Blood brain barrier chip for screening brain disease drug carrier and identification method thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a blood-brain barrier chip for screening brain disease drug carriers and an identification method of the blood-brain barrier chip. Preparation of the chip comprises the steps that a mold of the BBB chip is prepared through an SU-8 photoetching technology; manufacturing a PDMS rubber block with a pattern by using a mold; bonding the PDMS rubber block with the pattern with a slide to obtain a BBB chip; and carrying out pretreatment on the BBB chip. The identification standards of the identification method comprise transmembrane resistance value, survival cell apparent density, ZO-1 gene copy number and / or ZO-1 expression quantity. The blood-brain barrier chip provided by the invention has a good brain endothelial cell culture environment, brain endothelial cells and various nerve cells can be cultured by using the BBB chip, and the BBB crossing simulation effect of various brain disease drug carriers can be quickly and effectively compared and screened in vitro.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a blood-brain barrier chip for screening drug carriers for brain diseases and an identification method thereof. Background Art

[0002] The blood-brain barrier (BBB) ​​is one of the important protective mechanisms of the human body. It is a special cell barrier system formed by brain endothelial cells closely connected to each other through various junction proteins and interacting with astrocytes. It is used to ensure the energy supply of the brain and the stability of the microenvironment. However, many drugs for treating brain diseases are also difficult to achieve effective treatment due to the obstruction of the BBB. Drug carriers can change the way drugs enter the human body and their distribution in the body, control the release rate of drugs and deliver drugs to the target organ system. The use of drug carriers can increase the permeability of the BBB. Nano drug carriers are a submicron drug delivery system belonging to the nanoscale microscopic category. Encapsulating drugs in submicron particles can adjust the release rate of drugs, increase the permeability of biological membranes, change the distribution in the body, and improve bioavailability. Nanoparticles (NPs) can be used as carriers for conducting or delivering drugs. They are between 10 and 1000 nm in size and are generally composed of natural polymers or synthetic polymers. Current methods for testing drug delivery across the BBB use cell culture in a dish or animal testing, which lack sufficient predictive power, and about 90% of candidate drugs validated in these models fail in clinical trials due to excessive toxicity or ineffectiveness in humans. Therefore, the pharmaceutical industry needs more predictive tools.

[0003] Organ chips can be used as in vitro drug testing tools, and the original driving force came from the pharmaceutical industry. This technology uses chip manufacturing technology to build a real organ environment, implanting different cells to simulate the main structural and functional characteristics of different tissues and organs in the human body and the complex connections between organs. It is considered to be a very likely option to replace, at least partially replace animal testing, but the industrialization of organ chips is still in its infancy, and competition is still concentrated in chip design and manufacturing, especially chip pre-treatment, cell culture simulation and drug testing procedures are still in an immature and unstable stage. There is an urgent need to establish stable application programs, which is the biggest obstacle for all technologies to enter practicality and the most competitive field for organ chips in the future. BBB chip is an organ chip that simulates BBB in vitro, which can be used to test the effect of brain disease drugs crossing BBB. The development of stable BBB chips and their application programs can promote the transformation of BBB chip industry. Summary of the invention

[0004] The purpose of the present invention is to provide a BBB chip for screening drug carriers for brain diseases and a preparation method thereof, so as to provide more tools for screening candidate drugs.

[0005] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0006] The present invention provides a BBB chip for screening drug carriers for brain diseases. The BBB chip includes three cell flow channels: flow channel 1, flow channel 2, and flow channel 3. The three cell flow channels are used to inoculate cells to simulate cell interactions. Flow channel 2 is a channel structure with straight sides. Flow channel 1 and flow channel 3 are symmetrically arranged on both sides of flow channel 2 with flow channel 2 as the center. One side of the middle section of flow channel 1 and flow channel 3 is parallel to flow channel 2, and the two end sections form an angle α with flow channel 2. The α can be specifically 45°, and the other side of the middle section of flow channel 1 and flow channel 3 is convex. Injection holes are provided at both ends of each cell flow channel, and the injection holes are in the shape of a water drop. Isolation holes are opened between flow channel 2 and flow channel 1, flow channel 2 and flow channel 3, and the isolation holes are smaller than the cell size, which can prevent the flow of cells between different flow channels. The depth of the isolation hole does not exceed 1 / 5 of the depth of the cell flow channel. Specifically, the isolation holes are rectangular, each of which is designed to be 10-20 μm deep and 15-30 μm wide, and the depth of the three flow channels is 100 μm. The cell flow channel adopts a streamlined and smooth design without "sharp" corners to prevent dead corners and blockages during pretreatment and cell culture.

[0007] The present invention provides a method for preparing the above-mentioned BBB chip for screening drug carriers for brain diseases, comprising the following steps:

[0008] Step 1: Prepare the mold of the BBB chip by SU-8 photolithography technology; including:

[0009] (1) Use a coating machine to evenly coat a layer of SU-8 negative photoresist on the surface of the silicon wafer with a thickness of 20 μm, let it stand for 7 hours, and then bake it for 12 hours;

[0010] (2) Using a photolithography machine to transfer the pattern on the first photolithography mask to the photoresist layer by exposure, drying the photoresist, and using a spin coater to evenly coat the second layer of SU-8 photoresist on the first layer of photoresist with a thickness of 100 μm, leaving it to stand for 7 hours and then baking it for 12 hours;

[0011] (3) Cover the second mask layer on the photoresist and align the patterns on the two masks;

[0012] (4) The pattern on the second mask layer is transferred to the photoresist layer by a photolithography machine, and the photoresist is dried;

[0013] (5) Developing to remove the unexposed photoresist, and obtaining a two-layer BBB chip mold.

[0014] Step 2: Use the mold prepared in step 1 to make a PDMS block with a pattern;

[0015] Specifically include:

[0016] (1) Mix the PDMS prepolymer and curing agent in a volume ratio of 10:1 and remove bubbles. Clean the mold and blow dry it for later use.

[0017] (2) Slowly pour the mixture after removing bubbles in step (1) into the mold and apply vacuum to remove bubbles.

[0018] (3) Dry the mold in step (2) at 80°C for 3 hours, take it out, and place it in a room temperature environment to cool for 3 hours until it returns to room temperature.

[0019] (4) Cutting the PDMS glue into blocks with a length of 3.4 cm and a width of 1.8 cm.

[0020] (5) A hole with a diameter of 1.0 mm was punched at the widest end of the injection hole to make a patterned PDMS block.

[0021] Step 3: Bond the patterned PDMS block to a glass slide to obtain a BBB chip and identify its patency;

[0022] Specifically include:

[0023] (1) Clean the PDMS block and cover glass and blow dry.

[0024] (2) The washed PDMS glue block and cover glass are placed in a plasma machine, covered with plasma for 45 seconds, taken out, and the PDMS glue block is immediately covered on the cover glass for bonding to obtain a BBB chip.

[0025] Step 4: pre-treating the BBB chip, including internal room temperature sterilization treatment and internal surface modification;

[0026] The room temperature aseptic treatment refers to flushing the cell flow channel and the injection hole with sterile water and anhydrous ethanol in sequence. Specifically, sterile ultrapure water is injected into the BBB chip at a flow rate of 1.5 μL / min and flushed for more than 6 hours; then, anhydrous ethanol is injected into the BBB chip at a flow rate of 1.5 μL / min and flushed for more than 6 hours.

[0027] The inner surface modification refers to coating the cell flow channel and injection hole with fibronectin (FN) solution and matrigel solution in sequence. Specifically, the cell flow channel and injection hole of the BBB chip are first coated with 40 μg / mL FN solution for 45 minutes at 37°C and 5% CO2. Then, the cell flow channel of the BBB chip is coated with matrigel solution for 1 hour at 37°C and 5% CO2. The matrigel solution is: 200 μL of DMEM without FBS is added to every 100 μL (8-12 mg / mL) of matrigel and diluted to 300 μL (2.7-4.0 mg / mL). DMEM: DMEM containing 1% double antibodies (penicillin and streptomycin).

[0028] The present invention also provides a method for identifying a BBB chip, wherein the identification criteria of the method include: trans-epithelial electrical resistance (TEER) value, cell surface density, characteristic gene ZO-1 gene copy number and / or BBB key characterization factor connexin (Zonula occludens-1, ZO-1) expression level. The above criteria can be used to identify the structural integrity and function of the BBB chip.

[0029] hCMEC / D3 cells were cultured using the BBB chip. The TEER value refers to the TEER value of the BBB chip at 150Ω·cm when the cells were dynamically cultured for more than 72 hours. 2 Above; taking the Transwell model as the control, the TEER value of the BBB chip model was significantly greater than that of the Transwell model.

[0030] hCMEC / D3 cells were cultured using the BBB chip. The apparent density of surviving cells reached 2.5×10 after dynamic culture for more than 72 hours. 4 cell / mm 2 above.

[0031] hCMEC / D3 cells were cultured using the BBB chip. When the cells were dynamically cultured for more than 72 hours, the copy number of the ZO-1 gene was significantly upregulated, and the difference was statistically significant (****p<0.0001).

[0032] hCMEC / D3 cells were cultured using the BBB chip. Regarding the expression of ZO-1 protein, the ZO-1 protein formed a network-like connection structure when the cells were dynamically cultured for more than 72 hours.

[0033] The beneficial effects of the present invention are:

[0034] The BBB chip provided by the present invention has a good environment for culturing brain endothelial cells. The BBB chip can be used to culture brain endothelial cells and various nerve cells, and can quickly and effectively compare and screen the effects of various brain disease drug carriers crossing the simulated BBB in vitro.

[0035] The BBB chip pretreatment operation provided by the present invention can be carried out at room temperature, the operation is simple and convenient, and the stability is good; the hCMEC / D3 cells can be cultured quickly, effectively and stably using the BBB chip of the present invention; the present invention provides a detection standard for the BBB chip, and whether the BBB chip is feasible can be determined according to the standard of the present invention; the BBB chip of the present invention can be used to compare the effects of different drug carriers crossing the BBB. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The design diagram of the BBB chip in Example 1. (a) is an overall overview; (b) is an enlarged view of point A in (a). The numbers in the figure are: 1 is flow channel 1, 2 is flow channel 2, 3 is flow channel 3, 4 is injection hole, and 5 is isolation hole.

[0037] Figure 2 The BBB chip mold and the PDMS block with pattern in Examples 2 and 3. (a) BBB chip mask; (b) BBB chip mold; (c) PDMS block.

[0038] Figure 3 The structure and patency of the BBB chip obtained by bonding in Example 4 are identified. (a) Different bonding times of the BBB chip; (b) Using colored liquid to detect the patency of the BBB chip; (c) Using a microscope to identify the structure and patency of the BBB chip (20x magnification).

[0039] Figure 4 The BBB chip in Example 5 was sterilized. (a) BBB chip was sterilized at room temperature (b) BBB chip was sterilized at high temperature in an autoclave (c) BBB chip patency test after sterilization at room temperature (d) Cell apoptosis in a BBB chip that was not sterilized, treated with 75% ethanol or UV sterilization.

[0040] Figure 5 Surface modification and cell culture of the flow channel in the BBB chip in Example 5. (a) Unmodified BBB chip after sterilization at room temperature; (b) BBB chip after surface modification; (c) The cultured cells in the surface-modified BBB chip are relatively dense; (d) Cells in the unmodified BBB chip undergo apoptosis.

[0041] Figure 6 The cell density in the BBB chip was cultured using DMEM medium containing different FBS concentrations in Example 6.

[0042] Figure 7 The TEER value measurement results in the BBB chip, one of the simulated BBB identification standards in Example 6. (a) TEER measurement operation diagram in the BBB chip; (b) TEER detection results in the BBB chip and Transwell model.

[0043] Figure 8 This is the change in cell density in the BBB chip, which is the second criterion for simulating BBB identification in Example 6.

[0044] Fig. 9 The copy number change of the characteristic gene ZO-1 in the BBB chip of the third BBB identification standard simulated in Example 6 (****p<0.0001).

[0045] Fig.10 The immunofluorescence staining images of the characteristic protein factor ZO-1 in the BBB chip of the fourth BBB identification standard simulated in Example 6 (scale: 20 μm). (a) Immunofluorescence staining of ZO-1 in the BBB chip; (b) Immunofluorescence staining of ZO-1 in the Transwell.

[0046] Fig.11 Results of NaFL crossing the simulated BBB in Example 7. (a) Fluorescence image of NaFL penetration in BBB chip; (b) NaFL penetration data in BBBchip and Transwell model. (*p<0.05).

[0047] Fig.12 This is a schematic diagram of the pipeline connection settings during permeability testing in Example 8.

[0048] Fig.13 Comparison of the permeability of siRNA nanocapsules as drug carriers in Transwell and BBB chips in Example 8. (****p<0.0001). DETAILED DESCRIPTION

[0049] To make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme in the embodiment of the present invention will be clearly described below in conjunction with the accompanying drawings in the embodiment of the present invention. Obviously, the described embodiment is a part of the embodiment of the present invention, rather than all the embodiments. Based on the embodiment in the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative work premise belong to the scope of protection of the present invention. In addition, the equipment and reagents involved in the following embodiments are all commercially available equipment and reagents unless otherwise specified.

[0050] Example 1

[0051] This embodiment provides a simple BBB chip. According to the microstructural characteristics of the brain BBB, the chip template pattern is designed using software such as AutoCAD or 3DMax.

[0052] Specifically, Figure 1 As shown: The BBB chip of this embodiment includes three cell flow channels: flow channel 1, flow channel 2, and flow channel 3. The three cell flow channels are used to inoculate cells to simulate the interaction of cells. Flow channel 2 is a channel structure with straight sides. Flow channel 1 and flow channel 3 are symmetrically arranged on both sides of flow channel 2 with flow channel 2 as the center. One side of the middle section of flow channel 1 and flow channel 3 is parallel to flow channel 2 (the distance between the middle section of flow channel 1 and flow channel 3 and flow channel 2 is 0.016mm), and the two end sections form an angle α with flow channel 2, and the α is 45°. The other side of the middle section of flow channel 1 and flow channel 3 is convex arc, and the middle section of flow channel 1 and flow channel 3 is 5mm long and the two end sections are 8mm long. Except for the middle section of flow channel 1 and flow channel 3, each cell flow channel has a width of 0.2mm, and the widest part of the middle section of flow channel 1 and flow channel 3 is 0.3mm. The cell flow channel depth (corresponding to the photoresist height on the mold) is 0.1mm. Each cell flow channel is provided with an injection hole 4 at both ends. The injection hole 4 is in a water drop shape, and the length from the widest point to the narrowest point is 3.4mm, and the width of the widest point of the water drop-shaped injection hole 4 is 1mm. In specific applications, the number of injection holes 4 at the end of each cell flow channel can be 2, and the distance between the widest points of adjacent injection holes 4 is 4.9mm. The angle between the two injection holes 4 at the end of each cell flow channel is 95°, and one injection hole is used for injection, and the other injection hole is used to measure the TEER value.

[0053] In this embodiment, cell isolation holes 5 are provided between flow channel 2 and flow channel 1, and between flow channel 2 and flow channel 3, with a total of 38 holes, so that a fence structure is formed between the cell flow channels. The isolation holes 5 are rectangular holes, each of which has a width of 10 to 30 μm and a depth of 10 to 20 μm. The depth of the isolation holes 5 does not exceed 1 / 5 of the depth of the flow channel, which can prevent the fence structure between the flow channels from collapsing and deforming; the isolation holes 5 are smaller than the cells, which can prevent the flow of cells between different flow channels. Compared with the commercialized Synvivo chip, the chip design has more resistance measurement ports, which is convenient for subsequent structural determination, and the spacing between adjacent injection ports of this chip is larger, which is convenient for intubation injection.

[0054] Example 2

[0055] According to the structure of the BBB chip described in Example 1, a BBB chip mold was prepared by two SU-8 deep photolithography (the BBB chip mold has a two-layer photoresist structure), such as Figure 2 (a) and (b). The method comprises the following steps:

[0056] (1) Use a coating machine to evenly coat a layer of SU-8 negative photoresist on the surface of the silicon wafer with a thickness of 20 μm, let it stand for 7 hours, and then bake it for 12 hours;

[0057] (2) Using a photolithography machine to transfer the pattern on the first photolithography mask to the photoresist layer by exposure, drying the photoresist, and using a spin coater to evenly coat the second layer of SU-8 photoresist on the first layer of photoresist with a thickness of 100 μm, leaving it to stand for 7 hours and then baking it for 12 hours;

[0058] (3) Cover the second mask layer on the photoresist and align the patterns on the two masks;

[0059] (4) The pattern on the second mask layer is transferred to the photoresist layer by a photolithography machine, and the photoresist is dried;

[0060] (5) Developing to remove the unexposed photoresist, and obtaining a two-layer BBB chip mold.

[0061] More than 6 BBB chip modules are made on each silicon wafer to ensure that patterned PDMS blocks can be printed in small batches.

[0062] Example 3

[0063] The mold described in Example 2 was used to prepare a PDMS block with a pattern. The prepared PDMS block with a pattern is as follows: Figure 2 (c). The specific steps are as follows:

[0064] (1) Take a 50 mL centrifuge tube, add Dow Corning 184 optical adhesive PDMS prepolymer and curing agent in a volume ratio of 10:1 to prepare a 33 mL mixture, wrap the centrifuge tube with tin foil, protect it from light, and place it on a rotator to rotate for 1 hour at a speed of 20 r / min.

[0065] (2) After mixing by rotation, remove the centrifuge tube and place it in an ultrasonicator for sonication at 100% power, 20°C, and 20 min to mix it thoroughly. Then place it in a vacuum drying oven and evacuate for 30 min to remove bubbles.

[0066] (3) Clean the chip mold prepared in Example 2, add detergent, rinse with tap water, rinse the bubbles away, then rinse with ultrapure water 10 times, then add anhydrous ethanol and rinse 10 times, and then blow dry the chip mold with nitrogen.

[0067] (4) The mixture prepared in the centrifuge tube after evacuation in step (2) is evenly and slowly inverted on the chip mold cleaned in step (3), and the mold is placed in a vacuum drying oven and evacuated for 30 minutes to remove bubbles.

[0068] (5) Take out the mold from step (4) and place it in a constant temperature drying oven at 80°C for 3 hours.

[0069] (6) Take out the solidified PDMS glue on the mold and place it at room temperature for 3 hours.

[0070] (7) Cutting the PDMS glue into blocks with a length of 3.4 cm and a width of 1.8 cm.

[0071] (8) In order to insert the sample tube, a hole with a diameter of 1.0 mm was punched at the widest end of the teardrop-shaped injection hole using a hole puncher to make a patterned PDMS block.

[0072] Example 4

[0073] The patterned PDMS block described in Example 3 was bonded to a glass slide and the patency was assessed. Figure 3 Specifically including:

[0074] (1) Take out 25×75mm 2 , a rectangular cover glass with a thickness of 0.13 to 0.17 mm, clean the glue block and cover glass, add detergent, rinse with tap water until clean, then wash with ultrapure water 10 times, wash with anhydrous ethanol 10 times, finally blow dry with nitrogen, and put it in a culture dish.

[0075] (2) The washed glue block and cover glass were placed in a plasma machine, and the machine was preheated for 2 min. After being covered with plasma for a certain period of time (5, 15, 25, 35 or 45 seconds respectively from the start plasma button), the two were taken out, and the PDMS glue block was immediately covered on the cover glass for bonding. The PDMS glue block was pressed for 1 min in a 50 mL centrifuge tube filled with 30 mL of water to obtain a BBB chip.

[0076] The patency of the BBB chip was tested using colored liquid to detect the effect of plasma coverage time on the sealing of the BBB chip. Specifically, the BBB chip was microscopically identified using a microscope. The results showed that 25 to 45 seconds from the start of the plasma button was an acceptable plasma coverage time, with 45 seconds being the optimal plasma coverage time.

[0077] Example 5

[0078] This embodiment provides a BBB chip pretreatment method, specifically, including internal room temperature aseptic treatment and internal surface modification.

[0079] 1. Internal normal temperature sterile treatment

[0080] The cell living environment must be sterile. During the production process of the BBB chip, more impurities and bacteria will be introduced. Therefore, the BBB chip must be pre-treated to ensure a clean and sterile environment for cell survival. The channels in the BBB chip are narrow and there are corners, which cause impurities and bacteria to accumulate and are difficult to clean. The sterile treatment steps are as follows:

[0081] (1) Place the BBB chip and syringe pump in a clean bench.

[0082] (2) Place a syringe loaded with 1 mL of sterile ultrapure water on the syringe pump.

[0083] (3) Connect the BBB chip to the syringe on the injection pump through the microfluidic tube, adjust the injection pump speed, inject into the BBB chip at a flow rate of 1.5 μL / min, and flush for 6 hours.

[0084] (4) After rinsing, replace the sterile ultrapure water in the syringe with anhydrous ethanol and rinse for 6 hours to ensure the cleanliness and sterile environment of the chip.

[0085] Through this aseptic treatment method, the following Figure 4 (a) shows the BBB chip after aseptic treatment at room temperature. In this embodiment, after ultrapure water and anhydrous ethanol are injected into the clean bench with a syringe pump, the internal channels of the BBB chip can be flushed and all impurities and bacteria accumulated in the corners can be flushed and sterilized. The patency and sealing of the microchip are tested with colored liquid. The BBB chip has good patency and sealing, which can meet the requirements of cell culture and drug testing under aseptic conditions. Through comparative analysis, Figure 4 (b) The isolation holes between channel 1 and channel 2, and between channel 3 and channel 2 of the BBB chip sterilized at high temperature have been destroyed and cannot be used for subsequent research. In addition, cell apoptosis in the BBB chip that is not sterilized thoroughly by sterilization such as 75% ethanol or UV sterilization, such as Figure 4 (d) Therefore, the room temperature aseptic processing of the BBB chip in this embodiment is a relatively good method.

[0086] 2. Surface modification of BBB chip

[0087] 1. Reagent Preparation

[0088] (1) FN solution: In a clean bench, take 1 mL (0.2 mg / mL) of FN stock solution and add 4 mL of sterile balanced salt solution (0.9% NaCl solution) to dilute to 5 mL (40 μg / mL). Take 500 μL enzyme-free centrifuge tubes, divide and store at -20°C.

[0089] (2) DMEM: ordinary DMEM containing 1% double-antibody (penicillin and streptomycin) mixture.

[0090] (3) Matrigel: Transfer the matrigel from -20℃ to an ice box, place it in a 4℃ refrigerator overnight to melt, take it out and centrifuge it, centrifuge it at 4℃ 14000rpm for 20min, place it in an ice box after centrifugation, and then place it in a clean bench for sterilization for 30min together with the ice box. After sterilization, take a pre-cooled pipette tip, carefully aspirate the supernatant, transfer the supernatant to a 50mL centrifuge tube pre-cooled with enzyme removal, and then divide the supernatant into 1mL pre-cooled centrifuge tubes with enzyme removal, 100μL per tube. After dividing, store it at -20℃. Take 100μL (8-12mg / mL) of matrigel and add 200μL of DMEM without FBS to dilute it to 300μL (2.7-4.0mg / mL).

[0091] 2. Surface modification

[0092] (1) Place the BBB chip, 400 μL FN, and 300 μL Matrigel that have been sterilized at room temperature in an ultra-clean bench.

[0093] (2) Take a 1 mL syringe and draw 400 μL of FN into the three channels of the BBB chip at a flow rate of 1.5 μL / min, so that the three channels are filled with FN. Place the BBB chip in a 37°C, 5% CO2 incubator for 45 minutes. Take out the BBB chip, place it in a clean bench, and aspirate the excess FN solution.

[0094] (3) Take a 1 mL pre-cooled syringe and draw 300 μL of matrix gel into the three channels of the BBB chip at a flow rate of 1.5 μL / min, so that the matrix gel fills the three channels. The BBB chip is placed in a 37°C, 5% CO2 incubator for 1 hour. The surface modification of the BBB chip is completed. The modified effect is as follows: Figure 5 (b) In addition, the cultured cells in the surface-modified BBB chip are relatively dense ( Figure 5 (c)), cells cultured on the unmodified BBB chip underwent apoptosis ( Figure 5 (d)).

[0095] Example 6

[0096] This example provides a method for culturing hCMEC / D3 cells using a BBB chip and identification criteria for simulating the BBB.

[0097] 1. Method for culturing hCMEC / D3 cells using BBB chip. The specific steps are as follows:

[0098] 1. DMEM preparation: Traditional Transwell usually uses 10% FBS ordinary culture medium for culture. BBB chip culture cells need to optimize the FBS concentration in DMEM. Prepare DMEM containing 10%, 15%, 20%, 25%, 30% FBS and 1% double antibody mixture respectively, and inoculate cells in five identical chips. Observe the survival density of cells in different FBS concentrations at the same time (12h). The results are as follows Figure 6 As shown in Figure 2, the cell survival density was the highest in 20% FBS. Therefore, when culturing cells in the BBB chip, 20% FBS was added to DMEM.

[0099] 2. Cell recovery: Take out the hCMEC / D3 cells frozen in the liquid nitrogen tank, place them in a 37°C water bath until the frozen cells are thawed, transfer the thawed frozen cells to a 15mL centrifuge tube, add 4mL of DMEM medium containing 20% ​​FBS, gently blow and mix, place in an ultra-high speed centrifuge, speed 1000rpm, centrifuge for 3min, remove the supernatant, add 1mL of DMEM medium containing 20% ​​FBS, and slowly blow and mix. Add 8mL of DMEM medium containing 20% ​​FBS to a 10cm culture dish, transfer the mixed solution in the centrifuge tube to the 10cm culture dish, shake it back and forth ten times, mix the cells, and place it in a 37°C, 5% CO2 incubator.

[0100] For BBB chip: count the cells after cell recovery and before use, optimize the cell count to 1 × 10 6 Then the cells were taken out and sucked into the BBB chip with a 1 mL syringe, placed in a 37°C, 5% CO2 incubator, cultured with DMEM containing 20% ​​FBS for 3 h, connected to a syringe pump, and cultured dynamically at a flow rate of 1.5 μL / min.

[0101] For Transwell chambers: Add 100 μL NaCl to the Transwell chamber and place it in the incubator for 5 min for hydrophilic treatment. Remove the NaCl and add 2 mg / mL Matrigel for 30 min, then rinse with DMEM. Count the digested hCMEC / D3 cells and dilute the cell suspension to a concentration of 2 × 10 5 / mL, take out 200μL of cells and inoculate them in the Transwell chamber, add 800μL of DMEM medium on the outside. Observe the cell inoculation status under a microscope, place in a 37℃, 5% CO2 incubator, and measure the transendothelial resistance after 48h, which should reach 200Ω / cm 2 It can be used for subsequent experiments.

[0102] 2. The identification standards of simulated BBB are as follows:

[0103] Standard 1: TEER value.

[0104] The cell TEER value is currently recognized as a relatively sensitive and accurate indicator for judging the integrity of the BBB. By adding an appropriate potential difference to the entire cell layer, the resistance of the cell ion current in the area can be measured. The TEER value mainly reflects the resistance of substances in the process of paracellular transport, that is, the closer the cells are, the smaller the space between the cells that ions and other charged substances can cross, the greater the resistance, the higher the resistance value, and the better the barrier function. The TEER values ​​of the Transwell model and the BBB chip model were monitored for five consecutive days. Figure 7 As shown in the figure, the TEER value of the BBB chip model is significantly greater than that of the Transwell model, which indicates that the fluid shear force, culture medium replacement, and metabolic substance discharge provided by the dynamic culture based on the BBB chip have a very important influence on the formation of the barrier. The TEER test results show that the TEER value of the BBB chip on the third day (72 hours) was 157Ω·cm 2 , using only endothelial cells to simulate BBB meets the test requirements. Through the results of TEER value, we can know that the BBB chip model has a good barrier function, and the TEER value can be used to judge the expected effect of simulating BBB structure.

[0105] Criterion 2: Apparent density of viable cells.

[0106] Based on the above TEER detection, the cell density of the BBB chip was observed, such as Figure 8 As shown in Figure 2, the initial structure of the BBB was formed on the third day (72 hours) in the chip, and the apparent density reached 2.8×10 4 cell / mm 2 On the fifth day, the cell density reached (4±0.9)×10 4 cell / mm 2 In actual use, this density can be used as a criterion for constructing a BBB simulation chip structure.

[0107] Standard three: the copy number of ZO-1 gene (the number of ZO-1 genes).

[0108] In the BBB, there are many tight junction proteins and adhesion molecules at the junctions between endothelial cells to maintain the integrity of the barrier. Tight junction protein ZO-1 can be formed between hCMEC / D3 cells, which can limit the passive transmission of molecules to the brain. Genes guide protein synthesis. The more ZO-1 genes there are, the higher the expression of ZO-1 protein. Therefore, the number of ZO-1 genes can be used to judge the barrier function of the BBB. The gene number (copy number) of ZO-1 protein in the BBB chip was determined by RT-PCR. The gene copy number is expressed in 2 -ΔΔCt The method was used to calculate the change in the number of genes at each time point, and the number of control groups was set as "1". The changes in the number of genes at each time point were calculated one by one, and 3 replicates were set for each group. The calculation results were expressed as mean and standard deviation (n=3). The results showed that with the extension of dynamic culture time, the ZO-1 gene was significantly upregulated. At 96h of dynamic culture, the number of ZO-1 genes was statistically significantly different (****p<0.0001). Fig. 9 Therefore, the cell culture time point (72h) and the amount of ZO-1 gene can be used to determine the barrier function of BBB.

[0109] Standard 4: ZO-1 protein expression.

[0110] Immunofluorescence staining can be used to observe the expression of specific markers. Tight junction protein ZO-1 was selected as a marker for hCMEC / D3 cell identification, and BBB formation was observed by immunofluorescence staining of ZO-1 protein. Fig.10 As shown, blue fluorescence represents the cell nucleus, and ZO-1 protein is green fluorescence. After 72 hours of culture, ZO-1 is highly expressed in the hCMEC / D3 cell culture model based on the BBB chip, and a tight junction structure is formed. Compared with the hCMEC / D3 cell monoculture model based on Transwell, ZO-1 is significantly increased and evenly distributed, proving that the fluid shear force, culture medium replacement, and excretion of metabolic substances provided by the dynamic culture based on the BBB chip play an important role in the integrity of the BBB function. Therefore, the immunofluorescence image of the ZO-1 reticular connection structure after 72 hours of culture can be used as a functional judgment standard when simulating the BBB.

[0111] Example 7

[0112] This example provides a test for the permeability of sodium fluorescein (NaFL) across the BBB.

[0113] The structural and functional integrity of the BBB was determined using the standards in Example 6 above, and the permeability of the Transwell model and the BBB chip model to the compound was tested by measuring the diffusion rate of the fluorescent tracer across the membrane.

[0114] 500 μg / mL NaFL solution was added to the BBB chip and Transwell model at a flow rate of 1.5 μL / min. After 4 hours, the fluorescence data graph was taken using an inverted fluorescence microscope in the BBB chip, and the test solution was taken out from the lower chamber of the Transwell model, diluted 2 times with culture medium, and then the fluorescence intensity of the sample was detected using a fluorescence spectrophotometer. Fig.11 As shown in the figure, the NaFL permeability in the BBB chip is lower than that in the Transwell chip. The NaFL permeability in the BBB chip is 1.35×10 -5 cm / s. The permeability in this example also provides a reference for testing the permeability of brain disease drug carriers across the BBB.

[0115] Example 8

[0116] This example provides a test for the permeability of drug carriers for brain diseases across the BBB.

[0117] The permeability of siRNA nanocapsules was tested using the BBB chip, with the traditional Transwell as a control.

[0118] The drug carrier is a brain disease drug carrier in siRNA nanocapsules (patent number: ZL 2020 1 0319659.9). After cytotoxicity tests, the concentration of siRNA nanocapsules can be selected from 25 to 400 nM. Due to the small change in fluorescence, the fluorescence value is measured by an enzyme marker, and the permeability (Papp) is calculated using the following formula:

[0119]

[0120] In the above formula, V is the volume of the sample solution, dC / dt is the change of the fluorescence concentration in the cavity with time, and ΔC is the fluorescence concentration difference between the compartments.

[0121] The test method includes the following steps:

[0122] (1) Take the BBB chip and follow Fig.12 Connect the pipelines and culture hCMEC / D3 cells in channel 1 and channel 2 of the BBB chip to simulate the BBB.

[0123] (2) Use a syringe to draw 1 mL of siRNA nanocapsules (250 μg / mL) and 1 mL of blank culture medium, and use a syringe pump to inject the siRNA nanocapsules and blank culture medium into the culture chamber of flow channel 1 and the culture chamber of flow channel 2 at a speed of 1.5 μL / min.

[0124] (3) Collect the outflow liquid from the culture chamber of channel 2 at 4, 8, and 12 hours, and conduct quantitative analysis on the collected solution.

[0125] (4) Calculate the permeability P according to the formula app .

[0126] The results show that from Fig.13 It can be seen that the permeability of brain disease drug carriers through the simulated BBB in the BBB chip is 5.17×10 -7 cm / s, the permeability measured by the BBB chip is much lower than that in the Tranwell, which shows that the BBB chip can provide fluid shear force, culture medium replacement, and the discharge of metabolites to form a positive barrier. The in vitro BBB chip provided by the embodiment of the present invention can replace the traditional Transwell model for high-throughput drug screening and provide a reference number for drug modification.

[0127] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A BBB chip for screening drug carriers for brain diseases, characterized in that: The invention comprises a cell flow channel, wherein the cell flow channel comprises a flow channel 1 (1), a flow channel 2 (2), and a flow channel 3 (3). The flow channel 2 (2) is a channel structure with straight sides. The flow channel 1 (1) and the flow channel 3 (3) are symmetrically arranged on both sides of the flow channel 2 (2) with the flow channel 2 (2) as the center. One side of the middle section of the flow channel 1 (1) and the flow channel 3 (3) is parallel to the flow channel 2 (2), and the two end sections form an angle α with the flow channel 2 (2). The other side of the middle section of the flow channel 1 (1) and the flow channel 3 (3) is in an outwardly convex arc shape. Injection holes (4) are arranged at both ends of each cell flow channel. Isolation holes (5) are arranged between the flow channel 2 (2) and the flow channel 1 (1), the flow channel 2 (2) and the flow channel 3 (3). The isolation holes (5) are smaller than the size of the cell, and the depth of the isolation holes (5) does not exceed 1 / 5 of the depth of the cell flow channel.

2. A BBB chip for screening drug carriers for brain diseases according to claim 1, characterized in that: The α is 45°.

3. A BBB chip for screening drug carriers for brain diseases according to claim 1, characterized in that: The injection hole (4) is in the shape of a water drop.

4. A BBB chip for screening drug carriers for brain diseases according to claim 1, characterized in that: The isolation hole (5) is designed to have a depth of 10 to 20 μm and a width of 15 to 30 μm, and the depths of flow channel 1 (1), flow channel 2 (2), and flow channel 3 (3) are all 100 μm.

5. The method for preparing the BBB chip according to any one of claims 1 to 4, characterized in that: include: Step 1: preparing a mold of the BBB chip according to any one of claims 1 to 4 by SU-8 photolithography technology; Step 2: Use the mold prepared in step 1 to make a PDMS block with a pattern; Step 3: Bond the patterned PDMS block to a glass slide to obtain a BBB chip; Step 4: pre-treating the BBB chip, including internal room temperature sterilization treatment and internal surface modification; The room temperature aseptic treatment refers to flushing the cell flow channel and the injection hole with sterile water and anhydrous ethanol in sequence; The inner surface modification refers to coating the cell flow channel and the injection hole with fibronectin solution and matrix gel solution in sequence.

6. The method for identifying a BBB chip according to any one of claims 1 to 4, characterized in that: The identification criteria of the method include: transmembrane resistance value, apparent density of viable cells, copy number of ZO-1 gene and / or expression level of ZO-1 protein.

7. The identification method according to claim 6, characterized in that: For transmembrane resistance, when hCMEC / D3 cells were dynamically cultured for more than 72 h, the TEER value of the BBB chip was 150 Ω·cm 2 Above; taking the Transwell model as the control, the TEER value of the BBB chip model was significantly greater than that of the Transwell model.

8. The identification method according to claim 6, characterized in that: Regarding the apparent density of surviving cells, when hCMEC / D3 cells were dynamically cultured for more than 72 h, the cell density reached 2.5×10 4 cell / mm 2 above.

9. The identification method according to claim 6, characterized in that: As for the ZO-1 gene copy number, when hCMEC / D3 cells were dynamically cultured for more than 72 hours, the ZO-1 gene copy number was significantly upregulated, and the difference was statistically significant.

10. The identification method according to claim 6, characterized in that: Regarding the expression of ZO-1 protein, when hCMEC / D3 cells were dynamically cultured for more than 72 h, ZO-1 protein formed a network-like connection structure.

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