A method for constructing an in vitro brain lymphoid system chip and a brain lymphoid system chip

Abstract

This invention belongs to the field of organ-on-a-chip technology, proposing a method for constructing an in vitro brain lymphatic system chip and a brain lymphatic system chip, filling the gap in the construction of in vitro models of brain lymphatic systems. First, the microfluidic channels in the chip are rinsed, sterilized, washed, and cultured with cells. Then, vascular channel cells and brain parenchyma channel cells are seeded in the treated microfluidic channels. Finally, in the vascular channels, perfusion is performed at a preset flow rate and at preset intervals to provide shear force stimulation mimicking pulsed blood flow. Continuous perfusion achieves the basic formation of in vitro brain blood vessels. The intermittent fluid flow mimicking arterial pulsations is achieved through this preset flow rate and preset interval perfusion.

Description

Technical Field

[0001] This invention belongs to the field of organ-on-a-chip technology, and particularly relates to a method for constructing an in vitro brain lymphatic system chip and a brain lymphatic system chip. Background Technology

[0002] Various brain organ-on-a-chip technologies have been developed, including blood-brain barrier chips, neurovascular unit organ-on-a-chips, and neurodegenerative disease chips, for research in areas such as drug evaluation, metabolomics, viral invasion, and bacterial infection. However, for the newly discovered brain lymphoid system, methods for constructing in vitro models remain lacking.

[0003] In the brain-like lymphatic system, the blood flow velocity in the arteries is not uniform but fluctuates periodically due to the contraction and relaxation of the heart, generating pulsatile blood flow. Simultaneously, these pulsatile fluctuations pump the solution from the periarterial spaces to the brain tissue and perivenous spaces, propelling the overall fluid flow and facilitating the drainage of cerebrospinal fluid and brain parenchymal metabolic products. Therefore, the unique fluid flow characteristics of the brain-like lymphatic system limit the construction of in vitro models. Furthermore, crosstalk during cell migration between different channels within the chip also restricts the construction of in vitro models of the brain-like lymphatic system. Summary of the Invention

[0004] To address the aforementioned problems, this invention proposes an in vitro brain lymphoid system chip construction method and a brain lymphoid system chip, filling the gap in the construction of in vitro brain lymphoid system models. During continuous perfusion of the vascular channel, the culture medium in the brain parenchyma channel is replaced intermittently. Through co-culture of various cells in the vascular channel and the brain parenchyma channel, an in vitro lymphoid model is formed through direct contact between different cells or the transmission of signal molecules. Perfusion is performed at a preset flow rate and at preset intervals to provide shear force stimulation that mimics pulsed blood flow, thereby achieving intermittent fluid flow that mimics arterial pulsations.

[0005] To achieve the above objectives, in a first aspect, the present invention provides a method for constructing an in vitro brain-like lymphatic system chip, employing the following technical solution:

[0006] A method for constructing an in vitro brain lymphatic system chip, comprising:

[0007] The microfluidic channels in the chip are subjected to rinsing, sterilization, washing, and cell culture.

[0008] In the processed microfluidic channels, vascular channel cells and brain parenchyma channel cells were seeded.

[0009] In the vascular channel, perfusion is performed at a preset flow rate and at preset intervals to provide shear force stimulation that mimics pulsed blood flow. After continuous perfusion, in vitro cerebral blood vessels are formed. During the continuous perfusion of the vascular channel, the culture medium in the brain parenchyma channel is replaced at intervals.

[0010] Through co-culturing of various cells in vascular and brain parenchymal channels, an in vitro lymphoid model is formed through direct contact between different cells or the transmission of signal molecules.

[0011] Furthermore, the microfluidic channels in the chip were rinsed with deionized water and ethanol respectively. After the microfluidic channels were free of impurities, the chip was sterilized by UV irradiation. After the preset sterilization time, the microfluidic channels were rinsed with PBS solution. 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.

[0012] Further, cell seeding includes: digesting pre-cultured human brain microvascular endothelial cells with trypsin to adjust cell density; thoroughly washing the pretreated microfluidic channels with cell culture medium, and then introducing the digested endothelial cells into the arterial and venous channels respectively; placing the chip upright in a cell culture incubator for a preset time, then reintroducing cells into the arterial and venous channels, and finally inverting the chip in the cell culture incubator for a preset time, introducing fresh culture medium, and removing non-adherent cells.

[0013] Further, the brain parenchyma channel cell seeding process includes: digesting astrocytes and neurons, adjusting 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, then adding 12 μL of 10×PBS solution, and thoroughly mixing 380 μL of the cell mixture to obtain a cell gel mixture; and introducing the gel mixture into the brain parenchyma channel for cell culture on the chip.

[0014] Furthermore, in the vascular channel, a flow rate of 1 μL / min was used to perfuse 500 nL of volume every 1 second to provide shear force stimulation that mimics pulsed blood flow; after continuous perfusion for 24 hours, the growth status of endothelial cells was observed to confirm the basic formation of in vitro cerebral blood vessels.

[0015] Furthermore, during the vascular channel process, the culture medium in the brain parenchyma channel is changed every 6 hours to ensure the supply of nutrients required for normal cell growth and the removal of metabolic waste.

[0016] Furthermore, the length × width × height dimensions of the interstitial channel used to connect different vascular channels, as well as the interstitial channel between the vascular channel and the brain parenchyma channel, are 40 μm × 5 μm × 3 μm.

[0017] To achieve the above objectives, in a second aspect, the present invention also provides an in vitro brain-like lymphatic system chip, employing the following technical solution:

[0018] An in vitro brain-like lymphatic system chip, obtained by the in vitro brain-like lymphatic system chip construction method as described in the first aspect, includes a substrate and an upper polydimethylsiloxane layer, wherein the substrate and the upper polydimethylsiloxane layer are bonded together by silicon-oxygen bonds.

[0019] Furthermore, the channels in the chip include vascular channels on both sides, perivascular space channels, and brain parenchyma channels, which are connected by vertical gap channels. The length × width × height dimensions of the gap channels are 40 μm × 8 μm × 3.

[0020] Furthermore, the length × width × height dimensions of the gap channel were adjusted to 40 μm × 5 μm × 3 μm.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0022] 1. This invention fills the gap in the construction of in vitro models of brain-like lymphatic systems. First, the microfluidic channels in the chip are rinsed, sterilized, rinsed, and cultured. Then, vascular channel cells and brain parenchyma channel cells are seeded in the treated microfluidic channels. Finally, in the vascular channels, perfusion is performed at a preset flow rate and at preset intervals to provide shear force stimulation mimicking pulsed blood flow. Continuous perfusion achieves the basic formation of in vitro cerebral blood vessels. During the continuous perfusion of the vascular channels, the culture medium in the brain parenchyma channels is replaced intermittently. Through the co-culture of various cells in the vascular and brain parenchyma channels, an in vitro lymphatic model is formed through direct contact between different cells or the transmission of signal molecules. Perfusion at a preset flow rate and at preset intervals provides shear force stimulation mimicking pulsed blood flow, achieving intermittent fluid flow mimicking arterial pulsations.

[0023] 2. This invention provides shear force stimulation that mimics pulsed blood flow by adjusting the flow rate of the injection pump and performing perfusion at preset intervals at a preset flow rate, thus achieving intermittent fluid flow that mimics arterial pulsations. However, this improvement in flow stimulation leads to significant crosstalk between cell channels. Therefore, this invention improves the chip structure to address the flow rate and cell distribution, adjusting the gap channel size to 40 μm × 5 μm × 3 μm, which effectively improves the crosstalk problem between different cells in the main channels. Attached Figure Description

[0024] The accompanying drawings, which form part of this embodiment, are used to provide a further understanding of this embodiment. The illustrative embodiments and their descriptions are used to explain this embodiment and do not constitute an improper limitation of this embodiment.

[0025] Figure 1 This is a flowchart of Embodiment 1 of the present invention;

[0026] Figure 2 This is a microfluidic chip structure design diagram of Embodiment 1 of the present invention;

[0027] Figure 3 This is a bright-field image of the microchannel in the first-generation microfluidic chip of Embodiment 1 of the present invention;

[0028] Figure 4 This is a partial enlarged view of the small channel between the main channels of the first-generation chip in Embodiment 1 of the present invention;

[0029] Figure 5 This is a partial enlarged view of the small channel between the main channels of the second-generation chip in Embodiment 1 of the present invention;

[0030] Figure 6 This is a flowchart illustrating the construction process of the in vitro lymphoid organ-on-a-chip model according to Embodiment 1 of the present invention.

[0031] Figure 7 The flow velocity changes with time under the uniform flow stimulus condition of Embodiment 1 of the present invention;

[0032] Figure 8 The flow velocity changes over time under intermittent cyclic flow stimulation conditions as described in Embodiment 1 of the present invention;

[0033] Figure 9 This is a bright-field image of a first-generation chip cell from Embodiment 1 of the present invention;

[0034] Figure 10 This is a bright-field image of a second-generation chip cell from Embodiment 1 of the present invention;

[0035] Figure 11 This is a confocal fluorescence imaging image of live and dead cells stained with the first-generation chip in Embodiment 1 of the present invention; green: live cells; red: dead cells;

[0036] Figure 12 This is a confocal fluorescence imaging image of live and dead cells stained with the second-generation chip in Embodiment 1 of the present invention; green: live cells; red: dead cells;

[0037] Figure 13 This is a bar chart showing the cell survival rate of the first-generation chip in Embodiment 1 of the present invention.

[0038] Figure 14 This is a bar chart showing the cell survival rate of the second-generation chip in Embodiment 1 of the present invention;

[0039] Figure 15 This is a confocal fluorescence imaging image of the second-generation brain-like lymphoid organ-on-a-chip model of Embodiment 1 of the present invention; green: live cells; red: dead cells;

[0040] Figure 16 This is a magnified fluorescence imaging image of a portion of the core of the second-generation brain-like lymphoid organoid of Embodiment 1 of the present invention;

[0041] Figure 17 This is a three-dimensional reconstruction image of the vascular channel in Embodiment 1 of the present invention; green: living cells; red: dead cells;

[0042] Figure 18 This is a fluorescence image of the vascular channel from the main perspective in Embodiment 1 of the present invention; green: live cells; red: dead cells;

[0043] Figure 19 This is a side-view fluorescence image of the vascular channel in Embodiment 1 of the present invention; green: live cells; red: dead cells;

[0044] Figure 20 This is a three-dimensional reconstruction of the brain parenchyma pathway in Embodiment 1 of the present invention; green: living cells; red: dead cells. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0046] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, 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 pertains.

[0047] Previously, it was generally believed that the central nervous system lacked a lymphatic system. However, in recent years, researchers have discovered for the first time in the mouse brain a gap structure formed between the terminal appendages of astrocytes and the blood vessel walls, which possesses a metabolic waste removal function similar to that of the lymphatic system. This reveals a novel clearance pathway in the central nervous system and has been named the "glymphatic system." Current research has confirmed that the glymphatic system can effectively remove soluble proteins and metabolic waste. In addition, researchers have found that the glymphatic system can facilitate the drainage of certain cells. For example, by enhancing the drainage function of the dura mater lymphatic system, the process of clearing red blood cells is accelerated, which is beneficial for hematoma absorption and brain function recovery. Although some progress has been made in the study of the brain's glymphatic system, many questions remain to be explored, such as the impact of central nervous system diseases on the glymphatic system, the formation and development mechanism of the glymphatic system, and the glymphatic metabolic process of drugs. Therefore, in-depth research on these issues is needed using reliable glymphatic system models.

[0048] Current research on brain lymphoid systems still primarily relies on animal models. However, animal models have several limitations. First, they are costly, time-consuming to model, and raise ethical concerns. Furthermore, animal models differ significantly from human physiology, potentially producing entirely different physiological responses and affecting research accuracy. Second, animal models are complex physiological systems, and the generation of certain key signals may be masked by synergistic effects from other organs or systems, hindering precise research into the key mechanisms of brain lymphoid systems. More importantly, limitations imposed by the luminescence intensity of commercially available fluorescent probes and the imaging depth of existing imaging techniques make in-situ, real-time observation of exosome drainage into the brain and key molecules in brain lymphoid systems difficult in animal models. Therefore, there is a need to develop a more realistic in vitro brain lymphoid system model to replace animal models for related research.

[0049] In recent years, organ-on-a-chip technology has provided a highly promising tool for in vitro simulation of human organs. This microfluidic chip-based in vitro model can reproduce the physiological environment and function of human organs on a chip by simulating key tissue structures and functions, extracellular matrix, biochemical factors, and physicochemical parameters, without raising ethical concerns. Secondly, organ-on-a-chip technology is reproducible and mass-producible, which can reduce the differences between different models and enable the simultaneous construction of multiple models efficiently and with high throughput, greatly shortening the lengthy modeling time of animal models and improving research efficiency. Furthermore, in the study of the brain's lymphatic system clearance process, the high transparency of organ-on-a-chip technology enables high-resolution in-situ real-time monitoring of exosomes and key cellular molecules, facilitating rapid analysis of different proteins and nucleic acids and providing important information for research on brain-like lymphatic systems.

[0050] Various brain organ-on-a-chip technologies have been developed, including blood-brain barrier chips, neurovascular unit organ-on-a-chips, and neurodegenerative disease chips, for research in areas such as drug evaluation, metabolomics, viral invasion, and bacterial infection. However, for the newly discovered brain lymphoid system, methods for constructing in vitro models remain lacking.

[0051] To address at least one of the aforementioned problems, this invention provides a method for constructing an in vitro brain lymphoid system chip, and also provides a brain lymphoid system chip that reproduces blood flow stimulation and lymphoid system drainage function. It restores the true physiological structure of the brain lymphoid system in terms of molecular expression and spatial distribution, providing a reliable tool for further in-depth exploration of issues such as drug response, disease effects, and developmental mechanisms of the lymphoid system.

[0052] An in vitro brain lymphoid system chip construction method is disclosed, specifically targeting the dynamic flow stimulation, three-dimensional co-culture of multiple cells, and in vitro reconstruction of the basic spatial structure of the brain lymphoid system. The in vitro model constructed based on this method can reproduce the real-world brain lymphoid system to a certain extent, providing a powerful tool for related research. The method steps are as follows:

[0053] S1, Chip preprocessing.

[0054] First, the five microfluidic channels in the chip were rinsed with deionized water and ethanol, respectively. After microscopic inspection revealed no impurities in the microfluidic channels, the chip was sterilized by irradiation under a UV lamp. After 24 hours, the microfluidic channels were rinsed with PBS solution. Then, a mixture of 100 μg / mL fibronectin and type IV collagen was introduced into all microfluidic channels, and the microfluidic chip was 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 promotes the adhesion and growth of subsequently seeded cells within the chip.

[0055] S2, vascular channel cell seeding.

[0056] Human brain microvascular endothelial cells that had grown in culture flasks were digested with trypsin to adjust the cell density to 5 × 10⁶ cells / year. 5 Cells / mL. The pretreated microfluidic chip channels were thoroughly rinsed with cell culture medium, and then the digested endothelial cells were introduced into the arterial and venous channels, respectively. After placing the microfluidic chip 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 then introduced to remove any non-adherent cells. The microfluidic chip was then placed upright in the incubator, and cell growth was observed and the culture medium in the channels was replaced every 6 hours. Once the four inner walls of the channels were fully covered with cells, the next step was performed.

[0057] S3, brain parenchymal channel cell inoculation.

[0058] First, pre-cool all necessary tools at 4°C, including syringes, tubing, and pipette tips. Digest and resuspend astrocytes and neurons in EP tubes, adjusting the cell density to 1×10⁻⁶. 5The three cell types were mixed thoroughly and pre-cooled at 4°C for half an hour. Then, under ice bath conditions, 100 μL of type I collagen was added to 6 μL of 0.1 mol / L NaOH solution, and 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 types were thoroughly mixed to obtain a cell gel mixture. This gel mixture was introduced into the brain parenchyma channel, and the chip was placed in a 37°C cell culture incubator and allowed to stand for 1 hour. The gel mixture completely polymerized into a semi-solid gel, and the two cell types were uniformly dispersed on the gel scaffold, forming a three-dimensional spatial distribution.

[0059] S4, flow stimulation simulation and multi-cell co-culture.

[0060] In the vascular channel, a high-precision syringe pump was used to perfuse 500 nL of blood at a flow rate of 1 μL / min every 1 second, providing shear force stimulation to mimic 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. During the continuous perfusion of blood vessels, the culture medium in the brain parenchyma channel was changed every 6 hours to ensure the supply of nutrients necessary for normal cell growth and the removal of metabolic waste. After 24 hours of co-culture of various cells in the vascular channel and the brain parenchyma channel, a relatively complete in vitro lymphoid model could be formed through direct contact between different cells or the transmission of signaling molecules.

[0061] S5. Basic structural characterization of the brain-like lymphatic system.

[0062] After the aforementioned cell seeding and co-culture processes are completed, it is necessary to verify, through cell viability and basic structure, that the constructed in vitro lymphoid model accurately replicates the real in vivo lymphoid system to a certain extent. First, the cell viability in the nuclear model construction method was analyzed using the Calcein-AM / PI live / dead cell staining kit combined with confocal fluorescence imaging technology, and the cell viability rate was statistically determined. Then, the spatial distribution of cells in different channels of the constructed model was observed using three-dimensional reconstruction technology of the fluorescence imaging, confirming that the basic physiological structure of the in vivo lymphoid system was replicated in vitro.

[0063] This invention provides a brain-like lymphatic system chip constructed using the above-described method, specifically for simulating three-dimensional multicellular brain-like lymphatic structures with fluid flow stimulation and drainage functions. Furthermore, it improves upon the problem of cell migration crosstalk between different channels in this chip, developing a more realistic second-generation lymphatic organ-like chip by optimizing the fluid flow and internal structure within the channels. The chip mainly consists of a lower glass substrate with good light transmittance and an upper polydimethylsiloxane (PDMS) layer, which are firmly bonded together by silicon-oxygen bonds. The glass substrate has high light transmittance, enabling the acquisition of high-resolution fluorescence images, which is beneficial for real-time monitoring of molecular dynamic changes.

[0064] The first-generation lymphoid organ-on-a-chip mainly comprises five cuboid channels: a lateral vascular channel (length × width × height: 10000 μm × 1000 μm × 150 μm), a perivascular space channel (10000 μm × 200 μm × 150 μm), and a brain parenchyma channel (10000 μm × 2000 μm × 150 μm). These five main channels are connected by vertical interstitial channels measuring 40 μm × 8 μm × 3 μm, with a 100 μm spacing between the interstitial channels. This microchannel structure design enables direct cell contact between different channels, reproducing the interactions between cells in a real brain lymphoid system. Furthermore, previous organ-on-a-chip studies often used high-precision injection pumps to continuously perfuse culture medium within the channels at a uniform flow rate, providing shear force stimulation that mimics blood flow.

[0065] However, in the brain's lymphatic system, the blood flow velocity in arteries is not uniform but fluctuates periodically due to the contraction and relaxation of the heart, generating pulsatile blood flow. Simultaneously, this pulsatile flow pumps the solution in the periarterial spaces to the brain tissue and perivenous spaces, propelling the overall fluid flow and facilitating the drainage of cerebrospinal fluid and brain parenchymal metabolic products. Therefore, due to the unique characteristics of fluid flow in the brain's lymphatic system, arterial fluid flow cannot be simulated solely by a single flow velocity. In this organ-on-a-chip, by adjusting the infusion pump flow rate, the fluid in the arterial channels is perfused at a rate of 1 μL / min to achieve a volume of 60 nL, with cycles repeating every 1 second, achieving intermittent fluid flow similar to arterial pulsations. However, this improved flow stimulation leads to significant inter-channel crosstalk in cell distribution. Therefore, this invention improves the chip structure to address this flow velocity and cell distribution, adjusting the size of the small inter-channel gaps to 40 μm × 5 μm × 3 μm, effectively mitigating crosstalk between different cells in the main channels.

[0066] The in vitro lymphoid organ-on-a-chip constructed in this invention fills a gap in the field. Targeting the characteristics of fluid flow in brain lymphoid microarrays, it improves upon the uniform fluid flow velocity achieved in blood flow stimulation in previous organ-on-a-chip methods, realizing cyclic pulsed fluid flow stimulation. Furthermore, by optimizing the internal dimensions of the first-generation organ-on-a-chip, a second-generation lymphoid organ-on-a-chip model was developed, resolving the problem of cell crosstalk between different channels under intermittent flow stimulation. On the other hand, the model construction method is relatively simple and convenient, effectively shortening the model construction time and improving research efficiency. This construction method also has a certain degree of universality, allowing for expansion to the in vitro simulation of other human organs by changing cell types and culture methods, demonstrating broad application prospects. In addition, this in vitro lymphoid organ-on-a-chip exhibits high flexibility, allowing for cascading with various analytical systems, including confocal fluorescence imaging, mass spectrometry, and PCR sequencing, providing a convenient tool for the analysis of various biomolecules such as reactive oxygen species, various ions, proteins, and genes.

[0067] Example 1:

[0068] like Figure 1 As shown in the figure, this embodiment provides a method for constructing an in vitro brain lymphoid system chip, which can be used to simulate the molecular expression, basic structure and drainage function of the in vitro lymphoid system, and can be used for dynamic real-time monitoring of the lymphoid system response under various subsequent stimulation conditions.

[0069] S1. Microfluidic chip structure design:

[0070] like Figures 2-5 As shown, the first-generation microfluidic chip contains five channels, connected by multiple vertical small channels to ensure inter-cell communication between different channels. The five main channels are a vascular channel, a perivascular space channel, and a brain parenchyma channel. The vascular channel includes the two outermost arterial channels and a venous channel, where human umbilical artery endothelial cells directly adhere to the four inner walls of the channel for vascular simulation. The two adjacent channels are perivascular space channels, an important component of the lymphatic drainage system. The middle channel is the brain parenchyma channel, where astrocytes and neurons are dispersed on a three-dimensional gel scaffold for simulating the complex three-dimensional structure of the brain parenchyma. The second-generation chip improved the small channel structure, reducing its size 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.

[0071] Microfluidic chip structure design diagram. The dimensions of the vascular channels on both sides are 10000 μm × 1000 μm × 150 μm, the dimensions of the channels around the two adjacent blood vessels are 10000 μm × 200 μm × 150 μm, the dimensions of the channel in the middle of the brain parenchyma are 10000 μm × 2000 μm × 150 μm, the dimensions of the small channels connecting different channels are 40 μm × 8 μm × 3 μm, and the spacing between the channels is 100 μm.

[0072] S2. Construction of in vitro lymphoid organ-on-a-chip model:

[0073] First, human umbilical artery endothelial cells and human umbilical vein endothelial cells were added to the arterial and venous channels, respectively. Cells were then introduced into all four inner walls of the channels using a flip-chip method. Next, a mixture of astrocytes, neurons, and type I collagen was added to the brain parenchyma channel. After complete gel polymerization, the two cell types were spatially dispersed in three dimensions. Finally, a continuously perfused culture medium was introduced into both vascular channels. Through a period of fluid flow stimulation and co-culture of multiple cell types within different channels, the in vitro lymphoid organoid model was successfully constructed.

[0074] Figure 6 The diagram shown is a flowchart of the in vitro lymphoid organ-on-a-chip model construction process; optional, Day 1: 5×10 5 Human umbilical artery endothelial cells and human umbilical vein endothelial cells (1 × 10⁶ / mL) were introduced into the arterial and venous channels, respectively. After being placed upright in a cell culture incubator for 1 hour, the cells were reintroduced, and the chip was inverted for 1 hour of culture. Fresh culture medium was then introduced to remove adherent cells. On day 2, the endothelial cell culture medium was continuously perfused into the venous channel at a flow rate of 1 μL / min for 24 hours, while simultaneously being continuously perfused into the arterial channel with intermittent circulation for 24 hours, allowing the endothelial cells to form a complete and continuous monolayer on the four inner walls of the channel. On day 3, 1 × 10⁶ cells were used. 5 A mixed solution of astrocytes, neurons, and type I collagen per mL was introduced into the intermediate brain parenchyma channel and placed in a 37°C cell culture incubator to allow the two cell types to disperse evenly on the solidified gel scaffold. On day 4, the various cell types in the brain parenchyma channel and the vascular channel were co-cultured for 24 hours to form a complete in vitro lymphoid model. Figure 7 The change of flow velocity over time under uniform flow stimulus conditions. Figure 8 The flow velocity changes over time under intermittent cyclic flow stimulation conditions.

[0075] To characterize the successful formation of the complete lymphoid model, the lymphoid structure was observed using bright-field imaging, live and dead cell staining, and fluorescence three-dimensional reconstruction. Firstly, as... Figure 9The bright-field imaging shown indicates that the cells are growing well in the chip. Additionally, the live / dead cell staining image shows that most cells in the channels exhibit green fluorescence, such as... Figure 11 As shown, only a few cells exhibited red fluorescence, indicating that most cells maintained good viability in this microfluidic chip. Statistical analysis revealed that cell viability in the bilateral vascular channels and the central brain parenchyma channel were 98.23±1.41%, 97.51±2.06%, and 95.33±2.58%, respectively, all exceeding 95%. Figure 13 As shown. However, during co-culture, a small number of cells also appeared in the paravascular pathways, such as... Figure 9 As indicated by the red arrows, these cells likely arose because the flow stimulation in the arterial channels, after being adjusted from a uniform to an intermittent cyclic flow stimulation, not only pumped the periaqueductal fluid into adjacent channels but also caused crosstalk between different cells. Therefore, to mitigate the negative impacts of the improved flow rate adjustment, a more realistic lymphoid organ-on-a-chip was constructed, such as... Figure 10 , Figure 12 and Figure 14 As shown, we improved the first-generation chip by adjusting the size 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 brain parenchyma channels caused by intermittent fluid disturbance. In the second-generation lymphoid organ-on-a-chip, we found through live-dead cell staining imaging that no cells appeared in the paravascular channels, indicating that the structural optimization successfully inhibited the cross-channel migration of cells, and the constructed structure is more consistent with the real lymphoid system. In addition, the cell viability in the bilateral vascular channels and the intermediate brain parenchyma channel were 97.33±1.07%, 97.39±3.01%, and 96.95±2.76%, respectively, indicating that the structural change did not affect the cell viability in the chip.

[0076] like Figure 15 and Figure 16 As shown, a more realistic second-generation brain lymphoid organ-on-a-chip was then used for overall imaging. Through confocal fluorescence imaging of live and dead cells, it can be observed that the second-generation model exhibits cellular structures of blood vessels on both sides and brain parenchyma in the middle, consistent with the cell arrangement in a real brain lymphoid system. Furthermore, as... Figure 17 , Figure 18 and Figure 19 As shown, the three-dimensional reconstruction characterization of cells in the second-generation lymphoid organ-on-a-chip reveals that the endothelial cells in the vascular channels are arranged in a cuboid shape with a cavity in the middle, a structure very similar to the actual cavity-like structure of blood vessel walls. Figure 20As shown, the three types of cells in the intermediate brain parenchyma channels exhibit a three-dimensional spatial dispersion, similar to the three-dimensional structure of tissues in the brain parenchyma. Notably, compared to the first-generation lymphoid organ-on-a-chip, the second-generation organ-on-a-chip did not exhibit cell migration crosstalk between different channels in the perivascular space channels, a key aspect of in vitro replication of the lymphoid system. The improved structure of the second-generation organ-on-a-chip not only successfully simulated the pulse oscillations of arteries in the brain's lymphoid system but also improved upon traditional chips, enabling the structure to adapt to the cell crosstalk problem between different channels caused by the overall fluid flow in the lymphoid system. Furthermore, the growth of astrocytes in channels connected to the perivascular space replicated the pseudopodia extended from astrocytes in real lymphoid structures, one of the most important structures in lymphoid systems. These results demonstrate that the constructed in vitro lymphoid system model not only ensures the normal growth, survival, and basic molecular expression of various cells but also, to a certain extent, replicates the key physiological structures of the real lymphoid system.

[0077] The above description is merely a preferred embodiment of this practice and is not intended to limit the scope of this practice. Various modifications and variations can be made to this practice by those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this practice should be included within the protection scope of this practice.

Claims

1. A method for constructing an in vitro brain-like lymphatic system chip, characterized in that, include: The microfluidic channels in the chip are subjected to rinsing, sterilization, washing, and cell culture. In the processed microfluidic channels, vascular channel cells and brain parenchyma channel cells were seeded. In the vascular channel, perfusion is performed at a preset flow rate and at preset intervals to provide shear force stimulation that mimics pulsed blood flow. After continuous perfusion, in vitro cerebral blood vessels are formed. During the continuous perfusion of the vascular channel, the culture medium in the brain parenchyma channel is replaced at intervals. Through co-culture of various cells in vascular channels and brain parenchyma channels, an in vitro lymphoid model is formed through direct contact between different cells or the transmission of signal molecules. The microfluidic channels in the chip were rinsed with deionized water and ethanol respectively. After the microfluidic channels were free of impurities, the chip was sterilized by UV irradiation. After the sterilization time was preset, the microfluidic channels were rinsed with PBS solution. 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. Rinse all microfluidic channels with PBS solution to remove excess fibronectin and collagen mixture; Cell seeding includes: digesting pre-cultured human brain microvascular endothelial cells with trypsin to adjust cell density; thoroughly rinsing the pretreated microfluidic channels with cell culture medium, and then introducing the digested endothelial cells into the arterial and venous channels respectively; placing the chip upright in a cell culture incubator for a preset time, then reintroducing cells into the arterial and venous channels, and finally inverting the chip in the cell culture incubator for a preset time, introducing fresh culture medium, and removing non-adherent cells; Brain parenchymal channel cell seeding includes: digesting astrocytes and neurons, adjusting cell density, and thoroughly mixing and pre-cooling the cells; The length × width × height dimensions of the interstitial channel used to connect different vascular channels, as well as the interstitial channel between the vascular channel and the brain parenchyma channel, are 40 μm × 5 μm × 3 μm.

2. The method for constructing an in vitro brain-like lymphatic system chip as described in claim 1, characterized in that, Add 100 μL of type I collagen to 6 μL of 0.1 mol / L sodium hydroxide solution and mix well. Then add 12 μL of 10×PBS solution and mix thoroughly with 380 μL of cell mixture to obtain cell gel mixture. The gel mixture was introduced into the brain parenchyma channels to culture cells on the chip.

3. The method for constructing an in vitro brain-like lymphatic system chip as described in claim 1, characterized in that, In the vascular channel, a flow rate of 1 μL / min was used to perfuse 500 nL of volume every 1 second to provide shear force stimulation that mimics pulsatile blood flow; after continuous perfusion for 24 hours, the growth status of endothelial cells was observed to confirm the basic formation of cerebral blood vessels in vitro.

4. The method for constructing an in vitro brain-like lymphatic system chip as described in claim 3, characterized in that, During the vascular channel process, the culture medium in the brain parenchyma channel is changed every 6 hours to ensure the supply of nutrients required for normal cell growth and the removal of metabolic waste.

5. An in vitro brain-like lymphatic system chip, characterized in that, The chip is obtained by the in vitro brain-like lymphatic system chip construction method as described in any one of claims 1-4, comprising a substrate and an upper polydimethylsiloxane layer, wherein the substrate and the upper polydimethylsiloxane layer are bonded together by silicon-oxygen bonds.

6. The in vitro brain-like lymphatic system chip as described in claim 5, characterized in that, The dimensions of the gap channel (length × width × height) have been adjusted to 40 μm × 5 μm × 3 μm.

Images