Cell culture method for inhibiting cell migration based on ultrasonic waves

Abstract

The invention discloses a cell culture method for inhibiting cell migration based on ultrasonic waves. The method comprises the following steps: putting a cell glass slide into a cell culture dish, adding a PDL solution, completely absorbing the PDL solution, cleaning, airing for later use, putting the cell culture dish into an ultrasonic device, emitting ultrasonic waves by using the ultrasonic device to enable the ultrasonic waves to form a standing wave field for inhibiting cell migration in the cell culture dish, preparing a starvation culture medium, and culturing the starvation culture medium in the cell culture dish. And suspending the cells in a prepared starvation culture medium to obtain a cell suspension, then adding the cell suspension into a cell culture dish, removing an external ultrasonic sound field after a period of time, and transferring the cell culture dish into a carbon dioxide cell culture box for standing culture. According to the method, the one-dimensional ultrasonic standing wave field is applied in the early stage of the cell culture process, so that cell migration is effectively inhibited, cell arrangement is accurately controlled by adopting a non-contact method, damage to cells is reduced, and operation safety and cell activity are ensured.

Description

Technical Field

[0001] The invention belongs to the technical field of cell culture, and particularly relates to a cell culture method based on ultrasound to inhibit cell migration. Background Art

[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 pseudopodia of the cell head, the formation of new adhesion points, and the contraction of the cell body tail, and these activities are performed alternately in time and space. Cell migration is a key component of cell function. It not only plays a physiological role in the normal growth and development of organisms, but is also a ubiquitous form of movement for living cells. It plays a vital role in many physiological and pathological processes such as tissue damage repair, embryonic development, immune defense, infection response, and cancer metastasis. Therefore, cell migration has become a core topic in current cell biology research. Scientists are committed to making breakthrough progress in medical fields such as preventing cancer metastasis and promoting damage repair by studying the mechanism of cell migration.

[0003] Currently, 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 signaling 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, it is urgent to explore new treatment methods to provide more effective and safer treatment strategies for clinical treatment. Summary of the invention

[0004] In order to solve the problems existing in the background technology, the purpose of the present invention is to provide a cell culture method based on ultrasound to inhibit cell migration, so as to reduce damage to cells and ensure the safety of operation and cell activity.

[0005] The technical solution adopted by the present invention is as follows, comprising the following steps: Step S1, first, placing a cell slide into a cell culture dish in a clean bench, adding a poly-D-lysine (PDL) solution to immerse the cell slide, and storing it at room temperature away from light, then manually aspirating the poly-D-lysine solution, and then washing the cell slide twice with sterile water and drying the cell slide; The cell slide in step S1 uses a custom-made glass slide of 8mm*8mm.

[0006] Specifically, before step S1, the cell slides and cell culture dishes need to be sterilized, and the sterilization steps are as follows: put the clean cell slides and cell culture dishes into a sterilizer for high temperature and high pressure sterilization, dry them in an oven at 60°C, and then take them into a clean bench for ultraviolet light exposure.

[0007] Step S2, in a clean bench, placing the cell culture dish with the dried cell slide in the cavity of the sound field generator in the ultrasonic device, and adding sterile water in the gap between the cavity of the sound field generator in the ultrasonic device and the cell culture dish; Before step S2, the ultrasonic device needs to be disinfected and sterilized.

[0008] Step S3, using an ultrasonic device to emit ultrasonic waves, and allowing the emitted ultrasonic waves to form a standing wave field in the cell culture dish for patterning cells and inhibiting cell migration, thereby arranging the cells added in step S4 and inhibiting migration; Step S4, suspending the cells in a starvation medium to obtain a cell suspension, and then adding the cell suspension to a cell culture dish; The cells in step S4 are either human gastric cancer cells (HGC-27) or human kidney epithelial cells (293t) isolated from the body.

[0009] Step S5: After static culture for 1-2 hours, remove the external ultrasound and transfer the cell culture dish to a carbon dioxide cell culture incubator at 37° C. and a CO 2 concentration of 5% for static culture.

[0010] In step S1, the concentration of the poly-D-lysine solution is 80-120 ug / mL.

[0011] In the step S1, the cell slide is stored in the dark for at least 2 hours or overnight.

[0012] The ultrasonic device in step S2 is mainly composed of a rectangular sound field generator, a piezoelectric ceramic and a signal generator. A cavity for placing a cell culture dish is opened in the middle of the sound field generator. Four piezoelectric ceramics are evenly spaced and attached to the periphery of the sound field generator around the circumference of the sound field generator. Each piezoelectric ceramic is connected to the signal generator. The signal generator is used to generate sinusoidal wave signals of 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: In the clean bench, the cell culture dish with the dried cell slides is placed in the cavity of the sound field generator in the ultrasonic device, and sterile water is added to the gap area between the cavity of the sound field generator and the cell culture dish.

[0013] The ultrasonic device also includes a power amplifier, the piezoelectric ceramic is connected to the power amplifier, the power amplifier is used to amplify the sinusoidal wave signal generated by the signal generator, and then transmit the amplified sinusoidal wave signal to the piezoelectric ceramic.

[0014] The step S3 is specifically as follows: First, two piezoelectric ceramics symmetrically arranged along the central axis of the sound field generator (i.e., two non-adjacent piezoelectric ceramics) are used as a pair of piezoelectric ceramics. The signal generator is started to generate a sinusoidal 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 cells in the cell culture dish. The ultrasonic waves form a standing wave field in the cell culture dish that is used to pattern the cells and thereby inhibit cell migration.

[0015] In the step S3, the signal generator generates a sine wave signal with a signal frequency of 3.0-3.5 MHz, and the peak voltage of the ultrasonic wave generated by the piezoelectric ceramic is 10-12 Vpp.

[0016] In step S4, the starvation medium mainly consists of 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 any chemical inhibitors.

[0017] The cell culture medium adopts one of DMEM culture medium and RPMI 1640 culture medium; DMEM culture medium is a basic cell culture culture medium containing various amino acids, glucose and buffering agents, and RPMI 1640 culture medium contains reducing agent glutathione, high-concentration vitamins, vitamin choline, inositol and buffering agents and other ingredients.

[0018] In step S4, the density of cells in the cell suspension is 25-28 w / mL.

[0019] In step S4, the volume of the cell suspension is 250-300 μL.

[0020] In the step S5, the cells in the cell culture dish are statically cultured in a carbon dioxide cell culture incubator for 1-24 hours.

[0021] The ultrasonic device of the present invention is used to create a one-dimensional ultrasonic standing wave field in a cell culture dish. The ultrasonic device includes an acoustic field generator, a piezoelectric ceramic, a signal generator and / or a power amplifier. The acoustic field generator is designed with a cavity to accommodate the cell culture dish, and a square empty groove is arranged around the acoustic field generator, and the piezoelectric ceramic is attached to the empty groove of the acoustic field generator. The piezoelectric ceramic converts the electrical signal of the signal generator into ultrasonic waves, and enhances the signal through the power amplifier.

[0022] When the cell culture dish is placed in the cavity of the acoustic field generator, the four walls of the cell culture dish are parallel to the four walls of the acoustic field generator chamber and there is a gap between them. The gap between the two is filled with sterile water to ensure the conduction of ultrasonic waves. The two opposing piezoelectric ceramics receive signals and generate ultrasonic waves. Two 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. This standing wave field has the characteristics of periodic changes in spatial amplitude, fixed positions of nodes (minimum amplitude) and antinodes (maximum amplitude). By adjusting the frequency and peak voltage of the ultrasonic waves emitted by the piezoelectric ceramics, precise control of the one-dimensional ultrasonic standing wave field is achieved, thereby controlling the migration of cells.

[0023] The one-dimensional ultrasonic standing wave field utilizes the mechanical pressure and radiation force exerted by ultrasound on cells, as well as the characteristics that the cell density is slightly greater than that of the liquid culture medium. When cells are in a one-dimensional ultrasonic standing wave field, the acoustic radiation force generated by ultrasound will push the cells to gather at the nodes of the standing wave field, thereby causing the originally disordered cells in the cell suspension to form a periodic one-dimensional linear cell array along the x-axis or y-axis direction on the xoy plane of the cell culture dish, that is, a one-dimensional ultrasonic standing wave field patterned cell. Among them, 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 sides of the cell culture dish, respectively. This patterned linear array promotes focused adhesion between cells, effectively inhibits cell migration, and provides a new experimental means for cell behavior research.

[0024] In the present invention, a starvation medium is specially designed to precisely control cell migration. The starvation medium includes 1% fetal bovine serum (FBS). Compared with conventional complete medium (containing 10% fetal bovine serum FBS), the starvation medium used in the present invention significantly reduces the serum concentration, aiming to inhibit cell proliferation. By reducing the growth factors and nutrients in the serum, the starvation medium of the present invention can effectively slow down the metabolic activity and division rate of cells, thereby inhibiting cell proliferation. This inhibitory effect is crucial for clearly observing the phenomenon of cell migration, because it reduces the interference of proliferating cells on the experimental results and avoids the visual confusion caused by proliferating cells on the observation results in the later stage.

[0025] The method of the present invention can guide the cell patterning to form a one-dimensional ordered linear array by applying a one-dimensional ultrasonic standing wave field in the early stage of cell culture, thereby inhibiting the migration behavior of the cells. The specific implementation steps include: first, treating the cell crawling sheet with a poly-D-lysine solution, then setting specific parameters in the ultrasonic device to generate a standing wave field, and then adding the cell suspension to the cell culture dish and culturing under appropriate conditions. This method uses non-contact acoustic field operation to reduce damage to the cells, ensure high cell survival rate, and has little effect on the viability of human gastric cancer cells HGC-27 and human renal epithelial cells 293t. Experimental results confirm that the method of the present invention can significantly inhibit cell migration while maintaining cell activity, providing a safe and effective new technical approach for cell behavior research and medical applications.

[0026] The beneficial effects of the present invention are: 1. The method of the present invention applies a one-dimensional ultrasonic standing wave field in the early stage of the cell culture process to pattern the cells into a one-dimensional linear array, which effectively inhibits cell migration and provides a new approach for cell behavior research.

[0027] 2. The present invention uses a non-contact method to accurately control cell arrangement, reduce damage to cells, and ensure the safety of operation and cell activity.

[0028] 3. The present invention provides a new method for inhibiting cell migration, which has broad application prospects in scientific research and clinical treatment, and provides a new strategy for exploring the regulatory mechanism of cell migration.

[0029] 4. The present invention applies ultrasound during the cell culture process and provides a starvation medium, which can effectively slow down the metabolic activity and division rate of cells, thereby inhibiting the proliferation activity of cells. The combined effect of ultrasound and the starvation medium can effectively inhibit cell migration and reduce damage to cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Statistical graphs of cell numbers, wherein (a) is a statistical graph of the number of human gastric cancer cells (HGC-27), and (b) is a statistical graph of the number of human kidney epithelial cells (293t); Figure 2 The microscope images of cells after the acoustic field was applied for 1 hour, where (a) is a microscope image of HGC-27 cells and (b) is a microscope image of 293t cells; Figure 3 Live-dead staining fluorescence microscopy images of HGC-27 cells after 24 hours of culture, where (a) is the microscopy image of the control group and (b) is the microscopy image of the ultrasound group; Figure 4Live-dead staining fluorescence microscopy images of 293t cells after 24 hours of culture, where (a) is the microscopy image of the control group and (b) is the microscopy image of the ultrasound group; Figure 5 The figures are cell viability statistics after 24 hours of cell culture, where (a) is the cell viability statistics of HGC-27, and (b) is the cell viability statistics of 293t. DETAILED DESCRIPTION

[0031] The content of the present invention is further described below in conjunction with the accompanying drawings and specific examples, but it should not be construed as limiting the present invention. If not specifically indicated, the technical means used in the examples are conventional means well known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0032] Example 1: Method for culturing human gastric cancer cells (HGC-27) to inhibit cell migration S1: Sterilize the cell slides, cell culture dishes, and ultrasonic devices: Place the clean cell slides, cell culture dishes, and ultrasonic devices in a sterilizer for high temperature and high pressure sterilization, dry them in an oven at 60°C, and then place them in a clean bench for ultraviolet irradiation.

[0033] S2: Place the cell slide in a cell culture dish in a clean bench, add 100 ug / mL poly-D-lysine (PDL) solution to immerse the cell slide, store it in the dark at room temperature overnight, then manually aspirate the PDL solution, wash the cell slide twice with sterile water, and then dry the cell slide for later use; S3: In a clean bench, a cell culture dish containing dried cell slides is placed in the cavity of an acoustic field generator in an ultrasonic device, and sterile water is added to the gap between the cavity of the acoustic field generator and the cell culture dish; wherein the ultrasonic device of the present invention is mainly composed of an acoustic field generator, piezoelectric ceramics and a signal generator, a cavity for placing the cell culture dish is provided in the middle of the acoustic field generator, four piezoelectric ceramics are evenly spaced and attached to the periphery of the acoustic field generator around the circumference of the acoustic field generator, each piezoelectric ceramic is connected to a signal generator, and the signal generator is used to generate a sinusoidal wave signal and act on the piezoelectric ceramic, so that ultrasonic waves are generated between two non-adjacent piezoelectric ceramics.

[0034] S4. In the clean bench, connect the piezoelectric ceramics attached to 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 parameters to 11 dB, the signal generator parameters to 3.0 MHz, 10 Vpp, the signal generator generates a sine wave signal and acts only 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 to inhibit cell migration.

[0035] S5. First, prepare RPMI 1640 starvation medium, which contains RPMI 1640 medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin. Then prepare HGC-27 cell suspension: discard the cell culture medium supernatant from the T25 culture flask containing adherent HGC-27 cells, then add phosphate buffered saline (PBS) to the T25 culture flask for rinsing twice, add 1 mL 0.25% trypsin Trypsin for digestion for 3 minutes, add 1 mL RPMI1640 starvation medium to terminate digestion, discard the supernatant after centrifugation, add 2 mL RPMI 1640 starvation medium, resuspend, count using a cell counter and adjust the cell density to 25 w / mL.

[0036] S6: Take 250 μL of HGC-27 cell suspension with a cell density of 25 w / mL and inoculate it into the cell culture dish. Incubate it for 13 min, then turn off the power amplifier and directly connect it to the signal generator. Place it in a carbon dioxide incubator at 37°C and a CO2 concentration of 5% and incubate it for 1 hour. Then turn off the signal generator, transfer the cell culture dish into the well plate, and continue to incubate it in the carbon dioxide incubator for 23 hours.

[0037] S7: Live and dead staining: Prepare a mixed solution of cell staining reagents Calcein AM and propidium iodide PI. After the culture is completed, aspirate the supernatant in the cell culture dish, gently rinse the cells twice with PBS, add 200μL of Calcein AM and PI mixed solution to the cell culture dish and keep it in the dark for 15 minutes. After the staining is completed, aspirate the staining solution, wash it with PBS, and finally add PBS to the cell culture dish to maintain the cell morphology and moist state for subsequent fluorescence microscopy observation.

[0038] Example 2: Method for culturing human renal epithelial cells (293t) to inhibit cell migration Example 2 Human renal epithelial cells (293t) were selected as cells to be inhibited from migrating, and the culture steps were the same as those in Example 1, except for S5 to S6. The specific implementation steps of S5 to S6 in Example 2 are as follows: S5. First, prepare DMEM starvation medium, which contains DMEM medium, 1% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin; then prepare 293t cell suspension: discard the cell culture medium supernatant from the T25 culture flask containing adherent 293t cells, then add phosphate buffered saline (PBS) to the T25 culture flask to rinse twice, add 1 mL 0.25% trypsin Trypsin to digest for 3 minutes, add 1 mL DMEM starvation medium to terminate digestion, discard the supernatant after centrifugation, add 2 mL DMEM starvation medium, resuspend, count using a cell counter and adjust the cell density to 25 w / mL.

[0039] S6: Take 250 μL of 293t cell suspension with a cell density of 25 w / mL and inoculate it into the cell culture dish. Incubate it for 13 min, then turn off the power amplifier and directly connect it to the signal generator. Place it in a carbon dioxide incubator at 37°C and a CO2 concentration of 5% and incubate it for 1 hour. Then turn off the signal generator, transfer the cell culture dish into the well plate, and continue to incubate it in the carbon dioxide incubator for 23 hours.

[0040] In Example 1-2, after being exposed to the acoustic field generated by the ultrasonic device for 1 hour, HGC-27 cells and 293t cells formed a clear and orderly linear array on the glass slide. The microscope image of HGC-27 cells is as follows: Figure 2 As shown in (a), the microscope image of 293t cells is as follows Figure 2 (b) shows the advantages of this array in that it promotes the focused adhesion between cells and effectively inhibits cell migration. Compared with disordered arrangement, acoustic field treatment significantly improves the orderliness of cell arrangement.

[0041] After further culturing for 23 hours, Figure 3 The live-dead staining fluorescence microscopy image in (b) reveals the cell survival status of HGC-27 cells in the ultrasound group after 24 hours of culture. Figure 4 The live-dead staining fluorescence microscopy image in (b) reveals the cell survival status of 293t cells in the ultrasound group after 24 hours of culture. 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 suggests that cell migration is inhibited to a certain extent, thereby maintaining the initial arrangement pattern, further confirming the advantages of the array in promoting intercellular focal adhesion and inhibiting cell migration.

[0042] Comparative Example 1: A culture method for inhibiting cell migration without an ultrasonic device: S1: Place the cell slide into a cell culture dish, coat the slide with PDL solution, keep it away from light overnight, then aspirate the solution, wash it twice with sterile water and dry it for later use.

[0043] S2: Preparation of HGC-27 and 293t cell suspensions: Prepare HGC-27 cell suspensions and 293t cell suspensions according to the method of S5 in Example 1-2.

[0044] 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 and inoculate it into the cell culture dish, and place it in a carbon dioxide incubator at 37°C and a CO2 concentration of 5% for 24 hours.

[0045] S4: Live and dead staining: Cell staining was performed according to the method of S7 in Example 1.

[0046] In this comparative example, Figure 3 (a) is a live-dead staining fluorescence microscope image of the control group HGC-27 cells without the action of the ultrasonic device. Figure 4 (a) is a live-dead staining fluorescence microscope image of 293t cells in the control group without the action of the ultrasonic device. The layout of the HGC-27 cells and 293t cells in the control group without the action of the ultrasonic device on the glass slide is disordered and lacks obvious directional arrangement characteristics. Based on the live-dead staining fluorescence microscope images, the cell viability of the HGC-27 cells and 293t cells in the control group and the ultrasound group was quantitatively analyzed. Figure 5 (a) shows the statistical results of the viability of HGC-27 cells in the control group and the ultrasound group. Figure 5 (b) shows the statistical results of the viability of 293t cells in the control group and the ultrasound group. After 24 hours of culture, the viability of HGC-27 cells in the control group of the comparative example was 83.41%, while that of the ultrasound group of Example 1 reached 90.29%; for 293t cells, the viability of the control group was 68.70%, while that of the ultrasound group of Example 2 treated with ultrasound 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.

[0047] 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 acoustic field can prompt the cells to form an orderly directional arrangement on the glass slide and achieve focused adhesion in the local area. After another 23 hours of culture, the cells can still maintain a clear array, indicating that this method is significantly effective in inhibiting cell migration. Compared with the control group without the acoustic field, this method has little effect on cell viability, proving its safety and effectiveness.

[0048] In addition, to verify the effect of culture medium containing 1% FBS on the proliferation of HGC-27 and 293t cells, the following culture method was used: Comparative Example 2: Normal expansion culture of HGC-27 and 293t cells: The culture process of human gastric cancer cells (HGC-27) is as follows: S1: RPMI 1640 complete medium, including RPMI 1640 medium, 10% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin.

[0049] S2: Cell inoculation and culture: Take out the frozen cells from liquid nitrogen, place them in a 37°C water bath for 1 minute, add 3mL RPMI 1640 complete medium, discard the supernatant after centrifugation, and add RPMI 1640 complete medium to prepare a cell suspension. Add the cell suspension to a T25 culture flask and culture it in a cell culture incubator with 5% CO2 and 37°C. Change the medium every 2 days, and use cells of the same generation for experimental testing.

[0050] The culture process of human renal epithelial cells (293t) is as follows: S1: DMEM complete medium, including DMEM medium, 10% fetal bovine serum (FBS), 100 IU / mL streptomycin and 100 IU / mL penicillin.

[0051] S2: Cell inoculation and culture: Take out the frozen cells from liquid nitrogen, place them in a 37°C water bath for 1 minute, add 3 mL of DMEM complete medium, discard the supernatant after centrifugation, and add DMEM complete medium to prepare a cell suspension. Add the cell suspension to a T25 culture flask and culture it in a cell culture incubator with 5% CO2 and 37°C. Change the medium every 2 days and use cells of the same generation for experimental testing.

[0052] Comparative Example 3: Effect of 1% FBS in the culture medium on cell proliferation S1: Starvation medium preparation: 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.

[0053] S2: Prepare cell suspension: Select HGC-27 cells and 293t cells of the same generation, discard the supernatant of the cell culture medium in the T25 culture flask, rinse twice with PBS, add 1 mL of 0.25% Trypsin for digestion for 3 minutes, add 1 mL of the corresponding starvation medium to terminate the digestion, remove the supernatant after centrifugation, add 2 mL of the corresponding starvation medium, resuspend, count using a cell counter and adjust the cell density.

[0054] S3: Cell seeding and culture: HGC-27 and 293t cells were cultured at 10×10 4 The cells were seeded at a density of 100 μg / well in a 6-well plate, with 3 replicate wells set up. The cells were cultured in a cell culture incubator at 5% CO2 and 37°C for 24 hours.

[0055] S4: Cell counting: After the culture is completed, the cells are digested using the steps in S2, and the cells are counted using a cell counter to detect the cell number per well of HGC-27 and 293t cells after 24 hours.

[0056] Comparative Example 4: Effect of 10% FBS in the culture medium on cell proliferation S1: Preparation of complete culture medium: Prepare RPMI 1640 complete culture medium (for HGC-27 cells) and DMEM complete culture medium (for 293t cells) in the same manner as in Comparative Example 2.

[0057] S2: Prepare cell suspension: Select HGC-27 and 293t cells of the same generation, discard the cell culture supernatant in the T25 culture flask, rinse twice with PBS, add 1 mL of 0.25% Trypsin for 3 minutes, add 1 mL of the corresponding complete culture medium to terminate the digestion, remove the supernatant after centrifugation, add 2 mL of the corresponding complete culture medium, and resuspend. Use a cell counter to count and adjust the cell density.

[0058] S3: Cell seeding and culture: HGC-27 and 293t cells were cultured at 10×10 4 The cells were seeded at a density of 100 μg / well in a 6-well plate, with 3 replicate wells set up. The cells were cultured in a cell culture incubator at 5% CO2 and 37°C for 24 hours.

[0059] S4: Cell counting. After the culture is completed, the cells are digested using the steps in S2, and the cells are counted using a cell counter to detect the number of cells per well of HGC-27 and 293t cells after 24 hours.

[0060] Comparative Examples 2-4 were statistically analyzed. Comparative Example 2 showed the effect of the original inoculum size on cell proliferation, Comparative Example 3 showed the effect of the culture medium containing 1% FBS on cell proliferation, and Comparative Example 4 showed the effect of the culture medium containing 10% FBS on cell proliferation. The number statistics of HGC-27 in Comparative Examples 2-4 are shown in FIG. Figure 1 As shown in (a), the number statistics of 293t in Comparative Example 2-4 are 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 4 / hole and 11.36×10 4 / well, compared with the original cell inoculation amount, the proliferation effect was average, and the cell number was close to the initial inoculation level. After culturing HGC-27 cells and 293t cells in complete medium containing 10% FBS for 24 hours, the cell proliferation was 17.80×10 4 / hole and 14.81×10 4 / well, showing a significant proliferation effect compared with the original cell inoculation amount. After 24 hours of culture in starvation medium, the proliferation effect of HGC-27 cells and 293T cells was significantly worse than that of the complete medium group, and the number of cells after 24 hours was similar to the original inoculation amount. This result shows that starvation medium can effectively inhibit cell proliferation, which is of great significance for clearly observing cell migration phenomena, because it avoids the visual interference of proliferating cells on the observation results in the later stage.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. However, 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 invention.

Claims

1. A cell culture method based on ultrasound to inhibit cell migration, characterized in that: Step S1, first, placing a cell slide into a cell culture dish in a clean bench, adding a poly-D-lysine solution to immerse the cell slide, and storing it at room temperature away from light, then absorbing the poly-D-lysine solution, washing the cell slide with sterile water, and then drying the cell slide; Step S2, in a clean bench, placing a cell culture dish in the cavity of an ultrasonic device, and adding sterile water into the gap between the cavity of the ultrasonic device and the cell culture dish; Step S3, using an ultrasonic device to emit ultrasonic waves, and allowing the emitted ultrasonic waves to form a standing wave field in the cell culture dish for inhibiting cell migration; Step S4, suspending the cells in a starvation medium to obtain a cell suspension, and then adding the cell suspension to a cell culture dish; Step S5: After static culture for a preset time, the external ultrasonic wave is removed and the cell culture dish is transferred to a carbon dioxide cell culture incubator for static culture.

2. The cell culture method based on ultrasound inhibition of cell migration according to claim 1, characterized in that: In step S1, the concentration of the poly-D-lysine solution is 80-120 ug / mL.

3. The cell culture method based on ultrasound inhibition of cell migration according to claim 1, characterized in that: In the step S1, the cell slide is stored in the dark for at least 2 hours.

4. The cell culture method based on ultrasound inhibition of cell migration according to claim 1, characterized in that: The ultrasonic device in step S2 is mainly composed of an acoustic field generator, a piezoelectric ceramic and a signal generator. A cavity for placing a cell culture dish is provided in the middle of the acoustic field generator. Four piezoelectric ceramics are evenly spaced and attached to the periphery of the acoustic field generator around the circumference of the acoustic field generator. Each piezoelectric ceramic is connected to a signal generator. The signal generator is used to generate a sinusoidal wave signal and act on the piezoelectric ceramic, so that an ultrasonic wave is generated between two non-adjacent piezoelectric ceramics. Step S2 is specifically as follows: In the clean bench, the cell culture dish with the dried cell slides is placed in the cavity of the sound field generator in the ultrasonic device, and sterile water is added to the gap area between the cavity of the sound field generator and the cell culture dish.

5. The cell culture method based on ultrasound inhibition of cell migration according to claim 4, characterized in that: The step S3 is specifically 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 wave signal and act on the pair of piezoelectric ceramics, so that the pair of piezoelectric ceramics generate ultrasonic waves in the cavity of the sound field generator and act on the cell culture dish. The ultrasonic waves form a standing wave field in the cell culture dish to inhibit cell migration.

6. The cell culture method based on ultrasound inhibition of cell migration according to claim 4, characterized in that: In the step S3, the signal generator generates a sine wave signal with a signal frequency of 3.0-3.5 MHz, and the peak voltage of the ultrasonic wave generated by the piezoelectric ceramic is 10-12 Vpp.

7. The cell culture method based on ultrasound inhibition of cell migration according to claim 1, characterized in that: In step S4, the starvation medium mainly consists of cell culture medium, 1-1.2% fetal bovine serum (FBS), 80-120 IU / mL streptomycin and 80-120 IU / mL penicillin.

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

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

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

Images