A cell culture method for inhibiting cell migration based on ultrasonic waves

Abstract

The application discloses a cell culture method based on ultrasonic wave for inhibiting cell migration. The method comprises the following steps: placing a cell climbing sheet into a cell culture dish, adding a PDL solution, cleaning and air-drying the cell climbing sheet after the PDL solution is absorbed, placing the cell culture dish into an ultrasonic device, emitting ultrasonic waves by the ultrasonic device, forming a standing wave field in the cell culture dish for inhibiting cell migration, preparing a starvation medium, suspending cells in the prepared starvation medium to obtain a cell suspension, adding the cell suspension into the cell culture dish, removing the external ultrasonic wave field after a period of time, and transferring the cell culture dish into a carbon dioxide cell culture box for static culture. By applying a one-dimensional ultrasonic standing wave field in the early stage of the cell culture process, the application effectively inhibits cell migration, accurately controls cell arrangement by a non-contact method, reduces damage to the cells, and ensures the safety of the operation and the activity of the cells.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of cell culture, and particularly relates to a cell culture method based on ultrasonic wave inhibition of cell migration. BACKGROUND

[0002] Cell migration, also known as cell crawling, cell movement or cell motility, refers to the movement process of cells in response to specific chemical gradient signals. This process includes the extension of cell head pseudopods, the formation of new adhesion points and the contraction of cell body tails, which are alternately performed in time and space. Cell migration is a key component of cell function, not only playing a physiological role in the normal growth and development of organisms, but also being a universal movement form of living cells. It plays a crucial role in various physiological and pathological processes such as tissue injury repair, embryonic development, immune defense, infection response and cancer metastasis. Therefore, cell migration has become a core issue in current cell biology research, and scientists are committed to making breakthroughs in the medical field such as preventing cancer metastasis and promoting injury repair by studying the mechanism of cell migration.

[0003] At present, there are various means to inhibit cell migration, including non-drug and drug intervention. The most common method is to use specific drugs, such as chemotherapy drugs, targeted therapy drugs, immunosuppressants, etc., to inhibit the signal pathways related to cell migration. However, long-term use of certain drugs may cause cells to develop drug resistance, thereby reducing the effectiveness of the drugs. This phenomenon suggests that in the study of inhibiting cell migration, new treatment methods need to be explored to provide more effective and safer treatment strategies for clinical treatment. SUMMARY

[0004] In order to solve the problems in the background art, the purpose of the present application is to provide a cell culture method based on ultrasonic wave inhibition of cell migration, in order to reduce the damage to cells and ensure the safety of operation and cell activity.

[0005] The technical solution adopted by the present application is as follows, comprising the following steps:

[0006] Step S1, first, place the cell crawling slide in the cell culture dish in the clean bench, add poly-D-lysine (PDL) solution to immerse the cell crawling slide, store at room temperature in the dark, then manually suck the poly-D-lysine solution, then wash the cell crawling slide with sterile water twice and dry the cell crawling slide;

[0007] The cell crawling slide in step S1 is a customized glass slide with a size of 8mm * 8mm.

[0008] Specifically, before step S1, the cell slide and cell culture dish need to be sterilized, and the sterilization steps are as follows: put the clean cell slide and cell culture dish into the sterilization pot for high-temperature and high-pressure sterilization, dry in the oven at 60℃, and then take them into the ultraclean bench and irradiate them with ultraviolet light.

[0009] Step S2: In the ultraclean bench, place the cell culture dish with the dried cell slide in the cavity of the sound field generator of the ultrasonic device, and add sterile water in the gap between the cavity of the sound field generator of the ultrasonic device and the cell culture dish.

[0010] Before step S2, the ultrasonic device needs to be disinfected and sterilized.

[0011] Step S3: Use the ultrasonic device to emit ultrasonic waves, and make the emitted ultrasonic waves form a standing wave field in the cell culture dish for patterning cells and inhibiting cell migration, thereby arranging and inhibiting the migration of the cells added in step S4;

[0012] Step S4: suspend the cells in the starvation medium to obtain a cell suspension, and then add the cell suspension to the cell culture dish.

[0013] In step S4, the cells are one of in-vitro human gastric cancer cells (HGC-27) and human renal epithelial cells (293t).

[0014] Step S5: After standing for 1-2 hours, remove the external ultrasonic waves, and transfer the cell culture dish to a carbon dioxide cell culture box at 37℃ and with a carbon dioxide concentration of 5%.

[0015] In step S1, the concentration of the poly-D-lysine solution is 80-120 ug / mL.

[0016] In step S1, the light-protected storage time of the cell slide is at least 2 hours or overnight.

[0017] The ultrasonic device in step S2 mainly consists of a rectangular sound field generator, piezoelectric ceramics, and a signal generator. The middle part of the sound field generator is provided with a cavity for placing the cell culture dish. Four pieces of piezoelectric ceramics are uniformly and spacedly arranged around the circumference of the sound field generator and attached to the outer circumference of the sound field generator. Each piece of piezoelectric ceramic is connected with the signal generator, which is used to generate sinusoidal wave signals with different frequencies and amplitudes and act on the piezoelectric ceramics, so that ultrasonic waves are generated between two non-adjacent piezoelectric ceramics. Step S2 is specifically as follows:

[0018] In the ultraclean bench, place the cell culture dish with the dried cell slide in the cavity of the sound field generator of the ultrasonic device, and add sterile water in the gap between the cavity of the sound field generator and the cell culture dish.

[0019] The ultrasonic device further comprises a power amplifier, the piezoelectric ceramic is connected with the power amplifier, and the power amplifier is used for amplifying the sinusoidal wave signal generated by the signal generator and then transmitting the amplified sinusoidal wave signal to the piezoelectric ceramic.

[0020] The step S3 is specifically:

[0021] Firstly, two piezoelectric ceramics symmetrically arranged along the axis of the sound field generator (i.e. two non-adjacent piezoelectric ceramics) are taken as a pair of piezoelectric ceramics, the signal generator is started to generate a sinusoidal wave signal and only acts on the pair of piezoelectric ceramics, so that the pair of piezoelectric ceramics generates ultrasonic waves in the cavity of the sound field generator and acts on the cells in the cell culture dish, and the ultrasonic waves form a standing wave field in the cell culture dish for patterning cells and inhibiting cell migration.

[0022] In the step S3, the signal frequency of the sinusoidal wave signal generated by the signal generator is 3.0-3.5 MHz, and the peak voltage of the ultrasonic waves generated by the piezoelectric ceramic is 10-12 Vpp.

[0023] In the step S4, the starvation medium mainly comprises a cell culture medium, 1-1.2% fetal bovine serum FBS, 80-120 IU / mL streptomycin and 80-120 IU / mL penicillin, and does not contain a chemical inhibitor.

[0024] The cell culture medium adopts one of DMEM medium and RPMI 1640 medium; the DMEM medium is a cell culture basic medium containing various amino acids, glucose and buffer agents, and the RPMI 1640 medium contains reducing agent glutathione, high-concentration vitamins, vitamin choline, inositol and buffer agents.

[0025] In the step S4, the density of the cells in the cell suspension is 25-28 w / mL.

[0026] In the step S4, the volume of the cell suspension is 250-300 μL.

[0027] In the step S5, the culture time of the cells in the cell culture dish in the carbon dioxide cell culture box is 1-24 hours.

[0028] The ultrasonic device of the application is used for creating a one-dimensional ultrasonic standing wave field in a cell culture dish. The ultrasonic device comprises a sound field generator, a piezoelectric ceramic, a signal generator and / or a power amplifier. The sound field generator is designed with a cavity to accommodate the cell culture dish, and square grooves are arranged around the sound field generator, and the piezoelectric ceramic is attached to the grooves of the sound field generator. The piezoelectric ceramic converts the electrical signal of the signal generator into ultrasonic waves, and the signal is enhanced through the power amplifier.

[0029] When the cell culture dish is placed into the acoustic field generator cavity, the four walls of the cell culture dish are parallel to the four walls of the acoustic field generator cavity with a gap, and sterile water is filled in the gap of the two to ensure the transmission of ultrasonic waves. The opposite two pieces of piezoelectric ceramic receive signals and generate ultrasonic waves, and two columns of ultrasonic waves with the same frequency but opposite directions interfere in the culture medium in the cell culture dish to form a stable standing wave field, thereby creating a one-dimensional ultrasonic standing wave field. The standing wave field has the characteristics of spatial amplitude periodicity, fixed position of nodes (minimum amplitude) and antinodes (maximum amplitude). By adjusting the frequency and peak voltage of the ultrasonic waves emitted by the piezoelectric ceramic, the one-dimensional ultrasonic standing wave field is accurately controlled, and the migration of cells is controlled.

[0030] The one-dimensional ultrasonic standing wave field utilizes the mechanical pressure and radiation force exerted on the cells by ultrasonic waves, as well as the characteristics of the cell density being slightly greater than the liquid culture medium. When the cells are in the one-dimensional ultrasonic standing wave field, the acoustic radiation force generated by the ultrasonic waves will push the cells to the nodes of the standing wave field, thereby forming a one-dimensional linear cell array with periodicity in the xoy plane of the cell culture dish along the x-axis or y-axis direction. That is, the one-dimensional ultrasonic standing wave field patterns the cells. The xoy plane of the cell culture dish is parallel to the bottom surface, and the x-axis and y-axis are parallel to the two edges of the cell culture dish. This patterned linear array promotes focused adhesion between cells and effectively inhibits cell migration, providing a new experimental method for cell behavior research.

[0031] In the present application, in order to accurately control cell migration, a starvation medium is specially designed. The starvation medium composition includes 1% fetal bovine serum (FBS). Compared with the conventional complete medium (containing 10% fetal bovine serum FBS), the starvation medium used in the present application significantly reduces the serum concentration, aiming to inhibit the proliferation of cells. By reducing the growth factors and nutrients in the serum, the starvation medium of the present application can effectively slow down the metabolic activity and division speed of the cells, thereby inhibiting the proliferation of the cells. This inhibitory effect is crucial for clearly observing the cell migration phenomenon, as it reduces the interference of proliferating cells on the experimental results and avoids visual confusion caused by proliferating cells in the later observation results.

[0032] The method can guide cells to form a one-dimensional ordered linear array by applying a one-dimensional ultrasonic standing wave field in an early stage of cell culture, and further inhibit the migration behavior of cells.

[0033] The present application has the following advantages:

[0034] 1. The method can effectively inhibit cell migration by applying a one-dimensional ultrasonic standing wave field in the early stage of cell culture to pattern cells into a one-dimensional linear array, providing a new approach for cell behavior research.

[0035] 2. The method precisely controls cell arrangement using a non-contact method, reducing damage to cells and ensuring the safety and activity of the operation.

[0036] 3. The method provides a new method for inhibiting cell migration, which has wide application prospects in scientific research and clinical treatment, and provides a new strategy for exploring the regulation mechanism of cell migration.

[0037] 4. The method applies ultrasonic waves during cell culture and provides a starvation medium, which can effectively slow down the metabolic activity and division speed of cells, thereby inhibiting cell proliferation. The combined effect of ultrasonic waves and starvation medium can effectively inhibit cell migration and reduce damage to cells. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 Figure 1 is a cell number statistical diagram, wherein (a) is a statistical diagram of human gastric cancer cells (HGC-27), and (b) is a statistical diagram of human renal epithelial cells (293t);

[0039] Figure 2 Figure 2 is a microscope image of cells after 1 hour of sound field action, wherein (a) is a microscope image of HGC-27 cells, and (b) is a microscope image of 293t cells;

[0040] Figure 3 Figure 3 is a live and dead fluorescent microscope image of HGC-27 cells after 24 hours of culture, wherein (a) is a microscope image of the control group, and (b) is a microscope image of the ultrasonic group;

[0041] Figure 4 Live / Dead fluorescent microscope images of 293t cells after 24 hours of culture, wherein (a) is a microscope image of the control group, and (b) is a microscope image of the ultrasound group;

[0042] Figure 5 Cell viability graphs of cells after 24 hours of culture, wherein (a) is a cell viability graph of HGC-27, and (b) is a cell viability graph of 293t. DETAILED DESCRIPTION

[0043] The present application will be further described in conjunction with the accompanying drawings and specific examples, but should not be construed as a limitation on the present application. If not specifically indicated, the technical means used in the examples are conventional means known to those skilled in the art. Unless specifically stated, the reagents, methods and equipment used in the present application are conventional reagents, methods and equipment in the technical field.

[0044] Example 1: Inhibition of cell migration culture method of human gastric cancer cells (HGC-27)

[0045] S1: Sterilize the cell slides, cell culture dishes, and ultrasonic devices: Place clean cell slides, cell culture dishes, and ultrasonic devices in a sterilization pot for high-temperature and high-pressure sterilization, dry in an oven at 60°C, and then irradiate with ultraviolet light in a clean bench.

[0046] S2: Place the cell slides in the cell culture dishes in the clean bench, immerse the cell slides in 100 ug / mL poly-D-lysine (PDL) solution, store at room temperature in the dark overnight, then manually aspirate the PDL solution, and then wash the cell slides twice with sterile water and dry the cell slides for use;

[0047] S3: In the clean bench, place the cell culture dishes with the dried cell slides in the cavity of the sound field generator of the ultrasonic device, and add sterile water in the gap between the cavity of the sound field generator and the cell culture dishes; wherein the ultrasonic device of the present application mainly consists of a sound field generator, piezoelectric ceramics, and a signal generator, the middle part of the sound field generator is provided with a cavity for placing the cell culture dishes, four pieces of piezoelectric ceramics are uniformly and spacedly arranged around the circumference of the sound field generator and attached to the outer circumference of the sound field generator, each piece of piezoelectric ceramic is connected with the signal generator, the signal generator is used to generate a sinusoidal signal and act on the piezoelectric ceramics, so that ultrasonic waves are generated between two non-adjacent piezoelectric ceramics.

[0048] S4, in the clean bench, connect the piezoelectric ceramic attached in the cavity of the sound field generator to the power amplifier, connect the power amplifier to the signal generator, start the signal generator and the power amplifier, set the power amplifier parameter to 11 dB, the signal generator parameter to 3.0 MHz, 10Vpp, the signal generator generates a sine wave signal and only acts on a pair of piezoelectric ceramics, so that the pair of piezoelectric ceramics generates ultrasonic waves in the cavity of the sound field generator and acts on the cell culture dish, and the ultrasonic waves form a standing wave field in the cell culture dish for inhibiting cell migration.

[0049] S5, first, prepare the RPMI 1640 starvation medium, which comprises RPMI 1640 medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin; then prepare the HGC-27 cell suspension: discard the cell culture medium supernatant from the T25 culture flask containing adherent HGC-27 cells, then rinse twice with phosphate buffer PBS in the T25 culture flask, add 1 mL 0.25% trypsin, digest for 3 minutes, add 1 mL RPMI 1640 starvation medium to terminate digestion, after centrifugation, discard the supernatant, add 2 mL RPMI 1640 starvation medium, resuspend, count using a cell counter and adjust the cell density to 25 w / mL.

[0050] S6: take 250 μL of the HGC-27 cell suspension with a cell density of 25 w / mL and inoculate it in the cell culture dish, and incubate it for 13 min, then turn off the power amplifier, directly connect the signal generator, and place it in a 37°C, 5% CO2 carbon dioxide incubator for 1 hour, then turn off the signal generator, and continue to incubate it in the carbon dioxide incubator for 23 hours after transferring the cell culture dish into the well plate.

[0051] S7: Live / Dead staining: prepare a mixed solution of cell staining reagents Calcein AM and propidium iodide PI. After the incubation is completed, aspirate the supernatant in the cell culture dish, gently rinse the cells twice with PBS, add 200 μL of the mixed solution of Calcein AM and PI in the cell culture dish, and incubate it in the dark for 15 min. After the staining is completed, aspirate the staining solution, wash it with PBS, and finally add PBS in the cell culture dish to maintain the cell morphology and wet state for subsequent fluorescence microscope observation.

[0052] Example 2: Inhibition of cell migration culture method of human renal epithelial cells (293t)

[0053] Example 2 selects human kidney epithelial cells (293t) as cells to be inhibited migration, the culture steps are the same as example 1, the difference is S5~ S6, the specific implementation steps of S5~ S6 in example 2 are as follows:

[0054] S5, first prepare DMEM starvation medium, DMEM starvation medium contains DMEM medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin; then prepare 293t cell suspension: from the T25 culture flask containing adherent 293t cells, discard the cell culture supernatant, then rinse twice with phosphate buffered saline PBS in T25 culture flask, add 1 mL 0.25% trypsin, digest for 3 minutes, add 1 mL DMEM starvation medium to terminate digestion, centrifuge, discard the supernatant, add 2 mL DMEM starvation medium, resuspend, count and adjust the cell density to 25 w / mL using a cell counter.

[0055] S6: take 250 μL of 293t cell suspension with a cell density of 25 w / mL and inoculate in a cell culture dish, stand for 13 min, then turn off the power amplifier, change to direct connection of signal generator, and put into 37℃, CO2 concentration of 5% carbon dioxide incubator for 1 hour, then turn off the signal generator, transfer the cell culture dish into the hole plate, and continue to stand for 23 hours in the carbon dioxide incubator.

[0056] In examples 1-2, after the sound field generated by the ultrasonic device acts for 1 hour, HGC-27 cells and 293t cells form a clear and orderly linear array on the slide, the microscope image of HGC-27 cells is shown in Figure 2 (a), and the microscope image of 293t cells is shown in Figure 2 (b). The advantage of this array is to promote the focused adhesion between cells, effectively inhibit cell migration, and compared with disordered arrangement, the sound field treatment significantly improves the orderliness of cell arrangement.

[0057] Further culture for 23 hours, Figure 3 the live and dead fluorescent microscope image in (b) of Figure 4Live / Dead fluorescent microscopy images of (b) reveal the cell survival status of 293t cells after 24 hours of incubation. Green fluorescence represents live cells, and red fluorescence represents dead cells. The results show that HGC-27 cells and 293t cells affected by the acoustic field maintain a clear and orderly linear array, which implies that cell migration is inhibited to some extent, thereby maintaining the initial arrangement pattern, further confirming the advantages of the array in promoting intercellular focal adhesion and inhibiting cell migration.

[0058] Comparative Example 1, a cell migration inhibition culture method without an ultrasonic device:

[0059] S1: Place the cell climbing sheet into a cell culture dish, coat the sheet with PDL solution, and incubate in the dark overnight. After aspirating the solution, wash twice with sterile water and air dry for use.

[0060] S2: Prepare HGC-27 and 293t cell suspensions: Prepare HGC-27 cell suspensions and 293t cell suspensions according to the method of S5 in Examples 1-2.

[0061] S3: Place the cell culture dish in the well plate, take 250 μL of HGC-27 or 293t cell suspension with a cell density of 25 w / mL, inoculate the cell culture dish, and place it in a 37°C, 5% CO2 concentration carbon dioxide incubator for 24 hours of static culture.

[0062] S4: Live / Dead staining: Stain the cells according to the method of S7 in Example 1.

[0063] In this comparative example, Figure 3 (a) is a live / dead fluorescent microscopy image of the control group HGC-27 cells without the action of the ultrasonic device, Figure 4 (a) is a live / dead fluorescent microscopy image of the control group 293t cells without the action of the ultrasonic device. The control group HGC-27 cells and 293t cells without the action of the ultrasonic device show disordered layout on the glass sheet, lacking obvious directional arrangement characteristics. According to the live / dead fluorescent microscopy images, the cell viability of HGC-27 cells and 293t cells in the control group and the ultrasonic group was quantitatively analyzed. Figure 5 (a) shows the cell viability statistical results of the control group and the ultrasonic group HGC-27 cells, Figure 5(b) shows the statistical results of the viability of the control group and the ultrasonic group 293t cells. After a 24-hour culture period, the viability of HGC-27 cells in the control group of the comparative example was 83.41%, and the ultrasonic group of Example 1 reached 90.29%; for 293t cells, the viability of the control group was 68.70%, and the viability of the ultrasonic group of Example 2 treated by ultrasonic waves was 80.39%. From these data, it can be observed that the method of culturing using an ultrasonic device has little effect on cell viability, indicating that the technology is safe and effective in maintaining cell activity.

[0064] Therefore, the culture method based on the ultrasonic device can effectively inhibit the migration of HGC-27 cells and 293t cells. Only 1 hour of sound field action can promote the formation of ordered directional arrangement of cells on the slide and achieve focused adhesion in the local area. After 23 hours of culture, the cells can still maintain a clear array, indicating the significant effect of the method in inhibiting cell migration. Compared with the control group without sound field action, the method has little effect on cell viability, proving its safety and effectiveness.

[0065] In addition, to verify the effect of 1% FBS in the culture medium on the proliferation of HGC-27 and 293t cells, the following culture method was used:

[0066] Comparative Example 2: Normal expansion culture of HGC-27 and 293t cells:

[0067] The culture of human gastric cancer cells (HGC-27) is as follows:

[0068] S1: Preparation of RPMI 1640 complete medium, including RPMI 1640 medium, 10% fetal bovine serum (FBS), 100 IU / mL streptomycin, and 100 IU / mL penicillin.

[0069] S2: Cell inoculation and culture: Take the frozen cells from liquid nitrogen and place them in a 37°C water bath for 1 minute. Add 3 mL of RPMI 1640 complete medium, centrifuge and discard the supernatant, and then add RPMI 1640 complete medium to prepare a cell suspension. Add the cell suspension to a T25 culture flask and culture in a cell culture incubator at 5% CO2 and 37°C. Replace the medium every 2 days, and use cells of the same generation for experimental testing.

[0070] The culture of human renal epithelial cells (293t) is as follows:

[0071] S1: Preparation of DMEM complete medium, including DMEM medium, 10% fetal bovine serum (FBS), 100 IU / mL streptomycin, and 100 IU / mL penicillin.

[0072] S2: Cell inoculation and culture: The frozen cells were taken out from liquid nitrogen and placed in a 37°C water bath for 1 minute. 3 mL of DMEM complete medium was added, and after centrifugation, the supernatant was discarded, and DMEM complete medium was added to prepare a cell suspension. The cell suspension was added to a T25 culture flask, and cultured in a cell incubator with 5% CO2 and 37°C. The medium was changed every 2 days, and cells of the same generation were used for experimental testing.

[0073] Example 3: Effect of 1% FBS-containing medium on cell proliferation

[0074] S1: Preparation of starvation medium: RPMI 1640 starvation medium contains RPMI 1640 medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin, and 100 IU / mL penicillin, which is used by HGC-27 cells; DMEM starvation medium contains DMEM medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin, and 100 IU / mL penicillin, which is used by 293t cells.

[0075] S2: Preparation of cell suspension: Select HGC-27 cells and 293t cells of the same generation, discard the cell culture medium supernatant in the T25 culture flask, rinse twice with PBS, then add 1 mL of 0.25% Trypsin and digest for 3 minutes, add 1 mL of corresponding starvation medium to stop digestion, after centrifugation, take out the supernatant, add 2 mL of corresponding starvation medium, resuspend, count and adjust the cell density using a cell counter.

[0076] S3: Cell inoculation and culture: HGC-27 and 293t cells were inoculated in a 6-well plate at a density of 10 x 10 4 / well, with 3 replicate wells each. The cells were cultured in a cell incubator with 5% CO2 and 37°C for 24 hours.

[0077] S4: Cell counting: After the culture was completed, the cells were digested using the steps in S2, and counted using a cell counter to detect the number of HGC-27 and 293t cells per well after 24 hours.

[0078] Example 4: Effect of 10% FBS-containing medium on cell proliferation

[0079] S1: Preparation of complete medium: The same as in Example 2, RPMI 1640 complete medium (used by HGC-27 cells) and DMEM complete medium (used by 293t cells) were prepared.

[0080] S2: Preparation of cell suspension: Select HGC-27 and 293t cells of the same passage number, discard the cell culture supernatant in the T25 culture flask, wash twice with PBS, add 1 mL of 0.25% Trypsin for 3 minutes, add 1 mL of the corresponding complete culture medium to stop digestion, centrifuge, remove the supernatant, add 2 mL of the corresponding complete culture medium, and resuspend. Use a cell counter to count and adjust the cell density.

[0081] S3: Cell Seeding and Culture: HGC-27 and 293t cells were seeded and cultured at a rate of 10 × 10⁶ cells / year. 4 Cells were seeded at a density of 3 wells in 6-well plates, with 3 replicate wells in each well. Cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 24 hours.

[0082] S4: Cell Counting. After culture, the cells were digested using the steps in S2, and the cells were counted using a cell counter to determine the number of HGC-27 and 293t cells per well after 24 hours.

[0083] Statistical analysis was performed on Comparative Examples 2-4. Comparative Example 2 showed the effect of the initial inoculum size on cell proliferation, Comparative Example 3 showed the effect of 1% FBS in the culture medium on cell proliferation, and Comparative Example 4 showed the effect of 10% FBS in the culture medium on cell proliferation. The statistical graph of HGC-27 count in Comparative Examples 2-4 is shown below. Figure 1 As shown in (a), the statistical chart of the quantity of 293t in Comparative Example 2-4 is as follows. Figure 1 As shown in (b), after culturing HGC-27 cells and 293t cells in starvation medium containing only 1% FBS for 24 hours, the cell proliferation rates were 10.10 × 10⁻⁶ and 10⁻⁶, respectively. 4 / hole and 11.36×10 4 / well, compared with the original cell seeding amount, the proliferation effect was moderate, and the cell number was close to the initial seeding level. After culturing HGC-27 cells and 293t cells in complete medium containing 10% FBS for 24 hours, the cell proliferation was 17.80 × 10⁶ cells / well. 4 / hole and 14.81×10 4 The culture medium, compared to the original seeding density, showed a significant proliferation effect. After 24 hours of starvation culture, the proliferation of HGC-27 and 293T cells was significantly less than that of the complete culture medium group, and the cell count after 24 hours was similar to the original seeding density. This result indicates that starvation culture medium can effectively inhibit cell proliferation, which is of great significance for clearly observing cell migration, as it avoids visual interference from proliferating cells in the later stages of observation.

[0084] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, and are not intended to limit the present application; although the present application has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions recorded in the above embodiments can still be modified, or some or all of the technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A cell culture method based on ultrasound-inhibited cell migration, characterized in that: Step S1: First, place the cell slide into a cell culture dish in a laminar flow hood, add poly-D-lysine solution to immerse the cell slide, store it at room temperature in the dark, then aspirate the poly-D-lysine solution, wash the cell slide with sterile water, and then air dry the cell slide. Step S2: Inside the laminar flow hood, place the cell culture dish inside the cavity of the ultrasonic device, and add sterile water to the gap area between the cavity of the ultrasonic device and the cell culture dish. Step S3: Use an ultrasonic device to emit ultrasonic waves, and make the emitted ultrasonic waves form a standing wave field in the cell culture dish to inhibit cell migration. The specific steps of step S3 are as follows: First, two piezoelectric ceramics symmetrically arranged along the central axis of the sound field generator are used as a pair of piezoelectric ceramics. The signal generator is started to generate a sinusoidal signal and act on the pair of piezoelectric ceramics, so that the pair of piezoelectric ceramics generates ultrasonic waves in the cavity of the sound field generator and acts on the cell culture dish. The ultrasonic waves form a standing wave field in the cell culture dish to inhibit cell migration. Step S4: Suspend the cells in starvation medium to obtain a cell suspension, and then add the cell suspension to a cell culture dish; In step S4, the starvation medium contains DMEM or RPMI 1640 medium, 1-1.2% fetal bovine serum (FBS), 80-120 IU / mL streptomycin, and 80-120 IU / mL penicillin. Step S5: After the preset time for static culture, remove the external ultrasound and transfer the cell culture dish to a carbon dioxide cell culture incubator for static culture. S5 specifically involves: seeding the cell suspension into a cell culture dish, incubating for 13 minutes, then turning off the power amplifier and directly connecting it to the signal generator, with only the signal generator maintaining the sine wave signal, and placing it in a carbon dioxide incubator at 37°C and 5% CO2 concentration for 1 hour, then turning off the signal generator, transferring the cell culture dish into a well plate, and continuing to incubate in the carbon dioxide incubator for 23 hours. In step S1, the concentration of the poly-D-lysine solution is 80-120 ug / mL; In step S1, the cell smears are stored in the dark for at least 2 hours. In step S3, the signal frequency of the sinusoidal signal generated by the signal generator is 3.0-3.5 MHz, and the peak voltage of the ultrasonic wave generated by the piezoelectric ceramic is 10-12 Vpp.

2. The cell culture method based on ultrasound-induced cell migration inhibition according to claim 1, characterized in that: The ultrasonic device in step S2 consists of a sound field generator, piezoelectric ceramics, and a signal generator. The sound field generator has a cavity in its center for placing cell culture dishes. Four piezoelectric ceramics are evenly spaced around the outer periphery of the sound field generator. Each piezoelectric ceramic is connected to the signal generator, which generates a sinusoidal signal and applies it to the piezoelectric ceramics, causing ultrasonic waves to be generated between two non-adjacent piezoelectric ceramics. Step S2 specifically involves: Inside the clean bench, place the cell culture dish containing the dried cell slides into the cavity of the sound field generator in the ultrasonic device, and add sterile water to the gap area between the sound field generator cavity and the cell culture dish.

3. The cell culture method based on ultrasound-induced cell migration inhibition according to claim 1, characterized in that: In step S4, the cell density in the cell suspension is 25-28 w / mL.

4. The cell culture method based on ultrasound-induced cell migration inhibition according to claim 1, characterized in that: In step S4, the volume of the cell suspension is 250-300 μL.

5. The cell culture method based on ultrasound-inhibited cell migration according to claim 1, characterized in that: In step S5, the cells in the cell culture dish are cultured statically in a carbon dioxide cell culture incubator for 1-24 hours.

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