Volume ultrasonic cell assembling device based on porous chip, cell assembling method and application

Abstract

The invention discloses a body ultrasonic cell assembling device based on a porous chip, a cell assembling method and application. The cell assembling method comprises the following steps: step 1, placing the porous chip in a cell assembling chamber; step 2, placing a cell assembly chamber at the top of an ultrasonic transducer; step 3, injecting a cell solution into the cell assembly chamber; step 4, adjusting frequency and amplitude according to resonant frequency and working voltage of a piezoelectric ceramic piece of the ultrasonic transducer, keeping the signal for a preset time, and gathering the cells to a standing wave sound field node in the porous chip so as to complete target assembly of the cells; the porous chip and the ultrasonic transducer are utilized to generate a sound field, so that the cells are orderly assembled in the micropores, the cells or micro / nano-scale objects can be efficiently and controllably enriched in a high-flux manner, and the requirements of diversified biological research and cell assembly in tissue engineering are met.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical engineering, and in particular relates to a bulk ultrasound cell assembly device based on a porous chip, a cell assembly method and an application thereof. Background Art

[0002] In the field of tissue engineering, the orderly arrangement and three-dimensional assembly of cells are the key to achieving tissue repair and functional reconstruction. Cell-to-cell communication, migration, and the realization of specific functions usually rely on precise spatial arrangement and geometric characteristics of the extracellular matrix. Bulk ultrasound, a type of ultrasound propagating in elastic solids, has been widely used in the field of biomanufacturing with the development of microelectromechanical systems and microfluidics. By generating ultrasound waves of a specific frequency through piezoelectric crystals and coupling them into microfluidics, cells move and aggregate under the action of acoustic radiation force to achieve orderly assembly. Bulk ultrasound has the characteristics of high precision, non-contact, and non-destructiveness, and has broad application prospects in the fields of tissue engineering and regenerative medicine.

[0003] Controllable enrichment of cells or micro / nanoscale objects plays an important role in many biomedical and biochemical applications, such as improving the structure of bioengineered tissues or increasing the sensitivity of detection methods. However, the technologies that can achieve this process efficiently, with high throughput and good biocompatibility are limited and need further improvement. Summary of the invention

[0004] The present invention provides a bulk ultrasound cell assembly device based on a porous chip, a method for cell assembly and an application thereof, which can improve the structure of bioengineering tissues and enhance the sensitivity of detection methods.

[0005] To solve the above problems, the technical solution provided by the present invention is as follows:

[0006] The embodiment of the present invention provides a bulk ultrasound cell assembly device based on a porous chip, comprising an arbitrary waveform function generator (1), an ultrasonic transducer (2), a porous chip (3) and a cell assembly chamber (4); two electrodes of the arbitrary waveform function generator (1) are electrically connected to two electrodes of the ultrasonic transducer (2) through wires, respectively; the cell assembly chamber (4) is arranged on the top of the ultrasonic transducer (2), and the cell assembly chamber (4) is used to accommodate a cell solution; the porous chip (3) is arranged inside the cell assembly chamber (4), and a plurality of micropores (3-1) are distributed on the porous chip (3) in the form of a ring array, and the micropores (3-1) are used to modulate the sound field, and the shape of the micropores (3-1) matches the target assembly pattern of the cell;

[0007] The arbitrary waveform function generator (1) is used to provide an excitation signal to the ultrasonic transducer (2), and is capable of adjusting the frequency, amplitude and phase of the signal within a preset range; the ultrasonic transducer (2) converts a high-frequency electrical signal into a corresponding high-frequency acoustic signal through the piezoelectric effect; the micropores (3-1) in the porous chip (3) form a standing wave field by modulating the acoustic field to achieve controllable enrichment of cells or micro / nano-scale objects;

[0008] When cell assembly is performed, the radio frequency signal emitted by the arbitrary waveform function generator (1) is transmitted to the ultrasonic transducer (2), and the ultrasonic transducer (2) is used to generate a normal sound field. The normal sound field is modulated by the micropores (3-1) to form a standing wave field inside the micropores (3-1). At this time, the cells in the cell solution are affected by the acoustic radiation force in the standing wave field and are gathered into the acoustic potential well of the porous chip (3), thereby achieving orderly assembly and arrangement of the cells.

[0009] In an optional embodiment of the present invention, the material of the porous chip (3) is polymethyl methacrylate, quartz glass or metal.

[0010] In an optional embodiment of the present invention, the porous chip (3) is processed by any one of a laser engraving method, a CNC machine tool processing method or a 3D printing method.

[0011] In an optional embodiment of the present invention, the ultrasonic transducer (2) comprises a first polymethyl methacrylate layer, a second polymethyl methacrylate layer, a third polymethyl methacrylate layer and a piezoelectric ceramic sheet, wherein the third polymethyl methacrylate layer is a base, the first polymethyl methacrylate layer is located on the third polymethyl methacrylate layer, the piezoelectric ceramic sheet is embedded in the middle of the first polymethyl methacrylate layer, the second polymethyl methacrylate layer covers the piezoelectric ceramic sheet, and a through hole corresponding to the piezoelectric ceramic sheet is provided in the middle of the second polymethyl methacrylate layer, wherein the resonant frequency of the piezoelectric ceramic sheet is 20k-20MHz.

[0012] The embodiment of the present invention provides a method for cell assembly using a bulk ultrasound cell assembly device based on a porous chip, comprising the following steps:

[0013] Step 1, placing the porous chip (3) inside the cell assembly chamber (4);

[0014] Step 2, placing the cell assembly chamber (4) on top of the ultrasonic transducer (2);

[0015] Step 3, injecting the cell solution into the cell assembly chamber (4);

[0016] Step 4, adjusting the frequency and amplitude according to the resonant frequency and operating voltage of the piezoelectric ceramic piece of the ultrasonic transducer (2), maintaining the signal for a preset time, and the cells are gathered at the nodes of the standing wave acoustic field in the porous chip (3) to complete the target assembly of the cells.

[0017] In an optional embodiment of the present invention, step 3 includes: using an ultrasonic coupling agent to bond the cell assembly chamber (4) to the top of the ultrasonic transducer (2), ensuring that there are no bubbles in the ultrasonic coupling agent.

[0018] In an optional embodiment of the present invention, the target assembly of cells in step 4 includes at least one of microparticles, cells, hydrogel-encapsulated cells, cell spheres or microtissues.

[0019] An embodiment of the present invention also provides an application of a bulk ultrasound cell assembly device based on a porous chip, which is used for the orderly assembly and arrangement of cells in micropores, wherein the orderly assembly and arrangement of cells includes at least one of microparticles, cells, hydrogel-encapsulated cells, cell spheres or microtissues.

[0020] Compared with the prior art, the embodiments of the present invention provide a bulk ultrasound cell assembly device based on a porous chip, a method for cell assembly and application, which have the following beneficial effects:

[0021] The present invention precisely controls the acoustic field generated by the ultrasonic transducer by designing and processing a porous chip with a specific structure, thereby achieving orderly assembly and arrangement of cells in the micropores, thereby improving the sensitivity of the detection method. The bulk ultrasonic cell assembly device can efficiently and high-throughput controllably enrich cells or micro / nano objects, meeting the needs of cell assembly in diverse biological research and tissue engineering, showing broad application prospects and significant commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 A schematic diagram of a bulk ultrasound cell assembly device based on a porous chip provided in an embodiment of the present application.

[0024] Figure 2 A schematic diagram of the processing of a porous chip in a porous chip-based bulk ultrasound cell assembly device provided in an embodiment of the present application.

[0025] Figure 3This is a diagram showing the result of assembling polystyrene microparticles using a bulk ultrasound cell assembly device based on a porous chip in Example 1 of the present invention - the pattern of microparticles before assembly.

[0026] Figure 4 This is a diagram showing the result of assembling polystyrene microparticles using a porous chip-based bulk ultrasound cell assembly device in Example 1 of the present invention - the pattern of microparticles after assembly. DETAILED DESCRIPTION

[0027] The technical solutions in the embodiments of the present application will be described clearly and completely below in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present application.

[0028] Based on the information contained in this application, it is easy for those skilled in the art to make various changes to the precise description of the present invention without departing from the spirit and scope of the appended claims. It should be understood that the scope of the present invention is not limited to the defined process, property or component, because these embodiments and other descriptions are only for illustrating specific aspects of the present invention. In fact, various changes that the technicians in this field or related fields can obviously make to the embodiments of the present invention are all covered within the scope of the appended claims.

[0029] like Figure 1 and Figure 2 As shown, an embodiment of the present invention provides a bulk ultrasound cell assembly device based on a porous chip, which belongs to the field of tissue engineering and biomanufacturing technology under biomedical engineering. The bulk ultrasound cell assembly device based on a porous chip includes an arbitrary waveform function generator 1, an ultrasonic transducer 2, a porous chip 3 and a cell assembly chamber 4. The two electrodes of the arbitrary waveform function generator 1 are electrically connected to the two electrodes of the ultrasonic transducer 2 through wires, respectively. The cell assembly chamber 4 is arranged on the top of the ultrasonic transducer 2, and the cell assembly chamber 4 is used to accommodate the cell solution; the porous chip 3 is arranged inside the cell assembly chamber 4, and a plurality of micropores 3-1 are distributed on the porous chip 3 in the form of a ring array, and the micropores 3-1 are used to modulate the sound field effect, and the shape of the micropores 3-1 matches the target assembly pattern of the cell.

[0030] The arbitrary waveform function generator 1 is used to provide an excitation signal to the ultrasonic transducer 2, and can adjust the frequency, amplitude and phase of the signal within a preset range; the ultrasonic transducer 2 converts the high-frequency electrical signal into a corresponding high-frequency acoustic signal through the piezoelectric effect, which is used to generate an acoustic field of a specific frequency; the micropores 3-1 in the porous chip 3 achieve controllable enrichment of cells or micro / nano-scale objects through a modulated standing wave field.

[0031] When cell assembly is performed, the radio frequency signal emitted by the arbitrary waveform function generator 1 is transmitted to the ultrasonic transducer 2. The ultrasonic transducer 2 is used to generate a normal sound field. The normal sound field is modulated by the micropore 3-1 to form a standing wave field inside it. At this time, the cells in the cell solution are affected by the acoustic radiation force in the standing wave field and are gathered into the acoustic potential well of the porous chip 3 (in the micropore 3-1), thereby realizing the orderly assembly and arrangement of the cells.

[0032] The porous chip 3 is made of polymethyl methacrylate, quartz glass or metal. The porous chip 3 is processed by any one of laser engraving, CNC machine tool processing or 3D printing.

[0033] The ultrasonic transducer 2 includes a first polymethyl methacrylate layer, a second polymethyl methacrylate layer, a third polymethyl methacrylate layer and a piezoelectric ceramic sheet, wherein the third polymethyl methacrylate layer is a base, the first polymethyl methacrylate layer is located on the third polymethyl methacrylate layer, the piezoelectric ceramic sheet is embedded in the middle of the first polymethyl methacrylate layer, the second polymethyl methacrylate layer covers the piezoelectric ceramic sheet, and a through hole corresponding to the piezoelectric ceramic sheet is provided in the middle of the second polymethyl methacrylate layer. The resonant frequency of the piezoelectric ceramic sheet is 20k-20MHz.

[0034] The ultrasonic transducer 2 in this embodiment is formed by encapsulating a piezoelectric ceramic sheet with three layers of polymethyl methacrylate. Preferably, the piezoelectric ceramic sheet is encapsulated with three layers of polymethyl methacrylate of a specific height processed by a laser engraving machine, and the air backing layer of the base concentrates the sound energy on both sides of the piezoelectric ceramic sheet to one side, thereby improving the sound directivity and sound energy.

[0035] like Figure 2 As shown, the preparation method of the porous chip 3 includes the following steps: designing the target micropore array pattern 5 by AutoCAD software; importing the designed drawing into the system of the CO2 laser engraving machine; cutting the polymethyl methacrylate sheet to manufacture a functional porous chip 3, on which a plurality of micropores 3-1 are distributed in the form of a ring array. The porous chip 3 is placed inside the cell assembly chamber 4.

[0036] The embodiment of the present invention also provides a method for cell assembly using a bulk ultrasound cell assembly device based on a porous chip, comprising the following steps:

[0037] Step 1, placing the porous chip 3 inside the cell assembly chamber 4;

[0038] Step 2, placing the cell assembly chamber 4 on top of the ultrasonic transducer 2;

[0039] Step 3, injecting the cell solution into the cell assembly chamber 4;

[0040] Step 4, adjust the frequency and amplitude according to the resonant frequency and working voltage of the piezoelectric ceramic piece of the ultrasonic transducer 2, keep the signal for a preset time, and the cells are gathered at the node of the standing wave field in the porous chip 3 (in the micropore 3-1) to complete the target assembly of the cells.

[0041] Step 3 includes: using an ultrasonic coupling agent to bond the cell assembly chamber 4 to the top of the ultrasonic transducer 2, and ensuring that there are no bubbles in the ultrasonic coupling agent. Step 3 also includes: resuspending the cells using a buffer to obtain a cell solution. The target assembly of the cells in step 4 includes at least one of microparticles, cells, hydrogel-encapsulated cells, cell spheres, or microtissues.

[0042] An embodiment of the present invention also provides an application of a bulk ultrasound cell assembly device based on a porous chip, which is used for the orderly assembly and arrangement of cells in micropores, wherein the orderly assembly and arrangement of cells includes at least one of microparticles, cells, hydrogel-encapsulated cells, cell spheres or microtissues.

[0043] Example 1, taking polystyrene microparticle assembly as an example, further illustrates a bulk ultrasound cell assembly device based on a porous chip, a method for cell assembly and its application, as follows:

[0044] like Figure 1 As shown, the device for assembling and arranging polystyrene particles by processing an acoustic matching layer using soft lithography technology according to an embodiment of the present invention comprises an arbitrary waveform function generator 1, an ultrasonic transducer 2, a porous chip 3 and a cell assembly chamber 4. In this embodiment, the cell assembly chamber 4 is composed of a cell culture dish, the porous chip 3 is located inside the cell assembly chamber 4, and the ultrasonic transducer 2 is located at the bottom of the cell assembly chamber 4. The model of the arbitrary waveform function generator 1 is AFG3000, which has a dual-channel design, a maximum sampling rate of 1GS / s, an output frequency of up to 100MHz, and a maximum amplitude output of 10Vpp. In this embodiment, the arbitrary waveform function generator 1 outputs a 1.9MHz sine wave signal with an amplitude of 10Vpp and a phase of 0. The ultrasonic transducer 2 is formed by encapsulating a piezoelectric ceramic sheet with three layers of polymethyl methacrylate of a specific height and shape. The piezoelectric ceramic sheet is a Fujicera circular plate piezoelectric ceramic sheet with a diameter of 2.5cm, a thickness of 1mm, and a resonant frequency of 1.9MHz.

[0045] Figure 2 The schematic diagram of the manufacturing of the porous chip 3 is shown, and the manufacturing method thereof is as follows:

[0046] Step 1: Design the size of the porous chip according to the target assembly pattern, with a micropore diameter of 0.8 mm, a micropore height of 1.2 mm, and a micropore spacing of 1.6 mm.

[0047] Step 2: Import the porous chip drawing (target micropore array pattern 5) designed by AutoCAD into the system of the CO2 laser engraving machine, and select polymethyl methacrylate sheet for cutting.

[0048] Step 3: Set the parameters of the CO2 laser engraving machine, including power, speed, focal length, etc. These parameters will affect the depth and fineness of the engraving.

[0049] Step 4: Place the work material on the working area of ​​the laser engraving machine and perform precise positioning to ensure the accuracy of laser engraving. Step 5: After cutting and surface cleaning, place the prepared porous chip 3 inside the cell assembly chamber 4.

[0050] The particles in the particle solution are made of polystyrene, white in color, 10 μm in diameter, and 1.05 g / cm in density. 3 , whose physical properties are similar to those of cells, and the concentration of the microparticle solution is 1 mg / ml.

[0051] The steps of the method for forming a standing wave acoustic field and assembling and arranging particles using the present invention are as follows:

[0052] Step 1: According to the required cell assembly style, the corresponding porous chip 3 is processed by laser engraving technology, and the porous chip 3 is placed inside the cell assembly chamber 4.

[0053] Step 2: Apply a thin layer of ultrasonic coupling agent (i.e., the ultrasonic coupling agent commonly used in hospitals for B-ultrasound) on the ultrasonic transducer 2, and bond the cell assembly chamber 4 and the ultrasonic transducer 2 together to ensure that no bubbles are generated in the middle of the ultrasonic coupling agent.

[0054] Step 3: resuspend the microparticles to be assembled using phosphate buffer, and inject the sample into the cell assembly chamber 4 using a pipette.

[0055] Step 4: According to the resonant frequency and working voltage of the piezoelectric ceramic, the corresponding frequency is adjusted to 1.9 MHz and the amplitude is 10 Vpp. The two electrodes of the piezoelectric ceramic correspond to the two electrodes of the arbitrary waveform function generator respectively. The signal duration is maintained for 3 minutes. The particles are gathered at the nodes of the standing wave acoustic field to form a corresponding pattern.

[0056] Figure 3 and Figure 4 The effect diagram of the actual assembly of particles in the porous chip 3 is shown. Figure 3 is the particle pattern before assembly, Figure 4 This is the particle pattern after assembly. The darker white areas are the nodes with lower potential energy, where the particles will reach a stable state.

[0057] The present invention utilizes laser engraving technology to process the porous chip 3 for cell assembly, and can realize the orderly assembly and arrangement of cells in the micropores. The bulk ultrasound cell assembly device can efficiently and high-throughput controllably enrich cells or micro / nano objects, has good stability, and does not require direct contact or damage, providing a new solution to the demand for cell assembly in tissue engineering and other biological research.

[0058] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A bulk ultrasound cell assembly device based on a porous chip, characterized in that: The invention comprises an arbitrary waveform function generator (1), an ultrasonic transducer (2), a porous chip (3) and a cell assembly chamber (4); the two electrodes of the arbitrary waveform function generator (1) are electrically connected to the two electrodes of the ultrasonic transducer (2) through wires, respectively; the cell assembly chamber (4) is arranged on the top of the ultrasonic transducer (2), and the cell assembly chamber (4) is used to contain a cell solution; the porous chip (3) is arranged inside the cell assembly chamber (4), and a plurality of micropores (3-1) are distributed on the porous chip (3) in the form of a ring array, and the micropores (3-1) are used to modulate the sound field, and the shape of the micropores (3-1) matches the target assembly pattern of the cell; The arbitrary waveform function generator (1) is used to provide an excitation signal to the ultrasonic transducer (2), and is capable of adjusting the frequency, amplitude and phase of the signal within a preset range; the ultrasonic transducer (2) converts a high-frequency electrical signal into a corresponding high-frequency acoustic signal through the piezoelectric effect; the micropores (3-1) in the porous chip (3) form a standing wave field by modulating the acoustic field to achieve controllable enrichment of cells or micro / nano-scale objects; When cell assembly is performed, the radio frequency signal emitted by the arbitrary waveform function generator (1) is transmitted to the ultrasonic transducer (2), and the ultrasonic transducer (2) is used to generate a normal sound field. The normal sound field is modulated by the micropores (3-1) to form a standing wave field inside the micropores. At this time, the cells in the cell solution are affected by the acoustic radiation force in the standing wave field and are gathered into the acoustic potential well of the porous chip (3), thereby achieving orderly assembly and arrangement of the cells.

2. The body ultrasound cell assembly device based on a porous chip according to claim 1, characterized in that: The material of the porous chip (3) is polymethyl methacrylate, quartz glass or metal.

3. The body ultrasound cell assembly device based on a porous chip according to claim 1, characterized in that: The porous chip (3) is processed by any one of a laser engraving method, a CNC machine tool method or a 3D printing method.

4. The body ultrasound cell assembly device based on a porous chip according to claim 1, characterized in that: The ultrasonic transducer (2) comprises a first polymethyl methacrylate layer, a second polymethyl methacrylate layer, a third polymethyl methacrylate layer and a piezoelectric ceramic sheet, wherein the third polymethyl methacrylate layer is a base, the first polymethyl methacrylate layer is located on the third polymethyl methacrylate layer, the piezoelectric ceramic sheet is embedded in the middle of the first polymethyl methacrylate layer, the second polymethyl methacrylate layer covers the piezoelectric ceramic sheet, and a through hole corresponding to the piezoelectric ceramic sheet is arranged in the middle of the second polymethyl methacrylate layer, wherein the resonant frequency of the piezoelectric ceramic sheet is 20k-20MHz.

5. A method for cell assembly using a bulk ultrasound cell assembly device based on a porous chip, characterized in that: The following steps are involved: Step 1, placing the porous chip (3) inside the cell assembly chamber (4); Step 2, placing the cell assembly chamber (4) on top of the ultrasonic transducer (2); Step 3, injecting the cell solution into the cell assembly chamber (4); Step 4, adjusting the frequency and amplitude according to the resonant frequency and operating voltage of the piezoelectric ceramic piece of the ultrasonic transducer (2), maintaining the signal for a preset time, and the cells are gathered at the nodes of the standing wave acoustic field in the porous chip (3) to complete the target assembly of the cells.

6. The method for cell assembly using a bulk ultrasound cell assembly device based on a porous chip according to claim 5, characterized in that: Step 3 comprises: using an ultrasonic coupling agent to bond the cell assembly chamber (4) to the top of the ultrasonic transducer (2), and ensuring that there are no bubbles in the ultrasonic coupling agent.

7. The method for cell assembly using a bulk ultrasound cell assembly device based on a porous chip according to claim 5, characterized in that: The target assembly of cells in step 4 includes at least one of microparticles, cells, hydrogel-encapsulated cells, cell spheroids, or microtissues.

8. An application of a bulk ultrasound cell assembly device based on a porous chip, characterized in that: It is applied to orderly assembly and arrangement of cells in micropores, and the orderly assembly and arrangement of cells includes at least one of microparticles, cells, hydrogel-wrapped cells, cell spheres or microtissues.

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