A spiral multi-cell microfluidic platform and high-throughput screening method thereof

Abstract

The application discloses a spiral multi-cell microfluidic platform and a high-throughput screening method thereof. The platform comprises four spiral flow channels and a liquid storage tray. The spiral flow channel comprises a longitudinal partition plate, a transverse partition plate and a center partition plate. The transverse partition plate is provided with a first groove and a second groove. The center partition plate is provided with a center groove. The longitudinal partition plate and the transverse partition plate are symmetrically and uniformly arranged in the liquid storage tray. The height of the center groove is lower than that of the liquid storage tray. The first groove and the second groove are mirror-distributed and cross-arranged in the spiral flow channel. The height of the first groove and the second groove is lower than that of the longitudinal partition plate. The liquid storage tray is provided with one liquid inlet and three liquid outlets. The high-throughput screening method can obtain a change gradient of grafting density in a short time, effectively improves the preparation efficiency of samples, greatly reduces the workload and solves the problems of a complicated process and time consumption of a traditional orthogonal experiment method.

Description

Technical Field

[0001] This invention belongs to the field of microfluidic chip technology, specifically relating to a spiral multi-cell microfluidic platform and its high-throughput screening method. Background Technology

[0002] The poor surface properties of biomaterials, leading to issues such as infection, incompatibility, and suboptimal functional performance, hinder their further development. To address the poor surface activity of titanium-based implants, surface modification with active molecules is employed. Peptides, due to their sequence diversity and functional tunability, allow for precise surface tuning, enabling the effective fabrication of high-performance functionalized surfaces through surface functionalization with different functional peptides. However, when multiple functional peptides coexist, their interactions can significantly impact surface properties, particularly the often contradictory relationship between antibacterial and repair-promoting properties. In practical research, orthogonal experimental design is typically used to optimize performance combinations, but this process is cumbersome, costly, and time-consuming. Furthermore, orthogonal experiments often only address the interaction of two properties, failing to reveal the complex nonlinear relationships and multiple interactions among various properties, thus affecting the accuracy of the conclusions.

[0003] High-throughput experimental platforms based on gradient surfaces overcome the limitations of orthogonal experiments, enabling the testing of numerous surface modification combinations in a short time and rapid, efficient screening of multifunctional surfaces. However, traditional high-throughput platforms suffer from insufficient data acquisition accuracy due to a lack of clear boundaries. Furthermore, when multiple functions are involved (especially the conflict between antibacterial and pro-repair properties), the sheer volume of data makes analyzing patterns and finding optimal performance combinations difficult. Current research, focusing on both antibacterial and pro-repair properties, typically only addresses two; and studying only pro-repair properties limits optimization to three functions. 3D printing technology, as an emerging method, enables the functional design of material surfaces at the microscale. Its high printing precision and accurate control of details allow for the creation of complex geometries, structures, and porosities, showing great potential in the preparation of personalized materials and finding wide application in constructing various experimental platforms. Simultaneously, by combining with machine learning techniques, it can efficiently process massive amounts of data, uncover underlying patterns, and achieve multi-dimensional data analysis, providing powerful tools for predicting and optimizing complex properties.

[0004] The invention platform previously developed by our research group could only combine the properties of a maximum of two peptides. In order to further improve efficiency, our research group has further developed a microfluidic platform that can test a large number of surface modification combinations in a short time and quickly and efficiently screen multifunctional surfaces.

[0005] In order to overcome the shortcomings and disadvantages of the prior art, the primary objective of this invention is to provide a spiral multi-cell microfluidic platform.

[0006] Another objective of this invention is to provide a high-throughput screening method for a spiral multi-cell microfluidic platform.

[0007] The primary objective of this invention can be achieved through the following technical solution:

[0008] A spiral multi-cell microfluidic platform includes four spiral flow channels and a liquid storage tray. Each spiral flow channel includes a longitudinal baffle, a transverse baffle, and a central baffle. The transverse baffle has a first groove and a second groove, and the central baffle has a central groove. The longitudinal and transverse baffles are uniformly installed in the liquid storage tray in a centrally symmetrical manner. The height of the central groove is lower than the height of the liquid storage tray. The first and second grooves are mirror images of each other and are arranged in a crisscross pattern within the spiral flow channels. The height of the first and second grooves is lower than that of the longitudinal baffles. The liquid storage tray has one inlet and three outlets.

[0009] Preferably, the height of the first groove and the second groove is 1% to 99% of the height of the longitudinal partition, and the length of the first groove and the second groove is 1% to 99% of the length of the longitudinal partition.

[0010] Another objective of this invention can be achieved through the following technical solution:

[0011] A high-throughput screening method for a spiral multi-cell microfluidic platform includes the following steps:

[0012] S1. Design and 3D print a spiral multi-cell microfluidic platform. The spiral multi-cell microfluidic platform is then subjected to stress-relief annealing and sandblasting to obtain the spiral multi-cell microfluidic platform.

[0013] S2. Clean the spiral multi-cell microfluidic platform and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain a surface-modified spiral multi-cell microfluidic platform.

[0014] S3. Inject the peptide solution into the surface-modified spiral multi-cell microfluidic platform. The peptide solution passes through the regional cells of the surface-modified spiral multi-cell microfluidic platform at a uniform speed and reacts. After the peptide solution has reacted with all the regional cells, the spiral multi-cell microfluidic platform is cleaned to obtain a peptide-grafted spiral multi-cell microfluidic platform.

[0015] S4. Culture bacteria or cells in the polypeptide-grafted helical multi-cell microfluidic platform to determine the optimal region cell for the grafting density of bioactive polypeptides.

[0016] S5. Determine the peptide grafting density of the cell in the optimal bioactive peptide grafting density region.

[0017] A better option is to perform hydroxylation treatment on the spiral multi-cell microfluidic platform described in step S2, as follows:

[0018] S211. The spiral multi-cell microfluidic platform is ultrasonically cleaned with deionized water, acetone, anhydrous ethanol and deionized water in sequence for 10 to 30 minutes, and then the spiral multi-cell microfluidic platform is dried with protective gas.

[0019] S212. Immerse the spiral multi-cell microfluidic platform in HF acid with a mass fraction of 2% to 8% for 4 to 18 minutes.

[0020] S213. Finally, clean the spiral multi-cell microfluidic platform treated with HF acid in step S212, dry the spiral multi-cell microfluidic platform again with protective gas, and immerse the spiral multi-cell microfluidic platform in concentrated nitric acid for 10-30 minutes to obtain a surface-modified spiral multi-cell microfluidic platform.

[0021] A more preferred option is that the polypeptide molecule in the polypeptide solution in step S3 has the molecular structure HS-R-Peptide, where R includes C(PA)nP, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and the peptide includes one of KRWWKWWRR or KRWWKWWRRK-FITC or IGKYKLQYLEQWTLK or IGKYKLQYLEQWTLK-FITC or YIGSR or YIGSRK-FITC or KIPKASSVPTELSAISTLYL or KIPKASSVPTELSAISTLYLK-FITC.

[0022] A better option is that the concentration range of the polypeptide solution in step S3 is 1 μM to 5 mM.

[0023] A better option is that the reaction time between the peptide solution and the surface-modified helical multicell microfluidic platform in step S3 is 5 minutes to 24 hours.

[0024] A better option is that, in step S4, culturing bacteria in the polypeptide-grafted helical multicell microfluidic platform includes the following steps:

[0025] S411. Prepare the required nutrient broth culture medium and sterilize the nutrient broth culture medium in an autoclave at 121°C for 2 hours.

[0026] S412. Place the polypeptide-grafted helical multi-cell microfluidic platform in a first multi-well culture plate, and sequentially add peptides with a concentration of 10-10 to the regional cells of the polypeptide-grafted helical multi-cell microfluidic platform. 9 1 μl to 100 μl of bacterial culture with CFU / ml was incubated for 10 minutes to 24 hours on the microfluidic platform of the polypeptide-grafted helical multi-cell structure.

[0027] S413. After co-incubation, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate. After the bacterial solution has evaporated, the polypeptide-grafted helical multi-cell microfluidic platform is placed in a biological cabinet and left to stand for 10-15 minutes.

[0028] S414. Add a solution of resazurin with a concentration of 10-1000 μg / ml sequentially to the regional cell of the microfluidic platform of the polypeptide grafted helical multi-cell structure, mix with nutrient broth, and incubate the resazurin solution in the microfluidic platform of the polypeptide grafted helical multi-cell structure for 10 minutes to 8 hours.

[0029] S415. After the polypeptide-grafted helical multi-cell microfluidic platform has been statically cultured, the colony growth of the polypeptide-grafted helical multi-cell microfluidic platform is observed under a fluorescence microscope to determine the region cell with the optimal bioactive polypeptide grafting density.

[0030] A better option is that, in step S4, culturing cells in the polypeptide-grafted helical multicell microfluidic platform includes the following steps:

[0031] S421. Place the polypeptide-grafted helical multi-cell microfluidic platform in a third multi-well culture plate, sterilize the polypeptide-grafted helical multi-cell microfluidic platform, and sequentially add a concentration of 10-10 to each cell of the helical multi-cell microfluidic platform. 9 Incubate 1 μl to 100 μl of cell suspension per 1 cell / mL for 1 hour to 30 days.

[0032] S422. The polypeptide-grafted helical multi-cell microfluidic platform is removed from the third multi-well culture plate, cleaned with PBS, placed into the fourth multi-well culture plate, and then paraformaldehyde is added to the fourth multi-well culture plate to fix the polypeptide-grafted helical multi-cell microfluidic platform.

[0033] S423. Add live cell F-actin microfilament staining reagent to the fourth multi-well culture plate containing the polypeptide-grafted helical multi-cell microfluidic platform for fluorescent labeling and wash it with PBS. Then add DAPI dye to the fourth multi-well culture plate to fluorescently label the polypeptide-grafted helical multi-cell microfluidic platform. Then remove the polypeptide-grafted helical multi-cell microfluidic platform and wash it with PBS.

[0034] S424. Under a laser confocal microscope, observe the cell distribution, cell adhesion, and cell differentiation on the surface of the polypeptide-grafted helical multicell microfluidic platform described in step 423 using the FITC channel to identify the biologically active cell regions.

[0035] The present invention has the following advantages and beneficial effects compared with the prior art:

[0036] This invention combines 3D printing technology to design and construct a helical multi-cell microfluidic platform. Previous research on serpentine high-throughput experimental platforms typically only allows for the study of one or two peptide properties, while the helical high-throughput platform described in this invention can construct unidirectional, bidirectional, tridirectional, and four-directional peptide surfaces. This platform overcomes the limitations of existing technologies in multifunctional surface modification research, enabling the simultaneous study of four functional property combinations. Furthermore, by incorporating machine learning methods, feature selection and fitting regression are used to predict grafting behavior under different experimental conditions. A data-driven model training method captures complex nonlinear relationships, revealing the intrinsic connections between different surface parameters (such as peptide grafting density) and surface functions. Efficient data analysis and optimization are performed across multiple functional indicators (such as antibacterial properties and osteogenic properties) to explore optimal grafting parameter combinations and generate various solutions that achieve a superior balance among different objectives. This platform successfully reveals the interrelationships between multiple functional properties, providing theoretical basis and practical support for rational design based on performance requirements. The application of this platform provides a new tool for preparing high-quality surfaces with diverse functions, offers new ideas for the development of biomedical materials, and is of great significance for promoting the development of the field of biomaterials. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the spiral-shaped multi-cell microfluidic platform (3×3 format) of the present invention;

[0038] Figure 2 This is a schematic diagram of the spiral-shaped multi-cell microfluidic platform (5×5 format) of the present invention;

[0039] Figure 3This is a schematic diagram of the spiral-shaped multi-cell microfluidic platform (7×7 format) of the present invention;

[0040] Figure 4 This is a schematic diagram illustrating the usage effect of the spiral-shaped multi-cell microfluidic platform (5×5 format) of the present invention;

[0041] Figure 5 This is a schematic diagram of the flow direction of the polypeptide solution in the longitudinal and transverse directions (arrows indicate the flow direction) of the spiral multi-cell microfluidic platform (5×5 format) of the present invention;

[0042] Figure 6 This is a schematic diagram of the structure of CPAPAPKRWWKWWRR in this invention;

[0043] Figure 7 This is a schematic diagram of the structure of CPAAPIGKYKLQYLEQWTLK in this invention;

[0044] Figure 8 This is a schematic diagram of the structure of CPAPASYIGSR in this invention;

[0045] Figure 9 This is a schematic diagram of the structural formula of CPAPAPKIPKASSVPTELSAISTLYL in this invention;

[0046] Figure 10 This is a bar chart showing the changes in MFI values ​​on the grafted surface of a unidirectional polypeptide grafted with CPAPAPKRWWKWWRRK-FITC in this invention.

[0047] Figure 11 This is a bar chart showing the changes in MFI values ​​on the grafted surfaces of bidirectional polypeptides grafted with CPCAPAPKRWWKWWRR and CPCAPAPIGKYKLQYLEQWTLK-FITC in this invention.

[0048] Figure 12 This is a bar chart showing the changes in MFI values ​​on the grafted surfaces of three-dimensional polypeptides grafted with CPCAPAPKRWWKWWRR, CPCAPAPIGKYKLQYLEQWTL, and CPCAPAPYIGSRK-FITC in this invention.

[0049] Figure 13 This is a bar chart showing the changes in MFI values ​​on the grafted surfaces of four-way polypeptides grafted with CPCAPAPKRWWKWWRR, CPCAPAPIGKYKLQYLEQWTL, CPCAPAPYIGSR, and CPCAPAPKIPKASSVPTELSAISTLYLK-FITC in this invention.

[0050] Figure 14This is a bar chart showing the antibacterial surface results of the four-way polypeptide grafted surface in this invention.

[0051] Figure 15 This is a bar chart showing the endothelial cell adhesion-promoting surface results of the four-way polypeptide grafting surface in this invention;

[0052] Figure 16 This is a bar chart showing the surface results of the four-way polypeptide grafting surface promoting neural differentiation in this invention.

[0053] Figure 17 This is a bar chart showing the osteogenic surface results of the four-dimensional polypeptide grafting surface in this invention.

[0054] The components in the attached diagram are labeled as follows: 1-Liquid storage tray; 101-Liquid inlet; 102-Liquid outlet; 103-Area cell; 2-Vertical partition; 3-Transverse partition; 301-First groove; 302-Second groove; 4-Central partition; 401-Central groove. Detailed Implementation

[0055] The present invention will be further described in detail below with reference to specific embodiments, but the implementation of the present invention is not limited thereto. All materials used in the examples of the present invention are commercially available.

[0056] The materials used, the spiral multi-cell microfluidic platform, and the high-throughput screening method of the spiral multi-cell microfluidic platform in the specific embodiments are described below:

[0057] Staphylococcus aureus (ATCC 6538P); human bone marrow mesenchymal stem cells (ATCC CRL-12424); vascular endothelial cells (CRL-2873); Schwann cells. Purchase channel: VWR International, LLC, Pennsylvania, USA.

[0058] The peptides CPCAPAPKRWWKWWRR, CPCAPAPKRWWKWWRRK-FITC, CPCAPAPIGKYKLQYLEQWTLK, CPCAPAPIGKYKLQYLEQWTLK-FITC, CPCAPAPYIGSR, CPCAPAPYIGSRK-FITC, CPCAPAPKIPKASSVPTELSAISTLYL, and CPCAPAPKIPKASSVPTELSAISTLYLK-FITC used in the embodiments of this application were purchased from Shanghai Qiangyao Biotechnology Co., Ltd.

[0059] like Figures 1 to 3As shown, the four faces of the spiral multi-cell microfluidic platform are labeled "1", "2", "3" and "4" respectively. The spiral multi-cell microfluidic platform includes four spiral flow channels and a liquid storage tray 1. The spiral flow channels include longitudinal baffles 2, transverse baffles 3 and a central baffle 4. The transverse baffles 3 are provided with a first groove 301 and a second groove 302. The central baffle 4 is provided with a central groove 401. The longitudinal baffles 2 and transverse baffles 3 are uniformly installed in the liquid storage tray 1 in a centrally symmetrical manner. The height of the central groove is lower than the height of the liquid storage tray. The first groove and the second groove are mirror images of each other and are arranged in a cross pattern in the spiral flow channels. The height of the first groove 301 and the second groove 302 is lower than the height of the edge of the longitudinal baffle 2. The height of the central groove 401 is lower than the height of the first groove 301 and the second groove 302. The liquid storage tray is provided with one inlet 101 and three outlets 102.

[0060] The longitudinal baffle 2 can change the flow direction of the peptide solution. After the liquid enters through the inlet 101, it flows to the central region and splits into three branches that simultaneously flow out of the three outlets 102, thus forming a spiral flow. The height of the first groove 301 and the second groove 302 is lower than the height of the storage tray 1. The first groove 301 and the second groove 302 are mirror images of each other and are evenly distributed in the spiral flow channel. The height of the first groove 301 and the second groove 302 is 1% to 99% of the length of the longitudinal baffle 2, and the height of the first groove 301 and the second groove 302 is 0.1 to 20 mm. The length of the first groove 301 and the second groove 302 is 1% to 99% of the length of the longitudinal baffle. When the peptide solution is higher than the height of the first groove 301 and the second groove 302, it flows out from the second groove 302 to form a passage. The storage tray 1 serves to store the liquid. The inlet 101 is used to inject the peptide solution, and the outlet 102 is used for the peptide solution to flow out after the reaction is completed.

[0061] like Figure 1 As shown, the microfluidic platform comprises nine regional cells, specifically nine regional cells within a spiral flow channel. The spiral multi-cell microfluidic platform has a 3×3 specification. The spiral flow channel includes four longitudinal baffles 2, four transverse baffles 3, and four central baffles 4. The transverse baffles 3, longitudinal baffles 2, and central baffles 4 are installed within the spiral flow channel, forming the nine regional cells. The side length of each regional cell ranges from 0.05 to 100 mm.

[0062] like Figure 2 As shown, the microfluidic platform has a specification of 5×5 (that is, a total of 25 regional cells). The spiral flow channel includes 20 transverse baffles 3, 8 longitudinal baffles 2 and 4 central baffles 4. The transverse baffles 3 are arranged crosswise in the spiral flow channel to form 25 regional cells.

[0063] like Figure 3 As shown, the microfluidic platform has a specification of 7×7 (i.e., a total of 49 regional cells). The spiral flow channel includes 44 transverse baffles 3, 12 longitudinal baffles 2 and 4 central baffles 4. The transverse baffles 2 are arranged crosswise in the spiral flow channel to form 49 regional cells.

[0064] like Figures 1 to 3 As shown, each of the aforementioned transverse partitions 2 is provided with a first groove 301. The second groove 302 has the same size as the first groove 301 and is lower than the height of the longitudinal partition 3 and the liquid storage tray 1. The height of the central groove 401 is lower than the height of the first groove 301, the second groove 302, the longitudinal partition 2, and the liquid storage tray 1.

[0065] The high-throughput screening method for the spiral multi-cell microfluidic platform described in this embodiment includes the following steps:

[0066] S1. A spiral multi-cell microfluidic platform drawing is obtained through design. Based on the spiral multi-cell microfluidic platform drawing, a spiral multi-cell microfluidic platform printout is made using Ti6Al4V powder as material through 3D printing. The spiral multi-cell microfluidic platform printout is then subjected to stress-relief annealing to obtain the spiral multi-cell microfluidic platform.

[0067] The spiral-shaped multi-cell microfluidic platform printout undergoes stress-relief annealing, which includes the following steps:

[0068] S101, the spiral multi-cell microfluidic platform printout is heated to 300℃~700℃ at a heating rate of 3~10℃ / min, and then the spiral multi-cell microfluidic platform printout is kept at this temperature for 1~3h. After heating is stopped, it is allowed to cool naturally to room temperature in the furnace.

[0069] S102. The oxide scale on the surface of the spiral multi-cell microfluidic platform printout from step S101 is removed using a sandblasting machine.

[0070] S2. Clean the spiral multi-cell microfluidic platform from step S1, and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain a surface-modified spiral multi-cell microfluidic platform.

[0071] The steps for hydroxylation treatment of the spiral multi-cell microfluidic platform described in step S2 are as follows:

[0072] The S211 spiral multi-cell microfluidic platform was ultrasonically cleaned with deionized water, acetone, anhydrous ethanol and deionized water for 10-30 minutes in sequence, and then dried with a protective gas (including nitrogen, argon or helium).

[0073] S212. Then immerse the spiral multi-cell microfluidic platform in HF at a mass ratio of 2% to 8% for 4 to 18 minutes.

[0074] S213. Finally, clean the microfluidic platform that has been soaked in HF spiral multi-cell microfluidic platform, dry the spiral multi-cell microfluidic platform again with protective gas, and soak the spiral multi-cell microfluidic platform in concentrated nitric acid for 10-30 minutes to obtain the surface-modified spiral multi-cell microfluidic platform.

[0075] S3. Inject the peptide solution into the surface-modified spiral multi-cell microfluidic platform from step S2. The peptide solution is injected into the surface-modified spiral multi-cell microfluidic platform at a uniform rate using a syringe. The peptide solution flows through all regions 103 of the surface-modified spiral multi-cell microfluidic platform. After the peptide solution has flowed through all regions 103 of the surface-modified spiral multi-cell microfluidic platform (grafting reaction time is 5 min to 24 h), the spiral multi-cell microfluidic platform is cleaned with deionized water or anhydrous ethanol to obtain the peptide-grafted spiral multi-cell microfluidic platform.

[0076] The polypeptide in the polypeptide solution used in step S3 has the molecular structure formula HS-R-Peptide, where R includes, but is not limited to, C(PA)nP, C represents cysteine, n is an integer from 1 to 100, P represents proline, and A represents alanine; the polypeptide includes, but is not limited to, KRWWKWWRR or KRWWKWWRRK-FITC or IGKYKLQYLEQWTLK or IGKYKLQYLEQWTLK-FITC or YIGSR or YIGSRK-FITC or KIPKASSVPTELSAISTLYL or KIPKASSVPTELSAISTLYLK-FITC, etc. The concentration range of the polypeptide solution is 1 μM to 5 mM, and the reaction time is 5 min to 24 h.

[0077] S4. Culture bacteria or cells in a polypeptide-grafted helical multi-cell microfluidic platform to determine the optimal region cell 103 for the grafting density of bioactive polypeptides.

[0078] Step S4, which involves culturing bacteria in a polypeptide-grafted helical multi-cell microfluidic platform and then identifying cell 103 where antibacterial performance meets the standard, specifically includes the following steps:

[0079] S411. Prepare the required nutrient broth culture medium and sterilize the nutrient broth culture medium in an autoclave at 121°C for 2 hours.

[0080] S412. Place the polypeptide-grafted helical multi-cell microfluidic platform in a first multi-well culture plate, and sequentially add peptides with a concentration of 10-10 to the regional cells of the polypeptide-grafted helical multi-cell microfluidic platform. 9 1 μl to 100 μl of bacterial culture with CFU / ml was incubated for 10 minutes to 24 hours on the microfluidic platform of the polypeptide-grafted helical multi-cell structure.

[0081] S413. After co-incubation, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate. After the bacterial solution has evaporated, the polypeptide-grafted helical multi-cell microfluidic platform is placed in a biological cabinet and left to stand for 10-15 minutes.

[0082] S414. Add a solution of resazurin with a concentration of 10-1000 μg / ml sequentially to the regional cell of the microfluidic platform of the polypeptide grafted helical multi-cell structure, mix with nutrient broth, and incubate the resazurin solution in the microfluidic platform of the polypeptide grafted helical multi-cell structure for 10 minutes to 8 hours.

[0083] S415. After the polypeptide-grafted helical multi-cell microfluidic platform has been statically cultured, the colony growth of the polypeptide-grafted helical multi-cell microfluidic platform is observed under a fluorescence microscope to determine the region cell with the optimal bioactive polypeptide grafting density.

[0084] S42. The cell culture in step S4, which involves culturing cells (including bone marrow mesenchymal stem cells, vascular endothelial cells, or neural cells) in a polypeptide-grafted helical multicell microfluidic platform, using FITC fluorescent labeling and observing cell production using a laser confocal microscope, specifically includes the following steps:

[0085] S421. Place the peptide-grafted helical multicell microfluidic platform in a third-well culture plate (6-well plate). Sterilize the peptide-grafted helical multicell microfluidic platform with ethanol (75% ethanol solution) for 2 hours. Then add 10 cells / mL to the third-well culture plate. 9 Cell suspensions of 1 to 100 μL per mL were statically cultured at 37°C for 1 hour to 30 days in a 5% CO2 environment.

[0086] S422. Remove the peptide-grafted helical multi-cell microfluidic platform from the third multi-well culture plate, wash the peptide-grafted helical multi-cell microfluidic platform three times with PBS, and place the peptide-grafted helical multi-cell microfluidic platform into the fourth multi-well culture plate. The fourth multi-well culture plate is a 6-well culture plate. Then, add paraformaldehyde to the fourth multi-well culture plate to fix the peptide-grafted helical multi-cell microfluidic platform. The fixation conditions of paraformaldehyde are: fixation at 4°C for 12 hours.

[0087] S423. Add live cell F-actin microfilament protein staining reagent to the fourth multi-well culture plate containing the polypeptide-grafted helical multi-cell microfluidic platform for fluorescent labeling. After washing with PBS (phosphate buffered solution), add DAPI dye (DAPI, i.e., 4',6-diamidinyl-2-phenylindole, is a fluorescent dye that can strongly bind to DNA and is commonly used for fluorescence microscopy) for fluorescent labeling. Then, remove the polypeptide-grafted helical multi-cell microfluidic platform and wash it three times with PBS.

[0088] S424. Under a laser confocal microscope, the cell distribution, cell adhesion, and cell differentiation on the surface of the polypeptide-grafted helical multicell microfluidic platform in step 423 are observed using the FITC channel to identify the biologically active cell region 106.

[0089] S5. Determine the optimal bioactive peptide grafting density in cell 103.

[0090] The method for determining the peptide grafting density in step S5 is as follows: Fluorescent peptides such as CPAPAPKRWWKWWRR-FITC, CPAPAPIGKYKLQYLEQWTLK-FITC, CPAPAPYIGSRK-FITC, or CPAPAPKIPKASSVPTELSAISTLYLK-FITC are grafted onto the surface of a peptide-grafted helical multi-cell microfluidic platform. The fluorescence value of the peptide-grafted helical multi-cell microfluidic platform is measured, and a bar chart of MFI value changes is plotted. The relationship between grafting time and grafting density is determined through the MFI value change bar chart, thus determining the grafting density of each cell 103 of the peptide-grafted helical multi-cell microfluidic platform.

[0091] A surface-modified helical multi-cell microfluidic platform was prepared by immersing it in an HS-R-Peptide solution to obtain the target bioactive peptide grafting density. The immersion parameters, namely the concentration A of the bioactive peptide solution and the immersion time B, were determined as follows:

[0092] The surface-modified spiral multi-cell microfluidic platform with gradient grafting density of bioactive peptides in step S3 is divided into n regions along the flow direction of the peptide solution. For an N×L chip, it needs to be divided into N×L regions 103, with the region cells 103 numbered from 1 to n, where 1≤n≤N×L. The concentration of the peptide solution in step S3 is aμM, and the time for the peptide solution to flow through all region cells 103 in step S3 is bmin. Therefore, A / μM=a, B / min=nb / N×L, N≥3, L≥3, and both N and L are odd numbers.

[0093] The grafting density of the target bioactive peptide is the same as the grafting density of the bioactive peptide in cell 103 of the optimal bioactive peptide grafting density region in step S4.

[0094] Example 1

[0095] The high-throughput screening method of the spiral multi-cell microfluidic platform in this embodiment includes the following steps:

[0096] S1. A square 5×5 spiral multi-cell microfluidic platform with a size of 1.62cm×1.62cm was obtained through design and 3D printing. The spiral multi-cell microfluidic platform was treated with 4% HF acid for 5 minutes and then cleaned with deionized water for 15 minutes to remove residual fluoride ions on the surface, thus obtaining the spiral multi-cell microfluidic platform.

[0097] S2. Then immerse the spiral multi-cell microfluidic platform in concentrated nitric acid for 15 minutes to obtain sufficient hydroxyl groups. Rinse it with deionized water and then dry it with nitrogen to obtain the surface-modified spiral multi-cell microfluidic platform.

[0098] S3. The peptide solution was prepared using anhydrous ethanol as the solvent to form an anhydrous ethanol solution of the CPAPAPKRWWKWWRRK-FITC peptide, with a concentration of 100 μM. The volume of the peptide solution drawn using a 1 ml syringe was 400 μl, the flow rate of the peptide solution was 100 μl / h, the grafting reaction time was 4 h, and the injection process was as follows: Figure 7 As shown (injected from inlet 101 at position "1"); after the reaction, the surface-modified helical multi-cell microfluidic platform grafted with peptides was taken out, cleaned with anhydrous ethanol, and then dried with nitrogen to obtain the peptide-grafted helical multi-cell microfluidic platform.

[0099] S4. Drop a water film onto the surface of the polypeptide-grafted helical multicell microfluidic platform and observe it under a fluorescence microscope;

[0100] S5. Statistical analysis of fluorescence values: Plot a bar chart of MFI (Mean fluorescence intensity) value changes, such as... Figure 10 As shown, this demonstrates that the device can successfully construct a graft density gradient.

[0101] Example 2

[0102] In this embodiment, the anhydrous ethanol solution of the CPCAPAPKRWWKWWRRK-FITC peptide in Example 1 is replaced with anhydrous ethanol solution of the CPCAPAPKRWWKWWRRK peptide. After performing the steps in Example 1, the sample is cleaned, rotated 90° clockwise, and anhydrous ethanol solution of the CPCAPAPIGKYKLQYLEQWTLK-FITC peptide is introduced from outlet 102 at point "2" to prepare the anhydrous ethanol solution of the CPCAPAPIGKYKLQYLEQWTLK-FITC peptide. (The subsequent steps are the same as S3-S5. This embodiment is mainly to demonstrate that it can construct...) A bidirectional peptide grafting density (concentration 100 μM) was established. 400 μL of peptide solution was drawn using a 1 ml syringe, the flow rate was 100 μL / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified helical multi-cell microfluidic platform was removed, cleaned with anhydrous ethanol, and then dried with nitrogen. A water film was added to the surface of the platform, and the microfluidic platform was observed under a fluorescence microscope. Fluorescence values ​​were statistically analyzed, and a bar chart of MFI (Mean fluorescence intensity) values ​​was plotted. Figure 11 As shown, this demonstrates that the device can successfully construct a bidirectional peptide grafting density gradient.

[0103] Example 3

[0104] In this embodiment, the anhydrous ethanol solution of the CPAAPIGKYKLQYLEQWTLK-FITC peptide in Example 2 is replaced with anhydrous ethanol solution of the CPAAPIGKYKLQYLEQWTLK peptide. After performing the steps of Example 2, the sample is cleaned, rotated 90° clockwise, and anhydrous ethanol solution of the CPAAPIGKYKLQYLEQWTLK peptide is introduced through outlet 102 at point "3" to prepare the anhydrous ethanol solution of the CPAAPIGKYKLQYLEQWTLK peptide. (The subsequent steps are the same as S3-S5. This embodiment is mainly to demonstrate that it can construct...) The three-dimensional peptide grafting density was established at a concentration of 100 μM. 400 μL of peptide solution was drawn using a 1 ml syringe, the flow rate of the peptide solution was 100 μL / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified spiral multi-cell microfluidic platform grafted with peptides was removed, cleaned with anhydrous ethanol, and then dried with nitrogen gas to obtain the peptide-grafted spiral multi-cell microfluidic platform. A water film was dropped onto the surface of the peptide-grafted spiral multi-cell microfluidic platform, and observed under a fluorescence microscope. The fluorescence values ​​were statistically analyzed, and a bar chart of MFI (Mean fluorescence intensity) value changes was plotted. Figure 12 As shown, this demonstrates that the device can successfully construct a three-dimensional peptide grafting density gradient.

[0105] Example 4

[0106] In this embodiment, the anhydrous ethanol solution of the CPAPAYIGSR-FITC peptide in Example 3 is replaced with anhydrous ethanol solution of the CPAPAYIGSR peptide. After performing the steps in Example 3, the sample is cleaned, rotated 90° clockwise, and an anhydrous ethanol solution of the CPAPAYIGSR peptide is introduced from outlet 102 at position "4" to prepare the CPAPAYIGSR-FITC peptide solution using anhydrous ethanol as the solvent. (The subsequent steps are the same as S3-S5. This embodiment is mainly to demonstrate that a four-component PCR solution can be constructed.) The peptide grafting density was 150 μM; the volume of peptide solution drawn using a 1 ml syringe was 400 μl, the flow rate of the peptide solution was 100 μl / h, and the grafting reaction time was 4 h. After the reaction, the surface-modified spiral multi-cell microfluidic platform grafted with peptide was removed, cleaned with anhydrous ethanol, and then dried with nitrogen gas to obtain the peptide-grafted spiral multi-cell microfluidic platform; a water film was dropped onto the surface of the peptide-grafted spiral multi-cell microfluidic platform, and observed under a fluorescence microscope; the fluorescence values ​​were statistically analyzed and a bar chart of MFI (Mean fluorescence intensity) value changes was plotted, as shown below. Figure 13 As shown, this demonstrates that the device can successfully construct a four-way peptide grafting density gradient.

[0107] Example 5

[0108] In this embodiment, the anhydrous ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC peptide from Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL. After washing and drying with nitrogen, a gradient surface with four-dimensional peptide grafting densities of AMP-QK-YIGSR-BMP2 with different grafting densities was obtained. After performing steps S411-S415, the growth of the colonies was observed under a fluorescence microscope, and the bacterial survival rate was as follows: Figure 14 As shown, the bacterial survival rate varied between 23.16% and 79.39%.

[0109] Example 6

[0110] In this embodiment, the anhydrous ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC peptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL. After washing and drying with nitrogen, a gradient surface with four-dimensional peptide grafting density of AMP-QK-YIGSR-BMP2 with different grafting densities was obtained. Steps S421-S424 were performed to culture vascular endothelial cells in a peptide-grafted helical multi-cell microfluidic platform, label them with FITC fluorescence, and observe the cell production using a laser confocal microscope. Specifically, the following steps were included: the peptide-grafted helical multi-cell microfluidic platform was placed in a third multi-well culture plate (6-well plate), and the platform was sterilized with ethanol (75% ethanol solution) for 2 hours. Then, a solution of 8×10⁻⁶ ethanol was added to the helical multi-cell microfluidic platform. 4A cell suspension of 250 μL (number of cells / mL) was statically cultured for one hour, followed by the addition of 5 mL of culture medium to each well. The cell suspension was cultured at 37°C for 3 days in a 5% CO2 environment. The peptide-grafted helical multi-cell microfluidic platform was removed from the third well culture plate and washed three times with PBS. This platform was then placed into a fourth well culture plate (6 wells). Paraformaldehyde was added to the fourth well to immobilize the peptide-grafted helical multi-cell microfluidic platform at 4°C for 12 hours. The fourth well containing the immobilized peptide-grafted helical multi-cell microfluidic platform was then further immobilized. F-actin microfilament staining reagent for live cells was added to the culture plate for fluorescent labeling. After washing with PBS (phosphate buffered PBS), DAPI dye (DAPI, i.e., 4',6-diamidinyl-2-phenylindole, a fluorescent dye that strongly binds to DNA and is commonly used in fluorescence microscopy) was added for fluorescent labeling. Then, the polypeptide-grafted helical multi-cell microfluidic platform was removed and washed three times with PBS. Under a laser confocal microscope, the distribution and number of endothelial cells on the surface of the polypeptide-grafted helical multi-cell microfluidic platform from step 423 were observed using the FITC channel to identify the biologically active cell region 106. The endothelial cell adhesion was as follows. Figure 15 As shown, the number of adherent cells varied between 249 and 436.

[0111] Example 7

[0112] In this embodiment, the anhydrous ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC peptide in Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL. After washing and drying with nitrogen, a gradient surface with four-dimensional peptide grafting density of AMP-QK-YIGSR-BMP2 with different grafting densities was obtained. Steps S421-S424 were performed to culture Schwann cells in a peptide-grafted helical multi-cell microfluidic platform, label them with FITC fluorescence, and observe the cell production using a laser confocal microscope. Specifically, the following steps were included: the peptide-grafted helical multi-cell microfluidic platform was placed in a third multi-well culture plate (6-well plate), and the platform was sterilized with ethanol (75% ethanol solution) for 2 hours. Then, a solution of 8×10⁻⁶ ethanol was added to the microfluidic chip. 3A cell suspension of 250 μL / mL was statically cultured for one hour, followed by the addition of 5 mL of culture medium to each well. The cell suspension was cultured at 37°C for 3 days in a 5% CO2 environment. The peptide-grafted helical multi-cell microfluidic platform was removed from the third well culture plate and washed three times with PBS. This platform was then placed into a fourth 6-well culture plate, and paraformaldehyde was added to fix the microfluidic platform at 4°C for 12 hours. F-actin staining reagent for live cells was added to the fourth well of the microfluidic platform for fluorescent labeling. After washing with PBS (phosphate buffered saline), DAPI dye (DAPI, 4',6-diamidinyl-2-phenylindole, a fluorescent dye that strongly binds to DNA and is commonly used in fluorescence microscopy) was added for fluorescent labeling. Then, the polypeptide-grafted helical multi-cell microfluidic platform was removed and washed three times with PBS. Under a laser confocal microscope, the Schwann cell differentiation on the surface of the polypeptide-grafted helical multi-cell microfluidic platform from step 423 was observed using the FITC channel. The Schwann cell differentiation was as follows: Figure 16 As shown, the L / W value first increases and then decreases from region c1 to region c6, indicating that the high concentration region is suppressed. In region c4, the L / W value reaches the highest level of 9.91 (Figure 3c), at which point the grafting densities of AMP, QK, YIGSR, and BMP-2 are 0.80 grafts / nm. 2 0.44 per nm 2 2.21 per nm 2 0.41 per nm 2 .

[0113] Example 8

[0114] In this embodiment, the anhydrous ethanol solution of the CPAPAPKIPKASSVPTELSAISTLYLK-FITC peptide from Example 4 was replaced with CPAPAPKIPKASSVPTELSAISTLYL. After washing and drying with nitrogen, a gradient surface with four-dimensional peptide grafting densities of AMP-QK-YIGSR-BMP2 with different grafting densities was obtained. The prepared platform was placed in a 6-well plate, and 4 × 10⁴ μL of nitrogen was added to the platform. 4Human bone marrow mesenchymal stem cells (hBMSCs) were cultured at a density of [number] cells / mL for 3 days. Then, 50 μM ascorbic acid, 10 mM β-glycerophosphate, and 100 nM dexamethasone were added to the complete hBMSC medium, and the medium was replaced with osteogenic induction medium. After another 7 days of culture, the substrate was washed with PBS and fixed with 4% paraformaldehyde (4°C, overnight). The samples were then stained with ALP working solution for 15 min, followed by washing with PBS. The samples were then characterized using a digital fluorescence microscope (DM6M, Lecia, Germany) under bright-field conditions, and the parameters were quantified using Lecia's built-in software. The degree of osteogenic differentiation was quantified by ALP staining intensity; the higher the degree of differentiation, the greater the ALP staining intensity and the lower the value. Figure 17 As shown, the ALP staining intensity varied from 4320.19au to 6195.34au.

[0115] Comparative Example 1

[0116] The traditional method for grafting bioactive peptides onto the surface of titanium sheets is as follows: (1) Take 33 square titanium sheets with a size of 1cm×1cm, treat the titanium sheets with 4% HF acid for 5min, clean the titanium sheets with deionized water for 5min to remove residual fluoride ions on the surface; then clean the titanium sheets with deionized water for 3min; finally, soak the titanium sheets in concentrated nitric acid for 15min, clean the titanium sheets with concentrated nitric acid with deionized water for 3min, and dry the titanium sheets with nitrogen gas.

[0117] (2) The titanium sheet from step (1) was laid flat and fixed in a container. 300 μl of anhydrous ethanol solution of bioactive peptide CPAPAPKRWWKWWRR-FITC was added, with a concentration of 100 μM, to submerge the bottom of the titanium sheet. The 33 titanium sheets were divided into 11 groups, with 3 titanium sheets in each group. The soaking times of the titanium sheets in the bioactive peptide CPAPAPKRWWKWWRR-FITC solution for the 11 groups were 0 min, 18.5 min, 55.5 min, 74 min, 92.5 min, 111 min, 129.5 min, 148 min, 166.5 min, 203.5 min, and 240.5 min, respectively.

[0118] (3) Take out the titanium sheet from step (2) and wash the titanium sheet three times with anhydrous ethanol and deionized water respectively to obtain titanium sheets with different bioactive peptide grafting densities.

[0119] (4) The FITC channel of a fluorescence microscope was used to characterize the changes in surface fluorescence values.

[0120] Comparative Example 2

[0121] Staphylococcus aureus was cultured on the surface of Ti 6Al 4V sheets uniformly grafted with the CPCAPAPKRWWKWWRR peptide, and the inhibition of bacterial growth on the surface was observed using the agar mounting method. Meanwhile, Ti 6Al 4V sheets that underwent only hydroxylation treatment served as a control group. The results showed that, compared to the control group, the surface of the Ti 6Al 4V sheets uniformly grafted with the CPCAPAPKRWWKWWRR peptide exhibited 80.36% inhibition of Staphylococcus aureus growth.

[0122] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A spiral-shaped multi-cell microfluidic platform, characterized in that, The device includes four spiral flow channels and a liquid storage tray. Each spiral flow channel comprises a longitudinal baffle, a transverse baffle, and a central baffle. The transverse baffles have a first groove and a second groove, and the central baffle has a central groove. The longitudinal and transverse baffles are evenly installed in the liquid storage tray in a centrally symmetrical manner. The height of the central groove is lower than the height of the liquid storage tray. The first and second grooves are mirror images of each other and are arranged in a crisscross pattern within the spiral flow channels. The height of the first and second grooves is lower than that of the longitudinal baffles. The liquid storage tray has one inlet and three outlets. The spiral multi-cell microfluidic platform studies the properties of peptide liquids by injecting different peptide solutions into the inlet or outlet, and can study up to four peptide solutions. The specific working steps of the spiral multi-cell microfluidic platform are as follows: (1) After the first type of polypeptide liquid enters through the inlet, the longitudinal baffle can change the flow direction of the polypeptide solution. After the first type of polypeptide liquid flows to the central area, it is divided into three branches and flows out of the three outlets at the same time, thus forming a spiral flow direction. (2) Based on step (1), the second type of polypeptide liquid enters from one of the three outlets. The longitudinal baffle can change the flow direction of the second type of polypeptide solution. After the second type of polypeptide liquid flows to the central area, it is divided into three branches and flows out to the remaining inlet and the other two outlets at the same time, thus forming a spiral flow. (3) Based on step (2), the third type of polypeptide liquid is introduced from one of the remaining uninjected polypeptide liquids. The longitudinal partition can change the flow direction of the third type of polypeptide solution. After the third type of polypeptide liquid flows to the central region, it is divided into three branches and flows out to the remaining inlet and the other two outlets at the same time, thereby forming a spiral flow direction and constructing a three-dimensional polypeptide grafting density gradient. (4) Based on step (3), the fourth type of polypeptide liquid is introduced from the outlet of the last remaining uninjected polypeptide liquid. The longitudinal partition can change the flow direction of the fourth type of polypeptide solution. After the fourth type of polypeptide liquid flows to the central region, it is divided into three branches and flows out to the remaining inlet and the other two outlets at the same time, thereby forming a spiral flow direction and constructing a four-way polypeptide grafting density gradient.

2. The spiral multi-cell microfluidic platform according to claim 1, characterized in that, The height of the first groove and the second groove is 1% to 99% of the height of the longitudinal partition, and the length of the first groove and the second groove is 1% to 99% of the length of the longitudinal partition.

3. A high-throughput screening method for a spiral multi-cell microfluidic platform according to any one of claims 1 or 2, characterized in that, Includes the following steps: S1. Design and 3D print a spiral multi-cell microfluidic platform. The spiral multi-cell microfluidic platform is subjected to stress-relief annealing and sandblasting to obtain a spiral multi-cell microfluidic platform. S2. Clean the spiral multi-cell microfluidic platform and perform hydroxylation treatment on the spiral multi-cell microfluidic platform to obtain a surface-modified spiral multi-cell microfluidic platform. S3. Inject the peptide solution into the surface-modified spiral multi-cell microfluidic platform. The peptide solution passes through the regional cells of the surface-modified spiral multi-cell microfluidic platform at a uniform speed and reacts. After the peptide solution has reacted with all the regional cells, the spiral multi-cell microfluidic platform is cleaned to obtain a peptide-grafted spiral multi-cell microfluidic platform. S4. Culture bacteria or cells in the polypeptide-grafted helical multi-cell microfluidic platform to determine the optimal region cell for the grafting density of bioactive polypeptides. S5. Determine the peptide grafting density of the cell in the optimal bioactive peptide grafting density region.

4. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The steps for hydroxylation treatment of the spiral multi-cell microfluidic platform described in step S2 are as follows: S211. The spiral multi-cell microfluidic platform is ultrasonically cleaned with deionized water, acetone, anhydrous ethanol and deionized water in sequence for 10 to 30 minutes, and then the spiral multi-cell microfluidic platform is dried with protective gas. S212. Immerse the spiral multi-cell microfluidic platform in HF acid with a mass fraction of 2% to 8% for 4 to 18 minutes. S213. Finally, clean the spiral multi-cell microfluidic platform treated with HF acid in step S212, dry the spiral multi-cell microfluidic platform again with protective gas, and immerse the spiral multi-cell microfluidic platform in concentrated nitric acid for 10-30 minutes to obtain a surface-modified spiral multi-cell microfluidic platform.

5. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The polypeptide molecule in the polypeptide solution in step S3 has the molecular structure HS-R-Peptide, where R includes C(PA)nP, C is cysteine, n is an integer from 1 to 100, P is proline, A is alanine, and the peptide includes one of KRWWKWWRR or KRWWKWWRRK-FITC or IGKYKLQYLEQWTLK or IGKYKLQYLEQWTLK-FITC or YIGSR or YIGSRK-FITC or KIPKASSVPTELSAISTLYL or KIPKASSVPTELSAISTLYLK-FITC.

6. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The concentration range of the polypeptide solution in step S3 is 1 μM to 5 mM.

7. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, The reaction time between the polypeptide solution and the surface-modified helical multi-cell microfluidic platform in step S3 is 5 minutes to 24 hours.

8. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, Step S4, culturing bacteria in the polypeptide-grafted helical multi-cell microfluidic platform, includes the following steps: S411. Prepare the required nutrient broth culture medium, and sterilize the nutrient broth culture medium in an autoclave at 121°C for 2 hours. S412. Place the polypeptide-grafted helical multi-cell microfluidic platform in a first multi-well culture plate, and sequentially add peptides with a concentration of 10-10 to the regional cells of the polypeptide-grafted helical multi-cell microfluidic platform. 9 1 μl to 100 μl of bacterial culture with CFU / ml was incubated for 10 minutes to 24 hours on the microfluidic platform of the polypeptide-grafted helical multi-cell structure. S413. After co-incubation, the first multi-well culture plate is placed in a drying oven to allow the bacterial solution to evaporate. After the bacterial solution has evaporated, the polypeptide-grafted helical multi-cell microfluidic platform is placed in a biological cabinet and left to stand for 10-15 minutes. S414. Add a solution of resazurin with a concentration of 10-1000 μg / ml sequentially to the regional cell of the microfluidic platform of the polypeptide grafted helical multi-cell structure, mix with nutrient broth, and incubate the resazurin solution in the microfluidic platform of the polypeptide grafted helical multi-cell structure for 10 minutes to 8 hours. S415. After the polypeptide-grafted helical multi-cell microfluidic platform has been statically cultured, the colony growth of the polypeptide-grafted helical multi-cell microfluidic platform is observed under a fluorescence microscope to determine the region cell with the optimal bioactive polypeptide grafting density.

9. The spiral multi-cell microfluidic platform according to claim 3, characterized in that, Step S4, culturing cells in the polypeptide-grafted helical multicell microfluidic platform, includes the following steps: S421. Place the polypeptide-grafted helical multi-cell microfluidic platform in a third multi-well culture plate, sterilize the polypeptide-grafted helical multi-cell microfluidic platform, and sequentially add a concentration of 10-10 to each cell of the helical multi-cell microfluidic platform. 9 Incubate 1 μl to 100 μl of cell suspension per 1 cell / mL for 1 hour to 30 days. S422. The polypeptide-grafted helical multi-cell microfluidic platform is removed from the third multi-well culture plate, cleaned with PBS, placed into the fourth multi-well culture plate, and then paraformaldehyde is added to the fourth multi-well culture plate to fix the polypeptide-grafted helical multi-cell microfluidic platform. S423. Add live cell F-actin microfilament staining reagent to the fourth multi-well culture plate containing the polypeptide-grafted helical multi-cell microfluidic platform for fluorescent labeling and wash it with PBS. Then add DAPI dye to the fourth multi-well culture plate to fluorescently label the polypeptide-grafted helical multi-cell microfluidic platform. Then remove the polypeptide-grafted helical multi-cell microfluidic platform and wash it with PBS. S424. Under a laser confocal microscope, observe the cell distribution, cell adhesion, and cell differentiation on the surface of the polypeptide-grafted helical multicell microfluidic platform described in step 423 using the FITC channel to identify the biologically active cell regions.

Images