Dynamic micro-fluidic chip device for researching glioma CAR-T (Chimeric Antigen Receptor-T) therapy as well as preparation method and application of dynamic micro-fluidic chip device

Abstract

The invention relates to the technical field of micro-fluidic chips, in particular to a dynamic micro-fluidic chip device for studying glioma CAR-T therapy, a preparation method and application of the dynamic micro-fluidic chip device. The preparation method comprises the following steps: manufacturing the micro-fluidic chip; reconstruction of nervous tissues; reconstructing a blood brain barrier; and simulating a brain glioma microenvironment. The dynamic micro-fluidic chip device comprises a micro-fluidic chip; the micro-fluidic chip comprises a blood vessel channel, a brain parenchyma channel, a tumor channel and a subarachnoid space channel which are sequentially arranged from left to right; each of the blood vessel channel, the brain parenchyma channel, the tumor channel and the subarachnoid space channel comprises a cell culture chamber, and one side of each cell culture chamber is provided with a sample inlet; and the other sides of the cell culture chambers of the blood vessel channel and the subarachnoid space channel are respectively and correspondingly provided with an outlet. The application comprises the following steps: CAR-T cell infusion; carrying out transmembrane migration observation; and biomarker detection. According to the dynamic micro-fluidic chip device, the preparation method and the application, more accurate in-vitro evaluation of the CAR-T cell therapy is realized.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic chip technology, and in particular to a dynamic microfluidic chip device, a preparation method and an application thereof for studying glioma CAR-T therapy. Background Art

[0002] Glioma is the main primary intracranial tumor in the central nervous system, accounting for 75% of malignant brain tumors in adults. Its standard treatment includes surgical resection of the tumor, radiotherapy and chemotherapy. However, the prognosis of glioma patients receiving conventional treatment is poor. With the development of immunotherapy, immunotherapy based on chimeric antigen receptor T cells (CAR-T) has brought new hope for the treatment of glioma. CAR-T cells that have been expanded in vitro and genetically engineered can target and lyse cells carrying relevant tumor antigens, among which intravenous delivery is the simplest and most common way of administration. Although CAR-T cell therapy has made significant progress in the field of cancer treatment, its application in malignant tumors such as glioma is still limited by physical barriers such as the blood-brain barrier (BBB).

[0003] The blood-brain barrier is a complex structure composed of brain capillary walls and glial cells that strictly regulates the ability of immune cells to transport into the central nervous system. Therefore, the efficacy of CAR-T cell therapy depends particularly on whether therapeutic immune cells can effectively cross the blood-brain barrier to induce anti-tumor responses in the brain. The existence of the blood-brain barrier makes it challenging to accurately target and deliver CAR-T cells in brain tissue and maximize the therapeutic effect.

[0004] In order to simulate the blood-brain barrier model under physiological or pathological conditions in vitro, a variety of microfluidic chips have emerged. However, existing microfluidic chips still have shortcomings in simulating the complex physiological characteristics of the blood-brain barrier in the natural brain: CN106811408A discloses a method for establishing a three-dimensional blood-brain barrier model based on a microfluidic chip. This microfluidic chip solves the problems of secondary inoculation and long consumption time in non-contact co-culture between cells by constructing a collagen channel layer, but its co-culture system only contains brain astrocytes and brain microvascular endothelial cells, and cannot reproduce the multi-cellular, three-dimensional complex structural characteristics of the natural blood-brain barrier; CN108823145A discloses an in vitro construction method for simulating a blood-brain barrier by generating human brain microangiogenesis. This construction method sets a culture medium drop between an upper chamber and a lower chamber so that the culture medium maintains fluidity for a certain period of time due to a potential energy difference, but the liquid pressure depends on the potential energy difference. CN215162826U discloses a blood-brain barrier microfluidic chip, which promotes the flow of culture fluid through periodic air pressure control to simulate the fluid environment in blood vessels. However, the microporous membrane between the culture chambers of the device hinders the interaction between cells and affects the barrier function of the blood-brain barrier model.

[0005] After using microfluidic chips as a technical platform to establish a blood-brain barrier model under pathological conditions, it has great potential for further application in disease treatment. However, there is still a gap in the research on using microfluidic chips to simulate the physiological conditions of blood-brain barrier and tumor cells in CAR-T cell therapy in vitro. Therefore, it is necessary to develop a dynamic microfluidic chip device for blood-brain barrier-glioma CAR-T cell therapy to achieve more accurate in vitro evaluation of CAR-T cell therapy, and lay the foundation for the precise targeted delivery of CAR-T cells in brain tissue and the maximization of therapeutic effects. Summary of the invention

[0006] The first object of the present invention is to overcome the defects and shortcomings of the prior art and provide a method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy, which prepares a microfluidic chip that can simulate the glioma microenvironment.

[0007] The second object of the present invention is to provide a dynamic microfluidic chip device for studying glioma CAR-T therapy, which is used to simulate the glioma microenvironment.

[0008] The third object of the present invention is to provide an application of a dynamic microfluidic chip device for studying glioma CAR-T therapy, which provides a new research method for in vitro evaluation of CAR-T cell therapy for treating glioma.

[0009] The purpose of the present invention can be achieved through the following technical solutions: A method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy comprises the following steps: S1. Manufacturing of microfluidic chips: Computer-aided design software is used to design the channels of the microfluidic chip, including vascular channels, brain parenchyma channels, tumor channels and subarachnoid channels, and a master mold of the microfluidic chip is obtained by 3D printing technology; polydimethylsiloxane material is poured onto the master mold of the microfluidic chip for curing, and then demolding and punching are performed to finally obtain a microfluidic chip with the desired channel structure; S2. Reconstruction of neural tissue: Prepare a suspension of astrocytes and microglia; evenly mix the above two types of glial cells with soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; place the microfluidic chip in a low-temperature environment, then inject the gel-glial cell mixture into the brain parenchyma channel and place it in a cell culture incubator for gelation to achieve three-dimensional culture of glial cells; S3. Reconstruction of blood-brain barrier: prepare suspensions of human brain microvascular endothelial cells and human pericytes; mix the two cell suspensions and inject them into the vascular channel of the microfluidic chip, and co-culture with the established neural tissue; after a certain period of co-culture, tilt the microfluidic chip to allow the human brain microvascular endothelial cells and human pericytes to adhere to the interface between the vascular channel and the brain parenchyma channel, and dynamically incubate to form a blood-brain barrier microstructure; S4. Simulation of glioma microenvironment: prepare a glioma cell suspension and mix it with a soluble basement membrane (BME) hydrogel prepolymer to form a gel-glioma cell mixture; inject the gel-glioma cell mixture into the tumor channel of the microfluidic chip; prepare a fibroblast suspension at the same time, inject it into the subarachnoid channel, then tilt the microfluidic chip to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubate to form pia mater microstructures.

[0010] As a preferred embodiment, in step S2, the reconstruction of the neural tissue includes the following steps: S21. Prepare neural cell suspension: Use Eagle medium supplemented with fetal bovine serum (FBS) and EMEM medium to revive and culture immortalized human brain astrocyte cell line and immortalized human brain microglia cell line respectively; when the cell confluence reaches 80-90%, collect astrocyte suspension and microglia suspension respectively, and then evenly mix the collected astrocyte suspension and microglia suspension to form a neural cell mixed suspension; S22. Inoculating neural cells: uniformly mix the prepared mixed suspension of glial cells and soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; place the microfluidic chip on a cold bag, and then inject the gel-glial cell mixture into the brain parenchyma channel; after the injection, transfer the injected microfluidic chip to a cell culture incubator for constant temperature gelation; S23. Cultivate neural cells: After gelation, re-prepare Eagle medium and EMEM medium without adding FBS, and mix them to obtain serum-free glial cell medium; inject the prepared serum-free glial cell medium into the blood channel and subarachnoid channel respectively; replace the medium once a day within 5 days before the reconstruction of the blood-brain barrier.

[0011] As a preferred embodiment, in step S3, the reconstruction of the blood-brain barrier comprises the following steps: S31. Prepare blood-brain barrier cell suspension: use ECM culture medium and PM culture medium to resuscitate and culture immortalized human brain microvascular endothelial cell line and immortalized human brain pericyte cell line respectively; when the cell confluence reaches 80-90%, collect human brain microvascular endothelial cell suspension and human pericyte suspension respectively, and then evenly mix the collected human brain microvascular endothelial cell suspension and human pericyte suspension to form a blood-brain barrier cell mixed suspension; S32, inoculating blood-brain barrier cells: injecting the prepared mixed suspension of blood-brain barrier cells into the vascular channel to co-culture it with the neural tissue reconstructed in step S2; after 4 days of co-culture, tilting the microfluidic chip to allow human brain microvascular endothelial cells and human pericytes to adhere to the interface between the blood channel and the brain parenchymal channel, simulating the real structure of the blood-brain barrier in vivo; S33. Cultivate blood-brain barrier cells: without adding FBS, reconfigure ECM medium and PM medium, and mix them to obtain serum-containing endothelial cell medium; inject the prepared serum-containing endothelial cell medium into the blood channel, and use the flow effect to remove cells that have not successfully adhered and any possible debris; continue dynamic culture for 3 days, during which time replace the serum-containing endothelial cell medium in the blood channel and the serum-free glial cell medium in the subarachnoid channel daily.

[0012] As a preferred embodiment, in step S4, the reconstruction of the neural tissue includes the following steps: S41. Prepare brain glioma cell suspension: Use DMEM medium supplemented with 5% FBS and 1% penicillin / streptomycin solution to revive and culture immortalized human glioma cell line; when the cell confluence reaches 80-90%, collect the brain glioma cell suspension; S42. Inoculating glioma cells: uniformly mix the prepared glioma cell suspension and BME hydrogel prepolymer to form a gel-glioma cell mixture; place the microfluidic chip on a cold bag, and then inject the gel-glioma cell mixture into the tumor channel; after the injection, transfer the injected microfluidic chip to a cell culture incubator for constant temperature gelation; after gelation, inject serum-free glial cell culture medium into the subarachnoid channel, and continue to culture for 1-2 days, during which the culture medium is replaced once a day to maintain the growth state of the cells; S43. Construction of the pia mater: Use DMEM medium supplemented with 2 mM / L glutamine and 10% FBS to revive and culture immortalized human dermal fibroblast cell line; when the cell confluence reaches 80-90%, collect the fibroblast suspension; inject the fibroblast suspension into the subarachnoid channel of the microfluidic chip; tilt the microfluidic chip to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel; continue incubation to promote the fibroblasts to form stable pia mater microstructures.

[0013] Preferably, in step S42, replacing the culture medium includes the following steps: connecting a hose to the input end of the subarachnoid channel and connecting it to an automatic injection pump; injecting serum-free glial cell culture medium through the automatic injection pump to simulate the dynamic flow environment of cerebrospinal fluid in vivo; adjusting the flow rate of the automatic injection pump to ensure that the shear stress is controlled within 0.1dyn / cm² to truly reproduce the flow characteristics of cerebrospinal fluid in vivo.

[0014] A dynamic microfluidic chip device for studying glioma CAR-T therapy, comprising a microfluidic chip prepared by the above method; the microfluidic chip comprises a vascular channel, a brain parenchyma channel, a tumor channel and a subarachnoid space channel arranged in sequence from left to right; each of the vascular channel, the brain parenchyma channel, the tumor channel and the subarachnoid space channel comprises a cell culture chamber with an injection port on one side; the cell culture chambers of the vascular channel and the subarachnoid space channel have outflow ports correspondingly arranged on the other sides; The vascular channel is used to co-culture human brain microvascular endothelial cells and human pericytes to complete the reconstruction of the blood-brain barrier and the simulation of blood flow; The brain parenchyma channel is used for three-dimensional culture of astrocytes and microglia; The tumor channel is used for three-dimensional culture of brain glioma cells; The subarachnoid channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and the simulation of flowing cerebrospinal fluid.

[0015] As a preferred embodiment, the cell culture chamber of the tumor channel is a semicircular, circular or elliptical structure; the cell culture chamber of the subarachnoid channel is an arc-shaped tubular structure.

[0016] As a preference, an automatic injection pump is further included, wherein the output end of the automatic injection pump is connected to the microfluidic chip to inject a stable flowing liquid into the microfluidic chip and quantitatively control the flow rate of the injected liquid.

[0017] As a preferred embodiment, the automatic injection pump includes an injection head, a syringe, an injection hose and a base with a built-in driving motor; the injection head, the syringe and the injection hose are arranged in sequence, and the injection port on the vascular channel or the subarachnoid space channel is connected to the syringe through the injection hose; the syringe is fixedly mounted on the base, and the driving motor is transmission-connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to perform injection.

[0018] An application of the dynamic microfluidic chip device as described above for studying glioma CAR-T therapy comprises the following steps: S1. CAR-T cell infusion: A mixed solution of fluorescently labeled CAR-T cells and serum-containing endothelial cell culture medium was injected into the blood channel to simulate intravenous delivery of CAR-T cell therapy; S2. Transmembrane migration observation: Use confocal real-time imaging tracking technology to visualize the capture, adhesion and extravasation process of CAR-T cells and calculate related parameters; S3. Biomarker detection: The anti-tumor ability of CAR-T cells is evaluated by measuring the levels of specific biomarkers in the effluent of the subarachnoid channel.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The present invention forms a four-channel design by adding a subarachnoid channel. This design not only simulates the blood-brain barrier and cerebrospinal fluid flow environment in the body more comprehensively, but also can better reflect the restrictive effect of the pia mater on the growth, spread or metastasis of gliomas; and the present invention sets up a subarachnoid channel for monolayer culture of fibroblasts to complete the reconstruction of the pia mater, and simulates the dynamic microenvironment of the cerebrospinal fluid in the body by injecting serum-free glial cell culture medium, which enhances the complexity and authenticity of the model.

[0020] 2. The present invention sets the vascular channel into a slender parallel structure and the tumor channel into a wide and fat semicircle. The wide and fat semicircular tumor channel can better simulate the cerebral hemisphere and is closer to the growth environment of glioma cells in the ventricle under physiological conditions. While the structure of the vascular channel facilitates the precise control of blood flow shear force, the structure of the tumor channel is more suitable for the three-dimensional growth of glioma cells, and is conducive to timely feedback on the therapeutic effect, which helps to improve the authenticity and reliability of the experimental results.

[0021] 3. The present invention utilizes an automatic injection pump to achieve highly accurate laminar liquid delivery and adjust the shear stress range according to different pathological conditions; in addition, a detailed wall shear stress calculation formula is provided to ensure that the experimental conditions are close to the actual situation in vivo.

[0022] 4. The CAR-T cells of the present invention can migrate across the membrane under different fluid shear forces, and visualize this process through confocal real-time imaging tracking technology. At the same time, cerebrospinal fluid biomarker detection and analysis, such as VEGF, HGF, IL-6 and other level determination, are introduced to evaluate the anti-tumor ability of CAR-T cells. This method provides a more intuitive and quantitative evaluation method.

[0023] 5. The present invention constructs a more complete and realistic blood-brain barrier-glioma co-culture system, which shows higher accuracy and efficiency, especially in the study of CAR-T cell therapy. It can not only simulate complex physiological and pathological environments, but also provide a reliable platform for drug screening, thereby accelerating the development of new anti-cancer treatment strategies. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 is a top view of the microfluidic chip of the present invention; Figure 2 is a schematic structural diagram of a dynamic microfluidic chip device of the present invention; Figure 3 It is a schematic diagram of the structure of the microfluidic chip of the present invention; Figure 4 It is a schematic diagram of the preparation process of the microfluidic chip of the present invention; Figure 5 It is a structural simulation diagram of cell growth in the microfluidic chip of the present invention.

[0025] Among them: 1: blood channel, 2: brain parenchyma channel, 3: tumor channel, 4: subarachnoid channel, 5: blood channel inlet, 6: brain parenchyma channel inlet, 7: tumor channel inlet, 8: subarachnoid channel inlet, 9: blood channel outflow port, 10: subarachnoid channel outflow port, 11: injection head, 12: syringe, 13: base, 14: microscope slide lower layer, 15: PDMS upper layer. DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0027] As shown in the figure, a dynamic microfluidic chip device for studying glioma CAR-T therapy includes a microfluidic chip, which includes a vascular channel, a brain parenchyma channel, a tumor channel, and a subarachnoid space channel arranged from left to right; each of the vascular channel, the brain parenchyma channel, the tumor channel, and the subarachnoid space channel includes a cell culture chamber with an injection port on one side; the cell culture chambers of the vascular channel and the subarachnoid space channel have outflow ports correspondingly arranged on the other side; The vascular channel is used to co-culture human brain microvascular endothelial cells and human pericytes to complete the reconstruction of the blood-brain barrier and the simulation of blood flow; The brain parenchyma channel is used for three-dimensional culture of astrocytes and microglia; The tumor channel is used for three-dimensional culture of brain glioma cells; The subarachnoid channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and the simulation of flowing cerebrospinal fluid.

[0028] Wherein, the vascular channel comprises a vascular channel sample inlet, a vascular channel outflow port and a vascular channel cell culture chamber; The vascular channel inlet and outlet are both circular with a diameter of 2 mm. The vascular channel culture chamber is a strip-shaped tubular structure with a length of 10 mm, a width of 1 mm, and a height of 400 μm. The interface between the vascular channel and the brain parenchyma channel is the blood-brain barrier interface, and the length of the blood-brain barrier interface is 5 mm.

[0029] The brain parenchyma channel comprises a brain parenchyma channel sample inlet and a brain parenchyma channel cell culture chamber; The inlet of the brain parenchyma channel is a circle with a diameter of 2 mm, and the cell culture chamber of the brain parenchyma channel is a strip-shaped tubular structure with a length of 8 mm, a width of 2 mm, and a height of 100 μm.

[0030] The tumor channel comprises a tumor channel injection port and a tumor channel cell culture chamber; The tumor channel sample inlet is a circle with a diameter of 2 mm, and the tumor channel cell culture chamber is a semicircular structure with a diameter of 6 mm and a height of 100 μm.

[0031] The subarachnoid space channel comprises a subarachnoid space channel sample inlet, a subarachnoid space channel outflow outlet and a subarachnoid space channel culture chamber; The glioma cell mixed solution inlet and the glioma cell mixed solution outlet are circular with a diameter of 2 mm. The fibroblast culture chamber is an arc-shaped tubular structure with an inner diameter of 6 mm, an outer diameter of 8 mm, and a height of 400 μm. The interface between the tumor channel and the subarachnoid space channel is the pia mater interface, and the length of the pia mater interface is 1.6πmm.

[0032] Furthermore, the cell culture chamber of the tumor channel is a semicircular, circular or elliptical structure; and the cell culture chamber of the subarachnoid channel is an arc-shaped tubular structure.

[0033] As a preferred implementation scheme, the dynamic microfluidic chip device of this embodiment also includes an automatic injection pump, the output end of which is connected to the microfluidic chip to inject a stable flowing liquid into the microfluidic chip and quantitatively control the flow rate of the injected liquid.

[0034] Specifically, the automatic injection pump includes an injection head, a syringe, an injection hose and a base with a built-in driving motor; the injection head, the syringe and the injection hose are arranged in sequence, and the solution inlet on the vascular channel or the subarachnoid space channel is connected to the syringe through the injection hose; the syringe is fixedly mounted on the base, and the driving motor is transmission-connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to perform injection.

[0035] This embodiment controls the progress of the syringe, thereby controlling the flow rate of the liquid injected into the blood vessel channel or the subarachnoid space channel, thereby achieving regulation of the magnitude of the fluid shear force.

[0036] Specifically, the normal physiological shear effect at the blood-brain barrier is about 2dyn / cm². Considering the impact of different stages of tumor progression on the shear stress of blood flow at the blood-brain barrier, the shear stress applied to simulate the blood flow under pathological conditions in the vascular channel is set in the range of 1-6dyn / cm²; the space formed inside the vascular channel is a rectangular parallelepiped. Based on Newtonian fluid mechanics, the direction of the shear stress should be a parabola in the vertical direction of the fluid flow. The shear stress at the center of the fluid is zero, and the closer to the wall, the greater the shear stress. Since the diameter of the cells used is about 10μm, which is much smaller than the cavity height of 400μm, it can be considered that the shear stress on the center of mass of the cell is equal to the wall shear stress of the flow cavity. The wall shear stress calculation formula is as follows:

[0037] in, τ w is the wall shear stress, in dyn / cm 2 ; Q is the average flow rate of the fluid flowing through the cavity, in mL / min; μ is the viscosity coefficient of the fluid, in mPa·s; h and w are the height and width of the cavity cross section, respectively, in cm.

[0038] Through the fluid shear stress set in the experiment, the flow rate that needs to be set for the automatic injection pump is calculated and the experiment is carried out.

[0039] A method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy comprises the following steps: S1. Manufacturing of microfluidic chips: Computer-aided design software is used to design the channels of the microfluidic chip, including vascular channels, brain parenchyma channels, tumor channels and subarachnoid space channels, and the master mold of the microfluidic chip is obtained by 3D printing technology; polydimethylsiloxane material is poured onto the master mold of the microfluidic chip for curing, and then demolding and punching are performed to finally obtain a microfluidic chip with the desired channel structure.

[0040] Specifically, in step S1, the manufacture of the microfluidic chip includes the following steps: S11. Master mold design and manufacturing: Use AutoCAD software to design the channel of the microfluidic chip and obtain the design model; use a light-curing 3D printer to manufacture the resin master mold of the microfluidic chip, including spraying liquid photosensitive resin layer by layer according to the design model, UV curing, cleaning, and surface treatment; S12, preparation of PDMS chip: polydimethylsiloxane (PDMS) prepolymer and curing agent are mixed in a mass ratio of 10:1 to obtain a PDMS solution; the mixed PDMS solution is vacuumed to remove possible bubbles to ensure the uniformity and transparency of the final product; the vacuumed PDMS solution is poured into the prepared resin master mold, and the bubbles are further removed by a vacuum pump to ensure that there are no bubbles remaining inside the PDMS; the resin master mold filled with PDMS is placed in an oven for heating and curing at 65°C to 80°C for 2 to 4 hours until the PDMS is completely cured; after the curing is completed, the cured PDMS is peeled off from the resin master mold, and a corresponding injection port and outflow port are made on the PDMS using a puncher to ensure that the pore size meets the experimental requirements to obtain a PDMS upper layer with a channel structure; S13, assembly and bonding of microfluidic chip: plasma treatment is performed on the PDMS upper layer with the channel structure and the pre-cleaned microscope slide lower layer respectively to enhance the adhesion between the two; immediately after the plasma treatment, the PDMS upper layer is tightly attached to the microscope slide lower layer to form an irreversible bond to seal the channel structure of the microfluidic chip; S14. Sterilization and disinfection: The prepared microfluidic chip is sterilized at high temperature and high pressure to ensure a sterile environment, which is suitable for cell culture and other biological experiments. Before cell inoculation, the microfluidic chip is placed in a drying oven at 80°C for at least 24 hours to restore its hydrophobicity. It is disinfected by ultraviolet irradiation for 30 minutes to ensure that the surface of the microfluidic chip is sterile.

[0041] Ensure that the prepared microfluidic chip has high precision, good mechanical properties and a sterile environment, providing a reliable platform for subsequent experiments.

[0042] S2. Reconstruction of neural tissue: prepare a suspension of astrocytes and microglia; evenly mix the above two types of glial cells with soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; place the microfluidic chip in a low-temperature environment, then inject the gel-glial cell mixture into the brain parenchyma channel and place it in a cell culture incubator for gelation to achieve three-dimensional culture of glial cells.

[0043] Specifically, in step S2, the reconstruction of the neural tissue includes the following steps: S21. Prepare neural cell suspension: Use Eagle medium supplemented with 10% fetal bovine serum (FBS) to revive and culture immortalized human brain astrocyte cell line, and use EMEM medium supplemented with 10% fetal bovine serum (FBS) to revive and culture immortalized human brain microglia cell line; when the cell confluence reaches 80-90%, collect the astrocyte suspension and microglia suspension respectively, and then evenly mix the collected astrocyte suspension and microglia suspension at a molar ratio of 4:1-5:1 to form a neural cell mixed suspension; S22. Inoculation of neural cells: the prepared mixed suspension of glial cells and the soluble basement membrane (BME) hydrogel prepolymer are uniformly mixed at a volume ratio of 4:1 to form a gel-glial cell mixture; the microfluidic chip is placed on a cold bag to maintain a low temperature environment to prevent the gel from solidifying prematurely, and then the gel-glial cell mixture is injected into the brain parenchyma channel through the brain parenchyma channel injection port; after the injection is completed, the injected microfluidic chip is immediately transferred to the cell culture plate and moved to a cell culture incubator for constant temperature gelation; the conditions in the cell culture incubator are set to 37°C and 5% CO2 to ensure that the cells can gel in a suitable environment; S23. Cultivate neural cells: After gelation, reconfigure the above two glial cell culture media (Eagle medium and EMEM medium) without adding FBS, and mix them in corresponding proportions to obtain serum-free glial cell culture media; inject the prepared serum-free glial cell culture media into the blood channel and subarachnoid channel respectively; within 5 days before the reconstruction of the blood-brain barrier, replace the culture media once a day to ensure that the cells are always in a fresh and suitable growth environment, gradually complete the reconstruction of neural tissue, effectively promote the three-dimensional culture of glial cells, and also lay a solid foundation for the subsequent construction of a more complex blood-brain barrier model.

[0044] S3. Reconstruction of the blood-brain barrier: prepare a suspension of human brain microvascular endothelial cells and human pericytes; mix the two cell suspensions and inject them into the vascular channel of the microfluidic chip, and co-culture with the established neural tissue; after a certain period of co-culture, tilt the microfluidic chip to allow the human brain microvascular endothelial cells and human pericytes to adhere to the interface between the vascular channel and the brain parenchymal channel, and dynamically incubate to form a blood-brain barrier microstructure.

[0045] Specifically, in step S3, the reconstruction of the blood-brain barrier includes the following steps: S31. Prepare blood-brain barrier cell suspension: use ECM culture medium to revive and culture immortalized human brain microvascular endothelial cell line, and use PM culture medium to revive and culture immortalized human brain pericyte line; when the cell confluence reaches 80-90%, collect human brain microvascular endothelial cell suspension and human pericyte suspension respectively, and then evenly mix the collected human brain microvascular endothelial cell suspension and human pericyte suspension at a ratio of 9:1 to form a blood-brain barrier cell mixed suspension; S32, inoculating blood-brain barrier cells: injecting the prepared mixed suspension of blood-brain barrier cells into the vascular channel through the vascular channel injection port, so that it is co-cultured with the neural tissue reconstructed in step S2, and the conditions in the cell culture incubator are maintained at 37°C and 5% CO2 to continuously provide a suitable growth environment for the cells; after 4 days of co-culture, gently tilting the microfluidic chip so that human brain microvascular endothelial cells and human pericytes can adhere to the side wall of the hydrogel in the brain parenchymal channel, that is, adhere to the interface between the blood channel and the brain parenchymal channel, simulating the real structure of the blood-brain barrier in vivo; S33. Cultivate blood-brain barrier cells: without adding FBS, reconfigure the above two blood-brain barrier cell culture media (ECM culture medium and PM culture medium), and mix them in corresponding proportions to obtain serum-containing endothelial cell culture medium; inject the prepared serum-containing endothelial cell culture medium into the blood channel through the vascular channel inlet, and use the flow effect to remove cells that have not successfully adhered and any possible debris; continue dynamic culture for 3 days, during which time replace the serum-containing endothelial cell culture medium in the blood channel and the serum-free glial cell culture medium in the subarachnoid channel daily to ensure that the cells are always in a fresh and suitable growth environment, and finally form a stable blood-brain barrier microstructure, providing a reliable model basis for subsequent experiments.

[0046] S4. Simulation of glioma microenvironment: prepare a glioma cell suspension and mix it with a soluble basement membrane (BME) hydrogel prepolymer to form a gel-glioma cell mixture; inject the gel-glioma cell mixture into the tumor channel of the microfluidic chip; prepare a fibroblast suspension at the same time, inject it into the subarachnoid channel, then tilt the microfluidic chip to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubate to form pia mater microstructures.

[0047] Specifically, in step S4, the reconstruction of the neural tissue includes the following steps: S41. Prepare brain glioma cell suspension: Use DMEM medium supplemented with 5% FBS and 1% penicillin / streptomycin solution to revive and culture immortalized human glioma cell line; when the cell confluence reaches 80-90%, collect the brain glioma cell suspension; S42. Inoculation of glioma cells: Evenly mix the prepared glioma cell suspension and BME hydrogel prepolymer to form a gel-glioma cell mixture; place the microfluidic chip on a cold bag to maintain a low temperature environment to prevent the gel from solidifying prematurely, and then inject the gel-glioma cell mixture into the tumor channel through the tumor channel injection port; after the injection is completed, immediately transfer the injected microfluidic chip to the cell culture plate and move it to a cell culture incubator for constant temperature gelation; the conditions in the cell culture incubator are set to 37°C and 5% CO2 to ensure that the cells can gel in a suitable environment; after gelation, inject serum-free glial cell culture medium into the subarachnoid channel and continue to culture for 1-2 days, during which the culture medium is replaced once a day to maintain the growth state of the cells.

[0048] The dynamic flow environment of cerebrospinal fluid in vivo is simulated by regularly injecting serum-free glial cell culture medium into the injection port of the subarachnoid channel to replace the culture medium. The simulation method of the dynamic cerebrospinal fluid environment is as follows: a hose is connected to the injection port of the subarachnoid channel and connected to an automatic injection pump; serum-free glial cell culture medium is injected through the automatic injection pump to simulate the dynamic flow environment of cerebrospinal fluid in vivo; the flow rate of the automatic injection pump is adjusted to ensure that the shear stress is controlled within 0.1dyn / cm² to truly reproduce the flow characteristics of cerebrospinal fluid in vivo.

[0049] S43. Construction of the pia mater: Use DMEM medium supplemented with 2 mM / L glutamine and 10% FBS to revive and culture immortalized human dermal fibroblast cell line; when the cell confluence reaches 80-90%, collect the fibroblast suspension; inject the fibroblast suspension into the subarachnoid channel of the microfluidic chip through the subarachnoid channel inlet; tilt the microfluidic chip to allow the fibroblasts to adhere to the side wall of the hydrogel in the tumor channel, that is, to the interface between the tumor channel and the subarachnoid channel; continue incubation to promote the fibroblasts to form a stable pia mater microstructure.

[0050] A dynamic microfluidic chip device for studying glioma CAR-T therapy is used in the study of glioma CAR-T therapy, comprising the following steps: S1. CAR-T cell infusion: A mixed solution of fluorescently labeled CAR-T cells and serum-containing endothelial cell culture medium is injected into the blood channel to simulate the intravenous delivery of CAR-T cell therapy.

[0051] In step S1, CAR-T cell infusion, the specific operation steps are as follows: S11, connecting a hose to the blood channel inlet of the microfluidic chip and connecting it to an automatic injection pump to ensure that the flow rate and shear stress can be accurately controlled; S12, mixing the fluorescently labeled (e.g., using CellTracker™ Deep Red) CAR-T cells with serum-containing endothelial cell culture medium to form a cell mixed solution for infusion; S13. Inject the mixed solution into the blood channel of the microfluidic chip through an automatic injection pump to simulate intravenous delivery of CAR-T cell therapy; by adjusting the flow rate of the automatic injection pump and controlling different shear stresses, explore the effects of different fluid shear forces on blood-brain barrier permeability, CAR-T cell transmembrane migration, and tumor microenvironment construction; S14. After 2 hours of dynamic incubation, fresh serum-containing endothelial cell culture medium was injected into the blood channel to remove CAR-T cells that failed to adhere or migrate successfully to ensure the accuracy of subsequent observations.

[0052] S2. Transmembrane migration observation: Use confocal real-time imaging tracking technology to visualize the capture, adhesion, and extravasation processes of CAR-T cells and calculate related parameters.

[0053] In step S2, transmembrane migration observation, the specific operation steps are as follows: S21. Use confocal real-time imaging tracking technology to analyze the behavior of CAR-T cells using software such as Imaris and ImageJ. Since CAR-T cells are dyed red (CellTracker™ Deep Red), the trajectory, adhesion, and extravasation process of a single CAR-T cell captured on the blood-brain barrier wall can be clearly observed; S22. Calculate the flow rate of CAR-T cells; quantify the percentage of CAR-T cells that adhere to or migrate across the blood-brain barrier from all perfused cells during incubation to provide quantitative data support for evaluating the transmembrane migration ability of CAR-T cells.

[0054] S3. Biomarker detection: The anti-tumor ability of CAR-T cells is evaluated by measuring the levels of specific biomarkers in the effluent of the subarachnoid channel.

[0055] In step S3, biomarker detection, the specific operation steps for different grades of glioma are as follows: Low-grade glioma: Collect the effluent from the fluid injection port of the subarachnoid channel as a simulated cerebrospinal fluid sample; select VEGF and HGF in the effluent from the subarachnoid channel as key biomarkers; use human VEGF ELISA kit and human HGF ELISA kit for detection.

[0056] For low-grade gliomas, studies have shown that the levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and platelet-derived growth factor (PDGF) in the cerebrospinal fluid of patients with low-grade gliomas are significantly higher than normal levels. Among them, VEGF and HGF levels are independent influencing factors for postoperative recurrence.

[0057] High-grade glioma: Collect effluent from the fluid injection port of the subarachnoid channel as a simulated cerebrospinal fluid sample; select IL-6 and IL-2R in the effluent of the subarachnoid channel as key biomarkers; use a chemiluminescent immunoassay analyzer with the matching IL-6 and sIL-2R assay kits. This method can provide higher detection sensitivity and is suitable for the detection of low-concentration biomarkers.

[0058] For high-grade gliomas, compared with normal brain tissue, the expression levels of cytokines such as interleukin-6 (IL-6), soluble interleukin-2 receptor (sIL-2R), tumor necrosis factor-α (TNF-α), IL-8, IL-1β, and IL-10 in the cerebrospinal fluid of patients with high-grade gliomas are significantly increased and maintained for a longer time. In particular, the levels of IL-6 and sIL-2R are significantly positively correlated with the malignancy of the tumor.

[0059] On the surface of the brain, cerebrospinal fluid and brain tissue are separated by the pia mater. The highly permeable pia mater allows substances in the cerebrospinal fluid to easily enter the brain tissue. Therefore, in order to ensure that the microfluidic chip system can accurately reflect the actual situation in the body, the researchers used a cerebrospinal fluid (CSF) biomarker detection and analysis method similar to the clinical method to monitor the growth and mutation of tumors in the chip. This method can not only provide key information about tumor progression, but also evaluate the effect of CAR-T cell therapy.

[0060] In general, the dynamic microfluidic chip device of the present invention continuously injects culture fluid into the vascular channel and the subarachnoid channel to simulate the blood and cerebrospinal fluid environment flowing in the body. Under normal circumstances, cerebral blood flow is in a laminar state, and the automatic injection pump can provide highly accurate laminar liquid delivery, and ensure the stability of liquid flow and uniformity of velocity distribution. At the same time, the slender parallel vascular channels are also more convenient for controlling the shear force of laminar liquid. Glioma progression destroys the local hemodynamic environment, usually accompanied by microvascular hyperplasia and capillary damage with higher permeability, and increased plasma viscosity and blood viscosity. By controlling different fluid flow rates, the effects of different fluid shear forces on blood-brain barrier permeability, CAR-T cell transmembrane migration and tumor microenvironment construction are clarified. Mechanical force plays an indispensable role in various cell processes. Studies have shown that the migration and adhesion of immune cells in shear flow and dynamic cell-to-cell interactions will lead to different immune processes under the action of mechanical force. Therefore, compared with the static culture of cells, the dynamic culture of controllable shear force enables cells to grow and differentiate in a closer in vivo situation, which helps to better simulate the behavior and response of cells in a physiological mechanical environment.

[0061] The brain's waste removal and nutrient delivery functions rely on the flow of cerebrospinal fluid. The dynamic microfluidic chip device of the present invention takes into account the maintenance of glioma nutrition and metabolic balance by the cerebrospinal fluid environment and the restriction of glioma growth, spread or metastasis by the pia mater, and more realistically simulates the microenvironment in which gliomas grow in vivo. The wide, semicircular tumor channel can better simulate the cerebral hemisphere, which is a common site of gliomas with an oval shape, and is closer to the growth environment of glioma cells in the ventricles under physiological conditions. Human brain glioma cells are adherent cells, and a larger chamber area is not only conducive to tumor spread and monitoring of tumor growth, but also more convenient for timely feedback on the efficacy of CAR-T cell therapy, which can improve the dynamic visualization ability of confocal real-time imaging tracking combined with cerebrospinal fluid biomarker detection of different CAR-T cells' anti-tumor ability.

[0062] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy, characterized in that: The following steps are involved: S1. Manufacturing of microfluidic chips: Computer-aided design software is used to design the channels of the microfluidic chip, including vascular channels, brain parenchyma channels, tumor channels and subarachnoid channels, and the master mold of the microfluidic chip is obtained by 3D printing technology; Using polydimethylsiloxane material to pour onto the master mold of the microfluidic chip for curing, then demolding and drilling, finally obtaining a microfluidic chip with a desired channel structure; S2. Reconstruction of neural tissue: Prepare a suspension of astrocytes and microglia; evenly mix the above two types of glial cells with soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; place the microfluidic chip in a low-temperature environment, then inject the gel-glial cell mixture into the brain parenchyma channel and place it in a cell culture incubator for gelation to achieve three-dimensional culture of glial cells; S3. Reconstruction of blood-brain barrier: prepare suspensions of human brain microvascular endothelial cells and human pericytes; mix the two cell suspensions and inject them into the vascular channel of the microfluidic chip, and co-culture with the established neural tissue; after a certain period of co-culture, tilt the microfluidic chip to allow the human brain microvascular endothelial cells and human pericytes to adhere to the interface between the vascular channel and the brain parenchyma channel, and dynamically incubate to form a blood-brain barrier microstructure; S4. Simulation of glioma microenvironment: prepare a glioma cell suspension and mix it with a soluble basement membrane (BME) hydrogel prepolymer to form a gel-glioma cell mixture; inject the gel-glioma cell mixture into the tumor channel of the microfluidic chip; prepare a fibroblast suspension at the same time, inject it into the subarachnoid channel, then tilt the microfluidic chip to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubate to form pia mater microstructures.

2. The method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 1, characterized in that: In step S2, the reconstruction of the neural tissue includes the following steps: S21. Prepare neural cell suspension: Use Eagle medium supplemented with fetal bovine serum (FBS) and EMEM medium to revive and culture immortalized human brain astrocyte cell line and immortalized human brain microglia cell line respectively; when the cell confluence reaches 80-90%, collect astrocyte suspension and microglia suspension respectively, and then evenly mix the collected astrocyte suspension and microglia suspension to form a neural cell mixed suspension; S22. Inoculating neural cells: uniformly mix the prepared mixed suspension of glial cells and soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; place the microfluidic chip on a cold bag, and then inject the gel-glial cell mixture into the brain parenchyma channel; after the injection, transfer the injected microfluidic chip to a cell culture incubator for constant temperature gelation; S23. Cultivate neural cells: After gelation, re-prepare Eagle medium and EMEM medium without adding FBS, and mix them to obtain serum-free glial cell medium; inject the prepared serum-free glial cell medium into the blood channel and subarachnoid channel respectively; replace the medium once a day within 5 days before the reconstruction of the blood-brain barrier.

3. The method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 2, characterized in that: In step S3, the reconstruction of the blood-brain barrier includes the following steps: S31. Prepare blood-brain barrier cell suspension: use ECM culture medium and PM culture medium to resuscitate and culture immortalized human brain microvascular endothelial cell line and immortalized human brain pericyte cell line respectively; when the cell confluence reaches 80-90%, collect human brain microvascular endothelial cell suspension and human pericyte suspension respectively, and then evenly mix the collected human brain microvascular endothelial cell suspension and human pericyte suspension to form a blood-brain barrier cell mixed suspension; S32, inoculating blood-brain barrier cells: injecting the prepared mixed suspension of blood-brain barrier cells into the vascular channel to co-culture it with the neural tissue reconstructed in step S2; after 4 days of co-culture, tilting the microfluidic chip to allow human brain microvascular endothelial cells and human pericytes to adhere to the interface between the blood channel and the brain parenchymal channel, simulating the real structure of the blood-brain barrier in vivo; S33. Cultivate blood-brain barrier cells: without adding FBS, reconfigure ECM medium and PM medium, and mix them to obtain serum-containing endothelial cell medium; inject the prepared serum-containing endothelial cell medium into the blood channel, and use the flow effect to remove cells that have not successfully adhered and any possible debris; continue dynamic culture for 3 days, during which time replace the serum-containing endothelial cell medium in the blood channel and the serum-free glial cell medium in the subarachnoid channel daily.

4. The method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 3, characterized in that: In step S4, the reconstruction of the neural tissue includes the following steps: S41. Prepare brain glioma cell suspension: Use DMEM medium supplemented with 5% FBS and 1% penicillin / streptomycin solution to revive and culture immortalized human glioma cell line; when the cell confluence reaches 80-90%, collect the brain glioma cell suspension; S42. Inoculating glioma cells: uniformly mix the prepared glioma cell suspension and BME hydrogel prepolymer to form a gel-glioma cell mixture; place the microfluidic chip on a cold bag, and then inject the gel-glioma cell mixture into the tumor channel; after the injection, transfer the injected microfluidic chip to a cell culture incubator for constant temperature gelation; after gelation, inject serum-free glial cell culture medium into the subarachnoid channel, and continue to culture for 1-2 days, during which the culture medium is replaced once a day to maintain the growth state of the cells; S43. Construction of the pia mater: Use DMEM medium supplemented with 2 mM / L glutamine and 10% FBS to revive and culture immortalized human dermal fibroblast cell line; when the cell confluence reaches 80-90%, collect the fibroblast suspension; inject the fibroblast suspension into the subarachnoid channel of the microfluidic chip; tilt the microfluidic chip to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel; continue incubation to promote the fibroblasts to form stable pia mater microstructures.

5. According to the method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 4, in step S42, replacing the culture medium comprises the following steps: Connect a hose to the input end of the subarachnoid channel and connect it to an automatic injection pump; inject serum-free glial cell culture medium through the automatic injection pump to simulate the dynamic flow environment of cerebrospinal fluid in vivo; adjust the flow rate of the automatic injection pump to ensure that the shear stress is controlled within 0.1dyn / cm² to truly reproduce the flow characteristics of cerebrospinal fluid in vivo.

6. A dynamic microfluidic chip device for studying glioma CAR-T therapy, characterized in that: A microfluidic chip prepared by the method of claim 5; the microfluidic chip comprises a vascular channel, a brain parenchyma channel, a tumor channel and a subarachnoid space channel arranged in sequence from left to right; each of the vascular channel, the brain parenchyma channel, the tumor channel and the subarachnoid space channel comprises a cell culture chamber with an injection port on one side; the cell culture chambers of the vascular channel and the subarachnoid space channel have outflow ports correspondingly arranged on the other side; The vascular channel is used to co-culture human brain microvascular endothelial cells and human pericytes to complete the reconstruction of the blood-brain barrier and the simulation of blood flow; The brain parenchyma channel is used for three-dimensional culture of astrocytes and microglia; The tumor channel is used for three-dimensional culture of brain glioma cells; The subarachnoid channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and the simulation of flowing cerebrospinal fluid.

7. A dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 6, characterized in that: The cell culture chamber of the tumor channel is a semicircular, circular or elliptical structure; the cell culture chamber of the subarachnoid channel is an arc-shaped tubular structure.

8. A dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 6, characterized in that: It also includes an automatic injection pump, the output end of which is connected to the microfluidic chip to inject a stable flowing liquid into the microfluidic chip and quantitatively control the flow rate of the injected liquid.

9. A dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 8, characterized in that: The automatic injection pump comprises an injection head, a syringe, an injection hose and a base with a built-in driving motor; the injection head, the syringe and the injection hose are arranged in sequence, and the injection port on the vascular channel or the subarachnoid space channel is connected to the syringe through the injection hose; the syringe is fixedly mounted on the base, and the driving motor is transmission-connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to perform injection.

10. An application of a dynamic microfluidic chip device for studying glioma CAR-T therapy as described in any one of claims 6 to 9, characterized in that: The following steps are involved: S1. CAR-T cell infusion: A mixed solution of fluorescently labeled CAR-T cells and serum-containing endothelial cell culture medium was injected into the blood channel to simulate intravenous delivery of CAR-T cell therapy; S2. Transmembrane migration observation: Use confocal real-time imaging tracking technology to visualize the capture, adhesion and extravasation process of CAR-T cells and calculate related parameters; S3. Biomarker detection: The anti-tumor ability of CAR-T cells is evaluated by measuring the levels of specific biomarkers in the effluent of the subarachnoid channel.

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