A method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function and its application

Abstract

This invention discloses a method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function and its application, belonging to the biomedical field. The method includes: preparing a light-controlled biofunctionalized hydrogel with methacrylamide gelatin as the main framework; preparing a suspension of neurovascular matrix components using astrocytes and neurons; uniformly mixing the light-controlled biofunctionalized hydrogel and the neurovascular matrix component suspension to obtain a homogeneous cell-hydrogel composite solution, and preparing it into three-dimensional hydrogel microspheres; transferring the three-dimensional hydrogel microspheres to the bottom of a Transwell culture dish insert to form a stable neurolateral matrix layer; and adding a suspension of brain microvascular endothelial cells into the lower chamber of the Transwell culture dish. This invention achieves the synergistic effect of multiple bioactive substances, overcoming the shortcomings of the single bioactive substance loading method in the prior art.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function and its application. Background Technology

[0002] The blood-brain barrier (BBB) ​​is a crucial structure for maintaining homeostasis in the central nervous system. It is a complex neurovascular unit composed of vascular endothelial cells, astrocytes, pericytes, and neurons. Existing in vitro BBB models mostly employ monolayer endothelial cells or simple two-dimensional co-culture systems, which suffer from several drawbacks: structural limitations (lacking three-dimensional conformation and multi-cell synergy, failing to simulate the dynamic barrier characteristics of the in vivo BBB); functional deficiencies (traditional models struggle to maintain the long-term stability of tight junction proteins, leading to low transendothelial resistance (TEER) values ​​and inaccurate permeability predictions); and insufficient perfusion performance (existing hydrogel models have low mechanical strength, unable to support perfusion simulation of vascular lumens). Summary of the Invention

[0003] One of the objectives of this invention is to provide a method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function, so as to solve the problem that the existing technology lacks three-dimensional spatial conformation and multi-cell synergistic effect, and cannot simulate the dynamic barrier characteristics of the blood-brain barrier in vivo.

[0004] This invention is achieved through the following technical solution: a method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function, comprising the following steps: S100, preparing a photo-controlled biofunctionalized hydrogel with methacrylated gelatin as the main framework, wherein the photo-controlled biofunctionalized hydrogel is loaded with a functional bioactive peptide solution; S200, preparing a suspension of neurovascular matrix components using astrocytes and neuronal cells; S300, uniformly mixing the photo-controlled biofunctionalized hydrogel and the neurovascular matrix component suspension to obtain a homogeneous cell-hydrogel composite solution, injecting the cell-hydrogel composite solution into the channels of a microfluidic chip, and simultaneously... An oil phase is introduced into the transwell culture dish, allowing the cell-hydrogel composite solution to form droplets in the oil phase. The droplets are then irradiated with ultraviolet or blue light to form uniformly sized spherical three-dimensional hydrogel microspheres. S400: The three-dimensional hydrogel microspheres are transferred to the bottom of the Transwell culture dish insert to form a stable neural side matrix. A suspension of brain microvascular endothelial cells obtained according to standard cell culture procedures is added to the lower chamber of the Transwell culture dish, allowing the brain microvascular endothelial cells to attach to the lower surface of the Transwell membrane and grow into the hydrogel microspheres. The entire Transwell culture dish is then placed in a cell culture incubator at 37°C and 5% CO2 for culture.

[0005] Further, S100 includes the following sub-steps: S110, dissolving gelatin in deionized water to form a first mixed solution, continuously stirring and slowly adding methacrylic anhydride to prepare a second mixed solution, and reacting the second mixed solution at 45~55℃ for 3~4h to prepare a purified GelMA solution; S120, dissolving RGD cyclic peptide, nerve growth factor and brain-derived neurotrophic factor in phosphate buffered saline to prepare a functional bioactive peptide solution; S130, uniformly mixing the purified GelMA solution and the functional bioactive peptide solution, and after uniform mixing, adding a photoinitiator to prepare a photocontrolled biofunctionalized hydrogel.

[0006] Furthermore, the mass ratio of the functional bioactive peptide solution to phosphate-buffered saline is 0.001:1; the mass ratio of RGD cyclic peptide, nerve growth factor, and brain-derived neurotrophic factor in the functional bioactive peptide solution is 1000:0.1:0.1.

[0007] Further, S200 includes the following sub-steps: S210, thawing the frozen astrocytes and neurons, and centrifuging to remove the cryoprotectant; S220, resuspending the centrifuged cells in fresh culture medium, seeding them into cell culture flasks, and placing the cell culture flasks in a cell culture incubator at 37°C and 5% CO2 for culture; S230, collecting the cultured cells, resuspending them in serum-free culture medium or hydrogel precursor solution, and adjusting the cell concentration.

[0008] Furthermore, the concentration of astrocytes was 2 × 10⁻⁶. 6 ~5×10 6 Cells / mL.

[0009] Furthermore, the concentration of neurons was 5 × 10⁻⁶. 5 ~2×10 6 Cells / mL.

[0010] Furthermore, the cell-hydrogel composite solution is formed by mixing a light-controlled biofunctionalized hydrogel with a suspension of neurovascular matrix components at a volume ratio of 9:1 or 8:2. The mixing process is carried out in an ice bath or at 4°C to slow down the pre-crosslinking rate of the hydrogel while maintaining cell viability.

[0011] In another aspect, the present invention provides a three-dimensional in vitro blood-brain barrier model with barrier function, which is constructed according to the construction method of the three-dimensional in vitro blood-brain barrier model with barrier function described above.

[0012] The present invention also provides an application of a three-dimensional in vitro blood-brain barrier model with barrier function. The three-dimensional in vitro blood-brain barrier model constructed by the construction method described above includes the following applications: (1) application in brain immune response research; (2) application in evaluating blood-brain barrier penetration of macromolecular biological agents; (3) application in evaluating the neurotoxicity of environmental toxins and drugs; (4) application in research on the mechanism of blood-brain barrier dysfunction; (5) application in drug permeability assessment; and (6) application in personalized drug screening.

[0013] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0014] 1. This invention uses methacrylamide gelatin (GelMA) as the main framework, which naturally contains many bioactive sites. It can introduce bioactivity without further chemical modification, overcoming the problem of traditional biomimetic scaffolds lacking physiological activity. It can better simulate the complex biological environment in vivo and provide a scaffold that is closer to the in vivo environment for tissue engineering.

[0015] 2. This invention prepares a functional bioactive peptide solution by dissolving RGD cyclic peptide, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in phosphate-buffered saline, and then mixing it with GelMA, thereby achieving the synergistic effect of multiple bioactive substances and overcoming the shortcomings of the single loading method of bioactive substances in the prior art.

[0016] 3. This invention forms a photo-controlled biofunctionalized hydrogel with a controllable gradient structure by adding a photoinitiator after mixing GelMA and functional bioactive peptide solution for photocrosslinking. This solves the problem of uniform crosslinking degree and the same degradation rate of traditional UV or blue light cured hydrogels. At the same time, the prepared hydrogel has good biocompatibility and degradability, and realizes the sustained release of bioactive substances, meeting the needs of long-term gradual release of growth factors or drugs in the fields of tissue repair.

[0017] 4. This invention mixes hydrogel with a suspension of neurovascular matrix components and performs photocrosslinking in a microfluidic chip to form uniformly sized three-dimensional hydrogel microspheres, providing a biological scaffold that is closer to the in vivo environment for subsequent culture. Attached Figure Description

[0018] The accompanying drawings, which are included to provide a further understanding of embodiments of the invention and form part of this application, do not constitute a limitation thereof. In the drawings:

[0019] Figure 1 This is a diagram of a 4-hour leakage test provided in Embodiment 1 of the present invention.

[0020] Figure 2 This is a diagram of the FLU sodium leakage experiment provided in Example 1 of the present invention.

[0021] Figure 3 This is a diagram showing the continuity distribution of connective proteins in a laser confocal microscope provided in Embodiment 1 of the present invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated herein by reference to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended terms, meaning that they include but are not limited to. Unless the context clearly indicates otherwise, the expressions “a” and “an” as used herein include plural references. It should be noted that “first,” “second,” etc., are used merely for convenience of description and distinction and should not be construed as indicating or implying relative importance. The term “about” as used herein indicates a range of ±20% of the following numerical value. In some embodiments, the term “about” indicates a range of ±10% of the following numerical value. In some embodiments, the term “about” indicates a range of ±5% of the following numerical value.

[0024] Example 1

[0025] This embodiment discloses a method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function, including the following steps:

[0026] Step 1: First, prepare the photo-controlled biofunctionalized hydrogel. In this embodiment, the photo-controlled biofunctionalized hydrogel uses methacrylamide gelatin (GelMA) as the main framework. Compared with polyethylene glycol (PEG), GelMA naturally contains many bioactive sites, and bioactivity can be introduced without further chemical modification.

[0027] Specifically, in this embodiment, the photo-controlled biofunctionalized hydrogel can be prepared by the following method:

[0028] First, gelatin was dissolved in deionized water to form a first mixed solution. Methacrylic anhydride was slowly added while continuously stirring to prepare a second mixed solution. The second mixed solution was then reacted at 45-55°C for 3-4 hours to allow the methacrylic acid groups to fully react with the primary amine groups and hydroxyl groups of the gelatin. After the reaction was complete, unreacted methacrylic anhydride and byproducts were removed by dialysis to prepare a purified GelMA solution.

[0029] Then, RGD cyclic peptide, nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) were dissolved in phosphate-buffered saline to prepare a functional bioactive peptide solution.

[0030] The final loading concentrations of RGD cyclic peptide, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) in the functional bioactive peptide solution were 1.0 mM, 100 ng / mL, and 100 ng / mL, respectively.

[0031] Specifically, the mass ratio of the functional bioactive peptide solution to phosphate-buffered saline is 0.001:1, and the mass ratio of RGD cyclic peptide, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF) is 1000:0.1:0.1.

[0032] Finally, the purified GelMA solution and the functional bioactive peptide solution were mixed evenly. After the mixture was homogeneous, a photoinitiator was added to prepare the photo-controlled biofunctionalized hydrogel.

[0033] Specifically, the photoinitiator used in this embodiment is lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) or Irgacure 2959.

[0034] Step 2: Preparation of neurovascular matrix components. First, the frozen astrocytes (C8 / D1A strain) and neurons (HT22 strain) were removed from liquid nitrogen and rapidly thawed in a 37°C water bath. The thawed cells were then transferred to centrifuge tubes containing preheated culture medium and centrifuged to remove cryoprotectants such as DMSO.

[0035] Resuspend the centrifuged cells in fresh culture medium, inoculate them into suitable cell culture flasks (such as T-75 or T-25 culture flasks), and place the cell culture flasks in a cell culture incubator at 37°C and 5% CO2 for culture.

[0036] After culture, the cultured cells are digested and collected, centrifuged (1000 rpm, 5 min), the supernatant is removed, and the cells are resuspended in a small amount of serum-free culture medium or hydrogel precursor solution (without cross-linking agent). The cell density is adjusted to the preset concentration to prepare a suspension of neurovascular matrix components.

[0037] Specifically, before incorporating cells into the hydrogel, the cell density needs to be precisely adjusted to simulate the in vivo physiological environment and ensure successful model construction. In this embodiment, to simulate the density of astrocytes in brain tissue, the concentration of astrocytes (C8 / D1A strain) was set to 2 × 10⁻⁶. 6 ~5×10 6 Cells / mL. The concentration of neuronal cells (HT22 strain) was set at 5 × 10⁻⁶ cells / mL. 5 ~2×10 6 Cells / mL.

[0038] Step 3: Mix the photo-controlled biofunctionalized hydrogel and the neurovascular matrix component suspension at a volume ratio of 9:1 or 8:2 to form a homogeneous cell-hydrogel composite solution. Ensure that the cells are evenly distributed in the solution. The entire mixing process is carried out in an ice bath or at 4°C to slow down the pre-crosslinking rate of the hydrogel while maintaining cell viability.

[0039] The cell-hydrogel composite solution is injected into the channels of the microfluidic chip, while an oil phase is introduced into the intersecting channels, causing the cell-hydrogel composite solution to form droplets in the oil phase. After the droplets leave the chip, they are immediately irradiated with ultraviolet or blue light in the collection tank, thereby undergoing photocrosslinking in the microdroplet state to form uniformly sized spherical three-dimensional hydrogel microspheres.

[0040] Step 4: Remove the photocrosslinked three-dimensional hydrogel microspheres from the collection tank and transfer them to a sterile container. Wash the microspheres with sterile phosphate-buffered saline. After washing, remove the supernatant by low-speed centrifugation. This removes any residual oil phase, surfactants, unreacted photoinitiators, and free, unencapsulated cells that may have remained during the microfluidic process, ensuring a clean and biocompatible culture environment for subsequent operations.

[0041] The cleaned 3D hydrogel microspheres were transferred to the bottom of the Transwell culture dish insert, specifically the side below the membrane that is in direct contact with the culture medium, ensuring uniform distribution of the microspheres to form a stable "neural side" matrix. This placement method mimics the structure of neurons and astrocytes located outside blood vessels (i.e., the "neural side" of the blood-brain barrier) in vivo, providing a three-dimensional environment containing neurovascular unit components for subsequent seeding of brain microvascular endothelial cells.

[0042] Prepare brain microvascular endothelial cells for the experiment. These cells can be primary cells (such as mouse, rat, or human brain microvascular endothelial cells) or immortalized cell lines (such as bEnd.3 mouse brain endothelial cells). Ensure that the cell line is healthy and of appropriate passage number.

[0043] Culture BMECs according to standard cell culture procedures. When the cell confluence reaches 80-90%, digest (e.g., with trypsin) and collect the cells. Count the BMECs and adjust the cell density.

[0044] Typically, the inoculation density per well can be 1×10⁻⁶. 5 Up to 3×10 5 Within the range of cells / cm², a BMEC suspension with adjusted density was added to the lower chamber of a Transwell culture dish. BMECs attached to the lower surface of the Transwell membrane (i.e., above the hydrogel microsphere layer, but separated by the membrane) and grew into the hydrogel microspheres. This formed a co-culture system of the "vascular side" (above the Transwell membrane, but with BMECs attached to the lower surface of the membrane, representing the inner side of the blood vessel lumen) and the "neural side" (the region containing hydrogel microspheres and glial cells, representing the brain parenchyma side). The entire Transwell culture dish was placed in a cell culture incubator at 37°C and 5% CO2. After culture, a three-dimensional in vitro blood-brain barrier model was obtained.

[0045] Step 5: Conduct a control experiment on the three-dimensional in vitro blood-brain barrier model obtained after culture.

[0046] Figure 1 The figure shows a 4-hour leakage experiment. In the figure, blank represents the blank control group, BBB represents the three-dimensional in vitro blood-brain barrier model group prepared by the method in this embodiment, and co-culture represents the co-culture control group prepared by common methods.

[0047] Transendothelial resistance (TEER) was periodically measured in these three groups to observe the formation and maturation of the barrier. TEER values ​​were continuously monitored using a cell resistance meter, and a TEER value ≥ 200 Ω·cm² indicated that tight junctions were mature.

[0048] The permeability of a three-dimensional in vitro blood-brain barrier model was assessed by determining the apparent permeability coefficient (Papp) using a fluorescein-dextran (FITC-dextran) leakage assay. The FITC-dextran leakage assay results are shown in the figure below. Figure 2 As shown

[0049] Immunofluorescence staining was performed on the three-dimensional in vitro blood-brain barrier model using ZO-1 and Occludin as targets, and the expression of tight junction proteins in the three-dimensional in vitro blood-brain barrier model prepared in this embodiment was observed using laser confocal microscopy. Figure 3 This embodiment shows a map of tight junction protein expression under a laser confocal microscope. Figure 3The first row (top row) shows the expression of the ZO-1 (Zonula Occludens-1) tight junction protein, from left to right: DAPI staining image, ZO-1 immunofluorescence staining image, and the merge image of DAPI and ZO-1 synthesis. The second row (bottom row) shows the expression of the Occludin tight junction protein, from left to right: DAPI staining image, Occludin immunofluorescence staining image, and the merge image of DAPI and Occludin synthesis.

[0050] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function, characterized in that, The method for constructing the three-dimensional in vitro blood-brain barrier model includes: S100, a photo-controlled biofunctionalized hydrogel was prepared using methacrylamide gelatin as the main framework. The photo-controlled biofunctionalized hydrogel is loaded with a solution of functional bioactive peptides. S200, Preparation of a suspension of neurovascular matrix components using astrocytes and neurons; S300: A homogeneous cell-hydrogel composite solution is obtained by uniformly mixing a light-controlled biofunctionalized hydrogel with a suspension of neurovascular matrix components. The cell-hydrogel composite solution is injected into the channel of the microfluidic chip, and an oil phase is introduced into the intersecting channels, so that the cell-hydrogel composite solution forms droplets in the oil phase. The droplets are irradiated with ultraviolet or blue light to form uniformly sized spherical three-dimensional hydrogel microspheres. S400. The three-dimensional hydrogel microspheres are transferred to the bottom of the Transwell culture dish insert to form a stable neural side matrix. The suspension of brain microvascular endothelial cells obtained according to standard cell culture procedures was added to the lower chamber of the Transwell culture dish, so that the brain microvascular endothelial cells attached to the lower surface of the Transwell membrane and grew into the hydrogel microspheres. The entire Transwell culture dish was placed in a cell culture incubator at 37°C and 5% CO2 for culture. S100 includes the following sub-steps: S110. Dissolve gelatin in deionized water to form a first mixed solution. Stir continuously and slowly add methacrylic anhydride to prepare a second mixed solution. Place the second mixed solution at 45-55°C for 3-4 hours to prepare a purified GelMA solution. S120. A functional bioactive peptide solution was prepared by dissolving RGD cyclic peptide, nerve growth factor and brain-derived neurotrophic factor in phosphate-buffered saline. S130. The purified GelMA solution and the functional bioactive peptide solution are mixed evenly. After the mixture is evenly mixed, a photoinitiator is added to prepare a photo-controlled biofunctionalized hydrogel. The mass ratio of the functional bioactive peptide solution to the phosphate-buffered saline solution is 0.001:1; The mass ratio of RGD cyclic peptide, nerve growth factor, and brain-derived neurotrophic factor in the functional bioactive peptide solution is 1000:0.1:0.

1. S200 includes the following sub-steps: S210. Thaw the frozen astrocytes and neurons, and centrifuge to remove the cryoprotectant. S220. Resuspend the centrifuged cells in fresh culture medium, inoculate them into cell culture flasks, and place the cell culture flasks in a cell culture incubator at 37°C and 5% CO2 for culture. S230. Collect the cultured cells and resuspend them in serum-free medium or hydrogel precursor solution, and adjust the cell concentration.

2. The method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function according to claim 1, characterized in that, The concentration of the astrocytes was 2 × 10⁻⁶. 6 ~5×10 6 cells / mL; The concentration of the neurons was 5 × 10⁻⁶. 5 ~2×10 6 Cells / mL.

3. The method for constructing a three-dimensional in vitro blood-brain barrier model with barrier function according to claim 1, characterized in that, The cell-hydrogel composite solution is formed by mixing a light-controlled biofunctionalized hydrogel and a suspension of neurovascular matrix components at a volume ratio of 9:1 or 8:

2. The mixing process is carried out in an ice bath or at 4°C to slow down the pre-crosslinking rate of the hydrogel while maintaining cell activity.

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