CN112574877B - Cell assembling method and device based on medical B-ultrasonic machine - Google Patents
Cell assembling method and device based on medical B-ultrasonic machine Download PDFInfo
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Abstract
The invention discloses a cell assembly method, which comprises the following steps: resuspending the cells to be assembled and arranged by using a phosphate buffer solution, and injecting the cells to be assembled and arranged into a cell assembly chamber; step two: placing the cell assembly chamber right above a medical B-ultrasonic machine probe, and connecting by using an ultrasonic coupling agent; step three: starting the medical B-ultrasonic machine, adjusting the frequency to 1-10MHz, the MI to 0.6-1.4 and the TIS to 0.6-1.2 on a control panel of the machine, keeping the signals for 1-10 min, and assembling and arranging the cells. It can realize linear arrangement or complex pattern arrangement of cells, and is simple and easy to operate. The invention also provides an application of the medical B-ultrasonic machine in cell assembly.
Description
Technical Field
The invention belongs to the technical field of biological tissue engineering under biomedical engineering, and relates to a cell assembly method and a cell assembly device based on a medical B-ultrasonic machine.
Background
In the field of tissue engineering, since many cells communicate and function based on a certain shape and structure signal, in order to realize a specific tissue function and guide a specific differentiation direction, it is necessary to assemble and arrange a sample such as cells in a specific structure, that is, an orderly assembled and arranged cell plays an increasingly important role in the controlled growth of cells.
However, most of the conventional cell arrangement methods use a mold to perform passive arrangement on a cell solution, and such passive arrangement methods have disadvantages that they can only perform assembly arrangement on a cell solution, and cannot perform assembly arrangement on cells with smaller size, and therefore, the accuracy of the cell assembly arrangement is not sufficient, and at the same time, the mold contacting with the cells negatively affects the transmission of cell information and cell growth, and thus the conventional cell arrangement methods cannot meet the requirements of cell assembly arrangement.
The bulk ultrasonic wave is an ultrasonic wave which is transmitted in an elastic solid and is widely applied in recent years, and the bulk ultrasonic wave can also be applied to cell assembly and arrangement, and the specific process is as follows: the bulk ultrasonic wave generates ultrasonic waves with specific frequency on the piezoelectric crystal and couples the ultrasonic waves into the microfluid, and substances such as cells and the like move and gather under the action of the acoustic force, so that the bulk ultrasonic wave realizes the assembly and arrangement of the cells.
However, most of the conventional body ultrasonic cell assembly devices are formed by connecting two metal electrodes to a piezoelectric ceramic pressure transducer, the working frequency of the conventional body ultrasonic cell assembly device only covers the bandwidth of one frequency range, the impedance effect is strong outside the frequency range, the conventional body ultrasonic cell assembly device cannot perform effective ultrasonic assembly and arrangement on cells, and the requirements on cell arrangement in tissue engineering and other biological researches are difficult to meet.
Therefore, there is a need to provide a cell assembly and arrangement method that can meet the requirements of cell arrangement in tissue engineering and other biological research.
Disclosure of Invention
The invention aims to solve one of the technical problems in the prior art at least to a certain extent, and therefore, the invention deeply explores a cell assembly method and finds that the medical B-ultrasonic can be used for cell assembly, and has the advantages of wider adjustable frequency domain and better sound field stability.
Accordingly, in a first aspect, the present invention provides the use of a medical B-ultrasound machine for cell assembly.
In the technical scheme of the invention, the medical B-ultrasonic machine probe is a medical B-ultrasonic linear array probe or a medical B-ultrasonic phased array probe.
In a second aspect of the invention, the invention provides a method of cell assembly comprising the steps of,
the method comprises the following steps: resuspending the cells to be assembled and arranged by using a phosphate buffer solution, and injecting the cells to be assembled and arranged into a cell assembly chamber;
step two: placing the cell assembly chamber right above the medical B-ultrasonic probe;
step three: starting the medical B-ultrasonic machine, adjusting the frequency to 1-10MHz, the MI to 0.6-1.4 and the TIS to 0.6-1.2 on a control panel of the machine, keeping the signals for 1-10 min, and assembling and arranging the cells.
In a third aspect of the present invention, the present invention provides an apparatus used in the above cell assembly method, including a medical B-ultrasonic machine and a cell assembly chamber fixed directly above a probe of the medical B-ultrasonic machine, where the cell assembly chamber includes a cell culture dish, a polymethylmethacrylate chamber, and a glass sheet, the cell culture dish is fixed directly above the probe of the medical B-ultrasonic machine after being processed, the polymethylmethacrylate chamber is fixed on the cell culture dish, the chamber of the polymethylmethacrylate chamber is communicated with the inside of the cell culture dish, and the glass sheet is covered on the polymethylmethacrylate chamber.
In the technical scheme of the invention, the cell assembly chamber is fixed right above a probe of the medical B-ultrasonic machine.
In the technical scheme of the invention, the device also comprises a sucker type universal bench clamp for fixing the probe of the medical B-ultrasonic machine, wherein the sucker type universal bench clamp comprises a bottom sucker, a middle universal ball for adjusting the orientation of the probe of the medical B-ultrasonic machine and a top jaw for fixing the probe of the medical B-ultrasonic machine.
In the technical scheme of the invention, in the device, the probe of the medical B ultrasonic machine is a medical B ultrasonic linear array probe or a medical B ultrasonic phased array probe.
In the technical scheme of the invention, the thickness of the polymethyl methacrylate cavity (5) is 1-5 mm.
The invention also provides the application of the cell assembling method and the device in tissue engineering and biological research.
In the technical scheme of the invention, the cell assembly method and the device are applied to tissue engineering and biological research, and can realize linear arrangement or complex pattern arrangement of cells.
The invention has the beneficial effects that:
1. the medical B ultrasonic probe is applied to cell assembly for the first time, so that a more stable body ultrasonic sound field pattern is realized, the medical B ultrasonic probe can be more suitable for cell control requirements of biological research and tissue engineering, and has a certain application prospect;
2. the invention provides a cell assembly method, which can realize linear arrangement or complex pattern arrangement of cells and is simple and easy to operate;
3. the invention provides a cell assembling device which is simple in structure and convenient to operate.
Drawings
FIG. 1 is a schematic structural view of a cell assembly apparatus according to an embodiment of the present invention;
FIG. 2 is a schematic diagram of a cell assembly chamber according to an embodiment of the present invention;
FIG. 3 is a schematic view of a medical ultrasonic B-array probe used for assembling polystyrene microspheres in example 1 of the present invention; wherein, fig. 3a is a scanning mode of a medical B-ultrasonic linear array probe in embodiment 1 of the present invention; FIG. 3b is a polystyrene microsphere pattern before assembly; FIG. 3c is a pattern of polystyrene microspheres after assembly; FIG. 3d is the ultrasonic imaging diagram of the B-ultrasonic machine of the medical B-ultrasonic linear array probe;
FIG. 4 is a schematic view of a medical ultrasonic B-mode phased array probe used for assembling and arranging polystyrene microspheres in example 2 of the present invention; fig. 4a shows a scanning method of a medical B-mode ultrasound phased array probe in embodiment 2 of the present invention; FIG. 4b is a polystyrene microsphere pattern before assembly; FIG. 4c is a pattern of polystyrene microspheres after assembly; FIG. 4d is the ultrasonic imaging chart of the B-ultrasonic machine; FIG. 4e is a microsphere pattern after assembly; FIG. 4f is the ultrasonic imaging diagram of the B-ultrasonic machine of the medical B-ultrasonic phased array probe;
wherein the cell assembly chamber 1; a medical B-ultrasonic machine 2; a sucker type universal bench vice 3; a bottom suction cup 3-1; a middle universal ball 3-2; 3-3 parts of a top jaw; a glass sheet 4; a polymethylmethacrylate chamber 5; a cell culture dish 6.
Detailed Description
The scheme of the invention will be explained with reference to the examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples, where specific techniques or conditions are not indicated, are to be construed according to the techniques or conditions described in the literature in the art or according to the product specifications.
Application of medical B-ultrasonic machine in cell assembly
The invention provides an application of a medical B-ultrasonic machine in cell assembly. The medical B ultrasonic probe is applied to cell assembly for the first time, so that a more stable body ultrasonic sound field pattern is realized, the medical B ultrasonic probe can be more suitable for cell control requirements of biological research and tissue engineering, and has a certain application prospect.
In the technical scheme of the invention, the medical B-ultrasonic machine probe is a medical B-ultrasonic linear array probe or a medical B-ultrasonic phased array probe.
Cell assembly method
The invention provides a cell assembly method, comprising the following steps,
the method comprises the following steps: resuspending the cells to be assembled and arranged by using a phosphate buffer solution, and injecting the cells to be assembled and arranged into a cell assembly chamber;
step two: placing the cell assembly chamber right above the medical B-ultrasonic probe;
step three: starting the medical B-ultrasonic machine, adjusting the frequency to 1-10MHz, the MI to 0.6-1.4 and the TIS to 0.6-1.2 on a control panel of the machine, keeping the signals for 1-10 min, and assembling and arranging the cells.
The device used in the above cell assembly method
The invention provides a device used in the cell assembly method, which comprises a medical B-ultrasonic machine 2 and a cell assembly chamber 1 fixed right above a probe of the medical B-ultrasonic machine 2, wherein the cell assembly chamber 1 comprises a cell culture dish 6, a polymethyl methacrylate chamber 5 and a glass sheet 4, the cell culture dish 6 is fixed right above the probe of the medical B-ultrasonic machine 2 after being processed, the polymethyl methacrylate chamber 5 is fixed on the cell culture dish 6, the chamber of the polymethyl methacrylate chamber 5 is communicated with the interior of the cell culture dish 6, and the glass sheet 4 covers the polymethyl methacrylate chamber 5.
In the technical scheme of the invention, the cell assembly chamber 1 is fixed right above a probe of the medical B-ultrasonic machine 2.
In the technical scheme of the invention, the device also comprises a sucker type universal bench clamp 3 for fixing the probe of the medical B-ultrasonic machine 2, wherein the sucker type universal bench clamp 3 comprises a bottom sucker 3-1, a middle universal ball 3-2 for adjusting the orientation of the probe of the medical B-ultrasonic machine 2 and a top jaw 3-3 for fixing the probe of the medical B-ultrasonic machine 2.
In the technical scheme of the invention, in the device, the probe of the medical B-ultrasonic machine 2 is a medical B-ultrasonic linear array probe or a medical B-ultrasonic phased array probe.
In the technical scheme of the invention, the thickness of the polymethyl methacrylate cavity 5 is 1-5 mm.
The invention also provides the application of the cell assembling method and the device in tissue engineering and biological research.
In the technical scheme of the invention, the cell assembly method and the device are applied to tissue engineering and biological research, and can realize linear arrangement or complex pattern arrangement of cells.
The following embodiments of the invention describe a cell assembly method based on a medical B-ultrasonic machine, a device and an application thereof by taking a medical B-ultrasonic linear array probe and a phased array probe to assemble and arrange polystyrene microspheres as representative examples.
Example 1: medical B ultrasonic linear array probe for assembling and arranging polystyrene microspheres
The microsphere in the microsphere solution is made of polystyrene, has purple color, diameter of 10um and density of 1.05g/cm 3 The physical property is similar to that of cells, and the concentration of the microsphere solution is 1mg/ml. In this example, polystyrene microspheres were used to mimic cells.
As shown in FIG. 1, the polystyrene microsphere cell assembly device in the embodiment of the invention comprises three parts, namely a sucker type universal bench clamp 3, a medical B-ultrasonic machine 2 and a cell assembly chamber 1. The probe used by the medical B-ultrasonic machine is a medical B-ultrasonic linear array probe. The scanning mode of the medical B-ultrasonic linear array probe is shown in figure 3 a.
The model of the sucker type universal bench clamp 3 is MNT-608855, and the bottom sucker 3-1 can adsorb a flat desktop, so that the stability of the whole device is enhanced; the top jaw 3-3 can adjust the clamping distance within a certain range and is used for fixing the medical B ultrasonic linear array probe; the middle universal ball 3-2 can rotate 360 degrees and is used for adjusting the medical B ultrasonic linear array probe to keep the probe horizontal.
The model of the medical B-ultrasonic machine 2 is GE-Vivid 95, the model of the medical B-ultrasonic linear array probe is GE-9L, the size right above the probe is 1.4 multiplied by 5.6cm, the adjustable frequency domain is 3-10MHz, and the MI index range is 0.8-1.3.
As shown in fig. 2, the cell assembly chamber 1 is composed of three parts, the bottom is a processed 10cm cell culture dish 6, the model is NEST-704001, the culture dish is carved by a laser carving machine, and the carving size is 1.4 multiplied by 5.6cm; the middle part is a processed polymethyl methacrylate cavity 5, and an expected pattern drawn according to software is guided into a laser engraving machine for engraving, wherein the thickness is 1.8mm; the top is a glass sheet 4 with high transparency and the thickness is 0.13mm.
When the standing wave sound field is formed and the cells are assembled and arranged by using the method, the steps are as follows:
1. the suction cup type universal bench clamp 3 is fixed on a table top through a suction cup, the medical B ultrasonic linear array probe of the medical B ultrasonic machine 2 is fixed on the suction cup type universal bench clamp 3 with the front side facing upwards, the level meter is placed above the medical B ultrasonic linear array probe, and the medical B ultrasonic linear array probe is kept in a horizontal state by adjusting the middle universal ball 3-2. A thin layer of ultrasonic couplant is coated on the upper portion of the medical B ultrasonic linear array probe, the cell assembly chamber 1 and the medical B ultrasonic linear array probe are fixed together, and no bubbles are generated in the middle of the ultrasonic couplant.
2. The polystyrene microspheres to be assembled are resuspended in phosphate buffer, where we use purple polystyrene microspheres, and the concentration of the microsphere solution is kept at 1mg/ml for use. And then injecting the polystyrene microsphere solution into the cell assembly chamber 1 by using a liquid transfer device, covering a glass sheet to ensure that no bubbles are generated in the cell assembly chamber 1, and standing for 1min to ensure that the microspheres are settled to the bottom of the cell assembly chamber 1. The pattern is shown in figure 3 b. As can be seen, the purple polystyrene microspheres are dispersed.
3. Starting the medical B-ultrasonic machine, adjusting the frequency to be 3MHz, the MI to be 1.2 and the TIS to be 1.1 on a control panel of the machine, applying the machine to a corresponding medical B-ultrasonic linear array probe, keeping a signal for 3min, and gathering the polystyrene microspheres to the place of a standing wave sound field node to form a linear pattern, as shown in figure 3c, the figure shows that the linear pattern appears, which indicates that the polystyrene microspheres are assembled to form directional arrangement. The real-time echo signal image on the display of the medical B-ultrasonic machine is shown in figure 3 d.
Example 2: medical B ultrasonic phase control array probe for assembling and arranging polystyrene microspheres
The microsphere in the microsphere solution is made of polystyrene, has purple color, diameter of 10um and density of 1.05g/cm 3 The physical property is similar to that of cells, and the concentration of the microsphere solution is 1mg/ml. In this example, polystyrene microspheres were used to mimic cells.
As shown in FIG. 1, the polystyrene microsphere cell assembly device in the embodiment of the invention comprises three parts, namely a sucker type universal bench clamp 3, a medical B-ultrasonic machine 2 and a cell assembly chamber 1. The probe used by the medical B-ultrasonic machine is a medical B-ultrasonic phased array probe. The scanning mode of the medical B-ultrasonic phased array probe is shown in figure 4 a.
The model of the sucker type universal bench clamp 3 is Meinaite MNT-608855, and the bottom sucker 3-1 can adsorb a flat desktop, so that the stability of the whole device is enhanced; the top jaw 3-3 can adjust the clamping distance within a certain range and is used for fixing the medical B ultrasonic linear array probe; the middle universal ball 3-2 can rotate 360 degrees and is used for adjusting the medical B ultrasonic phased array probe to keep the probe horizontal.
The model of the medical B-ultrasonic machine is GE-Vivid 95, the model of the medical B-ultrasonic linear array probe is GE-MSSC, the size right above the probe is 1.9 multiplied by 3.0cm, the adjustable frequency domain is 2-4.5MHz, and the MI index range is 0.6-1.2.
As shown in FIG. 2, the cell assembly chamber is composed of three parts, the bottom is a processed 10cm cell culture dish 6, the model is NEST-704001, the culture dish is carved by a laser carving machine, and the carving size is 1.9 multiplied by 3.0cm; the middle part is a processed polymethyl methacrylate cavity 5, and an expected pattern drawn according to software is guided into a laser engraving machine for engraving, wherein the thickness is 1.8mm; the top is a glass sheet 4 with high transparency and the thickness is 0.13mm.
When the device is used for forming an acoustic field and assembling and arranging cells, the method comprises the following steps:
1. the suction cup type universal bench clamp 3 is fixed on a table top through a suction cup, a medical B ultrasonic phased array probe of the medical B ultrasonic machine 2 is fixed on the suction cup type universal bench clamp 3 with the front side facing upwards, a level meter is placed above the probe, and the medical B ultrasonic probe is kept in a horizontal state by adjusting a middle universal ball 3-2. A thin layer of ultrasonic coupling agent is coated above the medical B ultrasonic phased array probe, the cell assembly chamber 1 and the medical B ultrasonic phased array probe are fixed together, and no bubbles are generated in the middle of the ultrasonic coupling agent.
2. The polystyrene microspheres to be assembled are resuspended in phosphate buffer, where we use purple polystyrene microspheres, and the concentration of the microsphere solution is kept at 1mg/ml for use. And then injecting the polystyrene microsphere solution into the cell assembly chamber 1 by using a liquid transfer device, covering the glass sheet 4 to ensure that no bubbles are generated in the cell assembly chamber 1, and standing for 1min to ensure that the polystyrene microspheres are settled to the bottom of the cell assembly chamber 1. The pattern is shown in figure 4 b. As can be seen, the purple polystyrene microspheres are dispersed.
3. Starting the medical B-ultrasonic machine 2, adjusting the frequency to 1.7MHz, MI to 1.2 and TIS to 0.6 on a control panel thereof, applying the signals to a corresponding medical B-ultrasonic phased array probe to form a pattern as shown in figure 4c, wherein a complex pattern appears as seen from the figure, and a real-time echo signal image on a display of the medical B-ultrasonic machine 2 is shown in figure 4 d; the adjustment frequency was 2.3MHz, MI was 1.4, and TIS was 0.8, the corresponding pattern was formed as shown in FIG. 4e, and the real-time echo signal image on the display of the medical B-mode ultrasound machine is shown in FIG. 4 f. The signal is kept for 3min, and the cells are gathered to the place of the wave node of the standing wave sound field to form a corresponding pattern.
In conclusion, different medical B ultrasonic probes are used for cell assembly, so that a more complex sound field pattern can be realized, the stability is better, direct contact is not needed, and no damage is caused. Provides a completely new solution for the requirement of cell arrangement in tissue engineering and other biological researches.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (7)
1. A method for assembling cells in a linear or patterned arrangement of cells, comprising the steps of,
the method comprises the following steps: resuspending the cells to be assembled and arranged by using a phosphate buffer solution, and injecting the cells to be assembled and arranged into a cell assembly chamber;
step two: placing the cell assembly chamber right above a medical B-ultrasonic machine probe, and connecting by using an ultrasonic coupling agent;
step three: starting a medical B-ultrasonic machine, adjusting the frequency to be 1-10MHz, the MI to be 0.6-1.4 and the TIS to be 0.6-1.2 on a control panel of the B-ultrasonic machine, keeping signals for 1-10 min, and assembling and arranging cells;
the medical B-ultrasonic machine probe is a medical B-ultrasonic linear array probe or a medical B-ultrasonic phased array probe.
2. The device used in the cell assembly method according to claim 1, comprising a medical B-ultrasonic machine (2) and a cell assembly chamber (1) fixed right above a probe of the medical B-ultrasonic machine (2), wherein the cell assembly chamber (1) comprises a cell culture dish (6), a polymethyl methacrylate chamber (5) and a glass sheet (4), the cell culture dish (6) is fixed right above the probe of the medical B-ultrasonic machine (2) after being processed, the polymethyl methacrylate chamber (5) is fixed on the cell culture dish (6), the chamber of the polymethyl methacrylate chamber (5) is communicated with the inside of the cell culture dish (6), and the glass sheet (4) covers the polymethyl methacrylate chamber (5).
3. The device according to claim 2, characterized in that it further comprises a suction-cup type universal vice (3) for fixing the probe of the medical type-B ultrasonic machine (2), said suction-cup type universal vice (3) comprising a bottom suction cup (3-1), a middle universal ball (3-2) for adjusting the orientation of the probe of the medical type-B ultrasonic machine (2) and a top jaw (3-3) for fixing the probe of the medical type-B ultrasonic machine (2).
4. The device according to claim 2, characterized in that the probe of the medical B-ultrasonic machine (2) is a medical B-ultrasonic linear array probe or a medical B-ultrasonic phased array probe.
5. Device according to claim 2, characterized in that the thickness of the polymethylmethacrylate chamber (5) is 1-5 mm.
6. Use of the cell assembly method of claim 1 or the device of claim 2 in tissue engineering and biological research.
7. Use according to claim 6, characterized in that it allows the realization of linear arrangements or complex patterned arrangements of cells.
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