WO2013133485A1

WO2013133485A1 - Ultrasonic petri dish and ultrasonic monitoring system using same

Info

Publication number: WO2013133485A1
Application number: PCT/KR2012/005073
Authority: WO
Inventors: 최민주; 강관석; 조성찬; 테츠야코다마
Original assignee: 제주대학교 산학협력단
Priority date: 2012-03-05
Filing date: 2012-06-27
Publication date: 2013-09-12
Also published as: KR101441179B1; KR20130101340A

Abstract

Disclosed are an ultrasonic Petri dish used for experimenting on the reaction of a cell to ultrasonic waves or applying ultrasonic waves to a cell, and an ultrasonic monitoring system using same. The ultrasonic Petri dish is characterized by comprising: a body for containing a cell and a culture medium; and an ultrasonic sensing film attached to the body and sensing an ultrasonic signal. The body of the ultrasonic Petri dish consists of a material having an acoustic impedance similar to a body tissue and a low ultrasonic attenuation coefficient so as to increase the efficiency of ultrasonic wave penetration and reduce an attenuation effect. The sensing film is a generic term for a piezoelectric sensor film formed from a piezoelectric material having electrodes coated on both sides thereof, and can be attached to the inner bottom surface, the outer bottom surface, and/or the inner sidewall surface of the body of the Petri dish. The ultrasonic monitoring system is characterized by comprising: an ultrasonic Petri dish having an ultrasonic sensing film attached thereto; an ultrasonic wave generating unit for emitting an ultrasonic wave to the cell and the culture medium contained in the ultrasonic Petri dish; a signal obtaining unit for obtaining the signal sensed by the ultrasonic sensing film; a signal analyzing unit for analyzing the signal received from the signal obtaining unit, thereby estimating the power/intensity/pressure of the ultrasonic wave emitted to the cell and the culture medium, the height of the culture medium, the thickness of a cell layer, the cavitation generated in the culture medium/cell, the dynamic response of the cell, the temperature change of the culture medium/cell, etc.; a display unit for outputting the result analyzed by the signal analyzing unit; and a control unit for controlling the ultrasonic wave generating unit, the signal obtaining unit, the signal analyzing unit, and the display unit.

Description

Ultrasonic Petri Dishes and Ultrasonic Monitoring System Using Them

The present invention is an ultrasonic culture dish suitable for experimenting the response of a cell or tissue (hereinafter, referred to as a 'cell') to an ultrasonic wave or performing an ultrasonic treatment on the cell, while culturing a cell or experimenting with a cell response. And an ultrasonic monitoring system using the same.

Recently, the use of ultrasonic waves can effectively handle cell crushing, particle dispersion, particle crushing, homogenization, heterogeneous liquid emulsification, DNA fragmentation, active substance extraction, chemical reaction catalysis, and the like. Attempts have been actively made to use ultrasound to study the growth effects.

When experimenting with the response of a cell to ultrasound or performing ultrasound treatment on the cell, the cell activity against the ultrasound, the growth rate of the cell, and the cell's growth depending on the exposure conditions such as the frequency, pressure, intensity, and power of the ultrasound emitted to the cell. Life and death, abnormal cell development, etc. are different.

The power of the generated ultrasonic waves depends on the characteristics and performance of the ultrasonic transducer for the same driving conditions. The acoustic properties of the material of the petri dish affect the propagation efficiency of the ultrasonic waves passing through the petri dish. Therefore, non-invasive real-time monitoring of the power / pressure / intensity of the ultrasonic wave actually irradiated on the cell and the cavitation, which is a secondary acoustic phenomenon caused by the ultrasonic wave, can be performed to test the cell response to the ultrasonic wave or to perform ultrasonic wave treatment on the cell. It should be possible.

The size of a cell may change with respect to a physical stimulus such as ultrasound, which is information for interpreting the cell's sensitivity to the stimulus and the effect of cell growth. However, it is very difficult to observe changes in cell volume (cell layer thickness) or morphological response while irradiating ultrasound.

When the continuous wave ultrasound is irradiated, the sound field in the culture dish has standing wave characteristics, and the standing wave is sensitive to the height of the culture medium. When irradiating the culture dish with ultrasonic waves, the height of the culture solution is important information to accurately determine the characteristics of the ultrasonic waves applied to the cells. However, it is not easy to measure the height of the culture solution in real time and non-invasively while irradiating ultrasound.

When ultrasonic waves are irradiated to cells in the culture dish, the temperature rises due to the thermal effect of the ultrasonic waves. The temperature of the culture media affects cavitation, a biologically important environmental factor for cells and an important mechanism for the biological effects of ultrasound. When testing the cell's dynamic response to ultrasound or sonicating the cell, changes in temperature should be monitored in a non-invasive real time.

In conclusion, in order to experiment the reaction of the cells using ultrasonic waves or to efficiently perform the ultrasonic treatment on the cells, a culture dish made of a material having good ultrasonic permeability is required, and the exposure of the ultrasonic waves applied to the cells when the ultrasonic waves are irradiated. Characteristics (frequency, pressure, intensity, power, etc.), acoustic phenomena secondary to ultrasound (e.g. cavitation, dynamic response of cells), height of the culture medium, thickness of the cell layer, scattering / damping characteristics of the ultrasound to the cells , A monitoring device capable of observing a change in temperature of the culture medium / cell and the like, and a system for controlling the output of the ultrasound based thereon are required. Petri dishes and ultrasonic monitoring and control systems are not yet available for these ultrasonic experiments.

The present invention has been made to solve the above-mentioned problems, the information about the response of the cells due to the ultrasound and the power of the ultrasonic wave to be observed when experimenting the response of the cell to the ultrasound using a culture dish. Its purpose is to provide a technique that can be monitored in real time in a non-invasive manner. More specifically, it is an object of the present invention to specifically test the response of a cell or tissue (hereinafter referred to as a "cell") to ultrasonic waves or to perform an ultrasonic treatment on the cells while culturing the cells or testing the reaction of the cells. Ultrasonic culture dishes suitable for the following, and during the experiment of cell culture or the reaction of cells to ultrasound using such culture dishes, the power, cavitation, dynamic response of the cells, the height of the culture medium, To provide an ultrasonic monitoring system for non-invasive real-time monitoring of the thickness of the cell layer, the scattering / attenuation characteristics of the ultrasonic wave to the cell, and the temperature change of the culture medium / cell using an ultrasonic sensor. to be.

According to one embodiment for achieving the object of the present invention as described above, the ultrasonic culture dish, the body that can contain cells and culture medium; And an ultrasonic sensing film attached to the body and capable of sensing an ultrasonic signal.

The body of the culture dish may be formed of a material having a low acoustic attenuation coefficient and acoustic impedance similar to that of living tissue to increase the ultrasonic transmission efficiency and reduce the attenuation effect. At this time, the body is preferably formed of a transparent methylpentene polymer resin (TPX).

The ultrasonic sensing film may be attached to at least one of an inner bottom surface, an outer bottom surface, and an inner side wall surface of the body. Here, the ultrasonic sensing film may include a piezoelectric sensor film coated with electrodes on both surfaces of the piezoelectric material. In this case, the piezoelectric sensor film is preferably composed of a transparent polyvinylidene fluoride (PVDF) film.

Ultrasonic monitoring system according to another embodiment for achieving the object of the present invention comprises an ultrasonic culture dish with the ultrasonic sensing film described above; An ultrasonic generator for irradiating ultrasonic waves to the cells and the culture solution contained in the ultrasonic culture dish; A signal acquisition unit receiving a signal detected by the ultrasonic sensing film; Signal analysis unit for estimating the power / intensity / pressure of the ultrasonic wave irradiated to the cell, the height of the culture medium, the thickness of the cell layer, the cavitation, the dynamic response of the cell, the temperature change of the culture medium / cell by analyzing the signal received from the signal acquisition unit ; A display unit for outputting a result analyzed by the signal analyzer; And a controller for controlling the ultrasonic generator, the signal acquisition unit, the signal analyzer, and the display unit.

The ultrasonic generator may include one or more ultrasonic transducers in contact with the outer bottom surface of the ultrasonic culture dish and a driver for electrically driving the ultrasonic transducers.

The ultrasonic drive unit includes a function generator for generating an electric signal corresponding to the frequency, mode (continuous wave, pulse (pulse length and pulse repetition period)) of the ultrasonic wave set by the user, a power amplifier for amplifying the electric signal, and amplified electric power. It may include a matching network to allow energy to be transferred to the ultrasonic transducer well. In addition, the ultrasonic sensing film attached to the ultrasonic culture dish may further include an ultrasonic pulse / receiver to drive in the pulse echo mode. As a result, the A-mode RF signal for the cells and the culture medium may be obtained through the ultrasonic sensing film. In this case, the A-mode RF signal contains information related to the thickness of the cell layer and the ultrasonic scattering / damping characteristics of the cell through the signal processor.

The signal acquisition unit filters a preamplifier pre-amplifying a signal received from the ultrasonic sensing membrane and an ultrasonic signal amplified by the preamplifier to increase the S / N ratio of the signal detected by the ultrasonic sensing membrane attached to the ultrasonic culture dish. And an A / D converter for converting the output first filter and the filtered signal into a digital signal.

In addition, the signal acquisition unit may further include a second filter for filtering and outputting the pyroelectric signal portion of the signal amplified by the preamplifier.

The signal analysis unit analyzes the signal received from the signal acquisition unit to analyze the pressure of the ultrasonic wave in the culture dish and the standing wave characteristics formed in the culture dish, and based on this, the power / pressure / intensity of the ultrasonic wave irradiated into the cells in the culture dish. , Signals related to cavitation and cell dynamics and reactions, the height of the culture medium, the thickness of the cell layer, the ultrasonic scattering / damping characteristics of the cells, and changes in the temperature of the culture / cells. To this end, the signal analysis unit is an ultrasonic power analysis unit for extracting the ultrasonic intensity, power and pressure from the ultrasonic signal output from the signal acquisition unit, the pyroelectric signal analysis to estimate the temperature and ultrasonic power from the pyroelectric signal output from the signal acquisition unit A-mode RF signal for analyzing the height of the culture medium, the thickness of the cell layer, and the ultrasonic attenuation / scattering characteristics of the culture medium / cell from the A-mode RF signal received through the A / D converter of the signal acquisition unit from the pulser / receiver. It may include an analysis unit.

The display unit may display a setting window under the control of the controller so that a user may input a setting condition through the user interface.

As described above, the ultrasonic culture dish of the present invention may efficiently transmit ultrasonic energy to cells by using a material having high ultrasonic transmission efficiency and low attenuation.

In addition, the ultrasonic monitoring system configured by attaching an ultrasonic sensing film to the body of the culture dish is a signal related to the power / pressure / intensity, ultrasonic cavitation, and the dynamics and reactions of the ultrasonic waves applied to the cells while irradiating the ultrasonic waves, which are not previously possible, and the culture medium. The height of the layer, the thickness of the cell layer, the ultrasonic scattering / attenuation characteristics of the cells, the temperature change of the culture medium / cells can be observed in real time non-invasive.

1 is a plan view illustrating an ultrasonic culture dish with an ultrasonic sensing film according to an embodiment of the present invention;

FIG. 2 is a cross-sectional view in the direction of the arrow along the line A-A of FIG. 1; FIG.

Figure 3 is a photograph of the ultrasonic culture dish attached to the PVDF sensing membrane on the inner bottom surface produced according to an embodiment of the present invention;

4 is a block diagram schematically illustrating an ultrasonic monitoring system according to an embodiment of the present invention using the ultrasonic culture dish shown in FIG. 1;

FIG. 5 is a flow chart of the output extraction process obtained using the ultrasonic monitoring system shown in FIG. 4 when experimenting with the reaction of cells in the ultrasonic culture dish illustrated in FIG. 1 or irradiating ultrasonic waves for sonication of the cells; FIG.

FIG. 6 is a cross-sectional view illustrating a case where an ultrasonic culture dish is placed in a water tank in the ultrasonic monitoring system shown in FIG. 4; FIG.

FIG. 7 is a graph showing examples of ultrasonic waveforms measured through the ultrasonic sensing film of the ultrasonic culture dish shown in FIG. 3;

FIG. 8 is a graph illustrating the correlation between the measured ultrasonic magnitude and the electrical power driving the ultrasonic transducer, measured using the ultrasonic culture dish shown in FIG. 3;

FIG. 9 is a graph showing an example of an A-mode RF signal measured by driving an ultrasonic sensing film of the ultrasonic culture dish shown in FIG. 3 in a pulse / eco mode; FIG.

FIG. 10 is a graph showing an example of the change of the ultrasonic waveform measured in the ultrasonic sensing film while lowering the culture medium height from 8.6 mm to 7.64 mm in the ultrasonic culture dish shown in FIG. 3;

FIG. 11 is a graph illustrating the change in the ultrasonic pressure measured on the ultrasonic sensing membrane (left graph) compared with the theory (right graph) while varying the height of the culture medium in the range of one wavelength in the ultrasonic culture dish shown in FIG. 3;

FIG. 12 is a circuit diagram showing a pyroelectric potential induced by a temperature change of a culture solution due to ultrasonic irradiation and an equivalent electric circuit of PVDF used as an ultrasonic sensing film of the ultrasonic culture dish shown in FIG. 3;

FIG. 13 is a graph showing examples of a superelectric voltage signal induced in an ultrasonic sensing film by a temperature change of a culture solution raised when ultrasonic waves are irradiated to the ultrasonic culture dish shown in FIG. 3 for 10 seconds;

FIG. 14 is a graph showing an example of a time-integrated value of the superelectric voltage signal induced in the sensing membrane by the temperature of the culture solution raised when the ultrasonic culture dish shown in FIG. 3 is irradiated with ultrasonic waves for 10 seconds; FIG.

FIG. 15 illustrates the correlation between the maximum value of the superelectric voltage signal induced in the ultrasonic sensing membrane and the ultrasonic sound power when the ultrasonic culture dish shown in FIG. 3 is irradiated with ultrasonic waves for 10 seconds. graph; And

FIG. 16 is a flowchart illustrating a process when experimenting with a reaction of a cell to ultrasonic waves or ultrasonically treating a cell using an ultrasonic monitoring system using an ultrasonic culture dish according to an embodiment of the present invention.

Hereinafter, an ultrasonic culture dish and an ultrasonic monitoring system using the same according to preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 and 2 are a plan view and a cross-sectional view illustrating an ultrasonic culture dish according to an embodiment of the present invention.

As shown in FIG. 1 and FIG. 2, the ultrasonic culture dish 1 includes a body 10 and an ultrasonic sensing film 20.

The body 10 is for containing a culture solution 15 and cells containing cells to be treated by ultrasonic reaction experiment or ultrasonic wave, and is divided into a side portion 11 and a bottom portion 12.

The body 10 is formed in a cylindrical shape with a flat bottom and an open top so as to culture the cells with the culture solution 15 contained therein.

The body 10 is similar to the culture medium or the biological tissue, in order to increase the passage efficiency and reduce the attenuation effect of the ultrasonic wave when the ultrasonic wave generated outside the bottom 12 is irradiated to the culture medium 15 or the cell 16 in the body. It may be formed of a material having an acoustic impedance and low ultrasonic attenuation coefficient. In the present embodiment, the body 10 is formed of transparent methylpentene polymer resin (TPX).

As such, the body 10 formed of a material having a sound impedance similar to that of a living tissue or a material having a low ultrasonic attenuation coefficient may have ultrasonic waves generated from the ultrasonic transducer 30 (see FIG. 4), which will be described later, change in a medium, that is, the ultrasonic transducer 30. The culture plate bottom layer 12 allows delivery to the culture medium /

cells

15 and 16 in the body 10 with minimal propagation loss due to the impedance difference along the cells 16 / culture medium 15.

In the present embodiment, the ultrasonic sensing film 20 is attached to the inner surface, that is, the inner bottom surface of the bottom portion 12 of the body 10 by an adhesive 25 such as an epoxy resin.

The ultrasonic sensing film 20 may be composed of a piezoelectric sensor film coated with electrodes on both surfaces of the piezoelectric material. In this embodiment, the piezoelectric sensor film may be formed of a transparent polyvinylidene fluoride (PVDF) film.

The ultrasonic sensing film 20 includes

electrode connecting parts

20a and 20b extending through the side wall part 11 to electrically connect the electrodes coated on both sides with the outside. The

electrode connection parts

20a and 20b may be configured in a pair of strips arranged side by side at regular intervals. Optionally, the connecting

portions

20a and 20b may be formed in one strip form (see FIG. 3). The connecting

parts

20a and 20b connect the upper and lower surfaces of the sensing film 20 to an external electrode or a power source in an electrically insulated state. That is, the connecting portion 20a electrically connects the electrode on the upper surface of the sensing film 20 with an external electrode or a power supply, and the connecting portion 20b electrically connects the electrode on the lower surface of the sensing film 20 with an external electrode or power supply. Connect with

The ultrasonic sensing film 20 configured as described above is related to the cavitation, which is a dynamic or secondary acoustic phenomenon of the culture medium 15 and the cells 16 reacting by the ultrasonic wave and the waveform of the ultrasonic signal irradiated through the bottom part 12. Detect the signal. In addition, the ultrasonic sensing film 20 may detect the pyroelectric voltage reflecting the flow of charge induced by the temperature change of the culture solution 15 or the cell 16 around the sensing film by a pyroelectric effect.

In addition, the ultrasonic sensing film 20 may be driven in a pulse echo mode by the pulser 45 / receiver 46 of the ultrasonic driving unit 40 to be described later. As a result, the A-mode ultrasonic RF signal received through the ultrasonic sensing film. Contains information about the height of the culture, the morphological information of the cells or the ultrasonic scattering / attenuation of the culture or the cells.

On the ultrasonic sensing membrane 20, a thin protective layer 26 may be formed of a material having the same or similar material as that of the body 10 so as not to be in direct contact with the culture medium 15.

3 is an external photograph of an ultrasonic culture dish in which a PVDF sensing film is attached with an epoxy resin to an inner bottom surface manufactured according to the ultrasonic culture dish 1 according to one embodiment of the present invention shown in FIGS. 1 and 2.

Although the ultrasonic sensing film 20 is illustrated and described as being disposed only on the inner bottom surface of the body 10, the present invention is not limited thereto. For example, the ultrasonic sensing film may be configured to be attached to both the inner bottom surface and the outer bottom surface of the body 10 or to the inner side wall surface of the side wall portion 11. In this case, the sensing film (hereinafter, referred to as 'first sensing film') disposed on the inner bottom surface of the bottom part 12 or the inner side wall surface of the side wall part 11 is an ultrasonic wave wave and / or ultrasonic wave irradiated with a culture dish. Sensing signals generated by secondary acoustic phenomena (eg, cavitation, acoustic flow, dynamic response of cells, etc.) and disposed on the outer bottom surface of the bottom part 12 (hereinafter, 'second sensing film'). The ultrasonic wave detects an ultrasonic wave before entering the petri dish, and the detected signal does not include a secondary acoustic phenomenon caused by the ultrasonic wave inside the petri dish. Comparing and analyzing the signals measured from the first sensing film and the second sensing film, it is possible to efficiently separate the acoustic phenomenon secondary generated by the irradiated ultrasound.

4 is a block diagram schematically illustrating an ultrasonic monitoring system using the ultrasonic culture dish 1 shown in FIGS. 1 and 2. FIG. 5 is a diagram illustrating a reaction of a cell to ultrasonic waves or an ultrasonic treatment to cells using an ultrasonic monitoring system using an ultrasonic culture dish 1 manufactured according to an embodiment of the present invention shown in FIG. 4. When irradiating ultrasound, the characteristics of ultrasound (frequency, pressure, intensity, power, etc.) applied to cells, acoustic phenomena secondary to ultrasound (eg cavitation, acoustic flow, dynamic response of cells, etc.), cultures The flow chart of the process of extracting the height, the thickness of the cell layer, the scattering / attenuation characteristics of the ultrasonic waves to the cells, and the temperature change of the culture medium / cells is illustrated.

As shown in FIG. 4, the ultrasonic monitoring system includes an ultrasonic culture dish 1, an ultrasonic transducer 30, an ultrasonic transducer driver 40, a signal acquisition unit 50, a signal analyzer 60, and a display unit 70. , A user interface unit 80 and a control unit 90.

The ultrasonic culture dish 1 is configured in the same manner as described with reference to FIGS. 1 and 2.

The ultrasonic wave to be used to induce an ultrasonic reaction, for example, cell separation, cell growth, or the like, to the culture solution 15 or the cell 16 contained in the ultrasonic culture dish 1 may be an ultrasonic transducer constituting the ultrasonic generator ( 30) and the driving unit 40 for driving the same.

The ultrasonic transducer 30 is a portion for converting electrical signals, that is, energy into ultrasonic waves, under the body 11 of the ultrasonic culture dish 10, with an ultrasonic coupling layer 29 made of gel or liquid therebetween. Contact with (11).

Since the ultrasonic transducer 30 may generate heat in the process of converting electrical energy into ultrasonic waves, in order to obtain a cooling effect or to maintain a constant temperature in the culture dish, the ultrasonic culture dish 1 and the ultrasonic transducer 30 may be used. 6 may be configured to be located in a bath 35 containing a liquid 34 such as water. In this case, the ultrasonic culture dish 1 is supported on the support wall 38 disposed in the water tank 35, and the ultrasonic transducer 30 is disposed at a predetermined distance from the ultrasonic culture dish 1 in the support wall 38. In addition, the connection terminal 31 protrudes through the bottom of the tank 35 to be electrically connected to the matching network 43 of the ultrasonic driver 40 to be described later. The support wall 38 forms an opening 39 so that the liquid in the water tank 35 can flow into the space between the ultrasonic culture dish 1 and the ultrasonic transducer 30.

Optionally, although not shown in the figures, the ultrasonic culture dish 1 may be arranged on the support wall 38 in the water tank 35 and the ultrasonic transducer 30 may be located outside the water tank 35. In this case, the ultrasonic transducer 30 may contact the acoustic window and the coupling material having excellent ultrasonic transmission efficiency at the bottom of the tank 35, or the front surface of the ultrasonic transducer may enter the tank 35 to minimize ultrasonic transmission loss. Can be.

The ultrasonic driver 40 includes an ultrasonic transducer 30 to generate ultrasonic waves. The ultrasonic driver 40 includes a function generator 41, a power amplifier 42, and a matching network 43. The function generator 41 generates an electrical signal to supply to the ultrasonic transducer 30. The power amplifier 42 amplifies the generated electrical signal and outputs it to the ultrasonic transducer 30 through the matching network 43. The frequency and mode (continuous wave, pulse (pulse length, pulse generation period)) of the electrical signal that drives the ultrasonic transducer 30 are functioned through a user interface 80 such as an input device such as a mouse, keyboard, button, touch pad, or the like. It is input to the generator 41. The power / intensity / pressure of the generated ultrasonic waves is adjusted by controlling the voltage of the function generator 41 or the output of the power amplifier 42 via the user interface 80.

FIG. 7 shows an example of ultrasonic waveforms measured through an ultrasonic sensing film of an experimental ultrasonic culture dish prepared as shown in FIG. 3. At this time, while increasing the voltage of the function generator 41 while fixing the output of the power amplifier 42, the ultrasonic transducer 30 of 1 MHz was driven to irradiate ultrasonic waves to the culture dish located in the water tank 35.

FIG. 8 shows an example of the change in the size of the ultrasonic wave measured while increasing the electric power using the experimental ultrasonic culture dish prepared as shown in FIG. 3. At this time, while increasing the voltage of the function generator 41 while fixing the output of the power amplifier 42, the ultrasonic transducer 30 of 1 MHz was driven to irradiate ultrasonic waves to the culture dish located in the water tank 35. In FIG. 8, the ultrasonic pressure measured from the ultrasonic sensing film shows a high correlation with the electric power for driving the ultrasonic waves. This is the basis for estimating the power (corresponding to the ultrasonic power A of FIGS. 4 and 5) irradiated to the ultrasonic culture dish 1 with the measured ultrasonic pressure.

In addition, the ultrasonic driver 40 may include a pulser / receiver 45 for driving the ultrasonic sensing film 20 in a pulse echo mode and receiving and processing an A-mode RF signal through the ultrasonic sensing film 20. have. The A-mode RF signal received through the pulser / receiver 45 is sent to the signal analysis unit 60 through the A / D converter 53 of the signal acquisition unit 50 to measure the height of the culture medium, as will be described later. It can be used to obtain morphological information of cells or to evaluate ultrasonic scattering / damping characteristics of culture or cells. In this case, the A-mode RF signal may be output to the display unit 70 through the A / D converter 53.

FIG. 9 shows an example of a typical A-mode RF signal measured by driving an ultrasonic sensing membrane of a laboratory ultrasonic culture dish manufactured as shown in FIG. 3 in a pulse / eco mode. At this time, the height of the culture solution 15 is set to 7.64 mm, and the ultrasonic wave is irradiated to the culture dish located in the water tank 35 by driving the 1 MHz ultrasonic transducer 30 with the voltage of the function generator 41 at 100 kV. It was. The A-mode RF signal includes information on the height of the culture medium and the thickness of the cell layer, as well as information on the dynamic response of the cell to the ultrasonic wave and the scattering / damping effect of the ultrasonic wave on the cell / culture medium.

FIG. 10 shows an example of ultrasonic waveforms measured by the ultrasonic sensing film of the culture dish while decreasing the height of the culture medium from 8.6 mm to 7.64 mm in the experimental ultrasonic culture dish manufactured as shown in FIG. 3. At this time, the ultrasonic wave was irradiated to the culture dish located in the water tank 35 by driving an ultrasonic transducer of 1 MHz with the voltage of the function generator 41 at 100 kW. In FIG. 10, the measurement signal measured by the ultrasonic sensing membrane is changed according to the height of the culture medium under the same driving conditions, which experimentally shows that the standing wave affected by the height of the culture medium is formed due to the acoustic characteristics of the culture dish. .

FIG. 11 illustrates the change in the ultrasonic pressure (left graph) measured in the ultrasonic sensing membrane in comparison with the theory (right graph) when the culture medium height is changed in the range of one wavelength for the case of FIG. 10. The experimental results are characteristically similar to the theory, indicating that the ultrasonic culture dish of the present invention measures the characteristics of standing waves formed in the culture well.

The A-mode RF signal may be obtained by driving the ultrasonic transducer 30 in the pulse echo mode. In this case, the ultrasonic transducer 30 must stop the generation of ultrasonic waves for irradiating the cells 16 and the culture solution 15 and drive in the pulse echo mode. On the other hand, the A-mode RF signal measurement using the ultrasonic sensing film 20 can be performed in real time while irradiating ultrasonic waves to the cells.

The signal acquiring unit 50 includes amplifying a signal detected by the ultrasonic sensing film 20 and trimming the signal through an appropriate signal processing technique. In other words, the amplified signal includes a noise signal and an interference signal, and increases the signal-to-noise ratio through filtering (eg, high pass, low pass, band pass, etc.) to selectively remove and detect a signal in a predetermined frequency range. Can be. To this end, the signal acquisition unit 50 is a pre-amplifier 51 for amplifying the signal detected by the ultrasonic sensing film 20, the first filter 52a for filtering and outputting the ultrasonic signal amplified by the pre-amplifier 51 And a second filter 52b for filtering and outputting the pyroelectric signal amplified by the preamplifier 51. The signal after amplification is converted into a digital signal through the A / D converter 53 and then transmitted to the signal analyzer 60 or output to the display unit 70.

The signal analyzer 60 includes an algorithm for receiving the signal detected by the ultrasonic sensing film 20 through the signal acquisition unit 50 and extracting information necessary for the user. In one embodiment, the ultrasonic power analyzer 61 extracts the ultrasonic strength, power, pressure, etc. from the ultrasonic signal output from the signal acquisition unit 50, and the temperature and ultrasonic waves from the pyroelectric signal output from the signal acquisition unit 50. The height of the culture medium, the cell layer, from the A-mode RF signal received from the pyroelectric signal analyzer 62 for estimating the power, and the A / D converter 53 of the signal acquisition unit 50 from the pulser / receiver 45. A-mode RF signal analyzer 63 for analyzing the thickness, ultrasonic attenuation / scattering characteristics of the culture medium / cell may be included.

12 is an electrical circuit of a pyroelectric potential induced by a temperature change of a culture solution due to ultrasonic irradiation and an equivalent electric circuit of PVDF used as a sensing membrane of an experimental ultrasonic culture dish manufactured as shown in FIG. 3. The example modeled by FIG. That is, the voltage V due to the pyroelectric phenomenon induced in the PVDF sensing film by the change ΔT / Δt of the temperature T according to the surrounding time t is expressed by the following equation (1).

........................................... (One)

here

p _c : the pyroelectric coefficient [C / ㎠ ℃]

A: the electrode area [㎠]

C _T : the total capacitance

R _T : the total resistance

If equation (1) is rearranged with respect to a change in temperature, it is expressed as an integral form of superconducting voltage as shown in equation (2) below.

......................................... (2)

here

,

When ultrasound is irradiated to the culture, the rate of heat production (Q) due to ultrasound is proportional to the product of the intensity (I) of the ultrasound and the ultrasound attenuation coefficient (α) of the biological tissue as shown in Equation (3) below. do.

Ｑ = 2αI ......................................... ........... (3)

The temperature distribution formed in the culture medium 15 due to the heat generated by the ultrasonic wave may be calculated by the bioheat transfer equation of Equation 4 below.

........................(4)

In cell culture (15), it can be assumed that w _b = 0 (no blood perfusion) and ▽ ² T = 0 (thermal gradient is equal to zero), and equation (4) is as simple as equation (5) below. Is expressed.

............................. (5)

If equation (5) is expressed in terms of ultrasonic intensity using equation (3),

............................. (6)

Since the ultrasonic power is the product of the area A of the ultrasonic transducer 30 and the ultrasonic intensity, the ultrasonic power is expressed as a function of the rate of change of time with respect to temperature as shown in Equation (7) below.

(7)

Therefore, when the pyroelectric voltage induced by the temperature change of the culture solution 15 is measured, the temperature change of the culture solution 15 can be estimated by the above equation (2), and when the temperature change is measured, the above equation (7) is obtained. It is also possible to estimate the irradiated ultrasonic power. The estimated ultrasonic power corresponds to the ultrasonic power B of FIG. 4 or 5.

FIG. 13 illustrates a result of measuring the superconducting voltage signal induced on the ultrasonic sensing film by the temperature of the culture solution which is increased when the ultrasonic culture dish prepared as shown in FIG. 3 is irradiated with ultrasonic waves for 10 seconds. At this time, while the output of the power amplifier 42 is fixed, the ultrasonic transducer of 1 MHz while increasing the voltage of the function generator 41 to 50, 100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800 Hz Ultrasonic wave was irradiated to the culture dish located in the water tank 35 by driving 30.

As represented by equation (2) above, the temperature is expressed in the form of integral over time. FIG. 14 illustrates the integration of the pyroelectric voltage signal measured in FIG. 13 over time, with temperature characteristics induced by typical ultrasounds exponentially decreasing when the ultrasound function is raised or stopped logarithmically. Over temperature cooling characteristic curve.

FIG. 15 illustrates the correlation between the maximum value of the pyroelectric voltage signal measured in FIG. 14 and the ultrasonic power applied to the culture dish. As can be seen in Figure 15, the signal of the pyroelectric voltage shows a nearly linear relationship with the ultrasonic power at the power of less than 1,000 kHz mainly used in the experiment, which is the theoretical through the equation (2) and (7) It is a result of experimentally confirming the possibility of estimating the ultrasonic power (corresponding to the ultrasonic power B of FIG. 4 or 5) using the superelectric voltage signal shown in FIG.

Various information analyzed by the signal analyzer 60 may be displayed on the display unit 70 through the controller 90, and may be used as feedback information in an algorithm for controlling the ultrasonic driving conditions to maintain a preset ultrasonic exposure condition. Can be.

The display unit 70 displays a signal transferred directly from the signal acquisition unit 50 processing the signal sensed through the ultrasonic sensing film 20 to the controller 90 or outputs a signal output from the signal acquisition unit 50. The analysis result transmitted from the signal analysis unit 60 to be analyzed through the control unit 90 is displayed. In addition, the display unit 70 displays a setting input window for the user to set the condition of the ultrasound through the user interface unit 80 under the control of the controller 90, or displays the setting value of the ultrasound set by the user.

The controller 90 may be configured to expose ultrasonic conditions (eg, frequency, intensity, power, etc.) necessary for a user to perform a cell response experiment or ultrasonic processing through a user interface unit 80 including an input device such as a mouse or a keyboard. Or set a start / end command for operating the ultrasonic driver 40, or analyze the waveform of the signal received through the ultrasonic sensing film 20 or the signal analyzer 60, or analyze the same. The ultrasonic driver 40, the signal analyzer 60, and the display unit 70 are controlled to display the result.

The control unit 90 may be configured as a body of a laptop computer or a personal computer. However, the control unit 90 is not composed of a body of a laptop computer or a personal computer, but instead of setting up an ultrasonic exposure condition for performing a cell response experiment or an ultrasonic processing and starting / stopping commands for operating / stopping the ultrasonic driving unit 40. It can also be configured as a control box including a variety of buttons for inputting.

In addition, the controller 90 may control the ultrasonic driver 40, the signal analyzer 60, and the display unit 70 in a hardware form such as ASIC or included in a management program. have.

The display unit 70 may be configured as a monitor connected to a laptop computer or a personal computer having the controller 90 as a body. If the control unit 90 is configured as a control box, the display unit 70 receives the signal received through the control unit 90 from the signal acquisition unit 50 that processes the signal detected by the ultrasonic sensing film 20. It can consist of a signal converter such as an oscilloscope that displays an electrical signal of magnitude and phase. In this case, the ultrasonic frequency, intensity, power, pressure, temperature, the height of the culture medium, the thickness of the cell layer and the attenuation of the ultrasonic wave and the scattering characteristics of the culture medium and the cells analyzed by the signal analyzer 60 are displayed in numbers (digital). Or change over time in the form of a graph.

Hereinafter, an ultrasonic reaction experiment or an ultrasonic treatment process of the ultrasonic monitoring system according to an exemplary embodiment of the present invention configured as described above will be described in detail with reference to FIGS. 4 and 16.

First, the experimenter puts the culture solution 15 containing the cells 16 to be experimented or sonicated in the body 10 of the ultrasonic culture dish 1, and then puts the ultrasonic culture dish 1 at the bottom of the ultrasonic couple. It is arranged to contact the front portion of the ultrasonic transducer 30 via the ring 29.

Subsequently, the experimenter turns on the ultrasonic monitoring system and performs basic experiment conditions (frequency, mode, time) through a user interface 80 including input devices such as a mouse and a keyboard in a setting window displayed on the monitor of the display unit 70. , Energy, etc.) are input (S1), and the driving conditions (power, intensity, pressure, etc.) of the ultrasonic wave to be irradiated with the ultrasonic culture dish 1 and the temperature conditions of the culture solution 15 are set (S2).

Based on the input value, the controller 90 excites the ultrasonic wave into the ultrasonic culture dish 1 by exciting the ultrasonic transducer 30 through the ultrasonic driver 40 as described above (S3).

The ultrasonic sensing film 20 attached to the ultrasonic culture dish 1 detects a superconducting voltage signal induced by the ultrasonic wave and the superconducting phenomenon in the culture dish and / or is driven in the pulse echo mode. The mode RF signal is detected and transmitted to the signal analysis unit 60 through the signal acquisition unit 50 to extract necessary information (S4). The signal analyzer 60 analyzes the received signal to detect ultrasonic pressure, power, intensity, cavitation, dynamic response of the cell, height of the culture medium, thickness of the cell layer, scattering / attenuation characteristics of the cell with respect to the ultrasound, and temperature of the culture medium / cell. The analyzed result of the change is displayed through the display unit 70 (S5).

The controller 90 calculates a difference (E) from the set value with respect to the power, intensity, temperature, etc. which are variables controlling the ultrasonic output among the results output from the signal analyzer 60, and the value is a tolerance. The determination result is displayed on the display unit 70 (S6). If the determination result displayed by the display unit 7 is outside the allowable range, the experimenter goes to step S2 to change the set condition (S7). Thereafter, the controller 90 determines an end condition such as time elapsed (S8). If the determination result is not satisfied, the control unit 90 repeats the process after step S3, and if the end condition is satisfied, the display unit 70. Indicates that the termination condition is met. When it is indicated that the termination condition is satisfied, the experimenter turns on the power of the ultrasonic monitoring system and terminates the ultrasonic reaction experiment or the ultrasonic processing.

In the above, the present invention has been described and illustrated with reference to embodiments for illustrating the principle, but the present invention is not limited to the configuration and operation shown and described as such. Also, it will be understood by those skilled in the art that various changes and modifications can be made to the present invention without departing from the spirit and scope of the appended claims. Accordingly, all suitable changes, modifications, and equivalents to the present invention should be considered to be within the scope of the present invention.

Claims

A body capable of containing cells and culture medium; And

And an ultrasonic sensing film attached to the body to detect an ultrasonic signal.
The method of claim 1,

And the body is formed of a material having a sound impedance similar to living tissue and a low ultrasonic attenuation coefficient.
The method of claim 1,

The body is an ultrasonic culture dish, characterized in that formed of methylpentene polymer resin (TPX).
The ultrasonic culture dish of claim 1, wherein the ultrasonic sensing film is attached to at least one of an inner bottom surface, an outer bottom surface, and an inner side wall surface of the body.
The method of claim 1,

The ultrasonic sensing film is an ultrasonic culture dish, characterized in that it comprises a piezoelectric sensor membrane coated with electrodes on both sides of the piezoelectric material.
The method of claim 5,

The piezoelectric sensor membrane is an ultrasonic culture dish, characterized in that it comprises a polyvinylidene fluoride (PVDF) membrane.
An ultrasonic culture dish to which the ultrasonic sensing film according to any one of claims 1 to 6 is attached;

An ultrasonic generator for irradiating ultrasonic waves to the cells and the culture solution contained in the ultrasonic culture dish;

A signal acquisition unit receiving a signal detected by the ultrasonic sensing film;

Analyzing the signal received from the signal acquisition unit, the power / intensity / pressure of the ultrasonic wave irradiated to the cells and the culture medium, the height of the culture medium, the thickness of the cell layer, the cavitation, the dynamic reaction of the cells, the culture medium A signal analyzer estimating at least one of a temperature change of the cell;

A display unit for outputting a result analyzed by the signal analyzer; And

And a control unit for controlling the ultrasonic generator, the signal analyzer, and the display unit.
The method of claim 7, wherein

The ultrasonic generator,

One or more ultrasonic transducers in contact with the outer bottom surface of the ultrasonic culture dish; And

And an ultrasonic driver for electrically driving the ultrasonic transducer.
The method of claim 8,

The ultrasonic drive unit,

A function generator for generating an electrical signal corresponding to the frequency and mode of the ultrasonic wave set by the user;

A power amplifier for amplifying the electrical signal; And

And a matching network to facilitate delivery of the amplified electrical signal to the ultrasonic transducer.
The method of claim 9,

The ultrasonic drive unit,

And an ultrasonic pulse / receiver for driving the ultrasonic sensing membrane in a pulse echo mode to obtain an A-mode RF signal for the cells and the culture medium through the ultrasonic sensing membrane.
The method of claim 10,

The signal acquisition unit,

A preamplifier for preamplifying the signal received from the ultrasonic sensing film;

A first filter for filtering and outputting the ultrasonic signal amplified by the preamplifier; And

And an A / D converter for converting the filtered signal into a digital signal.
The method of claim 11,

The signal acquisition unit,

And a second filter for filtering and outputting a low frequency band pyroelectric signal amplified by the preamplifier.
The method of claim 12,

The signal analysis unit

An ultrasonic power analyzer for extracting ultrasonic intensity, power, and pressure from the ultrasonic signal output from the signal acquisition unit;

A pyroelectric signal analysis unit for estimating temperature and ultrasonic power from the pyroelectric signal output from the signal acquisition unit; And

A for analyzing the height of the culture medium, the thickness of the cell layer, and the ultrasonic attenuation / scattering characteristics of the culture medium / cell from the A-mode RF signal received through the A / D converter of the signal acquisition unit from the pulser / receiver. An ultrasonic monitoring system comprising a mode RF signal analyzer.
The method of claim 7, wherein

And the display unit displays a setting window under the control of the controller so that a user can input a setting condition through a user interface.