CN115386568B

CN115386568B - Cell regulation and control method, cell regulation and control device and cell mechanical property measurement method

Info

Publication number: CN115386568B
Application number: CN202211133863.7A
Authority: CN
Inventors: 胡意; 范真真; 王裕琳
Original assignee: Tianjin University
Current assignee: Tianjin University
Priority date: 2022-09-16
Filing date: 2022-09-16
Publication date: 2023-12-05
Anticipated expiration: 2042-09-16
Also published as: CN115386568A

Abstract

A cell regulation method, a device and a measurement method of cell mechanical properties, wherein the cell regulation method comprises the following steps: the cell solution was fed into the microchannel with fibronectin at the bottom. Applying a first ultrasonic surface standing wave signal along a first direction and a second ultrasonic surface standing wave signal along a second direction to the micro-channel to capture cells in the cell solution and adhere the captured cells; injecting microsphere solution into the micro-channel; applying a third ultrasonic surface standing wave signal and a fourth ultrasonic surface standing wave signal to the micro-channel to capture the microspheres; changing the third ultrasonic surface standing wave signal with the second pressure node offset in the first direction relative to the first pressure node such that the captured microspheres move toward the corresponding adherent cells; in the event that the second pressure node is offset in a second direction relative to the first pressure node, the fourth ultrasonic surface standing wave signal is altered such that the captured microspheres move along toward the corresponding adherent cells.

Description

Cell regulation and control method, cell regulation and control device and cell mechanical property measurement method

Technical Field

The invention relates to the field of cell regulation, in particular to a cell regulation method, a cell regulation device and a cell mechanical property measurement method.

Background

The cells can generate and sense mechanical force and act together with biochemical signals to regulate vital activities such as cell adhesion, cell migration, stem cell differentiation, organ development and the like. Various mechanical tools are not separated in the process of researching cell mechanical sensing and response: cell diffusion and growth can be induced by stretching the cell-attached matrix, and the viscoelasticity of the different cells can be measured using optical stretching. However, in a realistic ecological environment, the cells are not always subjected to global forces, and a dynamic anisotropic microenvironment is likely to act on the cells in a local force manner. Therefore, a new method for locally applying mechanical stimulus to cells based on ultrasound and targeted microbubbles is proposed for the first time in 2013, which is called an ultrasound tweezer cell regulation technique (Acoustic tweezing cytometry, ATC). The ultrasonic tweezers cell control technique (ATC) is a highly efficient tool that can apply controllable mechanical forces with subcellular resolution to cells.

The core of ATC is to drive RGD coated microbubbles attached to the cell membrane by using ultrasonic pulses of low sound pressure, and the radiation force generated by the ultrasonic action on the microbubbles is transmitted to the inside of the cells through the RGD-integrin-actin cytoskeleton, thereby causing rapid contraction of the cytoskeleton. At present, ATC has demonstrated its unique application potential in the biological fields of cell mechanical property detection, stem cell fate control and the like.

The main components of ATC are ultrasonic transducers and microbubbles. The ultrasound transducers used therein can be easily integrated with optical microscopes and are compatible with the workflow of biological experiments. The ultrasonic transducer is typically located at a distance from the cells at the bottom of the dish in order to drive the microbubbles using a far field acoustic field. Sound pressure in the far field acoustic field is relatively stable compared to the near field where sound pressure varies sharply with space. It can thus be assumed that the sound pressure is constant in the sound field action region aligned with the ultrasonic probe. The ultrasound transducer is typically tilted 45 deg. from the vertical to minimize interference with the reflected sound field while being compatible with the optical path of the microscope. Microbubbles (radius 1-3 μm) are bubbles encapsulated with lipids, polymers or proteins, and are a common in vivo ultrasound contrast agent. The use of ligands to modify the shell of the vesicle allows the microbubbles to bind to specific receptors on the cell membrane. By stimulating the targeted microbubbles attached to the cell membrane, ATC concentrates and amplifies the acoustic energy onto the cell membrane. The acoustic impedance mismatch between the internal gas of the microbubbles and the surrounding liquid allows the microbubbles to have strong acoustic scattering capability. When the microbubbles are subjected to ultrasonic action, the volumes of the microbubbles vibrate due to cavitation effect; at the same time, the nonlinear effect of the acoustic field will generate primary acoustic radiation forces on the microbubbles, which will translate in the direction of acoustic propagation. In order to effectively move the microbubbles while suppressing oscillation of the microbubbles, the ultrasonic signal applied by the ATC is a pulse train signal having a frequency close to the resonance frequency of the microbubbles themselves and a low sound pressure. In a 1MHz ultrasound field with a sound pressure of 0.05MPa, microbubbles with a radius of 2.3 μm were subjected to a primary acoustic radiation force of about 17nN. The acoustic field forces can be transmitted to the inside of the cell through the structure of the microbubble-receptor-cytoskeleton, and such mechanical forces are converted into various biological effects through coupling between mechanical and biochemical signals.

However, microbubbles themselves have poor reliability and stability; microbubbles vary in size from 0.7 μm to 10 μm and the stability of the microbubbles themselves is highly susceptible to rupture by ambient conditions such as temperature, pressure, etc. Therefore, the related ultrasonic forceps cell regulation and control method has more defects, and the microbubbles are randomly targeted to realize connection with cells by means of self buoyancy through inverting the cell culture dish, so that the connection position is uncontrollable. In addition, in the regulation and control process, microbubbles are excited by ultrasonic action, cavitation nuclei can expand and shrink sharply and burst even, local temperature and pressure in the nuclei rise sharply, and secondary effects such as strong shock waves, inscription force, high-speed microbeam current, free radicals and the like can be generated, so that the microvessels are ruptured, vascular endothelial cells shrink, cell gaps are widened, permeability of the walls of the microvessels is increased, and cell membrane integrity is damaged. Therefore, in the original ultrasonic tweezer cell regulation method, in order to reduce the damage to cells during microbubble cavitation, the sound field sound pressure is greatly limited. In addition, the ultrasonic probe is obliquely immersed in the cell culture solution at 45 degrees, the generated sound field is complex to propagate in the cell culture dish, and the sound field is difficult to express by numerical values through various refraction and reflection, so that the radiation force suffered by the microbubbles cannot be accurately calculated; the ultrasonic probe is immersed in the cell culture solution at an angle of 45 degrees, so that the cell culture dish is extremely easy to dye bacteria, long-term cell culture cannot be carried out, the cells grow extremely randomly in the cell culture dish, and the observation is carried out by adopting a 40-fold mirror, so that single experiment can only act and observe single cells or single cell colonies, and the experimental flux is low.

Disclosure of Invention

Accordingly, the present invention is directed to a cell control method, a cell control device and a cell mechanical property measurement method, which are used for solving at least one of the above-mentioned problems.

In order to achieve the above object, as one aspect of the present invention, there is provided a cell modulating method comprising:

and inputting the cell solution into a micro-channel with fibronectin at the bottom, and filling the micro-channel with the cell solution.

Applying a first ultrasonic surface standing wave signal along a first direction and a second ultrasonic surface standing wave signal along a second direction to the micro-channel to capture cells in the cell solution and adhere the captured cells to obtain adherent cells at a first pressure node of the first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal, wherein the first direction is perpendicular to the second direction;

injecting microsphere solution into the micro-flow channel to uniformly distribute microspheres in the microsphere solution in the micro-flow channel;

applying a third ultrasonic surface standing wave signal and a fourth ultrasonic surface standing wave signal to the fluidic channel to capture the microspheres at a second pressure node of the third ultrasonic surface standing wave signal and the fourth surface standing wave signal, wherein the second pressure node is offset in the first direction or the second direction relative to the first node;

In the case that the second pressure node is offset in the first direction relative to the first pressure node: changing the third ultrasonic surface standing wave signal so that the captured microspheres move towards the corresponding adherent cells until the adherent cells are adhered;

in the case that the second pressure node is offset in the second direction relative to the first pressure node: the fourth ultrasonic surface standing wave signal is altered such that the captured microspheres move along toward the corresponding adherent cells until the adherent cells are adhered.

According to an embodiment of the invention, the microsphere surface is modified with RGD polypeptides.

According to an embodiment of the invention, altering the third ultrasonic surface standing wave signal such that the captured microspheres move towards the corresponding adherent cells until the adherent cells are adhered, comprises:

changing the third ultrasonic surface standing wave signal so that the captured microsphere moves towards the corresponding adherent cell until the RGD polypeptide on the surface of the microsphere is connected with integrin on the cell membrane surface of the corresponding adherent cell;

changing the fourth ultrasonic surface standing wave signal such that the captured microspheres move along toward the corresponding adherent cells until the adherent cells are adhered, comprising:

Changing the fourth ultrasonic surface standing wave signal such that the captured microsphere moves towards the corresponding adherent cell until the RGD polypeptide of the microsphere surface is attached to the integrin of the cell membrane surface of the corresponding adherent cell.

According to an embodiment of the invention, the third ultrasonic surface wave signal is generated by two first ultrasonic surface wave signals having opposite propagation directions.

The altering the third ultrasonic surface standing wave signal comprises:

the phase difference between the two first ultrasonic surface wave signals with opposite propagation directions is changed.

The fourth ultrasonic surface standing wave signal is generated by two second ultrasonic surface wave signals with opposite propagation directions;

the altering the fourth ultrasonic surface standing wave signal comprises:

the phase difference between the two second ultrasonic surface wave signals with opposite propagation directions is changed.

According to an embodiment of the invention, the concentration of cells in the cell solution is 2.6-2.8X106 cells/mL.

According to an embodiment of the present invention, before the cell solution is input into the micro flow channel, the acoustic tweezer cell regulation method further includes:

injecting fibronectin solution into the micro flow channel, sealing and standing to enable the bottom of the micro flow channel to be provided with fibronectin;

Injecting ultrapure water into a micro-channel filled with a fibronectin solution, so that the micro-channel is filled with the ultrapure water;

injecting a cell solution from a first port of the micro flow channel to discharge the ultrapure water from a second port of the micro flow channel until the micro flow channel is filled with the cell solution.

According to the embodiment of the invention, the microsphere is made of polystyrene;

the concentration of the microspheres in the microsphere solution is 1.5 x 10 ⁷ And each mL.

As a second aspect of the present invention, there is also provided a cell regulation apparatus for realizing the cell regulation method as described above, comprising:

the signal generator module is suitable for emitting an electric signal;

an acoustic surface standing wave acoustic tweezer comprising:

a piezoelectric substrate;

the micro-flow channel is arranged on the piezoelectric substrate and is suitable for accommodating cell solution;

the two groups of electrode groups are arranged on the piezoelectric substrate in pairs and are positioned at the periphery of the micro-channel, wherein each group of electrode groups comprises two interdigital electrodes which are oppositely arranged; the two groups of electrodes transmit the electric signals sent by the first signal generator and the second signal generator to the piezoelectric substrate to generate a first ultrasonic surface standing wave signal, a second ultrasonic surface standing wave signal, a third ultrasonic surface standing wave signal and a fourth ultrasonic surface standing wave signal;

The first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal are suitable for capturing cells in the cell solution and enabling the captured cells to adhere to the wall to obtain adherent cells, the third ultrasonic surface standing wave signal and the fourth ultrasonic surface standing wave signal are suitable for capturing microspheres entering the micro flow channel, and the signal generator module is further suitable for enabling the third ultrasonic surface standing wave signal to be changed or enabling the fourth ultrasonic surface standing wave signal to be changed.

According to an embodiment of the present invention, the cell control device further includes:

a light source;

a microscope adapted to observe said cells and said cells under illumination from a light source;

and the computer is suitable for recording the microscope observation data.

As a third aspect of the present invention, there is also provided a method for measuring mechanical properties of cells, comprising:

step A: attaching microspheres to adherent cells using a cell control method as described above;

and (B) step (B): changing the third ultrasonic surface standing wave signal again in the case that the second pressure node is offset in the first direction relative to the first pressure node so that the microsphere moves to involve the adherent cells to deform the adherent cells, and changing the fourth ultrasonic surface standing wave signal in the case that the second pressure node is offset in the second direction relative to the first pressure node so that the microsphere moves to involve the adherent cells to deform the adherent cells; step C: removing the again modified third ultrasonic surface standing wave signal if the second pressure node is offset in the first direction relative to the first pressure node; removing the second modified ultrasonic surface standing wave signal if the second pressure node is offset in the second direction relative to the first pressure node;

Step D: repeating the step B-step C for preset times;

step E: and (3) obtaining a time-dependent change curve of the displacement of the microspheres in the process of the step B-the step C each time so as to measure the mechanical properties of the cells.

According to the embodiment of the invention, the first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal which are perpendicular to each other are applied to the micro-channel, so that the cell is attached to the pressure node of the first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal, and the position of the cell is fixed. The microspheres are captured at pressure nodes of the third ultrasonic surface standing wave signal and the fourth surface standing wave signal by applying the third ultrasonic surface standing wave signal and the fourth surface standing wave signal to the fluidic channel. The third ultrasonic surface standing wave signal or the fourth surface standing wave signal is different, and the captured positions of the microspheres are different, so that the connection positions of the microspheres and the cells are different when the third ultrasonic surface standing wave signal and the fourth surface standing wave signal are regulated, and the connection positions of the microspheres and the cells can be accurately controlled by using the third ultrasonic surface standing wave signal or the fourth surface standing wave signal. Compared with the prior ATC technology, the cell regulation method can realize high-flux and high-precision measurement of cells.

Drawings

FIG. 1 schematically shows a flow chart of a cell modulation method provided according to an embodiment of the invention;

FIG. 2 schematically illustrates a schematic diagram of a cell modulating device provided according to an embodiment of the invention;

FIG. 3 schematically illustrates a schematic diagram of a cell arrangement and adherence for 10min stimulated with a continuous electrical signal at a 20% duty cycle, 5V voltage, according to an embodiment of the present invention;

FIG. 4A schematically illustrates various steps in a microsphere-cell attachment process provided in accordance with an embodiment of the present invention;

FIG. 4B schematically illustrates a schematic diagram of the corresponding time axis for each step of FIG. 4A;

FIG. 5A schematically illustrates a schematic diagram of the relative positions of microspheres and adherent cells when the phase difference of the electrical signals from the first signal generator provided in accordance with an embodiment of the present invention is 150 °;

FIG. 5B schematically illustrates a schematic diagram of the relative positions of microspheres and adherent cells when the phase difference of the electrical signals from the first signal generator provided in accordance with an embodiment of the present invention is 30 °;

FIG. 6 schematically illustrates a schematic diagram of a measurement cell mechanics experimental device provided according to an embodiment of the invention;

FIG. 7A is a schematic diagram showing motion trace analysis of microspheres under the action of acoustic radiation force at different moments according to an embodiment of the present invention;

Fig. 7B schematically shows a graph of microsphere displacement versus time obtained by MATLAB processing according to an embodiment of the present invention.

FIG. 8A schematically illustrates a schematic diagram of a four-element model provided in accordance with an embodiment of the present invention;

FIG. 8B schematically illustrates a graph of creep curve fit using a four-element model provided in accordance with an embodiment of the present invention;

FIG. 8C schematically illustrates a relaxation curve fit using a four-element model provided in accordance with an embodiment of the present invention.

Reference numerals illustrate:

1. signal generator module

11. First signal generator

2. Acoustic surface standing wave acoustic tweezers

21. Piezoelectric substrate

22. Micro-channel

23. First group of electrode groups

24. Second group of electrode group

3. Light source

4. Microscope

5. Computer with a memory for storing data

6. First power amplifier

7. Second power amplifier

8. High-speed camera

Detailed Description

In the related research, the ultrasonic control particle is a particle control technology which does not need marking, is non-contact, is easy to integrate and miniaturize and has wide application range. It uses sound waves to manipulate biological particles ranging from nano-sized extracellular vesicles to millimeter-sized multicellular organisms. Ultrasonic control technology has shown good application value in the biomedical field, but different operating requirements require the use of different types of acoustic forceps platforms. The three main types of acoustic tweezers are standing wave acoustic tweezers, traveling wave acoustic tweezers and acoustic fluid tweezers. Standing wave and traveling wave acoustic tweezers manipulate particles directly by acoustic radiation forces, while acoustic flow tweezers manipulate particles indirectly by acoustically induced fluid flow.

The standing wave acoustic tweezers are classified into surface acoustic wave (Surface acoustic wave, SAW) acoustic tweezers and bulk wave (Bulk acoustic wave, BAW) acoustic tweezers according to the mode of generation of the acoustic waves. SAW acoustic tweezers convert electrical signals into surface acoustic waves by an interdigital transducer (Interdigital transducer, interdigital electrodes) deposited on the surface of a piezoelectric material. A surface acoustic wave is an acoustic wave that is generated on and propagates along the free surface of an elastic body, and is classified into a Rayleigh wave (Rayleigh wave), a Love wave (Love wave), and the like according to the vibration mode of the surface acoustic wave. The wavelength of SAW is typically on the order of microns, so the accuracy of SAW acoustic tweezers manipulation is very high. The resolution of the SSAW field-shifted microbubbles using 22.42MHz frequencies can reach 2.2 μm. The standing wave field formed by the two groups of interdigital electrodes can realize the movement and arrangement of cells and the movement and multicellular assembly of cells in a three-dimensional space. By increasing the number of pairs of interdigital electrodes, the degree of freedom in manipulation of the particles by the SAW acoustic tweezers can be increased. BAW tweezers can capture particles at pressure nodes or pressure antinodes by creating bulk waves using multiple sets of piezoelectric transducers, superimposed to form standing waves in the manipulation region. Since the wavelength of sound waves generated by the piezoelectric transducer is in millimeter level, the control accuracy of the BAW acoustic tweezers is low. But BAW operation throughput is high and particle placement in the whole cell culture dish can be achieved. The traveling wave acoustic tweezers realize particle manipulation through a traveling wave field. There are two modes of manipulation for BAW-based traveling wave acoustic tweezers: by controlling a plurality of pressure transducers to generate traveling waves with different phases at the same time, pressure nodes can be formed at any position in a three-dimensional space, so that particles are captured; the sound wave generated by the single transducer can form complex sound beams after passing through structures such as acoustic metamaterials, phononic crystals and the like. The travelling wave acoustic tweezers are mainly used for acoustic holography. Acoustic fluid tweezers are devices that use a stable flow of liquid created by absorption of acoustic energy to manipulate particles in the liquid. This flow is known as acoustic flow and is typically achieved by enhancing the acoustic flow with certain solid structures, such as phononic crystals, while high frequency acoustic waves may also be used to induce fluid to produce intense acoustic fluid motion. Acoustic streaming is a nonlinear phenomenon, and thus, the manipulation accuracy of acoustic streaming tweezers may be low. In addition, in order to generate strong acoustic streaming, it is necessary to prepare precise structures such as phonon crystals or acoustic wave devices requiring high frequencies, and the difficulty of preparing these devices is also high. These limit the widespread use of acoustic fluid forceps.

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

Fig. 1 schematically shows a flow chart of a cell modulation method provided according to an embodiment of the invention.

As shown in FIG. 1, the cell modulating method comprises steps S1 to S5.

Step S1: and inputting the cell solution into a micro-channel with fibronectin at the bottom, so that the micro-channel is filled with the cell solution.

Step S2: applying a first ultrasonic surface standing wave signal along a first direction and a second ultrasonic surface standing wave signal along a second direction to the micro-channel to capture cells in the cell solution at a first pressure node of the first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal and to adhere the captured cells to obtain adherent cells, wherein the first direction is perpendicular to the second direction;

step S3: injecting microsphere solution into the micro-flow channel to uniformly distribute microspheres in the microsphere solution in the micro-flow channel;

step S4: applying a third ultrasonic surface standing wave signal and a fourth ultrasonic surface standing wave signal to the fluidic channel to capture the microspheres at a second pressure node of the third ultrasonic surface standing wave signal and the fourth surface standing wave signal, wherein the second pressure node is offset in the first direction or the second direction relative to the first node;

Step S5: changing the third ultrasonic surface standing wave signal with the second pressure node offset in the first direction relative to the first pressure node such that the captured microspheres move toward the corresponding adherent cells until conforming to the adherent cells;

in the event that the second pressure node is offset in a second direction relative to the first pressure node, the fourth ultrasonic surface standing wave signal is altered such that the captured microspheres move along toward the corresponding adherent cells until conforming to the adherent cells.

According to an embodiment of the present invention, before inputting the cell solution into the micro flow channel, the acoustic tweezer cell regulation method further includes:

injecting fibronectin solution into the micro-flow channel, sealing and standing to enable the bottom of the micro-flow channel to be provided with fibronectin;

injecting ultrapure water into the micro-flow channel filled with the fibronectin solution to enable the micro-flow channel to be full of the ultrapure water;

injecting the cell solution from the first port of the micro flow channel to discharge the ultrapure water from the second port of the micro flow channel until the micro flow channel is filled with the cell solution.

According to an embodiment of the invention, the microsphere surface is modified with RGD polypeptides. Changing the third ultrasonic surface standing wave signal such that the captured microspheres move toward the corresponding adherent cells until conforming to the adherent cells, comprising:

changing the fourth ultrasonic surface standing wave signal such that the captured microspheres move along toward the corresponding adherent cells until conforming to the adherent cells, comprising:

the fourth ultrasonic surface standing wave signal is changed so that the captured microspheres move towards the corresponding adherent cells until the RGD polypeptides on the surfaces of the microspheres are linked to integrins on the cell membrane surfaces of the corresponding adherent cells.

According to an embodiment of the invention, the third ultrasonic surface wave signal is generated by two first ultrasonic surface wave signals with opposite propagation directions;

altering the third ultrasonic surface standing wave signal comprises:

changing a phase difference between two first ultrasonic surface wave signals with opposite propagation directions;

the fourth ultrasonic surface standing wave signal is generated by two second ultrasonic surface wave signals with opposite propagation directions.

Altering the fourth ultrasonic surface standing wave signal includes:

According to an embodiment of the present invention, the concentration of cells in the cell solution is 2.6-2.8X10 ⁶ And each mL.

According to an embodiment of the present invention, the material of the microspheres is polystyrene, and the concentration of the microspheres in the microsphere solution is 1.5×10 ⁷ And each mL.

Fig. 2 schematically shows a schematic diagram of a cell modulating device provided according to an embodiment of the invention.

As shown in fig. 2, the cell modulating device comprises: a signal generator module 1, an acoustic surface standing wave acoustic tweezer (SAW acoustic tweezer) 2 and microspheres.

The signal generator module 1 is adapted to emit an electrical signal. The signal generator module 1 comprises a first signal generator 11 and a second signal generator (not shown in the figures), the first signal generator 11 may comprise two channels and the second signal generator 2 may comprise one channel.

The acoustic surface standing wave acoustic tweezers 2 comprise: a piezoelectric substrate 21, a micro flow channel 22 and two groups of electrode groups. The micro flow channel 22 is provided on the piezoelectric substrate 21 and is adapted to contain a cell solution. The two sets of electrodes are a first set of electrodes 23 and a second set of electrodes 24, respectively. Two sets of electrodes are disposed on the piezoelectric substrate 21 and located at the periphery of the micro flow channel, wherein each set of electrodes includes two interdigital electrodes disposed opposite to each other. For example, the first set of electrodes may be disposed in the x-direction as shown in fig. 2 and the second set of electrodes may be disposed in the y-direction as shown in fig. 2. The two sets of electrodes transfer the electrical signals from the first and second signal generators to the piezoelectric substrate 21, producing first, second, third and fourth ultrasonic surface standing wave signals. The signal generator module is further adapted to change the third ultrasonic surface standing wave signal and the fourth ultrasonic surface standing wave signal.

According to an embodiment of the present invention, the cell control device further includes: a light source 3, a microscope 4 and a computer 5. The microscope 4 is adapted to observe cells and microspheres under illumination from the light source 3. The computer 5 is adapted to record microscopic observations. . The basic principle of the invention is as follows:

ultrasonic surface standing wave formation:

surface acoustic waves are acoustic waves that are generated at and propagate along the free surface of an elastomer, according to the acoustic surfaceThe vibration mode of waves is classified into Rayleigh waves (Rayleigh waves) and Love waves (Love waves). SAW acoustic tweezers convert an electrical signal into a surface acoustic wave by means of interdigital electrodes (Interdigital transducer, interdigital electrodes) deposited on the surface of a piezoelectric material. Rayleigh waves are generated by the vibration of particles on the surface of an elastic solid along an elliptical trajectory at the near surface, with energy concentrated mainly in the two wavelength ranges below the surface. Applying radio frequency electric signals to the interdigital electrodes, converting the electric signals into mechanical energy by the inverse piezoelectric effect of the piezoelectric material, taking the amplitude asThe form of an order of magnitude surface acoustic wave propagates along the surface of the material. When a surface acoustic wave propagates to a liquid, refraction occurs at the interface of the piezoelectric material and the liquid, and the SAW becomes a form of leaky surface acoustic wave (Leaky surface acoustic wave, LSAW). When two rows of opposing coherent LSAWs propagate to the liquid in the sealed chamber, an acoustic surface standing wave (Standing surface acoustic wave, SSAW) is formed. The acoustic surface standing wave field has periodically distributed pressure nodes (pressure minima) and/or pressure antinodes (pressure maxima), which, based on acoustic properties, capture particles to the pressure nodes or pressure antinode locations in the acoustic standing wave field, which is the basic principle behind the SAW acoustic tweezers 2 manipulating particles. When the particles are cells or microspheres, the standing acoustic surface wave captures the particles or microspheres at pressure nodes in the acoustic standing wave field.

In this embodiment, the interdigital electrode is connected to any one of the channels of the first signal generator 11 or the second signal generator to receive an electrical signal emitted from one of the signal generators 11 or the second signal generator. The interdigital electrodes can generate ultrasonic surface wave signals according to the electric signals, and when two interdigital electrodes which are oppositely arranged are used for receiving the electric signals with the same frequency for each group of electrode groups, ultrasonic surface wave signals with the same propagation direction and the same frequency are generated, and the ultrasonic surface wave signals with the opposite propagation direction and the same frequency form ultrasonic surface standing wave signals. When the phase difference of the electric signals received by the two interdigital electrodes of each electrode group is changed, the generated ultrasonic surface standing wave signal is changed.

Cell arrangement:

in order to increase the survival rate of cells, the influence of heat generated by the piezoelectric substrate upon excitation of SAW on cell growth is reduced, and the cells are arranged using a pulse wave. Channel 1, using the first signal generator 11 (33622 a, agilent), produces a sinusoidal signal of 44.8MHz that splits onto a first set of electrodes (e.g., two interdigital electrodes in the x-direction, the frequency of the channel 2 signal is 44.9MHz that splits onto a second set of electrodes (e.g., two interdigital electrodes in the y-direction), which ensures that cells are trapped at the pressure node at an initial phase of 0 (phase difference 0). Using a continuous pulse stimulus of low voltage high duty cycle (5V voltage, pulse cycle number 500k, pulse cycle 50ms, duty cycle 20%), the voltage is slowly reduced until shut off after 10min, and then the cell attachment is waited for, as shown in fig. 3.

Microsphere alignment and active adhesion:

after the cells are successfully adhered in the micro flow channel 22, injecting a DMEM solution, washing away suspended cells in the micro flow channel 22, leaving adhered living cells (i.e. adhered cells), then injecting a microsphere solution, stopping injecting the microsphere solution after the microspheres are uniformly dispersed in the micro flow channel 22, and standing for 60 seconds after the microspheres are static, wherein the purpose of doing so is to enable the microspheres to be deposited on the PDMS surface under the action of gravity, namely to be positioned at the same focal plane with the cells. Channel 1 of the first signal generator (signal generator 33622A) outputs continuous sinusoidal electric signals of 44.8MHz and 5V, the continuous sinusoidal electric signals are connected to the interdigital electrode at the left side in the x direction, channel 2 outputs signals of 44.8MHz and 5V to the interdigital electrode at the right side in the x direction, phase synchronization between two columns of signals is noted, the interdigital electrode at the left side in the x direction and the interdigital electrode at the right side generate two first ultrasonic surface wave signals with different directions under the action of the electric signals sent by the first signal generator, and the two first ultrasonic surface wave signals with different directions form a third ultrasonic surface standing wave signal; the second signal generator (signal generator 33250A) has only one channel, the channel of the second signal generator inputs electric signals of 44.9MHz and 5V to the two interdigital electrodes in the y direction, the two interdigital electrodes in the y direction generate two second ultrasonic surface wave signals with opposite directions, the two second ultrasonic surface wave signals with opposite directions obtain a fourth ultrasonic surface standing wave signal, and the microspheres are captured at the corresponding pressure node positions under the action of the third ultrasonic surface standing wave signal and the fourth ultrasonic surface standing wave signal.

The microspheres can be captured at any position by again applying a signal having a phase difference at the pressure node where the initial phase is 0 when the cells are arranged, i.e., the microspheres can be captured directly above the cells when a signal having a phase difference of 0 degrees is applied, and the microspheres can be captured at the pressure node near the edge position of the cells when a signal having a phase difference of 90 degrees is applied. After cell attachment, the microspheres were injected, first captured at a pressure node with an initial phase of 150 °, where the microspheres were located in the middle of two cells, where the captured position was 16.67 μm from the center of the left cell. Then the phase of the channel 1 is changed to be 30 degrees, at the moment, the microsphere moves leftwards under the action of the sound radiation force, and stays to the right side of the cell under the obstruction of the cell membrane, and at the moment, the microsphere is in a stress balance state under the action of the sound radiation force and the elasticity of the cell membrane. Note that the pressure exerted on the cell membrane at this time is in the horizontal direction. The surface area of the cells will be extended after attachment, and the microspheres will be blocked from the application of the greater acoustic radiation force. Microspheres with a diameter of 5 μm are subjected to an acoustic radiation force of about 2pN in a sound field generated by a voltage of 5V, and thus the cell membrane surface is subjected to a pressure of about 2 pN. If the inter-particle interaction between the cells and the microspheres is further considered, the cell membrane is subjected to a stronger force, because this force promotes aggregation of the cells and the microspheres. After being extruded, the integrins on the surface of the cell membrane start to express and can be connected with RGD on the surface of the microsphere in a short time, and the microsphere is connected to the right side of the cell. By controlling the initial phase, the location of the microsphere to cell attachment can be controlled.

The operation mode can control the connection position of the cells and the microspheres, and can ensure that the acting force between the cells and the microspheres is strong enough to ensure the connection of the cells and the microspheres. Moreover, this operation is high throughput and allows for the bulk manipulation of the attachment of multiple microspheres and cells.

According to an embodiment of the present invention, there is also provided a method for measuring mechanical properties of cells, including: step A-step D.

Step A: microspheres were attached to adherent cells using cell control methods as described above.

And (B) step (B): changing the third ultrasonic surface standing wave signal again in the case that the second pressure node is offset in the first direction relative to the first pressure node so that the microsphere moves to involve the adherent cells to deform the adherent cells, and changing the fourth ultrasonic surface standing wave signal in the case that the second pressure node is offset in the second direction relative to the first pressure node so that the microsphere moves to involve the adherent cells to deform the adherent cells;

step C: removing the again modified third ultrasonic surface standing wave signal if the second pressure node is offset in the first direction relative to the first pressure node; and removing the second changed fourth ultrasonic surface standing wave signal under the condition that the second pressure node is offset relative to the first pressure node in the second direction.

Step D: repeating the step B-step C for a preset number of times.

Step E: and (3) obtaining a time-dependent change curve of the displacement of the microspheres in the process of each step B-step C so as to measure the mechanical properties of the cells.

The following specific examples illustrate the method for measuring the mechanical properties of cells in detail, and the steps are as follows:

step H: single cell patterning and platform building process. Comprising the steps of H1-H2.

Step H1: and (5) early preparation.

The cells used in the experiments of this technology were mouse fibroblast cells NIH 3T3 (purchased from american standard cell bank ATCC). 3T3 cells are a kind of mechanical signal sensitive cells and are easy to culture, which are cultured in a 37℃5% carbon dioxide incubator using complete medium of DMEM+10% calf serum.On the day of the experiment, 75cm was treated with pancreatin at room temperature ² Three minutes after digestion of 3T3 cells in flask, adding complete medium to stop digestion, centrifuging, discarding supernatant, adding new medium, and suspending, wherein cell concentration is controlled to 2.6-2.8X10 ⁶ And each mL.

Step H2: and (5) a platform building process.

The experimental platform manipulated was shown in fig. 2, with the self-contained SAW acoustic tweezers laid flat on the stage of a copolymer Jiao Daozhi microscope (IX 83-FV3000, olympus). Channel 1 of the first signal generator (signal generator 33622a, agilent) produces a sinusoidal signal of 44.8MHz split into a first set of electrodes (e.g., two interdigital electrodes that may be in the x-direction); the frequency of the first signal generator channel 2 signal is 44.9MHz, split into a second set of electrodes (e.g. two interdigitated electrodes which may be y-direction). The first signal generator has a bandwidth of 120MHz, a maximum output voltage of 10V, and a power sufficient to drive the SAW to produce a surface wave. Therefore, the power amplifier is temporarily not connected in the cell manipulation experiment.

In order to improve the biocompatibility of the micro flow channel 22, the micro flow channel 22 is subjected to Plasma hydrophilic treatment, is soaked in alcohol for 4 hours, is washed by ultrapure water to remove the alcohol in the micro flow channel 22, is injected with a Fibronectin solution (Fibronectin 0895, sigma) to seal a pipe orifice, and is then kept at normal temperature for overnight. The concentration of fibronectin solution in the microchannel of cultured 3T3 cells was 50. Mu.g/mL. Before the experiment was started, the entire micro flow channel 22 was filled with ultrapure water in advance, and the purpose of this was to ensure that no bubbles were generated when the cell solution was injected, and that cell survival was not affected.

Step I: referring to fig. 4A and 4B, the experimental procedure includes steps I1-I5:

step I1: cells are injected and the cells are aligned.

The cell solution is fed into the micro flow channel 22 with fibronectin at the bottom, and a voltage signal is applied immediately after both ends of the micro flow channel 22 are filled with the cell solution and the cells are stationary in the micro flow channel 22. In order to increase the cell survival rate and reduce the effect of heat generated by the piezoelectric substrate 21 (for example, a lithium niobate substrate) upon excitation of SAW on cell growth, the cells are arranged using a pulse wave. Channel 1, using the first signal generator, produces a sinusoidal signal of 44.8MHz that is split across the two sets of interdigitated electrodes in the x-direction, with channel 2 signal frequency 44.9MHz, and split across the two sets of interdigitated electrodes in the y-direction, thus ensuring that the cells are captured at the pressure node with an initial phase of 0 (phase difference of 0). The ultrasonic parameters with the low voltage, the high duty ratio, the 5V voltage, the pulse cycle number of 500k, the pulse period of 50ms and the duty ratio of 20% are adopted for continuous pulse stimulation. After the cells were trapped at the pressure node, the voltage was maintained for 10 minutes, and then gradually reduced until the cells attached, the whole process was recorded using a confocal microscope at a recording interval of 10s.

At the 0 min-20 min, single cells are captured at the pressure node, forming a high-flux single cell patterning arrangement, which is that the used surface acoustic wave wavelength meets the condition of single cell patterning. There are few cells that are not blown open, and there are many cells at a part of a single pressure node because the cells are not particularly uniformly dispersed in the micro flow channel, but as a whole, one pressure node can be realized to capture one cell. Note that the position after cell attachment is actually biased from the pressure node, which is unavoidable because cells migrate. The direction of cell migration is not controllable in this experiment, but the location of the majority of cells is substantially near the pressure node location.

Step I2: the microspheres are injected after the cells adhere to the wall.

The existing ATC technology uses targeting microbubbles to act on the surface of a cell membrane through buoyancy and then are connected with receptors on the surface of the cell membrane, the whole process is random, and the connection position of the microbubbles and the cells is uncontrollable. The method for controlling the active connection of the microspheres and the cells by utilizing the sound radiation force provided by the embodiment of the invention can control the connection position of the microspheres and the cells, and can simultaneously control a plurality of microspheres to realize the targeted connection of the microspheres and the cells.

Streptavidin coated polystyrene microsphere (ZSH-SVP-50-5, spherech) has a diameter of 5.3 μm and a stock solution concentration of 0.5% w/v. Streptavidin on the microsphere surface can be connected with RGD polypeptide, and the cellThe integrins on the membrane surface can be connected with RGD polypeptide, so that the microsphere can be connected on the surface of a cell membrane only through simple one-step modification. Before injecting the cell solution into the micro flow channel, preparing RGD modified microsphere solution in advance, diluting to 0.1% solid concentration with DMEM culture solution, wherein the concentration of the microsphere is 1.5×10 ⁷ And each mL.

After cells are successfully adhered in the micro flow channel 22 in 20 min-21 min, DMEM solution is injected from another pipe orifice, suspended cells in the micro flow channel are washed away, adhered living cells are left, then microsphere solution is injected again, after microspheres are uniformly dispersed in the micro flow channel, the microsphere solution is stopped being injected, after the microspheres are static, the microspheres are kept stand for 60s, and the purpose of the method is to enable the microspheres to be deposited on the surface of PDMS under the action of gravity, namely, the microspheres and the cells are located at the same focal plane.

Step I3: the microspheres are arranged.

Channel 1 of signal generator 33622A outputs 44.8MHz, 5V continuous sine signal, connect to one interdigital electrode of x direction, channel 2 outputs 44.8MHz, 5V signal connect to another interdigital electrode of x direction, pay attention to the synchronization between two columns of signals; the interdigital electrode in the y direction inputs signals of 44.9MHz and 5V, and then 21 min-22 min are carried out, so that the microsphere is captured at the corresponding pressure node position.

The microspheres can be captured at any position by capturing the cells at the pressure node with the initial phase of 0 in the single cell patterning process and then applying the signals with the phase difference again, and the first signal generator 11 is used for illustration in the experiment by applying the signals with the phase difference of 150 degrees on the two interdigital electrodes in the x direction. The initial phases of the electric signals output to the interdigital electrodes in the x direction by the channels 1 and 2 of the signal generator 33622a are 150 ° and 0 °, respectively, the phase difference between the channels 1 and 2 is 150 °, the microspheres are injected after the cells are attached, the microspheres are first captured at the pressure node with the initial phase of 150 °, as shown in fig. 5A, at this time, the microspheres are located at the position between the two attached cells, and the captured position of the microspheres is 16.67 μm away from the center of the attached cells on the left side.

Step I4: the microspheres are moved.

At 22 min-26 min, the phase of the channel 1 is changed to 30 degrees, namely the phase difference between the channel 1 and the channel 2 is 30 degrees, at the moment, the microsphere can move leftwards under the action of sound radiation force, and the microsphere stays to the right side of a cell under the obstruction of the cell membrane, at the moment, the microsphere is in a stress balance state under the action of the sound radiation force and the elastic force of the cell membrane. The movement of the microspheres as indicated by the arrows in fig. 5A and 5B can be seen when the phase difference between the two channels is changed. Note that the pressure exerted on the cell membrane at this time is in the horizontal direction. The surface area of the cells will be extended after attachment, and the microspheres will be blocked from the application of the greater acoustic radiation force. Microspheres with a diameter of 5 μm are subjected to an acoustic radiation force of about 2pN in a sound field generated by a voltage of 5V, and thus the cell membrane surface is subjected to a pressure of about 2 pN. If the inter-particle interaction between the cells and the microspheres is further considered, the cell membrane is subjected to a stronger force, because this force promotes aggregation of the cells and the microspheres. After being extruded, the integrins on the surface of the cell membrane start to express and can be connected with RGD on the surface of the microsphere in a short time, and the microsphere is connected to the right side of the cell. By controlling the initial phase, the location of the microsphere to cell attachment can be controlled. The microsphere-to-cell connection process was recorded with a high speed camera 8 at a sampling rate of 1000fps and a resolution of 768 x 768 pixels.

The positions of the microspheres before and after changing the phase are shown in fig. 5A-5B, a plurality of microspheres in the visual field move leftwards to the cell edge under the action of the sound radiation force to be stationary in advance, and the microspheres without cell obstruction continue to move to the pressure node to be stationary, so that the whole process is completed within 2 seconds. The operation can simultaneously control a plurality of microspheres to contact cells, the microspheres actively squeeze the cells under the action of sound radiation force, and compared with the buoyancy of the microbubbles (the buoyancy of the microbubbles with the diameter of 5 mu m is about 0.65pN in water), the pressure of the microspheres acting on the surface of a cell membrane is larger through the sound radiation force, so that the method can increase the connection efficiency of the microspheres and the cells. Meanwhile, the connection position of the microsphere and the cell can be actively controlled by the operation.

Step I5: and removing the sound field.

Step J: cell mechanics measurements, including step J1-step J2.

The microsphere used in the method is connected to the cytoskeleton through the integrins on the cell membrane, so that the integral mechanical property of the microsphere-integrins-cytoskeleton can be measured by analyzing the movement of the microsphere under the action of sound radiation force. Compared with the prior art, the SAW-ATC technology can measure the mechanical response of a plurality of cells at the same time; the technique also provides a wider force measuring range, and the maximum traction force can reach 200pN; the operation is simpler and more convenient, and the force application mode can be controlled.

Step J1: experimental condition settings, specific experimental equipment is shown in fig. 6.

In a standing wave field excited by a 6V input voltage, a microsphere with a radius of 2.5 μm is subjected to an acoustic radiation force of about 4pN. Under this range of acoustic radiation forces, the movement of the microspheres attached to the cells is not captured by the optical microscope. Therefore, the section improves the sound pressure of the standing wave field by improving the input voltage, the SSAW sound pressure is about 0.5MPa under the voltage excitation of 30V, the sound radiation force of the microsphere with the radius of 2.5 mu m is about 100pN, and the sound radiation force of the microsphere under the voltage excitation of 40V is about 180pN.

In the work of joint patterning of microspheres and cells, the attachment of targeted microspheres to cells has been achieved. Therefore, the mechanical response of the cell can be measured by only applying a larger sound radiation force to the microsphere connected to the surface of the cell membrane and recording the movement condition of the microsphere. In order to provide a higher input voltage, the output signal of the first signal generator 11 (signal generator 33622 a) needs to be amplified. The signal of the first signal generator 11 is connected to the finger electrode on the left side in the x direction through a first power amplifier 6 (power amplifier 75A250A,Amplifier Research, U.S.), and the signal of the channel 2 is connected to the finger electrode on the right side in the x direction through a second power amplifier 7 (power amplifier LZY-22+, mini-Circuits, U.S.). Because two different power amplifiers are used, the phase difference between two rows of synchronous signals needs to be measured in advance, the initial phase of the signal of the channel 2 is adjusted to be 130 degrees, the phase of the two rows of signals when reaching the interdigital electrode can be ensured to be the same, and the phase of the output signal of the channel 1 is changed on the basis.

The output signals of the first signal generator have voltages of 300mVpp and 400mVpp, and the amplified voltages are 30V and 40V. The frequency of both columns of signals was 44.8MHz. In the operation of cell and microsphere combined patterning, the microspheres are already attached to the cell edge locations. It is desirable that the microsphere may be subjected to a sufficient force when the microsphere is pulled by the acoustic radiation force. The microspheres may be subjected to the greatest acoustic radiation force when they are positioned 10 μm from the pressure node. The initial phase of the signal of channel 1 is thus set to 180 deg., which ensures that the microsphere is subjected to a greater traction force. And simultaneously generating Trigger signals by using Labview to control two channels of the first signal generator, wherein the Trigger is triggered for 4 times, the interval is 4s, and the number of pulse trains is 10 x 106. The action time of the ultrasound is regulated and controlled by adjusting the number of pulse trains. No voltage is applied in the y-direction, i.e. only a one-dimensional standing wave acoustic field in the x-direction (as shown in fig. 6) is generated. Meanwhile, the high-speed camera 8 is used for recording the motion trail of the microsphere, the sampling rate is 1000fps, the resolution is 768×768, the triggering mode is set to be manual, 1000 pieces of microspheres are recorded before ultrasound, and the synchronous signal of the signal generator is used for triggering the shooting of the high-speed camera 8. The experimental platform is shown in figure 6.

Step J2: microsphere movement result analysis

The high speed camera records the movement of the microspheres throughout the ultrasound stimulation process as shown in fig. 7A. The microsphere is positioned at the edge of the cell, and moves slightly under the action of ultrasound, returns to the initial position after the ultrasound is removed, and moves repeatedly under the action of 4 times of ultrasound. The time-dependent change curve of the displacement of the microspheres obtained by processing the obtained Tiff image using MATLAB program is shown in fig. 7B, and it can be seen that the maximum displacement of the microspheres is less than 1 μm, so that it can be considered that the acoustic radiation force applied to the microspheres is a constant value in the whole motion process, that is, the microspheres move under the action of constant tension. Amplifying the motion of the microsphere under the action of any ultrasonic pulse, as shown in fig. 7B, it can be seen that the microsphere moves rapidly and then moves at a uniform speed; after the ultrasound is removed, it returns slowly, but not necessarily to the original position. The movement of the microspheres conforms to the "creep" law of the viscoelastic material.

The microsphere used in the technology is directly connected to the cytoskeleton through the integrins on the surface of the cell membrane. From previous studies, it was found that cells cannot be simply considered as elastic solid materials or viscous fluid materials, and that mechanical parameters of cell elasticity and viscosity can be extracted using a linear viscoelastic model based on a spring-loaded cuvette model. The present technique uses a four-element model to characterize the viscoelastic parameters of the microsphere-integrin-cytoskeleton system. A schematic of the four-element model is shown in fig. 8A.

The creep expression of the four-element model is:

the relaxation expression is:

wherein ε _creep For strain of creep, ε _recovery Epsilon for relaxed strain _creep And epsilon _recovery All correspond to the displacement of the microsphere; f is stress and corresponds to acoustic radiation force; k (k) ₁ And k ₂ Is the elastic coefficient of the model, eta ₁ And eta ₂ Is the viscosity coefficient of the model, t _c Is the end time of the ultrasound. And fitting the displacement data of the microspheres by using the formula to obtain the viscoelasticity parameter of the model.

First, it is necessary to accurately calculate the sound radiation force applied to the microspheres by the sound field. Taking the microsphere of FIG. 7A as an example, the cells adhered by the microsphere are positioned at the 16 th pressure of the sound field with an initial phase of 0 DEGThe microsphere moves to the cell from the 16 th pressure node of the sound field with the initial phase of 150 degrees, the data of the high-speed camera 8 are compared, so that the microsphere is 4.5 mu m away from the 16 th pressure node with the initial phase of 150 degrees, the initial phase of the sound field is 180 degrees when the microsphere is pulled, the position of the pressure node is shifted to the right by 3.33 mu m, the position of the 16 th pressure node is deltax=7.86 mu m, and the sound radiation force received by the microsphere is F=F _max sin(2k*Δx)，F _max The sound pressure of the microsphere is the sound pressure of the pressure antinode 16, the corresponding electroacoustic conversion efficiency is 15.178kPa/V, and the input voltage is 30V, and the sound radiation force received by the microsphere is 91.86pN.

The results of the fitting using the four-element model are shown in fig. 8B and 8C. Fitting values of the parameters are shown in table 1.

TABLE 1

According to the cell regulation method and the cell regulation device provided by the embodiment of the invention, as the microsphere is adopted, the reliability and the stability of the microsphere are better, and the defects that the size of the microbubble is random (different from 0.7 mu m to 10 mu m) and the stability of the microbubble is easily broken due to the influence of the surrounding environment (such as temperature, pressure and the like) caused by the use of the microbubble in the related technology are overcome. At the same time, the microbubbles are excited by the ultrasonic action, cavitation nuclei can expand and contract sharply, even burst, local temperature and pressure in the nuclei rise sharply, and secondary effects such as strong shock waves, inscription force, high-speed micro-beam current, free radicals and the like can be generated, so that the micro-blood vessels can be ruptured, vascular endothelial cells shrink, cell gaps are widened, permeability of the walls of the micro-blood vessels is increased, and cell membrane integrity is damaged. Therefore, in the original ultrasonic tweezer cell regulation experiment, in order to reduce the damage to cells during microbubble cavitation, the sound field sound pressure is greatly limited.

According to the cell regulation and control method and the cell regulation and control device provided by the embodiment of the invention, the connection position of the microsphere and the cell is controlled by utilizing the ultrasonic surface standing wave signal. Overcomes the defect that the connection with cells is realized by inverting the cell culture dish and leading the microbubbles to depend on the self buoyancy random targeting, and the connection position is uncontrollable in the prior art.

According to the cell regulation and control method and the cell regulation and control device provided by the embodiment of the invention, the defects that an ultrasonic probe is immersed in a cell culture solution at an inclination of 45 degrees in the prior art, a generated sound field is complex to spread in a cell culture dish, the sound field is difficult to express by numerical values through various refraction and reflection, and the radiation force suffered by microbubbles cannot be accurately calculated are overcome. And the ultrasonic probe is immersed in the cell culture solution at an inclination of 45 degrees, so that the defect that the cell culture cannot be carried out for a long time due to bacteria staining of a culture dish is very easy to occur.

According to the cell regulation method and the cell regulation device provided by the embodiment of the invention, the cell is captured by adopting the ultrasonic surface standing wave signal, so that the defects that in the prior art, the cell grows extremely randomly in a culture dish, and the observation is carried out by adopting a 40-fold mirror, only a single cell or a single cell colony can be acted and observed in a single experiment, and the experimental flux is low are overcome.

The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present invention, and are not meant to limit the scope of the invention, but to limit the invention thereto.

Claims

1. A method of cell regulation comprising:

inputting a cell solution into a micro-channel with fibronectin at the bottom, so that the micro-channel is filled with the cell solution;

changing the third ultrasonic surface standing wave signal with the second pressure node offset in the first direction relative to the first pressure node such that the captured microspheres move toward the corresponding adherent cells until the adherent cells are adhered;

In the event that the second pressure node is offset in the second direction relative to the first pressure node, the fourth ultrasonic surface standing wave signal is altered such that the captured microspheres move along toward the corresponding adherent cells until the adherent cells are adhered.

2. The cell modulating method of claim 1, wherein the microsphere surface is modified with an RGD polypeptide.

3. The cell conditioning method of claim 2, wherein altering the third ultrasonic surface standing wave signal such that the captured microspheres move toward the corresponding adherent cells until the adherent cells are adhered comprises:

4. The method for cell control according to claim 1, wherein,

the third ultrasonic surface standing wave signal is generated by two first ultrasonic surface wave signals with opposite propagation directions; the altering the third ultrasonic surface standing wave signal comprises:

the altering the fourth ultrasonic surface standing wave signal comprises:

5. The cell control method according to claim 1, wherein the cell concentration in the cell solution is 2.6 to 2.8X10 ⁶ And each mL.

6. The cell modulation method according to claim 1, wherein prior to inputting the cell solution into the micro flow channel, the cell modulation method further comprises:

7. The cell modulation method according to claim 1, wherein the microsphere is made of polystyrene;

8. A cell modulating device for performing the cell modulating method of any one of claims 1-7, comprising:

the signal generator module is suitable for emitting an electric signal;

an acoustic surface standing wave acoustic tweezer comprising:

a piezoelectric substrate;

The first ultrasonic surface standing wave signal and the second ultrasonic surface standing wave signal are suitable for capturing cells in the cell solution and enabling the captured cells to adhere to the wall to obtain adherent cells, the third ultrasonic surface standing wave signal and the fourth ultrasonic surface standing wave signal are suitable for capturing microspheres entering the micro-channel, and the signal generator module is further suitable for enabling the third ultrasonic surface standing wave signal to be changed or enabling the fourth ultrasonic surface standing wave signal to be changed.

9. The cell modulating device of claim 8, further comprising:

a light source;

a microscope adapted to observe the cells and the microspheres under illumination from a light source;

and the computer is suitable for recording the microscope observation data.

10. A method for measuring mechanical properties of cells, comprising:

step A: attaching microspheres to adherent cells using the cell modulating method of any one of claims 1-7;

Step C: removing the again modified third ultrasonic surface standing wave signal if the second pressure node is offset in the first direction relative to the first pressure node; removing the second modified ultrasonic surface standing wave signal if the second pressure node is offset in the second direction relative to the first pressure node;

step D: repeating the step B-step C for preset times;