Cell culture vessel and device adaptive to acoustic test and use method

Abstract

The invention discloses a cell culture vessel and device adaptive to acoustic testing and a using method, belongs to the technical field of cell culture devices, and can solve the problems that an existing cell culture vessel is poor in ultrasonic cell biological effect research accuracy, poor in repeatability and poor in sealing performance due to acoustic interference. The cell culture vessel comprises a cylindrical body, a sealing column, a sealing sheet and a sealing film, the cylindrical body is provided with an axial through cylindrical hole; a communicating hole for communicating the columnar hole with the outside is formed in the side wall of the columnar body; the sealing column is configured to be plugged into the communicating hole; the sealing sheet is attached to the bottom surface of the cylindrical body; the sealing film is attached to the top surface of the cylindrical body; the sound attenuation coefficients of the sealing piece and the sealing film are both smaller than or equal to 0.1. The device is high in sealing performance, can achieve uniform sound field irradiation, is small in acoustic interference, is good in sound field fidelity effect, and can be suitable for various acoustic tests.

Description

Technical Field

[0001] The present application relates to the technical field of cell culture devices, and in particular to a cell culture vessel, device and use method adapted for acoustic testing. Background Art

[0002] Ultrasonic technology can play a positive role in the treatment of diseases, and the basic experimental research on the biological effects of irradiating cell samples through ultrasonic cell irradiation systems has also increased significantly. Acoustic testing can be used to evaluate the effectiveness and safety of ultrasonic technology for cell samples, so the cell culture vessels used for acoustic testing need to provide precise ultrasonic parameter control and a stable culture environment to ensure that the cells in the cell culture vessels receive uniform ultrasonic irradiation in order to obtain accurate and reliable ultrasonic cell biological effects.

[0003] However, existing cell culture vessels do not fully consider the laws of acoustic energy propagation, and lack optimization and consideration of the sound wave propagation path during use, resulting in acoustic interference such as poor sound field fidelity, sound permeability, standing waves, sound wave reflection and scattering, sound wave refraction and sound energy attenuation in the experiment. Acoustic interference leads to poor accuracy and repeatability in the study of ultrasonic cell biological effects, and has also become a thorny problem in verifying the safety and effectiveness of ultrasound therapy. In addition, due to the poor sealing of existing cell culture vessels, it is difficult to conduct acoustic testing on them, especially there are great obstacles to the acquisition of in situ sound field data of cell samples, resulting in the inability to measure the ultrasonic irradiation received by cells in the actual cell culture vessel during use, which is a major problem hindering the safe and effective application of ultrasound technology. Summary of the invention

[0004] The embodiments of the present application provide a cell culture vessel, device and method of use adapted for acoustic testing, thereby solving the problems of poor accuracy and repeatability in ultrasonic cell biological effect research and poor sealing in existing cell culture vessels due to acoustic interference.

[0005] In order to achieve the above object, the technical solution of the embodiment of the present invention is:

[0006] In a first aspect, an embodiment of the present invention provides a cell culture vessel adapted for acoustic testing, comprising a columnar body, a sealing column, a sealing sheet, and a sealing film;

[0007] The columnar body is provided with a columnar hole which penetrates axially;

[0008] The side wall of the columnar body is provided with a connecting hole connecting the columnar hole and the outside world;

[0009] The sealing column is configured to be plugged into the communicating hole;

[0010] The sealing sheet is attached to the bottom surface of the columnar body;

[0011] The sealing film is attached to the top surface of the columnar body;

[0012] The sound attenuation coefficients of the sealing sheet and the sealing film are both less than or equal to 0.1.

[0013] In combination with the first aspect, in a possible implementation manner, the sealing sheet is a glass sheet, a cell climbing sheet or a plastic sheet.

[0014] In combination with the first aspect, in a possible implementation manner, the sealing film is made of one of a PET film, a PC film or a sound-permeable film.

[0015] In combination with the first aspect, in a possible implementation manner, the columnar hole is a quadrangular prism hole or a cylindrical hole.

[0016] In combination with the first aspect, in a possible implementation manner, an angle between a central axis of the communicating hole and a central axis of the columnar body in a counterclockwise direction is an acute angle.

[0017] In combination with the first aspect, in a possible implementation, the cell culture vessel adapted for acoustic testing further includes a fixing body;

[0018] A fixing body is respectively arranged at opposite positions on the outer side wall of the columnar body.

[0019] In combination with the first aspect, in a possible implementation manner, the columnar body includes a plurality of columnar sub-bodies that can be nested.

[0020] In a second aspect, another embodiment of the present invention provides a cell culture device adapted for acoustic testing, comprising the above-mentioned cell culture vessel adapted for acoustic testing, and also comprising a mounting bracket and a mounting sheet;

[0021] The mounting plate is fixedly arranged at the bottom of the cell culture vessel;

[0022] The mounting bracket includes two;

[0023] Each mounting bracket includes a mounting leg and a plurality of positioning tabs;

[0024] A plurality of the positioning pieces are arranged in a linear array on the side of the mounting leg;

[0025] The two mounting brackets are arranged opposite to each other so that the positioning pieces are opposite to each other;

[0026] The mounting piece is arranged on two positioning pieces with the same height.

[0027] In conjunction with the second aspect, in a possible implementation, the cell culture device adapted for acoustic testing further includes a first sound absorbing sheet and a second sound absorbing sheet;

[0028] A through hole is provided in the middle of the first sound absorbing sheet, and the area of ​​the through hole is consistent with the cross-sectional area of ​​the columnar hole;

[0029] The first sound absorbing sheet is arranged on the top surface of the columnar body, and the through hole corresponds to the position of the columnar hole;

[0030] The second sound absorbing sheet is disposed at the bottom of the mounting bracket and is parallel to and corresponding to the first sound absorbing sheet.

[0031] In a third aspect, another embodiment of the present invention provides a method for using a cell culture vessel adapted for acoustic testing, based on the above-mentioned cell culture vessel adapted for acoustic testing, comprising:

[0032] A sealing sheet is attached to the bottom surface of the column, and a sealing film is attached to the top surface of the column;

[0033] Place the column on its side with the communicating hole at the top;

[0034] Injecting the cell suspension from the communicating hole into the axially penetrating columnar hole provided on the columnar body until the columnar hole is filled with the cell suspension, and inserting the sealing column into the communicating hole;

[0035] The cell culture vessels were placed in an acoustic test scenario for testing.

[0036] One or more technical solutions provided in the embodiments of the present invention have at least the following technical effects or advantages:

[0037] The cell culture vessel adapted for acoustic testing provided by the embodiment of the present invention, in actual use, is affixed with a sealing sheet to the bottom surface of the columnar body, and a sealing film to the top surface of the columnar body. The columnar body is turned sideways and the connecting hole is located at the top. The cell suspension is injected from the connecting hole into the axially penetrating cylindrical hole set on the columnar body until the cylindrical hole is filled with the cell suspension, and the sealing column is inserted into the connecting hole, so that the cell culture vessel is a vessel with strong sealing. After that, the cell culture vessel is placed in an acoustic test scene for testing. The cell culture vessel provided by the embodiment of the present application is convenient for acquiring in-situ acoustic test data of cell samples due to its strong sealing, and can be adapted to underwater testing. In addition, since the sound attenuation coefficients of the sealing sheet and the sealing film are both less than or equal to 0.1, uniform sound field irradiation can be achieved, the acoustic interference is small, the sound field fidelity effect is better, and it can be adapted to a variety of acoustic tests. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for use in the description of the embodiments of the present invention will be briefly introduced below. Obviously, the accompanying drawings in the following description are some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying any creative work.

[0039] Figure 1 A stereoscopic cell culture vessel adapted for acoustic testing provided in an embodiment of the present application Figure 1 ;

[0040] Figure 2 for Figure 1 A top view of

[0041] Figure 3 for Figure 2 Middle AA view;

[0042] Figure 4 A stereoscopic cell culture vessel adapted for acoustic testing provided in an embodiment of the present application Figure 2 ;

[0043] Figure 5 for Figure 4 A top view of

[0044] Figure 6 A top view of another cell culture vessel adapted for acoustic testing provided in an embodiment of the present application;

[0045] Figure 7 A top view of another cell culture vessel adapted for acoustic testing provided in an embodiment of the present application;

[0046] Figure 8 A three-dimensional diagram of a cell culture device adapted for acoustic testing provided in an embodiment of the present application;

[0047] Fig. 9 A schematic diagram of a usage scenario of a cell culture device adapted for acoustic testing provided in an embodiment of the present application;

[0048] Fig.10 This is a microscopic image of the cell adhesion state of the cells cultured in Example 3;

[0049] Fig.11 This is a picture of the cell state under a microscope after underwater ultrasonic irradiation in Example 4.

[0050] Icons: 1-cell culture vessel; 11-column; 111-column hole; 112-connecting hole; 12-sealing sheet; 13-sealing film; 14-fixed body; 2-mounting bracket; 21-mounting leg; 22-positioning sheet; 3-mounting sheet; 4-first sound absorbing sheet; 5-second sound absorbing sheet; 6-rubber band. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] In the description of the embodiments of the present invention, it should be noted that the terms "center", "up", "down", "left", "right", "vertical", "horizontal", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, rather than indicating or implying that the devices or elements referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as limitations on the present invention. The terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance. In addition, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, or it can be an indirect connection through an intermediate medium, or it can be a connection between the two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the embodiments of the present invention can be understood according to the specific circumstances.

[0053] Please refer to Figures 1 to 3 As shown, the embodiment of the present invention provides a cell culture vessel 1 adapted for acoustic testing, comprising a column 11, a sealing column, a sealing sheet 12 and a sealing film 13. The column 11 can be cylindrical or prismatic. Figures 1 to 5 The columnar body 11 is a cylindrical structure diagram. The columnar body 11 can be made of acrylic material.

[0054] like Figure 4 and Figure 5 As shown, the columnar body 11 is provided with a columnar hole 111 which penetrates axially.

[0055] like Figure 1 and Figure 3 As shown, the side wall of the columnar body 11 is provided with a connecting hole 112 connecting the columnar hole 111 and the outside.

[0056] The sealing column is configured to be plugged into the communication hole 112 (the sealing column is not shown in the figure and can be a waterproof silicone rod). Figure 1As shown, the sealing sheet 12 is attached to the bottom surface of the column 11. The sealing film 13 is attached to the top surface of the column 11. That is, the cell culture vessel 1 provided in the embodiment of the present application is a three-layer structure. The acoustic attenuation coefficients of the sealing sheet 12 and the sealing film 13 are both less than or equal to 0.1. Therefore, the sealing sheet 12 and the sealing film 13 have good acoustic performance and good sound permeability, which can reduce the loss of acoustic energy in acoustic testing and reduce the acoustic interference of the cell culture vessel 1.

[0057] The cell culture vessel 1 adapted for acoustic testing provided in an embodiment of the present invention, in actual use, the sealing sheet 12 is attached to the bottom surface of the columnar body 11, and the sealing film 13 is attached to the top surface of the columnar body 11. The columnar body 11 is turned sideways and the connecting hole 112 is located at the top. The cell suspension is injected from the connecting hole 112 into the axially penetrating cylindrical hole 111 set on the columnar body 11 until the cylindrical hole 111 is filled with the cell suspension, and the sealing column is inserted into the connecting hole 112, so that the cell culture vessel 1 is a vessel with strong sealing. After that, the cell culture vessel 1 is placed in an acoustic test scene for testing. The cell culture vessel 1 provided in the embodiment of the present application is convenient for acquiring in-situ acoustic test data of cell samples due to its strong sealing, and can be adapted to underwater testing. In addition, since the acoustic attenuation coefficients of the sealing sheet 12 and the sealing film 13 are both less than or equal to 0.1, uniform sound field irradiation can be achieved, acoustic interference is small, and the sound field fidelity effect is better, which can adapt to a variety of acoustic tests.

[0058] Optionally, the sealing sheet 12 is made of a glass sheet, a cell climbing sheet or a plastic sheet, so that the sealing sheet 12 has good acoustic performance (sound permeability), light transmittance and biocompatibility, and can well seal the columnar pores 111. The glass sheet is non-cytotoxic and may be coated. The plastic sheet may be a Thermo Fisher plastic sheet with a special coating suitable for cell wall growth.

[0059] Optionally, the sealing film 13 is made of a PET film, a PC film or a sound-permeable film, so that the sealing film 13 has good acoustic performance (sound permeability), light transmittance and biocompatibility, and can well seal the columnar hole 111 .

[0060] The sealing sheet 12 and the sealing film 13 both have good sound and light transmittance, which can greatly improve the utilization rate of ultrasound, reduce sound attenuation, reduce refraction and reflection, and are used in acoustic testing to make cells more uniform during ultrasound irradiation.

[0061] The sealing sheet 12 and the sealing film 13 both have good biocompatibility and can also be used to culture cells. In addition, due to the good sealing performance, they can be used for underwater ultrasound experiments of adherent cells and suspended cells, and then further culture the cells for observation. At the same time, the sealing sheet 12 and the sealing film 13 both have good light transmittance and can also be directly used for microscopic observation, realizing the multiple functions of the cell culture vessel 1.

[0062] Furthermore, the cylindrical hole 111 is a quadrangular prism hole or a cylindrical hole. Of course, the cylindrical hole 111 can also be other shapes, and the shape of the cylindrical hole 111 can be flexibly adjusted to ensure the uniformity of the sound field during the acoustic test. The size of the cylindrical hole 111 can be customized according to the size of the transducer during the acoustic test, so as to facilitate the adaptation of transducers of various sizes. Quadrangular prism holes and cylindrical holes are easier to make and have a higher degree of adaptability when used.

[0063] like Figure 3 As shown, the counterclockwise angle between the central axis of the connecting hole 112 and the central axis of the columnar body 11 is an acute angle, that is, the connecting hole 112 is inclined on the side wall of the columnar body 11, which is convenient for subsequent injection of liquid substances and not easy to flow back.

[0064] Furthermore, the aperture of the communication hole 112 gradually decreases from the top to the bottom, that is, the shape of the communication hole 112 is a terrace, which can further prevent the backflow of the injected liquid substance. Compared with opening a hole in the sealing film 13, the communication hole 112 is provided on the side wall of the columnar body 11, and the columnar hole 111 is easier to seal after the liquid substance is injected, and the sealing is more convenient and simple, and the sealing performance is better.

[0065] like Figures 1 to 7 As shown, the cell culture vessel 1 adapted for acoustic testing further comprises a fixing body 14. A fixing body 14 is respectively arranged at opposite positions on the outer side wall of the columnar body 11.

[0066] In practice, when the cell culture vessel 1 is used for acoustic testing, it needs to be fixed to the mounting plate 3 and then placed on the mounting bracket 2. A fixing body 14 is respectively arranged at opposite positions on the outer wall of the columnar body 11, so that there are two symmetrical protrusions on the outer wall of the cell culture vessel 1, which is convenient for fixing the cell culture vessel 1 and the mounting plate 3 through the rubber band 6.

[0067] like Figure 6 and Figure 7 As shown, the columnar body 11 includes a plurality of columnar sub-bodies that can be nested. Figure 6 and Figure 7 The schematic diagram of the structure of two columnar sub-bodies nested in each other is shown. The inner diameter of the outer columnar sub-body is consistent with the outer diameter of the inner columnar sub-body, so that the outer columnar sub-body can be nested outside the inner columnar sub-body.

[0068] In practice, in order to fix the cell culture vessel 1 and the mounting plate 3 later, a fixing body 14 can be respectively arranged at a relative position on the outer wall of the columnar body 11, and then the fixing body 14 and the mounting plate 3 can be fixed by a rubber band 6, or a clamping tube can be arranged on the mounting plate 3 to clamp the columnar body 11 in the clamping tube. The columnar body 11 includes a plurality of columnar sub-bodies that can be nested, and only a fixing body 14 can be respectively arranged at a relative position on the outer wall of the outermost columnar sub-body, or only the size of the outermost columnar body 11 needs to be adapted to the size of the clamping tube, and then the internal columnar sub-bodies can be flexibly nested according to the actual transducer size and usage scenario to meet the transducer size, thereby making the cell culture vessel 1 more applicable and more cost-effective.

[0069] like Figure 8 and Fig. 9 As shown, another embodiment of the present invention provides a cell culture device adapted for acoustic testing, including the above-mentioned cell culture vessel 1 adapted for acoustic testing, and also includes a mounting bracket 2 and a mounting plate 3.

[0070] The mounting plate 3 is fixedly arranged at the bottom of the cell culture vessel 1. The mounting brackets 2 include two. Each mounting bracket 2 includes a mounting leg 21 and a plurality of positioning plates 22. A plurality of positioning plates 22 are arranged in a linear array on the side of the mounting leg 21. The two mounting brackets 2 are arranged opposite to each other so that the positioning plates 22 are opposite to each other. The mounting plate 3 is arranged on the two positioning plates 22 of the same height.

[0071] The cell culture device provided in the embodiment of the present application, the arrangement of the mounting bracket 2 and the plurality of positioning plates 22 can conveniently adjust the placement height of the cell culture vessel 1 and facilitate the placement of the cell culture device in an acoustic test scene.

[0072] The cell culture device adapted for acoustic testing provided in an embodiment of the present invention also includes a first sound absorbing sheet 4 and a second sound absorbing sheet 5. A through hole is provided in the middle of the first sound absorbing sheet 4, and the area of ​​the through hole is consistent with the cross-sectional area of ​​the columnar hole 111. The first sound absorbing sheet 4 is provided on the top surface of the columnar body 11, and the position of the through hole corresponds to the columnar hole 111. The second sound absorbing sheet 5 is provided at the bottom of the mounting bracket 2 and is parallel to the first sound absorbing sheet 4 and corresponds in position. The first sound absorbing sheet 4 and the second sound absorbing sheet 5 are made of sound absorbing material. The sound at the position other than the columnar hole 111 can be absorbed by the sound absorbing sheet, so that the use effect of the cell culture device can be better.

[0073] Another embodiment of the present invention provides a method for using a cell culture vessel 1 adapted for acoustic testing, based on the above-mentioned cell culture vessel 1 adapted for acoustic testing, comprising:

[0074] The sealing sheet 12 is attached to the bottom surface of the column 11, and the sealing film 13 is attached to the top surface of the column 11. Specifically, the sealing sheet 12 can be attached to the bottom surface of the column 11 with acrylic silicone resin composite adhesive, and the sealing film 13 can be attached to the top surface of the column 11 with acrylic silicone resin composite adhesive.

[0075] The columnar body 11 is placed on its side with the communication hole 112 located at the top.

[0076] The cell suspension is injected from the communicating hole 112 into the axially penetrating columnar hole 111 provided on the columnar body 11 until the columnar hole 111 is filled with the cell suspension, and the sealing column is inserted into the communicating hole 112 .

[0077] The cell culture vessel 1 is placed in an acoustic test scene for testing.

[0078] Several specific embodiments are provided below.

[0079] Example 1

[0080] The cell culture vessel 1 adapted for acoustic testing provided in the embodiment of the present invention can be used for acoustic field testing. The acoustic field test detects whether the acoustic field of the cell culture vessel 1 itself is uniform. A uniform acoustic field is used in underwater ultrasonic experiments. After cells are loaded, the acoustic field in the cell culture vessel 1 is relatively uniform everywhere, so that the experimental effect is better.

[0081] The glass sheet is adhered to the bottom surface of the columnar body 11 with an acrylic silicone resin composite adhesive, and the PET film is adhered to the top surface of the columnar body 11 with an acrylic silicone resin composite adhesive.

[0082] Three groups were set up, namely, a blank group, a control group and an experimental group. The blank group was in a free sound field in the sound field test scene. The control group was in a state where an existing cell culture vessel was placed between the transducer and the hydrophone in the sound field test scene. The experimental group was in a state where a cell culture vessel 1 according to an embodiment of the present invention was placed between the transducer and the hydrophone in the sound field test scene.

[0083] Connect the output of the signal generator to the input of the power amplifier, and the other end of the power amplifier to the ultrasonic transducer probe. Fix the transducer probe on the three-dimensional motion rack for the sound field test, install the hydrophone at the lower end, and add degassed water to the test water tank until the degassed water submerges the ultrasonic transmitting part of the transducer. Set the signal generator mode, rotate the signal generator button, observe the power on the power amplifier, and start scanning the sound field when the required parameters are adjusted. Adjust the relative parallel position of the transducer and the hydrophone, and record the relative position of the X, Y, and Z axes at this time.

[0084] Scan the X-axis first, and keep the Y and Z axes unchanged. Set the scanning range according to the initial value of the X-axis, set the scanning interval, set the scanning speed, click Auto-collection to start scanning the X-axis, record the coordinates of the position of the highest amplitude of the sound field waveform of the three-dimensional motion system connected to the hydrophone on the X-axis, and fix this coordinate. Start scanning the Z-axis, set the scanning range according to the initial value of the Z-axis, set the interval and speed, click Auto-collection to start scanning the Z-axis, record the coordinates of the position of the highest amplitude of the sound field waveform of the three-dimensional motion system connected to the hydrophone on the Z-axis, and fix this coordinate. Finally, scan the Y-axis, record the maximum value of the position coordinates of the amplitude of the sound field waveform of the three-dimensional motion system connected to the hydrophone on the Y-axis, set the distance between the Y-axis transducer and the hydrophone according to the experimental requirements, and start scanning here.

[0085] Blank group sound field scanning: Set the XZY surface scanning range according to the transducer size. This experiment uses a flat transducer. Set the X and Z axis scanning start and end values, speed, and spacing. Click Scan XZY Surface for automatic acquisition.

[0086] Scanning the sound field of the control group: After scanning the sound field of the blank group, click the X-axis and Z-axis to move the hydrophone to the initial center position, fix the existing cell culture vessel above the hydrophone, and click Scan XZY plane for automatic acquisition.

[0087] Scanning the sound field of the experimental group: After scanning the sound field of the control group, click the X-axis and Z-axis to move the hydrophone to the initial center position, fix the cell culture vessel 1 [sealing sheet 12 (glass), sealing film 13 (PET film), cell culture vessel 1 (sealing film 13 is PET film, sealing sheet 12 is glass)] of the embodiment of the present application above the hydrophone, click to scan the XZY plane, and automatically collect. Through the step scanning system, a two-dimensional cloud map of sound pressure and position is obtained, and parameter calculation is performed through data processing software (such as MATLAB). The specific values ​​of the sound field distribution diagram and related parameters are used to calculate the sound pressure and sound intensity for further application and research.

[0088] The average sound pressure and average sound intensity of the existing culture dish and the culture dish of the present invention, as well as the relative error of the sound pressure change and the relative error of the sound intensity change compared with the free sound field, were calculated. The sound pressure variance, mean square error, maximum deviation, and minimum deviation were calculated to evaluate the uniformity of the sound field of the existing cell culture vessel and the cell culture vessel 1 provided by the embodiment of the present invention. The average sound pressure and average sound intensity of each group, the relative error of the sound pressure change and the relative error of the sound intensity change compared with the free sound field are shown in Table 1 below.

[0089] Table 1

[0090]

[0091] The conditions of the received sound field in the cell culture vessel are shown in Table 1. It can be seen from Table 1 that the average sound pressure and average sound intensity of the cell culture vessel 1 of the embodiment of the present invention are closer to the free sound field than the existing cell culture vessels, indicating that the cell culture vessel 1 provided by the embodiment of the present invention has better acoustic performance underwater. Moreover, the relative errors of the sound pressure and sound intensity changes of the culture vessel of the present invention are smaller than those of the existing cell culture vessels compared with the free sound field, and the propagation characteristics of the sound field remain relatively stable before and after the cell culture vessel 1 provided by the embodiment of the present invention is added. The sound wave loses less energy when passing through the cell culture vessel 1 provided by the embodiment of the present invention, and the changes in sound pressure and sound intensity are also small. It shows that the sound field is more evenly distributed before and after the cell culture vessel 1 provided by the embodiment of the present invention is added, without obvious local enhancement or weakening, and the cell culture vessel 1 provided by the embodiment of the present invention has less influence on the sound field, and the stability of the sound field is higher.

[0092] The variance, mean square error, maximum deviation, and minimum deviation of the sound pressure of each group are shown in Table 2 below.

[0093] Table 2

[0094]

[0095] As can be seen from Table 2, the variance and mean square error of each group are small, the data are relatively uniform, and there is no obvious difference. The maximum deviation and minimum deviation of the cell culture vessel 1 of the embodiment of the present invention are both smaller than those of the existing cell culture vessel, indicating that the difference between the data points in the acoustic field of the cell culture vessel 1 of the embodiment of the present invention is small, and the data distribution is relatively uniform.

[0096] In addition, during actual use, the existing cell culture vessels cannot completely fill the vessel with liquid during ultrasonic irradiation, and there is a part of the interface between the culture liquid and the air. When the ultrasonic wave is transmitted from the liquid to the air, due to the huge difference in acoustic impedance between the two media of liquid and air, there are significant reflections and refractions on the interface, which makes the ultrasonic wave transmission performance poor, and may even form standing waves in the liquid, which not only causes the sound field to be uneven and difficult to calibrate, but also causes damage to the cells in the cell culture vessel. Therefore, since the cell culture vessel 1 provided in the embodiment of the present invention can completely fill the inner cavity of the cell culture vessel 1 with liquid, it has advantages over the existing cell culture vessels during ultrasonic irradiation.

[0097] Example 2

[0098] Sound attenuation test

[0099] The samples to be tested were set up as follows: control group: existing cell culture vessels, experimental group: cell culture vessels 1 according to the embodiment of the present invention (the sealing film 13 was a PET film, and the sealing sheet 12 was glass).

[0100] Prepare underwater sound source, hydrophone, signal generator, power amplifier, data acquisition device, computer and other equipment. Put the sample to be tested into the water and ensure that it is stably fixed in the predetermined position to avoid movement during the test.

[0101] Place the underwater sound source and hydrophone on both sides of the sample to be tested, with the sound source located in the far field of the sample to be tested and the hydrophone on the other side of the sample to be tested. Adjust the height and angle of the sound source and hydrophone so that they are on the same horizontal plane as the center line of the sample to be tested, ensuring that the sound waves can propagate in a straight line.

[0102] Connect the signal generator, power amplifier, data acquisition device and computer, debug the equipment parameters and ensure stable signal transmission. The signal generator generates a sound signal of a specific frequency, which is amplified by the power amplifier and drives the underwater sound source to emit sound waves. The sound waves propagate in the water and are received by the hydrophone after passing through the culture dish.

[0103] Table 3 Sound attenuation test results

[0104]

[0105] It can be seen from Table 3 that the acoustic attenuation coefficient of the existing cell culture vessel is greater than that of the cell culture vessel 1 according to the embodiment of the present invention, which indicates that when ultrasonic waves propagate inside the cell culture vessel 1 provided by the embodiment of the present invention, there is less energy loss, good sound transmission performance, higher ultrasonic utilization efficiency, and more conducive to the propagation of acoustic energy, which can reduce acoustic energy loss in the sound field test.

[0106] Example 3

[0107] The cell culture vessel 1 adapted for acoustic testing provided in the embodiment of the present invention can also be used for cell culture.

[0108] First, prepare a cell suspension.

[0109] The process of preparing the cell suspension is as follows: taking MDA-MB-231 cells (Shanghai Fuheng Biotechnology Co., Ltd.) as an example, take out the adherent cells from the cell bank, inoculate them in a culture bottle or culture dish, add an appropriate amount of culture medium, and place them in a cell culture incubator to culture until the cells reach 70% to 80% confluence. Aspirate the old culture medium in the culture bottle, and gently wash the cells 2 to 3 times with PBS buffer to remove the residual culture medium and dead cells. Add an appropriate amount of trypsin-EDTA solution (0.25% trypsin and 0.02% EDTA) to the culture bottle, and gently shake the culture bottle to evenly cover the cell surface with the trypsin solution. Place the culture bottle in a 37°C incubator and incubate for 3 to 5 minutes until the cells begin to fall off the wall of the culture bottle. Cell morphological changes can be observed under a microscope when the cells become round and begin to fall off. Aspirate the trypsin-EDTA solution, add an appropriate amount of serum-containing culture medium, and gently shake the culture bottle to terminate the digestion of the cells by trypsin. Use a pipette to gently blow the wall of the culture flask to completely remove the cells from the wall of the culture flask to form a cell suspension. Transfer the cell suspension to a centrifuge tube, centrifuge at 1000-1500rpm for 3-5 minutes, discard the supernatant, and collect the cell pellet. Add an appropriate amount of fresh culture medium to the centrifuge tube, and use a pipette to gently blow the cell pellet to evenly resuspend the cells in the culture medium to form a cell suspension.

[0110] Then, the glass sheet is adhered to the bottom surface of the column 11 with an acrylic silicone resin composite adhesive, and the PET film is adhered to the top surface of the column 11 with an acrylic silicone resin composite adhesive. The column 11 is placed on its side with the connecting hole 112 located at the top. The cell suspension is injected into the axially penetrating columnar hole 111 set on the column 11 from the connecting hole 112 with a pipette or a syringe until the columnar hole 111 is filled with the cell suspension, and a waterproof silicone rod is inserted into the connecting hole 112 to seal the connecting hole 112. The column 11 is placed in a constant temperature box and cultured at 37°C for 24 hours. After the cells are attached to the wall, they are placed on a microscope to observe the cell attachment and growth status.

[0111] Take the cell culture vessel 1 out of the incubator and place it on the microscope stage. Align the eyepiece and objective lens of the microscope to the center of the circle and observe the cells. Fig.10 shown. Fig.10 This is a microscopic image of the cell adhesion state of the cells cultured in Example 3. Fig.10 It can be seen that the cells adhered well to the wall as observed under the microscope, which indicates that the cell culture vessel 1 provided in the embodiment of the present invention can be used for cell culture.

[0112] Example 4

[0113] The cell culture vessel 1 adapted for acoustic testing provided in the embodiment of the present invention can also be used for underwater ultrasonic experiments.

[0114] First, a cell suspension is prepared. The process of preparing the cell suspension is the same as that in Example 3.

[0115] Then, a glass sheet is attached to the bottom surface of the columnar body 11 with an acrylic silicone resin composite adhesive, and a PET film is attached to the top surface of the columnar body 11 with an acrylic silicone resin composite adhesive. The columnar body 11 is placed on its side with the connecting hole 112 located at the top. The cell suspension is injected from the connecting hole 112 into the axially penetrating columnar hole 111 set on the columnar body 11 with a pipette or a needle until the columnar hole 111 is filled with the cell suspension, and a waterproof silicone rod is inserted into the connecting hole 112 to seal the connecting hole 112.

[0116] Then, the first sound absorbing sheet 4 made of sound absorbing material with a serrated surface is placed on the top surface of the columnar body 11, and the through hole set in the middle of the first sound absorbing sheet 4 is aligned with the columnar hole 111. The cell culture vessel 1 and the first sound absorbing sheet 4 are fixed on the mounting plate 3 (such as Figure 8 and Fig. 9 As shown, the cell culture vessel 1, the first sound absorbing sheet 4 and the mounting sheet 3 can be fixed into one piece using a rubber band 6).

[0117] Finally, the ultrasonic probe is connected to a multi-channel pulsed ultrasonic diagnostic instrument, and the experimental parameters are adjusted according to the experimental requirements. The pulse mode is selected to adjust the duty cycle, irradiation time, and power. Fig. 9 As shown, the mounting bracket 2 is placed in the water tank, and the mounting plate 3 is set on the two positioning plates 22 of the same height. The bottom of the mounting bracket 2 is parallel to the first sound absorbing plate 4 and the second sound absorbing plate 5 is set at the corresponding position. Clamp the ultrasonic probe with a test tube clamp. Adjust the placement of the test tube clamp so that the ultrasonic transducer probe on it is immersed in water, and the middle of the ultrasonic transducer probe is facing the columnar hole 111 in the middle of the cell culture vessel 1. Adjust the device to set the ultrasonic parameters and click the output button to start ultrasonic irradiation.

[0118] Take the cell culture vessel 1 out of the water and place it on the microscope stage. Align the eyepiece and objective lens of the microscope to the center of the circle and observe the cell morphology after ultrasonic irradiation. Fig.11 shown. Fig.11 This is a cell state diagram under a microscope of cells after underwater ultrasound irradiation in Example 4. Fig.11 It can be seen that there is no leakage inside the cell culture vessel 1 before and after ultrasonic irradiation, which shows that the cell culture vessel 1 has a good sealing effect and can be used in conjunction with the ultrasonic irradiation system to realize underwater ultrasonic experiments on cells.

[0119] The various embodiments in this specification are described in a progressive manner, and the same or similar parts between the various embodiments can be referenced to each other. Each embodiment focuses on the differences from other embodiments.

[0120] The above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit the present application. Although the present application has been described in detail with reference to the aforementioned embodiments, a person of ordinary skill in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the present application.

Claims

1. A cell culture vessel adapted for acoustic testing, characterized in that: It includes a columnar body, a sealing column, a sealing sheet and a sealing film; The columnar body is provided with a columnar hole which penetrates axially; The side wall of the columnar body is provided with a connecting hole connecting the columnar hole and the outside world; The sealing column is configured to be plugged into the communicating hole; The sealing sheet is attached to the bottom surface of the columnar body; The sealing film is attached to the top surface of the columnar body; The sound attenuation coefficients of the sealing sheet and the sealing film are both less than or equal to 0.

1.

2. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: The sealing sheet is made of one of a glass sheet, a cell climbing sheet or a plastic sheet.

3. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: The sealing film is made of one of PET film, PC film or sound-permeable film.

4. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: The columnar holes are quadrangular prism holes or cylindrical holes.

5. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: The counterclockwise angle between the central axis of the communicating hole and the central axis of the columnar body is an acute angle.

6. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: Also includes a fixed body; A fixing body is respectively arranged at opposite positions on the outer side wall of the columnar body.

7. The cell culture vessel adapted for acoustic testing according to claim 1, characterized in that: The columnar body includes a plurality of columnar sub-bodies that can be nested.

8. A cell culture device adapted for acoustic testing, characterized in that: A cell culture vessel adapted for acoustic testing according to any one of claims 1 to 7, further comprising a mounting bracket and a mounting sheet; The mounting plate is fixedly arranged at the bottom of the cell culture vessel; The mounting bracket includes two; Each mounting bracket includes a mounting leg and a plurality of positioning tabs; A plurality of the positioning pieces are arranged in a linear array on the side of the mounting leg; The two mounting brackets are arranged opposite to each other so that the positioning pieces are opposite to each other; The mounting piece is arranged on two positioning pieces with the same height.

9. The cell culture device adapted for acoustic testing according to claim 8, characterized in that: Also includes a first sound absorbing sheet and a second sound absorbing sheet; A through hole is provided in the middle of the first sound absorbing sheet, and the area of ​​the through hole is consistent with the cross-sectional area of ​​the columnar hole; The first sound absorbing sheet is arranged on the top surface of the columnar body, and the through hole corresponds to the position of the columnar hole; The second sound absorbing sheet is disposed at the bottom of the mounting bracket and is parallel to and corresponding to the first sound absorbing sheet.

10. A method for using a cell culture vessel adapted for acoustic testing, characterized in that: The cell culture vessel adapted for acoustic testing according to any one of claims 1 to 7, comprising: A sealing sheet is attached to the bottom surface of the column, and a sealing film is attached to the top surface of the column; Place the column on its side with the communicating hole at the top; Injecting the cell suspension from the communicating hole into the axially penetrating columnar hole provided on the columnar body until the columnar hole is filled with the cell suspension, and inserting the sealing column into the communicating hole; The cell culture vessels were placed in an acoustic test scenario for testing.

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