A dynamic microfluidic chip device for studying CAR-T therapy for gliomas, its preparation method and its application

Abstract

This invention relates to the field of microfluidic chip technology, specifically to a dynamic microfluidic chip device, its preparation method, and its applications for studying CAR-T therapy for gliomas. The preparation method includes: fabrication of the microfluidic chip; reconstruction of neural tissue; reconstruction of the blood-brain barrier; and simulation of the glioma microenvironment. The dynamic microfluidic chip device includes a microfluidic chip; the microfluidic chip includes, from left to right, a vascular channel, a brain parenchyma channel, a tumor channel, and a subarachnoid channel; each of the vascular channel, brain parenchyma channel, tumor channel, and subarachnoid channel includes a cell culture chamber with an inlet on one side; the cell culture chambers of the vascular channel and the subarachnoid channel have corresponding outlets on the other side. The applications include: CAR-T cell infusion; observation of transmembrane migration; and detection of biomarkers. This invention, through the dynamic microfluidic chip device, its preparation method, and its applications, aims to achieve more accurate in vitro evaluation of CAR-T cell therapy.

Description

Technical Field

[0001] This invention relates to the field of microfluidic chip technology, specifically to a dynamic microfluidic chip device, its preparation method, and its application for researching CAR-T therapy for gliomas. Background Technology

[0002] Gliomas are the leading primary intracranial tumors of the central nervous system, accounting for 75% of malignant brain tumors in adults. Standard treatment regimens include surgical resection of the tumor, radiotherapy, and chemotherapy. However, patients with gliomas who receive conventional treatment have a poor prognosis. With the development of immunotherapy, chimeric antigen receptor T-cell (CAR-T) immunotherapy has brought new hope to glioma treatment. CAR-T cells, expanded in vitro and genetically engineered, can target and lyse cells carrying relevant tumor antigens, with intravenous delivery being the simplest and most common administration method. Although CAR-T cell therapy has made significant progress in cancer treatment, its application in malignant tumors such as gliomas remains limited by physical barriers such as the blood-brain barrier (BBB).

[0003] The blood-brain barrier (BBB) ​​is a complex structure composed of brain capillary walls and glial cells that tightly regulates the ability of immune cells to be transported to the central nervous system. Therefore, the efficacy of CAR-T cell therapy is particularly dependent on whether therapeutic immune cells can effectively cross the BBB to induce an anti-tumor response in the brain. The presence of the BBB presents challenges to the precise targeted delivery of CAR-T cells to brain tissue and maximizing therapeutic efficacy.

[0004] To simulate the blood-brain barrier under physiological or pathological conditions in vitro, various microfluidic chips have been developed. However, existing microfluidic chips still have shortcomings in simulating the complex physiological characteristics of the blood-brain barrier in the natural brain:

[0005] 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 seeding and long consumption time in non-contact co-culture between cells by constructing a collagen channel layer. However, its co-culture system only contains brain astrocytes and brain microvascular endothelial cells, and cannot reproduce the multicellular, three-dimensional complex structural features of the natural blood-brain barrier.

[0006] CN108823145A discloses an in vitro construction method for simulating the blood-brain barrier by human brain microvascularization. This construction method sets a height difference of culture medium between the upper and lower chambers, so that the culture medium maintains fluidity due to the potential energy difference for a certain period of time. However, the pressure of this liquid depends on the potential energy difference.

[0007] CN215162826U discloses a blood-brain barrier microfluidic chip. This chip uses periodic gas pressure control to promote the flow of culture medium to simulate the fluid environment within blood vessels. However, the microporous membrane between the culture chambers of this device hinders the interaction between cells, affecting the barrier function of the blood-brain barrier model.

[0008] The establishment of a blood-brain barrier model under pathological conditions using microfluidic chips as a technological platform holds immense potential for further application in disease treatment. However, research on using microfluidic chips to simulate the physiological state of the blood-brain barrier and tumor cells in CAR-T cell therapy in vitro is still lacking. 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, laying the foundation for precise targeted delivery of CAR-T cells in brain tissue and maximizing therapeutic efficacy. Summary of the Invention

[0009] The first objective of this invention is to overcome the defects and deficiencies 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 capable of simulating the microenvironment of glioma.

[0010] A second objective of this invention is to provide a dynamic microfluidic chip device for studying glioma CAR-T therapy, which is used to simulate the glioma microenvironment.

[0011] The third objective of this invention is to provide an application of a dynamic microfluidic chip device for studying CAR-T therapy for gliomas, which provides a new research method for in vitro evaluation of CAR-T cell therapy for gliomas.

[0012] The objective of this invention can be achieved through the following technical solutions:

[0013] A method for fabricating a dynamic microfluidic chip device for studying CAR-T therapy for gliomas includes the following steps:

[0014] S1. Fabrication of Microfluidic Chips: The channels of the microfluidic chip are designed using computer-aided design software. These channels include vascular channels, brain parenchyma channels, tumor channels, and subarachnoid channels. A master mold for the microfluidic chip is then manufactured using 3D printing technology. Polydimethylsiloxane material is poured onto the master mold for curing, followed by demolding and perforation to finally obtain a microfluidic chip with the required channel structure.

[0015] S2. Reconstruction of neural tissue: Prepare suspensions of astrocytes and microglia; uniformly mix the 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;

[0016] S3. Reconstruction of the 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 for 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 the blood-brain barrier microstructure;

[0017] S4. Simulation of the glioma microenvironment: A glioma cell suspension was prepared and mixed with a soluble basement membrane (BME) hydrogel prepolymer to form a gel-glioma cell mixture; the gel-glioma cell mixture was injected into the tumor channel of the microfluidic chip; at the same time, a fibroblast cell suspension was prepared and injected into the subarachnoid channel, and the microfluidic chip was tilted to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubated to form pia mater microstructures.

[0018] As a preferred embodiment, in step S2, the reconstruction of neural tissue includes the following steps:

[0019] S21. Preparation of nerve cell suspension: Immortalized human brain astrocyte cell line and immortalized human brain microglia cell line were revived and cultured using Eagle medium and EMEM medium supplemented with fetal bovine serum (FBS), respectively; when the cell confluence reached 80-90%, the astrocyte cell suspension and microglia cell suspension were collected separately, and then the collected astrocyte cell suspension and microglia cell suspension were mixed evenly to form a mixed nerve cell suspension;

[0020] S22. Inoculation of nerve cells: The prepared glial cell suspension is uniformly mixed with the soluble basement membrane (BME) hydrogel prepolymer to form a gel-glial cell mixture; the microfluidic chip is placed on a cold bag, and the gel-glial cell mixture is injected into the brain parenchyma channel; after injection, the injected microfluidic chip is transferred to a cell culture incubator for isothermal gelation.

[0021] S23. Culturing nerve cells: After gelation, Eagle medium and EMEM medium were reconstituted without the addition of fetal bovine serum and mixed to obtain serum-free glial cell culture medium; the prepared serum-free glial cell culture medium was injected into the vascular channel and the subarachnoid channel respectively; the culture medium was changed once a day for 5 days before the blood-brain barrier remodeling.

[0022] As a preferred embodiment, in step S3, the reconstruction of the blood-brain barrier includes the following steps:

[0023] S31. Preparation of blood-brain barrier cell suspension: Immortalized human brain microvascular endothelial cell line and immortalized human brain pericyte line were revived and cultured using ECM medium and PM medium, respectively; when the cell confluence reached 80-90%, the human brain microvascular endothelial cell suspension and the human pericyte suspension were collected separately, and then the collected human brain microvascular endothelial cell suspension and human pericyte suspension were mixed evenly to form a blood-brain barrier cell mixed suspension;

[0024] S32. Inoculation of blood-brain barrier cells: The prepared blood-brain barrier cell suspension was injected into the vascular channel and co-cultured with the neural tissue reconstructed in step S2. After co-culturing for 4 days, the microfluidic chip was tilted so that human brain microvascular endothelial cells and human pericytes adhered to the interface between the vascular channel and the brain parenchyma channel, simulating the real structure of the blood-brain barrier in vivo.

[0025] S33. Culture of blood-brain barrier cells: Without adding fetal bovine serum, reconstitute ECM and PM media and mix them to obtain serum-containing endothelial cell culture medium; inject the prepared serum-containing endothelial cell culture medium into the vascular channel and use the flow action to remove cells that have not adhered successfully and any possible debris; continue dynamic culture for 3 days, during which time the serum-containing endothelial cell culture medium in the vascular channel and the serum-free glial cell culture medium in the subarachnoid space channel are changed daily.

[0026] As a preferred embodiment, in step S4, the simulation of the glioma microenvironment includes the following steps:

[0027] S41. Preparation of glioma cell suspension: Immortalized human glioma cell lines were revived and cultured using DMEM medium supplemented with 5% fetal bovine serum and 1% penicillin / streptomycin solution; when the cell confluence reached 80-90%, the glioma cell suspension was collected.

[0028] S42. Inoculation of glioma cells: The prepared glioma cell suspension is uniformly mixed with the soluble basement membrane hydrogel prepolymer to form a gel-glioma cell mixture; the microfluidic chip is placed on a cold bag, and the gel-glioma cell mixture is injected into the tumor channel; after injection, the injected microfluidic chip is transferred to a cell culture incubator for isothermal gelation; after gelation, serum-free glioma cell culture medium is injected into the subarachnoid space channel and cultured for 1-2 days, changing the culture medium daily to maintain cell growth.

[0029] S43. Construction of pia mater: Immortalized human dermal fibroblast cell lines were revived and cultured using DMEM medium supplemented with 2 mM / L glutamine and 10% fetal bovine serum; when the cell confluence reached 80-90%, the fibroblast cell suspension was collected; the fibroblast cell suspension was injected into the subarachnoid channel of the microfluidic chip; the microfluidic chip was tilted to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel; incubation was continued to promote the formation of stable pia mater microstructures by fibroblasts.

[0030] As a preferred embodiment, in step S42, changing the culture medium includes the following steps: connecting a tubing 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 0.1 dyn / cm² to realistically reproduce the flow characteristics of cerebrospinal fluid in vivo.

[0031] A dynamic microfluidic chip device for studying CAR-T therapy for gliomas includes a microfluidic chip prepared using the method described above. The microfluidic chip includes, from left to right, a vascular channel, a brain parenchyma channel, a tumor channel, and a subarachnoid space channel. Each of the vascular channel, brain parenchyma channel, tumor channel, and subarachnoid space channel includes a cell culture chamber with an inlet on one side. The cell culture chambers of the vascular channel and the subarachnoid space channel have outlets on the other side, respectively.

[0032] 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;

[0033] The brain parenchyma channel is used for three-dimensional culture of astrocytes and microglia;

[0034] The tumor channel is used for three-dimensional culture of glioma cells;

[0035] The subarachnoid space channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and to simulate the flow of cerebrospinal fluid.

[0036] As a preferred option, the cell culture chamber of the tumor channel has a semi-circular, circular, or elliptical structure; the cell culture chamber of the subarachnoid channel has an arc-shaped tubular structure.

[0037] As a preferred embodiment, the system also includes an automatic injection pump, the output of which is connected to the microfluidic chip to inject a stable flow of liquid into the microfluidic chip and to quantitatively control the flow rate of the injected liquid.

[0038] As a preferred embodiment, the automatic injection pump includes an injection head, a syringe, an injection tubing, and a base with a built-in drive motor; the injection head, syringe, and injection tubing are arranged in sequence, and the inlet on the vascular channel or subarachnoid channel is connected to the syringe through the injection tubing; the syringe is fixedly mounted on the base, and the drive motor is connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to inject.

[0039] An application of a dynamic microfluidic chip device for researching CAR-T therapy for gliomas, characterized by comprising the following steps:

[0040] S1. CAR-T cell infusion: Fluorescently labeled CAR-T cells mixed with serum-containing endothelial cell culture medium are injected into the vascular channel to simulate intravenous delivery of CAR-T cell therapy;

[0041] S2. Observation of transmembrane migration: The capture, adhesion and extravasation process of CAR-T cells were visualized using confocal real-time imaging tracking technology, and relevant parameters were calculated;

[0042] S3. Biomarker detection: The anti-tumor ability of CAR-T cells is assessed by measuring the levels of specific biomarkers in the effluent from the subarachnoid passage.

[0043] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0044] 1. This invention incorporates a subarachnoid space channel, creating a four-channel design. This design not only more comprehensively simulates the blood-brain barrier and cerebrospinal fluid flow environment in vivo, but also better reflects the restrictive effect of the pia mater on the growth, spread, or metastasis of gliomas. Furthermore, by setting up the subarachnoid space channel for monolayer fibroblast culture to reconstruct the pia mater, and by injecting serum-free glial cell culture medium to simulate the dynamic microenvironment of cerebrospinal fluid in vivo, this invention enhances the complexity and realism of the model.

[0045] 2. This invention sets the vascular channel as a slender parallel structure and the tumor channel as a wide, thick semicircle. The wide, thick semicircular tumor channel can better simulate the cerebral hemisphere and is closer to the growth environment of glioma cells in the ventricles under physiological conditions. While the structure of the vascular channel facilitates 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 facilitates timely feedback of therapeutic effects, which helps to improve the authenticity and reliability of experimental results.

[0046] 3. This invention utilizes an automated syringe pump to achieve highly accurate laminar flow fluid delivery and adjusts the shear stress range according to different pathological conditions; in addition, it provides detailed formulas for calculating wall shear stress to ensure that experimental conditions closely approximate real-world conditions.

[0047] 4. The present invention demonstrates the transmembrane migration behavior of CAR-T cells under different fluid shear forces, and visualizes this process using confocal real-time imaging tracking technology. Simultaneously, cerebrospinal fluid biomarker detection and analysis, such as the measurement of VEGF, HGF, and IL-6 levels, are introduced to assess the anti-tumor ability of CAR-T cells. This method provides a more intuitive and quantitative evaluation tool.

[0048] 5. This invention constructs a more complete and realistic blood-brain barrier-glioma co-culture system, which demonstrates higher precision and efficiency, particularly in CAR-T cell therapy research. It not only simulates complex physiological and pathological environments but also provides a reliable platform for drug screening, thereby accelerating the development of novel anti-cancer treatment strategies. Attached Figure Description

[0049] Figure 1 This is a top view of the microfluidic chip of the present invention;

[0050] Figure 2 This is a schematic diagram of the structure of the dynamic microfluidic chip device of the present invention;

[0051] Figure 3 This is a schematic diagram of the microfluidic chip of the present invention;

[0052] Figure 4 This is a schematic diagram of the fabrication process of the microfluidic chip of the present invention;

[0053] Figure 5 This is a structural simulation diagram of cell growth in the microfluidic chip of the present invention.

[0054] Among them: 1: Vascular channel, 2: Brain parenchyma channel, 3: Tumor channel, 4: Subarachnoid channel, 5: Vascular channel inlet, 6: Brain parenchyma channel inlet, 7: Tumor channel inlet, 8: Subarachnoid channel inlet, 9: Vascular channel outlet, 10: Subarachnoid channel outlet, 11: Injector head, 12: Syringe, 13: Base, 14: Lower layer of microscope slide, 15: Upper layer of PDMS. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0056] As shown in the figure, a dynamic microfluidic chip device for studying CAR-T therapy for glioma includes a microfluidic chip comprising, from left to right, a vascular channel, a brain parenchyma channel, a tumor channel, and a subarachnoid space channel; each of the vascular channel, brain parenchyma channel, tumor channel, and subarachnoid space channel includes a cell culture chamber with an inlet on one side; the cell culture chambers of the vascular channel and the subarachnoid space channel have outlets on the other side respectively.

[0057] 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;

[0058] The brain parenchyma channel is used for three-dimensional culture of astrocytes and microglia;

[0059] The tumor channel is used for three-dimensional culture of glioma cells;

[0060] The subarachnoid space channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and to simulate the flow of cerebrospinal fluid.

[0061] The vascular channel includes a vascular channel inlet, a vascular channel outlet, and a vascular channel cell culture chamber;

[0062] Both the vascular channel inlet and outlet are circular with a diameter of 2 mm. The vascular channel culture chamber is a strip-shaped lumen 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, which has a length of 5 mm.

[0063] The brain parenchyma channel includes a brain parenchyma channel inlet and a brain parenchyma channel cell culture chamber;

[0064] The brain parenchyma channel inlet is a circle with a diameter of 2 mm, and the brain parenchyma channel cell culture chamber is a strip-shaped lumen structure with a length of 8 mm, a width of 2 mm, and a height of 100 μm.

[0065] The tumor channel includes a tumor channel inlet and a tumor channel cell culture chamber;

[0066] The tumor channel inlet is a circle with a diameter of 2 mm, and the tumor channel cell culture chamber is a semi-circular structure with a diameter of 6 mm and a height of 100 μm.

[0067] The subarachnoid channel includes a subarachnoid channel inlet, a subarachnoid channel outlet, and a subarachnoid channel culture chamber;

[0068] The glioma cell mixed solution inlet and 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, with a length of 1.6π mm.

[0069] Furthermore, the cell culture chamber of the tumor channel has a semi-circular, circular, or elliptical structure; the cell culture chamber of the subarachnoid channel has an arc-shaped tubular structure.

[0070] As a preferred embodiment, the dynamic microfluidic chip device of this embodiment further includes an automatic injection pump. The output end of the automatic injection pump is connected to the microfluidic chip and is used to inject a stable flow of liquid into the microfluidic chip and quantitatively control the flow rate of the injected liquid.

[0071] Specifically, the automatic injection pump includes an injection head, a syringe, an injection tubing, and a base with a built-in drive motor; the injection head, syringe, and injection tubing are arranged in sequence, and the solution inlet on the vascular channel or subarachnoid channel is connected to the syringe through the injection tubing; the syringe is fixedly installed on the base, and the drive motor is connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to inject.

[0072] This embodiment controls the flow rate of the liquid injected into the vascular channel or subarachnoid channel by controlling the progress of the syringe, thereby achieving regulation of the fluid shear force.

[0073] Specifically, the normal physiological shear stress at the blood-brain barrier is approximately 2 dyn / cm². Considering the influence of different tumor progression stages on the blood flow shear stress at the blood-brain barrier, the shear stress applied to simulate blood flow under pathological conditions in the vascular channel is set within the range of 1-6 dyn / cm². The internal space of the vascular channel is a cuboid. Based on Newtonian fluid dynamics, the direction of shear stress in the direction perpendicular to fluid flow should be parabolic, with zero shear stress at the fluid center and increasing towards the wall. Since the cell diameter used is approximately 10 μm, much smaller than the cavity height of 400 μm, the shear stress at the cell centroid can be considered equal to the shear stress on the wall of the flow cavity. The formula for calculating the wall shear stress is as follows:

[0074]

[0075] in, τ w This represents the wall shear stress, expressed in dyn / cm. 2 Q represents the average flow rate of the fluid flowing through the cavity, expressed in mL / min. μ ν is the viscosity coefficient of the fluid, expressed in mPa·s; h and w These represent the height and width of the cavity's cross-section, respectively, in cm.

[0076] By setting the fluid shear stress in the experiment, the required flow rate of the automatic injection pump was calculated and the experiment was carried out.

[0077] A method for fabricating a dynamic microfluidic chip device for studying CAR-T therapy for gliomas includes the following steps:

[0078] S1. Fabrication of microfluidic chips: The channels of the microfluidic chip are designed using computer-aided design software. The channels of the microfluidic chip include vascular channels, brain parenchyma channels, tumor channels, and subarachnoid channels. 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 and cured. Then, the mold is demolded and holes are punched to finally obtain a microfluidic chip with the required channel structure.

[0079] Specifically, in step S1, the fabrication of the microfluidic chip includes the following steps:

[0080] S11. Master mold design and manufacturing: Use AutoCAD software to design the channels of the microfluidic chip and obtain the design model; manufacture the resin master mold of the microfluidic chip using a photopolymerization 3D printer, including spraying liquid photosensitive resin layer by layer according to the design model, UV curing, cleaning, and surface treatment.

[0081] S12. Preparation of PDMS Chip: Polydimethylsiloxane (PDMS) prepolymer and curing agent are mixed uniformly at a mass ratio of 10:1 to obtain PDMS solution; the mixed PDMS solution is vacuumed to remove any possible air bubbles, ensuring the uniformity and transparency of the final product; the vacuumed PDMS solution is poured into a prepared resin master mold, and air bubbles are further removed using a vacuum pump to ensure that no air bubbles remain 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 curing, the cured PDMS is peeled off from the resin master mold, and corresponding inlet and outlet ports are made on the PDMS using a punch, ensuring that the pore size meets the experimental requirements, to obtain the upper layer of PDMS with channel structure;

[0082] S13. Microfluidic chip assembly and bonding: The upper PDMS layer with channel structure and the lower pre-cleaned microscope slide are subjected to plasma treatment to enhance the adhesion between the two; immediately after plasma treatment, the upper PDMS layer and the lower microscope slide are tightly bonded to form an irreversible bond, sealing the channel structure of the microfluidic chip.

[0083] S14. Sterilization and disinfection: The prepared microfluidic chip is subjected to high temperature and high pressure sterilization to ensure a sterile environment suitable for cell culture and other biological experiments; before cell seeding, the microfluidic chip is placed in an 80°C drying oven for at least 24 hours to restore its hydrophobicity; disinfection is performed by ultraviolet irradiation for 30 minutes to ensure that the surface of the microfluidic chip is sterile.

[0084] This ensures that the fabricated microfluidic chip has high precision, good mechanical properties, and a sterile environment, providing a reliable platform for subsequent experiments.

[0085] S2. Reconstruction of neural tissue: Prepare suspensions of astrocytes and microglia; mix the 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 to gel, thereby achieving three-dimensional culture of glial cells.

[0086] Specifically, in step S2, the reconstruction of neural tissue includes the following steps:

[0087] S21. Preparation of nerve cell suspension: Immortalized human brain astrocytes were revived and cultured using Eagle medium supplemented with 10% fetal bovine serum (FBS), and immortalized human brain microglia were revived and cultured using EMEM medium supplemented with 10% fetal bovine serum (FBS). When the cell confluence reached 80-90%, the astrocyte suspension and microglia suspension were collected separately. Then, the collected astrocyte suspension and microglia suspension were mixed evenly in a molar ratio of 4:1 to 5:1 to form a mixed nerve cell suspension.

[0088] S22. Inoculation of nerve cells: The prepared glial cell suspension and soluble basement membrane (BME) hydrogel prepolymer are mixed uniformly 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 and prevent premature gel solidification. The gel-glial cell mixture is then injected into the brain parenchyma channel through the brain parenchyma channel inlet. After injection, the injected microfluidic chip is immediately transferred to a cell culture plate and then to a cell culture incubator for isothermal 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.

[0089] S23. Culturing nerve cells: After gelation, without adding fetal bovine serum, the two glial cell culture media (Eagle medium and EMEM medium) were reconstituted and mixed in the appropriate proportions to obtain serum-free glial cell culture medium. The prepared serum-free glial cell culture medium was injected into the vascular channel and the subarachnoid space channel, respectively. During the 5 days before the blood-brain barrier reconstruction, the culture medium was changed once a day to ensure that the cells were always in a fresh and suitable growth environment, gradually completing the reconstruction of neural tissue, effectively promoting the three-dimensional culture of glial cells, and laying a solid foundation for the subsequent construction of more complex blood-brain barrier models.

[0090] S3. Reconstruction of the 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 channels of the microfluidic chip for 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 channels and the brain parenchyma channels, and dynamically incubate to form the blood-brain barrier microstructure.

[0091] Specifically, in step S3, the reconstruction of the blood-brain barrier includes the following steps:

[0092] S31. Preparation of blood-brain barrier cell suspension: Immortalized human brain microvascular endothelial cell line was revived and cultured using ECM medium, and immortalized human brain pericyte cell line was revived and cultured using PM medium; when the cell confluence reached 80-90%, human brain microvascular endothelial cell suspension and human pericyte cell suspension were collected separately, and then the collected human brain microvascular endothelial cell suspension and human pericyte cell suspension were mixed evenly in a molar ratio of 9:1 to form a blood-brain barrier cell mixed suspension;

[0093] S32. Inoculation of blood-brain barrier cells: The prepared blood-brain barrier cell suspension was injected into the vascular channel through the vascular channel inlet, and co-cultured with the neural tissue reconstructed in step S2. The conditions in the cell culture incubator were maintained at 37°C and 5% CO2 to provide a suitable growth environment for the cells. After co-culturing for 4 days, the microfluidic chip was gently tilted so that human brain microvascular endothelial cells and human pericytes could adhere to the sidewall of the hydrogel in the brain parenchyma channel, that is, adhere to the interface between the vascular channel and the brain parenchyma channel, simulating the real structure of the blood-brain barrier in vivo.

[0094] S33. Culturing Blood-Brain Barrier Cells: Without adding fetal bovine serum, the two blood-brain barrier cell culture media (ECM and PM media) were reconstituted and mixed in the appropriate proportions to obtain serum-containing endothelial cell culture medium. The prepared serum-containing endothelial cell culture medium was injected into the vascular channel through the inlet, and the flow action was used to remove cells that failed to adhere and any possible debris. The cells were then cultured dynamically for 3 days. During this period, the serum-containing endothelial cell culture medium in the vascular channel and the serum-free glial cell culture medium in the subarachnoid space channel were replaced daily to ensure that the cells were always in a fresh and suitable growth environment, ultimately forming a stable blood-brain barrier microstructure, providing a reliable model basis for subsequent experiments.

[0095] S4. Simulation of the glioma microenvironment: A glioma cell suspension was prepared and mixed with a soluble basement membrane (BME) hydrogel prepolymer to form a gel-glioma cell mixture; the gel-glioma cell mixture was injected into the tumor channel of the microfluidic chip; at the same time, a fibroblast cell suspension was prepared and injected into the subarachnoid channel, and the microfluidic chip was tilted to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubated to form pia mater microstructures.

[0096] Specifically, in step S4, the simulation of the glioma microenvironment includes the following steps:

[0097] S41. Preparation of glioma cell suspension: Immortalized human glioma cell lines were revived and cultured using DMEM medium supplemented with 5% fetal bovine serum and 1% penicillin / streptomycin solution; when the cell confluence reached 80-90%, the glioma cell suspension was collected.

[0098] S42. Inoculation of glioma cells: The prepared glioma cell suspension is uniformly mixed with the soluble basement membrane hydrogel prepolymer to form a gel-glioma cell mixture. The microfluidic chip is placed on a cold bag to maintain a low temperature environment and prevent premature gel solidification. The gel-glioma cell mixture is then injected into the tumor channel through the tumor channel inlet. After injection, the injected microfluidic chip is immediately transferred to a cell culture plate and then to a cell culture incubator for isothermal 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, serum-free glioma cell culture medium is injected into the subarachnoid space and cultured for 1-2 days, changing the culture medium daily to maintain cell growth.

[0099] The dynamic flow environment of cerebrospinal fluid (CSF) in vivo is simulated by periodically injecting serum-free glial cell culture medium into the inlet of the subarachnoid channel and replacing the culture medium. The simulation method involves connecting a tubing to the inlet of the subarachnoid channel and then to an automated syringe pump; injecting serum-free glial cell culture medium through the automated syringe pump to simulate the dynamic flow environment of CSF in vivo; and adjusting the flow rate of the automated syringe pump to ensure that the shear stress is controlled within 0.1 dyn / cm² to realistically reproduce the flow characteristics of CSF in vivo.

[0100] S43. Construction of pia mater: Immortalized human dermal fibroblast cell lines were revived and cultured using DMEM medium supplemented with 2 mM / L glutamine and 10% fetal bovine serum; when the cell confluence reached 80-90%, the fibroblast cell suspension was collected; the fibroblast cell suspension was injected into the subarachnoid channel of the microfluidic chip through the subarachnoid channel inlet; the microfluidic chip was tilted to allow the fibroblasts to adhere to the sidewalls of the hydrogel in the tumor channel, i.e., to adhere to the interface between the tumor channel and the subarachnoid channel; incubation was continued to promote the formation of stable pia mater microstructures by fibroblasts.

[0101] The application of a dynamic microfluidic chip device in glioma CAR-T therapy research includes the following steps:

[0102] S1. CAR-T cell infusion: Fluorescently labeled CAR-T cells are injected into a vascular channel in a mixture of serum-containing endothelial cell culture medium to simulate intravenous delivery of CAR-T cell therapy.

[0103] In step S1, CAR-T cell infusion, the specific operational steps are as follows:

[0104] S11. Connect a tubing to the vascular channel inlet of the microfluidic chip and connect it to an automatic injection pump to ensure precise control of flow rate and shear stress.

[0105] S12. Mix CAR-T cells that have been fluorescently labeled (e.g., using CellTracker™ Deep Red) with serum-containing endothelial cell culture medium to form a cell mixture solution for infusion;

[0106] S13. The above mixed solution was injected into the vascular channel of the microfluidic chip by an automated injection pump to simulate intravenous delivery of CAR-T cell therapy; by adjusting the flow rate of the automated injection pump and controlling different shear stresses, the effects of different fluid shear forces on blood-brain barrier permeability, CAR-T cell transmembrane migration and tumor microenvironment construction were investigated.

[0107] S14. After 2 hours of dynamic incubation, fresh serum-containing endothelial cell culture medium is injected into the vascular channel to remove CAR-T cells that have not successfully adhered or migrated, ensuring the accuracy of subsequent observations.

[0108] S2. Observation of transmembrane migration: The capture, adhesion and extravasation of CAR-T cells were visualized using confocal real-time imaging tracking technology, and relevant parameters were calculated.

[0109] In step S2, the specific operating steps for observing transmembrane migration are as follows:

[0110] S21. Using confocal real-time imaging tracking technology, the behavior of CAR-T cells is analyzed using software such as Imaris and ImageJ. Because CAR-T cells are stained red (CellTracker™ Deep Red), the trajectory, adhesion, and extravasation process of individual CAR-T cells captured on the blood-brain barrier can be clearly observed.

[0111] 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, providing quantitative data support for assessing the transmembrane migration ability of CAR-T cells.

[0112] S3. Biomarker detection: The anti-tumor ability of CAR-T cells is assessed by measuring the levels of specific biomarkers in the effluent from the subarachnoid passage.

[0113] In step S3, biomarker detection, the specific operational steps for different grades of gliomas are as follows:

[0114] Low-grade gliomas: Effluent was collected from the fluid inlet of the subarachnoid channel as a simulated cerebrospinal fluid sample; VEGF and HGF in the effluent from the subarachnoid channel were selected as key biomarkers; detection was performed using human VEGF ELISA kits and human HGF ELISA kits.

[0115] 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, the levels of VEGF and HGF are independent influencing factors for postoperative recurrence.

[0116] High-grade glioma: Effluent was collected from the fluid inlet of the subarachnoid passage as a simulated cerebrospinal fluid sample; IL-6 and IL-2R in the effluent from the subarachnoid passage were selected as key biomarkers; a chemiluminescent immunoassay analyzer was used in conjunction with the matching IL-6 and sIL-2R assay kit. This method can provide higher detection sensitivity and is suitable for the detection of low concentrations of biomarkers.

[0117] In 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 period of time. In particular, the levels of IL-6 and sIL-2R are significantly positively correlated with the degree of tumor malignancy.

[0118] On the surface of the brain, the cerebrospinal fluid (CSF) and brain tissue are separated by the pia mater. The highly permeable pia mater allows substances from the CSF to easily enter the brain tissue. Therefore, to ensure that the microfluidic chip system accurately reflects the actual situation in vivo, researchers used a CSF biomarker detection and analysis method similar to that used in clinical settings to monitor tumor growth and mutation status within the chip. This method not only provides crucial information about tumor progression but also evaluates the effectiveness of CAR-T cell therapy.

[0119] In summary, the dynamic microfluidic chip device of this invention continuously injects culture medium into vascular and subarachnoid channels to simulate the in vivo environment of flowing blood and cerebrospinal fluid. Under normal conditions, cerebral blood flow is laminar. An automated infusion pump can provide highly accurate laminar fluid delivery, ensuring the stability and uniformity of fluid flow velocity distribution. Simultaneously, the elongated, parallel vascular channels facilitate control of the laminar fluid shear force. Glioma progression disrupts the local hemodynamic environment, typically accompanied by increased permeability of microvessels and capillary damage, as well as increased plasma and blood viscosity. By controlling different fluid flow rates, the effects of varying fluid shear forces on blood-brain barrier permeability, CAR-T cell transmembrane migration, and tumor microenvironment construction are elucidated. Mechanical forces play an indispensable role in various cellular processes. Studies have shown that the migration and adhesion of immune cells in shear flow, as well as dynamic intercellular interactions, lead to different immune processes under mechanical forces. Therefore, compared to static cell culture, dynamic culture with controllable shear forces allows cells to grow and differentiate more closely to in vivo conditions, helping to better simulate cellular behavior and responses under physiological and mechanical environments.

[0120] The brain's waste removal and nutrient delivery functions depend on the flow of cerebrospinal fluid. This invention's dynamic microfluidic chip device considers the cerebrospinal fluid environment's role in maintaining glioma nutrition and metabolic balance, as well as the pia mater's restriction on glioma growth, spread, or metastasis, thus more realistically simulating the microenvironment in which gliomas grow in vivo. The wide, semi-circular tumor channel better simulates the cerebral hemispheres—the common site of gliomas with a shape similar to an oval—and more closely resembles the growth environment of glioma cells in the ventricles under physiological conditions. Since human glioma cells are adherent cells, the larger chamber area not only facilitates tumor spread and monitoring of tumor growth but also allows for timely feedback on the efficacy of CAR-T cell therapy. This enhances the dynamic visualization capabilities of confocal real-time imaging tracking combined with cerebrospinal fluid biomarker detection to assess the anti-tumor abilities of different CAR-T cells.

[0121] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A method for fabricating a dynamic microfluidic chip device for studying CAR-T therapy for gliomas, characterized in that, Includes the following steps: S1. Fabrication of microfluidic chips: The channels of microfluidic chips are designed using computer-aided design software. The channels of microfluidic chips include vascular channels, brain parenchyma channels, tumor channels and subarachnoid channels. The master mold of the microfluidic chip is obtained by manufacturing through 3D printing technology. Polydimethylsiloxane material is poured onto the master mold of the microfluidic chip and cured, then demolded and punched to finally obtain a microfluidic chip with the required channel structure. S2. Reconstruction of neural tissue: Prepare suspensions of astrocytes and microglia; mix the two types of glial cells with a soluble basement membrane 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 to gel, so as to achieve three-dimensional culture of glial cells; S3. Reconstruction of the 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 for 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 the blood-brain barrier microstructure; S4. Simulation of the glioma microenvironment: A glioma cell suspension was prepared and mixed with a soluble basement membrane hydrogel prepolymer to form a gel-glioma cell mixture; the gel-glioma cell mixture was injected into the tumor channel of the microfluidic chip; at the same time, a fibroblast cell suspension was prepared and injected into the subarachnoid channel, and the microfluidic chip was tilted to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel, and dynamically incubated 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 neural tissue includes the following steps: S21. Preparation of nerve cell suspension: Immortalized human brain astrocyte cell line and immortalized human brain microglia cell line were revived and cultured using Eagle medium and EMEM medium supplemented with fetal bovine serum, respectively; when the cell confluence reached 80-90%, the astrocyte cell suspension and microglia cell suspension were collected separately, and then the collected astrocyte cell suspension and microglia cell suspension were mixed evenly to form a mixed nerve cell suspension; S22. Inoculation of nerve cells: The prepared glial cell suspension is mixed evenly with the soluble basement membrane hydrogel prepolymer to form a gel-glial cell mixture; the microfluidic chip is placed on a cold bag, and the gel-glial cell mixture is injected into the brain parenchyma channel; after injection, the injected microfluidic chip is transferred to a cell culture incubator for isothermal gelation. S23. Culturing nerve cells: After gelation, Eagle medium and EMEM medium were reconstituted without the addition of fetal bovine serum and mixed to obtain serum-free glial cell culture medium; the prepared serum-free glial cell culture medium was injected into the vascular channel and the subarachnoid channel respectively; the culture medium was changed once a day for 5 days before the blood-brain barrier remodeling.

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. Preparation of blood-brain barrier cell suspension: Immortalized human brain microvascular endothelial cell line and immortalized human brain pericyte line were revived and cultured using ECM medium and PM medium, respectively; when the cell confluence reached 80-90%, the human brain microvascular endothelial cell suspension and the human pericyte suspension were collected separately, and then the collected human brain microvascular endothelial cell suspension and human pericyte suspension were mixed evenly to form a blood-brain barrier cell mixed suspension; S32. Inoculation of blood-brain barrier cells: The prepared blood-brain barrier cell suspension was injected into the vascular channel and co-cultured with the neural tissue reconstructed in step S2. After co-culturing for 4 days, the microfluidic chip was tilted so that human brain microvascular endothelial cells and human pericytes adhered to the interface between the vascular channel and the brain parenchyma channel, simulating the real structure of the blood-brain barrier in vivo. S33. Culture of blood-brain barrier cells: Without adding fetal bovine serum, reconstitute ECM and PM media and mix them to obtain serum-containing endothelial cell culture medium; inject the prepared serum-containing endothelial cell culture medium into the vascular channel and use the flow action to remove cells that have not adhered successfully and any possible debris; continue dynamic culture for 3 days, during which time the serum-containing endothelial cell culture medium in the vascular channel and the serum-free glial cell culture medium in the subarachnoid space channel are changed 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 simulation of the glioma microenvironment includes the following steps: S41. Preparation of glioma cell suspension: Immortalized human glioma cell lines were revived and cultured using DMEM medium supplemented with 5% fetal bovine serum and 1% penicillin / streptomycin solution; when the cell confluence reached 80-90%, the glioma cell suspension was collected. S42. Inoculation of glioma cells: The prepared glioma cell suspension is uniformly mixed with the soluble basement membrane hydrogel prepolymer to form a gel-glioma cell mixture; the microfluidic chip is placed on a cold bag, and the gel-glioma cell mixture is injected into the tumor channel; after injection, the injected microfluidic chip is transferred to a cell culture incubator for isothermal gelation; after gelation, serum-free glioma cell culture medium is injected into the subarachnoid space channel and cultured for 1-2 days, changing the culture medium daily to maintain cell growth. S43. Construction of pia mater: Immortalized human dermal fibroblast cell lines were revived and cultured using DMEM medium supplemented with 2 mM / L glutamine and 10% fetal bovine serum; when the cell confluence reached 80-90%, the fibroblast cell suspension was collected; the fibroblast cell suspension was injected into the subarachnoid channel of the microfluidic chip; the microfluidic chip was tilted to allow the fibroblasts to adhere to the interface between the tumor channel and the subarachnoid channel; incubation was continued to promote the formation of stable pia mater microstructures by fibroblasts.

5. The method for preparing a dynamic microfluidic chip device for studying glioma CAR-T therapy according to claim 4, wherein step S42, changing the culture medium includes the following steps: A tubing is connected to the input end of the subarachnoid space channel and then to an automated infusion pump. Serum-free glial cell culture medium is injected through the automated infusion pump to simulate the dynamic flow environment of cerebrospinal fluid in vivo. The flow rate of the automated infusion pump is adjusted to ensure that the shear stress is controlled within 0.1 dyn / cm² to realistically reproduce the flow characteristics of cerebrospinal fluid in vivo.

6. A dynamic microfluidic chip device for studying CAR-T therapy for gliomas, characterized in that, The invention includes a microfluidic chip prepared using the method described in claim 5; the microfluidic chip includes a vascular channel, a brain parenchyma channel, a tumor channel, and a subarachnoid space channel arranged sequentially from left to right; each of the vascular channel, brain parenchyma channel, tumor channel, and subarachnoid space channel includes a cell culture chamber with an inlet on one side; the cell culture chambers of the vascular channel and the subarachnoid space channel are respectively provided with outlets 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 glioma cells; The subarachnoid space channel is used for monolayer culture of fibroblasts to complete the reconstruction of the pia mater and to simulate the flow of cerebrospinal fluid.

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

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

9. A dynamic microfluidic chip device for studying CAR-T therapy for gliomas according to claim 8, characterized in that, The automatic injection pump includes an injection head, a syringe, an injection tubing, and a base with a built-in drive motor. The injection head, syringe, and injection tubing are arranged in sequence, and the inlet on the vascular channel or subarachnoid channel is connected to the syringe through the injection tubing. The syringe is fixedly installed on the base, and the drive motor is connected to the syringe through the injection head, thereby driving the injection head to drive the syringe on the base to inject.

10. The application of a dynamic microfluidic chip device as described in any one of claims 6-9 for studying CAR-T therapy for gliomas, characterized in that, Includes the following steps: S1. CAR-T cell infusion: Fluorescently labeled CAR-T cells mixed with serum-containing endothelial cell culture medium are injected into the vascular channel to simulate intravenous delivery of CAR-T cell therapy; S2. Observation of transmembrane migration: The capture, adhesion and extravasation process of CAR-T cells were visualized using confocal real-time imaging tracking technology, and relevant parameters were calculated; S3. Biomarker detection: The anti-tumor ability of CAR-T cells is assessed by measuring the levels of specific biomarkers in the effluent from the subarachnoid passage.

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