A method for constructing and evaluating a steady-state neuroinflammatory microenvironment model

Abstract

This invention discloses a method for constructing and evaluating a homeostatic neuroinflammatory microenvironment model. In this invention, brain microvascular endothelial cells and microglia are seeded into the methacrylamide gelatin layer and the paper fiber layer of a methacrylamide gelatin-paper fiber composite membrane (GCC), respectively, and co-cultured for 24–96 hours without LPS stimulation to form a homeostatic neuroinflammatory microenvironment model. The 24-hour GCC group… TNF-alpha Compared to the PET group, CX3CL1 decreased by about 60% and remained at a low level for 72 hours; CX3CL1 increased by nearly 3 times in the GCC group; based on the evaluation of 4 / 10 / 70 kDa graded probes, GCC showed better restriction on 10 kDa than PET, exhibiting molecular weight-dependent permeation characteristics opposite to PET. The homeostatic neuroinflammatory microenvironment model proposed in this application is suitable for simulating the blood-brain barrier in early pathological conditions, and can be used for anti-inflammatory drug screening and research on neurodegenerative diseases, showing good application prospects.

Description

Technical Field

[0001] This invention belongs to the field of in vitro model construction technology, specifically relating to a method for constructing and evaluating a homeostatic neuroinflammatory microenvironment model. Background Technology

[0002] Neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and multiple sclerosis) and cerebrovascular diseases pose significant health challenges to the global aging society. The blood-brain barrier (BBB), acting as the "gatekeeper" of the central nervous system (CNS), is closely associated with neuroinflammation due to its dysfunction, making it a crucial link in the development and progression of these diseases. Constructing high-fidelity in vitro models that can simulate the interactions of neurovascular units (NVUs) under physiological and pathological conditions is invaluable for understanding disease mechanisms, screening CNS-targeted drugs, and assessing neurotoxicity. However, existing in vitro models still have significant limitations in terms of physiological relevance, making it difficult to accurately predict in vivo drug responses and disease progression.

[0003] Currently, the widely used Transwell co-culture system mostly uses porous polymer membranes such as polyethylene terephthalate (PET) or polycarbonate (PC) as cell growth substrates. These materials have the following inherent defects: (1) Mechanical mismatch: The elastic modulus of PET membrane is about GPa (~8800 kPa), while the physiological stiffness of brain tissue is only 1-10 kPa. The difference of three orders of magnitude leads to abnormal cytoskeleton tension and dysregulation of tight junction protein expression; (2) Structural limitations: The physical thickness (~10 μm) and rigid microporous structure of commercial Transwell membranes hinder direct paracrine / procrine signal transduction between cells, and cannot simulate the three-dimensional microenvironment in which microglia infiltrate the basement membrane through protrusions and interact with endothelial cells; (3) Single evaluation method: Traditional transendothelial resistance (TEER) detection relies on rigid insulating support. For hydrogel-type soft biomimetic membranes, the data is distorted due to interference from ionic conductivity, and it cannot provide permeability information based on molecular weight grading, which is difficult to meet the screening needs of complex drugs (such as antibodies, peptides, and nano-formulations).

[0004] Microglia, as resident immune cells of the central nervous system, maintain a "homeostatic surveillance" phenotype (manifested as low expression of pro-inflammatory factors such as TNF-α) in healthy brain tissue through the regulation of the CX3CL1-CX3CR1 axis signaling pathway. However, they transform into a pro-inflammatory phenotype under pathological conditions. In existing in vitro models, microglia spontaneously activate under rigid two-dimensional substrates or without endothelial cell co-culture (even without LPS stimulation, they highly express TNF-α), making it impossible to distinguish the regulatory effects of drugs on "pathological inflammation" and "physiological homeostasis," resulting in a large number of false positive results. Therefore, developing novel in vitro models that can maintain the microglia homeostatic phenotype, support quantitative detection of inflammatory factors, and possess physiologically relevant permeability is a pressing technical challenge in the field of neuroimmunology research.

[0005] Chinese invention patent CN202410048033.7 discloses a preparation process of GelMA / paper fiber composite membrane (GCC) and its method for co-culturing brain microvascular endothelial cells (bEnd.3) and microglia (N9), confirming that the membrane supports cell survival, expression of basic barrier protein (PECAM-1) and its three-dimensional spatial distribution. However, this technology only involves morphological observation (immunofluorescence staining, live / dead staining, tissue section staining) and fails to reveal the following key biological effects: (1) Quantitative differences in homeostatic inflammatory phenotypes: Existing technologies do not detect differences in the expression levels of inflammatory factors (TNF-α) and chemokines (CX3CL1, CCL2), and fail to disclose the quantitative relationship between GCC soft substrates (~10 kPa) and PET hard substrates (~GPa) in inducing microglial inflammatory phenotypes; (2) Pathological features of selective leakage: Existing technologies (PET) can only construct "all-or-nothing" barriers (either completely restricted or completely permeable), and cannot simulate the selective permeability features under physiological or pathological conditions; (3) Temporal immunomodulatory mechanisms: Existing technologies do not disclose the temporal expression patterns of CX3CL1 and CCL2 in the co-culture system (such as dynamic changes in 24h vs 72h) and their correlation with basement membrane stiffness. Therefore, there is an urgent need for a method that can quantitatively evaluate the regulatory effect of soft substrates on the neuroinflammatory microenvironment, as well as a standardized barrier function detection method applicable to soft biomimetic membranes. Summary of the Invention

[0006] Therefore, the purpose of this invention is to provide a method for constructing and evaluating a homeostatic neuroinflammatory microenvironment model, thereby addressing the technical deficiencies in the prior art, such as the lack of quantitative inflammatory factor data and the lack of permeability evaluation standards applicable to soft membranes.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] In a first aspect, the present invention provides a method for constructing an in vitro homeostatic neuroinflammatory microenvironment model, comprising the following steps: fixing a methacrylamide gelatin-paper fiber composite membrane to the bottom of a Transwell chamber; seeding a suspension of brain microvascular endothelial cells onto the methacrylamide gelatin layer of the methacrylamide gelatin-paper fiber composite membrane; seeding a suspension of microglia onto the paper fiber layer of the methacrylamide gelatin-paper fiber composite membrane; and co-culturing for 24 to 96 hours without LPS stimulation to form a homeostatic neuroinflammatory microenvironment model.

[0009] Based on the above technical solution, further, the microglia in the obtained homeostatic neuroinflammatory microenvironment model TNF-α The expression level was significantly lower than that of the polyethylene terephthalate (PET) film control group, and the expression level of CX3CL1 was significantly higher than that of the PET control group.

[0010] Based on the above technical solution, the thickness of the methacrylamide gelatin layer in the methacrylamide gelatin-paper fiber composite film is ≤10μm, preferably 1~2μm, and the elastic modulus is 5~15 kPa; the methacrylamide gelatin layer is cured on the surface of the paper fiber layer by ultraviolet light crosslinking.

[0011] Based on the above technical solution, the paper fiber layer is further described as Whatman 105 lens cleaning paper, with a fiber diameter of 10~50μm and a thickness of 20~60μm.

[0012] Based on the above technical solution, furthermore, the density of both the brain microvascular endothelial cell suspension and the microglial cell suspension is 1~9×10⁻⁶. 5 cells / mL; the co-culture specifically refers to steady-state co-culture at 37℃ and 5% CO2.

[0013] Based on the above technical solution, the LPS-free stimulation condition is further achieved by using a culture medium with an endotoxin level ≤0.1 EU / mL.

[0014] Based on the above technical solution, further, the microglia are N9 cells, and the brain microvascular endothelial cells are bEnd.3 cells.

[0015] Based on the above technical solution, a silicone ring is further used to assist in fixing the methacrylamide gelatin-paper fiber composite membrane, so that the brain microvascular endothelial cells and microglia are in a micron-level ultra-close distance.

[0016] Based on the above technical solution, the preparation method of the methacrylamide gelatin-paper fiber composite film further includes the following steps: (1) Sodium alginate aqueous solution and calcium chloride aqueous solution are sequentially added to the paper fiber layer in a volume ratio of 1:2 to 2:1. After they crosslink to form calcium alginate, they are used as sacrificial templates. The concentration of sodium alginate aqueous solution is 3 to 5 wt%, and the concentration of calcium chloride aqueous solution is 4 to 6 wt%. (2) The GelMA hydrogel solution containing photoinitiator is uniformly sprayed onto the surface of the paper fiber layer containing the sacrificial template obtained in step (1), and then crosslinked with ultraviolet light. The sacrificial template is removed by placing it in an aqueous solution containing disodium ethylenediaminetetraacetate to restore the paper fiber support function, and then air-drying to form a film.

[0017] Based on the above technical solution, further, in step (2), the concentration of the GelMA hydrogel solution is 5~15% (w / v), and the working parameters of ultraviolet crosslinking are as follows: wavelength: 395nm, irradiation time 10~40s; the concentration of disodium ethylenediaminetetraacetate aqueous solution is 1~5wt%; the spraying process parameters are: pump pressure 20~60psi, temperature 25~40℃, humidity 20~40%, and the distance from the nozzle to the surface of the paper fiber layer is 6~10cm.

[0018] Based on the above technical solution, further, the air-drying conditions in step (2) are 20~30℃ and relative humidity below 50%, and natural dehydration and air drying.

[0019] Secondly, the present invention provides a homeostatic neuroinflammatory microenvironment model obtained by the above-described construction method.

[0020] Thirdly, the present invention provides a method for evaluating the paracrine communication efficiency of vascular-immune units in the above-mentioned homeostatic neuroinflammatory microenvironment model. The method assesses the inflammatory state of endothelial cells by detecting the concentration of CCL2 protein in the culture medium of brain microvascular endothelial cells in the homeostatic neuroinflammatory microenvironment model; and assesses the transmembrane signal transduction efficiency by detecting the concentration of CX3CL1 protein in the culture medium of microglia in the homeostatic neuroinflammatory microenvironment model, thereby obtaining the paracrine communication efficiency of vascular-immune units in the homeostatic neuroinflammatory microenvironment model.

[0021] Based on the above technical solution, the CX3CL1 concentration in the culture medium of microglia in the GCC group was significantly higher than that in the PET control group.

[0022] Fourthly, the present invention provides a method for evaluating the permeability of the above-mentioned homeostatic neuroinflammatory microenvironment model, comprising the following steps: adding a culture medium containing fluorescently labeled dextran with molecular weights of 10 kDa and 70 kDa to the side of the brain microvascular endothelial cells of the homeostatic neuroinflammatory microenvironment model, incubating for 30-150 min, detecting the fluorescence intensity in the solution on the side of the paper fiber layer, and calculating the apparent permeability coefficient and barrier function index.

[0023] Based on the above technical solution, further, the ratio of the barrier index of the steady-state neuroinflammatory microenvironment model to that of 10 kDa fluorescently labeled dextran to that of 70 kDa fluorescently labeled dextran is greater than 1.0, exhibiting a molecular weight-dependent permeation characteristic opposite to that of PET membrane.

[0024] Based on the above technical solution, it further includes adding 4 kDa fluorescently labeled dextran as a molecular weight control.

[0025] Based on the above technical solution, further, the barrier index of the homeostatic neuroinflammatory microenvironment model for 10 kDa fluorescently labeled dextran is 3.0~3.5, and the barrier index of the homeostatic neuroinflammatory microenvironment model for 70 kDa fluorescently labeled dextran is 1.7~2.5.

[0026] Furthermore, the homeostatic neuroinflammatory microenvironment model constructed in this invention, because it can maintain the homeostatic phenotype of microglia (low TNF-α, high CX3CL1) and support graded permeability detection, is expected to be used for in vitro screening and evaluation of neuroprotective drugs or anti-inflammatory drugs, providing an experimental platform for the development of drugs for the central nervous system.

[0027] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention quantitatively reveals the dose-response relationship between softness / stiffness and inflammatory phenotype: For the first time, this invention uses RT-q-PCR and ELISA to quantitatively detect that GCC soft substrates (~10 kPa) can significantly inhibit spontaneous inflammatory activation of microglia (TNF-α decreased to 60% of the PET group, p<0.0001) and significantly promote the expression of the anti-inflammatory chemokine CX3CL1 (increased to 3 times that of the PET group). This quantitative difference has not been revealed in the prior art, providing a molecular-level gold standard for assessing the biocompatibility of materials.

[0028] 2. This invention establishes an evaluation method for the selective permeability characteristics of soft biomimetic membranes: Addressing the technical bottleneck of the TEER method's inapplicability to hydrogel membranes, this invention establishes a differentiated detection system based on 4 / 10 / 70 kDa graded tracers. Compared to rigid PET membranes (high restriction for macromolecules, relatively low restriction for medium-sized molecules), GCC exhibits a unique selective permeability pattern: its restriction ability for 10 kDa medium-molecular-weight substances is significantly better than PET (barrier index increased by approximately 52%), while its permeability to 70 kDa macromolecules is relatively high (barrier index approximately 46% of PET). This characteristic more closely resembles the selective filtering function of the leaky blood-brain barrier in early pathological states (such as early Alzheimer's disease and early Parkinson's disease)—that is, the tight junctions loosen to allow certain macromolecules to pass through, while still maintaining selective blocking of medium-sized molecules. This provides a differentiated evaluation platform that PET membranes cannot achieve for studying angiogenesis-immune interactions under pathophysiological conditions, simulating the drug cross-barrier delivery window, and screening drugs that restore barrier function.

[0029] 3. This invention reveals temporal immune regulatory characteristics: This invention reveals the temporal expression pattern of CX3CL1 and CCL2 in a GCC environment (high CX3CL1 / low CCL2 expression is established within 24 hours). TNF-α The condition (which remains stable for 72 hours) can be used to simulate different pathological stages, such as early neuroprotection or late inflammation resolution. Attached Figure Description

[0030] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0031] Figure 1 The graph shows the Young's modulus results of the GelMA layer of PET and GCC films determined by atomic force microscopy.

[0032] Figure 2 A schematic diagram of a three-dimensional co-culture system with micron-level ultra-close distance formed on both sides of a GelMA / paper fiber composite membrane.

[0033] Figure 3 This is a frozen section of a three-dimensional co-culture system on a GCC membrane stained with hematoxylin and eosin (HE). Scale bar: 50 micrometers.

[0034] Figure 4 To determine the secretion of N9 cells under four different culture conditions TNF-α The results of mRNA expression levels are shown in the figure, where (a) is the result of 24-hour detection and (b) is the result of 72-hour detection.

[0035] Figure 5The images show the ELISA results of CCL2 protein secreted by bEnd.3 cells under four different culture conditions. (a) shows the results after 24 hours, and (b) shows the results after 72 hours.

[0036] Figure 6 The figures show the ELISA results of CX3CL1 transmembrane transport under four different culture conditions, where (a) is the result after 24 hours and (b) is the result after 72 hours.

[0037] Figure 7 The graph shows the apparent permeability of GCC and PET membrane systems measured using classic tracers (4 kDa, 10 kDa, and 70 kDa FITC-labeled dextran).

[0038] Figure 8 Scanning electron microscope image of the GelMA hydrogel layer on the surface of the GCC membrane (after freeze-drying), scale bar: 10 μm. Detailed Implementation

[0039] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0040] Example 1: A method for constructing a three-dimensional homeostatic neuroinflammatory microenvironment 1.1 Material Preparation The GelMA / paper fiber composite membrane (GCC membrane) used in this embodiment was prepared according to the method described in the invention patent (application number: 202410048033.7), and the specific steps are as follows: Step 1: Accurately weigh 0.8g of sodium alginate and 1.2g of calcium chloride dihydrate, and dissolve them separately in 20mL of deionized water at room temperature to prepare sodium alginate solution and calcium chloride solution, respectively. Step 2: Accurately weigh 1g of disodium ethylenediaminetetraacetate dihydrate and dissolve it in 50mL of deionized water at room temperature as a complexing agent solution; use a 20μl pipette to uniformly coat the sodium alginate solution and calcium chloride solution onto the surface of paper fibers (fiber diameter 20-50 μm, porosity >100 μm, Whatman 105 lens paper), with a mixing ratio of 1:1; and immediately form a calcium alginate gel as a sacrificial template; Step 3: Accurately weigh an appropriate amount of methacrylated hydrogel solid, add 0.4-0.6 ml of photoinitiator solution, heat in a 60°C water bath to dissolve, and shake several times during the process; prepare a GelMA hydrogel solution with a concentration of approximately 12% (w / v); Step 4: Adjust the pressure of the pressure pump to 40 psi and the spraying distance to 6-10 cm, and spray the methacrylic hydrogel solution evenly onto the surface of the paper containing the sacrificial template. Step 5: Irradiate with 395nm wavelength ultraviolet light for 30s, then transfer the film to a 3%-5% EDTA disodium salt solution, set the shaker temperature to 37℃, the rotation speed to 50-100rpm, and the time to 1-2h to remove the sacrificial template layer. Step 6: Take out the sample from Step 5 and air dry it naturally at 20℃~30℃ with a relative humidity of less than 50% to form a film.

[0041] A GelMA hydrogel layer with a thickness of approximately 1-2 μm was constructed on the surface of Whatman 105 lens paper using the sacrificial template method. Atomic force microscopy analysis showed that the elastic modulus in the swollen state was 9.8 ± 1.2 kPa. Figure 1 As shown.

[0042] 1.2 Cell Seeding and Steady-State Culture The GCC membrane was attached to the bottom of the Transwell chamber (Getetra), and fixed using an auxiliary fixation device (such as a silicone ring). Cell seeding was then performed on both sides: first, N9 cells (microglia N9 at 6 × 10⁶ cells / year) were seeded onto the paper fiber layer. 5 (Cells / mL were seeded into the paper fiber layer of the GCC membrane), and after culturing for 12 h to allow them to immobilize, the chamber was inverted, and cerebral vascular endothelial cells bEnd.3 were seeded onto the surface of the GelMA layer (bEnd.3 cells were seeded at a density of 3 × 10⁶ cells / mL). 5 Cells were seeded at a density of 1 / mL in the GelMA layer of the GCC membrane; culture conditions: serum-free medium with endotoxin levels <0.1 EU / mL was used, and no LPS or other inflammatory stimulants were added throughout the process. Steady-state co-culture was carried out at 37℃ and 5% CO2 (quiescent co-culture); this design allows the two cell types to be located on opposite sides of the GelMA / paper fiber interface, forming a three-dimensional co-culture system with a micron-level ultra-close distance (<10 μm). Figure 2 ).

[0043] 1.3 Validation of the three-dimensional spatial structure of co-cultured cells After 72 hours of incubation, the three-dimensional co-culture on the GCC membrane was frozen sectioned and stained with hematoxylin and eosin (HE) to further verify that bEnd.3 formed a continuous monolayer in the GelMA layer and that N9 exhibited a three-dimensional spatial distribution in the paper fiber layer (e.g., ...). Figure 3 (As shown). This structural morphology is consistent with the typical features observed in previous studies (invention patent application number 202410048033.7), indicating that the model construction method is stable and reproducible.

[0044] 1.4 Control Group Setup A PET membrane control group was set up simultaneously: a 24-well Transwell PET membrane (Getex) with a pore size of 0.4 μm was used, and the cell seeding density, culture medium formulation, and culture time were completely consistent with the GCC group. A commercially available Transwell standard co-culture method was used: bEnd.3 cells were seeded on the upper surface of the PET membrane (0.4 μm pore size), and N9 cells were seeded in a 24-well plate (Polystyrene, PS) below the Transwell chambers. There was approximately a physical distance of 1 mm between the two cell types, and substance exchange mainly relied on diffusion through the PET membrane micropores.

[0045] Example 2: Quantitative detection of microglial homeostatic phenotypes This embodiment uses real-time quantitative PCR (RT-qPCR) to quantitatively analyze the expression levels of inflammatory factors in microglia (N9) in the steady-state co-culture model constructed in Example 1, verifying the direct effect of the GCC three-dimensional paper fiber scaffold on the homeostasis maintenance of N9 cells, and the indirect effect on cerebral vascular endothelial cells (bEnd.3) growing on soft matrix in the co-culture system.

[0046] The specific experimental design and results are as follows: 2.1 Experimental Grouping and Sampling The experiment was divided into the following four groups (n=3): ① GCC co-culture group: bEnd.3 and N9 cells were seeded on both sides of the GCC membrane respectively (seeding method and culture conditions are the same as in Example 1); ② GCC N9 group alone: ​​N9 cells were seeded only in the paper fiber layer of the GCC membrane; ③ PET co-culture group: bEnd.3 and N9 cells were seeded on Transwell PET membranes (0.4 μm pore size, Jet) and then seeded in 24-well plates (Polystyrene, PS) below the chambers. The method and culture conditions were the same as in Example 1. ④ PET N9 group alone: ​​N9 cells were seeded in 24-well plates (Polystyrene, PS) below the Transwell chamber.

[0047] Supernatant and cell samples were collected from each group after 24 and 72 hours of incubation. The entire process was performed under LPS-free conditions, with endotoxin levels in the culture medium <0.1 EU / mL.

[0048] 2.2 RT-qPCR detection of inflammatory factors TNF-α Express 2.2.1 RNA Extraction and Reverse Transcription Total RNA was extracted from N9 cells in each group using RNAiso reagent (Takara). After purity was determined by NanoDrop (A260 / A280 ratio 1.8-2.0), it was reverse transcribed into cDNA using the PrimeScript™ RT kit (Takara) with gDNA Eraser, following the manufacturer's instructions.

[0049] 2.2.2 Real-time quantitative PCR Using TB Green ® qPCR detection was performed using Premix Ex Taq™ (Tli RNaseH Plus) reagents. The primer sequences are as follows: TNF-α Upstream primer: 5'-TCTCAGCCTCTTCTCATTCCTG-3'; TNF-α Downstream primer: 5'-TACAGGCTTGTCACTCGAATT-3'; GAPDH Upstream primer: 5'-CATGGCCTTCCGTGTTCCTA-3'; GAPDH Upstream primer: 5'-GCGGCACGTCAGATCCA-3'; The 25 μL reaction mixture contained: 12.5 μL of 2× Premix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), 1 μL of cDNA template, 0.5 μL of ROX Reference Dye (50×, add according to instrument model), and sterile water to a final volume of 25 μL. Reaction conditions: 30 s pre-denaturation at 95°C, followed by 40 cycles, each cycle consisting of 5 seconds of reaction at 95°C and 30 seconds of reaction at 60°C; using 2... -ΔΔCt The relative expression level is calculated by the method, in order to GAPDH For internal reference.

[0050] 2.3 Experimental Results and Analysis Under steady-state conditions without LPS stimulation, the relative gene expression of microglia N9 cells in the four experimental groups after 24 hours (a) of incubation: After 24 hours, the two co-culture groups TNF-α The levels are generally higher than in single-layer cultures, indicating that bEnd.3 and N9 have begun to "communicate." Among them, the PET co-culture group... TNF-α The highest levels of *bEnd.3* cells, cultured on a rigid PET substrate, indicate that early microglial inflammatory activation was induced. In contrast, the response was significantly weakened in the GCC co-culture group. TNF-α The level decreased by about 60% (p <0.001). Meanwhile, the GCC co-culture group... TNF-α The expression level was comparable to that of N9 cells in the GCC isolated group. This indicates that N9 cells grown in the flexible GCC paper fiber layer did not elicit a response to the same extent as bEnd.3-driven pro-inflammatory signals. Figure 4 a).

[0051] Under steady-state conditions without LPS stimulation, the relative gene expression of microglia N9 cells in the four experimental groups after 72 hours of incubation (b): At 72 h of culture, the PET N9 group alone... TNF-α The expression level was the highest among all experimental groups, while it was the lowest in the GCC co-culture group, with a difference of nearly 5 times. p <0.0001), and compared with the PET co-culture group, the GCC co-culture group and the GCC N9 group alone TNF-α The relative expression level was significantly lower ( p <0.001). Within the GCC group, there was no significant difference between co-culture and solitary culture (Figure 4b). Significant differences appeared after 72 hours of culture, in the PET group containing only N9. TNF-α The expression of [a specific cell type] was significantly increased, indicating a persistent activation state after prolonged incubation on a hard surface. Notably, this increase was suppressed in the PET co-culture group containing bEnd.3. In the PET co-culture group, the presence of bEnd.3 cells had a certain inhibitory effect on the expression of N9 inflammatory factors.

[0052] Example 3: Validation of paracrine communication between vascular and immune units This embodiment verifies the effectiveness of vascular-immune paracrine communication in the GCC co-culture system (endothelial cells-microglia) constructed in Example 1. Neurovascular units (NVUs) rely on complex molecular dialogues to maintain brain homeostasis, among which the chemokines CCL2 (MCP-1) and CX3CL1 (Fractalkine) are key factors in endothelial cell-microglia communication. CCL2, as a major mediator of microglia chemotaxis and blood-brain barrier (BBB) ​​permeability, promotes rapid recruitment and activation of microglia under environmental disturbances. Conversely, CX3CL1 plays a crucial homeostatic "checkpoint" role; its unique membrane-bound and soluble states enable it to regulate the resting state of microglia and inhibit their overactivation via the CX3CL1-CX3CR1 axis.

[0053] This embodiment demonstrates, by detecting the chemokines (CX3CL1, CCL2) secreted by endothelial cells and their transmembrane transport efficiency, that the micron-scale structure and selective permeability of GCCs facilitate bidirectional signal transduction, while the millimeter-scale distance of traditional Transwells hinders this process.

[0054] The specific experimental design and results are as follows: 3.1 Experimental Grouping The experiment was divided into the following four groups (n=3): ① GCC co-culture group: bEnd.3 and N9 cells were seeded on both sides of the GCC membrane respectively (seeding method and culture conditions are the same as in Example 1); ② GCC-only bEnd.3 group: bEnd.3 cells were seeded only in the GelMA layer of the GCC membrane; ③ PET co-culture group: bEnd.3 cells were seeded on Transwell PET membranes (0.4 μm pore size, Jettex), and N9 cells were seeded in 24-well plates (Polystyrene, PS) below the chambers. The method and culture conditions were the same as in Example 1. ④ PET-only bEnd.3 group: bEnd.3 cells were seeded only on the PET membrane in the Transwell chamber.

[0055] 3.2 Sample processing and detection After culturing in an incubator for 24 hours and 72 hours, the supernatant and subnatant of each culture chamber were collected and centrifuged at 4°C (500g, 10 min) to remove cell debris. The concentrations of CCL2 and CX3CL1 proteins in the collected supernatant and subnatant of the culture chambers were detected using the Mouse CCL2 ELISA Kit (E-MSEL-M0012, E-Lite) and the Mouse CX3CL1 ELISA Kit (E-EL-M0267, E-Lite), respectively, strictly following the instructions.

[0056] 3.3 Experimental Results and Analysis We used enzyme-linked immunosorbent assay (ELISA) to test and analyze CCL2 and CX3CL1 in the four experimental groups at 24 and 72 hours (Figures 5-6). The results showed that at 24 hours, the CCL2 expression level in the PET co-culture group was slightly higher than that in the GCC co-culture group, and the PET bEnd.3 group alone was also higher than the GCC bEnd.3 group alone. CCL2 is a typical pro-inflammatory and chemokine secreted by vascular endothelial cells. Because the Transwell control group (PET membrane) had a stiffness of GPa, it may have caused a kind of "mechanical stress" on the endothelial cells in the early stages, leading to the secretion of more CCL2. In contrast, the GCC group, due to the soft matrix environment of GelMA, may have had a more homeostatic cell state. The higher CCL2 concentrations in both co-culture groups compared to the single endothelial cell culture group indirectly indicate the inflammatory effect produced by N9 cells at 24 hours. TNF-α ) can also induce endothelial secretion of CCL2 through liquid phase factors. At this time, the corresponding CX3CL1 (lower supernatant) expression level is: the expression level in the GCC co-culture group is significantly higher than that in the PET co-culture group ( p<0.0001), while the PET group inoculated with bEnd.3 alone had higher values ​​than the PET co-culture group, and the GCC group inoculated with bEnd.3 alone had significantly lower values ​​than the GCC co-culture group. Figure 6 a). This result directly demonstrates that spatial proximity is key to establishing vascular-immune microenvironment homeostasis. During the early 24-hour response phase, the GCC co-culture group, with its micrometer-scale thickness, established a highly efficient paracrine circuit. The CX3CL1 (homeostasis signal) highly expressed in endothelial cells bEnd.3 rapidly acted on the underlying N9 cells, effectively inhibiting the production of pro-inflammatory factors by N9 cells. TNF-α (like Figure 5 (as shown in a). In contrast, the Transwell (PET) group, due to the millimeter-scale physical spacing, experienced a "spatial delay" and "dilution effect" in the transmission of CX3CL1 signaling, resulting in N9 cells being in a state of ineffective regulation in the early stages, exhibiting significant inflammatory stress. TNF-α High expression, such as Figure 5 (as shown in a).

[0057] At 72 hours of culture, the CCL2 expression levels in the GCC and PET co-culture groups tended to be consistent, indicating that as the culture time increased, the system may have entered a relatively stable plateau phase, or the secretion level may have reached the detection limit. The CX3CL1 level in the GCC co-culture group showed a decreasing trend at 72 hours. Figure 6 b), combined with the low-inflammatory state of N9 ( Figure 5 b) reflects that the system has entered the "steady-state maintenance" stage from the "early response" stage (the factor is captured by the receptor and enters the dynamic cycle); while the factor accumulation in the PET group at 72h reflects a disordered accumulation lacking feedback regulation.

[0058] Example 4: Evaluation method for selective barrier function of soft biomimetic membranes This embodiment establishes a graded permeability evaluation method suitable for soft biomimetic membranes. By comparing the permeation behavior of fluorescent tracers of different molecular weights on GCC and PET membranes, the selective barrier characteristics of GCC for medium molecular weight substances are verified.

[0059] 4.1 Experimental grouping and tracer preparation Set up four groups (n=3): GCC+ cell group: Following the construction method of the GCC co-culture model described in Example 1, only bEnd.3 was seeded on the GelMA layer of the GCC membrane and cultured for 72 h to form an endothelial monolayer; GCC cell-free group: GCC membrane only, no cell seeding; PET+ cell group: Following the construction method of the PET membrane control group described in Example 1, only bEnd.3 was seeded onto the PET membrane (0.4 μm pore size) and cultured for 72 h; Cell-free PET group: PET membrane only, without cell inoculation.

[0060] Fluorescent tracers: FITC-labeled dextran (FITC-dextran, MCE) with molecular weights of 4 kDa, 10 kDa and 70 kDa were prepared at a working concentration of 10 μM.

[0061] 4.2 Permeability Testing Methods At 72 h of cell culture, the upper chamber medium of the Transwell and GCC baskets was aspirated, and 200 μL of the above-mentioned FITC-dextran tracer solutions of different molecular weights were added to the upper chamber; 1 mL of blank medium was added to the lower chamber. The baskets were incubated at 37°C in a 5% CO2 incubator. Samples (100 μL) were taken from the lower chamber at time points of 30 min, 60 min, 90 min, 120 min, and 150 min, and an equal volume of blank medium was added.

[0062] The fluorescence intensity of the sample was detected using a microplate reader (excitation wavelength 485 nm, emission wavelength 530 nm). The concentration of the lower chamber fluorescent tracer was calculated based on the standard curve, and the apparent permeability coefficient (Papp) was calculated using the following formula: Papp = ×

[0063] in, dQ / dt A is the amount of fluorescent material entering the lower chamber per unit time (μg / min), and A is the membrane area (cm²). 2 ), C 0 represents the initial concentration in the upper chamber (μg / mL).

[0064] Simultaneously calculate the Barrier Index (BI), which is the ratio of permeability without a cell membrane to that with a cell membrane. P blank / P cell ), used to characterize the barrier integrity of the endothelial cell layer.

[0065] 4.3 Experimental Results and Analysis 4.3.1 Analysis of the selective barrier characteristics of GCC and PET After endothelial cells bEnd.3 formed barriers on GCC and PET membranes, the permeability coefficients of cell-free and cell-containing membranes under two different membrane systems were obtained by testing the permeation restriction of fluorescent tracers of different molecular weights. The results are as follows: Figure 7 As shown in Table 1, to further compare and analyze the barrier characteristics of the two different membrane systems, the barrier function index of tracers with different molecular weights was calculated and obtained.

[0066] Table 1. Barrier function index of GCC and PET membrane systems for tracers of various molecular weights ( P blank / P cell (Mean±SD, n=3)

[0067] The results show that GCC and PET have fundamentally different molecular weight-dependent barrier characteristics: (1) Small molecules (4 kDa): equivalent barrier The two groups of barrier indices were similar (2.3 vs 2.7, p>0.05), indicating that for small molecule nutrients (such as glucose and ions), the endothelial cell layers on both basement membranes can provide basic barrier function, and the difference is not significant.

[0068] (2) Medium molecular weight (10 kDa): GCC shows advantages The GCC barrier index was significantly higher than that of PET (3.2±0.1 vs 2.1±0.3, p<0.001), indicating that endothelial cells grown on a soft substrate (~10 kPa) have a stronger ability to restrict medium molecular weight substances (such as small peptides and cytokines).

[0069] (3) Macromolecules (70 kDa): GCC barrier is significantly enhanced The PET barrier index (4.7±0.2) was approximately 2.2 times that of GCC (2.1±0.4) (p<0.0001), indicating that rigid porous membranes are more effective at physically blocking macromolecules (such as antibodies and plasma proteins). The GCC group showed relatively weaker barrier function against macromolecules, possibly due to the porous network structure of the GelMA hydrogel itself (wider pore size distribution). Figure 8 Although endothelial cells form tight junctions on the soft substrate, the hydrogel layer beneath the cell layer exhibits some permeability to 70 kDa molecules. Furthermore, differences in cell-base interactions lead to differences in paracellular pathways.

[0070] In summary, the graded permeability evaluation method (4 / 10 / 70 kDa) established in this embodiment reveals that GCC flexible biomimetic membrane and PET rigid membrane have different molecular weight selectivity: that is, GCC has a significantly better ability to confine medium molecular weight substances (10 kDa) than PET (barrier index increased by 52%), exhibiting the characteristics of an "intermediate molecular sieve"; PET has a better ability to confine macromolecular substances (70 kDa) than GCC (barrier index 2.2 times higher), exhibiting the characteristics of a "macromolecular barrier".

[0071] This difference in selectivity stems from the fundamental difference between the two materials: PET relies on a rigid physical pore size (0.4 μm) for mechanical filtration, while GCC relies on the cell layer density induced by a soft substrate to achieve a biological barrier. Although GCC's absolute barrier against macromolecules is weaker than PET's, its high restriction on medium-sized molecules, combined with the efficient paracrine communication demonstrated in Example 3, indicates that GCC is more suitable for mimicking physiological or early pathological microenvironments with selective permeability, rather than a simple "all or nothing" barrier.

[0072] This grading evaluation method can distinguish the permeability spectrum characteristics of different biomimetic membranes, providing a standardized screening platform for BBB penetration studies of drugs with different molecular weights (small molecule chemotherapy drugs vs. antibody drugs).

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; modifications or equivalent substitutions may be made to the technical solutions described in the foregoing embodiments, and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for constructing a homeostatic neuroinflammatory microenvironment model, characterized in that, The process includes the following steps: fixing a methacrylated gelatin-paper fiber composite membrane to the bottom of a Transwell chamber; seeding a suspension of brain microvascular endothelial cells onto the methacrylated gelatin layer of the methacrylated gelatin-paper fiber composite membrane; seeding a suspension of microglia onto the paper fiber layer of the methacrylated gelatin-paper fiber composite membrane; and co-culturing the membrane for 24-96 hours without LPS stimulation to form a homeostatic neuroinflammatory microenvironment model. The thickness of the methacrylamide gelatin layer in the methacrylamide gelatin-paper fiber composite film is ≤10μm, and the elastic modulus is 5~15 kPa; the methacrylamide gelatin layer is cured on the surface of the paper fiber layer by ultraviolet light crosslinking.

2. The construction method of claim 1, wherein, The density of both the brain microvascular endothelial cell suspension and the microglial cell suspension is 1~9×10⁻⁶. 5 cells / mL; the co-culture specifically refers to steady-state co-culture at 37°C and 5% CO2; the LPS-free stimulation condition is achieved by using a culture medium with an endotoxin level ≤0.1 EU / mL.

3. The construction method of claim 1, wherein, A silicone ring was used to fix a methacrylamide gelatin-paper fiber composite membrane, bringing brain microvascular endothelial cells and microglia into a micron-level ultra-close distance.

4. The construction method according to claim 1, characterized in that, The preparation method of the methacrylamide gelatin-paper fiber composite film includes the following steps: (1) Sodium alginate aqueous solution and calcium chloride aqueous solution are sequentially added to the paper fiber layer in a volume ratio of 1:2 to 2:

1. After they crosslink to form calcium alginate, they are used as sacrificial templates. The concentration of sodium alginate aqueous solution is 3 to 5 wt%, and the concentration of calcium chloride aqueous solution is 4 to 6 wt%. (2) The GelMA hydrogel solution containing photoinitiator is uniformly sprayed onto the surface of the paper fiber layer containing the sacrificial template obtained in step (1), and then crosslinked with ultraviolet light. The sacrificial template is removed by placing it in an aqueous solution containing disodium ethylenediaminetetraacetate to restore the paper fiber support function, and then air-drying to form a film.

5. The construction method according to claim 4, characterized in that, In step (2), the concentration of the GelMA hydrogel solution is 5-15% (w / v), and the working parameters for UV crosslinking are as follows: wavelength: 395nm, irradiation time 10-40s; concentration of disodium ethylenediaminetetraacetate aqueous solution is 1-5wt%; the spraying process parameters are: pump pressure 20-60psi, temperature 25-40℃, humidity 20-40%, and distance from nozzle to paper fiber layer surface 6-10cm.

6. The homeostatic neuroinflammatory microenvironment model obtained by the construction method according to any one of claims 1-5.

7. A method of evaluating the efficiency of the paracrine communication of the vascular-immune unit of the steady-state neuroinflammatory microenvironment model of claim 6, characterized in that, The inflammatory status of brain microvascular endothelial cells was assessed by detecting the concentration of CCL2 protein in the culture medium of brain microvascular endothelial cells in a homeostatic neuroinflammatory microenvironment model; the transmembrane signal transduction efficiency was assessed by detecting the concentration of CX3CL1 protein in the culture medium of microglia in the homeostatic neuroinflammatory microenvironment model, thereby obtaining the paracrine communication efficiency of vascular-immune units in the homeostatic neuroinflammatory microenvironment model.

8. A method of evaluating the permeability of the steady-state neuroinflammatory microenvironment model of claim 6, characterized by, The steps include: adding culture medium containing fluorescently labeled dextran with molecular weights of 10 kDa and 70 kDa to the side of brain microvascular endothelial cells in a homeostatic neuroinflammatory microenvironment model, incubating for 30-150 min, detecting the fluorescence intensity in the solution on the paper fiber layer side, and calculating the apparent permeability coefficient and barrier function index.

9. The method of claim 8, wherein, The ratio of the barrier index of the homeostatic neuroinflammatory microenvironment model to that of 10 kDa fluorescently labeled dextran to that of 70 kDa fluorescently labeled dextran is greater than 1.

0.

10. The method of claim 8, wherein, It also includes the addition of 4 kDa fluorescently labeled dextran as a molecular weight control.

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