WO2020091464A1 - Method for increasing viability of cell by irradiating cell with ultrasonic waves and ultrasonic irradiation apparatus using same - Google Patents

Abstract

Disclosed are a method for increasing the viability of a cell by irradiating the cell with ultrasonic waves, and an ultrasound irradiation apparatus using same, wherein in a state where each of ultrasonic parameters is preconfigured in a predetermined range, the ultrasonic irradiation apparatus places an ultrasonic generation unit within a threshold range from the epidermis of an object to irradiate the epidermis of the object with ultrasonic waves, the ultrasonic parameters include at least a pressure of ultrasonic waves and a duty percentage of the ultrasonic waves, the pressure of the ultrasonic waves is 0.5MPa to 1MPa, and the duty percentage of the ultrasonic waves is 1% to 5%.

Description

Method for increasing cell viability by irradiating ultrasound and ultrasound irradiation device using same

The present invention relates to a method for increasing the survival rate of cells by irradiating ultrasound and an ultrasound irradiation apparatus using the same.

Typical materials used as hair growth agents are Minoxidil and Propecia. Minoxidil is a formulation applied to the scalp and was developed as a treatment for hypertension due to the effect of expanding blood vessels at Pfizer in the past, but was sold as a treatment for hair loss after studying side effects of hair on the forehead and hands. Although the mechanism of action of minoxidil has not been clarified, it is known that it theoretically expands the blood vessels of the scalp, opens the potassium channel of the cell membrane, supplies oxygen and nutrients to the hair follicle, suppresses hair loss, promotes hair growth, and thickens hair. However, side effects such as erythema, scalp dryness, chest palpitations, tachycardia, and arrhythmia may occur.

The ingredient of Propecia sold by Merck is finasteride, which was originally developed to treat prostatic hyperplasia, but has been used as a hair loss treatment agent because it promotes hair growth. 5α-reductase converts the male hormone testosterone to dihydrotestosterone (DHT), which is a major component of hair loss. Finasteride inhibits this 5α-reductase enzyme and lowers the concentration of DHT that causes hair loss. Typical side effects include erectile dysfunction, decreased libido, sexual dysfunction, and sexual dysfunction, dizziness, headache, edema, and skin rash. Men with low infertility or sperm count should be cautious about taking medication. In addition, there is a fear of birth defects, so women of childbearing age should not take or contact with medications, and there are restrictions on prescribing.

In addition, avodot is a component of the dutasteride family, and was developed as a treatment for prostate hyperplasia, like finasteride, but has been found to be used as a treatment for hair loss because of its anti-hair loss effect. In general, it is known that dutasteride has a slightly stronger hair loss suppression effect than finasteride. However, it is known to have strong side effects such as decreased libido and decreased kidney function, so it is used in a limited way compared to finasteride, and has not been approved by the FDA as a treatment for hair loss in the United States.

Although there are various raw materials included in the functional cosmetics for preventing hair loss, such as reddensyl, these are mostly peptides and growth factors, and most of them have high molecular weight (e.g., 0.5 to 10 kDa), so skin absorption rate when used directly It is difficult to transfer raw materials through the skin, which is difficult.

Even with the existing drug delivery system (DDS), this also has a low skin absorption rate, so there is a limit to absorbing only small molecules (<500Da) raw materials, depending on the raw material characteristics (hydrophilicity, hydrophobicity, poor solubility, etc.) There is a disadvantage that the absorption deviation is large.

Iontophoresis is a technique that promotes the absorption of drugs by using a potential difference by generating a micro-current through a device such as a patch on an application site. It has the advantage of being available for a wide range of applications, non-invasive and painless, but if the polarity of the drug is not sufficient, the application may be limited, and the size or depth of application may be limited. In addition, erythema may occur when an excessively strong current is applied, and there may be side effects such as itching.

The microneedle creates a hole hundreds of micrometers deep in the skin and delivers it. In addition, the laser system (laser system) can be applied to the patient's skin by shaving the skin to a depth of about 5-10 micrometers on the surface through laser irradiation, but the stomach system may cause pain, It can irritate the skin and cause erythema. In addition, there are disadvantages that it is difficult to apply repeatedly, and difficult to apply a wide area.

Given the limitations of such a drug delivery system, there is a need for a method of increasing the survival rate of cells without side effects in a short application time, non-invasive and painless.

The present invention aims to solve all of the above-mentioned problems.

In addition, another object of the present invention is to increase the survival rate of cells without pain, non-invasively by irradiating ultrasound.

In addition, another object of the present invention is to increase the survival rate of cells by irradiating only ultrasonic waves without using expensive drugs.

The characteristic configuration of the present invention for achieving the objects of the present invention as described above and for realizing the characteristic effects of the present invention described below is as follows.

According to an aspect of the present invention, in a method of increasing the survival rate of cells by irradiating ultrasound, in a state in which each of the ultrasound parameters is preset to a predetermined range, the ultrasound irradiation apparatus, the ultrasound generating unit is a critical range from the epidermis of the subject The ultrasound parameters are irradiated to the epidermis of the object by positioning within, but the ultrasound parameters include at least the pressure of the ultrasound and the duty percentage of the ultrasound, the pressure of the ultrasound is 0.5 MPa to 1 MPa, and the duty percentage of the ultrasound is 1%. A method characterized by being between 5% and 5% is disclosed.

As an example, the ultrasonic parameters further include an intensity of ultrasonic waves, and the intensity of the ultrasonic waves is 166.7 mW / cm ² to 416.7 mW / cm ² .

As an example, the ultrasound parameters further include a frequency of ultrasound, and the frequency of the ultrasound is 0.5 MHz to 4.6 MHz.

As an example, the ultrasonic parameters further include a total irradiation time of ultrasonic waves, and a method characterized in that the total irradiation time of ultrasonic waves is within 10 minutes.

As an example, a method characterized by a method characterized in that the cell is an outer root sheath cell is disclosed.

According to another aspect of the present invention, an ultrasonic irradiation apparatus for increasing the survival rate of cells by irradiating ultrasound, comprising: an ultrasonic generator; And a controller configured to position the ultrasound generating unit within a critical range from the epidermis of an object to cause the ultrasound generating unit to irradiate ultrasound to the epidermis of the object, while each of the ultrasound parameters is preset to a predetermined range. The ultrasonic parameters include at least the pressure of the ultrasonic wave and the duty percentage of the ultrasonic wave, the pressure of the ultrasonic wave is 0.5 MPa to 1 MPa, and the ultrasonic irradiation device is characterized in that the duty percentage of the ultrasonic wave is 1% to 5%. .

As an example, the ultrasonic parameters further include the intensity of ultrasonic waves, and the ultrasonic intensity of the ultrasonic waves is 166.7 mW / cm ² to 416.7 mW / cm ² .

As an example, the ultrasonic parameters further include a frequency of ultrasonic waves, and the ultrasonic wave frequency is 0.5 MHz to 4.6 MHz.

As an example, the ultrasonic parameters further include a total irradiation time of the ultrasound, and the total irradiation time of the ultrasound is disclosed within 10 minutes.

As an example, the ultrasonic irradiation device is characterized in that the cells are outer root sheath (outer root sheath) cells.

According to the present invention, there are the following effects.

The present invention has an effect of increasing the survival rate of cells without pain non-invasively by irradiating ultrasound.

In addition, the present invention has an effect of increasing the survival rate of cells by irradiating only ultrasound without using expensive drugs.

1A and 1B are schematic views for explaining the concept of parameters of ultrasound in a method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention,

Figure 2 schematically shows the skin stability test results for a method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention,

3A and 3B schematically illustrate the survival rate of human lateral myocardial cell according to the concentration of the existing drug when only the existing drug is applied,

FIG. 4 schematically shows the experimental results of the survival rate of human lateral root sheath cells by the method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention together with the experimental results by other drugs,

Figure 5 schematically shows the experimental results of human outer root cell viability for the control group,

Figure 6 schematically shows the results of the experiment immediately after irradiation with ultrasound (pressure: 0.5 MPa, duty percentage: 1%) to human lateral root sheath cells;

Figure 7 schematically shows the results of the experiment 12 hours after the ultrasound (pressure: 0.5 MPa, duty percentage: 1%) was irradiated to human lateral root sheath cells,

8 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 0.5 MPa, duty percentage: 2%) to human lateral root sheath cells;

Figure 9 schematically shows the results of the experiment 12 hours after the irradiation of ultrasound (pressure: 0.5 MPa, duty percentage: 2%) to human lateral root sheath cells,

10 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 0.5 MPa, duty percentage: 3%) to human lateral root sheath cells;

FIG. 11 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 0.5 MPa, duty percentage: 3%) to human lateral root sheath cells;

FIG. 12 schematically shows the experimental results immediately after irradiating ultrasound (pressure: 1 MPa, duty percentage: 1%) to human lateral root sheath cells;

Figure 13 schematically shows the results of the experiment 12 hours after the ultrasound (pressure: 1 MPa, duty percentage: 1%) was irradiated to human lateral root hair cells.

FIG. 14 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1 MPa, duty percentage: 2%) to human lateral root sheath cells;

FIG. 15 schematically shows the results of the experiment 12 hours after the irradiation of ultrasound (pressure: 1 MPa, duty percentage: 2%) to human lateral root sheath cells;

FIG. 16 schematically shows the experimental results immediately after irradiation of ultrasound (pressure: 1 MPa, duty percentage: 3%) to human lateral root sheath cells;

Figure 17 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 1 MPa, duty percentage: 3%) to human lateral root sheath cells,

Figure 18 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1.5 MPa, duty percentage: 5%) to human lateral root sheath cells;

Figure 19 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 1.5 MPa, duty percentage: 5%) to human lateral root sheath cells,

Figure 20 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1.5 MPa, duty percentage: 10%) to human lateral root sheath cells,

Figure 21 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 1.5 MPa, duty percentage: 10%) to human lateral root sheath cells,

22 is a diagram schematically showing the results of experiments on the survival rate of human outer root sheath cells by the method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention with the pressure and duty percentage of ultrasound as variables;

23 and 24 schematically show the results of experiments on the survival rate of human lateral root sheath cells by the method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention with the intensity of ultrasound as a variable Did,

FIG. 25 is an ultrasound (pressure: 0.5 MPa, duty percentage: 2%, intensity: 166.7 mW / cm ² ) irradiated to human lateral root sheath cells, schematically showing experimental results according to various frequencies of ultrasound,

FIG. 26 schematically shows experimental results according to various frequencies of ultrasound while irradiating ultrasound (pressure: 1 MPa, duty percentage: 1%, intensity: 333.3 mW / cm ² ) to human lateral root sheath cells,

FIG. 27 is an ultrasound (pressure: 0.5 MPa, duty percentage: 5%, intensity: 416.7 mW / cm ² ) irradiated to human lateral root sheath cells, schematically showing experimental results according to various frequencies of ultrasound,

28 to 31 schematically show experimental results of gene expression when ultrasonic waves are irradiated to human lateral myocardial cell.

For a detailed description of the present invention, which will be described later, reference is made to the accompanying drawings that illustrate, by way of example, specific embodiments in which the present invention may be practiced. These examples are described in detail enough to enable those skilled in the art to practice the present invention. It should be understood that the various embodiments of the present invention are different, but need not be mutually exclusive. For example, certain shapes, structures, and properties described herein may be implemented in other embodiments without departing from the spirit and scope of the invention in relation to one embodiment. In addition, it should be understood that the location or placement of individual components within each disclosed embodiment can be changed without departing from the spirit and scope of the invention. Therefore, the following detailed description is not intended to be taken in a limiting sense, and the scope of the present invention, if appropriately described, is limited only by the appended claims, along with all ranges equivalent to those claimed. In the drawings, similar reference numerals refer to the same or similar functions across various aspects.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings in order to enable those skilled in the art to easily implement the present invention.

1A and 1B are diagrams for explaining the concept of parameters of ultrasound in a method of increasing the viability of cells by irradiating ultrasound according to an embodiment of the present invention.

The ultrasonic parameters are as follows.

Frequency: the frequency of the ultrasound itself

Duty percentage: The percentage value divided by one cycle of the actual irradiation time at which the ultrasound is irradiated

PRF (pulse repetition frequency): The number of times an ultrasonic square wave is irradiated for 1 second

PRP (pulse repetition period): 1 / PRF

Pressure: Pressure of ultrasound

Intensity = duty * pressure ² / (2 * c * rho): Energy of ultrasonic waves per unit irradiation area (c: sound velocity in water = 1500m / s, rho: density of water = 1000kg / m ³ )

For example, in FIG. 1A, since the square wave of ultrasonic waves having a frequency of 1 MHz is repeated 3 times for 1 second, the PRF value becomes 3 Hz, and the PRP value is 1 / PRF, resulting in 0.3333 seconds. In addition, the duty percentage in FIG. 1B has a value of Ton / (Ton + Toff) * 100 as a value for a time (Ton) at which ultrasonic waves are actually irradiated among one cycle (Ton + Toff) in which ultrasonic waves are irradiated.

For reference, Table 1 below shows intensity values according to pressure and duty percentage of each ultrasonic wave.

Pressure (MPa)	Intensity (W / m 2 )	Intensity with 100% duty (W / ㎠)	Intensity with 1% duty (mW / ㎠)	Intensity with 2% duty (mW / ㎠)	Intensity with 3% duty (mW / ㎠)	Intensity with 5% duty (mW / ㎠)
0.5	83333.3	8.3	83.3	166.7	250.0	416.7
0.7	163333.3	16.3	163.3	326.7	490.0	816.7
One	333333.3	33.3	333.3	666.7	1000.0	1666.7

Next, Figure 2 schematically shows the results of a skin stability test for a method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention.

Specifically, as a result of monitoring the temperature change while irradiating ultrasound to the skin of an 8-week-old male rat, the skin temperature increased by about 1.2 degrees Celsius for 30 minutes, and only an average temperature change of 0.05 degrees Celsius per minute was observed. . That is, it can be confirmed that the degree of temperature change due to irradiation with ultrasonic waves does not reach the extent to damage the skin.

Next, experimental conditions for observing cell viability according to ultrasonic irradiation will be described.

For reference, Table 2 below shows cell seeding density.

Recommended seeding density	5 * 10 ^ 3 cells / cm 2
Dish	Surface area (cm 2 )	Seeding density
60 mm	21	1.05 * 10 ^ 5 cells
100 mm	55	2.75 * 10 ^ 5 cells
T75	75	3.75 * 10 ^ 5 cells

In addition, Table 3 below shows the required amount of cells according to the number of conditions.

	Passage 9
	Per 1 well of ultrasound	10min	Well can	Cell seeding number (total amount)	T75 flask
Cell viability assay	96well plate (3 * 10 ^ 3 cell / well)	Condition	22	6.6 * 10 ^ 4	8.4 * 10 ^ 6
		3well	66	1.98 * 10 ^ 5
	6well plate (8 * 10 ^ 4 cell / well)	Condition	20	1.6 * 10 ^ 6
		3well	60	4.8 * 10 ^ 6
PCR	6well plate	Drug treatment conditions	22	1.76 * 10 ^ 6
		Sonication conditions	20	1.6 * 10 ^ 6
		3well	126	1 * 10 ^ 7

First, when explaining the material, cell culture is human hair outer root sheath cells (HHORSC), mesenchymal stem cell medium (MSCM), FBS 0.25% trypsin / EDTA solution, trypsin neutralization solution, dulbecco's phosphate-buffered saline (DPBS), It consists of poly-L-lysine, T75 flask.

And WST-1 cell proliferation assay system is used for WST-1 assay, and Trizol, sensiFAST probe Hi-ROX one step kit, PrimeTime qPCR assay is used for PCR.

First, in culture dish coating (based on T75 flask), 10 ml of D.W. is added to the flask, and then 15 ul of poly-L-lysine (10 mg / ml) is added. Then, put in a 37-degree incubator and coat for 1 hour. Then wash twice with D.W.

Then, MSCM 500ml basal medium, 25ml FBS, 5ml Mesenchymal stem cells are used to make whole medium.

Then, the frozen cell vial is dissolved in a 37 degree water bath, the dissolved cell is placed in 3 ml of a medium, and centrifuged at 3000 rpm for 3 minutes to cell down. Then, after washing with DPBS, centrifugation is performed at 3000 rpm for 3 minutes, and the cell is down. This process is repeated twice. Then, 8 ml of the medium is added to a T75 flask coated with Poly-L-lysine, the cell pellet is released, seeded, and cultured in a 37 ° C 5% CO2 incubator.

And, in the cell subculture, when it is confirmed by a microscope that the cell has grown to 90% or more, wash it with 3 ml of DBPS, add 3 ml of trypsin / EDTA solution, and incubate for 3 minutes in a 37-degree incubator. After confirming, add 3 ml each of the medium and trypsin neutralization solution to neutralize the trypsin / EDTA solution, transfer the neutralized cell solution to a conical tube, centrifuge it at 3000 rpm for 3 minutes, and cell down, T75 coated with poly-L-lysine 8 ml of medium is added to the flask, the cell pellet is released, seeded, and cultured in a 37% 5% CO2 incubator.

In addition, in the 6-well plate / 96-well plate cell seeding, poly-L-lysine coating is added according to Table 4 for each plate, and then the culture dish coating method is performed.

Required amount of poly L lysine per area (ug / cm 2 )			2
Dish	Surface area (cm 2 )	Required amount of Poly L lysine (ug)	10mg / mlPoly L lysine (ul)
T75	75	150	15
100 mm	55	110	11
6well	4.8	9.6	2
12well	3.9	7.8	0.78 (1/10 diluted to 7.8ul)
96well	0.3	0.6	0.06 (1/100 diluted 6ul)

At this time, if it is confirmed under the microscope that the cells grew more than 90% in the cell subculture, wash with 3 ml of DBPS, add 3 ml of trypsin / EDTA solution, incubate for 3 minutes in a 37 degree incubator, and confirm that the cells have fallen out of the cell flask. Neutralize the trypsin / EDTA solution by adding 3 ml each of the medium and trypsin neutralization solution, transfer the neutralized cell solution to the conical tube, centrifuge for 3 minutes at 3000 rpm, and then cell down, then count the cells using trypan blue and hemocytometer. . Then, seed the cells as much as 8 * 10 ^ 4 for 6 well plates and 4 * 10 ^ 4 for 96 well plates.

In the WST assay, in the case of an experiment in which only ultrasonic waves are irradiated, the cells seeded in a 6 well plate are cultured for 12 to 18 hours and then checked under a microscope. Then, after removing the media, wash it twice with DPBS, switch to growth factor free media, and irradiate the cells for 10 minutes. Then, incubated for 24 hours in a 37% 5% CO2 incubator, the medium was removed, washed with DPBS, treated with 0.2 ml of WST-1 drug, and incubated for 3 hours, and then, in a 6 well plate, 3 wells of 96 well plates per well The solution is transferred and the 96 well plate is measured at 450 nm with an absorbance meter.

In the WST assay, in the case of an experiment in which only drugs are applied, the cells seeded in a 96 well plate are cultured for 12 to 18 hours, and then confirmed whether they are well seeded. And after removing the media, wash it twice with DPBS, change it to growth factor free media, and apply the drug to the cells. Then, incubated for 24 hours in a 37% 5% CO2 incubator, the medium was removed, washed with DPBS, treated with 0.2 ml of WST-1 drug, incubated for 3 hours, and measured at 450 nm with a 96 well plate absorbance meter.

Meanwhile, FIGS. 3A and 3B schematically illustrate the survival rate of human lateral myocardial cell according to the concentration of the existing drug when only the existing drug is applied.

Specifically, FIG. 3a shows the cell survival rate according to the concentration of lidensil when the hair loss treatment drug lidensil is applied, and FIG. 3b shows the cell survival rate according to the concentration of minoxidil when the hair loss treatment drug minoxidil is applied. City.

For reference, Table 5 below shows the experimental conditions for the case where only the drug is applied. At this time, the solubility of minoxidil is 0.2% dissolved in PBS, and the condition is measured 24 hours after application of the drug.

	96 well plate
Control	Positive control (EGF)	Minoxidil	Reddensil
medium	50ng / 100ul	0.00002%	0.01%
		0.0002%	0.02%
		0.002%	0.04%
		0.05%	0.1%
		0.01%	0.2%
		0.02%	0.5%
		0.05%	One%
		0.15%	2%
		0.2%	3%
			4%
			5%
			10%

Next, Figure 4 shows the results of experiments with the survival rate of human lateral root sheath cells by the method of increasing the survival rate of cells by irradiating ultrasound according to an embodiment of the present invention together with the results of experiments with other drugs.

Referring to Figure 4, (i) when compared to the case of applying 50 ng EGF and (ii) when applying a 0.5% concentration of reddensil, ultrasound (pressure: 0.5 MPa, duty: 5%) without application of drugs Only the investigation showed that the cell viability was higher.

On the other hand, in the cell experiment, MTT and WST-1 comparison experiments were performed, and in the case of MTT, there was a problem in that the absorbance range of Redensyl and the absorbance range of MTT overlapped each other, resulting in poor data accuracy. On the other hand, in the case of WST-1, since the absorbance range of Redensyl and the absorbance range of WST-1 did not overlap, the experiment was conducted using WST-1.

In addition, a media comparison experiment was performed. In the first experiment, after seeding stabilization, complete media was treated with Redensyl and observed for 24 hours. However, the time is not limited to 24 hours, and the time may be changed depending on the cell state. In addition, in the second experiment, after stabilization of seeding, redensyl treatment was performed on media that did not contain growth factors and observed for 24 hours. As in the first experiment, the second experiment may be able to change the time according to the cell state.

As a result of the first experiment and the second experiment, the effect of the ultrasound could be better observed when the media was processed without growth factor. Therefore, experiments were performed by processing Redensyl on media that does not contain growth factors.

After that, an ultrasonic irradiation experiment was conducted, and before and after sonication, quantitative analysis was performed through an assay.

Tables 6 to 9 show the conditions of ultrasonic irradiation experiments to evaluate the physical effects of ultrasound on cells.

Specifically, Tables 6 and 7 show the conditions observed by redensyl treatment on serum free media, overnight, and complete media during the stabilization process after seeding.

96Well-1	A	B	C	D	E	F	G	H
One	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT
2	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT
3	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT
4	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT
5	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT
6	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT
7	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST
8	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST
9	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST
10	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST
11	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST
12	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST

96Well-2	A	B	C	D	E	F	G	H
One	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT
2	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT
3	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT
4	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT
5	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT
6	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT
7	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST
8	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST
9	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST
10	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST
11	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST
12	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST

In addition, Table 8 and Table 9 show the conditions observed by stabilization by seeding and then treated with Redensyl in serum free media.

96Well-3	A	B	C	D	E	F	G	H
One	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT	M-24-XTT
2	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT	P-24-XTT
3	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT	R0.01-24-XTT
4	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT	R0.04-24-XTT
5	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT	R0.2-24-XTT
6	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT	R1-24-XTT
7	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST	M-24-WST
8	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST	P-24-WST
9	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST	R0.01-24-WST
10	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST	R0.04-24-WST
11	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST	R0.2-24-WST
12	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST	R1-24-WST

96Well-4	A	B	C	D	E	F	G	H
One	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT	M-48-XTT
2	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT	P-48-XTT
3	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT	R0.01-48-XTT
4	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT	R0.04-48-XTT
5	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT	R0.2-48-XTT
6	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT	R1-48-XTT
7	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST	M-48-WST
8	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST	P-48-WST
9	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST	R0.01-48-WST
10	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST	R0.04-48-WST
11	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST	R0.2-48-WST
12	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST	R1-48-WST

In addition, Table 10 shows experimental conditions for irradiating ultrasonic waves while varying the pressure and duty percentage of ultrasonic waves.

6Well-1	One	2	3
A	1MPa, 3%	1MPa, 2%	1MPa, 1%
B	0.5MPa, 3%	0.5MPa, 2%	0.5MPa, 1%
6Well-2	One	2	3
A	1MPa, 3%	1MPa, 2%	1MPa, 1%
B	0.5MPa, 3%	0.5MPa, 2%	0.5MPa, 1%
6Well-3	One	2	3
A	1MPa, 3%	1MPa, 2%	1MPa, 1%
B	0.5MPa, 3%	0.5MPa, 2%	0.5MPa, 1%
6Well-4	One	2	3
A	Control	Control	control
B	1.5MPa, 10%	1.5MPa, 5%
6Well-5	One	2	3
A
B
6Well-6	One	2	3
A
B

Referring to Table 10, while varying the pressure (0.5 MPa, 1 MPa, 1.5 MPa) and duty percentage (1%, 2%, 3%) of the ultrasound, an experiment was performed to irradiate the cells with ultrasound for 10 minutes.

Specifically, in wells 1 to 3, ultrasonic waves were irradiated with a pressure of 0.5 MPa or 1 MPa and a duty percentage of ultrasonic waves of 1% to 3%.

However, in well 4, the cell death was induced by increasing the pressure (1.5 MPa) and the duty percentage (5%, 10%) for comparison with the results of different pressures and different duty percentages.

The results are shown in FIGS. 5 to 21.

First, FIG. 5 schematically shows the survival rate at a specific time point and the survival rate after 12 hours from a specific time point for human lateral root sheath cells that have not been irradiated with ultrasound, and a control group for comparison with the group irradiated with ultrasound is shown. it means.

Referring to FIG. 5, when ultrasound is not irradiated, no significant change in cell viability after 12 hours is observed from a specific time point indicated by the right figure, compared to a specific time point indicated by the left figure.

FIG. 6 schematically shows the results of the experiment immediately after irradiation with ultrasound (pressure: 0.5 MPa, duty percentage: 1%) to human lateral hair root sheath cells.

FIG. 7 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 0.5 MPa, duty percentage: 1%) to human lateral root sheath cells.

Referring to FIGS. 6 and 7, it can be seen that cell viability increased to a certain degree when 12 hours elapsed after irradiation with ultrasound (pressure: 0.5 MPa, duty: 1%) compared to the result of the control group after 12 hours.

FIG. 8 schematically shows the experimental results immediately after irradiating ultrasound (pressure: 0.5 MPa, duty percentage: 2%) to human lateral capillary cells.

Figure 9 schematically shows the results of the experiment 12 hours after the irradiation of ultrasound (pressure: 0.5MPa, duty percentage: 2%) to human lateral root root cells.

Referring to FIGS. 8 and 9, it can be confirmed that cell viability was significantly increased when 12 hours elapsed after irradiation with ultrasound (pressure: 0.5 MPa, duty: 2%) compared to the result of the control group after 12 hours.

FIG. 10 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 0.5 MPa, duty percentage: 3%) to human lateral hairy root cells.

FIG. 11 schematically shows the results of an experiment 12 hours after irradiation of ultrasound (pressure: 0.5 MPa, duty percentage: 3%) to human lateral root sheath cells.

Referring to FIGS. 10 and 11, it can be seen that cell viability was significantly increased when 12 hours elapsed after irradiation with ultrasound (pressure: 0.5 MPa, duty: 3%) compared to the control group results after 12 hours.

FIG. 12 schematically shows the experimental results immediately after irradiation with ultrasonic waves (pressure: 1 MPa, duty percentage: 1%) to human lateral capillary cells.

FIG. 13 schematically shows the results of the experiment 12 hours after irradiation of ultrasound (pressure: 1 MPa, duty percentage: 1%) to human lateral hair root cells.

Referring to FIGS. 12 and 13, it can be seen that cell viability was significantly increased when 12 hours elapsed after irradiation with ultrasound (pressure: 1 MPa, duty: 1%) compared to the result of the control group after 12 hours.

FIG. 14 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1 MPa, duty percentage: 2%) to human lateral hair root cells.

FIG. 15 schematically shows the results of the experiment 12 hours after the irradiation of ultrasound (pressure: 1 MPa, duty percentage: 2%) to human lateral root sheath cells.

Referring to FIGS. 14 and 15, when 12 hours elapsed after irradiation with ultrasound (pressure: 1 MPa, duty: 2%) when compared with the control group results, it was confirmed that the cell viability decreased, such as cell bursting or deformation. Can be.

FIG. 16 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1 MPa, duty percentage: 3%) to human lateral hairy root cells.

FIG. 17 schematically shows the results of an experiment 12 hours after irradiation of ultrasound (pressure: 1 MPa, duty percentage: 3%) to human lateral hairy root cells.

Referring to FIGS. 16 and 17, when compared with the control group results, cell viability, such as cells bursting or deforming when 12 hours elapsed immediately after irradiation with ultrasound (pressure: 1 MPa, duty: 3%) and after irradiation You can see that it has decreased.

FIG. 18 schematically shows the experimental results immediately after irradiation with ultrasound (pressure: 1.5 MPa, duty percentage: 5%) to human lateral hairy root cells.

Figure 19 schematically shows the results of the experiment 12 hours after irradiation of human lateral root root cells with ultrasound (pressure: 1.5 MPa, duty percentage: 5%).

Referring to FIGS. 18 and 19, when compared to the control group results, cells burst or deform at a high rate immediately after irradiation with ultrasound (pressure: 1.5 MPa, duty: 5%) and 12 hours after irradiation. It can be seen that the cell viability decreased.

FIG. 20 schematically shows the experimental results immediately after irradiation with ultrasonic waves (pressure: 1.5 MPa, duty percentage: 10%) to human lateral capillary cells.

FIG. 21 schematically shows the results of an experiment 12 hours after irradiation of ultrasound (pressure: 1.5 MPa, duty percentage: 10%) to human lateral root sheath cells.

20 and 21, when compared with the control group results, most of the cells burst or deform after the ultrasound (pressure: 1.5 MPa, duty: 10%) irradiation and 12 hours after irradiation. It can be seen that the cell viability decreased.

5 through 21, it was confirmed that the energy of the pressure of 1MPa or less, the duty of 5% or less, and the intensity of 416.7 mW / cm ² or less is safe for the cells. In addition, when the pressure was set to 1.5 MPa or more, it was observed that the cells burst or deform. In addition, it was observed that the cell growth was increased compared to the control group when irradiating ultrasonic waves in a safe energy range.

For example, in the method of increasing the viability of a cell by irradiating ultrasound, in a state in which each of the ultrasound parameters is preset to a predetermined range, the ultrasound irradiation apparatus positions the ultrasound generator within a critical range from the epidermis of the subject, Irradiating ultrasound to the epidermis, the ultrasound parameters include at least the pressure of the ultrasound and the duty percentage of the ultrasound, the pressure of the ultrasound is 0.5 MPa to 1 MPa, and the duty percentage of the ultrasound may be 1% to 5%.

Further, the parameters of the ultrasound may further include the intensity of the ultrasound, and the intensity of the ultrasound may be 166.7 mW / cm ² to 416.7 mW / cm ² .

In addition, the parameters of the ultrasound may further include the total irradiation time of the ultrasound, and the total irradiation time of the ultrasound may be 10 minutes, but is not limited thereto, and may be an irradiation time within 10 minutes or more than 10 minutes. have.

Also, the ultrasound generating unit may irradiate ultrasound to the epidermis of the subject in a state in contact with the epidermis of the subject or spaced apart from the epidermis of the subject. In addition, the ultrasonic generator may have a shape surrounding the epidermis of the object in the form of a helmet or head gear.

In addition, the cells may be outer root sheath cells.

FIG. 22 schematically shows the survival rate test results of human lateral myocardial cell derived by irradiating ultrasound according to pressure and duty percentage of various ultrasounds, and FIGS. 23 and 24 show ultrasounds according to the intensity of various ultrasounds. It is a schematic illustration of the survival rate test results of human lateral root sheath cells derived by examining.

22 to 24, among the ultrasonic parameters (i) the pressure of the ultrasonic wave is 0.5MPa to 1MPa, (ii) the duty percentage of the ultrasonic wave is 1% to 5%, and (iii) the intensity of the ultrasonic wave is 166.7 mW / cm ^It can be seen that the survival rate of cells at ² to 416.7 mW / cm ² is significantly higher than that of cells in other ultrasound ranges.

For reference, Table 11 below shows the cell viability measured for each pressure and duty percentage of ultrasonic waves irradiated to the cells.

	av	sd
Control	100%	0.01
0.3MPa1%	95%	0.01
0.5MPa1%	117%	0.09
0.5MPa2%	124%	0.05
0.5MPa5%	125%	0.02
1MPa1%	118%	0.04
1MPa2%	68%	0.08
1MPa3%	32%	0.09

On the other hand, the parameters of the ultrasound may further include the frequency of the ultrasound, the frequency of the ultrasound may be 0.5MHz to 4.6MHz.

25 to 27 are diagrams schematically showing experimental results obtained by varying the frequency of ultrasonic waves while irradiating ultrasonic waves to human lateral root sheath cells while maintaining the pressure and the duty percentage of ultrasonic waves.

As an example, FIG. 25 shows ultrasonic waves (pressure: 0.5 MPa, duty percentage: 2%, intensity: 166.7 mW / cm ² ) to human lateral root sheath cells, but the frequencies of ultrasonic waves are 0.2 MHz, 0.5 MHz, 1 MHz, and 4.6. The experimental results obtained by changing to MHz and 10 MHz are shown.

As can be seen in FIG. 25, it can be seen that the cell survival rate in the section where the frequency of the ultrasound is 0.5 MHz to 4.6 MHz is significantly higher than the cell survival rate in the other section.

As another example, FIG. 26 irradiates ultrasonic waves (pressure: 1 MPa, duty percentage: 1%, intensity: 333.3 mW / cm ² ) to human outer root sheath cells, but the frequencies of ultrasonic waves are 0.2 MHz, 0.5 MHz, 1 MHz, The experimental results obtained by changing to 4.6 MHz and 10 MHz are shown.

As can be seen in FIG. 26, it can be seen that the cell survival rate in the section where the frequency of the ultrasound is 0.5 MHz to 4.6 MHz is significantly higher than the cell survival rate in the other section.

As another example, FIG. 27 irradiates ultrasonic waves (pressure: 0.5 MPa, duty percentage: 5%, intensity: 416.7 mW / cm ² ) to human outer root sheath cells, but the frequencies of ultrasonic waves are 0.2 MHz, 0.5 MHz, 1 MHz, The experimental results obtained by changing to 4.6 MHz and 10 MHz are shown.

As can be seen in FIG. 27, it can be seen that the cell survival rate in the section where the frequency of the ultrasound is 0.5 MHz to 4.6 MHz is significantly higher than the cell survival rate in the other section.

28 to 31 schematically illustrate the results of gene expression when irradiated to human lateral root sheath cells while changing the pressure and duty percentage of ultrasonic waves having a frequency of 1 MHz.

Specifically, FIG. 28 schematically shows the results of WNT10B and Beta Catenin-related gene expression when ultrasonic waves having a frequency of 1 MHz were irradiated to human lateral root sheath cells while changing pressure and duty percentage. The intensity of the ultrasound was set to 166.7 mW / cm ² (0.5 MPa, 2%), 333.3 mW / cm ² (1MPa, 1%) and 416.7 mW / cm ² (0.5MPa, 5%) to human lateral root sheath cells. When the ultrasound was examined, it was confirmed that the observed WNT10B and Beta Catenin-related gene expression levels were significantly higher than those in the Control group.

In addition, FIG. 29 schematically shows the results of Keratin 15 and VDR-related gene expression when ultrasonic waves having a frequency of 1 MHz are irradiated to human lateral root sheath cells while changing pressure and duty percentage. As in FIG. 28, the intensity of ultrasonic waves was set to 166.7 mW / cm ² (0.5 MPa, 2%), 333.3 mW / cm ² (1 MPa, 1%) and 416.7 mW / cm ² (0.5 MPa, 5%). When ultrasonic waves were irradiated to human lateral root sheath cells, it can be seen that the observed Keratin 15 and VDR-related gene expression levels were significantly higher than those in the Control group.

In addition, FIG. 30 schematically shows the results of gene expression related to PCNA and Ki67 when irradiated to human lateral root sheath cells while changing the pressure and duty percentage of ultrasonic waves having a frequency of 1 MHz. As in FIG. 28, the intensity of ultrasonic waves was set to 166.7 mW / cm ² (0.5 MPa, 2%), 333.3 mW / cm ² (1 MPa, 1%) and 416.7 mW / cm ² (0.5 MPa, 5%). When ultrasonic waves were irradiated to human lateral root sheath cells, it was confirmed that the observed PCNA and Ki67-related gene expression levels were significantly higher than in the Control group.

In addition, FIG. 31 schematically shows the results of BCL2-related gene expression when ultrasonic waves having a frequency of 1 MHz are irradiated to human lateral root sheath cells while changing pressure and duty percentage. As in FIG. 28, the intensity of ultrasonic waves was set to 166.7 mW / cm ² (0.5 MPa, 2%), 333.3 mW / cm ² (1 MPa, 1%) and 416.7 mW / cm ² (0.5 MPa, 5%). When ultrasonic waves were irradiated onto human lateral root sheath cells, it can be seen that the observed BCL2-related gene expression level was significantly higher than that of the Control group.

Meanwhile, the ultrasonic parameters may further include PRF or PRP, PRF may be 1 Hz to 100 Hz, and PRP may be 0.01 to 1 second.

In the above, the present invention has been described by specific matters such as specific components, etc. and limited embodiments and drawings, which are provided to help the overall understanding of the present invention, but the present invention is not limited to the above embodiments , Those skilled in the art to which the present invention pertains can make various modifications and variations from these descriptions.

Therefore, the spirit of the present invention is not limited to the above-described embodiment, and should not be determined, and all claims that are equally or equivalently modified as well as the claims below will fall within the scope of the spirit of the present invention. Would say

Claims (10)

1. In the method of increasing the viability of cells by irradiating ultrasound,

   While each of the ultrasound parameters is preset to a predetermined range, the ultrasound irradiation apparatus irradiates ultrasound to the epidermis of the subject by placing the ultrasound generator within a critical range from the epidermis of the subject,

   The ultrasound parameters include at least the pressure of the ultrasound and the duty percentage of the ultrasound, the pressure of the ultrasound is 0.5MPa to 1MPa, and the duty percentage of the ultrasound is 1% to 5%.
2. According to claim 1,

   The ultrasound parameters further include the intensity of the ultrasound,

   The intensity of the ultrasound is characterized in that 166.7 mW / cm ² to 416.7 mW / cm ² .
3. According to claim 1,

   The frequency of the ultrasound is further included in the ultrasound parameters,

   The frequency of the ultrasound is characterized in that 0.5MHz to 4.6MHz.
4. According to claim 1,

   The ultrasound parameters further include the total irradiation time of the ultrasound,

   The total irradiation time of the ultrasound, characterized in that within 10 minutes.
5. According to claim 1,

   The cell is characterized in that the outer root sheath (outer root sheath) cells.
6. In the ultrasound irradiation device for increasing the viability of the cells by irradiation with ultrasound,

   An ultrasonic generator; And

   A control unit configured to position the ultrasonic generator within a threshold range from the epidermis of the object to cause the ultrasonic generator to irradiate ultrasound to the epidermis of the object, while each of the ultrasonic parameters is preset to a predetermined range;

   Including,

   The ultrasound parameters include at least the pressure of the ultrasonic wave and the duty percentage of the ultrasonic wave, the pressure of the ultrasonic wave is 0.5 MPa to 1 MPa, and the duty percentage of the ultrasonic wave is 1% to 5%.
7. The method of claim 6,

   The ultrasound parameters further include the intensity of the ultrasound,

   The ultrasound intensity is 166.7 mW / cm ² to 416.7 mW / cm ² .
8. The method of claim 6,

   The frequency of the ultrasound is further included in the ultrasound parameters,

   The ultrasonic frequency is 0.5MHz to 4.6MHz, characterized in that the ultrasonic irradiation device.
9. The method of claim 6,

   The ultrasound parameters further include the total irradiation time of the ultrasound,

   The ultrasonic irradiation device, characterized in that the total irradiation time of the ultrasonic waves is within 10 minutes.
10. The method of claim 6,

    The cell is an ultrasound irradiation device, characterized in that the outer root sheath (outer root sheath) cells.

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