KR102188025B1 - Composition for regulation of cell cycle comprising plasma-activated media - Google Patents

Abstract

The present invention provides a cell cycle control composition comprising a plasma-activated culture medium (PAM) produced by irradiating plasma to a cell culture medium, and a method for adjusting the cell cycle using the composition. The method has the property of reversibly regulating the cell cycle without cell death or damage.

Description

Composition for regulation of cell cycle comprising plasma-activated media

The present invention relates to a composition for controlling a cell cycle including a plasma active culture medium and a method for controlling the cell cycle using a plasma active culture medium.

Cell division is an essential process performed to compensate for the growth and natural loss of living organisms, and plays a very important role in maintaining living organisms. Therefore, cell division is carried out through a very sophisticated and complex process, and such cell division abnormalities cause diseases such as cancer.

In the case of eukaryotic cells, when a signal for cell growth is received from the outside, it proliferates according to the cell cycle through a signaling system. Eukaryotic cells proliferate by periodically going through the process of S phase and somatic cell division (M phase), and G1 and G2 phases exist between S and M phases, respectively. In other words, it divides by rotating the cycle of the G1-S-G2-M groups in sequence. After cell division is completed, it returns to the G1 phase, and the G1 phase is the most active period of intracellular metabolism. Most cells spend a lot of time in the G1 phase, and if there is no external signal, the cell cycle stops progressing and exists as the G0 phase (rest phase), which stops at the G1 phase.

The cell cycle is a fundamental process in the proliferation and growth of individual cells. Cell cycle progression is regulated by both internal and external factors. Internally, each phase of the cell cycle is regulated by cyclin dependent protein kinases (CDKs) such as CDK-1, CDK-2, and CDK-4. Additionally, several external factors, including nutrients, growth factors, and hormones, are involved in the cell cycle, and these factors act through intracellular signal transduction neetworks. ROS has a decisive effect on the cell cycle, and specifically, it is involved in the cell cycle through phosphorylation and ubiquitination of CDK. Additionally, ROS inhibits the NF-kB pathway and is involved in the alteration of cyclins D, A, and p21.

When a disorder occurs in the regulation of the cell cycle, cell growth is not regulated, resulting in diseases such as cancer. Therefore, there is a need to develop a technology that regulates the cell cycle.

It is an object of the present invention to provide a composition for cell cycle control, comprising a plasma active culture medium (PAM).

Another object of the present invention is to provide a method for controlling the cell cycle by using a plasma active culture medium.

In order to achieve the object of the present invention, the present invention provides a composition for controlling the cell cycle, comprising a plasma-activated culture medium (PAM) prepared by irradiating plasma to a cell culture medium.

The present invention also provides a method for stopping the cell cycle, comprising the step of culturing cells and treating the cultured cells with a composition for controlling a cell cycle including a plasma active culture medium.

Hereinafter, the present invention will be described in more detail.

The composition for controlling a cell cycle provided by the present invention includes a plasma-activated culture medium (PAM) prepared by irradiating a plasma with a cell culture medium.

By using the plasma culture solution of the present invention, it is possible to compensate for the disadvantage that plasma activation effects cannot be obtained evenly on all cells when plasma is directly irradiated to cells in culture.

In the present invention, "cell cycle regulation" means cell cycle arrest, and preferably, it means reversible cell cycle arrest.

In the present invention, "plasma" refers to an ionized gas that satisfies Debye shielding. It is regarded as one of the three basic states of matter, gas, liquid, and solid, and is expressed as the fourth state. In the plasma of the present invention, a neutral gas is converted to a plasma by an external voltage, and electrons and cations may be generated by excitation and ionization of the neutral gas, and radicals excited by molecular gas may exist.

The plasma of the present invention may be generated under normal pressure. For example, it may be a normal temperature and normal pressure plasma (NTAPP), but is not limited thereto. Room temperature atmospheric pressure plasma of the present invention may be used in the same meaning as room temperature atmospheric pressure plasma.

The plasma generating apparatus of the present invention can use a known plasma generating apparatus without limitation, as long as it can generate the plasma active culture solution according to the object of the present invention. In particular, it is used in which arc and static electricity are inhibited to generate plasma for biological purposes. It is characterized. For example, the plasma generating apparatus of the present invention includes an apparatus main body; A needle or rod-type power electrode in which a flat ground electrode provided on one side of the device body is disposed to face the flat ground electrode in the body; And a high voltage power supply device for supplying power to the power electrode. In an embodiment of the present invention, plasma generation was performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating apparatus and the medium were separated by 0.35cm.

In the'room temperature normal pressure plasma' of the present invention, room temperature is a temperature range that does not correspond to a high-temperature plasma, and the temperature conditions considered as room temperature plasma in the art can be applied without limitation, for example, 60°C or less, 40°C Hereinafter, it means 0 to 60°C, 5 to 45°C, 20 to 40°C, 20 to 30°C, or RT (room temperature).

The carrier gas may be used without limitation as long as it is suitable for the purpose of producing a plasma and plasma active liquid that achieves the object of the present invention, but preferably at least one selected from at least one combination selected from the group consisting of helium and argon, and more Preferably, argon may be used as a carrier gas, and plasma may be generated by supplying a high voltage current to the gas.

On the other hand, plasma activated water, which is a liquid activated using plasma, exhibits similar characteristics to gaseous plasma, and is used in agriculture and medical fields. Plasma activated water is produced by generating plasma on the surface of the medium or in water in a liquid medium.

The plasma-activated culture medium of the present invention may be used in the same sense as a plasma-conditioned medium (PCM). Such a plasma treatment medium has the same or improved or similar anticancer effect through the same mechanism as the irradiated plasma, and has the advantage of being easy to move and apply compared to plasma.

For the production of the plasma-activated culture medium of the present invention, the medium irradiated with plasma can be used without limitation any known medium used for cell culture, and the medium is suitable for culturing the corresponding cells according to the type of cells selected for culture. It is preferable to select and use an appropriate medium. The culture medium of the cells may be a culture medium of animal or plant cells, preferably a culture medium of mammalian cells.

Specifically, in the case of a medium suitable for epithelial tissue of the skin, for example, melanocytes or keratinocytes, M254 medium may be used as a basal medium. The M254 medium may not contain calcium, and 20 amino acids, vitamins, inorganic salts, Adenine.HCl, D-Glucose (Dextrose), DL-alpha-lipoic Acid, Ethanolamine, HEPES, O-Phosphorylethanolamine , Putrescine 2HCl, Sodium Pyruvate, and Thymidine. The 20 amino acids are glycine, alanine, alanyl glutamine, arginine hydrochloride, asparagine, aspartic acid, cysteine, glutamic acid, histidine hydrochloride, isoleucine, leucine, lysine hydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine. Includes. The vitamins include choline chloride, pantothenic acid, folic acid, myo-inositol, niacinamide, pyridoxal hydrochloride, vitamin B12, and d-Biotin. The inorganic salts are NaVO3, ((NH4)6(Mo7O24-4H2O), CuSo4-5H2O, FesO4-7H2O, MgCl2-6H2O, MnSO4-H2O, NiCl2-6H2O, KCl, NaCH3COO-3H2O, NaHCO3, NaCl, Na2SiO3-9H2O. , Ha2HPO4-7H2O, Na2SeO3, SnCl2-2H2O, and ZnSO4-7H2O.

In order to prepare the plasma-activated culture medium of the present invention, the plasma-irradiated culture medium may additionally contain an additive required for culturing cells, and may include, for example, human melanocyte growth supplement (HMGS). have.

The plasma may be generated using microwave power, and the microwave power may be appropriately selected and used by a person skilled in the art according to the purpose and method of the experiment. For example, 1.0W to 5.0W, 2.0W to 4.0 It may be W or 2.5W to 3.5W, preferably 3.0W.

The plasma may be irradiated in a state in which the culture medium to be irradiated with plasma and the plasma gas (plume) are in contact or at a distance from a predetermined distance. When the plasma electrode is separated from the medium and the plasma is irradiated, the separation distance is 0.1 to 15cm, 0.1 to 10cm, 0.1 to 5cm, 0.1 to 3cm, 0.1 to 1cm, 0.1 to 0.5cm, 0.3 to 15cm, 0.3 to 10cm, 0.3 to It may be 5cm, 0.3 to 3cm, 0.3 to 1cm, or 0.3 to 0.5cm, preferably 0.3 to 0.4cm, more preferably 0.35cm. When the electrode is irradiated in contact with the culture solution, the electrode may be positioned on the surface of the culture solution or in the culture solution.

The time for irradiating the plasma to the culture medium is 4 minutes to 30 minutes, 4 minutes to 25 minutes, 4 minutes to 20 minutes, 5 minutes to 30 minutes, 5 minutes to 25 minutes, 5 minutes to 20 minutes, or 8 minutes to 20 minutes. It can be minutes.

In an embodiment of the present invention, a plasma-activated culture solution was prepared by applying plasma at room temperature and pressure to M254 medium. Plasma-activated culture solution was prepared by irradiating plasma for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes with the electrode 0.35 cm away from the surface of the 10ml M254 medium contained in a Petri dish. The conditions of the room temperature and atmospheric pressure plasma were performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating apparatus and the medium were separated by 0.35 cm. 10 mL of cell-free medium was placed in a Petri dish, and plasma was irradiated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes, respectively, to prepare a final plasma active culture solution. Microwave accuracy was adjusted to ±2.0%, gas pressure was 0 to 3 Bar, and gas flow was adjusted to 0 to 5 SLM.

The spectrum emitted from the injected argon gas atom was 690 to 990 nm and the peaks due to N2 and O were measured between 337.2 and 690 to 900 nm, respectively. The OH band was observed in the range of 307 to 309 nm.

The plasma-activated culture medium provided by the present invention has the advantage of being able to stop the cell cycle without causing cell death or cell damage. The stoppage of the cell cycle is a reversible reaction, and when cells are cultured in a normal culture medium not irradiated with plasma, the stoppage of the cell cycle may be recovered.

The composition for cell cycle control comprising the plasma active culture medium provided by the present invention can control the cell cycle of eukaryotic cells, preferably animal cells, and more preferably human-derived cells. The cells are, for example, melanocytes, cervical cancer cells, skin cancer cells, oral cancer cells, gastric cancer cells, colon cancer cells, lung cancer cells, egg cells, sperm cells, fertilized eggs, hair cells, nail root cells , Skin cells, sweat gland (apocrine sweat gland) cells, neurons, and may be one or more cells selected from the group consisting of immune cells, and the immune cells are, for example, lymphocytes, macrophages, plasma cells, eosinophils, or It may be an NK cell.

In one embodiment of the present invention, as a result of using the plasma active culture medium as a cell culture medium, no damage or death of melanocytes was observed (Example 3). More specifically, as a result of examining the melanocytes under a microscope, there was no apparent damage to the cells, such as dendritic processes peculiar to melanocytes were confirmed (FIG. 1). Therefore, the method of regulating the cell cycle according to the present invention can temporarily stop the cell cycle without damaging normal cells. The composition for controlling a cell cycle including a plasma active culture medium provided by the present invention may arrest the cell cycle through the control of G0/G1 and G2/M checkpoints.

In one embodiment of the present invention, as a result of measuring the number of live cells after using the plasma-activated culture medium as a cell culture medium, the number of harvested cells decreased as the plasma exposure time increased, but compared to the initial inoculation amount of cells. Since the number of cells did not decrease (FIG. 2), it was confirmed that the plasma-activated culture medium had the effect of somewhat inhibiting cell proliferation, but no cell death or cell damage occurred.

In one embodiment of the present invention, as a result of culturing melanocytes in PAM, the G0/G1 phase decreased, the G2/M phase increased, and the cell cycle was stopped, but it was found that this is a temporary phenomenon. In addition, PAM-treated cells did not show significant cell death by the control group, so only cell cycle arrest was observed without cell death or damage.

In one embodiment of the present invention, as a result of culturing the melanocytes treated with PAM in a general M254 medium that was not irradiated with plasma again, the cell cycle recovered as time passed. Specifically, DNA synthesis was inhibited in the group where the total incubation time was 72 hours after 48 hours incubation in the plasma-activated culture medium, but the treatment of the plasma-activated medium was terminated in the group cultured for 96 hours. After that, DNA synthesis gradually increased. In addition, compared to the control group, the cell cycle of the G0/G1 phase decreased, and it was found that the recovery of the cell cycle is achieved through a decrease in cell cycle arrest and an increase in somatic cell division. Was confirmed.

The plasma active culture medium may increase intracellular active oxygen (ROS). In the present invention, active oxygen in a cell refers to a chemically reactive molecule including an oxygen atom, and has a property of inducing oxidative stress in cells. The active oxygen is, for example, peroxide (H ₂ O ₂ ), superoxide anion radical (O ₂ ), hydroxyl radical (OH), and singlet oxygen (singlet oxygen). , ¹ O ₂ ), but is not limited thereto.

In the case of culturing cells in the plasma-activated culture solution of the present invention, when the plasma-activated culture solution is treated as compared to the control cells not treated with the plasma-activated culture solution, the concentration of active oxygen in the cell is 1.1 to 3 times, 1.5 to 3 times, 1.7 times to 3 times, 1.1 times to 2.7 times, 1.5 times to 2.7 times, 1.7 times to 2.7 times, 1.1 times to 2.5 times, 1.5 times to 2.5 times, 1.7 times to 2.5 times, 1.1 times to 2.3 times, 1.5 times To 2.3 times or 1.7 to 2.3 times.

In one embodiment of the present invention, the plasma active culture medium is treated and intracellular active oxygen is measured by flow cytometry to determine the positive control H ₂ O ₂ treatment group, the negative control a-MSH or arbutin treatment group and intracellular active oxygen. As a result of comparison of the amounts, H ₂ O ₂ showed a similar increase in intracellular free radicals as when treated with 100 uM. Specifically, it showed an increase in intracellular active oxygen at a level of 1.8 to 2.1 times compared to the control group not treated with the plasma active culture medium, and this increase is interpreted as a level that inhibits cell proliferation without inducing cell death.

The method of stopping the cell cycle provided by the present invention includes the steps of (1) culturing the cells and (2) treating the cultured cells with a composition for cell cycle control including a plasma active culture medium.

Each step is described in detail below.

The method of stopping the cell cycle provided by the present invention includes the step of (1) culturing cells.

The cell refers to a eukaryotic cell, preferably an animal cell, more preferably a cell isolated from a mammal including a human. The cells are, for example, melanocytes, cervical cancer cells, skin cancer cells, oral cancer cells, gastric cancer cells, colon cancer cells, lung cancer cells, egg cells, sperm cells, fertilized eggs, hair cells, nail root cells, It may be a skin cell, a sweat gland cell (apocrine sweet gland), a nerve cell, an immune cell, and preferably a melanocyte. The immune cells may be, for example, lymphocytes, macrophages, plasma cells, eosinophils, or NK cells.

The step of culturing the cells may be performed using a general culture medium not irradiated with plasma, and the culture medium may be appropriately selected and used by a person skilled in the art according to common technical knowledge, which is considered suitable for cell culture. It may be to further include an additive required for.

In one embodiment of the present invention, 1% (v/v) of HGMS was added to M254 medium that was not irradiated with plasma and used for cell culture, and melanocytes were used as cells to be cultured.

The method for stopping the cell cycle provided by the present invention includes the step of (2) treating the cultured cells with a cell cycle control composition comprising a plasma active culture medium.

The step of treating the cultured cells with the cell cycle control composition may be performed by culturing the cells by replacing the culture medium of the cultured cells with a plasma active culture medium.

The method for preparing a composition for controlling the cell cycle including the plasma-activated culture medium is as described above.

The plasma-activated culture medium is preferably used immediately after preparation, and specifically within 1 week, within 5 days, within 3 days, within 2 days, within 24 hours, within 12 hours, within 8 hours, within 4 hours after preparation, It may be used within 2 hours after manufacture, within 1 hour after manufacture, within 30 minutes after manufacture, within 15 minutes after manufacture, within 10 minutes after manufacture, or within 5 minutes after manufacture, but is not limited thereto. The time required for use after the preparation refers to the time required to replace the cell culture solution after plasma irradiation to the plasma active culture solution is completed.

In one embodiment of the present invention, 1% (v/v) of human melanocyte growth supplement (HMGS, Gibco Cascade biologics, USA), 100 U/ml penicillin and 50 ug/ml streptomycin were added to M254 in human skin melanocytes. After culturing in the medium, the culture medium was replaced with the M254 medium irradiated with plasma for 4 minutes, 8 minutes, 12 minutes, 16 minutes or 20 minutes, respectively, and cultivation was performed thereafter.

The cell cycle stopped by the method of stopping the cell cycle provided by the present invention has a temporary effect, and the cell cycle can be reversibly restored. In the recovery of the cell cycle arrest, for example, the cell cycle arrest may be restored by culturing the cells in a cell culture solution not treated with plasma.

In an embodiment of the present invention, the melanocytes are cultured in a plasma-activated culture solution for 48 hours, and then cultured for 24 hours (in total culture for 72 hours) or 48 hours (in total culture for 96 hours) in a normal culture solution not irradiated with plasma. As a result of observing the distribution of, it was confirmed that DNA synthesis was still inhibited in the group cultured for a total of 72 hours, but the synthesis of DNA increased in the group cultured for 96 hours, and the DNA synthesis function for cell cycle progression was restored.

The method of stopping the cell cycle provided by the present invention can be used for storage of cells and prevention or treatment of cell cycle related diseases such as cancer. For example, it may be used for the following purposes, but it will be apparent to those skilled in the art that it is not limited thereto.

Application example 1. The cell cycle is stopped until the melanin stored in melanocytes is consumed and used for skin whitening purposes.

Application example 2. It is used to inhibit cancer progression by stopping the cell cycle of cancer cells.

Use case 3. It is used for preservation of reproductive cells or fertilized eggs to solve the cell damage problem caused by the existing cryopreservation method.

Application Example 4. It is used to prolong the duration of cosmetic procedures such as hair or nails by causing the hair or nail cell cycle to stop.

Application Example 5. It is used to prevent recurrence of ingrown toenails by inhibiting the cell cycle after treatment by surgical resection of ingrown toenails.

Application Example 6. After the wound has recovered to a certain level, it is used to prevent keloid generation by inhibiting the cell cycle and overgrowing the fibrous tissue in the affected area.

Use Case 7. Relieve symptoms by inhibiting the production of apocrine sweat gland cells in patients with hyperhidrosis.

Application Example 8. It is used to relieve the phantom pain of the limb amputation caused by the growth of nerve cells by stopping the cell cycle of nerve cells.

Application example 9. It is used for contraception by inhibiting cell division of fertilized eggs.

Application example 10. It is used for suppressing autoimmunity and hypersensitivity reaction by stopping the cell cycle of immune cells.

Application Example 11. Used for maintaining the freshness of foods that can be damaged by cell division such as fruits and vegetables.

By treating the cells with the plasma-activated culture solution provided by the present invention, the cell cycle can be reversibly stopped. The plasma-activated culture medium provided by the present invention does not cause cell death and has an effect of temporarily stopping the cell cycle without damaging the cells.

The method of stopping the cell cycle using the plasma-activated culture medium provided by the present invention is a reversible method, and does not cause damage or death of cells, and thus has excellent safety.

1 is a photograph of melanocytes cultured for 24 hours or 48 hours by adding a plasma-activated culture medium irradiated with plasma for 20 minutes under a microscope.
FIG. 2 is a graph showing the results of measuring the number of viable cells after culturing for 24 hours or 48 hours by adding each plasma active culture solution.
3A is a schematic diagram of a normal temperature and normal pressure plasma generator.
3B is a graph showing an optical emission spectrum in the range of 200 to 100 nm during discharge of the plasma jet at room temperature and pressure according to Example 1. FIG.
4 is a graph showing the cell distribution for each cell cycle according to the treatment time of the plasma-activated culture medium. 4A is a sub-G1, FIG. 4B is a G0/G1 group, FIG. 4C is an S group, and FIG. 4D is a distribution graph of a G2/M group.
5 is a graph showing whether or not the cell cycle is recovered by culturing cells in the plasma-untreated culture medium after treatment or non-treatment of the plasma-activated culture medium in Example 6. 5A is a graph showing the distribution by cell cycle of the group cultured for a total of 72 hours, and FIG. 5B is a graph showing the distribution by cell cycle of the group cultured for a total of 96 hours. The upper part shows the graph of the control group without using the plasma-activated culture medium, and the lower part shows the result of the group treated with the plasma-activated culture medium irradiated with plasma for 8 minutes. 5C is a graph comparing the distribution of G0/G1 and G2/M phases by total culture time with a control group. 5D is a graph showing the results of measuring the number of viable cells in the control group and the plasma-activated culture solution-treated group according to the culture time.
6 is a graph showing the amount of active oxygen (ROS) in cells after treatment with a plasma active culture medium relative to a control without plasma active culture medium.
7 is a graph showing the results of flow cytometry for analyzing the amount of ROS in cells according to the treatment of the plasma active culture medium. 7A is a graph showing the results of gating a population of actual healthy cells for flow cytometry. 7B is a graph showing the ratio of fluorescence signals between M1 and M2 of a control group not treated with a plasma-activated culture medium (top) and a diagram showing corresponding values (bottom). 7C is a graph showing the ratio of M2 groups between the plasma-activated culture solution-treated group and the control group.

Hereinafter, the present invention will be described in more detail through examples. However, the following examples are for illustrating the present invention and do not limit the scope of the present invention.

Example 1. Preparation of plasma activated media (PAM)

In order to compensate for the disadvantages such as the inability to obtain a plasma activation effect evenly on all cells when plasma is directly irradiated to the cells in culture, a plasma-activated culture solution was prepared and used in the experiment by directly irradiating plasma to the liquid medium for cell culture. .

M254 medium (GIBCO Cascade Biologics, Portland, OR, USA) was used as the melanocyte culture medium, and a plasma activation medium was prepared by applying normal temperature and atmospheric pressure plasma to the M254 medium.

Specifically, the room temperature and atmospheric pressure plasma conditions according to the present invention were performed at room temperature and atmospheric pressure using 3.0W microwave power and argon (Ar) gas, and the plasma generating apparatus and the medium were separated by 0.35 cm. The plasma irradiation temperature was not to exceed 40°C. 10 mL of cell-free medium was placed in a Petri dish, and plasma was irradiated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes, respectively, to prepare a final plasma active culture solution. Microwave accuracy was adjusted to ±2.0%, gas pressure was 0 to 3 Bar, and gas flow was adjusted to 0 to 5 SLM. Fig. 3A shows a schematic diagram of the normal temperature and normal pressure plasma generator, and Fig. 3C shows the distance between the plasma generator electrode and the medium.

3B shows the optical emission spectrum during discharge of the plasma jet at room temperature and pressure of the present invention in the range of 200 to 1000 nm. The spectrum emitted from the injected argon gas atom was 690 to 990 nm and the peaks due to N2 and O were measured between 337.2 and 690 to 900 nm, respectively. The OH band was observed in the range of 307 to 309 nm.

After culturing the melanocytes first, the experiment was carried out by replacing the culture medium with a plasma-activated medium, and plasma-activated medium (PAM) was used for culturing melanocytes immediately after plasma irradiation.

Example 2. Culture of melanocytes

The melanocytes of normal human skin used in the present invention were purchased from ATTC (Manassas, VA, USA) and used. The melanocytes used in the present invention were M254 Medium (GIBCO Cascade Biologics, USA) in a medium to which 1% of human melanocyte growth supplement (HMGS, Gibco Cascade biologics, USA), 100U/ml penicillin and 50ug/ml streptomycin were added. Cultured.

Dispense the cultured cells into a 60mm cell culture dish at 1 Х 10 ⁶ cells/dish and adapt for 24 hours, then 24 in 10 mL of plasma-activated culture medium irradiated with plasma for 4 minutes, 8 minutes, 12 minutes, 16 minutes, or 20 minutes. Or 48 hours incubation. As a positive control, α-MSH (α-melanocyte stimulating hormone), which is known to have an effect of increasing melanogenesis instead of plasma active culture, was treated in 10 mL of medium to a final concentration of 1 μM, and as a negative control Arbutin, which is known to inhibit melanin production, was added to 1 mM.

After incubation, cells were harvested after treatment with trypsin-EDTA solution (JBI, Seoul, Korea) and washing with PBS containing 5% (v/v) FBS. All experiments were repeated 3 times.

Example 3. Confirmation of cell death or damage

3-1. Cell culture

Dispense melanocytes into a 60mm cell culture dish at 5 Х 10 ⁵ cells/dish and incubate for 24 hours, then plasma-activated culture solution (plasma irradiation time of 4 minutes, 8 minutes, 12 minutes, 16 minutes or 20 minutes, respectively) or 1 uM a -MSH, 1mM arbutin was added, hydrogen peroxide (H ₂ O ₂ ), which is known to form ROS, was treated to a final concentration of 100 uM or 200 uM, followed by incubation for 3 hours. Afterwards, each cell was washed with PBS, and M254 containing 20 uM 2, 7-dichlorofluorescin diacetate (2, 7-dichlorofluorescin diacetate; DCFH-DA) (Sigma-Aldrich, St. Louis, MO, USA). The medium was treated with HMGS at 37°C for 30 minutes and used for flow cytometry.

3-2. Microscopic observation

The results of microscopic observation of cells cultured for 24 hours or 48 hours in a plasma-activated medium irradiated with plasma for 20 minutes are shown in FIG. 1, respectively. As shown in FIG. 1, as the culture time increased, the number of melanocytes increased, and dendritic projections peculiar to melanocytes were identified, so that almost no damaged cells were observed. Therefore, it can be seen that the plasma-activated culture medium of the present invention does not cause the death or damage of melanocytes, unlike when the amount of reactive oxygen species is increased by irradiating plasma to cancer cells.

3-3. Cell count

Apoptosis or cell damage was confirmed by measuring the number of cells. Specifically, after attaching 1×10 ⁶ cells to a 100 mm culture dish for 24 hours, the culture solution was removed, and melanocytes were cultured using PAM treated for 4 minutes, 8 minutes, 12 minutes, 16 minutes, and 20 minutes, respectively. Subsequently, after 24 and 48 hours, the cells were removed by treatment with trypsin-EDTA solution (JBI, Seoul, Korea), and the enzyme was inhibited with 5% FBS. Then, it was washed twice with phosphate buffered saline (PBS, pH 7.4) and the number of cells was measured using an automatic cytometer ADAM-MC cell counter (NanoEnTeK, Seoul, Korea).

To determine whether melanocytes are killed or damaged, the live cells in the case of using plasma activated water with plasma exposure times of 0, 4, 8, 12, 16, or 20 minutes, respectively. The number was measured. The results are shown in FIG. 2. As a result of measuring the number of cells, the number of harvested cells gradually decreased as the plasma exposure time increased, but there was no decrease in the number of cells compared to the amount of inoculated cells, indicating that there was no cell death or cell damage. That is, the plasma-activated culture medium of the present invention has the effect of somewhat inhibiting cell proliferation, but does not cause cell death or cell damage.

Example 4. Reactive oxygen (ROS) level change in cells

4-1. How to measure active oxygen production

In order to analyze the change of M2 by the plasma active culture medium, the amount of ROS in melanocytes was analyzed. In melanocytes, the increase of intracellular ROS by plasma-activated culture was measured by flow cytometry using 2', 7'-dichlorofluorocein acetate (DCFH-DA).

Specifically, the increase in ROS levels after exposure to H ₂ O ₂ as a positive control was verified, and the effects of a-MSH and arbutin as negative controls were also investigated. H2O2 itself was used as a ROS and as a positive control. In addition, the group that was not treated with the plasma active culture medium was used as a control.

First, melanocytes were aliquoted into a 6cm cell culture dish at 5x 10 ⁵ cells/dish, and then cultured for 24 hours to attach to the dish. Attached melanocytes were treated with a-MSH (1uM), arbutin (1uM), and H ₂ O ₂ (100uM Ehsms 200 uM) and used as a control. As the experimental group, a plasma-activated culture solution was used according to the plasma irradiation time, and specifically, the plasma irradiation time of the plasma-activated culture solution was non-plasma treatment (0 minutes), 4 minutes, 8 minutes, 12 minutes, 16 minutes, and 20 minutes, respectively.

Plasma-activated culture solution or a-MSH, arbutin, H ₂ O ₂ was treated and cultured for 3 hours, and then intracellular ROS measurement was performed. The ROS is peroxide (H ₂ O ₂ ), superoxide anion radical (O ₂ ), hydroxyl radical (OH), and singlet oxygen ( ¹ O ₂ ) Includes.

Each cell was washed with PBS and incubated at 37° C. for 30 minutes in M254 medium containing HMGS in which 20uM DCFH-DA (Sigma-Aldrich) was diluted. Immediately after the culture, the intracellular ROS level was measured using flow cytometry.

4-2. ROS change in melanocytes according to plasma-activated culture medium treatment

7 shows a flow cytometric analysis result graph for analyzing the amount of ROS in cells according to the treatment of the plasma active culture medium. First, gating results for excluding cells that interfere with analysis, such as cell debris or excessively differentiated, are shown in FIG. 7A. 7A is a graph showing the results of gating a population of actual healthy cells for flow cytometry, and the results of flow cytometry are indicated by the fluorescence ratio of the M2 phase in FIGS. 7B to 12C, and M1 to the left of the baseline is under stress conditions. It means cells that are not present, and M2 to the right of the baseline represents the percentage of cells with increased ROS production. As can be seen in FIG. 7C, it was confirmed that the ratio corresponding to M2 increased in the experimental group treated with the plasma-activated culture solution, and thus the production of ROS increased relatively much compared to the control group of FIG. 7B.

As a result of treatment of melanocytes with H ₂ O ₂ 100 and 200 uM, intracellular active oxygen (ROS) levels increased rapidly, respectively, resulting in an increase of 1.8 and 2.2 times, respectively, compared to the H ₂ O ₂ untreated cell group. a-MSH did not affect ROS production. As shown in Fig. 6, the ROS level was increased in all cells cultured in plasma active culture. The increase of ROS was not proportional to the plasma irradiation time, and the increase of ROS by the plasma-activated culture medium was similar to the increase by H2O2 100 uM treatment (FIG. 6).

In the plasma-activated culture medium treatment in the present invention, the increase in ROS in melanocytes is 1.8 to 2.1 times higher than that of the control group not treated with the plasma-activated culture medium, and the increase is small. This low level of increase in ROS cannot induce apoptosis, but is predicted to inhibit cell proliferation.

Example 5. Cell cycle arrest effect of PAM

5-1. Experimental method

In order to confirm the mechanism of action of the inhibitory effect of cell proliferation identified in Example 3, flow cytometric analysis was performed to investigate the effect of the plasma-activated culture medium on the cell cycle distribution. Melanocytes were cultured for 24 hours or 48 hours using plasma-activated culture medium irradiated with plasma for various plasma exposure times (0 minutes, 4 minutes, 8 minutes, 12 minutes, 16 minutes and 20 minutes) between 0 and 20 minutes. .

As a control, cells not treated with a plasma active culture medium were used, and the criteria for apoptosis and dead cells were set based on this.

After cell culture, the cells were washed twice with PBS buffer, treated with 10 ug/mL of RNaseA enzyme, and stained with 50 ug/mL of propidium iodide (Sigma-Aldrich). The cell cycle was then analyzed using FACSCalibur flow cytometer (Becton Dickinson) and CellQuest 6.0 software.

5-2. PAM and cell cycle arrest

4 shows a graph of cell distribution for each cell cycle step according to the treatment time of the plasma active culture medium. 4A is a graph of a sub-G1 group, FIG. 4B is a G0/G1 group, FIG. 4C is an S group, and FIG. 4D is a graph of a G2/M group.

Specifically, after culturing melanocytes in a plasma-activated culture medium for 24 hours, the number of cells in the sub-G1 phase increased statistically significantly according to the plasma irradiation time (FIG. 4A). The distribution of the ratio of G0/G1 groups also increased with the plasma irradiation time (Fig. 4b). This increase in the distribution of melanocytes in the G0/G1 phase is accompanied by a decrease in the distribution of cells corresponding to the G2/M phase (FIG. 4D). The S group was reduced by plasma treatment (Fig. 4c). The above results suggest that the proliferation of melanocytes was inhibited by an increase in the growth phase of cells, inhibition of DNA synthesis and somatic cell division.

When melanocytes were cultured in a plasma-activated culture medium for 48 hours, the sub-G1 phase increased, but did not show a statistically significant increase rate (FIG. 4A). The plasma-activated culture medium reduced the G0/G1 phase and increased the G2/M phase (FIGS. 4b and 4d). This result means that the cell cycle is arrested, but this arrest does not last long. DNA synthesis and somatic division increased as cell cycle arrest began to decrease. The S phase was also not remarkable but appeared to increase.

The above results suggest that the plasma-activated culture medium may induce apoptosis of melanocytes. However, apoptosis did not appear at a significant level, and did not last long and was only a temporary phenomenon. These results are consistent with the cell proliferation and damage results of Example 3.

Example 6. Cell cycle switching

6-1. Cell cycle monitoring to confirm cell cycle switching

In order to confirm the cell cycle switching of the plasma-activated culture medium, melanocytes were cultured in a plasma-activated culture medium irradiated with plasma for 8 minutes and monitored for an extended period.

Specifically, cells were cultured for 48 hours in a plasma-activated culture medium irradiated with plasma for 8 minutes, and cells not treated with a plasma-activated culture medium were used as a control. Thereafter, the culture solution was replaced with a new culture solution not irradiated with plasma, and the cells were cultured for 24 or 48 hours. Cell cycle analysis was performed in substantially the same manner as in Example 5-1.

6-2. Recovery of cell cycle arrest

In FIG. 5, a graph is shown as a result of confirming whether or not the cell cycle is recovered by culturing cells in the plasma-untreated culture medium after treatment or non-treatment of the plasma active culture medium. Figures 5a to 10b show the cell cycle analysis results of each group according to the culture time. Each group refers to a group cultured for a total of 72 hours (48 hours in PAM and then 24 hours in plasma untreated medium, Fig. 5A) or a total of 96 hours (48 hours in PAM and 48 hours in plasma untreated medium, Fig. 5B). .

Compared to the control group not treated with the plasma-activated culture medium, cell death in the sub-G1 phase was slightly increased, but this change was not judged to be a significant increase in both groups.

Regarding the change of the S phase, DNA synthesis was inhibited in the 72-hour culture group, but the DNA synthesis increased in the 96-hour culture group (FIGS. 5a and b). Therefore, it is interpreted that the DNA synthesis gradually increased at a specific time point after treatment with the plasma activation medium.

5C shows a graph comparing the distribution of G0/G1 and G2/M phases by total culture time with the control group. As shown in Fig. 5c, the cell cycle distribution of the control cells varied from group to group. The distribution of the G0/G1 phase in the 96-hour culture group was 13% higher than that of the 72-hour culture group. In the case of the G2/M phase, the distribution of the 96-hour culture group was 53% lower than that of the 72-hour culture group. The cause of the difference is still unclear, but it is predicted that this difference is partially affected by the physiological state of the cell at the time the experiment is performed. In general, 0.3 to 2% of cells correspond to the sub-G1 phase, 70 to 74% of the cells correspond to the G0/G1 phase, 15-20% of the S phase, and 7 to 14% of the cells correspond to the G2/M phase. Is known. Therefore, the present inventors considered the difference between the control group and the experimental group at the same time. Compared with the control group, the cell cycle distribution in the G0/G1 phase decreased by 20% and 17% in the 72-hour and 96-hour culture groups, respectively. The above results clearly show that cell cycle recovery is achieved through decreased cell cycle arrest and increased somatic division.

4 to 5, the cells were found to return to their original state through recovery of cell cycle progression within 48 hours after treatment with the plasma-activated culture medium. However, it takes a long time to fully recover to a normal state, and many cells stayed in an arrest state. Therefore, even after 96 hours, the cell proliferation rate treated with the plasma-activated culture solution was 1.0 x 10 ⁴ cells/h, which was lower than that of the control group, 3.0 x 10 ⁴ cells/h (FIG. 5D).

It is presumed that the cell cycle arrest observed in the present invention is related to the increased reactive oxygen (ROS) by plasma activated culture. The effect of free radicals on the cell cycle was proportional to the plasma irradiation time, and ranged from temporary growth arrest to permanent cell death such as apoptosis or necrosis. Compared with previous studies, ROS caused transient multi-phase cell cycle arrest in the G1, S, and G2 phases of mouse fibroblasts, but did not affect the M phase. In another study, it has been reported that the accumulation of ROS induces arrest of G2/M phase and subsequent apoptosis.

In the present invention, the plasma-activated culture medium induced cell cycle arrest through the regulation of the G0/G1 and G2/M phases, but did not cause significant cell death. Furthermore, cell cycle arrest is a transient phenomenon lasting for 48 hours or shorter, and the cell cycle slowly recovered.

The half-life of the active oxygen produced by the normal temperature and atmospheric pressure plasma in the culture medium is several hours to several days depending on the temperature, but the ROS level decreases with time. Therefore, the increase in intracellular ROS level in melanocytes also decreases with time, and is predicted to affect the recovery of cell cycle progression. The above can be summarized as follows. Treatment of the plasma-activated culture medium (PAM) at an appropriate concentration may cause cell proliferation and cell cycle arrest, but the cell proliferation and cell cycle arrest may be recovered after a certain period of time. The above results suggest that the cell cycle can be turned on and off without intracellular toxicity, that is, switching is possible using normal temperature and atmospheric pressure plasma.

Claims (15)

A composition for controlling cell cycle, comprising a plasma activated media (PAM) prepared by irradiating plasma with a liquid medium for cell culture that does not contain cells,
The cells are one or more cells selected from the group consisting of melanocytes, germ cells, fertilized eggs, hair root cells, nail root cells, neurons, and immune cells,
The plasma-activated culture medium will reversibly stop the cell cycle without inducing cell death. The composition of claim 1, wherein the plasma is a normal temperature and normal pressure plasma, and the normal pressure is atmospheric pressure. The composition of claim 1, wherein the plasma is irradiated for 4 to 30 minutes. The composition of claim 1, wherein the plasma-activated culture medium increases intracellular active oxygen by 1.1 to 3 times as compared to a culture solution not containing the plasma-activated culture medium. delete The composition of claim 1, wherein the plasma-activated culture medium stops the cell cycle through control of the G0/G1 or G2/M checkpoint. delete The composition of claim 1, wherein the cell culture liquid medium is a mammalian cell culture liquid medium. Culturing one or more cells selected from the group consisting of melanocytes, germ cells, fertilized eggs, hair root cells, nail root cells, neurons, and immune cells; And
Comprising the step of treating the cultured cells with a composition for controlling a cell cycle including a plasma activated media (PAM) prepared by irradiating plasma with a liquid medium for cell culture that does not contain cells,
A method of reversibly arresting the cell cycle. The method of claim 9, wherein the plasma is a normal temperature and normal pressure plasma, and the normal pressure is atmospheric pressure. The method of claim 9, wherein the plasma is irradiated for 4 to 30 minutes. delete The method of claim 9, wherein the plasma active culture medium increases intracellular active oxygen. The method of claim 9, wherein the cell cycle arrest is recovered by culturing cells in a liquid medium for cell culture not irradiated with plasma. The method of claim 9, wherein the cell culture liquid medium is a mammalian cell culture liquid medium.

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