A cell processing device based on surface acoustic wave

Abstract

This invention provides a cell treatment device based on surface acoustic waves (SAWs), comprising a SAW excitation device and a cell treatment pool. The SAW excitation device transmits SAWs into the cell treatment pool for cell culture, proliferation, and lysis. Simultaneously, nanowires are disposed on the pool wall. Under the action of SAWs, cell lysis is achieved through the nanowires. The cell lysis effect is further enhanced by optimizing the material and placement of the nanowires. This device, through the combination of SAWs and nanowires, significantly improves cell lysis without adversely affecting cell culture and proliferation. Furthermore, by controlling the output power of the SAWs, they can be used for both efficient cell culture and proliferation, and efficient cell lysis, demonstrating broad application prospects.

Description

[0001] This application claims priority to the earlier Chinese application, application number 2025101392075, filed on February 8, 2025; all its contents are part of this invention. Technical Field

[0002] This invention relates to the field of cell proliferation and lysis technology, and more specifically, to a cell processing device based on surface acoustic waves. Background Technology

[0003] Cell processing includes processes such as cell culture, proliferation, and lysis. After lysis, DNA, RNA, proteins, etc., can be extracted and collected from the cells using traditional biological methods for further detection.

[0004] Cell proliferation is a crucial cornerstone of modern bioengineering and medical research. Existing cell proliferation methods primarily utilize various stimuli to promote cell growth, including growth factor therapy, electrostimulation therapy, and ultrasound therapy. Growth factor therapy typically involves specially formulated culture media or specific organic compounds extracted from living organisms, resulting in high costs, short drug half-lives, and unsustainable effects. While electrostimulation promotes cell proliferation at a lower cost (see existing technology CN105505773A (a magnetic-electric stimulation cell culture device) for direct magnetic-electric stimulation of cells), the proliferation effect is difficult to control, and direct application can easily damage cells. Compared to the former, ultrasound therapy is safer, more durable, and more economical. However, traditional medical ultrasound therapy often uses body waves to generate ultrasound, making it difficult to concentrate energy on the area requiring healing. To address this energy loss, which is difficult to standardize and quantify, external equipment may become complex, and treatment time needs strict control to prevent excessive temperature in the treatment area and tissue thermal damage while ensuring treatment effectiveness.

[0005] Unlike ultrasound, surface acoustic waves propagate in a two-dimensional plane, with more concentrated and easily controlled energy. They also have multiple applications such as driving, sensing, and sorting. For example, CN202410381191.4 (an application of a surface acoustic wave sensor in DMMP gas detection) describes a method where a sensing layer is directly coated on two interdigital transducers, and sensing of the sensing layer can be achieved by detecting the frequency response.

[0006] Most biomarkers, such as DNA, RNA, and proteins, reside inside cells. Detection requires cell lysis to analyze these intracellular biomarkers and further identify various diseases. However, cell samples are scarce and require culture and proliferation before lysis and detection. Therefore, cell processing devices need to simultaneously perform culture, proliferation, and lysis. CN108414303B discloses a particle-collision cell lysator based on surface acoustic waves (SAWs). This lysator utilizes the acoustic microfluidic effect of SAWs to drive droplets and the cells and particles within them at high speeds, achieving cell lysis. However, its cell lysis effect is not ideal, often resulting in incomplete lysis. Furthermore, the colliding particles are completely dispersed in the cell solution, rendering the solution unusable for further cell culture and proliferation, limiting its application to cell lysis. For cell solutions with low cell content, the processing effect is difficult to guarantee.

[0007] Therefore, there is an urgent need to find a cell processing device that can both culture and proliferate cells and lyse cells, so as to achieve more efficient cell culture, proliferation and lysis. Summary of the Invention

[0008] To address the aforementioned problems, this invention provides a cell treatment device based on surface acoustic waves (SAWs), comprising a SAW excitation device and a cell treatment pool. The SAW excitation device transmits SAWs into the cell treatment pool for cell culture, proliferation, and lysis. Simultaneously, nanowires are disposed on the pool wall. Under the influence of SAWs, cell lysis is achieved through the nanowires. The cell lysis effect is further enhanced by optimizing the material and placement of the nanowires. This device, through the combination of SAWs and nanowires, significantly improves cell lysis without adversely affecting cell culture and proliferation. Furthermore, by controlling the output power of the SAWs, they can be used for both efficient cell culture and proliferation, as well as efficient cell lysis, demonstrating broad application prospects.

[0009] On one hand, the present invention provides a cell processing device, the device comprising a surface acoustic wave excitation device and a cell processing pool, the surface acoustic wave excitation device being used to transmit surface acoustic waves to the cell processing pool; the cell processing pool being used for cell culture, proliferation and lysis.

[0010] The surface acoustic wave (SAW) described in this invention is an elastic wave that is generated and propagates on the surface of a piezoelectric substrate material, and whose amplitude decreases rapidly with increasing depth into the substrate material. When a voltage is applied to the electrodes of a piezoelectric crystal (lithium niobate, piezoelectric ceramic), mechanical distortion is formed in the crystal lattice of the piezoelectric crystal due to the piezoelectric effect, thereby generating a SAW.

[0011] In existing technologies, ultrasound is commonly used for cell culture and proliferation. Ultrasound is a high-frequency mechanical wave that propagates in three-dimensional space, while surface acoustic waves (SAWs) are mechanical waves that propagate in two-dimensional space. Therefore, for cells growing on a two-dimensional plane, such as adherent cells and human dermal fibroblasts, SAWs provide higher energy utilization, are more concentrated, efficient, and controllable when stimulating their proliferation. Thus, SAWs are more preferred for the proliferation culture of cells growing on a two-dimensional plane, such as human retinal epithelial cells, human lung cancer cells, and rat myoblasts.

[0012] This invention creatively employs surface acoustic waves (SAWs) to proliferate and culture cells, and designs a corresponding SAW cell processing device. This device can achieve cell culture, proliferation, and lysis through the driving of SAWs. Moreover, when switching between the two functions of cell culture, proliferation, and cell lysis, no structural changes or additional components need to be made. Only the output power of the SAW excitation device needs to be adjusted to achieve the effect of both efficient cell culture and proliferation and efficient cell lysis.

[0013] Furthermore, the walls of the cell treatment pool contain nanowires.

[0014] Surface acoustic waves used for cell proliferation and culture refer to surface acoustic waves of a specific wavelength that propagate unidirectionally on a plane using interdigital transducers with a specific structure. Corresponding cell culture devices have been designed based on this. Initially, the device only has a single function, such as being used only for cell culture and proliferation. If it is to be used for cell lysis, it is necessary to change some of the device structure or the composition of the formula in order to switch between the two functions of cell culture and proliferation and cell lysis. For example, after being used for cell culture and proliferation, if it is to be used for cell lysis, it is necessary to add components such as collision particles to the cell culture medium. In other words, the composition of the cell culture medium is increased in order to achieve cell lysis.

[0015] In order to enable the surface acoustic wave cell treatment device to be used for both efficient cell culture and proliferation and efficient cell lysis, the present invention further improves the cell treatment device by setting nanowires on the walls of the cell treatment pool.

[0016] Nanowires can be defined as a one-dimensional structure with a transverse dimension limited to less than 100 nanometers, including metal nanowires, semiconductor nanowires, and insulator nanowires.

[0017] This invention achieves cell lysis by adding zinc oxide nanowires to the inner wall of a cell treatment tank. After the cells are driven to detach from their growth surface by surface acoustic waves, they continue to rotate in the culture medium under the influence of acoustic flow, continuously colliding with the nanowires on the tank wall. This generates a huge interaction force and momentum transfer between the two, thereby destroying the cell membrane function and structure and achieving the purpose of lysis.

[0018] Studies have shown that by fixing nanowires to the inner wall of a cell treatment pool, when used for cell culture and proliferation, the nanowires do not adversely affect cell culture and proliferation; when used for cell lysis, the nanowires, due to their finer diameter, sharper edges, and higher mechanical strength, can lyse cells more efficiently.

[0019] Furthermore, the cell treatment pool is any one or more of the following shapes: cylindrical, spherical, square, rectangular, and frustum-shaped, and the nanowires are located on all or part of the inner wall surface of the cell treatment pool.

[0020] Studies have shown that nanowires need to be located on the vertical inner wall surface of the cell treatment tank, while nanowires do not need to be placed on the bottom surface. This is because during cell lysis, cells rotate under the influence of surface acoustic waves, and the rotation generates centrifugal force, causing cells to adhere as closely as possible to the vertical inner wall. Nanowires on the bottom surface are unlikely to play a role and cannot improve the cell lysis effect. On the contrary, placing nanowires on the bottom surface may also affect the cell culture process, since most cells grow adherently to the bottom of the tank. If there are a large number of nanowires on the bottom surface of the tank, it may be detrimental to cell adhesion and growth. Therefore, nanowires do not need to be placed on the bottom surface of the tank, and nanowires are preferably placed on the vertical inner wall surface of the cell treatment tank.

[0021] In some embodiments, when the cell treatment pool is square, the inner wall includes a front wall, a rear wall, a left wall, and a right wall, and the nanowires are located on one or more portions or all of the surfaces of the front wall, the rear wall, the left wall, and the right wall.

[0022] In some embodiments, the nanowires are uniformly arranged on the surfaces of the front, rear, left, and right walls.

[0023] Furthermore, the nanowires are prepared using any one or more of zinc oxide, tin dioxide, silicon carbide, silver, copper, gold, iron, aluminum, silicon, germanium, and gallium arsenide.

[0024] Understandably, nanowires made from any material, as long as they have sufficient strength and fineness, can be used to achieve cell lysis.

[0025] In some embodiments, the nanowires are zinc oxide nanowires, which help improve cell lysis without affecting the cell culture process.

[0026] In some methods, the zinc oxide nanowires are prepared by chemical bath deposition on a flexible stainless steel substrate, and then the prepared flexible stainless steel substrate is laid on the vertical inner wall surface of the cell treatment pool.

[0027] In some methods, the specific method for chemical bath deposition of the zinc oxide nanowires involves: preparing a zinc oxide seed layer required for this growth method using atomic layer deposition (ALD), and growing it for 15 hours in a precursor solution (30 mM) composed of zinc nitrate hexahydrate and hexamethylenetetramine (HMTA) in a 1:1 hydration ratio, at a water bath temperature of 85°C. This invention, by adjusting the precursor solution concentration, growth time, and water bath temperature, can prepare zinc oxide nanowires with diameters of 50-200 nm and lengths of 5-15 μm, thereby optimizing cell lysis efficiency.

[0028] In some methods, the nanowires are recycled after growth. Specifically, after growth, the substrate is treated with ultrasonic oscillation, which suspends the nanowires in the solution for later use. Then, the solution is dropped onto a rectangular PDMS film with an area consistent with the inner wall area of ​​the cell treatment pool, and the film is then attached to the inner wall surface of the cell treatment pool.

[0029] Furthermore, the nanowires have a diameter of 50–200 nm and a length of 5–15 μm; the distribution density of the nanowires on the pool wall is 500–5000 wires / m. 2 .

[0030] Studies have shown that when the diameter, length, and distribution density of nanowires are kept within a suitable range, it helps to improve cell lysis efficiency without adversely affecting the cell culture process.

[0031] Furthermore, the cells include any one or more of prokaryotic cells, animal cells, plant cells, and fungal cells.

[0032] It is understood that the cell processing device provided by the present invention can be used to process any type of cell.

[0033] In some methods, animal cells are preferred for treatment because they do not have cell walls and respond better to surface acoustic wave stimuli.

[0034] In some embodiments, the cell processing apparatus provided by the present invention preferably culturees cells suitable for growth in a two-dimensional plane, such as human retinal epithelial cells, human lung cancer cells, rat myoblasts, etc.

[0035] In some methods, the present invention uses human dermal fibroblasts for cell treatment. It has been found that when human dermal fibroblasts are used, the culture process is not adversely affected, the lysis effect is more complete, and it is more advantageous than other cells.

[0036] Furthermore, the surface acoustic wave excitation device includes a piezoelectric substrate; the cell treatment pool is formed by combining a pool wall and a portion of the piezoelectric substrate, and the portion of the piezoelectric substrate of the surface acoustic wave excitation device constitutes the bottom of the cell treatment pool.

[0037] The cell treatment pool consists of a pool wall and a pool bottom. The pool bottom refers to the piezoelectric substrate surface, the interdigital transducer at the acoustic stimulation end, and the acoustic stimulation end reflector grid. The pool wall is bonded to the piezoelectric substrate surface through different methods such as bonding and adhesion, forming a pool bottom locally on the piezoelectric substrate surface for cell culture.

[0038] Two sets of interlaced, periodically distributed metal strips are deposited on the surface of a piezoelectric substrate. Each set of electrodes is connected to a busbar. When an alternating voltage is applied to the busbar of the input interdigital transducer, a periodically distributed electric field is generated on the surface of the piezoelectric substrate. Due to the inverse piezoelectric effect of the piezoelectric material, this periodic electric field will produce corresponding elastic deformation near the surface of the piezoelectric substrate, i.e., surface acoustic waves (SAWs). Simultaneously, due to the strip shape, the SAWs propagate from both sides of the interdigital transducer. When the interdigital width and interdigital spacing are the same, and the interdigital period is much smaller than the thickness of the piezoelectric substrate, a common type of SAW—Rayleigh wave—can be generated. The wavelength of this wave is equal to four times the interdigital width, which is the interdigital period. In this case, the acoustic frequency f = v / λ determined by the interdigital transducer can be calculated from the acoustic wavelength and the acoustic propagation speed at the substrate. Using Y-128 tangential lithium niobate as the piezoelectric substrate, surface acoustic waves (SAWs) with frequencies in the range of 1–100 MHz performed well in fluid actuation, while SAWs with frequencies in the range of 100–1000 MHz performed well in sensing. Therefore, a 16 MHz driven SAW and a 160 MHz sensing SAW were designed. Since the sound velocity of lithium niobate is around 3600 m / s, the interdigital transducer for the driven SAW has an interdigital transducer width of 56.25 micrometers, and the interdigital transducer for the sensing SAW has an interdigital transducer width of 5.625 micrometers.

[0039] Meanwhile, since surface acoustic waves (SAWs) propagate along both sides of the interdigital transducer, a reflective grating is needed for driven SAWs to concentrate energy on one side for cell processing. The reflective grating is a periodically distributed metal strip on one side of the interdigital transducer. Its main function is to reflect the SAWs generated by the interdigital transducer back to the transducer, forming a standing wave, thereby enhancing the propagation efficiency of the sound wave and strengthening the SAW propagating to the other side of the transducer. Therefore, structurally, its metal strip is not connected to any electrodes. Structurally, since it is responsible for reflecting the SAWs generated by the interdigital transducer and forming a standing wave, its interdigital width and interdigital spacing are the same as those of the transducer. Furthermore, the distance between the interdigital transducer and the reflective grating is 10 to 30 times the interdigital period. If this distance is too low, the SAWs may not develop sufficiently before reaching the reflective grating, affecting the wave reflection efficiency and the overall performance of the device. If this distance is too high, the SAWs will experience more attenuation during propagation, reducing the intensity of the reflected wave.

[0040] Driven surface acoustic waves propagate into the cell culture chamber, gently agitating the culture medium and cells at low power to promote cell proliferation; at high power, the cells are driven to detach from the cell wall and rotate in the chamber with the culture medium, and are subsequently lysed under the influence of nanowires.

[0041] Furthermore, it also includes a cell number sensing device, which comprises a surface acoustic wave sensing device and a cell sensing area, for monitoring cell proliferation in the cell treatment pool.

[0042] Furthermore, the walls of the cell treatment pool are made of polydimethylsiloxane and a coagulant, wherein the ratio of polydimethylsiloxane to coagulant is greater than 10:0.9.

[0043] This invention uses surface acoustic wave (SAW) sensors on both sides of a cell treatment pool. When the cell density in the cell treatment pool changes, the SAW sensors can immediately detect the change in the frequency of high-frequency SAW waves, thereby providing feedback on the change in the number of cells and monitoring the cell proliferation in the cell treatment pool.

[0044] In some embodiments, the cell processing apparatus provided by the present invention includes a surface acoustic wave excitation device, a cell processing pool, a cell quantity sensing device, and a power supply device.

[0045] The surface acoustic wave excitation device includes a piezoelectric substrate, an interdigital transducer at the acoustic stimulation end, and an acoustic stimulation end reflective grating.

[0046] The piezoelectric substrate is made of lithium niobate with various tangential orientations. This material not only has stable physical and chemical properties, but its high electromechanical coupling coefficient means high electro-acoustic conversion efficiency, which can provide higher energy utilization for the device. In device fabrication, to generate surface acoustic waves (SAWs), the thickness of the piezoelectric substrate must be much greater than the wavelength of the SAWs. However, excessive thickness can affect the overall size of the device. For example, in this invention, the wavelengths of the SAWs are designed to be 225 micrometers and 22.5 micrometers. The main purpose of controlling the wavelength of the SAWs within this range is to balance the efficiency of promoting proliferation and the efficiency of fragmentation, as the power and energy required for each are different. This work requires the same device to perform two tasks. For the same reason, the thickness of the piezoelectric substrate is determined to be 400-500 micrometers. Of course, it is understandable that different SAWs can be used to design different piezoelectric substrate thicknesses. The wavelength control range can be adjusted by adjusting the substrate thickness in combination with the interdigital transducer's interdigital width. The 400-500 micrometer substrate thickness and 56.25 micrometer interdigital width used in this invention correspond to a wavelength adjustment range of 20-250 micrometers, which covers the wavelength adjustment range designed in this invention. Different substrate thicknesses combined with different interdigital widths can also adjust the wavelength, but this will present economic and compactness issues in terms of the overall size and thickness of the interdigital transducer. Therefore, the numerical combination provided by this invention is a relatively optimal solution.

[0047] The interdigital transducer at the acoustic stimulation end is a key structure of the surface acoustic wave (SAW) device, comprising a layer of metallized interdigital patterns and wiring distributed on a piezoelectric substrate. The metal is typically aluminum, gold, or other conductive materials. The interdigital pattern consists of multiple parallel metal fingers (electrodes) of a certain width, called the interdigital width. These metal fingers are spaced equidistantly from adjacent metal fingers, called the interdigital spacing. The metal fingers and spacing are arranged alternately, forming a comb-like structure. Adjacent metal fingers are connected to two electrodes via wiring, and an external AC voltage is applied, converting the electrical signal into a mechanical wave through the inverse piezoelectric effect. When the interdigital width and interdigital spacing are the same, and the interdigital period is much smaller than the thickness of the piezoelectric substrate, a Rayleigh wave, one of the most common types of SAW, can be generated. In this case, the acoustic frequency f = v / λ, determined by the interdigital transducer, can be calculated from the acoustic wavelength and the propagation speed of the acoustic wave on the substrate. Using Y-128 tangential lithium niobate as the piezoelectric substrate, surface acoustic waves (SAWs) with frequencies between 1 and 100 MHz showed good performance in fluid actuation. Therefore, a 16 MHz driven SAW was designed. Since the sound velocity on lithium niobate is around 3600 m / s, the interdigital transducer for generating the driven SAW had an interdigital width of 56.25 μm. The overlap length of the two interdigital pairs is called the acoustic aperture, with a value of 1 cm. SAWs are generated on the overlapping interdigital pairs and propagate along both ends of the interdigital transducer. The SAW at one end is reflected by a reflective grating, reinforcing the SAW at the other end. Simultaneously, the acoustic aperture determines the beamwidth of the excited SAW. Here, the length of the acoustic aperture is consistent with the width of the subsequent cell culture chamber, ensuring that the bottom of the cell processing chamber is stimulated by SAWs.

[0048] The acoustic stimulation reflection grating is a layer of metallized interdigital pattern distributed at one end of the interdigital transducer at the acoustic stimulation end. Its main function is to reflect the surface acoustic waves (SAWs) generated by the interdigital transducers back to the transducers to form standing waves, thereby enhancing the propagation efficiency of the sound waves and increasing the SAW propagation into the cell culture chamber. Simultaneously, the SAWs slightly disturb the culture medium and cells in the cell culture chamber, promoting cell proliferation. Therefore, its metal fingers are not connected to any electrodes. Furthermore, the width and spacing of the interdigital fingers are related to the wavelength of the SAWs. In SAW devices that generate Rayleigh waves, the width and spacing of the interdigital fingers are the same as those of the interdigital transducers. In addition, the distance between the interdigital transducers and the reflection grating is 20 times the interdigital period, i.e., 4500 micrometers. If this distance is too low, the SAWs may not develop sufficiently before propagating to the reflection grating, thus affecting the wave reflection efficiency and the overall performance of the device. If this distance is too high, the SAWs will experience more attenuation during propagation, reducing the intensity of the reflected wave.

[0049] The cell treatment tank includes a tank wall and a tank bottom. The tank wall is a square hollow cylinder, 1 cm long and wide and 5 mm high, formed by injection molding of polydimethylsiloxane (PDMS), a bio-harmless material. During injection molding, PDMS and a coagulant are mixed at a mass ratio of 10:0.9, making the molded PDMS tank wall softer than PDMS material molded at a conventional ratio of 10:1, which helps reduce the loss of surface acoustic waves when passing through the tank wall into the culture chamber. The tank bottom refers to the surface of a piezoelectric substrate. The PDMS tank wall is bonded to the piezoelectric substrate surface using various methods such as bonding and adhesion, forming a localized tank bottom on the piezoelectric substrate surface for cell culture.

[0050] The cell number sensing device includes a surface acoustic wave (SAW) sensor and a cell sensing area. By placing the SAW sensor near the cell sensing area, the SAW sensor can sense changes in cell density in the cell sensing area and emit high-frequency SAW waves. The frequency of the high-frequency SAW waves changes with the cell density, thereby providing feedback on changes in cell number and monitoring cell proliferation in the cell treatment pool.

[0051] The surface acoustic wave (SAW) sensing device includes a SAW excitation device at the sensing signal generation end and an acoustic-to-electric conversion device at the sensing signal receiving end.

[0052] The surface acoustic wave (SAW) excitation device for the sensing signal generation end includes a signal generator, a piezoelectric substrate, an interdigital transducer, and a sensing signal reflection grid. Its basic structure is the same as the SAW excitation device in the acoustic stimulation cell proliferation module, featuring a piezoelectric substrate, an interdigital transducer for the sensing signal generation end, and a reflection grid. However, its interdigital period is much smaller, at 5.625 micrometers, corresponding to a center frequency of 160 MHz for the SAW device, while the output power of the signal generator is controlled between 0.001 watts and 0.01 watts. Compared to the SAW device used for driving, this sensing SAW device has a higher center frequency, resulting in higher information sensitivity accuracy while reducing the driving function and minimizing the impact of SAW on the cells.

[0053] The acoustic-to-electric conversion device at the sensing signal receiving end includes a piezoelectric substrate, an interdigital transducer at the sensing signal receiving end, and a sensing signal receiving computer. This acoustic-to-electric conversion device has an interdigital transducer with the same structure as the surface acoustic wave (SAW) excitation device at the sensing signal generating end, but it does not have a reflective grating structure, nor does it apply an AC signal to it via electrodes. The environmentally sensitive high-frequency SAW generated in the SAW excitation device at the sensing signal generating end changes its frequency due to the characteristics of the medium as it propagates on the surface of the medium. When the SAW propagates to the interdigital transducer at the sensing signal receiving end, an AC signal with its frequency characteristics is generated due to the piezoelectric effect. By detecting the frequency change of the SAW on the sensing signal receiving computer, the changing characteristics of its propagation surface can be detected.

[0054] The cell sensing region is the two-dimensional planar space between the interdigital transducer of the surface acoustic wave (SAW) excitation device at the sensing signal generation end and the interdigital transducer of the acoustic-to-electric conversion device at the sensing signal receiving end, which refers to the bottom of the cell culture chamber within the device. When adherent cells grow at different densities on the bottom of the chamber, the high-frequency SAW waves emitted from the SAW excitation device at the sensing signal generation end will experience varying degrees of frequency change as they pass through this region. These changes then propagate to the interdigital transducer at the sensing signal receiving end, are converted into alternating current signals, and are recorded by the computer. By detecting these changes, the growth density information of the adherent cells at the bottom of the chamber can be obtained.

[0055] The power supply device includes an acoustic stimulation end signal generator and an acoustic drive signal connector.

[0056] The acoustic stimulation signal generator is used to generate a sinusoidal electrical signal with a specific frequency and adjustable power. The specific frequency refers to the center frequency of the surface acoustic wave (SAW) device. By inputting an AC signal with the same center frequency to the device, the SAW is excited. By adjusting the power of the electrical signal, the amplitude of the SAW is controlled, thereby regulating the degree of stimulation of cells affected by the SAW. The power ranges from 0.5 watts to 5 watts. When the power is too low, the effect on the cells is weak and cannot promote their proliferation. When the power is too high, the strong mechanical wave can cause cells to detach from their attached growth surface.

[0057] The acoustic drive signal connector is an SMA RF connector. It includes contact pairs, an insulator, and a threaded connection. Each contact pair consists of a center conductor (inner core) and an outer conductor (outer shell). The center conductor is typically in the form of a pin or hole, while the outer conductor is a mating hole or pin, along with the threaded connection. The contact pair's function is to establish a connection between two circuits. The insulator supports and secures the contact pairs, ensuring electrical insulation between the contact pairs and between the contact pairs and the outer shell. It is typically made of polytetrafluoroethylene (PTFE), a material with good dielectric properties and chemical stability. The threaded connection uses imperial threads, providing high strength and good shock resistance. The electrical signal generated by the signal generator is input to the surface acoustic wave excitation device via the acoustic drive signal connector, generating surface acoustic waves for stimulating cells.

[0058] In some embodiments, the cell processing pool is further equipped with a microfluidic channel. After cell lysis is completed, the cells and culture medium can be driven by surface acoustic waves to leave the cell processing pool from the microfluidic channel and enter the proteomics analysis module, where differentially expressed genes in the lysate are quantitatively detected through nucleic acid purification.

[0059] On the other hand, a cell lysis device includes a surface acoustic wave (SAW) excitation device and a cell processing pool, wherein the SAW excitation device is used to transmit SAW waves to the cell processing pool; the cell processing pool is used for cell lysis; and the pool wall of the cell processing pool contains nanowires.

[0060] It is understood that the cell processing device provided by the present invention can also be used directly as a cell lysis device. After the cells and cell culture medium are loaded into the cell processing pool, efficient cell lysis can be achieved based on surface acoustic waves and under the action of the pool wall containing nanowires.

[0061] In another aspect, the present invention provides a cell treatment method, wherein the method uses the cell treatment device described above for treatment, and includes the following steps:

[0062] (1) Place the cells and culture medium in the cell treatment pool, turn on the surface acoustic wave excitation device, the output power of the surface acoustic wave excitation device is 0.5 watts to 5 watts, and carry out cell proliferation;

[0063] (2) After cell proliferation is completed, the output power of the surface acoustic wave excitation device is increased to 10 watts to 20 watts to perform cell lysis.

[0064] The surface acoustic wave excitation device uses an acoustic stimulation end signal generator to generate a sinusoidal electrical signal with a specific frequency and adjustable power to excite surface acoustic waves.

[0065] The specific frequency referred to here is the center frequency of the surface acoustic wave (SAW) device. SAW waves are excited by inputting an AC signal with the same center frequency to the device. Whether for cell culture and proliferation or cell lysis, a specific frequency of 16MHz is preferred. This frequency should not be too high, as high-frequency SAW waves have a weaker stimulating effect on cells; at the same time, the frequency should not be too low, as this would reduce the energy utilization efficiency of the cells.

[0066] By adjusting the power of the electrical signal, the amplitude of the surface acoustic waves (SAWs) can be controlled, thereby regulating the degree to which the SAWs stimulate cells. When used for cell culture and proliferation, the power is preferably 0.5 watts to 5 watts. If the power is too low, the effect on cells is weak and cannot promote proliferation; if the power is too high, the strong mechanical waves will cause cells to detach from their growth surface, making further culture and proliferation impossible. Therefore, the power needs to be controlled within the 0.5 watts to 5 watts range. When used for cell lysis, the power should be higher than that used for stimulating cell proliferation, ranging from 10 watts to 20 watts. This higher-powered SAW can cause cells to detach from their growth surface, suspending them in the culture medium. With the continuous driving of the SAW, the cells will gain acceleration in the culture medium along the direction of SAW propagation. Simultaneously, the power is limited to below 20 watts to ensure that the cells are not killed by excessively strong SAWs.

[0067] In another aspect, the present invention provides a cell lysis method, wherein the method uses the cell lysis device described above for processing.

[0068] In some embodiments, the lysis method includes the following steps: placing a cell solution in a cell treatment pool, turning on a surface acoustic wave excitation device, increasing the output power of the surface acoustic wave excitation device to 10 watts to 20 watts, and performing cell lysis.

[0069] The present invention has the following beneficial effects:

[0070] 1. A cell processing device based on surface acoustic waves is provided, which can be used for both cell culture and proliferation and cell lysis.

[0071] 2. The cell processing device provided by the present invention can achieve the effect of both high-efficiency cell culture and proliferation and high-efficiency cell lysis by simply adjusting the output power of the surface acoustic wave excitation device without changing any structure or adding any additional components when switching between cell culture and proliferation and cell lysis.

[0072] 3. It was found that when nanowires are fixed to the surface of the cell processing pool wall, the cell processing device prepared by combining surface acoustic waves and nanowires does not have an adverse effect on cell culture and proliferation when used for cell culture and proliferation; when used for cell lysis, the nanowires, due to their finer diameter, sharper and more pointed edges, and higher mechanical strength, can lyse cells more efficiently.

[0073] 4. By controlling the output power of surface acoustic waves, surface acoustic waves can be used for both efficient cell culture and cell lysis, showing broad application prospects. Attached Figure Description

[0074] Figure 1 This is a three-dimensional schematic diagram of the entire device;

[0075] Figure 2 This is a top view of the entire device;

[0076] Figure 3 This is a top view of an interdigital transducer;

[0077] Figure 4 A top view showing details of the cell culture pool. Detailed Implementation

[0078] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings. It should be noted that the following embodiments are intended to facilitate understanding of the present invention and are not intended to limit it in any way. The raw materials and equipment used in the specific embodiments of the present invention are all known products and were obtained by purchasing commercially available products.

[0079] In the description of this invention, it should be understood that the terms "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0080] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "connected" and "linked" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0081] Example 1: Cell processing device based on surface acoustic waves provided by the present invention

[0082] The cell processing device 1 based on surface acoustic waves provided in this embodiment is as follows: Figures 1-4 As shown, where Figure 1 A three-dimensional view of the cell processing device; Figure 2 This is a top view of the cell processing device; Figure 3 This is a top view of an interdigital transducer; Figure 4 This is a top view of the cell treatment pool.

[0083] like Figure 1 This embodiment provides a cell processing device 1, including a surface acoustic wave (SAW) excitation device 2 and a cell processing pool 3. The SAW excitation device 2 is used to transmit SAW waves to the cell processing pool 3; the cell processing pool 3 is used for cell culture, proliferation, and lysis. This device can achieve cell culture, proliferation, and lysis by driving SAW waves. Moreover, when switching between cell culture, proliferation, and cell lysis functions, only the output power of the SAW excitation device needs to be adjusted to achieve both efficient cell culture and proliferation and efficient cell lysis.

[0084] like Figures 1-4 It can be seen that the cell treatment tank 1 contains nanowires 5 on its tank wall 4. When the cells continue to rotate in the cell treatment tank 3 under the influence of acoustic flow, they continuously collide with the nanowires 5 on the tank wall 4, generating huge interaction forces and momentum transfer between them, thereby disrupting the cell membrane function and structure and achieving the purpose of lysis. However, when used for cell culture and proliferation, the nanowires 5 do not have an adverse effect on cell culture and proliferation.

[0085] like Figure 4The cell treatment pool 3 can be any one or more of the following shapes: cylindrical, spherical, square, rectangular, and frustum-shaped. Nanowires 5 are located on all or part of the surface of the pool wall 4 of the cell treatment pool 3. The nanowires 5 need to be located on the surface of the vertical inner wall 6 of the cell treatment pool 3, while the bottom 7 does not need to be equipped with nanowires 5. Preferably, in this embodiment, the cell treatment pool 3 is square, and the inner wall 6 includes a front wall 8, a rear wall 9, a left wall 10, and a right wall 11. The nanowires 5 are located on part or all of the surface of one or more of the front wall 8, rear wall 9, left wall 10, and right wall 11 and are uniformly arranged.

[0086] Preferably, the nanowires 5 are prepared using any one or more of zinc oxide, tin dioxide, silicon carbide, silver, copper, gold, iron, aluminum, silicon, germanium, and gallium arsenide. In fact, nanowires 5 prepared from any material, as long as they possess sufficient strength and fineness, can be used to achieve cell lysis. In this embodiment, zinc oxide nanowires 12 are used as the nanowires 5, which helps improve the cell lysis effect without affecting the cell culture process. The zinc oxide nanowires 12 are prepared by chemical bath deposition on a flexible stainless steel substrate. Maintaining the diameter, length, and distribution density of the nanowires 5 within a preferred and suitable range helps improve the cell lysis efficiency without adversely affecting the cell culture process.

[0087] In this embodiment, zinc oxide nanowires are preferably prepared by chemical bath deposition: the zinc oxide seed layer required for this growth method is prepared using atomic layer deposition (ALD). The preparation of the zinc oxide (ZnO) seed layer mainly relies on atomic layer deposition (ALD) technology, and the specific process is as follows: In the experiment, most of the ZnO seed layer is deposited using an ALD instrument, and the reaction is completed in the main reaction chamber; precursor A is deionized water (H2O), precursor B is diethyl zinc (Zn(C2H5)2), the precursor delivery temperature is about 150°C, the growth temperature of the ZnO seed layer is 250°C, argon (Ar) is used as the carrier gas, and the flow rate is 40 standard cubic centimeters per minute (sccm); through repeated precursor pulses and cleaning steps, the zinc oxide seed layer is gradually thickened until the required thickness is reached. This method can precisely control the film thickness and uniformity, obtain a high-quality zinc oxide seed layer, and can be carried out at a relatively low temperature, which helps to avoid thermal damage to the substrate material. Zinc oxide nanowires with diameters of 50-200 nm and lengths of 5-15 μm were prepared by growing them for 15 hours in a precursor solution (30 mM) composed of zinc nitrate hexahydrate and hexamethylenetetramine (HMTA) in a 1:1 hydration ratio at a water bath temperature of 85 °C. These nanowires (preferably 100 nm in diameter and 10 μm in length in this embodiment) exhibited better cell lysis efficiency when used as substrates for cell treatment pool 3. After growth, the nanowires were recycled. Specifically, after growth, the substrate was treated with ultrasonic oscillation to suspend the nanowires in the solution for later use. The solution was then dropped onto a rectangular PDMS film with an area equal to the inner wall area of ​​the cell treatment pool, and the film was then attached to the inner wall 6 of the cell treatment pool 3. In this embodiment, the distribution density of the nanowires 5 on the pool wall 4 was 500-5000 wires / m. 2 (In this embodiment, the preferred density is 2000 roots / m) 2 ).

[0088] The cell processing device 1 can be used to process any type of cell, including any one or more of prokaryotic cells, animal cells, plant cells, and fungal cells. Preferably, animal cells are used for processing, as they do not have cell walls and have a better response to surface acoustic wave stimulation. More preferably, cells suitable for two-dimensional planar growth are used, such as human dermal fibroblasts. When using these cells for processing, the culture process is not adversely affected, the lysis effect is more complete, and they have advantages over other cells.

[0089] like Figure 1 and Figure 2 The surface acoustic wave excitation device 2 includes a piezoelectric substrate 13; the cell treatment pool 3 is formed by combining the pool wall 4 and part of the piezoelectric substrate 13, and part of the piezoelectric substrate 13 of the surface acoustic wave excitation device 2 constitutes the bottom of the cell treatment pool 3.

[0090] Preferably, the cell treatment pool 3 consists of a pool wall 4 and a pool bottom 7. The pool bottom 7 refers to the upper surface 14 of the piezoelectric substrate 13. The pool wall 4 and the upper surface 14 of the piezoelectric substrate 13 are bonded together using various methods such as bonding and adhesion, forming the pool bottom 7 locally on the upper surface 14 of the piezoelectric substrate 13 for cell culture. Additionally, cell number sensing devices 15 are provided on both sides of the cell treatment pool 3 to monitor cell proliferation in the cell treatment pool 3. When the cell density in the cell treatment pool 3 changes, the surface acoustic wave sensing device 2 can immediately sense the change in the frequency of the high-frequency surface acoustic wave, thereby providing feedback on the change in cell number and monitoring cell proliferation in the cell treatment pool 3.

[0091] like Figure 1 and Figure 2 The cell processing device 1 includes a surface acoustic wave excitation device 2, a cell processing pool 3, a cell quantity sensing device 15, and a power supply device 17.

[0092] The surface acoustic wave (SAW) excitation device 2 includes a piezoelectric substrate 13, an interdigital transducer 18 at the acoustic stimulation end, and an acoustic stimulation end reflective grating 19. The piezoelectric substrate 13 is made of Y-128 tangential lithium niobate (purchased from the Shanghai Institute of Optics and Fine Mechanics, Chinese Academy of Sciences, model Dia4"*0.5mm). This material not only possesses stable physical and chemical properties, but its high electromechanical coupling coefficient also implies high electro-acoustic conversion efficiency, providing higher energy utilization for the cell processing device 1. During fabrication, to generate SAW waves, the thickness of the piezoelectric substrate 13 must be much greater than the wavelength 39 nm of the SAW waves. However, excessive thickness would affect the overall size of the cell processing device 1; therefore, the thickness of the piezoelectric substrate 13 is preferably 500 micrometers. The interdigital transducer 18 at the acoustic stimulation end is a key structure of the SAW excitation device 2, including components distributed on the piezoelectric substrate 13. A layer of metallized interdigitated pattern and wiring is provided. The metal is typically aluminum, gold, or other conductive materials (gold is preferred in this embodiment). The interdigitated pattern consists of multiple parallel metal fingers 20 (electrodes) with a certain width, referred to as the interdigitated width 30. These metal fingers 20 are spaced equidistantly from adjacent metal fingers 20, referred to as the interdigitated spacing 16. The metal fingers 20 and the spacing are arranged alternately, forming a comb-like structure. Adjacent metal fingers 20 are connected to two electrodes 21 via wiring and an external AC voltage is applied, converting the electrical signal into a mechanical wave through the inverse piezoelectric effect. When the interdigitated width 30 and the interdigitated spacing 16 are the same, and the size of the interdigitated period is much smaller than the thickness of the piezoelectric substrate 13, it is possible to... This generates Rayleigh waves, one of the most common surface acoustic waves. The wavelength 39 of this wave is equal to four times the interdigital width, which is the interdigital period (wavelength 39). The center frequency is calculated by dividing the sound velocity of the piezoelectric substrate by the wavelength 39. The wavelength 39 of the interdigital transducer 18 at the acoustic stimulation end is designed to be 36–360 micrometers, while the sound velocity of lithium niobate tangentially in Y-128 is around 3600 m / s, corresponding to a center frequency of 10 MHz–100 MHz, which is the operating frequency of the surface acoustic wave. The overlap length of the two sets of interdigital pairs 22 is called the acoustic aperture 23, with a value of 1 cm. The surface acoustic wave is generated on the overlapping interdigital pairs 22 and propagates along both ends of the interdigital transducer 18 at the acoustic stimulation end. The surface acoustic wave at one end is reflected by the acoustic stimulation end reflector. The acoustic aperture 19 reflects and strengthens the surface acoustic waves at the other end. Simultaneously, the acoustic aperture determines the beam width of the excited surface acoustic waves. Here, the length of the acoustic aperture 23 is consistent with the width 24 of the subsequent cell treatment pool 3, ensuring that the bottom 25 of the cell treatment pool 3 is stimulated by surface acoustic waves. The acoustic stimulation end reflection grating 19 is a layer of metallized interdigital pattern distributed at one end of the acoustic stimulation end interdigital transducer 18. Its main function is to reflect the surface acoustic waves generated by the acoustic stimulation end interdigital transducer 18 back to the acoustic stimulation end interdigital transducer 18 to form a standing wave, thereby enhancing the propagation efficiency of the sound waves and strengthening the surface acoustic waves propagating towards the cell treatment pool 3. Simultaneously, the surface acoustic waves slightly disturb the culture medium and cells in the cell treatment pool 3, promoting cell proliferation.Therefore, its metal finger 26 is not connected to any electrode. Meanwhile, its interdigital width 30 and interdigital spacing 16 are related to the wavelength 39 of the surface acoustic wave (SAW). In the SAW excitation device 2 that generates Rayleigh waves, the interdigital width 30 and interdigital spacing 16 are the same as those of the interdigital transducer 18 at the acoustic stimulation end. Furthermore, the distance between the interdigital transducer 18 at the acoustic stimulation end and the acoustic stimulation end reflector grating 19 is 30 times the interdigital period. If this distance is too low, the SAW may not develop sufficiently before propagating to the acoustic stimulation end reflector grating 19, thus affecting the wave reflection efficiency and the overall performance of the SAW excitation device 2. If this distance is too high, the SAW will experience more attenuation during propagation, reducing the intensity of the reflected wave.

[0093] The cell treatment tank 3 includes a tank wall 4 and a tank bottom 7. The tank wall 4 is a square hollow cylinder with a length and width of 1 cm and a height of 5 mm, formed by injection molding of polydimethylsiloxane (PDMS), a bio-harmless material. During injection molding, PDMS and a coagulant are mixed at a mass ratio of 10:0.9, making the molded PDMS tank wall softer than PDMS material molded at a conventional ratio of 10:1, which helps reduce the loss of surface acoustic waves when passing through the tank wall 4 into the cell treatment tank 3. The tank bottom 7 refers to the upper surface 14 of the piezoelectric substrate 13. The PDMS tank wall 4 is bonded to the upper surface 14 of the piezoelectric substrate 13 using various methods such as bonding and adhesion, forming the tank bottom 7 locally on the upper surface 14 of the piezoelectric substrate 13 for cell culture.

[0094] The cell number sensing device 15 includes a surface acoustic wave (SAW) sensor 26 and a cell sensing region 27. By placing the SAW sensor 15 near the cell sensing region 27, the SAW sensor 15 can sense changes in cell density in the cell sensing region 27 and emit high-frequency SAW waves. The frequency of these high-frequency SAW waves changes with the cell density, thereby providing feedback on changes in cell number and monitoring cell proliferation in the cell treatment pool 3. The SAW sensor 26 includes a SAW excitation device 28 at the sensing signal generation end and a sound-to-electric conversion device 29 at the sensing signal receiving end. The basic structure of the SAW excitation device 28 at the sensing signal generation end is the same as that of the SAW excitation device 2, having a piezoelectric substrate 13, an interdigital transducer 31 at the sensing signal generation end, a sensing signal generator 32, and a sensing signal reflection grating 33. However, its interdigital period is much smaller, ranging from 3.6 to 36 micrometers, corresponding to a surface acoustic wave center frequency of 100 MHz to 1 GHz, while the output power of the signal generator 32 is controlled at 0.001 watts to 0.01 watts. Compared to the surface acoustic wave excitation device 2 used for driving, this surface acoustic wave sensing device 26 has a higher center frequency, which brings higher information sensitivity accuracy, while weakening the driving function and reducing the impact of surface acoustic waves on cells. The sensing signal receiving end acoustic-to-electric conversion device 29 includes a piezoelectric substrate 13, a sensing signal receiving end interdigital transducer 34, and a sensing end signal receiving computer 35. The sensing signal receiving end acoustic-to-electric conversion device 29 has an interdigital transducer with the same structure as the sensing signal generating end surface acoustic wave excitation device 28, but it does not have a reflective grating structure 33, nor does it apply an alternating current signal to it through electrodes. The high-frequency surface acoustic waves (SAWs) generated in the SAW excitation device 28 at the sensing signal generation end are sensitive to the environment. As they propagate along the surface of the medium, their frequency changes due to the medium's properties. When the SAWs reach the interdigital transducer 34 at the sensing signal receiving end, an alternating current signal with its own frequency characteristics is generated due to the piezoelectric effect. By detecting the frequency change of the SAWs on the signal receiving computer 35 at the sensing end, the changes in the propagation surface can be detected. The cell sensing area 27 is the two-dimensional planar space 36 between the interdigital transducer 31 of the SAW excitation device 28 at the sensing signal generation end and the interdigital transducer 34 of the acoustic-to-electric conversion device 29 at the sensing signal receiving end, which corresponds to the bottom 7 of the cell treatment pool 3 within the device. When adherent cells grow at different densities on the bottom of the pool 7, the high-frequency surface acoustic waves emitted from the surface acoustic wave excitation device 28 at the sensing signal generation end will change in frequency to varying degrees as they pass through this area. These waves will then continue to propagate to the interdigital transducer 29 at the sensing signal receiving end, where they will be converted into alternating current signals and recorded by the computer 35. By detecting these changes, the growth density information of the adherent cells on the bottom of the pool 7 can be obtained.

[0095] The power supply device 17 includes an acoustic stimulation end signal generator 36 and an acoustic drive signal connector 37. The acoustic stimulation end signal generator 36 generates a sinusoidal electrical signal with a specific frequency and adjustable power. The specific frequency refers to the center frequency of the surface acoustic wave excitation device 2. An AC electrical signal with the same center frequency is input to it to excite the surface acoustic waves. By adjusting the power of the electrical signal, the amplitude of the surface acoustic waves is controlled, thereby regulating the degree of stimulation of cells affected by the surface acoustic waves. The power is between 0.5 watts and 5 watts. When the power is too low, the effect on the cells is weak and cannot promote their proliferation. When the power is too high, the strong mechanical waves will cause the cells to detach from their attached growth surface. The acoustic drive signal connector 37 is an SMA RF connector. It includes contact pairs, an insulator, and a threaded connection. The contact pair includes a center conductor (inner core) and an outer conductor (outer jacket). The center conductor is usually in the form of a pin or hole, while the outer conductor consists of mating holes or pins, and a threaded connection. The function of the contact pair is to achieve the connection between two circuits. The insulator serves to support and secure the contact pairs, ensuring electrical insulation between the contact pairs and between the contact pairs and the housing. It is typically made of polytetrafluoroethylene (PTFE), a material with good dielectric properties and chemical stability. The threaded connection uses imperial threads, providing high strength and good shock resistance. The electrical signal generated by the acoustic stimulation end signal generator 36 is input to the surface acoustic wave excitation device 2 via the acoustic drive signal connector, generating surface acoustic waves for stimulating cells.

[0096] Preferably, the cell processing pool 3 is further provided with a microchannel (not shown in the figure). After cell lysis is completed, the cells and culture medium can be driven by surface acoustic waves to leave the cell processing pool 3 from the microchannel and enter the proteomics analysis module, where differentially expressed genes in the lysate are quantitatively detected through nucleic acid purification.

[0097] The cell processing device 1 provided in this embodiment can also be used directly as a cell lysis device 38. After the cells and cell culture medium are loaded into the cell processing pool 3, efficient cell lysis can be achieved based on surface acoustic waves and under the action of the pool wall 4 containing nanowires 5.

[0098] Example 2: Methods for cell culture and lysis

[0099] This embodiment uses the cell processing device 1 provided in Example 1 for cell culture, proliferation, and lysis. The specific steps are as follows:

[0100] 1. Cell proliferation

[0101] 1.1 Clean cell treatment tank 3 with anhydrous ethanol and PBS buffer.

[0102] 1.2 Add 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Prologis) to cell treatment tank 3, and seed human dermal fibroblasts to achieve a cell density of 6000 cells / cm³. 2 .

[0103] 1.3 Place the cell processing device 1 into the incubator and incubate at 37°C and 5% carbon dioxide for two hours to allow the cells to adhere to the wall (most of them adhere to the bottom of the pool 7).

[0104] 1.4 Connect the acoustic stimulation end signal generator 36 to the acoustic drive signal connector 37 on the cell processing device 1, and place the cell processing device 1 in an incubator at 37°C and 5% carbon dioxide concentration. Set the sensor signal generator 32 to output a sinusoidal electrical signal with a frequency of 16MHz and a power of 1 watt. Control the sensor signal generator 32 to drive for 20 seconds, pause for 280 seconds, drive again, repeat 8 times, stop for 2 hours, and this is one set, for a total of 4 hours. Then repeat this set of driving schemes 6 times.

[0105] 1.5 The acoustic propagation process ends after 24 hours.

[0106] 2. Cell growth number sensing

[0107] 2.1 Connect the sensor signal generator 32 to the sensor signal input connector 38 on the cell processing device 1, and connect the sensor signal receiving computer 35 to the sensor signal output connector 39 on the cell processing device 1.

[0108] 2.2 The sensor signal generator 32 is configured to output a sinusoidal electrical signal with a frequency of 160MHz and a power of 0.005W, and to output it continuously.

[0109] 2.3 During cell growth, changes in electrical signals are continuously monitored and recorded on the sensor signal receiving computer 35.

[0110] 2.4 When the cells have grown throughout the entire bottom of the pool, a set of electrical signals is recorded every 5% of the cells removed, and finally calibration data related to cell density and electrical signals are obtained.

[0111] 2.5 Calibration data were used to process data during cell growth to obtain the cell growth count at different time points. Calculations showed that after cell culture, the cell density reached 24,000 cells / cm³. 2 .

[0112] 3. Cell lysis

[0113] 3.1 Connect the sensor signal generator 32 to the acoustic drive signal connector 37 on the cell processing device 1, and place the cell processing device 1 in an incubator at 37°C and 5% carbon dioxide concentration. Configure the sensor signal generator 32 to output a sinusoidal electrical signal with a frequency of 16MHz and a power of 15W.

[0114] 3.2 The culture medium in cell treatment tank 3 begins to rotate violently driven by surface acoustic waves, and the adherent cells detach from the bottom of the tank 7 and rotate with the culture medium.

[0115] 3.3 The cells collided rapidly with and were ruptured by the nanowires 5 on the pool wall 4.

[0116] 3.4 Wait 2 hours to allow the cells to fully lyse, and collect the culture medium and the lysed material within it.

[0117] Example 3: Cell lysis method

[0118] This embodiment uses the cell processing device 1 provided in Example 1 to perform cell lysis. The specific steps are as follows:

[0119] 1. Clean cell treatment tank 3 with anhydrous ethanol and PBS buffer.

[0120] 2. Add 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Pronosei) to cell treatment tank 3, and seed human dermal fibroblasts to achieve a cell density of 6000 cells / cm³. 2 Place cell processing device 1 in an incubator at 37°C and 5% carbon dioxide concentration, and wait for cell growth.

[0121] 3. Connect the sensor signal generator 32 to the acoustic drive signal connector 37 on the cell processing device 1, and place the cell processing device 1 in an incubator at 37°C and 5% carbon dioxide concentration. Configure the sensor signal generator 32 to output a sine wave electrical signal with a frequency of 16MHz and a power of 15W.

[0122] 4. Driven by surface acoustic waves, the culture medium in cell treatment pool 3 begins to rotate violently, and the adherent cells detach from the bottom 7 of the pool and rotate with the culture medium.

[0123] 5. The cells collide rapidly with and are broken down by the nanowires 5 on the pool wall 4.

[0124] 6. Wait 2 hours to allow the cells to fully lyse, and collect the culture medium and the lysed material inside.

[0125] Example 4: Comparison of the culture effects of different cell culture methods

[0126] This example uses the following four cell culture methods: 1. Cells are cultured using a surface acoustic wave cell treatment device (Example 1) according to the method provided in Example 2; 2. Cells are cultured using electrical stimulation, where the electrical power is 0.1 mW / cm². 2 (100μA / cm 2 3. Cells are cultured using ultrasound, with a power of 30 mW / cm². 2 4. Standard culture method: as shown in 1.1 to 1.3 of Example 2. The initial sample was 200 μL of culture medium containing human dermal fibroblasts (the culture medium was complete human dermal fibroblast culture medium, purchased from Prologis), with a cell density of 5000 cells / cm³. 2 With all other culture conditions being the same, the cells were cultured for three days. The cell density was examined after the culture was completed, and the effects of different culture methods on cell culture proliferation were investigated. The results are shown in Table 1.

[0127] Table 1. Effects of different culture methods on cell culture proliferation

[0128] Training methods <![CDATA[Cell density (cells / cm 2 )]]> 1 (Example 1) <![CDATA[1.52×10 5 ]]> 2 <![CDATA[1.37×10 4 ]]> 3 <![CDATA[1.21×10 4 ]]> 4 <![CDATA[1.01×10 4 ]]>

[0129] As can be seen from Table 1, different culture methods have completely different effects on cell culture and proliferation. Since human dermal fibroblasts are cells that grow adherently on a two-dimensional plane, surface acoustic waves can maximize their culture and proliferation effect and have the highest energy utilization rate. Therefore, surface acoustic wave culture is the most preferred cell culture method.

[0130] Example 5: Effects of surface acoustic wave output frequency and power on cell culture and lysis.

[0131] This embodiment uses the cell treatment device provided in Example 1. The output frequency and power of the sensor signal generator are set according to Table 1. 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Procyte) is added to the cell treatment tank, and human dermal fibroblasts are seeded to achieve a cell density of 6000 cells / cm³ in the cell treatment tank. 2 Cells were cultured and proliferated according to the method provided in Example 2, and cell density was detected using the cell growth number sensor in Example 2. The effects of different nanowire distribution patterns on cell culture and proliferation were investigated, and the results are shown in Table 2.

[0132] Table 2. Effects of surface acoustic wave output frequency and power on cell culture proliferation

[0133] Output frequency (MHz) Power (watts) <![CDATA[Cell density (cells / cm 2 )]]> 16 (Example 1) 1 24000 8 1 18750 32 1 16330 16 0.5 12210 16 5 16850 16 10 10302

[0134] As can be seen from Table 2, the output frequency and power of surface acoustic waves directly affect the cell culture and proliferation effect. Therefore, it is necessary to select appropriate output frequency and power. The optimal output frequency is 16MHz and the power is 1W.

[0135] With an output frequency of 16MHz selected, this embodiment further investigated the effect of different powers on cell lysis. In this embodiment, 200μL of culture medium (complete human dermal fibroblast culture medium, purchased from Procyte) was added to the cell treatment tank provided in Example 1, and human dermal fibroblasts were seeded. Cell proliferation was allowed to occur until the cell density reached 24,000 cells / cm³. 2 Then, the cells were lysed using the method provided in Example 3, and the lysed samples were collected. The expression of TGF-β1 mRNA in the cells was quantitatively detected using real-time RT-PCR. The primer and probe set for detection was as follows:

[0136] Primer 1: 5'-CGCGTGCTAATGGTGGAAA-3'

[0137] Primer 2: 5'-CGCTTCTCGGAGCTCTGATG-3'

[0138] Fluorescently labeled probe: 5'-FAM-CCACAACGAAATCTAT-MGB-3'

[0139] The cell lysis rate was calculated by comparing the quantitative detection data of the experimental lysed samples with those of the externally sent lysed samples. The detection results are shown in Table 3.

[0140] Table 3. Effect of surface acoustic wave power on cell lysis

[0141]

[0142] As can be seen from Table 3, the power of surface acoustic waves directly affects the cell lysis effect. When used for cell lysis, the optimal output power is 15 watts.

[0143] Example 6: The effect of nanowire distribution on cell culture and lysis.

[0144] This embodiment uses the cell treatment device provided in Example 1, wherein the nanowires are distributed in the following four ways: 1. Uniformly distributed on the pool wall (as in Example 1); 2. Uniformly distributed on the pool bottom; 3. Simultaneously distributed on both the pool wall and the pool bottom, while magnetic beads (CN108414303B, with an addition amount of 4×10⁻⁶) are directly added to the cell treatment pool. 6A comparison was made using a method involving individual magnetic beads. 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Pronosei) was added to the cell treatment tank, and human dermal fibroblasts were seeded to achieve a cell density of 6000 cells / cm³ in cell treatment tank 3. 2 Cells were cultured and proliferated according to the method provided in Example 2, and cell density was detected using the cell growth number sensor in Example 2. The effects of different nanowire distribution patterns on cell culture and proliferation were investigated, and the results are shown in Table 4.

[0145] Table 4. Effects of nanowire distribution patterns on cell culture proliferation

[0146] Distribution method <![CDATA[Cell density (cells / cm 2 )]]> 1 (Example 1) 24000 2 18100 3 17900 Nanowire-free 24150 Magnetic bead (CN108414303B) 5650

[0147] As can be seen from Table 4, when nanowires are distributed at the bottom of the pool under the action of surface acoustic waves, the cell culture proliferation effect is adversely affected, and the cell proliferation rate is significantly slowed down.

[0148] In addition, the presence of magnetic beads significantly affects cell proliferation, making cell culture difficult. This may be because cells struggle to adhere to the cell wall when magnetic beads are present.

[0149] To investigate the effects of different nanowire distribution patterns on cell lysis and to compare this with the method of directly adding magnetic beads (CN108414303B) to the cell treatment tank, this embodiment added 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Prologis) to cell treatment tanks with different nanowire distribution patterns, and then seeded human dermal fibroblasts, waiting for cell proliferation to reach a cell density of 24,000 cells / cm³. 2 Then, the cells were lysed using the method provided in Example 3, and the lysed samples were collected. The expression of TGF-β1 mRNA in the cells was quantitatively detected using real-time RT-PCR. The primer and probe set for detection was as follows:

[0150] Primer 1: 5'-CGCGTGCTAATGGTGGAAA-3'

[0151] Primer 2: 5'-CGCTTCTCGGAGCTCTGATG-3'

[0152] Fluorescently labeled probe: 5'-FAM-CCACAACGAAATCTAT-MGB-3'

[0153] The cell lysis rate was calculated by comparing the quantitative detection data of the experimental lysed samples with those of the externally sent lysed samples. The detection results are shown in Table 5.

[0154] Table 5. Effect of nanowire distribution pattern on cell lysis effect

[0155] Distribution method TGF-β1 mRNA (copies / mL) Cell lysis rate (%) 1 <![CDATA[1×10 12 ]]> 99.8 2 <![CDATA[6.98×10 11 ]]> 70 3 <![CDATA[9.05×10 11 ]]> 90.7 Nanowire-free <![CDATA[1.19×10 10 ]]> 1.2 Magnetic bead (CN108414303B) <![CDATA[9.51×10 11 ]]> 95.3

[0156] As shown in Table 5, under the action of surface acoustic waves, the lysis effect of human dermal fibroblast samples with the same initial density after being lysed in cell treatment cells with three different nanowire distribution patterns was significantly different. Whether or not nanowires were distributed at the bottom of the cell did not improve the lysis effect.

[0157] In addition, compared with the method of adding particulate matter to the cell treatment pool for lysis, the lysis effect of nanowires is significantly better and more thorough.

[0158] In summary, the distribution of nanowires at the bottom of the pool not only has an adverse effect on cell culture and proliferation, but also does not improve the lysis effect. Therefore, it is preferable to place the nanowires on the pool wall rather than at the bottom.

[0159] Example 7: The effect of nanowire material selection on cell culture and lysis.

[0160] This embodiment uses the cell processing device provided in Example 1. The nanowires are prepared using the materials shown in Table 6. Zinc oxide and copper nanowires are prepared by chemical bath deposition, tin dioxide and silicon carbide nanowires are prepared by thermal evaporation, and silver, aluminum, and iron nanowires are prepared by template method. Human dermal fibroblasts are seeded to achieve a cell density of 6000 / cm³ in cell processing pool 3. 2 Cells were cultured and proliferated according to the method provided in Example 2, and cell density was detected using the cell growth number sensor in Example 2 to investigate the effect of different nanowire materials on cell culture and proliferation. The detection results are shown in Table 6.

[0161] Table 6. Effects of nanowire materials on cell culture proliferation

[0162] Nanowire materials <![CDATA[Cell density (cells / cm 2 )]]> Nanowire-free 24000 Zinc oxide 23920 Tin dioxide 20030 silicon carbide 19900 silver 23900 copper 20100 aluminum 21100 iron 21200

[0163] As shown in Table 6, when nanowires prepared with zinc oxide or silver are distributed on the pool wall under the action of surface acoustic waves, they do not have an adverse effect on cell culture proliferation. The cell culture proliferation effect is significantly better than that of nanowires prepared with other materials.

[0164] To investigate the effects of different nanowire materials on cell lysis, in this embodiment, 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Procyte) was added to cell treatment pools containing different nanowire materials, and human dermal fibroblasts were seeded and allowed to proliferate until the cell density reached 24,000 cells / cm³. 2Then, the cells were lysed using the method provided in Example 3, and the lysed samples were collected. The expression of TGF-β1 mRNA in the cells was quantitatively detected using real-time RT-PCR. The primer and probe set for detection was as follows:

[0165] Primer 1: 5'-CGCGTGCTAATGGTGGAAA-3'

[0166] Primer 2: 5'-CGCTTCTCGGAGCTCTGATG-3'

[0167] Fluorescently labeled probe: 5'-FAM-CCACAACGAAATCTAT-MGB-3'

[0168] The cell lysis rate was calculated by comparing the quantitative detection data of the experimental lysed samples with those of the externally sent lysed samples. The detection results are shown in Table 7.

[0169] Table 7. Effects of nanowire materials on cell lysis

[0170]

[0171]

[0172] As shown in Table 7, under the action of surface acoustic waves, the lysis effects of human dermal fibroblast samples with the same initial density after being lysed in cell treatment pools with different nanowire materials also showed significant differences. Among them, the nanowires prepared with zinc oxide material had the best lysis effect, with a cell lysis rate of 99.8%.

[0173] In summary, nanowires prepared using zinc oxide not only have no adverse effects on cell culture and proliferation, but also exhibit the best cell lysis effect.

[0174] Example 8: Effects of nanowire size and density on cell culture and lysis.

[0175] In this embodiment, following the method provided in Example 1, three different sizes of zinc oxide nanowires were prepared, and the nanowires were produced at densities of 500, 2000, and 5000 nanowires / m, respectively. 2The zinc oxide nanowires were arranged in a density on the pool wall. The preparation methods for three different sizes of zinc oxide nanowires were as follows: 1. Zinc oxide nanowires with a diameter of 50 nm and a length of 15 μm were prepared by growing them in a precursor solution (30 mM) composed of zinc nitrate hexahydrate and hexamethylenetetramine (HMTA) in a 1:1 hydration ratio for 15 hours at a water bath temperature of 85 °C; 2. Zinc oxide nanowires with a diameter of 100 nm and a length of 10 μm were prepared by growing them in a precursor solution (30 mM) composed of zinc nitrate hexahydrate and hexamethylenetetramine (HMTA) in a 1:2 hydration ratio for 18 hours at a water bath temperature of 85 °C (Example 1); 3. Zinc oxide nanowires with a diameter of 200 nm and a length of 5 μm were prepared by growing them in a precursor solution (30 mM) composed of zinc nitrate hexahydrate and hexamethylenetetramine (HMTA) in a 2:1 hydration ratio for 10 hours at a water bath temperature of 85 °C. Cell culture and proliferation were performed according to the method provided in Example 2, and cell density was detected using the cell growth number sensor in Example 2. The effects of different nanowire materials on cell culture and proliferation were investigated, and the detection results are shown in Table 8.

[0176] Table 8. Effects of nanowire size and density on cell culture proliferation

[0177] Nanowire size <![CDATA[Nanowire density (number / m 2 )]]> <![CDATA[Cell density (cells / cm 2 )]]> 50nm in diameter and 15μm in length 500 24000 50nm in diameter and 15μm in length 2000 24100 50nm in diameter and 15μm in length 5000 23800 100nm in diameter and 10μm in length 500 24100 100nm in diameter and 10μm in length 2000 24200 100nm in diameter and 10μm in length 5000 23990 200nm in diameter and 5μm in length 500 24010 200nm in diameter and 5μm in length 2000 23900 200nm in diameter and 5μm in length 5000 23800

[0178] As shown in Table 8, under the action of surface acoustic waves, the diameter is 50-200 nm, the length is 5-15 μm, and the density is 500-5000 strands / m. 2 The zinc oxide nanowires at that time did not have a significant impact on cell culture because the cells at the bottom of the pool did not come into contact with the nanowires on the pool wall during the cell growth stage.

[0179] To investigate the effects of different nanowire sizes and densities on cell lysis, this embodiment added 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Prologis) to cell treatment pools with different nanowire sizes and densities, and seeded human dermal fibroblasts, waiting for cell proliferation to reach a cell density of 24,000 cells / cm³. 2 Then, the cells were lysed using the method provided in Example 3, and the lysed samples were collected. The expression of TGF-β1 mRNA in the cells was quantitatively detected using real-time RT-PCR. The primer and probe set for detection was as follows:

[0180] Primer 1: 5'-CGCGTGCTAATGGTGGAAA-3'

[0181] Primer 2: 5'-CGCTTCTCGGAGCTCTGATG-3'

[0182] Fluorescently labeled probe: 5'-FAM-CCACAACGAAATCTAT-MGB-3'

[0183] The cell lysis rate was calculated by comparing the quantitative detection data of the experimental lysed samples with those of the externally sent lysed samples. The detection results are shown in Table 9.

[0184] Table 9. Effects of nanowire size and density on cell lysis.

[0185]

[0186] As shown in Table 9, under the action of surface acoustic waves, human dermal fibroblast samples with uniform initial density showed significant differences in lysis effects after being treated in cell treatment chambers with zinc oxide nanowires of different sizes and densities. Specifically, samples with diameters of 50-200 nm, lengths of 5-15 μm, and densities of 255-5000 nanowires / m showed the most significant differences. 2 The nanowires exhibit good pyrolysis performance, with the optimal size being a diameter of 100 nm, a length of 10 μm, and a density of 2000 wires / m. 2 .

[0187] In summary, nanowires prepared using zinc oxide not only have no adverse effects on cell culture and proliferation, but also exhibit the best cell lysis effect.

[0188] Example 9: Comparison of different cell culture and lysis effects

[0189] This embodiment uses the cell treatment device provided in Example 1, in which human dermal fibroblasts, human chondrocytes, and human cardiomyocytes are seeded respectively, so that the cell density in the cell treatment pool is 6000 cells / cm³. 2 Cells were cultured and proliferated according to the method provided in Example 2, and cell density was detected using the cell growth number sensor in Example 2. The effects of different nanowire distribution patterns on cell culture and proliferation were investigated, and the results are shown in Table 10.

[0190] Table 10. Comparison of cell culture and proliferation of different cells

[0191] cell <![CDATA[Cell density (cells / cm 2 )]]> Human dermal fibroblasts 24200 Human chondrocytes 22010 human cardiomyocytes 22500

[0192] As can be seen from Table 10, the cell processing device provided by the present invention has good culture effect when used to culture these three types of cells, and the zinc oxide nanowires do not have any obvious adverse effects. Among them, the culture effect of human fibroblasts is the best, which may be because human dermal fibroblasts are least affected by nanowires during the culture process (at lower surface acoustic wave output power).

[0193] To compare different cell lysis effects, in this example, 200 μL of culture medium (complete human dermal fibroblast culture medium, purchased from Pronosei) was added to the cell treatment tank, and human dermal fibroblasts were seeded. After cell proliferation and reaching a cell density of 24,000 cells / cm², cell lysis was performed using the method provided in Example 3. The lysed samples were collected, and the expression of TGF-β1 mRNA in the cells was quantitatively detected by real-time RT-PCR. The primer and probe set for detection was as follows:

[0194] Primer 1: 5'-CGCGTGCTAATGGTGGAAA-3'

[0195] Primer 2: 5'-CGCTTCTCGGAGCTCTGATG-3'

[0196] Fluorescently labeled probe: 5'-FAM-CCACAACGAAATCTAT-MGB-3'

[0197] The cell lysis rate was calculated by comparing the quantitative detection data of the experimental lysed samples with those of the externally sent lysed samples. The detection results are shown in Table 11.

[0198] Table 11. Comparison of lysis effects of different cell types

[0199] cell Genes (copies / mL) Cell lysis rate (%) Human dermal fibroblasts <![CDATA[10 12 ]]> 99.7 Human chondrocytes <![CDATA[6.99×10 11 ]]> 70 human cardiomyocytes <![CDATA[9.73×10 11 ]]> 95.7

[0200] As can be seen from Table 11, the three cell samples with the same initial density can all be lysed by the cell processing device provided by the present invention. However, there are some differences in the lysis effect. The lysis effect is best for human dermal fibroblasts, followed by human cardiomyocytes. The reason may be that human dermal fibroblasts are most easily lysed by zinc oxide nanowires under the action of higher surface acoustic wave power.

[0201] In summary, the cell processing device provided by this invention can be used for the culture, proliferation, and lysis of different cells. However, different cells have slight differences. Under specific surface acoustic wave power, the lysis effect of zinc oxide nanowires on specific cells varies, and the degree of influence on the cell culture process also varies.

[0202] The distribution of nanowires at the bottom of the pool not only has an adverse effect on cell culture and proliferation, but also does not improve the lysis effect. Therefore, it is preferable to place the nanowires on the pool wall rather than at the bottom.

[0203] The technical features of the embodiments described above can be combined arbitrarily. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as the combination of these technical features does not contradict each other, it should be considered within the scope of this specification. Although the present invention has been disclosed above, the present invention is not limited thereto. It can be extended according to its application scope in the field of microfluidics. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A cell processing device, characterized by, It includes a surface acoustic wave excitation device and a cell treatment pool, wherein the surface acoustic wave excitation device is used to transmit surface acoustic waves to the cell treatment pool; and the cell treatment pool is used for cell culture, proliferation and lysis.

2. The cell processing device of claim 1, wherein, The walls of the cell treatment pool contain nanowires.

3. The cell processing device of claim 2, wherein the cell processing device is configured to be used in a closed system. The cell treatment pool is any one or more of the following shapes: cylindrical, spherical, square, rectangular, and frustum-shaped, and the nanowires are located on all or part of the inner wall surface of the cell treatment pool.

4. The cell treatment device as described in claim 2, characterized in that, The nanowires are prepared using any one or more of the following: zinc oxide, tin dioxide, silicon carbide, silver, copper, gold, iron, aluminum, silicon, germanium, and gallium arsenide.

5. The cell processing device of claim 2, wherein the cell processing device is configured to be used in a closed system. The nanowire has a diameter of 50-200 nm and a length of 5-15 μm; the distribution density of the nanowire on the pool wall is 500-5000 roots / m 2 .

6. The cell processing device of claim 1, wherein, The cells include any one or more of prokaryotic cells, animal cells, plant cells, and fungal cells.

7. The cell processing device of claim 1, wherein, The surface acoustic wave excitation device includes a piezoelectric substrate, an interdigital transducer at the acoustic stimulation end, and an acoustic stimulation end reflector grid; the cell treatment pool is formed by a combination of a pool wall and a portion of the piezoelectric substrate, and a portion of the piezoelectric substrate of the surface acoustic wave excitation device constitutes the bottom of the cell treatment pool.

8. The cell processing device of claim 1, wherein, It also includes a cell number sensing device, which comprises a surface acoustic wave sensor and a cell sensing area, for monitoring cell proliferation in the cell treatment pool.

9. A cell lysis device, characterized by, The device includes a surface acoustic wave (SAW) excitation device and a cell treatment pool. The SAW excitation device is used to transmit SAW waves to the cell treatment pool. The cell treatment pool is used for cell lysis. The walls of the cell treatment pool contain nanowires.

10. A method of processing cells, comprising: The cell processing device according to any one of claims 1 to 9 includes the following steps: (1) Place the cells and culture medium in the cell treatment pool, turn on the surface acoustic wave excitation device, the output power of the surface acoustic wave excitation device is 0.5 watts to 5 watts, and carry out cell proliferation; (2) After cell proliferation is completed, the output power of the surface acoustic wave excitation device is increased to 10 watts to 20 watts to perform cell lysis.

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