Three-dimensional ultrasonic sound field device and method for arranging cells in 3d

Abstract

The application discloses a three-dimensional ultrasonic sound field device and a method for arranging cells in 3D, and relates to the technical field of cell culture. The three-dimensional ultrasonic sound field device comprises a sound field generating device, a cell culture pool and a device base. The cell culture pool is detachably arranged in the chamber and used for placing experimental samples to be arranged. The signal generator generates sine wave signals with different frequencies and amplitudes and independently applies the sine wave signals to each piezoelectric ceramic. Different piezoelectric ceramics generate different interference patterns according to the received sine wave signals with different amplitudes, so that the cells in the cell culture pool generate different arrangement modes. The three-dimensional ultrasonic sound field device uses multi-wave interference in three-dimensional directions, realizes a sound field mode in three-dimensional space, and can satisfy various configurations of suspended cells in space.

Description

Technical Field

[0001] This invention relates to the field of tissue engineering technology, and in particular to a three-dimensional ultrasound sound field device and a method for 3D cell arrangement. Background Technology

[0002] Arranging cells and other biological samples into desired patterns plays an important role in many biological and biomedical studies, such as cell-cell interactions, tissue engineering, and regenerative medicine.

[0003] Cells are immobilized within an acoustic potential trap formed by the interference of multiple ultrasonic beams, and the cell arrangement changes according to the acoustic field model. This has become a relatively common method of cell manipulation. For example, Chinese patent document CN110643486A discloses an ultrasonic acoustic field device and a method for fabricating a digital PCR droplet array chip. The device applies a sine wave to a piezoelectric ceramic device with the same resonant frequency through a signal generator. The radiation force generated by the sound wave can capture droplets in an emulsion, causing them to arrange in a two-dimensional pattern at a specific location. For example, Chinese patent document CN202010061217.9 discloses a hexagonal surface wave acoustic tweezers chip for cell arrangement and assembly. This chip includes hexagonal acoustic tweezers and a microfluidic cavity, which are connected together by plasma bonding. The microfluidic cavity is used to contain cell solution, and a microfluidic channel is provided at the bottom of the microfluidic cavity. The microfluidic cavity also has at least one cell solution sample inlet and at least one cell solution sample outlet. The hexagonal acoustic tweezers are used to generate a coherent beam and are disposed around the microfluidic cavity. The hexagonal acoustic tweezers include a Z-cut lithium niobate substrate and a [missing information - likely a component or material] disposed on the Z-cut lithium niobate substrate. The bottom has six interdigital transducers; each of the two poles of the interdigital transducer is led out by a wire, which is connected to a power amplifier and a signal source in sequence; when cell assembly and arrangement are required, the signal source generates a radio frequency signal, which is amplified to a specific power by the power amplifier and then applied to a specific interdigital transducer. According to different signal configurations, different transducers are activated or their phases are modulated. The surface acoustic waves generated by each transducer are transmitted to the microfluidic cavity and interfere to produce different sound field patterns; the cells in the cell solution sample are gathered into the acoustic potential trap by acoustic force in the sound field, thereby generating the corresponding structural pattern and realizing the assembly and arrangement of cells.

[0004] Traditional acoustic tweezers used for cell manipulation mostly employ surface waves, which can only manipulate cells on a limited scale. They use two pairs of mutually perpendicular piezoelectric ceramics to generate standing wave fields and manipulate cells or other particles by forming a square lattice acoustic field through the standing wavelength. However, this method is limited to particles on the substrate surface and does not involve arranging and assembling cells in three-dimensional space to achieve a certain configuration in the spatial dimension of cells. This makes it difficult to meet the needs of arranging suspended cells in three-dimensional space. Summary of the Invention

[0005] This invention addresses the shortcomings of existing ultrasonic sound field devices in arranging cells in three-dimensional space by providing a three-dimensional ultrasonic sound field device for 3D cell arrangement. This sound field device not only enables cells to arrange themselves in space, but also allows cells to have different arrangement patterns by modulating the amplitude of different piezoelectric ceramics.

[0006] The technical solution of the present invention is as follows:

[0007] A three-dimensional ultrasonic sound field device for 3D cell alignment, comprising:

[0008] A sound field generating device has a chamber for accommodating a cell culture pool. Piezoelectric ceramics are respectively arranged on the inner walls and bottom walls of the chamber. The two poles of each piezoelectric ceramic are connected to a signal generator. The five piezoelectric ceramics generate a coherent ultrasonic beam, which generates a three-dimensional ultrasonic sound field in the chamber.

[0009] The cell culture pool can be detachably set in the chamber for placing experimental samples that need to be arranged.

[0010] The device base is detachably connected to the sound field generator and is used to fix the sound field generator.

[0011] The signal generator produces sinusoidal signals of different frequencies and amplitudes according to the needs of cell arrangement and applies them independently to each piezoelectric ceramic. Different piezoelectric ceramics produce different interference patterns according to the different amplitude sinusoidal signals they receive, causing the cells in the cell culture pool to produce different arrangement patterns.

[0012] The sound waves generated by the piezoelectric ceramic on the bottom surface of the sound field generating device of the present invention are incident on the solution in the cell culture tank and then on the air. Since the gas-liquid interface is a good sound wave reflecting layer, a standing wave sound field can be generated in the Z-axis direction of the cell culture tank through the piezoelectric ceramic at the bottom. The three-dimensional ultrasonic sound field device of the present invention places piezoelectric ceramics on the four sides and bottom surface of the cell culture tank, which enables the cells to be arranged in three dimensions in the cell culture tank.

[0013] When cells need to be arranged, a signal generator produces a sinusoidal signal of a certain frequency and amplitude and applies it to the piezoelectric ceramic. Different piezoelectric ceramics can produce different interference patterns according to the different amplitude signals they receive, thereby causing the cells in the cell culture pool to be arranged in different ways.

[0014] Let the piezoelectric ceramics on the inner walls of the chamber be P1-P4, and the piezoelectric ceramic on the bottom wall of the chamber be P5, then:

[0015] When the amplitudes of P1-P5 are simultaneously at their maximum values, the cells are arranged in a three-dimensional lattice pattern within the cell culture tank.

[0016] When the amplitude of P1-P4 is at its maximum and the amplitude of P5 is 0, the cells will be arranged in a linear pattern parallel to the Z-axis in the cell culture tank.

[0017] When the amplitude of P5 is at its maximum value and the other amplitudes are 0, the cells will be arranged in a planar pattern parallel to the xoy plane in the cell culture pool; the xoy plane is parallel to P5.

[0018] Arranged in multiple parallel xoy planes, with the plane spacing being half the wavelength of the sound wave generated by the P5 piezoelectric ceramic;

[0019] When the amplitudes of P1 and P2 are at their maximum values ​​and the remaining amplitudes are 0, the cells in the cell culture tank will obtain a planar arrangement in a direction parallel to the angle bisector of the angle formed by P1 and P2.

[0020] When the amplitudes of P1 and P3 are at their maximum values ​​and the remaining amplitudes are 0, the cells will achieve a planar arrangement parallel to the xoz plane in the cell culture pool; the xoz plane is parallel to P1 and P3.

[0021] Preferably, the sound field generating device and the device base are made of resin material.

[0022] Preferably, the cell culture pool is made of a transparent material.

[0023] Furthermore, the cell culture pool is made of quartz glass.

[0024] Preferably, a limiting groove is provided at the bottom of the chamber to limit the arrangement of the culture pools.

[0025] More preferably, the limiting groove includes at least two L-shaped limiting brackets.

[0026] The L-shaped limiting support and the cell culture pool should ideally fit together tightly, meaning that there needs to be a certain amount of friction between them to prevent the cell culture pool from shaking and affecting the cell arrangement.

[0027] After the cell culture pool is placed into the chamber of the sound field generator, the four walls of the cell culture pool should be parallel to the four inner walls of the chamber, and the space between the inner wall of the chamber and the cell culture pool should be filled with pure water to ensure normal transmission of ultrasound.

[0028] The method for 3D cell arrangement using the three-dimensional ultrasonic sound field device of the present invention includes the following steps:

[0029] (1) Place the sound field generator on the device base, then place the cell culture pool into the chamber of the sound field generator, and add pure water to the chamber;

[0030] (2) Add the cell dispersion to the cell culture tank;

[0031] (3) Based on the sound field pattern required for cell arrangement, the corresponding piezoelectric ceramic is modulated with the corresponding amplitude so that the generated sound waves produce different interference sound waves in the space of the cell culture pool, thereby arranging the cells in 3D.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] The three-dimensional ultrasonic sound field device of the present invention is the first to use multi-wave interference in three-dimensional direction, realizing a sound field mode in three-dimensional space, which can meet the various configurations of suspended cells in space and has great application prospects. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the equiaxed exploded structure of the three-dimensional ultrasonic sound field device of the present invention;

[0035] Figure 2 This is a schematic diagram of the sound field generating device; where (a) is a top view and (b) is a cross-sectional view of (a) along the AA direction;

[0036] Figure 3 This is a structural diagram of the device base; where (a) is the front view and (b) is the top view.

[0037] Figure 4 This is a structural diagram of a cell culture pool;

[0038] Figure 5 When the amplitudes of P1-P5 are simultaneously at their maximum values, the cells acquire a lattice-like arrangement in three-dimensional space; where (a) is the xoz plane, (b) is the yoz plane, and (c) is the xoy plane;

[0039] Figure 6 When the amplitude of P1-P4 is at its maximum value and the amplitude of P5 is 0, a line-like arrangement parallel to the Z-axis direction will be obtained, where (a) is the xoz plane, (b) is the yoz plane, and (c) is the xoy plane.

[0040] Figure 7 When the amplitude of P5 is at its maximum value and the other amplitudes are 0, a planar arrangement parallel to the xoy plane will be obtained; where (a) is the xoz plane, (b) is the yoz plane, and (c) is the xoy plane;

[0041] Figure 8 When the amplitudes of P1 and P2 are at their maximum values ​​and the remaining amplitudes are 0, a planar arrangement will be obtained in the direction parallel to the angle bisector of the angle formed by P1 and P2; where (a) is the xoz plane, (b) is the yoz plane, and (c) is the xoy plane.

[0042] Figure 9 When the amplitudes of P1 and P3 are at their maximum values ​​and the remaining amplitudes are 0, a planar arrangement parallel to the xoz plane will be obtained; where (a) is the xoz plane, (b) is the yoz plane, and (c) is the xoy plane. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be noted that the embodiments described below are intended to facilitate the understanding of the present invention and do not limit it in any way.

[0044] An ultrasonic sound field device for 3D cell alignment, such as Figure 1-4 As shown, the device consists of three parts: a sound field generating device 1, a device base 2, and a cell culture tank 3. The sound field generating device has slots around its perimeter, with dimensions corresponding to the piezoelectric ceramic used for attaching it. There is a square slot in the center, with L-shaped supports at the four corners to fix the cell culture tank 3. The bottom of the sound field generating device 1 also has a square slot slightly larger than the piezoelectric ceramic used for attaching the bottom piezoelectric ceramic. The device base 2 has baffles on both sides to restrict the position of the sound field generating device 1. Its upper surface has a through-slot with a thickness of 1.5mm, ensuring the sound field generating device 1 is placed flat on the device base without tilting or shaking. When the sound field generating device 1 is placed on the device base 2, the device base 2 can accommodate the solder joints of the bottom piezoelectric ceramic, and the wires connecting the bottom piezoelectric ceramic can also be connected to the signal generator through the slot in the device base 2.

[0045] Cell culture tank 3 is used to hold cell solutions. When in use, place it in the sound field generating device 1 and add the cell solutions to cell culture tank 3 using a pipette.

[0046] The sound field generating device 1 and the device base 2 are printed from resin material, and the cell culture tank 3 is sintered from quartz glass. The five piezoelectric ceramic pieces measure 12.6mm × 12.6mm × 0.66mm, as shown below. Figure 1 As shown, paste it into the corresponding groove.

[0047] Figure 3 The device base 2 shown has a through groove in the middle to accommodate the solder joints of the piezoelectric ceramic at the bottom and the connecting wires. The function of the device base 1 is to place the sound field generating device 1 stably and prevent tilting caused by the wires and solder joints.

[0048] When assembling the entire device, place the sound field generator 1 (with the piezoelectric ceramics attached) on the device base 2, then place it in the cell culture tank 3, and fill the gap between the cell culture tank 3 and the sound field generator 1 with pure water. Connect each of the five piezoelectric ceramics to a signal generator, set the signal generator to the corresponding piezoelectric ceramic resonant frequency, and adjust the amplitude of the corresponding piezoelectric ceramic as needed. Add cell solution to the cell culture tank, and output a sine wave signal using the signal generator to obtain the corresponding arrangement.

[0049] P1-P4 are four piezoelectric ceramics around the cell culture tank 3, and P5 is the piezoelectric ceramic at the bottom of the cell culture tank 3. P1 and P3 are pasted parallel to the yoz plane, P2 and P4 are pasted parallel to the xoz plane, and P5 is pasted parallel to the xoy plane.

[0050] The sound field generating device 1 of the present invention has piezoelectric ceramics placed on each of the five faces of a hexahedron, enabling cells to be arranged on the corners of the cube. The sound waves generated by the piezoelectric ceramics on the bottom surface of the sound field generating device 1 of the present invention are incident into the solution in the cell culture tank and then into the air. Since the gas-liquid interface is a good sound wave reflecting layer, a standing wave sound field can be generated in the Z-axis direction by a single piece of piezoelectric ceramic.

[0051] During cell arrangement, a signal generator produces a sinusoidal signal of a certain frequency and amplitude, which is applied to the piezoelectric ceramic. Different piezoelectric ceramics can produce different interference patterns according to the different amplitude signals they receive, thereby causing the cells in the cell culture pool to arrange in different ways.

[0052] like Figure 5 As shown, when the amplitudes of P1-P5 are all at their maximum values, a lattice-like arrangement will be obtained in three-dimensional space.

[0053] like Figure 6 As shown, when the amplitudes of P1-P4 are at their maximum values ​​and the amplitude of P5 is 0, a linear arrangement parallel to the Z-axis direction will be obtained.

[0054] like Figure 7 As shown, when the amplitude of P5 is at its maximum value and the other amplitudes are 0, a planar arrangement parallel to the xoy plane will be obtained.

[0055] like Figure 8 As shown, when the amplitudes of P1 and P2 are at their maximum values ​​and the remaining amplitudes are 0, a planar arrangement will be obtained in the direction parallel to the angle bisector of the angle formed by P1 and P2. The difference between this and other planar arrangements is that the plane area is different at different positions.

[0056] like Figure 9As shown, when the amplitudes of P1 and P3 are at their maximum values ​​and the remaining amplitudes are 0, a planar arrangement parallel to the xoz plane will be obtained.

[0057] The method for 3D cell alignment using the ultrasonic sound field device of the present invention includes the following steps:

[0058] Step 1: Place the sound field generator on the base of the device, ensuring the entire device is stable. Then place the cell culture medium in the sound field generator, and add purified water around it to ensure that the sound waves can be properly incident into the cell culture tank.

[0059] Step 2: Add the cell solution to the cell culture tank using a pipette.

[0060] Step 3: Modulate the amplitude of the corresponding piezoelectric ceramic according to the required sound field pattern, so that the generated sound waves produce different interference sound waves in the cell culture pool space, and use the corresponding interference sound waves to arrange the cells.

[0061] The ultrasonic sound field device of this invention is the first to utilize multi-wave interference in three dimensions, realizing a sound field mode in three-dimensional space. This allows it to accommodate various configurations of suspended cells in space, demonstrating significant application potential. The above-described embodiments provide a detailed explanation of the technical solution and beneficial effects of this invention. It should be understood that the above descriptions are merely specific embodiments of this invention and are not intended to limit the invention. Any modifications, additions, and equivalent substitutions made within the scope of the principles of this invention should be included within the protection scope of this invention.

Claims

1. A three-dimensional ultrasonic sound field device for 3D cell alignment, characterized in that, include: A sound field generating device has a chamber for accommodating a cell culture pool. Piezoelectric ceramics are respectively arranged on the inner walls and bottom walls of the chamber. The two poles of each piezoelectric ceramic are connected to a signal generator. The five piezoelectric ceramics generate a coherent ultrasonic beam, which generates a three-dimensional ultrasonic sound field in the chamber. The cell culture pool can be detachably set in the chamber for placing experimental samples that need to be arranged. The device base is detachably connected to the sound field generator and is used to fix the sound field generator. The signal generator generates sinusoidal signals of different frequencies and amplitudes according to the needs of cell arrangement and applies them independently to each piezoelectric ceramic. Different piezoelectric ceramics generate different interference patterns according to the different amplitude sinusoidal signals they receive, causing the cells in the cell culture pool to arrange in different ways. Let the piezoelectric ceramics on the inner walls of the chamber be P1-P4, and the piezoelectric ceramic on the bottom wall of the chamber be P5, then: When the amplitudes of P1-P5 are simultaneously at their maximum values, the cells are arranged in a three-dimensional lattice pattern within the cell culture tank. When the amplitude of P1-P4 is at its maximum and the amplitude of P5 is 0, the cells will be arranged in a linear pattern parallel to the Z-axis in the cell culture tank. When the amplitude of P5 is at its maximum value and the other amplitudes are 0, the cells will be arranged in a planar pattern parallel to the xoy plane in the cell culture pool; the xoy plane is parallel to P5. When the amplitudes of P1 and P2 are at their maximum values ​​and the remaining amplitudes are 0, the cells in the cell culture tank will obtain a planar arrangement in a direction parallel to the angle bisector of the angle formed by P1 and P2. When the amplitudes of P1 and P3 are at their maximum values ​​and the remaining amplitudes are 0, the cells will achieve a planar arrangement parallel to the xoz plane in the cell culture pool; the xoz plane is parallel to P1 and P3.

2. The three-dimensional ultrasonic sound field device for 3D cell alignment according to claim 1, characterized in that, The sound field generating device and its base are made of resin.

3. The three-dimensional ultrasonic sound field device for 3D cell alignment according to claim 1, characterized in that, The cell culture pool is made of transparent material.

4. The three-dimensional ultrasonic sound field device for 3D cell alignment according to claim 3, characterized in that, The cell culture tank is made of quartz glass.

5. The three-dimensional ultrasonic sound field device for 3D cell alignment according to claim 1, characterized in that, The bottom of the chamber is provided with a limiting groove to limit the arrangement of the culture pools.

6. The three-dimensional ultrasonic sound field device for 3D cell alignment according to claim 5, characterized in that, The limiting groove includes at least two L-shaped limiting brackets.

7. A method for 3D cell arrangement using a three-dimensional ultrasonic sound field device as described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Place the sound field generator on the device base, then place the cell culture pool into the chamber of the sound field generator, and add pure water to the chamber; (2) Add the cell dispersion to the cell culture tank; (3) Based on the sound field pattern required for cell arrangement, the corresponding piezoelectric ceramic is modulated with the corresponding amplitude so that the generated sound waves produce different interference sound waves in the space of the cell culture pool, thereby arranging the cells in 3D.

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