Cell ball assembling method based on ultrasonic driving microneedle

Abstract

The invention relates to a cell ball assembling method based on an ultrasonic driving microneedle, which comprises the following steps: capturing and transferring cell balls by adopting a microneedle provided with a PZT transducer, and arranging the cell balls in two-dimensional or three-dimensional manner by utilizing an acoustic streaming effect to assemble into a specific pattern; the device can be used for assembling a sphere with a required space pattern, and has the advantages of good compatibility, fast response, no mark, no contact and the like.

Description

Technical Field

[0001] The present invention relates to a technology in the field of medical devices, specifically a cell sphere assembly method based on ultrasound-driven microneedles. Background Art

[0002] Cell spheroids are three-dimensional tissue structures formed by the aggregation of a large number of cells of the same or different types. Compared with traditional two-dimensional 2D monolayer cells, cells in spheroids can naturally form complex cell-cell and cell-matrix interactions. 3D structures are closer to natural tissue microenvironments and are important detection targets for tissue engineering. These cell spheroids can form reproducible tissues such as intestines, liver, brain, bones, and kidneys through co-culture, which is of great significance for in vitro detection of human development and diseases. Cell spheroids are often used to detect interactions between different cell types in 3D environments such as proliferation, migration, and invasion, especially in tumor models. By detecting the behavior of tumor cells in cell spheroids, we can better understand the mechanisms of tumor occurrence and development. Therefore, the ability to construct tumor sphere assemblies with desired spatial patterns or even heterogeneous structures is crucial. Summary of the invention

[0003] In view of the limitations of existing 3D bioprinting technology in bioprinting materials, such as being limited to two-dimensional planes, lacking flexibility in shape and being unsuitable for making complex patterns, and complicated operation, the present invention proposes a cell sphere assembly method based on ultrasonically driven microneedles, which can assemble spheres with the desired spatial pattern and has the advantages of good compatibility, fast response, no labeling, and no contact.

[0004] The present invention is achieved through the following technical solutions:

[0005] The invention relates to a cell ball assembly method based on ultrasound-driven microneedles, which uses microneedles equipped with PZT transducers to capture and transfer cell balls, and uses acoustic streaming effect to arrange the cell balls in two or three dimensions to assemble into specific patterns.

[0006] In the two-dimensional or three-dimensional arrangement, when the cell spheres are assembled at a separation distance of 20 μm, they will not fuse but will be close to each other after 24 hours of culture; when the cell spheres are assembled at a separation distance of 50 μm, the cell spheres will not affect each other and will grow independently.

[0007] The microneedle is preferably made using PμSL 3D printing technology.

[0008] The PZT transducer is attached to one side of the metal plate, and then the microneedle is glued to the center of the PZT transducer.

[0009] For the capture and transfer, the microneedles are preferably placed on a three-dimensional mobile platform, and the power amplifier amplifies the signal to the PZT transducer, and then drives the microneedles to adsorb and release the cell spheres. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 Schematic diagram of the cell ball capture principle;

[0011] Figure 2 Schematic diagram of the cell spheroid patterning process;

[0012] Figure 3 Schematic diagram of cell spheroid patterning application;

[0013] Figure 4 Schematic diagram for assembling cell spheroids in two and three dimensions;

[0014] Figure 5 Schematic diagram of the ring-shaped patterned fusion process of cell spheres;

[0015] Figure 6 Schematic diagram of the cell spheroid invasion process;

[0016] Figure 7 Schematic diagram of the microneedle vibration characteristics and the acoustic pressure distribution in the liquid;

[0017] Figure 8 Schematic diagram of the force distribution of microspheres in liquid;

[0018] Fig. 9 Schematic diagram of the motion trajectory and velocity change of microspheres in liquid;

[0019] Fig.10 Schematic diagram of the cell spheroid fusion process. DETAILED DESCRIPTION

[0020] like Figure 1 and Figure 2 As shown, this embodiment relates to a cell spheroid assembly method based on ultrasound-driven microneedles, which uses microneedles equipped with PZT transducers to capture and transfer cell spheroids, and uses the acoustic streaming effect to arrange the cell spheroids in two or three dimensions to assemble into a specific pattern.

[0021] The transfer cell sphere is preferably encapsulated by gelatin methacrylate (GelMA) or hyaluronic acid methacrylate (HAMA) solution, specifically, 5wt.% GelMA or HAMA powder is dissolved in PBS containing 0.25wt.% lithium phenyl-2,4,6-trimethylbenzoylphosphonate photoinitiator, and heated in a 50°C water bath for 30 minutes to obtain.

[0022] like Figure 3As shown, the specific pattern described refers to: osteospheres were assembled into rings and a tumor invasion model was developed to demonstrate the application of the acoustic bioprinting system.

[0023] To verify the practicality of the acoustic bioprinting system, cell spheroids were assembled into Figure 4 The pre-designed two-dimensional pattern and three-dimensional pyramid structure are shown. Figure 4 B and Figure 4 C shows that multiple spheres are deposited in a pyramid-shaped 3D structure. The results show that this method can not only assemble spheres into 2D and 3D structures, but also control the heterogeneity of the structures. This ability is crucial for developing functional tissue structures that closely resemble the structure and properties of natural tissues.

[0024] like Figure 5 As shown, MC3T3-E1 cell spheroids were arranged into rings as specific patterns in a hyaluronic acid methacrylate (HAMA) solution, and the cell spheroid patterns were fixed to a three-dimensional matrix after UV curing. After further culture, the ring-shaped cell spheroids fused into a whole.

[0025] The MC3T3-E1 cell spheroids are formed by inoculating 10,000 cells into the wells of an ultra-low attachment 96-well plate and adding 200 μL α-MEM, and culturing for 2 to 3 days. The cell spheroids are cultured in a plastic culture dish at a temperature of 37° C., a CO2 concentration of 5%, and a culture medium of α-MEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin.

[0026] The hyaluronic acid methacrylate (HAMA) solution is obtained by dissolving HAMA powder in PBS containing lithium phenyl-2,4,6-trimethylbenzoylphosphonate photoinitiator and heating in a water bath.

[0027] like Figure 6 As shown, this example further constructed a model of human breast cancer cells (MCF-7) and normal human skin fibroblasts (NHDFs) cells to detect the invasiveness of three-dimensional cell spheroids, which has great application potential in tissue engineering and disease model construction.

[0028] The MCF-7 cells and NHDF cells are formed by inoculating 10,000 cells into the wells of an ultra-low attachment 96-well plate and adding 200 μL DMEM. The cells are cultured for 2 to 3 days. The cell spheroids are cultured in a plastic culture dish at a temperature of 37° C. and a CO2 concentration of 5%. The culture medium is DMEM, which contains 10% fetal bovine serum and 1% penicillin-streptomycin.

[0029] To better observe the tumor invasion process, MCF-7 and NHDFs were labeled with red DiI dye and green DiO dye at 37°C and 5% CO2 concentration for about 20 minutes, so that MCF-7 was labeled with red signals and NHDF was labeled with green signals. The labeled cells were washed three times with fresh culture medium and then mixed with culture medium to prepare spheroids.

[0030] like Figure 7 As shown in the figure, the results obtained after numerical simulation of the acoustic and force distribution characteristics by FEM software COMSOL Multiphysics. It can be seen that the ability of the microneedle to capture cell spheres is mainly caused by acoustic streaming and acoustic radiation force (ARF).

[0031] like Figure 7 A, Microneedle vibration displacement distribution results, the amplitude of the cone microneedle vibration increases gradually from the root to the tip. Figure 7 From the sound pressure distribution diagram of the water area shown in B, it can be found that the area with the strongest local sound pressure corresponds to the maximum vibration in the microneedle.

[0032] By analyzing the force acting on the microsphere in the liquid, we can get the following Figure 8 The drag force, acoustic radiation force, and total force results shown in the figure show that the drag force on the microsphere near the tip of the microneedle is small, while the acoustic radiation force is relatively large, resulting in the total force acting on the microsphere being consistent with the acoustic radiation force and pointing to the tip of the needle. Since the range of the acoustic radiation force is smaller than that of the acoustic flow, the microspheres farther away are mainly affected by the drag force.

[0033] like Fig. 9 As shown in the figure, the initial motion trajectory of the sphere moves along the acoustic streamline, and only when it is close enough to the tip of the microneedle does it change direction to point to the tip. At the same time, according to the instantaneous speed of the particles at different times, it can be found that at the beginning, due to the small drag force, the speed and acceleration are small. When approaching the needle tip, the speed and acceleration increase instantly.

[0034] like Fig.10 As shown in the figure, cells are arranged at different spacings under ultrasound drive. If the spacing between cells is too large, effective fusion cannot be formed. When the cell spheres are assembled at a separation distance of 20 μm, no fusion occurs after 24 hours of culture, but they are close to each other; when the cell spheres are assembled at a separation distance of 50 μm, the cell spheres do not affect each other and grow independently.

[0035] The above-mentioned specific implementation can be partially adjusted in different ways by those skilled in the art without departing from the principle and purpose of the present invention. The protection scope of the present invention shall be based on the claims and shall not be limited by the above-mentioned specific implementation. Each implementation scheme within its scope shall be subject to the constraints of the present invention.

Claims

1. A cell spheroid assembly method based on ultrasound-driven microneedles, characterized in that: Microneedles equipped with PZT transducers are used to capture and transfer cell spheroids, and the cell spheroids are arranged in two or three dimensions to assemble into specific patterns using the acoustic streaming effect; In the two-dimensional or three-dimensional arrangement, when the cell spheres are assembled at a separation distance of 20 μm, no fusion occurs but the cells are close to each other after 24 hours of culture; when the cell spheres are assembled at a separation distance of 50 μm, the cell spheres do not affect each other and grow independently.

2. The method for assembling cell spheres based on ultrasound-driven microneedles according to claim 1, characterized in that: The microneedles are made using PμSL 3D printing technology.

3. The cell spheroid assembly method based on ultrasound-driven microneedles according to claim 1, characterized in that: The PZT transducer is attached to one side of the metal plate, and then the microneedle is glued to the center of the PZT transducer.

4. The method for assembling cell spheres based on ultrasound-driven microneedles according to claim 1, characterized in that: The capture and transfer are achieved in the following manner: the microneedles are placed on a three-dimensional mobile platform, and the power amplifier amplifies the signal to the PZT transducer, which then drives the microneedles to adsorb and release the cell balls.

5. The cell spheroid assembly method based on ultrasound-driven microneedles according to claim 1 or 4, characterized in that: The capture and transfer are achieved by encapsulating with methacrylated gelatin (GelMA) or hyaluronic acid methacrylate (HAMA) solution, wherein the solution is obtained by dissolving GelMA or HAMA powder in PBS containing lithium phenyl-2,4,6-trimethylbenzoylphosphonate photoinitiator and heating in a water bath.

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