KR102115360B1 - Adult stem cell culture fluid and a producing method thereof - Google Patents

Abstract

Task: Making mesenchymal stem cell culture in an economical and easy way
Solution: The present invention can obtain mesenchymal stem cell culture medium containing high content cytokines and cell growth factors for a long period of time by culturing mesenchymal stem cells in a serum-free medium using a microcarrier as an attachment support. .

Description

Adult stem cell culture fluid and its producing method

The present invention relates to a method for culturing adult stem cells in three dimensions for a long time using a microcarrier, an adult stem cell culture prepared by the method, and a cosmetic composition containing the culture medium.

Adult stem cells have been found to exist in many tissues of our body as cells that can self-proliferate in an undifferentiated state and multiply differentiate into cells of other tissues. The first known of these is adult stem cells derived from bone marrow. It has since been found that adult stem cells are isolated not only from umbilical cord blood but also from peripheral blood, and also exist anywhere in our body, including the placenta, skin and nerve tissue. Bone marrow stem cells (BMSCs) have been identified as mesenchymal stem cells through many studies, but there are pains when collecting them, and there is a disadvantage that large amounts of stem cells cannot be obtained, and umbilical cord blood is difficult to obtain in a timely manner. It has problems such as long-term storage for use as an autologous tissue. The origin of the fact that stem cells are also present in adipose tissue was found in 1992 by microvascular endothelial cells, von Willebrand factor (vWF), a-smooth muscle cell actin and cytokeratin stained cells in the SVF layer isolated from human adipose tissue. After Han [Young C, Jarrell BE, Hoying JB, Williams SK. Cell Transplant 1992; 1: 293-298.] A recent study confirmed that cells similar to stem cells exist in these tissues and that these cells are multipotent stem cells [Zuk PA, Zhu M, Ashjian P, De Ugarte DA, Huang JI, Mizuno H, Alfonso ZC, Fraser JK, Benhaim P, Hedrick MH. Mol Biol Cell 2002; 13: 4279-4295].

Adipose tissue-derived stem cells play an important role in wound healing and tissue regeneration through functions such as angiogenesis, immune regulation, anti-inflammatory action, cell death prevention, and cell attraction by secreting differentiation capacity and various cytokines and cell growth factors. It is said to.

It is known that there are at least 30 and as many as 470 proteins in the secretions secreted by adipose tissue-derived stem cells (S. Zvonic, et al., Molecular & Cellular Proteomics 6 (2007) 18-28 ., J. Kim, et al., Proteomics 10 (2010) 394-405., J. Zhong, et al., Journal of Proteome Research 9 (2010) 5228-5238.DM Mutch, et al., International Journal of Obesity (London) 33 (2009) 354-363.A. Rosenow, et al., Journal of Proteome Research 9 (2010) 5389-5401) It is reported that the composition varies according to the culture period and conditions of stem cells. Is becoming.

Recently, several researchers have reported a method of obtaining a high concentration of culture supernatant through culturing of mesodermal stem cells. In Korean Patent Publication Nos. 10-2009-0001163 (2009. 01. 08) and International Publication WO 2007/086637 (2007. 08. 02), mesenchymal stem cells are hypoxic cultured, irradiated with UV light, lack of nutrients, or mechanical friction. Disclosed is a method of producing a high concentration of several cell growth factors by adding a physical stimulus such as and adding vitamin A, B, C, or D to the culture medium. However, in this method, after the stem cells are simply two-dimensional cultured, in order to increase the content of VEGF and the like, the two-dimensional culture is performed under low oxygen conditions only at a specific time. That is, in the two-dimensional culture, even when low oxygen conditions are given, there is a limit in increasing the content of VEGF contained in the culture supernatant, and there is a problem that the culture supernatant can be obtained only once.

On the other hand, in Korean Patent Publication No. 10-2012-0126284, a scaffold using fibrinogen as a raw material was prepared to culture mesenchymal stem cells in a bottle or in a culture bag in three dimensions to culture EGF, VEGF, HGF, and TGF-b1. It is reported that the content is significantly higher than that of 2D culture. Also, in Korean Patent Publication No. 10-2013-0127329, the concentration of IGF-1, BMP2, CXCL2, IL-8, VEGF, etc. was significantly higher than that of 2D culture by making a 3D culture by making a scaffold using collagen, and the concentrated culture supernatant was It has been reported that it has an effect on bone formation. Seok-Ho Bang (Molecular Therapy, vol. 22 no.4 apr. 2014) found that 3D spheroid culture of human adipose tissue-derived stem cells (hADSC) in a spinner flask resulted in concentrations of VEGF, FGF-2, HGF, and CXCL2. Was significantly higher than 2D culture, and 3D culture supernatant was reported to be effective in angiogenesis.

Comparing the above reports, it can be seen that the 3D culture method is more effective than the 2D culture method to obtain a culture supernatant containing a high concentration of cell growth factor.

Korean Patent Publication No. 10-2012-0126284 discloses a change in the EGF, VEGF, HGF, and TGF-b1 contents of the culture medium on the 3rd, 6th, and 9th days using a 3D culture method using a scaffold. Other ingredients tended to decrease in content over time. Therefore, it is necessary to improve the culture method in order to obtain a higher concentration of culture medium for a long time using limited stem cell resources.

The culture methods used by the above researchers have some problems to be solved to be used as a general 3D culture method of hADSC.

In other words, since the 3D culture methods using the scaffold are randomly constructed, the physical conditions such as size and number are not standardized, making it difficult to repeatedly create the same conditions when culturing stem cells. It is a method of floating culture while stirring in a spinner flask by forming a spherical colony of stem cells without attaching to an attachment support, which takes into account the fact that hADSC is a cell that adheres and grows. Their loss is expected, and the method of separating the cells from the culture supernatant is ambiguous, so it is expected that there will be difficulties in the process of replacing the culture solution or obtaining the culture supernatant.

The present invention provides a method for obtaining a human adipose tissue-derived mesenchymal stem cell culture medium containing a high content of cytokines and cell growth factors for a long time by culturing in three dimensions in a serum-free medium using a microcarrier as an attachment support. The purpose.

In order to achieve the above object, the present invention

(a) isolating adult stem cells from adipose tissue;

(b) passaging the isolated adult stem cells;

(c) adding two or more passages of stem cells to a culture vessel containing a serum-free medium and a microcarrier to leave the stem cells attached to the microcarriers;

(d) after the stem cells are attached to the microcarrier, adding the medium to the culture vessel, repeating stirring and stopping, and culturing the stem cells in three dimensions; And

(e) adding one or more of a microcarrier or a medium to the culture container to continuously maintain the culture of the stem cells for 2 weeks or more to continuously obtain a culture medium; a method for preparing a stem cell culture medium is provided.

In addition, the present invention is characterized in that the step of (d) repeating the stirring and stopping and the step of culturing the stem cells in three dimensions is repeated for 10 minutes to 1 hour and 10 minutes to 1 hour rest.

In addition, the present invention is characterized in that the step of culturing the stem cells in three dimensions by repeating the stirring and stopping of (d) is 20 to 80 rpm, 20 to 1 hour stirring, and 10 to 1 hour rest.

In addition, the present invention is characterized in that the step (c) is cultured under hypoxic conditions of 5-10%.

In addition, in the present invention, step (e) is characterized in that a culture medium is obtained at intervals of 3 to 5 days and medium is added.

In addition, in the present invention, the step (e) is a method of preparing a stem cell culture medium for culturing for 6 weeks or more and obtaining a culture medium.

In addition, the present invention is prepared by the above method, and provides an adult stem cell culture medium containing VEGF 8 ng / ml or more, HGF 7 ng / ml or more, procollagen type 1 peptide 9 mg / ml or more, TGFb-1 7 ng / ml or more. (Figure 7).

In addition, the present invention provides an adult stem cell culture medium characterized by containing VEGF 5-7 times and HGF 15-20 times as compared to an adult stem cell two-dimensional culture medium.

In addition to this, the present invention provides a cosmetic composition comprising an adult stem cell culture.

The serum-free medium used in the present invention means a commercially available serum-free medium for mesenchymal stem cells, and the microcarrier of the present invention is a non-animal protein as a spherical cell attachment support having a diameter of 100 to 200 µm made of polystyrene. It means that the charged material is coated.

In the three-dimensional culture of the stem cells, it is preferable to obtain a culture medium while exchanging the medium at intervals of 3 to 5 days by adding a microcarrier according to the propagation time of the stem cells from the start date of the 3D culture.

The obtained stem cell culture medium has a characteristic that the concentration of VEGF (Vascular Endothelial Growth Factor) is about 7 times, and the hepatocyte growth factor (HGF) is about 20 times higher than that of 2D culture, and can be used in cosmetic compositions and the like.

In the present invention, a 3D culture using a spinner flask of 3D culture method using a scaffold or Moocular Therapy, vol.22 no.4 apr.2014 reported in Korean Patent Registration No. 10-2012-0126284 (spheroid culture) By improving and standardizing, we propose a method that can easily and stably cultivate stem cells for a long time to mass-produce a high-concentration culture solution more easily.

Specifically, the three-dimensional culture of adipose tissue-derived stem cells using the microcarrier of the present invention can secure a large surface area capable of attaching cells with a small amount of microcarriers (360 cm 2 / g), and the culture medium during cell culture. By homogenizing the culture environment through agitation, it is possible to maintain a constant environment in which all cells are in contact, so it is possible to repeatedly predict the proliferation of cells, and it is possible to continuously proliferate without using proteolytic enzymes such as trypsin to delay cell aging. This allows long-term cell culture.

In addition, the method of the present invention is a simple method of cultivation, so that only by exchanging the medium and adding microcarriers, stem cells can be proliferated, thereby reducing cost and labor, and scale-up for mass cultivation is possible.

In addition to this, the culture solution obtained by the method of the present invention contains a high concentration of the cell active material, so that no separate operation is required to increase the concentration of the cell active material.

According to the three-dimensional culture method of the present invention, a high concentration of culture solution can be more stably obtained using limited stem cells.

The method of the present invention will be described in detail in order.

(1) Isolation of stem cells derived from human adipose tissue

Isolation of stem cells from human adipose tissue follows a conventional method. That is, after washing the separated fat inhalation to obtain only adipose tissue, treated with collagenase, and then centrifuged to obtain a stromal vascular fraction containing stem cells. The stromal vascular fraction in the form of the obtained cell pellet was washed, and then tissue other than cells was removed by passing through a cell filter, and then cultured in a flask for one day. The non-adherent cells were then removed. The flask was coated with an attached protein, and the stem cells were cultured using a serum-free medium.

(2) Stem cell culture

The isolated adherent stem cells were exchanged for medium every 3 days, maintained until the cells in the flask were 70-80% full, and then passaged. In the initial culture, it is preferable to add an antifungal agent and a reagent to prevent the growth of mycoplasma causing contamination. The general conditions of the initial culture were temperature 37 ° C. and 5% CO ₂ .

(3) passage culture

When the cells filled 70-80% of the bottom of the flask, the growth medium was removed and trypsin treated to remove the cells from the flask. For passage culture, 10,000 cells / cm 2 of cells were dispensed into a new flask coated with an attached protein. After passage, the culture conditions were 37 ° C, 5% CO ₂ , and 5-10% O ₂ .

(4) 3D culture using microcarriers

The cultured cells were removed and dispensed with a small amount of serum-free medium in a spinner flask together with a microcarrier, and then stagnated for one day so that stem cells could adhere to the microcarrier. Subsequently, the medium was added and cultured continuously by allowing the microcarrier to float while rotating the rotating rod in the spinner flask using a magnetic stirrer. When the density of cells attached to the microcarrier was increased, the cells were cultured while adding the microcarrier and medium. The culture conditions were temperature 37 ° C., 5% CO ₂ , and 5-10% O ₂ .

(5) Obtaining the culture solution

Nutrients required for cell growth in serum-free medium are almost exhausted in 3-4 days after supply, so a certain amount of medium must be exchanged for stable and continuous cell culture. In the present invention, it is preferable to replace 50 to 75% of the culture medium with fresh medium.

The present invention has been proposed for the purpose of producing a high-density stem cell culture solution for a long period of time in an easy manner. Compared to the existing three-dimensional culture methods, there is a common point that a high concentration of stem cell culture medium can be obtained, but the method of the present invention can obtain a high concentration culture medium with little variation over a long period of time, and can easily mass-produce the culture medium simply by adding microcarriers. It is possible to reduce the cost for cell replacement by stably culturing limited cells for a long period of time, to produce more high-concentration culture medium, and to facilitate the standardization of culture medium production. Therefore, according to the method of the present invention, it can be said that the method is suitable for the needs of mass production of the culture medium when the active ingredient in the culture medium is prepared as a cosmetic composition and applied to commercialization.

1 shows the appearance of stem cells that adhere and proliferate to the microcarriers on the first and seventh days of the three-dimensional culture of the present invention.
Figure 2 shows the appearance of stem cells that proliferate when inoculated into a two-dimensional culture vessel agglomerated microcarrier mass on the 32nd day with the three-dimensional culture of the present invention.
FIG. 3 is a diagram showing cytokines detected in a three-dimensional culture medium of the present invention using 102 types of cytokine array kits (Proteome Profiler Array; Human XL Cytokine Array Kit; R & D system).
Figure 4 is a method of the present invention, after the start of the three-dimensional culture, 50% of the culture medium is replaced with a new medium without the addition of new microcarriers (3 days, 7 days, 10 days, 14 days, 28 days, 22 days, 26 days) It is a graph showing the relative content change of VEGF by date in the culture medium obtained.
5 is a culture obtained for 50 days by adding microcarriers on the 7th, 17th, and 27th days after the 3D cultivation with the method of the present invention, and increasing the volume of the medium to 1.5 times the initial level on the 17th and 2 times on the 27th, respectively. It is a graph showing the relative content change of VEGF within.
Figure 6 is a graph comparing the VEGF content in the three-dimensional culture solution of the present invention obtained under low oxygen conditions and 21% oxygen culture conditions.
7 is a graph showing the content of VEGF, HGF, TGFb-1 and type 1 procollagen in a culture medium obtained during 2D or 3D culture of adipose tissue-derived stem cells.
8 is a graph showing the results of measuring the number of cells after 48 hours by adding 50% of the culture medium obtained by the method of the present invention and the culture medium obtained by the conventional two-dimensional culture method to the culture medium of adipose tissue-derived stem cells.

Hereinafter, the configuration of the present invention will be described in more detail through examples. However, the following examples are only to illustrate the present invention, it is obvious to those skilled in the art to which the present invention pertains that the scope of the present invention is not limited only to the description of the examples.

Example  1: Isolation of adipose stem cells

; Gibco)로 37℃, 5% CO₂ 배양기에서 24시간 동안 배양한 후 기질 배지와 함께 비접착성 세포들을 제거하여 지방 줄기세포를 단리하였다.Fat tissue donor consent was obtained, and fat tissue was collected through a liposuction method. The obtained adipose tissue was washed three times with PBS containing 2% AbAm (Antibiotics-Antimycotics) to obtain pure adipose tissue (fat tissue). After treating 0.1% collagenase in pure adipose tissue, centrifugation was performed at 1500 rpm for 5 minutes to obtain a stromal vascular fraction pellet. After washing the obtained pellets with PBS and removing other tissue through a 100 μm nylon cell filter, serum-free medium containing 0.05% gentamicin (StemPro® MSC SFM CTS

; Adipose stem cells were isolated by removing non-adherent cells together with the substrate medium after incubation for 24 hours in a 37 ° C, 5% CO ₂ incubator with Gibco).

Example  2: Stem cell culture

Attached stem cells isolated in Example 1, while proliferating until the cells in the flask were 70-80% confluent while exchanging the proliferation medium at 3 day intervals, the proliferation medium was removed and trypsin was treated to treat the cells in the flask. Removed. For passage culture, 10,000 cells / cm 2 of cells were dispensed into a new flask coated with an attached protein, and then propagated until 70-80% of fullness was used for 2D or 3D culture. After passage, the culture conditions were 37 ° C., 5% CO ₂ , and 5% to 10% O ₂ .

Example  3: Microcarriers  Stem cell 3D culture

The two or more passages of stem cells obtained in Example 2 were removed and dispensed together with a microcarrier in a spinner flask with a small amount of serum-free proliferation medium (5 × 10 ⁴ to 1 × 10 ⁵ cells / ml). Thereafter, the stem cells were stagnated in the incubator for 24 hours to allow the stem cells to adhere to the microcarriers. After 24 hours, the medium was repeatedly added, and the impeller in the spinner flask was repeatedly set by rotating the impeller (40 minutes at 35 to 50 rpm) and pause (10 minutes) using a magnetic automatic control device. It was made possible. When the density of stem cells attached to the microcarrier was increased, the cells were cultured while adding the microcarrier and medium.

As shown in FIG. 1, after 24 hours after the start of the 3D culture, the stem cells are stably attached to the microcarrier surface, and the surrounding microcarriers begin to aggregate by the attached protein secreted by the attached stem cells. After 7 days of cultivation, stem cells are distributed on the surface of most microcarriers to start division and proliferation, and the microcarriers aggregate more. At this time, microcarriers are replenished to increase the surface area to which the divided stem cells can adhere.

After a certain period of time, the number of single microcarriers was reduced and the number and size of aggregates were repeated. At this time, microcarriers and media were added to continuously culture.

As in the two-dimensional culture, stem cells can be proliferated only by adding a microcarrier without removing cells using trypsin, so it was determined that cell aging can be stably reduced and cell culture can be stably performed for a long period of time. 2 shows that after 30 days of 3D culture, some aggregates were inoculated into a flask for 2D culture, and after 3 days, a large amount of stem cells were proliferated from the aggregates. Normally, a long-term two-dimensional culture results in severe deformation of cells, but stem cells proliferated in aggregates after 30 days show the initial cell form. As a result, it was found that long-term stem cell three-dimensional culture is possible by the method of the present invention, and a high concentration of culture solution can be continuously obtained in this process.

Experimental Example  1: Selection of indicators through qualitative analysis of cytokines in culture

For long-term three-dimensional culture of stem cells, it is necessary to add a microcarrier for proliferation and replace or add a new medium. Stem cells that adhere to growth do not proliferate anymore and enter a resting state when there is no free space to attach. In the resting state, active proliferation does not occur, so the concentration of cytokines or cell growth factors they secrete is no longer high. Therefore, in order to induce active growth, it is necessary to add a microcarrier as an attachment support and supply a medium at an appropriate time.

In order to determine the periods, it was decided to look at various cytokine or cell growth factors related to cell proliferation during a long-term culture process.

In order to confirm the presence of many cytokines or cell growth factors in the culture medium and to determine appropriate indicators, the culture medium obtained on the 7th day of the 3D culture was qualitatively analyzed using a Western blot test (Human XL Cytokine Array Kit (R & D system)). (Figure 3).

The qualitative analysis of 102 sine tocaine or cell growth factors followed the following process. After preparing all reagents, control samples, and culture samples of the provided product at room temperature in advance, Array buffer 6 and a membrane were added to each well of a pre-designed 96-well plate and reacted for 1 hour at a rocking platform shaker at room temperature. After that, the reacted solution was discarded, and a control sample and a culture sample were added together with Array Buffer 6 so that the final volume was 1.5 ml. (The amount of the culture solution sample was set to 200-500 µl or more.) After that, the mixture was reacted for 12 hours or more while maintaining 2 to 8 ° C in a rocking platform shaker and washed three times with washing solution for 10 minutes. After adding 120 µL of Detection Antibody Cocktail to 6 ml of a mixture of 4 ml of Array Buffer 4 in 8 ml of Array Buffer 6 provided, 1.5 ml was added to each well and reacted at room temperature for 1 hour. After washing 3 times for 10 minutes each with washing solution, Streptavidin-HRP was added to each well and reacted for 30 minutes. Thereafter, the cells were washed three times for 10 minutes each with a washing solution, and then, after adding a Chemi Reagent Mix solution and reacting for 1 minute, a photograph was taken (Chemi-doc).

3 is a view showing the relative content of each cytokine by analyzing the observed spots. A total of 43 cytokines or cell growth factors out of 102 were detected, and HGF and VEGF were high in cell growth factors. Among them, VEGF is known as a representative cell growth factor secreted by stem cells related to angiogenesis. Therefore, it was decided to use VEGF as an indicator in the analysis related to the proliferation of stem cells.

Experimental Example  2: In the culture medium due to periodic medium replacement VEGF  Content change measurement

The medium used in the present invention is a serum-free medium commercialized for culturing mesodermal stem cells. In general, serum-free medium contains limited components necessary for cell growth and proliferation. Therefore, after a certain period of time, a new medium should be replaced for smooth cell culture. Proper medium replacement cycle is an important factor for stably culturing cells.

In 2D culture, medium is usually replaced at intervals of 3 to 4 days, so the possibility of long-term cultivation was examined in the 3D culture of the present invention using a replacement cycle of 3 to 4 days. Determine the amount of medium through the known surface area of the microcarrier as an attachment support, and incubate for 30 days while maintaining the initial microcarrier amount by replacing 50-75% with a new medium every 3-4 days. Concentration changes were measured.

4 is a graph measuring the concentration of VEGF by date while replacing with a new medium on the 3rd, 7th, 11th, 15th, 18th, 22nd and 26th. The concentration increased steadily until the 7 days after the initial 3 days. This is because the cells continued to proliferate due to the large number of attachable surfaces provided by the microcarriers at the beginning of the culture. After 7 days, the concentration was reduced immediately after the medium was replaced, and after 3-4 days, the concentration level before replacement was restored, but no significant change in concentration was observed. This is because, unlike the early stage of culture, microcarriers are not added, and thus, the adhesion surface is insufficient, so that the cells are not proliferated and the number of cells is not increased during the culture period.

As a result of this experiment, it was found that a certain number of stem cells can be cultured for a long period of time by replacing with a new medium while obtaining 50 to 75% of the culture medium at intervals of 3 to 4 days.

In addition, it was determined that the addition of microcarriers during the incubation period can induce cell proliferation and, accordingly, the number of cells increases, thereby increasing the concentration of cell growth factors including VEGF.

Experimental Example  3: Change of medium Microcarrier  In the culture medium according to the addition VEGF  Content change measurement

In order to further examine the contents determined in Experimental Example 2, 3D culture was performed for 50 days while adding microcarriers on the 7th, 17th, and 27th days after cultivation, and the medium was obtained by replacing the medium at intervals of 3-4 days. The VEGF content in the culture medium was measured. In order to supply sufficient nutrients to the proliferated cells, the amount of the medium was increased 1.5 times and 2 times, respectively, on the 7th and 17th days, and the amount of the medium was maintained twice on the 27th day. The culture conditions were temperature 37 ° C, 5% CO ₂ , and 21% O ₂ . From the 7th day to the 17th day, the concentration of VEGF decreased gently, and on the 17th, it decreased to 80% of the 7th day. This is judged to be because the number of cells was increased on the 7th day because the amount of the medium was increased 1.5 times, but the concentration was diluted. After the 17th day, although the amount of the medium was doubled to the initial level, the concentration temporarily increased, and then slowly decreased until the 27th day to 76% of the 7th day. This is judged to be because cell proliferation occurred by the microcarrier added on the 17th day. On the other hand, the concentration of VEGF increased significantly after the 27th day when only the microcarrier was added, and increased to 128% and 190% on the 7th day, respectively, on the 31st and 50th days. This is judged to be because the amount of medium does not increase and cell proliferation occurs and the dilution effect does not appear.

As a result, it was determined that the addition of microcarriers induces the proliferation of stem cells, thereby increasing the concentration of cytokines or cell growth factors in the culture medium.

Experimental Example  4: In the culture medium obtained under hypoxic conditions and 21% oxygen conditions VEGF  Content comparison

In two-dimensional culture, it is known that the concentration of cell growth factor acting on wound healing is higher in hypoxic conditions than in 21% oxygen conditions. In order to find out whether such a phenomenon appeared in the three-dimensional culture of the present invention, the VEGF concentration in the culture medium was measured after culturing under hypoxic conditions (5-10% O ₂ ) and 21% oxygen conditions. Microcarriers were added on the 7th day after the start of cultivation, and the medium was 1.5-fold, and cultured for 21 days to obtain 50-75% of the culture solution at 3-4 day intervals (7, 10, 14, 17, 21) 1) Replaced with fresh medium.

6 is a graph showing the average value of the VEGF concentration in the culture medium obtained over 5 times. VEGF concentration was 8.5% higher than that of the 21% hypoxic condition, so it was judged that the hypoxic condition was more efficient to obtain a high concentration of culture.

Example  4: Obtaining the culture solution

In order to synthesize the results of Experimental Example 3 and Experimental Example 4, to obtain a high-concentration culture, 50-75% of the culture was obtained at intervals of 3 to 5 days using hypoxic conditions, and a new medium of the same amount was supplied. Microcarriers were added to induce the proliferation of stem cells, and the amount of medium added was also increased. As the incubation time increased, the amount of the culture solution obtained at one time also increased. Some of the obtained culture broth was filtered using a 0.25 to 0.45 μm filter and then stored at -80 ° C.

Example  5: Measurement of cell growth factor content

The stem cells (passage 2) obtained in Example 3 were subjected to two-dimensional culture and three-dimensional culture at the same time, so that the two-dimensional culture was obtained after three days, and the three-dimensional culture was used for the culture obtained in Example 4.

To measure the content of cell growth factors in each culture, an enzyme-linked immunosorbent assay (ELISA) was used (Quantikine ELISA Kit, R & D system; Procollagen type 1 peptide kit (Takara Bio Inc., Japan)). That is, 100-200 µl of the standard material and the culture sample was added to each well of a 96-well plate, and 50 µl of the first reaction solution provided in the product was added to each well and reacted for 2 hours. The culture sample was diluted 1:10 to 1:20 as needed. After the reaction, the solution was removed, washed 3-4 times with a washing solution, and 100-200 µl of conjugate was added to each well, followed by reaction for 2 hours. Thereafter, the reaction solution was removed, and after washing 3-4 times with a washing solution, 200 µl of a substrate solution was added and reacted at room temperature for 15-25 minutes. After the reaction, 50 to 100 µl of a stop solution was added to each well, and then mixed well, and absorbance was measured at 450 to 540 nm within 30 minutes.

The content of each growth factor was obtained by diluting the provided standard solution step by step to obtain the formula of the standard curve for each absorbance and the corresponding contents and substituting the absorbance of the samples to the formula.

7 is a table showing the concentration of various cell growth factors in the culture medium obtained for 32 days in Example 4. The cell growth factor content of the culture solution obtained by the three-dimensional culture method of the present invention was significantly increased than that of the two-dimensional culture. In addition, during the 32-day culture period, the content of each cell growth factor was maintained at a high concentration without significant deviation. In the case of VEGF, high concentrations appeared from the culture medium obtained on the 7th day after culture, and the concentration was maintained during the culture period, and the concentration was about 5 to 7 times higher than that of the 2D culture medium. In the case of HGF, a high concentration was maintained during the entire 3D culture, and after 7 days, the culture medium had a content of about 15 to 20 times higher than that of the 2D culture. Procollagen increased 2-4 fold over 2D culture. TGFb-1 increased more than 3-fold.

Experimental Example  5: Effect of culture medium on proliferation of adipose tissue-derived stem cells

The stem cell culture contains large amounts of cytokines that act on wound healing and various growth factors that act on cell proliferation. The following experiment was performed to examine the effect of the three-dimensional culture obtained in the present invention on the proliferation of cells. First, 5 × 10 ⁴ cells were dispensed into the prepared 6 wells (3 repetitions), and 10% bovine serum medium was used as a control group, and a medium containing 50% 3D culture medium and 50% 2D culture medium were used as a control group. Each medium was used. The culture conditions were temperature 37 ° C., 5% CO ₂ , and 5-10% O _2. After 48 hours, cells were separated with trypsin and the number was measured.

The number of cells to which the culture medium prepared by the method of the present invention was added was more than 1.8 times that of the control group, and it was found that 15% higher than the two-dimensional culture medium, the high concentration of the three-dimensional culture medium was effective for cell proliferation.

Recently, several researchers have reported how to obtain a high concentration of culture supernatant through the cultivation of mesodermal stem cells. In Korean Patent Publication No. 10-2009-0001163 (2009. 01. 08) and International Publication WO 2007/086637 (2007. 08. 02), mesenchymal stem cells are cultured with hypoxia, ultraviolet irradiation, lack of nutrients, or mechanical friction. Disclosed is a method for producing a high concentration of several cell growth factors by applying the same physical stimulation and adding vitamin A, B, C, or D to the culture medium. However, in this method, after the stem cells are simply two-dimensional cultured, in order to increase the content of VEGF and the like, the two-dimensional culture is performed under low oxygen conditions only at a specific time. In addition, the serum-free culture medium used is a basic culture medium in which stem cells are difficult to stably grow and proliferate, and there is a problem in that the physiological activity of cell growth factors secreted in the culture supernatant is reduced. That is, in the two-dimensional culture, there are limitations in increasing the content of VEGF and the like contained in the culture supernatant even when low oxygen conditions are given, and there is a problem in that the culture supernatant can be obtained only once.

On the other hand, in Korean Patent Publication No. 10-2012-0126284, a scaffold using fibrinogen as a raw material was used to culture mesenchymal stem cells in a culture vessel in three dimensions, so that the contents of EGF, VEGF, HGF, and TGF-b1 were superior to those of two-dimensional culture. I am reporting an increase. In addition, in Korean Patent Publication No. 10-2013-0127329, when 3D culture was made by making a scaffold using collagen, concentrations of IGF-1, BMP2, CXCL2, IL-8, and VEGF were significantly higher than 2D culture, and the concentrated culture supernatant was It has been reported to be effective in bone formation. Seok-Ho Bang (Molecular Therapy, vol.22 no.4 apr.2014) showed that the concentration of VEGF, FGF-2, HGF, and CXCL2 was significantly higher than that of 2D culture as a result of 3D spheroid culture of hADSC in a spinner flask. It has been reported that 3D culture supernatant is effective for angiogenesis.

Comparing the above reports, it can be seen that the 3D culture method is more effective than the 2D culture method to obtain a culture supernatant containing a high concentration of cell growth factor. However, there are some problems to be solved in order to apply the above cultivation methods as a general 3D culture method of hADSC. In other words, 3D culture methods using scaffolds lacked a review of the material of scaffolds most suitable for mesenchymal stem cell attachment, growth, and proliferation, and because scaffolds were randomly made, physical conditions such as size and number were not standardized. It is difficult to make the same conditions when culturing stem cells. The 3D suspension culture method by Bok Seok-ho et al. Is a method of forming a spherical colony without attaching hADSC to an attachment support, and culturing it while stirring in a spinner flask, which is a single cell that failed to form colonies considering the fact that hADSC is a cell that grows attached Loss of (single cells) is expected. In addition, the method of separating the cells from the culture supernatant is ambiguous, and thus it is expected that there will be difficulties in the process of replacing the culture supernatant or obtaining the culture supernatant.

The three-dimensional culture method using the microcarrier of the present invention has the following advantages compared to the two-dimensional culture method.

First, it is possible to secure a large surface area to which stem cells can adhere with a small amount of microcarriers. For example, 3mg / ml of Cytodex1 corresponds to 176 flasks of 75 cm 2 with a surface area of 1.32 × 10 ⁴ cm 2 / ℓ.

Second, the culture environment can be homogenized through agitation of the culture medium during cell culture, so that long-term culture is possible.

Third, it is possible to scale up for mass culture of stem cells.

Fourth, continuous proliferation of stem cells is possible without using proteolytic enzymes such as trypsin, and thus aging of stem cells is prevented.

Fifth, the concentration of growth factors in the culture supernatant is 10 to 20 times higher than in conventional culture methods.

Sixth, since the cultivation method is simple, it can reduce cost and labor.

In addition, the three-dimensional culture method of the present invention has the following differences from the existing two-dimensional culture method or three-dimensional culture method.

First, the concentration of the main cell growth factor in the culture supernatant is 13 to 20 times higher than that of the two-dimensional culture.

Second, it is possible to obtain a culture supernatant containing a constant high concentration of cell growth factor for at least 40 days from a cultured cell, thereby producing a large amount of culture supernatant from limited cells.

Third, since the detachment of cells is not necessary, the cultivation process is easy and the use of consumer goods is rarely used, thus reducing labor and production costs.

Fourth, since the cultivation process is simple, the cell growth factor concentration in the culture supernatant can be maintained relatively uniformly throughout the cell culture, thereby standardizing the product.

Fifth, mass production is possible only by expanding production facilities.

Claims (9)

(a) isolating adult stem cells from adipose tissue;
(b) passaging the isolated adult stem cells;
(c) adding two or more passages of stem cells to a culture vessel containing a serum-free medium and a microcarrier to leave the stem cells attached to the microcarriers;
(d) after the stem cells are attached to the microcarrier, adding the medium to the culture vessel, repeating stirring and stopping, and culturing the stem cells in three dimensions; And
(e) adding a microcarrier and a medium to the culture vessel, without detaching the cells, repeating stirring and stopping, and continuously obtaining a culture solution while maintaining a three-dimensional suspension culture of stem cells for 2 weeks or more; Method for preparing cell culture
The method according to claim 1,
The steps of (d) and (e) repeating the agitation and stop, and the step of culturing the stem cells in three dimensions is a method of preparing a stem cell culture solution for 10 minutes to 1 hour stirring and 10 minutes to 1 hour rest.
The method according to claim 1,
The steps of (d) and (e) repeating agitation and stopping, and the step of culturing the stem cells in three dimensions is 20 to 80 rpm for 20 minutes to 1 hour with stirring and 10 minutes to 1 hour with a method of preparing stem cell culture. .
The method according to claim 1,
Step (c) is a method of preparing a stem cell culture medium for culturing under 5-10% hypoxic conditions.
The method according to claim 1,
Step (e) is a method of preparing a stem cell culture medium in which a culture medium is obtained and the medium is added at intervals of 3 to 5 days.
The method according to claim 1,
Step (e) is a method of preparing a stem cell culture medium in which culture is performed for 6 weeks or more to obtain a culture medium.
An adult stem cell culture medium prepared by the method of any one of claims 1 to 6, containing VEGF 8 ng / ml or more, HGF 7 ng / ml or more, procollagen type 1 peptide 9 mg / ml or more, TGFb-1 7 ng / ml or more .
The method according to claim 7,
Adult stem cell culture, characterized in that it contains VEGF 5-7 times, HGF 15-20 times compared to the adult stem cell two-dimensional culture.
A cosmetic composition comprising the adult stem cell culture of claim 7.

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