to simulate the molecular expression, basic structure and drainage function of the in vitro lymphatic system, and can be used for dynamic real-time monitoring of the response of the lymphatic system under various subsequent stimulation conditions.

[0041] S1. Microfluidic chip structure design: like Figures 2 to 5 As shown, the first-generation microfluidic chip contains five channels, interconnected by multiple vertical channels to ensure intercellular communication between different channels. The five main channels are the vascular channel, the perivascular space channel, and the parenchymal channel. The vascular channel includes two outermost arterial channels and a venous channel. Human umbilical artery endothelial cells and human umbilical artery endothelial cells are grown directly on the upper, lower, left, and right inner walls of the channel to simulate blood vessels. The two adjacent channels are the perivascular space channels, which are important components of the lymphatic drainage system. The central channel is the parenchymal channel. Astrocytes and neurons are dispersed on a three-dimensional gel scaffold to simulate the complex three-dimensional structure of the brain parenchyma. The second-generation chip improves the structure of the interstitial channels, reducing their dimensions from 40 μm × 8 μm × 3 μm in the first generation to 40 μm × 5 μm × 3 μm, effectively reducing crosstalk between different cells in the main channels.

[0042] Microfluidic chip design. The dimensions of the vascular channels on either side are 10,000 μm × 1,000 μm × 150 μm, the dimensions of the adjacent perivascular channels are 10,000 μm × 200 μm × 150 μm, the dimensions of the central parenchymal channel are 10,000 μm × 2,000 μm × 150 μm, and the small channels connecting the different channels are 40 μm × 8 μm × 3 μm. The spacing between the channels is 100 μm.

[0043] S2. Construction of in vitro lymphoid organ-on-a-chip model: First, human umbilical artery endothelial cells and human umbilical vein endothelial cells were added to the arterial channel and venous channel, respectively. By flipping the chip, the cells were allowed to grow all four inner walls of the channel. Subsequently, a mixture of astrocytes, neurons, and type I collagen was added to the brain parenchymal channel. After the gel was completely polymerized, a three-dimensional spatial dispersion of the two cell types was achieved. Next, a continuous perfusion of culture medium was introduced into the two vascular channels. Through a period of fluid flow stimulation and co-culture of multiple cells in different channels, the in vitro lymphoid organoid model was finally constructed.

[0044] Figure 6 The figure shows the flow chart for constructing an in vitro lymphoid organ chip model; Optional, Day 1: Place 5×10 5 Human umbilical artery endothelial cells (100 μL / mL) and human umbilical vein endothelial cells (100 μL / mL) were introduced into the arterial channel and venous channel, respectively. The cells were placed upright in a cell culture incubator for 1 hour, then reintroduced and the chip was incubated upside down for 1 hour. Fresh culture medium was then introduced to remove adherent cells. Day 2: Endothelial cell culture medium was continuously perfused into the venous channel at a constant flow rate of 1 μL / min for 24 hours. At the same time, intermittent circulation was continuously perfused into the arterial channel for 24 hours, allowing the endothelial cells to form a complete and continuous monolayer of cells on the upper, lower, left, and right inner walls of the channel. Day 3: 1×10 5 A mixed solution of astrocytes, neurons, and type I collagen (1000 cells / mL) was introduced into the middle parenchymal channel and placed in a 37°C cell culture incubator to allow both cell types to be evenly dispersed throughout the solidified gel scaffold. Day 4: Multiple cells were co-cultured in the parenchymal and vascular channels for 24 hours to establish a complete in vitro lymphoid model. Figure 7 is the change of flow velocity over time under uniform flow stimulation conditions, Figure 8 The flow velocity changes over time under intermittent cyclic flow stimulation conditions.

[0045] In order to characterize the successful formation of the complete lymphoid model, the lymphoid structure was observed by bright field imaging, live-dead cell staining, and fluorescence three-dimensional reconstruction. Figure 9In the bright field imaging shown, it can be observed that the cells are growing well in the chip. In addition, the live-dead cell staining image shows that most cells in the channel show green fluorescence, as shown in the figure below. Figure 11 As shown in the figure, only a few cells showed red fluorescence, indicating that most cells were in a good survival state in the microfluidic chip. Statistics showed that the cell survival rates in the vascular channels on both sides and the middle brain parenchyma channel were 98.23±1.41%, 97.51±2.06% and 95.33±2.58%, respectively, all above 95%. Figure 13 However, during the co-culture process, a small number of cells also appeared in the paravascular channels, such as Figure 9 As shown by the red arrows in the figure, these cells are likely due to the fact that the flow stimulation in the arterial channel is adjusted from a uniform speed to an intermittent cyclic flow stimulation, which not only pumps the fluid around the artery into the adjacent channel, but also causes crosstalk between different cells. Therefore, in order to improve the negative impact of the flow rate adjustment, a more simulated lymphoid organ chip is constructed, such as Figure 10 、 Figure 12 and Figure 14 As shown, we improved the first-generation chip by adjusting the dimensions of the small channels between the main channels from 40 μm × 8 μm × 3 μm to 40 μm × 5 μm × 3 μm to limit the cross-channel migration of cells in the vascular and parenchymal channels caused by intermittent fluid disturbances. In the second-generation lymphoid organ-chip, live-dead cell staining imaging revealed that no cells were present in the paravascular channels, indicating that the structural optimization successfully inhibited cross-channel migration of cells, making the constructed structure more consistent with a realistic lymphoid system. Furthermore, the cell viability rates in the bilateral vascular channels and the central parenchymal channel were 97.33±1.07%, 97.39±3.01%, and 96.95±2.76%, respectively, indicating that the structural changes did not affect cell survival in the chip.

[0046] like Figure 15 and Figure 16 As shown in the figure, the second-generation brain lymphatic organ chip, which is more realistic, was imaged as a whole. Through the live-dead cell confocal fluorescence imaging, it can be observed that in the second-generation model, the cell structure of blood vessels on both sides and the middle brain parenchyma was formed, which is consistent with the arrangement of cells in the real brain lymphatic system. Figure 17 、 Figure 18 and Figure 19 As shown in the figure, the three-dimensional reconstruction of cells in the second-generation lymphoid organ chip shows that the endothelial cells in the vascular channel are arranged in a rectangular shape with a cavity in the middle. This structure is very similar to the real vascular wall structure with a cavity. Figure 20As shown, the three cell types in the central parenchymal channel exhibit a three-dimensional spatially dispersed distribution, similar to the three-dimensional structure of brain parenchyma tissue. Notably, compared to the first-generation lymphoid organ-chip, the second-generation organ-chip exhibited no migration crosstalk between cells in the perivascular spaces within the channels, a key feature of in vitro replication of the lymphoid system. The improved structure of the second-generation organ-chip not only successfully simulated the arterial pulsation of the brain lymphoid system but also improved upon the traditional chip, enabling it to adapt to the crosstalk between cells in the different channels caused by the overall fluid flow in the lymphoid system. Furthermore, the growth of astrocytes in the channels connected to the perivascular spaces replicated the extended pseudopodia of astrocytes in the real lymphoid structure, one of the most important structures in the lymphoid system. These findings demonstrate that the constructed in vitro lymphoid system model not only ensures the normal growth, survival, and expression of essential molecules of multiple cells, but also recreates the key physiological structures of the real lymphoid system to a certain extent.

[0047] The above description is merely a preferred embodiment of this embodiment and is not intended to limit this embodiment. Those skilled in the art will readily appreciate that this embodiment may be modified and varied in various ways. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this embodiment shall be within the scope of protection of this embodiment.

Claims

1. A method for constructing an in vitro brain lymphatic system chip, characterized in that: include: The microfluidic channels in the chip are flushed, sterilized, rinsed and cultured with cells; In the treated microfluidic channels, vascular channel cell seeding and brain parenchymal channel cell seeding were performed; In the vascular channel, perfusion is performed at a preset flow rate and at intervals of preset duration, providing shear force stimulation that simulates pulsatile blood flow. After continuous perfusion, the formation of in vitro cerebral blood vessels is achieved. During the continuous perfusion of the vascular channel, the culture medium of the brain parenchyma channel is replaced at intervals. By co-culturing multiple cells in vascular channels and brain parenchymal channels, an in vitro lymphoid model is formed through direct contact between different cells or the transmission of signal molecules.

2. The method for constructing an in vitro brain lymphatic system chip according to claim 1, wherein: The microfluidic channels in the chip were rinsed with deionized water and ethanol respectively. After there were no impurities in the microfluidic channels, the chip was disinfected and sterilized by ultraviolet light. After the preset disinfection and sterilization time, the microfluidic channels were rinsed with PBS solution, and then a mixture of fibronectin and collagen was introduced into all microfluidic channels, and the chip was placed in a preset temperature environment for cell culture. PBS solution was added to rinse all microfluidic channels to remove excess fibronectin and collagen mixture.

3. The method for constructing an in vitro brain lymphatic system chip according to claim 1, wherein: Cell inoculation includes: digesting pre-cultured human brain microvascular endothelial cells with trypsin to adjust the cell density; thoroughly rinsing the pre-treated microfluidic channel with cell culture medium, and then introducing the digested endothelial cells into the arterial channel and venous channel respectively; placing the chip upright in a cell culture incubator for a preset time, then re-introducing cells into the arterial channel and venous channel, and then inverting the chip in the cell culture incubator for a preset time, introducing fresh culture medium, and removing cells that are not attached to the wall.

4. The method for constructing an in vitro brain lymphatic system chip according to claim 1, wherein: Cell seeding in the brain parenchymal channel includes: digesting astrocytes and neurons, adjusting the cell density, and thoroughly mixing and pre-cooling the cells; adding 100 μL of type I collagen to 6 μL of 0.1 mol / L sodium hydroxide solution and mixing; adding 12 μL of 10× PBS solution, and then thoroughly mixing 380 μL of the cell mixture solution to obtain a cell-gel mixture solution; passing the gel mixture solution into the brain parenchymal channel to culture cells on the chip.

5. The method for constructing an in vitro brain lymphatic system chip according to claim 1, wherein: In the vascular channel, 500 nL of volume was perfused every 1 second at a flow rate of 1 μL / min, providing shear force stimulation that simulated pulsatile blood flow; after 24 hours of continuous perfusion, the growth status of endothelial cells was observed to confirm the basic formation of in vitro cerebral blood vessels.

6. The method for constructing an in vitro brain lymphatic system chip according to claim 5, wherein: During the vascular channel process, the culture medium of the brain parenchymal channel was replaced every 6 hours to ensure the supply of nutrients required for normal cell growth and the removal of metabolic waste.

7. The method for constructing an in vitro brain lymphatic system chip according to claim 1, wherein: The length × width × height dimensions of the gap channel used to connect different vascular channels, as well as vascular channels and brain parenchymal channels, are 40 μm × 5 μm × 3 μm.

8. An in vitro brain lymphatic system chip, characterized in that: The chip is obtained by the in vitro brain lymphatic system construction method according to any one of claims 1 to 7, comprising a substrate and an upper layer of polydimethylsiloxane, wherein the substrate and the upper layer of polydimethylsiloxane are connected by silicon-oxygen bonds.

9. The in vitro brain lymphatic system chip according to claim 8, characterized in that: The channels in the chip include vascular channels on both sides, perivascular gap channels and brain parenchyma channels. The channels are connected by vertical gap channels, and the length × width × height dimensions of the gap channels are 40 μm × 8 μm × 3.

10. The in vitro brain lymphatic system chip according to claim 9, characterized in that: The length × width × height dimensions of the gap channel were adjusted to 40 μm × 5 μm × 3 μm.

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