KR20150049041A - Method for culturing adiposed derived stem cell - Google Patents

Abstract

The present invention relates to a culturing method for adipose-derived stem cells separated from human body. According to the present invention, the culturing method for adipose-derived stem cells separated from adipose tissue of the human body is a culturing method by completing a primary culturing step and a subculturing step under hypoxia conditions of the low density of oxygen than atmospheric air conditions. The primary culturing step is configured to seed and culture stem cells in first culture media comprising aqueous nutrient media containing glucose, blood plasma separated from the human body, platelet-rich plasma irradiated with visible light rays, and a first additive, and the subculturing step is configured to seed and culture stem cells after completing the primary culturing step in second culture media comprising nutrient media, bovine serum mixed by a ratio of 1 to 5 wt% of the nutrient media, and a second additive.

Description

[0001] The present invention relates to a method for culturing adipose derived stem cells,

The present invention relates to a method for culturing stem cells, and more particularly, to a method for culturing adipose derived stem cells for differentiation and proliferation of stem cells extracted from adipose tissue isolated from a human body as one of adult stem cells through primary culture and subculture will be.

Stem cells are undifferentiated cells that exist between differentiated cells in tissues or organs, and they have self-renewal ability (Self Renewal) and differentiation to specific tissues in the body It is a universal cell.

Adult stem cells are hematopoietic stem cells, mesenchymal stem cells, neuroblastoma, and epithelial cells. In the case of hematopoietic stem cells, hematopoietic cells and lymphocytes can be produced as stem cells, which are useful for treating immune system diseases including hematologic malignancies. Cells differentiate into bone, cartilage, fat, and fibrous tissue and are involved in the recovery of each tissue when damaged. However, these adult stem cells are present in small amounts in individual tissues and may remain silent for many years without splitting or multiplying. In other words, stem cells refer to undifferentiated cells that have this differentiation ability but do not yet undergo differentiation. In this undifferentiated state, stem cells can differentiate into various tissue cells when given appropriate conditions.

Meanwhile, stem cells can be divided into embryonic stem cells and adult stem cells. In other words, it can be divided into two types: adult stem cells existing in various kinds of tissues in our body since birth, and embryonic stem cells derived from embryonic stem cells, which are the beginning of human life. Adult stem cells refer to cells constituting specific tissues. For example, bone marrow cells are cells that are destined to differentiate into hematopoietic cells, skin stem cells to skin, and neural stem cells to neurons.

The term "stem cell" refers to a stem cell obtained from an adult other than embryonic stem cells, including stem cells originating from the base of blood and tissues including fat, bone marrow, umbilical cord blood and peripheral blood.

In particular, fat-derived stem cells are a type of adult stem cells that are easier to harvest than stem cells or cord blood stem cells and can be mass-cultured, but have a high content of stem cells. Therefore, fat-derived stem cells are used for various purposes Research is actively underway.

However, unlike embryonic stem cells, adipose-derived stem cells have a very short life span when cultured in vitro, and are only retained for a short period of time. The limited lifespan of these stem cells is a major constraint on the use of such cells.

For example, in cell therapy or regenerative medicine, in order to obtain the minimum stem cells necessary for the treatment, at least several subcultures must be carried out. During this subculture, the cells are aged and deformed and are no longer suitable for the concept of treatment .

In addition, since the utilization of the stem cells is greatly reduced when the stem cell cultures are grown above the standard size, there is a quantitative problem of how fast the stem cells can be proliferated in the stem cell culture, The quality problem of whether or not it can be restricted within a certain range is becoming an important task.

In order to solve these problems, various prior studies have been conducted. Among them, a method of culturing stem cells under hypoxia conditions has been studied. We also studied the effect of hypoxic conditions on stem cell culture. In this study, stem cells were cultured under the normal condition and hypoxic condition for two cells labeled with JSH and VAN, respectively. The general conditions are 5% of carbon dioxide and 21% of oxygen such as atmospheric conditions. Hypoxic conditions include 5% of carbon dioxide and 1% of oxygen. Experiments were carried out at 37 [deg.] C in an incubator, the results of which are shown in Figures 1 and 2.

1 is inde a result targeting JSH cells, were the initial number of cells starting at 3.75 × 10 ⁵ dog, general condition, whereas 8.40 × 10 ⁵ pieces increased, low-oxygen conditions, 1.60 × 10 increased to ^six. Fig. 2 shows the results of experiments on VAN cells. Initial cell counts started at 3.75 × 10 ⁵ , which increased to 1.02 × 10 ⁶ under normal conditions, but increased to 1.45 × 10 ⁶ under hypoxic conditions.

FIG. 3 is a photomicrograph of a cell that has undergone 4 passages in the above experiment, confirming that cells cultured under hypoxia conditions are smaller in size than cells cultured under normoxia.

These results suggest that the cell size is kept small and cell growth rate is increased 1.4 ~ 1.9 times under hypoxic conditions. However, the problem that the rate of cell culture does not increase more than 2 times in the hypoxic condition compared to the general condition remains a problem to be solved.

The present invention provides a method for culturing an adipose-derived stem cell capable of maintaining a small size of stem cells while rapidly growing stem cells using a culture solution containing various additives under hypoxic condition It has its purpose.

In order to accomplish the above object, the present invention provides a method for culturing adipose stem cells, which comprises culturing adipose-derived stem cells isolated from adipose tissue of a human under hypoxia conditions having lower oxygen content than atmospheric conditions, Wherein the first culture medium comprises a liquid nutrient medium containing glucose, a plasma separated from a human body and a platet rich plasma (PRP) irradiating visible light, Wherein the subculturing step comprises culturing the nutrient medium with the bovine serum mixed with the nutrient medium at a ratio of 1 to 5 wt% based on the weight of the nutrient medium, and culturing the second medium with the second Characterized in that a stem cell obtained through the primary culture step is inoculated into a second culture solution containing an additive and cultured.

According to the present invention, the first additive comprises transferrin, selenium, triiodothyronine (T3), and ornithine. The first additive is mixed with the first culture medium in a range of 0.2 to 0.6% by weight based on the weight of the nutrient medium.

In one embodiment of the present invention, it is preferable that EGF (Epidermal Growth Factor) is added to the first culture medium within 48 to 72 hours after the start of the primary culture.

In the present invention, the platelet-rich plasma irradiated with the visible light is mixed with the first culture liquid at a ratio of 1 to 8% by weight based on the weight of the nutrient medium.

In one embodiment of the present invention, the second culture medium comprises a reusing culture medium which has been used as a culture medium for culturing stem cells, and the reusing culture medium is present in an amount of 1 to 10 By weight in the second culture liquid.

The reusable culture medium contains glucose and an additive. Optionally, bovine serum may be mixed at a ratio of 1 to 5% by weight based on the weight of the basic culture medium.

The second additive is mixed with the second culture medium in a ratio of 0.3 to 1.7% by weight based on the weight of the nutrient medium. The second additive includes insulin, transferrin and selenium, and further contains ethanolamine and vitamin E. can do.

In one embodiment of the present invention, it is preferable that EGF (Epidermal Growth Factor) is added to the second culture solution within 48 to 72 hours after starting the subculture.

In the present invention, the plasma and the platelet-rich plasma are separated from the human body in the primary culture to perform pretreatment for irradiating visible light, and a first culture medium is prepared by mixing a first additive having a unique composition and a blending ratio. Cells are inoculated in a first culture medium and cultured under hypoxic conditions, thereby increasing the growth rate of the cells and keeping the size of the cells small.

In the subculture, a second culture medium is prepared by mixing the second additive while maintaining the minimum amount of bovine serum with the risk, and preferably by mixing the reusing medium, the culture speed of the cell is increased and the size of the cell is maintained .

That is, when the primary culture and the subculture are continuously performed using the culture method according to the present invention, the growth rate of the cells can be increased to at least 4 times as compared with the general culture method, and the stem cells can be cultured in a large amount. It is anticipated that this will provide an opportunity for industrial use of stem cells. Furthermore, since the size of the cells can be kept small, it is expected that the stem cells can be used more safely and effectively in the case of using them for therapeutic purposes or cosmetic purposes.

FIGS. 1 and 2 are tables showing the results of experiments on the effect of hypoxic conditions on stem cell culture.
FIG. 3 is a photomicrograph of a stem cell subcultured through the experiment shown in FIGS. 1 and 2. FIG.
FIG. 4 is a schematic flow chart of a method for culturing adipose-derived stem cells according to an embodiment of the present invention.
FIG. 5 is a composition table of the culture medium in which the effect of visible light on plasma and platelet-rich plasma when the cell culture was tested (hereinafter referred to as "visible light effect test").
6 is a table showing the results of the visible light effect test.
FIG. 7 and FIG. 8 are electron micrographs showing the size of the cultured cells according to whether or not they were irradiated with visible light.
9 is a photomicrograph showing the result of a culture experiment according to the concentration condition of the additive.
10 is a composition table of mediums 1 to 3 used in the primary culture experiment.
FIG. 11 is a table showing the conditions of the first culture experiment using the media shown in FIG. 10; FIG.
12 is a table showing the results of the primary culture experiment.
FIGS. 13 and 14 are graphs showing cell culture results of JOO cells and VAN cells, respectively.
15 is a table showing the size of primary cultured cells.
FIGS. 16 and 17 are tables and graphs showing the results of measurement of TGF in a stem cell culture after completion of stem cell culture. FIG.
18 is a composition table of Medium 1 to 4 used in the subculture experiment.
FIG. 19 is a table showing experimental conditions for subculturing using the media shown in FIG. 18; FIG.
20 is a table showing the results of subculture experiments using the media shown in Fig.
21 and 22 are graphs showing cell culture results of TIEN cells and NHJ cells, respectively.
23 is a table showing the size of subcultured cells.
24 is a composition table of mediums 1 to 5 used in the subculture experiment using the second culture medium containing the reusing medium.
25 is a table showing the experimental conditions for subculturing using the media shown in Fig.
26 is a table showing the results of subculture experiments using the media shown in Fig.
FIGS. 27 and 28 are graphs showing cell culture results of JOO cells and VAN cells, respectively.
29 is a table showing the size of subcultured cells.
FIG. 30 is a table showing experimental conditions for observing the influence of the addition timing of EGF.
31 is a graph showing the experimental result according to FIG.

The present invention relates to a method for culturing adipose derived stem cells, particularly adipose derived stem cells isolated from adipose tissue of a human body. However, the subject of the present invention is not limited to adipose-derived stem cells isolated from the human body, but also includes stem cells isolated from adipose tissue of other animals.

Hereinafter, a method for culturing adipose stem cells according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 4 is a schematic flow chart of a method for culturing adipose-derived stem cells according to an embodiment of the present invention.

Referring to FIG. 4, the method for culturing adipose stem cells according to an embodiment of the present invention is divided into two stages: primary culture and subculture. Of course, in the case of subculture, it is possible to perform successively in the same manner repeatedly.

The primary culture period is an important time for cells to separate from tissues and adapt to the outside of the body. Since the cells are small and weak, the environment in which the cells can be stably cultured is very important.

In the present invention, stem cells are inoculated into a flask containing a first culture medium in an incubator under hypoxic conditions to cultivate stem cells.

The hypoxia condition refers to an environmental condition having an oxygen content lower than the concentration of oxygen in the atmosphere (approximately 21%), and the same applies to the present invention in the present invention. However, from a more positive point of view, the hypoxic condition used in the present invention means that the oxygen concentration in the air in the incubator is about 1 to 5%. The incubator also maintains a temperature of approximately 37 ° C and maintains a concentration of carbon dioxide of approximately 5%.

The first culture solution is for supplying the nutrients necessary for stably growing the stem cells. In the present invention, an optimal first culture solution combination is prepared in order to increase the culture speed of the stem cells and to keep the cell size small.

The first culture medium includes a liquid nutrient medium containing glucose, plasma separated from the human body, and platelet rich plasma (PRP) irradiating visible light and a first additive.

In the present invention, DMEM (Dulbecco's Modified Eagle's Media), which is widely used for cell culture, was used as a liquid nutrient medium containing glucose.

According to the present invention, the two most important factors of the first culture used in the primary culture are plasma and platelet rich plasma (PRP) separated from the human body, and plasma and platelet rich plasma Rich plasma is irradiated beforehand with visible light.

In general, the culture medium for the proliferation of animal cells contains bovine serum. Because bovine serum can not exclude the effects of bovine diseases or unexpected immune responses, the plasma of the person (especially the patient) and platelet-rich plasma By using the most similar conditions in the body, the primary culture can be performed safely and stably.

Plasma and Platelet Rich Plasma is obtained by centrifuging human blood. In the present invention, blood is centrifuged to separate plasma and platelet-rich plasma from each other. Platelets contain large amounts of growth factors and cytokines that are necessary for cell proliferation.

Plasma in the first culture medium is mixed at a ratio of 10 to 20% by weight based on the weight of the nutrient medium, and the platelet-rich plasma is mixed at a ratio of 1 to 8% by weight based on the weight of the nutrient medium.

Particularly, in the present invention, plasma and platelet-rich plasma isolated from human blood are not used as they are in the culture medium, but are characterized in that pretreatment is performed to irradiate plasma and platelet-rich plasma with visible light.

We investigated the effect of visible light from an LED light source on plasma and platelet-rich plasma to affect cell growth.

FIG. 5 is a composition table of the culture medium in which the influence of visible light on plasma and platelet-rich plasma was examined for the effect on cell culture (hereinafter referred to as "visible light effect test"). FIG. This is the table shown.

5, the experiment was divided into three groups. In Group 1 to Group 3, nutrient medium containing glucose was commonly used, bovine serum was added to Group 1, and blood Platelet rich plasma was added to Group 3 and platelet rich plasma irradiated with visible light was added.

The experimental results in FIG. 6 are the results of two experiments (case 1 and case 2), in which the results of two experiments were the same in group 1 with bovine serum and in group 2 and 3 the results of two experiments were slightly different . Importantly, the cells were found to grow more rapidly in Group 2 and Group 3, supplemented with human blood plasma and platelet rich plasma, compared to Group 1 with bovine serum. However, the results of cell culture of Group 2 and Group 3 showed no significant difference in cell culture rate depending on the irradiation of visible light.

However, it was confirmed that there was a large difference in the size of the cultured cells depending on whether or not the irradiation was performed with visible light. FIG. 7 and FIG. 8 are electron micrographs showing the size of the cultured cells according to whether or not they were irradiated with visible light.

FIG. 7 is a photograph of cells cultured using platelet-rich plasma irradiated with visible light, and FIG. 8 shows a case where no visible light is irradiated. In the electron microscope photograph of FIG. 7, the average size of the cells was 14 .mu.m, whereas in the photograph of FIG. 8, the average size of the cells was 19.4 .mu.m.

The results of the experiments show that culturing the cells using platelet-rich plasma and platelet-derived plasma from human blood greatly increases the incubation rate of cells compared with the case of using bovine serum, and irradiating the platelet-rich plasma with visible light It was confirmed that the size of the cultured cells can be kept small.

As described above, the platelet-rich plasma obtained from the human body and the platelet-rich plasma irradiated with visible light under the hypoxic conditions showed a certain degree of progression in terms of cell size and speed, but the first additive was used .

However, the first additive induces the cell to be stably adaptable even in a small amount, rather than inducing too much function using a high concentration and a high dose.

In the present embodiment, the first additive is mixed with the first culture medium in a range of 0.2 to 0.6 wt.% Based on the weight of the nutrient medium, and includes transferrin, selenium, triiodothyronine (T3), and ornithine.

Transferrin promotes the storage and transport of extracellular matrix iron ions and plays a role as an important antioxidant. Control the amount of iron in the cell environment. If the amount of iron is insufficient, cell division may be inhibited and killed. Thus, the role of transferrin in primary culture prevents cell death and helps to facilitate cell division.

Triiodothyronine (T3, 3,3 ', 5-Triiodo-L-thyronine sodium salt) enhances protein synthesis and regulates protein expression. Essential to the growth of cells in primary culture is the stable adaptation of the cells. For this, the expression of the protein in the cell membrane is essential. T3 is necessary as a kind of stimulant because the role of protein in the cell membrane attached to the culture vessel is important and the transport protein that transports minerals and hormones is also important.

Ornithine (L-Ornithine) is an amino acid that helps in the synthesis of polyamines (Polyamine). Polyamines are essential substances involved in the differentiation and growth of almost all cells and play a pivotal role in cell development. By adding L-Ornithine in the primary culture, the synthesis of polyamines was promoted and the stable adaptation and growth of young stem cells were observed.

Selenium is required for normal cell growth in vivo. It has an antioxidant function that protects cells by reducing peroxides. It also promotes the activity of glutathione and is considered to be one of the essential nutrients. In addition, selenium is incorporated into selenoprotein P (SeP) and SeP carries the initial selenium and carries the protein to the cell. It appears outside the cell membrane and in association with the extracellular matrix, and protects the cell membrane from the peroxidation of the fat. SeP is present in human plasma at 50% of total selenium. The role of selenium is important because cell membrane protection is important for cell stabilization in primary culture and protein transport is also important.

In the present invention, human plasma was used instead of bovine serum in the primary culture, and it is expected that the SeP contained therein also activates the incorporation of selenium into the enzyme, thereby further promoting cell membrane protection of cells cultured at the early stage.

As described above, in the present embodiment, the first additive is mixed with the first culture solution in the range of 0.2 to 0.6% by weight based on the weight of the nutrient medium. If the content is less than the above range, the desired effect as an additive can not be obtained, Even if it exceeds this range, cell growth is inhibited. A number of experiments were conducted to find the appropriate concentration of the additive of the present inventor, and the concentration in the above range was found.

9 is a photomicrograph showing the result of a culture experiment according to the concentration condition of the additive. 5,000 cells per culture container area were inoculated and photographed at 4 days. (a) The photograph shows the case where the first additive is blended within the range according to the present invention, and (b) the case where the additive having the concentration of four times the blend is added as compared with (a). As shown in the photograph, it was confirmed that the number of cells (b) was significantly smaller than that of (a).

As described above, a culture medium containing a nutrient medium containing glucose, a plasma separated from a human body and irradiated with visible light, a platelet-rich plasma, and a first additive is placed in a culture container, Stem cells are inoculated. Then, the incubation container is placed in an incubator under hypoxic conditions and the incubation is continued.

EGF (Epidermal Growth Factor), known as a growth factor promoting the production of cells, is added to the first culture medium 2 days after the start of culture, that is, within 48 to 72 hours. According to our experiments, the cell growth rate did not increase even when EGF alone or FGF was added from the beginning of culture, but when EGF was added 48 hours after cell culture, cell growth was most effectively promoted .

We studied the effects of primary cultures on hypoxic conditions in primary cultures.

In the primary culture experiment, three mediums of media 1 to 3 were used. The compositions of medium 1 to medium 3 are shown in the table of FIG. 10, DMEM was commonly used for medium 1 to medium 3, while bovine serum (FBS) was used for medium 1, which is a comparative example, in medium 2 and medium 3 based on medium according to the present invention. Plasma separated from the human body and platelet - rich plasma were used instead of serum. Plasma and platelet - rich plasma were pretreated with visible light of green, blue, red and yellow wavelengths for 15 minutes using an LED light source. Medium 2 and medium 3 differ in whether ornithine and selenium are added.

In the primary culture experiment, the reproducibility was observed using two cells labeled JOO and VAN. Plasma (and platelet rich plasma) isolated from three blood plots labeled A, B and C was used. Experiments were carried out using different media under hypoxic conditions and normal conditions. A table for the conditions of the primary culture experiment is shown in Fig.

11, medium 1 supplemented with bovine serum was used in all of the comparative groups (group 1), and medium 2 and medium 3, which are the culture media according to the present invention, were used in the control groups 1 to 3 (groups 2 to 4). EGF was not added to medium 1, and EGF was added to medium 2 and medium 3.

Each group was inoculated with cells in a T75 flask at 10,000 cells / cm ² per area. When the cell concentration in the flask grew to about 80 to 90%, the cells were detached from the flask to confirm the number of cells, survival rate, and cell shape. The number of cells and the survival rate were measured using 'Luna' of 'Logosbiosystems'.

The results of the primary culture experiments are shown in the drawings. The table in Fig. 12 shows the results for the incubation rate, and is visualized in the graphs of Figs. 13 and 14. Fig. 15 is a table showing the sizes of the cultured cells.

The results of the experiment showed that the cell growth rate was faster than that of the conventional culture method and that the cell growth rate was faster than that of the conventional culturing method. In particular, when the cells were cultured under hypoxic condition in Group 4 using Medium 3 containing 0.5 ng / ml of T3, the growth rate was 2 to 3 times faster than that of the normal culture.

Especially, in JOO case, it can be seen that the cells do not show much difference from the initial cultured cells (plated cells) under the normal culture conditions. Human cells begin to be primary cultures and take about 7 days to reach 80-90% concentration in cell culture vessels. In case of JOO, the concentration after 6 days does not reach 50%. In this case, it takes considerable time to fill cells in the cell culture container. However, using the hypoxic conditions and culture medium developed by our team, it is possible to induce rapid growth as in the case of normal cells, even in such difficult growing cells.

For reference, hypoxic conditions using medium 1 were not marked differently in the result table since they were not substantially different from general conditions using medium 1.

As described above, when the primary culture is performed using the culture method according to the present invention, the cell growth rate is much faster than the conventional culture method (using the normal oxygen condition and the normal culture solution), and the cell size can be kept small have.

On the other hand, as described above, when the primary culture is completed, the stem cells are separated from the flask, transferred to another flask, and subcultured. The subculture is also carried out in an incubator under hypoxic conditions as in the case of the primary culture, but the addition of the second culture solution to the flask is different from that in the first culture solution in the primary culture.

During the subculture, the cells have been adapted from outside the body through the primary culture. Since only stable and healthy cells are transferred to the new culture container after the initial culture, the focus is on increasing the growth rate while maintaining a stable culture environment in the subculture. Thus, in the present invention, a second culture medium having a composition suitable for cell growth was derived.

The second culture medium used in the subculture comprises the same nutrient medium as used in the primary culture and the bovine serum and the second additive mixed at a ratio of 1 to 5 wt% based on the nutrient medium weight. Although it is optional, it is preferable that the reusing culture medium is mixed with the second culture solution.

Bovine serum (FBS) is used because it can not obtain a sufficient amount of blood to be suitable for the cultivation of the expanded cells in the subculture although the plasma separated from the human body is used because of the risk of the bovine serum in the primary culture. However, bovine serum was sought to reduce the use of bovine serum because of the high cost, the risk from cow disease, and the disadvantage of varying activity per production LOT. Therefore, in the present invention, the bovine serum is blended only in the range of 1 to 5 wt% based on the weight of the nutrient medium.

Many nutrients deficient by reducing the use of bovine serum were supplemented by the effective use of the second additive, and at the same time, by using hypoxic conditions, the stem cells were able to grow quickly and stably.

Because the most disadvantage of using commercially available stem cells is the high price, our researchers have been able to grow large numbers of stem cells in a short time with low serum, We propose a method to compensate for the drawbacks.

The second additive to be added to the second culture medium used in the present invention is mixed with the second culture medium in a ratio of 0.3 to 1.7% by weight based on the weight of the nutrient medium and includes lipid, insulin, transferrin, selenium, ethanolamine and vitamin E do. In addition, EGF is added to the second culture medium within 48 to 72 hours after starting the subculture. The reason why EGF is added after 2 days from the start of culture is the same as that described in the primary culture.

Lipids are responsible for biomembrane synthesis. It is an energy source of living cells and is an important component of the structure. By supplementing the deficient lipid component by reducing the use of bovine serum, the cells can be grown using an abundant lipid during in vitro culture.

Insulin promotes the uptake of glucose and amino acids into the cells, which can promote cell proliferation. In the culture medium lacking bovine serum, the addition of biochemical activity is necessary because of the rapid decrease of biochemical activity.

Transferrin promotes the growth of cells in a culture medium with reduced serum as in insulin.

Selenium induces the activity of the tumor suppressor gene p53, blocking the process of inducing or killing the abnormal cells that can develop into cancer cells themselves. It also promotes the growth of cells by promoting the biochemical signal of mitochondria in cells as well as enhancing antioxidative and cell membrane protection and enhancing the functional role.

Ethanolamine is a component of cell membrane phospholipids. There is a cell growth promotion effect.

Vitamin E acts as a powerful antioxidant to reduce oxidative stress and increase cell division. That is, it acts to speed up cell growth.

In addition, the second additive includes hydrocortisone, which promotes cell adhesion and proliferation. T3 promotes cell growth along with insulin and hydrocortisone in a serum-reduced culture.

Insulin, Transferrin, and Selenium are important components in the culture medium in which bovine serum is reduced. When this essential ingredient is added to the culture, cell growth is promoted in place of nutrients that are deficient from bovine serum.

The additives described above are also increased in their role by interrelation as described below.

For example, when ethanolamine is present in the presence of only one insulin, it is further activated that glucose is ingested intracellularly. This is because ethanolamine facilitates the transport of glucose.

Selenium also acts as an antioxidant and binds with Vitamin E to further promote the role of antioxidants. In other words, it effectively reduces the stress of the cell and helps the growth of the cell.

Lipids and ethanolamine also interact. That is, the two OH groups of glycerol contain the basic structure of a phospholipid in which a fatty acid and phosphoric acid are bonded, and a phosphatidyl ethanolamine in which a phosphate group is bonded to ethanolamine. These are important parts of cell membranes and mitochondria membranes.

On the other hand, it is preferable to mix a reusing culture medium with the second culture solution used in the present invention. The reusing medium means a culture medium used for culturing the stem cells, that is, a culture medium already used, which can be concentrated and added to the second culture medium.

The culture medium used for culturing the stem cells must be treated as waste, and the stem cell culture fluid contains a large amount of components useful for cell culture, and can be directly or after being concentrated and reused in the form of an additive.

That is, when the culture medium containing the cytokines and growth factors naturally secreted by the stem cells in the course of culturing the stem cells is concentrated without being discarded and used again as a culture medium additive, Stem cells could be more effective for growth.

It is believed that the number of growth factors and proteins secreted by stem cells is more than a dozen. In particular, they are safe and effective because they are natural and self-sustaining factors. The reusable culture medium is added in a range of 1 to 10% by weight based on the weight of the nutrient medium contained in the second culture medium. The reusable culture medium may be a form for reusing the second culture medium according to the present invention, or may be a culture medium for use in a general culture method. For example, a basic medium containing glucose and various additives may be included, and bovine serum may also be included. Even in the case of the culture medium containing the bovine serum, the bovine serum is preferably mixed in the range of 1 to 5% by weight based on the weight of the basic medium. More preferably, the reusing culture medium is a culture solution used for culturing the cells under hypoxic conditions.

(IGFBP-1), macrophage proliferative factor (M-CSF) and its receptors, platelet-derived growth factor beta (IGFBP-β), and blood vessels in the culture medium of adipose- Endothelial cell growth factor (VEGF) is significantly increased.

FIGS. 15 and 16 are tables and graphs showing the results of measurement of TGF in a stem cell culture fluid after completion of stem cell culture. FIG. As shown in the measurement results of FIG. 15 and FIG. 16, it was confirmed that TGF which was not added to the culture was detected at the end of cell culture even under the condition of the second culture solution developed by the present inventors. That is, despite the fact that TGF was not added to the stem cell culture medium, TGF was detected at 1 to 4 ng / ml at the end of the stem cell culture. In the table of FIG. 15, Samples 1 to 7 contained general oxygen and hypoxic conditions. Sample No. 7 was cultured under hypoxic conditions. It was confirmed that TGF was contained most in the culture medium after hypoxic conditions .

We conducted experiments on the effect of subculture using a second culture medium under hypoxic conditions.

18 is a composition table of Medium 1 to 4 used in the subculture experiment.

Referring to the table of FIG. 18, medium 1 to 5 were commonly used in DMEM. Medium 1 and medium 2 were mixed with 10 to 20% bovine serum, and media 3 and 4 were mixed with low to 1 to 5%. In Medium 2 to Medium 4, the second additive according to the present invention was mixed at 0.33 to 1.62 wt% with respect to the nutrient medium.

Subculture experiments were performed using two cells labeled NHJ and TIEN. Experiments were carried out using different media under hypoxic conditions and normal conditions. A table for the subculture experiment conditions is shown in FIG. In the subculture experiment, 5000 cells per area were placed in a culture container and cultured for 4 days.

The results of the subculture experiments are shown in Figs. 20 to 22. Fig. FIG. 20 is a table showing the results of subculture experiments using the media shown in FIG. 18, and FIGS. 21 and 22 are graphs showing cell culture results of groups of TIEN cells and NHJ cells, respectively.

As a result of the experimental results, it can be seen that the cells grown in the culture medium (Group 2, 3, 4) developed by the present inventors were more proliferated under the hypoxia condition than the conventional culture method (group 1, control). Survival rate was more than 99% in all groups.

Also, referring to the table in FIG. 23, when the cell shape was compared between the normal oxygen condition (CO ₂ incubator) and the hypoxic condition (1% O ₂ ), the hypoxic condition cells had smaller cell sizes Could.

In addition, the researchers added subculture solution to the second culture medium and subcultured under hypoxic conditions.

24 is a composition table of mediums 1 to 5 used in the subculture experiment using the culture medium supplemented with the reusing medium.

Referring to the table of FIG. 24, medium 1 to 5 were commonly used DMEM, medium 1 to 3 contained 10 to 20% bovine serum, medium 4 and 5 contained bovine serum at 1 to 5% Mixed. In Medium 2 to Medium 5, the second additive according to the present invention was mixed at 0.33 to 1.62 wt% with respect to the nutrient medium. Above all, in culture mediums 2 to 4, the culture medium was mixed with the culture medium in the range of 1 to 10% by weight based on the nutrient medium weight. Medium 2 and medium 4 contained bovine serum in the medium, and medium 3 and medium 5 did not contain bovine serum. The culture medium used for culturing the stem cells was concentrated and added.

In the subculture experiment using the culture medium containing the reusing medium, the reproducibility was observed using two cells designated as JOO and VAN. Experiments were carried out using different media under hypoxic conditions and normal conditions. A table for the experimental conditions for subculture is shown in FIG.

The results of the subculture experiments are shown in FIG. 26 to FIG. FIG. 26 is a table showing the results of subculture experiments using the media shown in FIG. 24, and FIGS. 27 and 28 are graphs showing cell culture results of JOO cells and VAN cells, respectively.

The results of the experiment using the culture medium without the reusing medium showed that the cell growth was faster when the culture solution developed by the present inventors under the hypoxic condition was used, but as the bovine serum decreased to 10%, 5% and 3% Growth did not appear prominently. However, as shown in this experiment, in the hypoxic condition, the growth rate was higher as the bovine serum concentration was decreased under the condition that the culture medium was added to the culture medium. And the growth rate of cell was remarkably increased 4 times or more in both JOO and VAN cells. In other words, it is found that the growth rate of cells is increased by using only a small amount of non - economic bovine serum, and using the culture medium of reusing.

A number of cytokines secreted during cell growth, such as growth factors, are used to concentrate the culture and add high growth promoters. It is thought that stem cell growth has been promoted because growth promoting substance lacking in the culture fluid enters.

Also, as shown in the table of FIG. 29, it was confirmed that the size of the cells was also kept small when the culture medium and method according to the present invention were used, even when the culture medium was used, as compared with the case using the general culture method.

The effects of primary culture and subculture culture developed by our team are summarized below.

In the first culture, the growth rate of the cells was increased when cultured under the hypoxic condition using the first culture solution developed by the present inventor. Also, the size of the cells could be kept small.

As described above, since the subculture has a limitation in culturing using human serum, the use of bovine serum is desired, but the use of bovine serum is a problem of stability. As a result of the first experiment, when the bovine serum was supplemented with 10%, 5%, and 3% of the second culture solution developed by the present inventor, the growth rate was higher than that of the general culture method under hypoxic condition. However, the lower the concentration of serum, the greater the proliferation rate did not increase.

In addition, it was confirmed that the concentration of bovine serum increased as the concentration of leucine supplemented to the cell culture increased, and the cell proliferation rate could be increased more than 4 times as compared with the general culture method.

This result is based on the commercial use of stem cells and it will be very helpful for the stem cell culture system which has to be mass - produced for the current treatment. Furthermore, the use of low - concentration bovine serum for stem cell culture system, which should be applied to humans, is considered to be an important basis for enhancing safety.

In the meantime, the present inventors proposed a method in which EGF is added to the first culture medium and the second culture medium in the first culture and the second culture medium within 48 to 72 hours after the start of the culture.

FIG. 30 is a table showing experimental conditions for observing the influence of the addition timing of EGF, and FIG. 31 is a graph showing experimental results according to FIG.

30, all of the culture samples were prepared using DMEM as the basic medium. When the FGF alone was added to the culture medium from the beginning (control), when EGF alone was added at the initial stage (EGF only), when EGF (FGF + EGF) and FGF were added to the culture medium in the early stage when FGF and EGF were added to the culture medium at the early stage (FGF and 2day FGF) + 2day EGF).

Referring to the graph of FIG. 31, it was confirmed that the culture speed was the fastest in cases where EGF was added after 2 days in both JOO and VAN cells.

As described above, in the present invention, the plasma and the platelet-rich plasma are separated from the human body in the primary culture to perform pretreatment for irradiating visible light, and a first culture medium is prepared by mixing a first additive having a unique composition and a blending ratio . Cells are inoculated in a first culture medium and cultured under hypoxic conditions, thereby increasing the growth rate of the cells and keeping the size of the cells small.

In the subculture, a second culture medium is prepared by mixing the second additive while maintaining the minimum amount of bovine serum with the risk, and preferably by mixing the reusing medium, the culture speed of the cell is increased and the size of the cell is maintained .

That is, when the primary culture and the subculture are continuously performed using the culture method according to the present invention, the growth rate of the cells can be increased to at least 4 times as compared with the general culture method, and the stem cells can be cultured in a large amount. It is anticipated that this will provide an opportunity for industrial use of stem cells. Furthermore, since the size of the cells can be kept small, it is expected that the stem cells can be used more safely and effectively in the case of using them for therapeutic purposes or cosmetic purposes.

For reference, in the E + Arabic numerals such as E + 06 and E + 07 shown in the diagram of the drawing, E means 10, and Arabic numbers mean several power. That is, E + 06 means 10 ⁶ and E + 07 means 10 ⁷ .

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be taken by way of limitation and that those skilled in the art will recognize that various modifications and equivalent arrangements may be made therein. It will be possible. Accordingly, the true scope of protection of the present invention should be determined only by the appended claims.

Claims (12)

A method for culturing adipose derived stem cells isolated from adipose tissue of a human body through a primary culture step and a subculture step in a hypoxia condition having a lower oxygen content than an atmospheric condition,
Wherein the first culture medium comprises a nutrient medium in the form of a liquid containing glucose, a plasma separated from the human body, a platelet rich plasma (PRP) irradiating visible light, and a first additive 1 < / RTI > culture medium,
Wherein the subculturing step comprises inoculating the first culture medium with the nutrient medium and a second culture medium containing bovine serum and a second additive mixed at a ratio of 1 to 5 wt% And culturing the stem cells. The method according to claim 1,
Wherein the first additive comprises transferrin, selenium, triiodothyronine (T3), and ornithine. 3. The method of claim 2,
Wherein the first additive is mixed with the first culture medium in a range of 0.2 to 0.6 wt% based on the weight of the nutrient medium. Wherein the EGF (Epidermal Growth Factor) is added to the first culture medium within 48 to 72 hours after the start of the primary culture. The method according to claim 1,
Wherein the platelet-rich plasma irradiated with the visible light is mixed with the first culture medium at a ratio of 1 to 8% by weight based on the weight of the nutrient medium. The method according to claim 1,
Wherein the plasma of the first culture medium is irradiated with visible light. The method according to claim 1,
Wherein the second culture liquid comprises a reusing culture medium which has been used as a culture medium for culturing stem cells. 8. The method of claim 7,
Wherein the reusing culture medium is contained in the second culture medium in a range of 1 to 10% by weight based on the weight of the nutrient medium contained in the second culture medium. 8. The method of claim 7,
Wherein the reusing medium comprises a basic medium containing glucose and an additive and optionally bovine serum is mixed at a ratio of 1 to 5% by weight based on the weight of the basic medium. The method according to claim 1,
The second additive is mixed with the second culture medium in a proportion of 0.3 to 1.7% by weight based on the weight of the nutrient medium,
Wherein the second additive comprises insulin, transferrin, and selenium. 11. The method of claim 10,
Wherein the second additive further comprises ethanolamine and vitamin E. 2. The method according to claim 1, The method according to claim 1,
Wherein the EGF (Epidermal Growth Factor) is added to the second culture medium within 48 to 72 hours after the passage of the subculture.

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