Rapid tissue cracking method and acoustic fluid device

Abstract

The invention discloses a rapid tissue lysis method, which comprises the following steps: adjusting the position of an acoustic fluid chip of an acoustic fluid device to directly face a tissue block, and immersing the tissue block and the acoustic fluid chip in liquid; the sound fluid chip generates special ultrasonic waves, and when the special ultrasonic waves are transmitted in liquid, the sound fluid effect can be triggered to form jet flow which is jetted forwards and vortex which is formed by convolution, so that jet flow force, shearing force and sound fluid vortex are generated; the tissue block in the liquid is rotationally captured in the vortex through the acoustic fluid vortex, the tissue block is impacted through the jet flow force, and the hydrolyzed product is stripped out of the tissue surface through the shearing force. The invention also correspondingly provides an acoustic fluid device. The method can shorten the time of tissue lysis.

Description

[0001] This application is a divisional application with an application date of July 12, 2022, Chinese patent application number CN202210815935.X, and invention name “Acoustic fluid device and automated lysis and biopsy device for rapid tissue lysis”. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to a method for rapid tissue lysis and an acoustic fluid device. Background Art

[0003] The rapid and effective lysis of biological tissues into single-cell suspensions is not only an essential part of biological research experiments, but also a key issue in the field of clinical medicine. In biological experiments, flow cytometry is often used to identify, count, and analyze parameters of cells in tissues. The key factor that determines whether the flow cytometer's analysis is accurate lies in the quality of the input single-cell suspension. Therefore, during the tissue lysis process, researchers will pursue lysis methods that have high cell yields, fast lysis rates, minimal cell damage, and strong cell activity. In clinical practice, the demand for rapid intraoperative testing is increasing, and related research is also gradually advancing. In single-cell-based detection methods, a convenient and rapid sample pretreatment method can greatly shorten the overall detection time and improve detection efficiency while ensuring high cell activity and high dispersion.

[0004] At present, traditional methods mostly use enzymatic and physical-mechanical methods to process tissue specimens to prepare single-cell suspensions. However, there are common problems such as long digestion time, severe mechanical damage to the tissue, and poor cell dispersion. Among them, the enzymatic method lyses tissue by destroying intertissue collagen fibers, intercellular connecting proteins, and polysaccharides. Compared with the physical-mechanical method, it greatly reduces the physical damage to cells, but there are problems such as slow digestion process and enzymes affecting cell activity. Therefore, a shaker is often used during the operation to achieve the purpose of shaking and mixing the tissue, but the digestion process generally still takes 1 to 2 hours (different types and sizes of tissue blocks will affect the digestion time), and it has certain requirements on the operator's skills. It is time-consuming and labor-intensive and cannot meet the needs of rapid lysis and preparation of single cells.

[0005] The disadvantages of the shaker enzymatic hydrolysis method are as follows: (1) The shaker plays a role in shaking and mixing the tissue during digestion, but the overall digestion time is still too long. Cells immersed in the enzyme solution for a long time can easily cause problems such as reduced cell activity or even death. (2) As can be seen from the operation steps, the shaker enzymatic hydrolysis method has many operation steps and a large workload. It is difficult to complete in a short time. All steps must be manually performed in a sterile environment, which requires high operator proficiency and operating experience. If the operator is not proficient, it is easy to cause problems such as contamination and excessive digestion leading to reduced cell activity.

[0006] The mechanical method is to cut and disperse the tissue blocks through mechanical contact or cutting force generated by liquid, so that the cells fall off and separate. The main methods are cutting, mesh squeezing, and blowing with a syringe or pipette. The disadvantages of the mechanical method are as follows: Although the operation steps of the mechanical method are simpler than the enzymatic method, since its principle is to use cutting force to forcibly break the connection between cells in the tissue, cells are very easy to die and break in this process, and the cell survival rate is much lower than that of the enzymatic method. A large amount of tissue sample support is required to obtain a sufficient number of living cells. Summary of the Invention

[0007] In order to make up for the deficiencies in the above fields, the main purpose of the present invention is to provide a method for rapid tissue lysis and an acoustic fluidic device, which helps to quickly and easily lyse tissue into a single cell suspension while ensuring high cell activity and dispersion.

[0008] The present application provides a method for rapid tissue lysis, comprising:

[0009] Adjusting the position of the acoustofluidic chip of the acoustofluidic device to face the tissue block, and immersing the tissue block and the acoustofluidic chip in the liquid;

[0010] The acoustofluidic chip generates a super-ultrasound wave, which, when propagating in the liquid, triggers an acoustofluidic effect, forming a forward jet and a vortex formed by a swirl, thereby generating a jet force, a shear force and an acoustofluidic vortex;

[0011] The tissue block in the liquid is rotated and captured in the vortex by the acoustic fluid vortex, the tissue block is impacted by the jet force, and the hydrolysis product is peeled off the tissue surface by the shear force.

[0012] Optionally, the liquid includes an enzyme solution; when the jet force impacts the tissue block, the diffusion of the enzyme between the solid and liquid media is accelerated; when the hydrolysis product is peeled off the tissue surface by the shear force, the deeper interstitial layer of the tissue surface is exposed to provide more enzyme binding sites.

[0013] Optionally, the liquid also includes antibodies that can bind to antigens on the cell membrane surface of cells of the tissue; when the tissue block in the liquid is rotated and captured in the vortex by the acoustic fluid vortex, the collision probability between the antibody and the antigen is increased, thereby promoting antigen-antibody binding.

[0014] Optionally, when the position of the acoustofluidic chip of the acoustofluidic device is adjusted to face the tissue block, the distance between the acoustofluidic chip and the tissue block is within 1 cm.

[0015] Optionally, the method further includes: the tissue block is placed in an inverted cone-shaped lysis chamber, the acoustic fluid chip extends into the lysis chamber, and the lysis chamber contains the liquid.

[0016] The present application also provides an acoustofluidic device, which is applied to any of the above-mentioned methods for rapid tissue lysis, wherein the acoustofluidic device comprises: an acoustofluidic chip, a PCB board, a tube shell, and an RF connecting line;

[0017] The acoustofluidic chip is arranged on the front of the PCB board, the signal terminal of the acoustofluidic chip is connected to the signal terminal on the front of the PCB board, and the ground terminal of the acoustofluidic chip is connected to the ground terminal of the PCB board; the tube shell is sleeved on the RF connecting wire; two wires extend from the end of the RF connecting wire close to the PCB board, namely a signal line and a ground line, which are respectively connected to the signal terminal and the ground terminal on the back of the PCB board, forming a power transmission path;

[0018] The acoustofluidic chip is used to generate ultra-ultrasound waves. When the ultra-ultrasound waves propagate in the liquid, they trigger the acoustofluidic effect, forming a forward-spraying jet and a vortex formed by the rotation, so as to generate jet force, shear force and acoustofluidic vortex; the acoustofluidic vortex is used to rotate and capture tissue blocks in the liquid in the vortex, the jet force is used to impact the tissue blocks, and the shear force is used to peel the hydrolysis products off the tissue surface.

[0019] Optionally, the acoustofluidic chip is a surface acoustic wave resonator chip, a bulk acoustic wave resonator chip or a piezoelectric ceramic chip.

[0020] Optionally, the BAW resonator chip is a solid-state mounted BAW resonator chip; the resonant frequency range of the solid-state mounted BAW resonator is 1 GHz to 10 GHz, and the applied power range is 0.1 W to 10 W.

[0021] Optionally, the acoustofluidic chip package is fixed on the front side of the PCB board.

[0022] Optionally, the signal end of the acoustofluidic chip and the signal end on the front of the PCB board are connected via a middle gold wire, and the ground end of the acoustofluidic chip and the ground end of the PCB board are connected via two left and right gold wires.

[0023] Optionally, the shape of the piezoelectric layer of the resonator may be circular, pentagonal, square, triangular, polygonal, etc. The length, width and height dimensions of the solid-state assembly type bulk acoustic wave resonator chip are 0.1 mm*0.1 mm*0.5 mm to 3 cm*3 cm*0.5 mm.

[0024] Optionally, the resonant frequency of the solid-assembly bulk acoustic wave resonator is 2.49 GHz, the applied power is 2 W, and the shape of the piezoelectric layer of the resonator is a pentagon.

[0025] The present invention uses SMR chips, but is not limited to them. BAW (Bulk Acoustic Wave) resonators (SMR belongs to BAW resonators), SAW (Surface Acoustic Wave Resonators), PZT (Piezoelectric) and other piezoelectric resonant devices can all achieve this operation.

[0026] In this packaging method, the SMR chip has dimensions of 0.1mm*0.1mm*0.5mm to 3cm*3cm*0.5mm (array resonator, increasing the processing area). The number of resonators on a chip ranges from 1 to 100.

[0027] In this packaging method, to adapt to the chip size, the length, width and height dimensions of the PCB board are 1mm*1mm*0.3mm to 4cm*4cm*0.3mm. The tube shell (300) is a cylindrical hollow tube with an inner diameter of 1 to 3mm, an outer diameter of 5 to 10mm, and a length of 3cm to 20cm. The length of the RF connecting line (400) is 3cm to 20cm.

[0028] The present invention also provides an automated lysis and biopsy device for rapid tissue lysis. The automated lysis and biopsy device for rapid tissue lysis provided by the present invention comprises a housing 11, wherein four vertical tubes are arranged in the housing 11, namely, a liquid inlet tube 12, a sample inlet tube 13, a sample outlet tube 14, and a waste liquid tube 15, wherein the injection tube 1 extends into the liquid inlet tube 12; a simple xy-axis displacement platform 3 is arranged at the upper end of the sample inlet tube 13, and the xy-axis displacement platform is used to fix the upper part of the acoustic fluid device 4, and the lower part of the acoustic fluid device 4 extends into the sample inlet tube 13; the liquid outlet tube 5 extends into the sample outlet tube 14; the waste liquid tube 6 extends into the waste liquid tube 15; the liquid inlet tube 12, the sample inlet tube 13, the sample outlet tube 14, the waste liquid tube 6 extends into the waste liquid tube 15; the liquid inlet tube 12, the sample inlet tube 13, the sample outlet tube 14, the waste liquid tube 15, ...15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 15, the waste liquid tube 1 13, the sample outlet tube 14, and the bottom of the waste liquid tube 15 are connected by a horizontal elbow 16, and the filter membranes 7, 8, 9 and 10 are respectively arranged in the horizontal elbow 16, wherein the filter membrane 7 is arranged between the liquid inlet tube 12 and the sample inlet tube 13; the filter membranes 8 and 9 are arranged between the sample inlet tube 13 and the sample outlet tube 14; the filter membrane 10 is arranged between the sample outlet tube 14 and the waste liquid tube 15; the bottom of the box body 11 is provided with a lysis chamber 17 at a position corresponding to the acoustic fluid device 4, and an observation chamber 18 is provided at a position corresponding to the sample outlet tube 14.

[0029] Preferably, the diameters of the injection tube 1, the outlet tube 5, and the waste liquid tube 6 are 1 to 10 mm, and are used to inject enzyme solutions and culture solutions; the diameters of the inlet tube 12, the sample injection tube 13, the sample outlet tube 14, and the waste liquid tube 15 are 10 mm to 30 mm; the lysis chamber 17 is an inverted conical chamber with an upper bottom diameter of 5 to 30 mm, a lower bottom diameter of 1 to 10 mm, and a depth of 5 to 30 mm; the observation chamber 18 is a cylindrical chamber with a diameter of 5 to 30 mm and a depth of 5 to 30 mm.

[0030] Preferably, the filter membrane 7 and the filter membrane 10 are 5-10 μm filter membranes, the filter membrane 8 is 70-100 μm filter membrane, and the filter membrane 9 is 30-50 μm filter membrane.

[0031] The present invention also provides a tissue lysis method. The tissue lysis method provided by the present invention comprises the following steps:

[0032] (1) placing the tissue to be lysed into the sample inlet tube of the automated lysis and biopsy device for rapid tissue lysis, and adjusting the position of the acoustofluidic device on the translation stage to face the tissue block to be lysed;

[0033] (2) introducing an enzyme solution from the injection tube 1 and immersing the tissue block to be lysed and the acoustic fluidic device in the solution;

[0034] (3) turning on the acoustofluidic device and waiting for the tissue block to swirl into the acoustic vortex to accelerate the enzymatic hydrolysis reaction, which lasts for 10 to 20 minutes;

[0035] (4) Filter and replace the enzyme solution. Inject the culture medium from the injection tube 1 and suck out the solution from the waste tube 6 at the same time. Continue the cycle for 2 to 5 minutes. The enzyme solution in the lumen will be replaced by the cell culture medium.

[0036] (5) Open the liquid outlet tube 5 and suck out the single cell suspension between the filter membranes 9 and 10.

[0037] The present invention also provides a tissue biopsy method. The tissue biopsy method provided by the present invention comprises the following steps:

[0038] (1) placing the tissue to be lysed into the sample inlet tube of the automated lysis and biopsy device for rapid tissue lysis, and adjusting the position of the acoustofluidic device on the translation stage to face the tissue block to be lysed;

[0039] (2) introducing a mixed solution of enzyme solution and fluorescent specific antibody from the injection tube 1, and immersing the tissue block to be lysed and the acoustofluidic device in the solution;

[0040] (3) turning on the acoustofluidic device and waiting for the tissue block to swirl into the acoustic vortex to accelerate the enzymatic hydrolysis reaction and antigen-antibody binding, and continuing the action for 10 to 20 minutes;

[0041] (4) Filter and replace the mixed solution of enzyme solution and fluorescent specific antibody. Inject culture fluid from injection tube 1 while aspirating the solution from waste tube 6. Continue the cycle for 2 to 5 minutes. The original mixed solution in the lumen will be replaced by cell culture fluid.

[0042] (5) The single cell morphology and fluorescence staining results are observed through the observation chamber 18 in combination with an inverted fluorescence microscope, and the presence of target cell types and the cell ratio are determined by the presence or absence of fluorescence and the intensity of fluorescence.

[0043] The present invention has the following beneficial effects:

[0044] 1. The present invention utilizes an acoustic fluidic device to accelerate the process of enzymatic hydrolysis reaction, shortens the time of tissue lysis, ensures high activity and high dispersion of cells, and solves the problems of long lysis time, poor cell activity, high cell mortality rate, and low cell acquisition rate in traditional methods.

[0045] 2. The present invention designs an easy-to-use automated biopsy device, which reduces manual operation, eases operational difficulty, and greatly simplifies the operating steps. This shortens the time required to prepare single-cell suspensions and mitigates contamination and other issues caused by improper operation. The device is suitable for use in scientific research experiments requiring rapid acquisition of single-cell suspensions and in clinical applications requiring rapid cell biopsy or cancer cell screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 Schematic diagram of the acoustofluidic device for rapid tissue lysis of the present invention; wherein, 100 is the acoustofluidic chip, 200 is the PCB board, 300 is the tube shell, and 400 is the RF connecting line.

[0047] Figure 2 Schematic diagram of the stacked structure of the SMR chip.

[0048] Figure 3 Schematic diagram of SMR digestion.

[0049] Figure 4 Schematic diagram of the automated lysis and biopsy device for rapid tissue lysis of the present invention; wherein: 1 - liquid injection tube, 2 - tube cover, 3 - xy-axis translation stage, 4 - acoustofluidic device, 5 - liquid outlet tube, 6 - waste liquid tube, 7 - filter membrane, 8 - filter membrane, 9 - filter membrane, 10 - filter membrane, 11 - housing, 12 - liquid inlet tube, 13 - sample inlet tube, 14 - sample outlet tube, 15 - waste liquid tube, 16 - transverse bend tube, 17 - lysis chamber, 18 - observation chamber.

[0050] Figure 5 This is a three-dimensional diagram of the automated lysis and biopsy device for rapid tissue lysis of the present invention.

[0051] Figure 6The following are the primary cell culture conditions after two lysis methods, where a is the cells lysed by the shaker group and b is the cells lysed by the SMR group.

[0052] Figure 7 The cell numbers and cell growth rates after treatment with shaker and lysis device, respectively.

[0053] Figure 8 The results are shown for lysis of 30 mg of renal tumor tissue under five different treatment conditions.

[0054] Figure 9 The fluorescence staining results at two different positions in the chamber 18 were observed under an inverted fluorescence microscope.

[0055] It should be understood that the sizes and shapes of the blocks in the above structural diagrams are for reference only and should not constitute an exclusive interpretation of the embodiments of the present invention. The relative positions and inclusion relationships between the blocks presented in the structural diagrams are merely schematic representations of the structural relationships between the blocks and do not limit the physical connection methods of the embodiments of the present invention. DETAILED DESCRIPTION

[0056] refer to Figure 1 , showing the acoustic fluid device for rapid tissue lysis of the present invention. 100 is an SMR chip with dimensions (length, width, height) of 0.8mm*0.5mm*0.5mm. 200 is a thin PCB board with dimensions (length, width, height) of 3mm*3mm*0.3mm. 100 and 200 are connected by three gold wires, the signal end of the SMR is connected to the signal end on the front of the PCB board (a gold wire in the middle), and the ground end is also connected (two gold wires on the left and right). 300 is a tube shell, which is a hollow cylinder made of copper with an inner diameter of 2mm and an outer diameter of 3mm. 400 is an RF connecting wire with a wire diameter of 1.3mm. Two wires extend from the end, which are a signal line and a ground line respectively. The tube shell 300 is put on the RF connecting wire 400. Two wires extend from the end of the RF connecting wire 400 close to the PCB board 200, and are respectively soldered to the two solder joints (signal end and ground end) on the back of the PCB board 200 using solder. The package forms a signal power transmission path for the SMR chip, and the RF connecting line 400 is connected to the signal generator to drive the SMR device to work.

[0057] The thin PCB acts as a transition piece in this acoustofluidic device, connecting the SMR chip on one side (gold wire connection) and the RF line on the other side (solder connection). Because the SMR chip is small and its electrodes are also small, it cannot be directly connected to the RF line to apply power.

[0058] The SMR chip is bonded to the designated position on the PCB board using AB glue, and the two are firmly connected.

[0059] The SMR ground, PCB ground, and RF line ground are connected. The SMR signal, PCB signal, and RF signal are connected.

[0060] RF cables are soft and easily bend, which can lead to structural instability and difficulty in adjusting device positions. Therefore, a hard tube shell is processed to cover the RF cable to ensure a stable connection.

[0061] First, the tube shell 300 is put on the RF connecting wire 400, and AB glue is used to bond the tube shell 300 and the RF connecting wire 400 at the connection end (SMR connector).

[0062] The two wires at the end of the RF connecting line 400 are a signal line and a ground line respectively. The two solder joints on the back of the thin PCB board 200 correspond to the signal end and the ground end of the SMR chip. The purpose of soldering is to conduct this path. Figure 1 The hexagon and the thread behind it are SMR connectors, which are part of the RF cable.

[0063] This package forms the signal and power transmission path for the SMR chip. Through the connection between the PCB and RF cables, a sinusoidal signal from a signal generator is transmitted to the chip, driving its operation. The specific principle of the signal generator's operation is that the piezoelectric material, based on the inverse piezoelectric effect, converts a sinusoidal voltage (a GHz sinusoidal signal provided by the signal generator) applied to the electrodes at a specific frequency into mechanical deformation of the piezoelectric thin film AlN. This periodic deformation caused by the alternating electrical signal generates bulk acoustic waves (BAUs) within the piezoelectric AlN film, propagating along the thickness of the film, known as hypersonic waves. Due to the use of a Bragg reflector structure, the hypersonic waves are totally reflected at the interface between the upper electrode and the air and the lower electrode and the Bragg reflector, generating acoustic waves traveling in the opposite direction of their original propagation. These waves then superimpose to form standing waves, leading to resonance. When hypersonic waves propagate in liquids, they trigger the acoustohydrodynamic effect, a nonlinear fluid effect caused by the attenuation of BAUs. This effect manifests as a forward-moving jet and swirling vortices.

[0064] The purpose of this package is: (1) to design it as an insertable device, reducing the package size and allowing it to work deep into pipes or cavities. (2) to facilitate clamping and enable precise manipulation when used with a translation stage.

[0065] The RF connecting line is used to transmit power, and one end of the RF connecting line is an SMA connector, which can be connected to the SMA connector transmission line extending from the signal generator.

[0066] The stacked structure of the SMR chip is as follows Figure 2 As shown, it consists of a silicon substrate, a Bragg reflection layer formed by Mo and SiO2, a bottom electrode Mo, a piezoelectric layer AlN, a top electrode Mo and a gold electrode.

[0067] like Figure 3 As shown, the SMR chip (gigahertz bulk acoustic wave resonator) in the acoustofluidic device of the present invention can generate strong jet force and strong shear force to lyse fresh tissue, and use the acoustic pressure field generated by gigahertz ultrasound to drive the enzyme solution to form an acoustofluid vortex, which rotates and captures the tissue block in the vortex to perform layer-by-layer interstitial digestion and cell peeling. The jet force impacts the tissue block, accelerates the diffusion of the enzyme between the solid and liquid media, increases the surface area of the tissue, and accelerates the hydrolysis rate of the interstitial tissue. The shear force quickly peels the hydrolysis product off the tissue surface, exposing deeper interstitial tissue and providing more binding sites for the enzyme. At the same time, the acoustofluidic vortex can also increase the collision probability of fluorescent antibodies and antigens on the cell membrane surface, promote antigen-antibody binding, and accelerate the process of immunofluorescence staining. Using the acoustofluidic device of the present invention to process tissue specimens greatly accelerates the lysis rate compared to traditional lysis methods, can quickly lyse tissue into a single cell suspension, and can maintain cell activity. Moreover, the acoustofluidic device can simultaneously accelerate the tissue lysis and antigen-antibody staining process. It only takes 10 to 30 minutes to prepare the tissue block into a single-cell suspension stained with immunofluorescence, which has promoted the development of liquid biopsy and cancer cell detection for clinical applications.

[0068] refer to Figure 4 and Figure 5, shows the automated lysis and biopsy device for rapid tissue lysis of the present invention. 1 is an injection tube with a diameter of 1 to 10 mm, which is used to inject enzyme solution and culture medium. 2 is a tube cover, which is used to fix the injection tube 1 and prevent debris from falling into the device. The lower end is a rubber plug to seal the pipeline. 3 is a simple xy-axis displacement stage, which is used to adjust the position of 4 (SMR device) to facilitate alignment of tissue blocks. It is made of transparent resin 3D printing. 4 is the above-mentioned acoustic fluid device, that is, the SMR device. 5 is an outlet pipe, which is used to extract the single cell suspension after the lysis is completed. 6 is a waste liquid pipe, which is used to suck out excess waste liquid and replace the enzyme solution. The injection tube 1, the outlet tube 5, and the waste liquid tube 6 are all connected to a peristaltic pump for injecting or extracting liquid. 7, 8, 9, and 10 are all filter membranes used to filter cells. Common cell sizes are generally 10 to 20 μm. Filter membranes 7 and 10 are 5-10 μm filters, filter membrane 8 is 70-100 μm, and filter membrane 9 is 30-50 μm. Filter membranes 7 and 8 confine the tissue mass to a certain area for lysis. Single cells after lysis cannot pass through filters 7 and 10, but can pass through filters 8 and 9. Filter membranes 8 and 9 also restrict the passage of cell clusters, so single cells are trapped between filters 9 and 10. Reference numeral 11 represents the device housing, within which the pipes, as shown, have a diameter of 15 mm. The housing is made of PMMA. Four openings are provided in the housing, and the pipes are contained within the housing. Reference numeral 17 represents an inverted conical lysis chamber with an upper base diameter of 5-30 mm, a lower base diameter of 1-10 mm, and a depth of 5-30 mm. A tissue mass is placed in the chamber, and the acoustofluidic device is moved into alignment and extends into the chamber to rapidly lyse the tissue mass. 18 is a cylindrical observation chamber with a diameter of 5 to 30 mm and a depth of 5 to 30 mm. The single cell suspension is located behind the sample tube 14, and the cell morphology and fluorescent staining can be observed online from the bottom of the observation chamber using an inverted microscope.

[0069] A simple xy-axis translation stage fixes the device by clamping. The translation stage has a clamping hole. The device is passed through the hole, and the screw is turned to tighten and narrow the hole, clamping the end of the device (away from the SMR chip end).

[0070] like Figure 4As shown, the automated lysis and biopsy device for rapid tissue lysis of the present invention comprises a housing 11, wherein four vertical tubes are respectively arranged in the housing 11, namely, a liquid inlet tube 12, a sample inlet tube 13, a sample outlet tube 14, and a waste liquid tube 15. The liquid injection tube 1 extends into the liquid inlet tube 12; a simple xy-axis translation stage 3 is provided at the upper end of the sample inlet tube 13, and the xy-axis translation stage is used to fix the upper part of the acoustofluidic device 4, and the lower part of the acoustofluidic device 4 extends into the sample inlet tube 13; the liquid outlet tube 5 extends into the liquid inlet tube 12; The sample inlet and outlet pipes 14 are connected; the waste liquid pipe 6 extends into the waste liquid pipe 15; the bottoms of the liquid inlet pipe 12, the sample inlet pipe 13, the sample outlet pipe 14, and the waste liquid pipe 15 are connected by a transverse elbow 16, and filter membranes 7, 8, 9, and 10 are respectively arranged in the transverse elbow 16, wherein the filter membrane 7 is arranged between the liquid inlet pipe 12 and the sample inlet pipe 13; the filter membranes 8 and 9 are arranged between the sample inlet pipe 13 and the sample outlet pipe 14; and the filter membrane 10 is arranged between the sample outlet pipe 14 and the waste liquid pipe 15. The bottom of the housing 11 is provided with a lysis chamber 17 at a position corresponding to the acoustofluidic device 4, and an observation chamber 18 is provided at a position corresponding to the sample outlet pipe 14.

[0071] Example 1: Rapid tissue lysis

[0072] The operating procedures of the automated lysis and biopsy device for rapid tissue lysis of the present invention are as follows:

[0073] ① Washing: Soak the tissue in DMEM containing penicillin (400 U / ml) and streptomycin (0.4 mg / ml) for 15 minutes. Mince the fresh renal tumor tissue into 1 mm x 1 mm pieces using a scalpel while in the culture medium. Wash the tissue two to three times with D-Hanks. Fresh renal tumor tissue was obtained from human renal tumors provided by the Second Hospital of Medical University. The pieces were approximately 5 x 5 x 5 mm in size and weighed approximately 100 mg.

[0074] ②Digestion:

[0075] 1. Place the tissue block from the sample injection tube 13 into the lysis chamber 17. Use the translation stage to adjust the position of the SMR device to face the tissue block within 1 cm.

[0076] Specifically, the tissue block is placed into the sampling tube 13 through the opening on the sampling tube 13 . The tissue block is placed first, and then the acoustofluidic device is clamped on the displacement stage and inserted into the sampling tube 13 to adjust the position for processing.

[0077] 2. Inject an appropriate amount of enzyme solution from injection tube 1 and immerse the tissue block and SMR device in the solution.

[0078] 3. Turn on the SMR device, and the signal generator outputs a 2.49 GHz sinusoidal signal. The power is adjusted to 2 W. The tissue block is rotated into the acoustic vortex to accelerate the enzymatic hydrolysis reaction. The reaction lasts for 15 minutes.

[0079] 4. Filter and replace the enzyme solution. Inject culture medium from injection tube 1 while aspirating the solution from waste tube 6. Cells will follow the flow through two filter membranes, filter 8 and filter 9 (removing incompletely digested tissue and cell clusters). Cell fragments will follow the flow through filter 10 and be aspirated with the waste liquid. This cycle continues for 3 minutes, replacing the enzyme solution in the lumen with cell culture medium.

[0080] 5. Open the liquid outlet tube 5 and aspirate the single cell suspension between the filter membranes 9 and 10.

[0081] Control experiment:

[0082] Shaking table enzymatic hydrolysis method, the specific method is as follows:

[0083] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 minutes.

[0084] ② Shearing: Use a sharp scalpel to cut fresh renal tumor tissue into small pieces of 1mm*1mm in size under culture medium.

[0085] ③ Washing: Soak and wash the tissue 2 to 3 times with D-Hanks or PBS buffer.

[0086] ④Digestion: Add enzyme solution (type and concentration depend on tissue type) into the centrifuge tube, place the tissue block into the centrifuge tube, and shake the tissue on a shaker at a constant temperature of 37°C for 1 to 2 hours according to standard operation (determine the digestion time according to the tissue digestion situation, and digestion is sufficient when the tissue becomes fluffy and flocculent).

[0087] ⑤ Centrifugation: After treatment, perform a rapid centrifugation at 200g for 5 minutes. Aspirate the enzyme solution above and replace it with culture medium. Repeat the entire centrifugation process three times to ensure complete removal of the enzyme solution.

[0088] ⑥ Filtration: Use an appropriate amount of culture medium to repeatedly blow and disperse the cells fully, and filter the cells with a cell strainer to remove undigested tissue and large impurities.

[0089] The control experiment using only enzyme is as follows:

[0090] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 minutes.

[0091] ② Shearing: Use a sharp scalpel to cut fresh tumor tissue into small pieces of 1mm*1mm in size under culture medium.

[0092] ③ Washing: Soak and wash the tissue 2 to 3 times with D-Hanks or PBS buffer.

[0093] ④Digestion: Add enzyme solution (type and concentration depend on tissue type) into the centrifuge tube, place the tissue block into the centrifuge tube, and incubate at a constant temperature of 37° for 1 to 2 hours.

[0094] The control experiment without enzyme and shaker is as follows:

[0095] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 minutes.

[0096] ② Shearing: Use a sharp scalpel to cut fresh tumor tissue into small pieces of 1mm*1mm in size under culture medium.

[0097] ③ Washing: Soak and wash the tissue 2 to 3 times with D-Hanks or PBS buffer.

[0098] ④Digestion: Add cell culture medium (depending on the tissue type) to the centrifuge tube, place the tissue block into the centrifuge tube, and use a shaker to shake the tissue at a constant temperature of 37° for 1 to 2 hours.

[0099] The control experiment of no enzyme + acoustic fluidic lysis device, the specific method is as follows:

[0100] ① Cleaning: Soak the tissue in DMEM culture medium containing penicillin (concentration 400U / ml) and streptomycin (concentration 0.4mg / ml) for 15 minutes. Use a scalpel to cut the fresh tumor tissue into small pieces of 1mm*1mm in size under the culture medium, and soak and clean the tissue with D-Hanks for 2 to 3 times.

[0101] ②Digestion:

[0102] 1. Place the tissue block from the sample injection tube 13 into the lysis chamber 17. Use the translation stage to adjust the position of the SMR device to face the tissue block within 1 cm.

[0103] Specifically, the tissue block is placed into the sampling tube 13 through the opening on the sampling tube 13 . The tissue block is placed first, and then the acoustofluidic device is clamped on the displacement stage and inserted into the sampling tube 13 to adjust the position for processing.

[0104] 2. Inject an appropriate amount of cell culture fluid from injection tube 1 and immerse the tissue block and SMR device in the solution.

[0105] 3. Turn on the SMR device, and the signal generator outputs a 2.49 GHz sinusoidal signal with the power adjusted to 2 W for 15 minutes.

[0106] Fresh renal tumor tissues of the same origin and size were lysed using the traditional shaker enzymatic hydrolysis method and the automated lysis and biopsy device of the present invention. The results are as follows: Figure 6 As shown, the experiment verified that the single-cell suspension lysed by the automated lysis and biopsy device of the present invention has successfully completed primary culture. The number of cells lysed in only 15 minutes reached the amount that the shaking group could lyse in 1 hour and 15 minutes. According to the processing results of the 11-day growth photos of cells at the same site (both groups randomly selected 5 sites for 1-11 days), the total number of cells per day was slightly higher than that of the shaking group, and the cell division rate was basically the same. The automated lysis and biopsy device of the present invention increased the lysis rate by 4 to 5 times compared to traditional methods while ensuring cell activity, demonstrating its feasibility in the application of rapid tissue lysis to prepare single-cell suspensions.

[0107] Figure 7 The left figure shows the change in cell number over the days after treatment with a shaker and a lysis device, respectively. The statistical source is Figure 6 The photographic results in the image were taken with a field of view of 2500 x 1900 μm. Five random locations were selected at the bottom of the culture flask to record the number of cells in each group each day. The average of these five locations was used as the final result. It can be seen that for tissues of the same mass, the number of cells treated with the lysis device was higher. Figure 7 The right graph shows the cell growth rate every two days. The growth rate of the lysis device group and the shaker group was almost the same in each cycle, demonstrating that the lysis device did not reduce cell activity or inhibit their normal division compared to traditional dissociation methods, but had a significant advantage in lysis speed.

[0108] Figure 8 30mg of renal tumor tissue was lysed under five different treatment conditions. 50ul of the solution was sampled at regular intervals during the treatment process. After staining with calcein, the cells were photographed and counted. The number of single cells lysed at different digestion times was counted. The data were then statistically analyzed and curve fitted to plot the changes in cell number and lysis rate under different treatment conditions with treatment time. Figure 8 It can be seen that the automated lysis device of the present invention can accelerate the enzymatic digestion process of tissue blocks, and the lysis efficiency is much greater than that of other methods.

[0109] Example 2: Tissue biopsy

[0110] The operating procedures of the automated lysis and biopsy device for tissue biopsy of the present invention are as follows:

[0111] ① Washing: Soak the tissue in DMEM containing penicillin (400 U / ml) and streptomycin (0.4 mg / ml) for 15 minutes. Mince the fresh renal tumor tissue into 1 mm x 1 mm pieces using a scalpel. Wash the tissue two to three times with D-Hanks. Fresh renal tumor tissue was obtained from human renal tumors provided by the Second Hospital of Medical University. The pieces were approximately 5 x 5 x 5 mm in size and weighed approximately 100 mg.

[0112] ②Digestion:

[0113] 1. Place the tissue block from the sample injection tube 13 into the lysis chamber 17. Use the translation stage to adjust the position of the SMR device to face the tissue block within 1 cm.

[0114] 2. Inject an appropriate amount of a mixed solution of enzyme solution and fluorescent-specific antibody from injection tube 1 and immerse the tissue block and SMR device in the solution. The fluorescent-specific antibody is Abcam CA9 antibody conjugated with a PE fluorescent group.

[0115] 3. Turn on the SMR device, and the signal generator outputs a 2.49 GHz sinusoidal signal. The power is adjusted to 2W. The tissue block is rotated into the acoustic vortex to accelerate the enzymatic reaction and antibody-antigen binding process. The action lasts for 15 minutes.

[0116] 4. Filter and replace the enzyme solution. Inject culture medium from injection tube 1 while aspirating the solution from waste tube 6. Cells will follow the flow through two filter membranes, filter 8 and filter 9 (removing incompletely digested tissue and cell clusters). Cell fragments will follow the flow through filter 10 and be aspirated with the waste liquid. This cycle continues for 3 minutes, replacing the enzyme solution in the lumen with cell culture medium.

[0117] 5. Use an inverted fluorescence microscope to observe the single cell morphology and fluorescence staining results through the observation chamber 18, and judge whether there are target cells and the approximate proportion of cells by the presence or absence of fluorescence and the intensity of fluorescence.

[0118] Figure 9To observe the fluorescence staining results at two different locations in chamber 18 under an inverted fluorescence microscope, calcein marks all living cells, and the CA9 antibody with PE fluorescence marks the CA9 protein that is highly expressed in clear renal cancer cells. As can be seen from the figure, the cells are fully dispersed into single cells, and the clear renal cancer cells with high CA9 expression are fully stained. The fluorescence intensity is much higher than that of other cells and can be easily distinguished. This proves that the SMR device can fully accelerate the enzymatic hydrolysis and antigen-antibody binding processes at the same time. The processing time of 15 minutes can complete the single cell preparation and sufficient staining of fluorescent antibodies. According to the imaging results of the two locations in the figure, it can be preliminarily determined that the cancer cell type of this tissue is clear renal cancer cells. Through image counting statistics, it is found that the proportion of cancer cells is about 4%, which can be inferred that the malignancy of the tissue at this location is not high.

[0119] The specific operation of image counting is to take images of cells at five different locations in the observation chamber 18 under a microscope and import the images into Image J. First, the RGB color channels are separated, then the images of different channels are binarized, and finally the cell count is counted using the built-in counting plug-in of Image J. The statistical results of the three different RGB color channels represent the number of cells emitting red, green, and blue fluorescence, respectively. For example, the statistical results of the R channel in this experiment are the number of cells emitting red fluorescence, that is, the number of clear renal cancer cells that show PE red fluorescence due to high expression of CA9.

[0120] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for rapid tissue lysis, characterized in that: include: Adjusting the position of the acoustofluidic chip of the acoustofluidic device to face the tissue block, and immersing the tissue block and the acoustofluidic chip in the liquid; The acoustofluidic chip generates a super-ultrasound wave, which, when propagating in the liquid, triggers an acoustofluidic effect, forming a forward jet and a vortex formed by a swirl, thereby generating a jet force, a shear force and an acoustofluidic vortex; The tissue block in the liquid is rotated and captured in the vortex by the acoustic fluid vortex, the tissue block is impacted by the jet force, and the hydrolysis product is peeled off the tissue surface by the shear force.

2. The method according to claim 1, characterized in that The liquid includes an enzyme solution; When the jet force impacts the tissue block, the diffusion of the enzyme between the solid and liquid media is accelerated; When the hydrolysis product is peeled off the tissue surface by shear force, the deeper interstitial layer of the tissue surface is also exposed to provide more enzyme binding sites.

3. The method according to claim 1 or 2, characterized in that Also includes: The liquid also includes antibodies that can bind to antigens on the cell membrane surface of cells of the tissue; When the tissue mass in the liquid is rotated and captured in the vortex by the acoustic fluid vortex, the collision probability between the antibody and the antigen is increased, thereby promoting antigen-antibody binding.

4. The method according to any one of claims 1 to 3, characterized in that: When the position of the acoustofluidic chip of the acoustofluidic device is adjusted to face the tissue block, the distance between the acoustofluidic chip and the tissue block is within 1 cm.

5. The method according to any one of claims 1 to 4, characterized in that: Also includes: The tissue block is placed in an inverted cone-shaped lysis chamber, the acoustic fluid chip extends into the lysis chamber, and the lysis chamber contains the liquid.

6. An acoustofluidic device, characterized in that: The method for rapid tissue lysis according to any one of claims 1 to 5, wherein the acoustofluidic device comprises: an acoustofluidic chip, a PCB board, a tube shell, and an RF connecting line; The acoustofluidic chip is arranged on the front of the PCB board, the signal terminal of the acoustofluidic chip is connected to the signal terminal on the front of the PCB board, and the ground terminal of the acoustofluidic chip is connected to the ground terminal of the PCB board; the tube shell is sleeved on the RF connecting wire; two wires extend from the end of the RF connecting wire close to the PCB board, namely a signal line and a ground line, which are respectively connected to the signal terminal and the ground terminal on the back of the PCB board, forming a power transmission path; The acoustofluidic chip is used to generate ultra-ultrasound waves. When the ultra-ultrasound waves propagate in the liquid, they trigger the acoustofluidic effect, forming a forward-spraying jet and a vortex formed by the rotation, so as to generate jet force, shear force and acoustofluidic vortex; the acoustofluidic vortex is used to rotate and capture tissue blocks in the liquid in the vortex, the jet force is used to impact the tissue blocks, and the shear force is used to peel the hydrolysis products off the tissue surface.

7. The acoustofluidic device according to claim 6, wherein: The acoustofluidic chip is a surface acoustic wave resonator chip, a bulk acoustic wave resonator chip or a piezoelectric ceramic chip.

8. The acoustofluidic device according to claim 7, wherein: The BAW resonator chip is a solid-state mounted BAW resonator chip; the resonant frequency range of the solid-state mounted BAW resonator is 1 GHz to 10 GHz, and the applied power range is 0.1 W to 10 W.

9. The acoustofluidic device according to claim 6, wherein: The acoustofluidic chip package is fixed on the front side of the PCB board.

10. The acoustofluidic device according to claim 6, wherein: The signal end of the acoustofluidic chip and the signal end on the front of the PCB are connected via a gold wire in the middle, and the ground end of the acoustofluidic chip and the ground end of the PCB are connected via two gold wires on the left and right.

Images