KR20210045103A - A method of preparing anti-aging essence comprising high-concentration cytokines through maturing and a system module for producing cytokines therefor - Google Patents

Abstract

The present invention relates to a method for obtaining high-concentration cytokines secreted from a cell and a system module for producing cytokines to monitor a cell nutrient solution in real time therefor. An embodiment of the present invention provides a method for producing anti-aging essence containing high-concentration cytokines through maturing, the method comprising: a) a step of separating a cell from adipose tissue; b) a step of maturing the separated cell in a clean nutrient solution; and c) a step of recovering a cytokine solution from the matured cell. Also, another embodiment of the present invention provides the system module for producing cytokines, the system module comprising: a clean booth unit in which a first filter and a first blower are formed; a biological safety cabinet unit in which a second filter and a second blower are formed; an incubator unit which includes a culture space; a flask unit which matures the cell; a microscope unit which enlarges and observes the cell; a photographing unit which photographs the cell; a main PC unit which receives and transmits image data photographed by the photographing unit in real time; and a communicating unit which is connected to the main PC unit and transmits the image data to a host PC unit positioned outside the clean booth unit.

Description

A METHOD OF PREPARING ANTI-AGING ESSENCE COMPRISING HIGH-CONCENTRATION CYTOKINES THROUGH MATURING AND A SYSTEM MODULE FOR PRODUCING CYTOKINES THEREFOR}

The present technology is an advanced engineering method for obtaining a high concentration of cytokines secreted from cells, a technology for applying a high-concentration cytokine solution purified by this method as an anti-aging essence, and monitoring the cell nutrient solution in real time for this purpose. It is a technology related to the cytokine production system module for

Conventionally, as a method of applying and using cells in preparations such as pharmaceuticals, cells derived from various tissues have been used as raw materials in preparations such as pharmaceuticals with the same concept and method as a therapeutic agent. There are two fatal limitations to formulations such as drugs to which this method was applied.First, it is impossible to maintain the lifespan of cells after they are added to drugs, etc., and the next is that the cells obtained due to safety concerns. If the various infectious agents that can exist are not completely removed, there is a possibility that the risks will be transmitted to the customers who use them, leading to infection of cosmetics. In particular, with regard to safety, in the case of infection with a viral infectious agent during the cell culture process, it is not easy to discriminate by the shape of the cell, so the probability that the infection is not recognized until the manufacture of the final drug, etc. is greatly increased. do. Therefore, the conventional methods disclosed later use a method of secreting various physiologically active substances produced by cells (eg, stem cells), and then using the secreted substances as raw materials for preparations such as pharmaceuticals. This method utilizes the properties of various cells secreting proteins, peptides and various physiologically active substances that can activate other cells during the cell growth process, and its biological effectiveness is proven when these secreted substances are treated to other cells. It is based on scientific evidence. In addition, since the cells themselves are not directly used as cosmetic ingredients, they can escape to some extent from ethical issues and direct limitations on safety.

In particular, among substances secreted by cells, cytokines are included, and cytokines are proteins of low molecular weight secreted by cells. Mainly produced by immune cells (T lymphocytes, B lymphocytes, white blood cells, macrophages), but also various cells such as fibroblasts, endothelial cells, and stromal cells can be produced. Molecular weight is usually 5~20kDa or less, and it is small, and it is active in cells mainly in the form of a monomer or a dimer. Depending on their function, these cytokines are interleukin series (collectively referred to as substances that act between immune cells), lymphokine series (collectively refer to substances from lymphocytes), monokine series (subjects from monocytes (including macrophages)), Interferon series (a substance that has antiviral cancer-repellant action), CSF series (a substance that acts on cell groups made in the bone marrow), chemokine series (called chemotaxis, a substance that mediates the action of a chemical substance) It can be classified as Tongqing). Cytokines are substances that are made in cells and affect other cells, including themselves, so they can be similar to growth factors, but they can be distinguished from cytokines. It is known that a cytokine is usually made by several types of cells, and its effects can also affect many types of cells. For example, it can affect the activation, growth, and differentiation of several immune cells, and it can affect not only natural and acquired immune responses, but also the maturation process of immune cells. In addition, it promotes or inhibits cell proliferation and differentiation, and is involved in inflammation and hematopoiesis. In particular, cytokines are a major target for the development of therapeutics because their role is very important in the human body, and many items are already being sold as therapeutics.

Most cytokines act on neighboring cells that interact with them, and cytokines that act in this way are called paracrine factors. The paracrine factor can locally control the function of nearby cells and persist rather briefly due to the degradation of the paracrine ligand.

However, since the amount of these bioactive substances is quite limited, it is difficult to obtain a high concentration that is practically applicable. In addition, in order to obtain physiologically active substances secreted from cells, it is necessary to first secrete such substances from cells. At this time, various kinds of antibiotics are added to prevent infection of bacteria, fungi, and yeasts. And, most of the antibiotics are not only prohibited items as raw materials for cosmetics, but are also harmful to the human body. Therefore, it is necessary to establish a technology for obtaining bioactive substances suitable for cosmetic raw materials by utilizing a technology that does not use antibiotics in the process of obtaining bioactive substances secreted from cells.

In accordance with this need, the present inventors have developed a technology capable of obtaining a high-concentration cytokine solution without using substances harmful to the human body.Based on this technology, the National Boramae Hospital and autologous cytokines (including stromal cells, if necessary) have been developed. ) To develop and popularize advanced regenerative medicine for degenerative and intractable diseases, and to establish complementary cooperation for health promotion and medical development of other people.

The technical problem to be achieved by the present invention is to provide a high-tech engineering method for obtaining cytokines secreted from cells at a high concentration without using substances harmful to the human body.

In addition, another technical problem to be achieved by the present invention is divided into two or more spaces, so that some spaces can be maintained in a sterile state regardless of whether or not an operator has entered or not, and the culture state of cells can be observed in real time even outside the space. It is to provide a module for the cytokine production system.

The technical problem to be achieved by the present invention is not limited to the technical problems mentioned above, and other technical problems that are not mentioned can be clearly understood by those of ordinary skill in the technical field to which the present invention belongs from the following description. There will be.

In order to achieve the above technical problem, according to one aspect of the present invention, a method for producing an anti-aging essence containing a high concentration cytokine through maturing is

Separating the cells from the adipose tissue;

Maturing the isolated cells in a clean nutrient solution; And

Recovering cytokine fluid from the matured cells;

It may include.

In an embodiment of the present invention, the maturing step,

First maturing the isolated cells in a clean nutrient solution;

Secondary maturing the primary matured cells in a clean nutrient solution; And

Tertiary maturing without providing a clean nutrient when the secondary matured cells are confluent growth of 60 to 90% in a state where a clean nutrient solution is provided;

It may include.

In an embodiment of the present invention, the clean nutrient solution contains 10% bovine serum, but may not contain antibiotics and phenol red.

In an embodiment of the present invention, in the step of providing a clean nutrient solution to the secondary matured cells, the clean nutrient solution may be provided for 2 to 5 days.

In an embodiment of the present invention, the step of recovering the cytokine solution,

Analyzing the cytokine content in the clean nutrient solution of the matured cells; And

Recovering a cytokine solution according to the analyzed cytokine content;

It may include.

In an embodiment of the present invention, the step of analyzing the cytokine content may measure TGF-β (Transforming growth factor beta) and VEGF (Vascular endothelial growth factor) concentrations in the clean nutrient solution of the matured cells. have.

In an embodiment of the present invention, in the step of recovering the cytokine solution, when the TGF-β concentration in the clean nutrient solution of the matured cells is 1 to 2 ppb and the VEGF concentration is 5 to 20 ppb, the cytokine solution is Can be recovered.

In an embodiment of the present invention, the step of maturing the cells may be performed for 10 to 20 days.

In order to achieve the above technical problem, in an embodiment according to another aspect of the present invention, a first internal space is formed therein, a first filter and a first blower are formed on one surface, and external air is passed through the first filter. A clean booth that flows into the interior, is installed in the first internal space, a second internal space is formed therein, and a second filter and a second blower are formed on one side of the air in the first internal space. A biosafety cabinet that flows into the interior through the second filter, an incubator section that is installed in the first interior space, and has a cell maturing space, and each cell maturing space. One or more flasks that are located and that receive cells and nutrients to mature the cells, and a microscope that is installed in the cell maturing space and observes magnified cells that are matured in the flask, An imaging unit installed in the microscope unit to image cells cultured in the flask unit, and an imaging unit installed in the first internal space, receives and transmits the image data captured by the imaging unit in real time, and captures images according to the input user's command data. Cytokine including a main PC unit that controls the unit and a communication unit that is connected to the main PC unit and transmits video data to the host PC unit located outside the clean booth unit, and transmits command data received from the host PC unit to the main PC unit. Provides a production system module.

In an embodiment of the present invention, a first counter unit installed in the first inner space to counter particles located in the first inner space, and a first counter unit installed in the second inner space to counter particles located in the second inner space The second counter unit may further include a control unit for controlling the first blower and the second blower according to the number of particles located in the first inner space and the second inner space.

In an embodiment of the present invention, when the number of particles located in the first inner space is greater than or equal to a preset first value, the control unit rotates the first blower at a first speed, and rotates the second blower at a second speed, When the number of particles located in the first inner space is less than a preset first value, the controller may rotate the first blower at the second speed and the second blower at the first speed.

In one embodiment of the present invention, the microscope unit includes a fixture to which the flask unit is detachably coupled, a stage to which the fixture is detachably coupled, and the image of cells accommodated in the flask unit located on the stage. An optical system that guides, and a lens driving means for driving the lens to perform focus alignment and aberration correction for cells, and the flask unit can be restrained from moving and rotating at a predetermined position on the stage by the fixture.

In an embodiment of the present invention, the stage has a first through hole formed with a protrusion, and the flask part has a plate-shaped plate in which a second through hole communicating with the first through hole is formed, and is formed on one surface of the plate. , A plurality of first fixing members formed to correspond to the outer circumferential surface of the flask part, and fixing the flask part, and a second fixing member formed on the other surface of the plate, inserted into the through hole of the stage, and having a groove corresponding to the protrusion, If the protrusion is inserted into the groove of the second fixing member, the plate may be prevented from rotating and moving.

In one embodiment of the present invention, the clean booth unit is detachably coupled, and may further include a moving unit for moving the clean booth unit.

According to an embodiment of the present invention, it is possible to recover a high-concentration cytokine solution from cells without using antibiotics or other harmful substances that have been conventionally used to prevent bacterial contamination.

In addition, according to another embodiment of the present invention, the user can easily observe the culture state of cells in real time inside or outside the clean booth, and does not directly manage the air quality inside the clean booth and the biosafety test loan. As a result, convenience can be improved.

The effects of the present invention are not limited to the above effects, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a view showing a method for producing an anti-aging essence containing high-concentration cytokines through maturing according to an embodiment of the present invention.
2 is a diagram showing a cytokine production system module according to an embodiment of the present invention.
3 is a block diagram of a cytokine production system module according to an embodiment of the present invention.
4 is a diagram illustrating a microscope unit and an imaging unit of a cytokine production system module according to an embodiment of the present invention.
5 is a view showing a flask portion coupled to the fixture of the cytokine production system module according to an embodiment of the present invention.
6 is a diagram showing a fixture of a cytokine production system module according to an embodiment of the present invention.
7 is a flowchart illustrating a method of controlling a cytokine production system module according to an embodiment of the present invention.
8 is a graph showing the wound regeneration effect of the cytokine solution obtained through the production method according to an embodiment of the present invention.
9 is a diagram showing the epidermal regeneration effect of the cytokine solution obtained through the production method according to an embodiment of the present invention.
10 is a graph showing the angiogenesis effect of cytokine solution obtained through the production method according to an embodiment of the present invention.
11 shows the results of analyzing the human colostrum component.
12 is a diagram showing the effect of treating burn wounds of a cytokine solution obtained through a production method according to an embodiment of the present invention.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be implemented in various different forms, and therefore is not limited to the embodiments described herein. In the drawings, parts irrelevant to the description are omitted in order to clearly describe the present invention, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is "connected (connected, contacted, bonded)" with another part, it is not only "directly connected", but also "indirectly connected" with another member in between "Including the case. In addition, when a part "includes" a certain component, this means that other components may be further provided, not excluding other components, unless specifically stated to the contrary.

The terms used in the present specification are only used to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, but one or more other features. It is to be understood that the presence or addition of elements or numbers, steps, actions, components, parts, or combinations thereof, does not preclude the possibility of preliminary exclusion.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

A method for producing anti-aging essence containing high-concentration cytokines through maturing according to an aspect of the present invention comprises: a) separating cells from adipose tissue; b) maturing the isolated cells in a clean nutrient solution; And c) recovering the cytokine solution from the matured cells. The maturation means to mature the cells.

In the present invention, "high concentration" may mean that the concentration of cytokines obtained according to the present invention is higher than that of human colostrum. Human colostrum refers to the milk secreted within a few days after delivery, and colostrum has a different composition from the milk secreted afterwards, and contains many solids, proteins, fats, ash, and lactose. The main components of colostrum are immune factors, antimicrobial activity and growth factors, and immune factors and antimicrobial active components may include immunoglobulins, lactoferrin, lysozyme, lactoperoxidase, and cytokines. , Growth factors may include insuline-like growth factor (IGF), epithermal growth factor (EGF), transforming growth factor (TGF-β), and fibroblast growth factor (FGF). These growth factors of colostrum play an important role in wound healing and play a physiologically active function of colostrum. There are EGFR (epithelial proliferation factor receptor) and FGFR (fibroblast proliferation factor), which are involved in cell-cell interactions such as cell differentiation, immune function, and nerve function due to the specific involvement of growth factors that induce the activity of tyrosine kinase receptor. . In addition, VEGF (vascular endothelial growth factor) and PDGF (platelet-derived growth factor) are also present. Growth factors showed synergistic effects on keratinocyte differentiation and cell migration for tissue recovery, and growth factor peptides contained in colostrum or colostrum were effective in treating intestinal diseases, thus showing the possibility of use as a therapeutic agent. The concentration of the cytokine obtained according to the present invention was higher than the concentration of the growth factors of the colostrum, which can indirectly show that the present invention is superior to the prior art. 11 is a result of analyzing the cytokine component contained in human colostrum, and the concentration of cytokine obtained according to the present invention was higher than that of the cytokine concentration of this colostrum.

The technology using cytokines according to an embodiment of the present invention is a next-generation technology superior to the conventional stem cell technology, and has a higher probability of success than the conventional technology using stem cells because it utilizes the effect of the paracrine factor.

In the present invention, "anti-aging essence" does not simply mean a cosmetic composition, but is a concept including a therapeutic composition.

The step of separating the cells from the adipose tissue is a step of obtaining cells from an adipose tissue sample of the subject subject, and the subject subject may be an animal, preferably a human. In addition, the cells may be somatic cells, bone cells, nerve cells, skin cells, muscle cells, blood cells, adipocytes, immune cells, or stem cells, and preferably stem cells. More preferably, it may be an autologous adipose stem cell.

The step of maturing the isolated cells in a clean nutrient solution is a step of allowing the isolated cells to secrete a high concentration of cytokines, and according to an embodiment of the present invention, the maturing step comprises: a) the isolated cells First maturing the in a clean nutrient solution; b) secondary maturing the primary matured cells in a clean nutrient solution; And c) tertiary maturing without providing a clean nutrient solution when the secondary matured cells are confluent growth of 60 to 90%.

According to an embodiment of the present invention, in the step of first maturing the isolated cells in a clean nutrient solution, cells isolated from adipose tissue can be proliferated in a clean nutrient solution, and the separated cells are confluent growth) When it reaches 100%, subculture can be performed and cultured with cells in passage 1.

According to another embodiment of the present invention, in the step of secondary maturing the first matured cells in a clean nutrient solution, the isolated cells are subcultured through the first maturing step. When the cells become passage 1, they can be transferred to another flask to provide a clean nutrient solution again, and the cells can be subcultured again to be cultured as cells in passage 2.

According to another embodiment of the present invention, when the secondary matured cells are confluent growth of 60 to 90% in a state where a clean nutrient is provided, the third maturation is performed without providing a clean nutrient. In the step, when the separated cells are subcultured through the second maturing step and become cells of passage 2, they are transferred to another flask to provide clean nutrients again, and the cells are proliferated to 60 to 90% of confluent growth. After that, the cells can be cultured without providing any more clean nutrients. Specifically, the cells may be proliferated until confluent 70 to 90%, preferably 75 to 90%, more preferably 75 to 85%.

In particular, the reason for the maturing of the cells without providing a clean nutrient solution is that the cells can secrete cytokines when they are under stress, in other words, in the process of the cells being killed or weakened in a nutrient-limited state. This is because it can secrete cytokines at high concentrations.

According to an embodiment of the present invention, the maturation can occur because all the steps according to the embodiment of the present invention are characterized in that it is performed in a perfect aseptic state.

According to an embodiment of the present invention, in the step of providing a clean nutrient solution to the secondary matured cells, the clean nutrient solution may be provided for 2 to 5 days, preferably for 2 to 4 days, further Preferably, it may be provided for 2 to 3 days.

According to an embodiment of the present invention, the clean spirit solution is a medium for culturing cells, and contains 10% bovine serum, but may not contain antibiotics and phenol red. It does not contain antibiotics and phenol red that are harmful to the human body, so that cytokines that are harmless to the human body can be obtained at a high concentration.

According to an embodiment of the present invention, the step of recovering the cytokine solution from the matured cells includes: a) analyzing the cytokine content in the clean nutrient solution of the matured cells; And b) recovering the cytokine solution according to the analyzed cytokine content.

Specifically, the step of analyzing the cytokine content may measure the concentration of Transforming growth factor beta (TGF-β) and Vascular endothelial growth factor (VEGF) in the clean nutrient solution of the matured cells, and the TGF-β And when the VEGF concentration is 1 to 2 ppb, the VEGF concentration is 5 to 20 ppb, the cytokine solution can be recovered. Preferably, the cytokine solution can be recovered when the concentration of TGF-β and VEGF is 1 to 1.7 ppb, and the concentration of VEGF is 5 to 15 ppb, and even more preferably, the concentration of TGF-β and VEGF is 1.5 to 1.7 ppb, and the cytokine solution can be recovered when the VEGF concentration is 10 to 15 ppb.

According to an embodiment of the present invention, the step of maturing the cells may be performed for 10 to 20 days. The period may refer to the total period of maturing the cell, and more specifically, when the step of maturing the cell includes a detailed step, the sum of the maturing periods of each detailed step is the total period Can mean That is, when the step of maturing the cells goes through the step of first, second, and third maturing, a period of undergoing all three maturing steps may be performed for 10 to 20 days.

According to another aspect of the present invention, the cytokine production system module 1 applied to the method for producing anti-aging essences containing high-concentration cytokines through maturing is a cytokine production system module capable of remote monitoring in real time in a sterile state. Can be The cytokine production system module 1 includes a clean booth unit 10, a biosafety laboratory unit 20, an incubator unit 30, a flask unit 40, a microscope unit 310, an imaging unit 315, and a main PC. It may include a unit 50 and a communication unit 60.

The clean booth unit 10 is a component having a space in which an environment such as particulate matter, air temperature, humidity, pressure, etc. is constantly maintained, and is formed in a box shape, and a first internal space is formed therein.

In addition, the clean booth unit 10 has a first suction path (not shown) that passes air located outside the clean booth unit 10 on one side, and the air located inside the clean booth unit 10 on the other side. A first discharge path (not shown) for exhausting the gas to the outside is formed, and a first filter 110 is coupled to the first suction path, so that air located outside may selectively pass. That is, air located outside the clean booth 10 may be introduced into the first internal space through the first filter 110, and air located in the first internal space may be discharged to the outside through the first discharge path. I can. At this time, a first blower 130 for inducing a flow of air is coupled to one surface of the first filter 110.

Meanwhile, the first suction path may be formed on the ceiling or side surfaces of the clean booth unit 10, and the first discharge path may be formed on the bottom surface of the clean booth unit 10.

The Biological Safety Cabinet unit 20 is a component having a sterile working space, and is disposed in a first internal space, and a second internal space may be formed therein. In this case, the second internal space may be divided into a plurality of spaces by the separating plate.

The user may move the cells matured in the incubator unit 30 to be described later to the second internal space of the biosafety laboratory unit 20 to separate the cytokines secreted by the cells.

The biosafety test loan 20 has a second suction path (not shown) that passes air located outside the biosafety test loan 20 on one side, and is located inside the biosafety test loan 20 on the other side. A second air discharge path (not shown) for exhausting air to the first internal space is formed, and a second filter 210 is coupled to the second suction path so that air located outside may selectively pass. In this case, a second blower 230 for inducing a flow of air may be coupled to one surface of the second filter 210.

That is, the air located in the first internal space can be introduced into the second internal space through the second filter 210, and the air located in the second internal space is discharged to the first internal space through the second air discharge path. I can.

In this way, the biosafety test loan 20 does not take a long time to create a sterile state because the air, which is primarily filtered by the first filter 110, is introduced through the second filter 210. . Meanwhile, the first filter 110 and the second filter 210 may be Hepa filters.

According to an embodiment of the present invention, particles in the first inner space of the clean booth unit 10 are provided so that the user can visually check the number of particles located in the clean booth unit 10 and the biosafety test unit 20. A first counter unit 120 for countering may be installed, and a second counter unit 220 for countering particles may be installed in a second inner space of the biological safety test unit 20.

At this time, the first counter unit 120 and the second counter unit 220 use the principle that the moving speed of the large particles and the small particles are different, and check the particle size or irradiate the particles with a laser to determine the particle size according to the laser scattering. You can check the size.

According to an embodiment of the present invention, the cytokine production system module 1 may further include a control unit 70 that controls the first blower 130 and the second blower 230. In this case, the control unit 70 is connected to the two counter units 120 and 220 to receive the number of particles measured from the counter units 120 and 220.

When the number of particles measured by the first counter unit 120 is greater than or equal to a preset first value, the control unit 70 rotates the first blower 130 at a first speed, and rotates the second blower 230 at a second speed. Can be rotated with

In addition, when the number of particles measured by the first counter unit 120 is less than a preset first value, the control unit 70 rotates the first blower 130 at a second speed and controls the second blower 230 It can be rotated at 1 speed. In this case, the first speed may be faster than the second speed.

In other words, when the air in the first internal space of the clean booth unit 10 contains more particles than the set level, the control unit 70 generates a strong air volume by the first blower 130 to rapidly increase the amount of air in the first internal space. It discharges air to the outside and purifies the first internal space. In addition, when the air in the first internal space contains particles less than the set level, the control unit 70 generates a minimum amount of air with the first blower 130 to maintain the cleanliness of the first internal space, and the second A strong air volume is generated by the blower 230 to purify the second internal space.

Thereafter, when the number of particles measured by the second counter unit 220 is less than a preset second value, the controller 70 may rotate the second blower 230 at the second speed. Meanwhile, the second counter unit 220 may rotate the second blower 230 at the second speed. When the number of particles measured by the unit 220 is greater than or equal to a preset second value, the second blower may be rotated at the first speed.

In other words, when the air in the second internal space of the biosafety test unit 20 contains more particles than the set level, the control unit 70 generates a strong air volume with the second blower 230 to rapidly generate the second internal space. Air is discharged to the first inner space, and the second inner space is cleaned. In addition, when the air in the second internal space contains particles less than the set level, the control unit 70 generates a minimum amount of air with the second blower 230 to maintain the cleanliness of the second internal space.

As described above, in the present invention, since the user does not have to directly manage the air quality inside the clean booth unit 10 and the biosafety test unit 20, the user's convenience may be improved. In addition, since the operation of the second blower 230 is controlled according to the number of particles in the first internal space, the biosafety test loan 20 can continue to maintain a sterile state once it becomes aseptic. That is, the present invention can achieve a more complete ring sterilization state.

The incubator unit 30 is a device for maturing cells, and has a cell maturing space in which certain conditions are maintained, and a flask part 40 containing cells and nutrients may be located in the cell maturing space. . In addition, a nutrient solution to be additionally supplied to the cells of the flask unit 40 may be located in the cell maturing space.

According to an embodiment of the present invention, the cytokine production system module 1 may be provided with two incubator units 30. At this time, one incubator unit 30 may be for maturing the sampling cells for observing the maturing state of the cells, and the other incubator unit 30 produces cytokines that are substantially injected into the human body. It may be maturing the producing cells that do.

At this time, since the two incubator units 30 are matured under the same conditions, the user can observe the maturing state of the cells through the sampling cells without directly observing the production cells. As described above, by observing the sampling cells, the present invention can prevent the production cells or cytokines from being contaminated in the process of observing the maturing state.

3 to 5, the microscope unit 310 is a device for observing cells accommodated in the flask unit 40, and may be installed in the cell maturing space of any one incubator unit 30. In this case, two microscope units 310 may be installed in the cell maturing space for the efficiency of observation, but the present invention is not limited thereto, and one may be installed.

In more detail, the microscope unit 310 includes a fixture 311 to which the flask unit 40 is detachably coupled, a stage 312 to which the fixture 311 is detachably coupled, and a lens. It may include an optical system 313 for guiding the image of the cells accommodated in the flask unit 40 located at, and a lens driving means 314 for focusing and correcting aberrations on the cells by driving the lens. In this case, the flask unit 40 may be fixed at a predetermined position on the stage 312 by the fixture 311.

Meanwhile, in the stage 312, a first through hole 3121 may be formed so that the optical system 313 located under the stage 312 can observe the cells on the stage 312. In addition, the stage 312 may have a protrusion 3122 protruding from the inner circumferential surface toward the center of the first through hole 3121, and the protrusion 3122 is inserted into an area of the fixture 311, 311) makes it impossible to move and rotate.

When describing the fixture 311 in detail, the fixture 311 includes a plate-shaped plate 3111, a plurality of first fixing members 3112 formed on one surface of the plate 3111 to correspond to the flask portion 40 And a second fixing member 3113 inserted into the first through hole 3121 of the stage 312 on the other surface of the plate 3111 and having a groove corresponding to the protrusion 3122 formed therein. At this time, when the protrusion 3122 is located in the groove of the second fixing member 3113, the plate 3111 cannot be moved or rotated. As described above, the user of the present invention can observe the same position every time by coupling the flask portion 40 to the fixture 311 and then coupling the fixture to the microscope portion 310.

Meanwhile, the plate 3111 has a second through hole 3114 corresponding to the first through hole 3121 so that the optical system 313 can observe the cells on the stage 312.

The imaging unit 315 is positioned on the stage 312 and is a device for observing cells accommodated in the flask unit 40, and may be coupled to the microscope unit 310. The imaging unit 315 is connected to the main PC unit 50 outside of the incubator unit 30, and driving can be controlled according to the command data input through the main PC unit 50, and the captured image is It may be transmitted to the main PC unit 50. In this case, the main PC unit 50 may be installed on the table 510 located near the incubator unit 30, and may be connected to the communication unit 60 for transmitting and receiving data with the host PC unit A located outside. .

The main PC unit 50 may transmit an image captured by the host PC unit A through the communication unit 60 and may receive command data from the host PC unit A. In this case, the host PC unit A may be a mobile terminal or a fixed terminal located outside the clean booth unit 10. Accordingly, the user can observe the culture state of the cells from the outside through the imaging unit 315 without entering the inside of the clean booth unit 10.

In addition, the main PC unit 50 may be connected to an externally located cloud data center (B) through the communication unit 60, and transmits an image captured to the cloud data center (B) at preset time intervals, thereby transmitting the image. It can be stored in the cloud data center (B). In this case, the communication unit 60 and the control unit 70 may be a communication module and a control module formed in the main PC unit 50, and in this case, it may be controlled by the processor of the main PC unit 50.

According to an embodiment of the present invention, any one of a moving unit (not shown) and an air cleaning unit 80 may be further included. The moving unit (not shown) is a component that moves the position of the clean booth unit 10 and may be a vehicle, and may be detachably coupled to the clean booth unit 10.

The air cleaning unit 80 circulates air in the first inner space of the clean booth unit 10 and removes particles, and may be disposed in the first inner space.

In the cytokine production system module 1 of the present invention, components for cell maturation are modularized and configured, and since it is formed to be movable, the user can use the cytokine production system of the present invention inside or outside the building. By providing the module 1, it is possible to easily form a cell maturing space.

6 is a flow chart showing a control method of the cytokine production system module 1 according to an embodiment of the present invention.

The control unit 70 drives the first counter unit 120 for counting the number of particles in the first inner space and the second counter unit 220 for counting the number of particles in the second inner space in step S100. In this case, the first counter unit 120 and the second counter unit 220 may transmit the number of counted particles to the control unit 70 at predetermined time intervals.

The control unit 70 determines whether the number of particles in the first internal space countered in step S110 is equal to or greater than a preset first value, and if it is determined that it is equal to or greater than the preset first value, the first blower 130 starts at a first speed in step S120. And the second blower can be rotated at a second speed. On the other hand, if it is determined that the counted number of particles in the first internal space is less than the preset first value, the control unit 70 rotates the first blower 130 at a second speed in step S130, and turns the second blower 230 It can be rotated at the first speed.

Thereafter, the control unit 70 determines whether the number of particles in the second inner space countered in step S140 is equal to or greater than a preset second value, and if it is determined that it is equal to or greater than the second preset value, the second blower 230 is selected in step S150. It can be rotated at 1 speed. On the other hand, if it is determined that the counted number of particles in the second inner space is less than the preset second value, the control unit 70 may rotate the second blower 230 at the second speed in step S160.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily implement the present invention. However, the present invention may be implemented in various different forms and is not limited to the embodiments described herein.

Example 1. Stem cell isolation and maturing process

1.1 Stem cell isolation from adipose tissue and continuous culture process up to Passage 1

(1) Local washing

end. Take out the syringe containing the adipose tissue sample and discard the lower layer, then transfer the remaining adipose tissue to a conical tube, add 30 ml of PBS, close the lid, shake, and when the lower layer and the fat layer are separated, remove the lower layer using a pipette. I did.

I. The above PBS washing process was repeated 2-3 times to remove the blood mixed with fat (at the time of the last washing, lift the tube vertically and remove as much PBS as possible).

(2) enzyme treatment

end. A collagen dissolving agent having a concentration of 0.3 unit/ml diluted with PBS was taken as much as 80% of the fat volume and then mixed well with the washed fat.

I. Solution 1 was shaken in a 50℃ CO ₂ incubator for 5 minutes to perform an enzyme reaction, and then transferred to a 40℃ incubator for an enzymatic reaction for 30 minutes (every 2-3 minutes, the degree of decomposition was visually checked to minimize treatment time. box).

All. When the adipose tissue was decomposed into a viscous solution, an equal amount of clean nutrient solution (including 10% bovine serum (BS)) was added to stop the reaction.

(3) cell filtration and cell seeding

end. After centrifugation (2500 rpm, 5 minutes), the supernatant containing the oil layer was removed, the pellet was released in 10 ml of PBS, and filtered step by step using a 100 μm, 40 μm cell strainer.

I. After centrifuging the filtered cell solution (2500 rpm, 5 minutes), the supernatant was removed, and a clean nutrient solution (including 10% BS) was added to release the cells.

All. After counting, the cells were seeded using a clean nutrient solution (including 10% BS) in a flask of an appropriate size, and replaced with the first clean nutrient solution (including 10% BS) the next day.

la. Cell proliferation was observed by adding an appropriate amount of the first clean nutrient solution according to the daily culture conditions.

hemp. When the cells at the bottom of the flask proliferated with 100% confluent growth (to be fully filled), passage 1 was continuously cultured by subculture.

1.2 P1 cell continuous culture process

end. A fixed number of cells were seeded in a flask using a clean nutrient solution (including 10% BS).

I. Cell proliferation was observed by maximizing the role of self-cytokine secreted by cells in the solution by adding an appropriate amount of clean nutrient solution (including 10% BS) according to the daily culture conditions. When the cells at the bottom of the flask proliferated by 80% confluent growth, cell recovery and cytokine recovery were performed.

All. Cytokine solution recovery-Nutrients used in culture were collected separately, centrifuged (4000 rpm, 5 minutes, 3 times), and then stored refrigerated or frozen.

la. Cell recovery-After washing the stem cells attached to the bottom of the flask with PBS, put 10 to 20 ml of trypsin solution (use an appropriate amount depending on the number of cells) into the flask, and treat the cells for 3 minutes in an incubator at 38°C. When it was confirmed, the bottom of the flask was washed with Blank α-MEM to stop the reaction by treating BS and collecting the cells once more.

la. After adding blank α-MEM to the cell solution in which the trypsin reaction was stopped, centrifugation (3500 rpm, 5 minutes) to remove the supernatant, obtain a cell pellet and dissolve in a certain amount of nutrients, count the number of cells, and then collect a fixed number of cells. A clean nutrient solution (including 10% BS) was used to seed the flask and cultured continuously.

Example 2. P2 ~ P3 continuous culture and Cytokine production process

2.1 Passage 2 Cytokine Solution Production-Cell Continuous Culture Process

end. Cells cultured up to Passage 1 by attaching to the flask were subjected to Trypsin treatment, counted with a predetermined number of cells with a cell counter, and sequentially seeded with P2 cells in the flask using a clean nutrient solution (including 10% BS).

I. Cell proliferation was observed by maximizing the role of self-cytokine secreted by cells in the solution by adding an appropriate amount of clean nutrient solution (including 10% BS) according to the daily culture conditions.

All. When the cells at the bottom of the flask proliferated with 100% confluent growth (to be fully filled), about 0.5 ml of the culture solution was sampled from Day 6 to analyze the contents of TGF-β and VEGF. After the clean nutrient solution was filled with 160 ml, the cytokine and cells were recovered when the culture medium TGF-β1.5ppb / VEGF: 10ppb or more (about D15 days) while performing maturing to increase the cytokine content without adding additional nutrients.

la. At an appropriate time point when the cytokine secretion amount reached the desired target, the culture solution was separated and collected, and the centrifugation process at 4000 rpm for 5 minutes was repeated twice, and then the cytokine solution was recovered. After packing the cytokine solution in a suitable container in a sterile environment, it was frozen and refrigerated, and stored until the time it was supplied to the patient.

hemp. Cytokine solution recovery- Nutrients used in culture were collected separately, centrifuged (4000 rpm, 5 minutes, 2 times), and then stored refrigerated or frozen. Depending on the number of flasks and cells to be continuously seeded, only some of the cells in the flask were recovered, and the cells in the remaining flasks were stored aseptically after purification for continuous flask use.

bar. Cell Recovery-Stem cells attached to the bottom of the flask are washed with PBS, and then 10 to 20 ml of trypsin solution (use an appropriate amount depending on the number of cells) is added to the flask and treated in a 38°C incubator for 3 minutes. When confirmed, the reaction was stopped by treatment with BS. The bottom of the flask was washed with Blank α-MEM to collect the cells once more.

four. After adding blank α-MEM to the cell solution in which the trypsin reaction was stopped, centrifugation (3500 rpm, 5 minutes) to remove the supernatant, obtain a cell pellet, dissolve in a certain amount of nutrients, count the number of cells, and then collect a fixed number of cells. A clean nutrient solution (including 10% BS) was used to seed the flask and cultured continuously. When the cells were recovered, the collagen mass was recovered and stored frozen, and non-freezing production was performed, but in some cases, some cells were frozen.

Example 3. Culture and cytokine production process after freezing and thawing

3.1 Cell freezing process

end. After seeding to the next passage, the remaining cells (or all cells) were collected in a conical tube and centrifuged (1500 rpm, 5 minutes), and the supernatant was removed.

I. For washing the cells, PBS was added to release the cell pellet, followed by centrifugation (1500 rpm, 5 minutes).

All. After removing the supernatant after centrifugation, the cell pellet was released with BS. At this time, the amount of BS was about half of the amount of solution required for one frozen vial, and BS was added by calculating according to the number of vial to be frozen.

la. After uniformly mixing the cells with BS, 0.5ml of cell solution was added to the vial.

hemp. Excluding the amount of BS previously added, prepare the necessary cell freezing solution (BS: DMSO = 4: 1) and mix uniformly, and 0.5ml each so that the seed solution per vial becomes 1ml with 5 million cells. It was put in a frozen vial (cell solution: cell freezing solution = 1: 1).

-2.5 million were put into each frozen vial with a total solution of 0.5ml / BS & DMSO 0.25ml.

-7.5 million were added to each frozen vial with a total solution of 1.5ml / BS & DMSO 0.75ml.

bar. 1 ml of the cell freezing solution in which the cells were uniformly mixed was put into a freezing vial, and quickly put in a freezing container containing isopropanol and placed in a cryogenic freezer.

four. The next day, the cell freezing vial in the cryogenic freezer was transferred to a liquid nitrogen tank and stored.

3.2 Thawed cell culture process

end. The cell freezing vial was taken out from the liquid nitrogen tank and quickly melted for 10 minutes in a 38°C incubator.

I. The thawed cell solution was removed with a pipette, diluted in 3 ml of PBS, and the cells buried in the vial were also recovered using 1 ml of PBS, followed by counting.

All. After centrifugation (1500 rpm, 5 minutes), the supernatant was removed, cells were released in a clean nutrient solution (including 10% BS), and seeded in a flask.

la. The next day, nutrients are replaced with a clean nutrient solution (including 10% BS), and a clean nutrient solution (including 10% BS) is added according to the daily culture conditions to observe cell proliferation that takes full advantage of the role of self-cytokine secreted by cells in the solution. I did.

hemp. When the cells at the bottom of the flask proliferate confluently (filled), cytokines and cell pellets are obtained by the same method as the continuous culture cell recovery method, and then a predetermined number of cells are seeded into the flask using a clean nutrient solution (including 10% BS). It was cultured continuously.

3.3 Self-cytokine after thawing cell culture| Production and recovery

end. According to the thawed cell culture method, the cells in culture are added with an appropriate amount of clean nutrient solution (including 10% BS) according to the daily culture conditions, and the cells in the solution secrete the self cytokine. Cell proliferation was observed to make the most of its role.

I. After culturing for a certain period of time, the amount of secretion of cytokines was confirmed while culturing was performed.

All. At an appropriate time point when the cytokine secretion amount reached the desired target, the culture solution was separated and collected, and the centrifugation process at 4000 rpm for 5 minutes was repeated twice, and then the cytokine solution was recovered.

la. Self-cytokine solution was packed in a suitable container in a sterile environment, then frozen, and stored frozen until the time it was supplied to the patient.

Example 4. Cytokine solution concentration

1. Aseptic concentration process

end. In the BSC, a warmer, thermometer, scale, time switch, dish for concentration, and cytokine solution to be concentrated were prepared (* dish for concentration: sanitized by hot water disinfection).

I. After the cytokine solution was slowly poured into the concentrated dish (decanting) and sampling was performed'before concentration', the starting weight was checked.

All. Place the heater facing the inside of the BSC and turn on the power (connect the time switch and the heater to adjust the heater ON/OFF every 15 minutes). The concentrated dish was placed next to the warmer, and the temperature was checked by placing a thermometer in the BSC (temperature range in BSC: 33 to 36°C).

la. 1) Early stage: It was checked from time to time whether the temperature was well controlled (the location of the heater and the time switch were adjusted).

2) Mid: Weight and temperature were measured and recorded at regular intervals.

3) Second half: The weight was checked from time to time.

hemp. Immediately before the start of recovery, the weight of the concentrated cytokine solution was checked, and'after concentration' sampling was performed. The weight of the container to be recovered was checked and adjusted to zero, and the concentrated cytokine solution was slowly poured into the container to be recovered (decanting), and the weight was checked (final loss is determined by the difference between the weight before the start of recovery and the weight finally recovered). ).

The above description of the present invention is for illustrative purposes only, and those of ordinary skill in the art to which the present invention pertains will be able to understand that other specific forms can be easily modified without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative and non-limiting in all respects. For example, each component described as a single type may be implemented in a distributed manner, and similarly, components described as being distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the claims to be described later, and all changes or modified forms derived from the meaning and scope of the claims and their equivalent concepts should be construed as being included in the scope of the present invention.

1: Cytokine production system module
10: Clean booth
20: Biosafety test loan
30: incubator unit
40: flask part
50: Main PC part
60: communication department

Claims (14)

Separating the cells from the adipose tissue;
Maturing the isolated cells in a clean nutrient solution; And
Recovering cytokine fluid from the matured cells;
Containing, high-concentration cytokine-containing anti-aging essence production method through maturing.
The method of claim 1,
The maturing step,
First maturing the isolated cells in a clean nutrient solution;
Secondary maturing the primary matured cells in a clean nutrient solution; And
Tertiary maturing without providing a clean nutrient when the secondary matured cells are confluent growth of 60 to 90% in a state where a clean nutrient solution is provided;
Characterized in that it comprises a, high-concentration cytokine-containing anti-aging essence production method through maturing.
The method of claim 1,
The clean nutrient solution contains 10% bovine serum, but does not contain antibiotics and phenol red. A method for producing anti-aging essence containing high-concentration cytokines through maturing.
The method of claim 2,
In the step of providing a clean nutrient solution to the secondary matured cells, the clean nutrient solution is provided for 2 to 5 days.
The method of claim 1,
The step of recovering the cytokine solution,
Analyzing the cytokine content in the clean nutrient solution of the matured cells; And
Recovering a cytokine solution according to the analyzed cytokine content;
Characterized in that it comprises a, high-concentration cytokine-containing anti-aging essence production method through maturing.
The method of claim 5,
The step of analyzing the cytokine content comprises measuring the concentrations of transforming growth factor beta (TGF-β) and vascular endothelial growth factor (VEGF) in the clean nutrient solution of the matured cells. Method for producing anti-aging essence containing high-concentration cytokines.
The method of claim 5,
The step of recovering the cytokine solution comprises recovering the cytokine solution when the TGF-β concentration in the clean nutrient solution of the matured cells is 1 to 2 ppb and the VEGF concentration is 5 to 20 ppb. A method of producing anti-aging essence containing high-concentration cytokines through chewing.
The method of claim 1,
The step of maturing the cells, characterized in that made for 5 to 20 days, high concentration cytokine-containing anti-aging essence production method through maturing.
As a cytokine production system module applied to the method of producing anti-aging essence containing high-concentration cytokines through maturing according to claim 1,
A first internal space is formed inside, a first filter and a first blower are formed on one surface, and a clean booth part through which external air is introduced into the interior through the first filter,
It is installed in the first internal space, a second internal space is formed therein, and a second filter and a second blower are formed on one surface, so that the air in the first internal space is introduced into the interior through the second filter. With the Department of Biological Safety Cabinet,
An incubator unit installed in the first inner space and having a cell maturing space,
At least one flask (Flask) part located in each of the cell maturing spaces and receiving cells and nutrients to maturate cells,
A microscope unit installed in the cell maturing space and for magnifying and observing cells maturing in the flask unit;
An imaging unit installed in the microscope unit and configured to capture cells grown in the flask unit;
A main PC unit installed in the first internal space, receiving and transmitting image data captured by the image capturing unit in real time, and controlling the image capturing unit according to input user command data;
A communication unit connected to the main PC unit, transmitting the video data to a host PC unit located outside the clean booth unit, and transmitting command data received from the host PC unit to the main PC unit,
Cytokine production system module, characterized in that it is possible to observe the maturation state of the cells in the incubator unit in real time from the outside of the clean booth unit by the communication unit.
The method of claim 9,
A first counter unit installed in the first inner space to counter particles located in the first inner space,
A second counter unit installed in the second inner space to counter particles located in the second inner space,
The cytokine production system module, further comprising a control unit for controlling the first blower and the second blower according to the number of particles located in the first inner space and the second inner space.
The method of claim 10,
When the number of particles located in the first inner space is greater than or equal to a preset first value, the control unit rotates the first blower at a first speed, and rotates the second blower at a second speed,
Saito, characterized in that when the number of particles located in the first inner space is less than a preset first value, the control unit rotates the first blower at a second speed, and rotates the second blower at a first speed. Cain production system module.
The method of claim 9,
The microscope unit,
A fixture to which the flask unit is detachably coupled,
A stage to which the fixtures are detachably coupled,
An optical system including a lens and guiding the image of cells accommodated in the flask portion located on the stage,
And a lens driving means for driving the lens to perform focus alignment and aberration correction on the cells,
The flask unit, characterized in that the movement and rotation at a predetermined position on the stage by the fixture is suppressed, cytokine production system module.
The method of claim 12,
The stage has a first through hole formed with a protrusion,
The flask part,
A plate-shaped plate in which a second through hole communicating with the first through hole is formed,
A plurality of first fixing members formed on one surface of the plate and formed to correspond to the outer circumferential surface of the flask part to fix the flask part,
A second fixing member formed on the other surface of the plate, inserted into the through hole of the stage, and having a groove corresponding to the protrusion,
When a projection is inserted into the groove of the second fixing member, the plate is characterized in that the rotation and movement is suppressed, cytokine production system module.
The method of claim 9,
Cytokine production system module, characterized in that the clean booth unit is detachably coupled and further comprises a moving unit for moving the clean booth unit.

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