KR101639988B1 - Method for preparation of mesenchymal stem cell culture comprising high concentration of cell growth factors and composition prepared therefrom - Google Patents

Abstract

The present invention relates to a method for producing a mesenchymal stem cell culture medium containing a high concentration of cell growth factors and a composition obtained therefrom, the method comprising: (a) culturing mesenchymal stem cells; (b) collecting mesenchymal stem cells cultured in two or more passages and culturing the cells in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And (c) collecting the culture medium. According to the method of the present invention, the cells can be stably maintained for a long period of time to produce a large amount of cell culture liquid. The stem cell culture liquid composition obtained according to the method of the present invention The content of the cell growth factor is markedly increased as compared with the composition produced according to the conventional method, so that it is more suitable for clinical use and particularly excellent in clinical use including wound healing and cosmetic composition.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for producing a mesenchymal stem cell culture solution containing a high concentration of a cell growth factor and a composition obtained therefrom, and a composition obtained therefrom. BACKGROUND ART < RTI ID = 0.0 >

The present invention relates to a method for producing a mesenchymal stem cell culture medium containing a high concentration of a cell growth factor and a composition obtained therefrom. The present invention improves the conventional two-dimensional culture method, The present invention also provides a method for stably culturing mesenchymal stem cells in a medium for a long period of time.

Several types of progenitors have been found in human bone marrow. Among them, precursor cells exhibiting multipotentiality are called mesenchymal stem cells. Mesenchymal stem cells are known to exist not only in bone marrow, but also in most organs of the body, including fat, liver, and muscle. The mesenchymal stem cells are self-renewing and have the ability to stimulate osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon-ligament fibroblasts, adipocytes adipocytes) and the like.

In addition, mesenchymal stem cells are known to secrete various growth factors and physiologically active substances during the culturing process. Biologically active factors secreted by mesenchymal stem cells have functions such as inhibition of immune response, inhibition of fibrosis and apoptosis in wound tissues, induction of angiogenesis and stimulation of cell division. Therefore, the stem cell culture solution can be used for clinical use such as medicines and cosmetic compositions for promoting wound healing.

However, in order to cultivate mesenchymal stem cells, bovine serum is usually used, but the culture medium containing bovine serum is inadequate for clinical application. That is, since the use of animal-derived proteins such as bovine serum is limited for clinical application of cell culture media, it is preferable to exclude bovine serum for culture of stem cells.

Bovine serum proteins are necessary for the proliferation and maintenance of stem cells. Therefore, a method of obtaining a culture solution containing a high concentration of physiologically active substance while culturing mesenchymal stem cells in a serum-free medium is required.

Korean Patent Laid-Open No. 10-2009-0001163 (2009. 01. 08) discloses that mesenchymal stem cells obtained from human bone marrow, umbilical cord blood or placenta tissue are cultured in serum medium, subcultured in serum-free medium, Discloses a method of mass-producing bFGF or TGF-? From mesenchymal stem cells by applying physical stimulation of hypoxic culture to the cells. Similarly, in International Publication WO 2007/086637 (Aug. 02, 2007), adult stem cells extracted from adipocytes of mammals are cultured in serum medium, subcultured in serum-free medium, cultured in hypoxic culture, A method of mass-producing bFGF, VEGF or TGF-? From mesenchymal stem cells by adding physical stimulation such as ultraviolet irradiation, nutrient deficiency, or mechanical friction and adding vitamin A, B, C, . In addition, in Korean Patent Laid-Open No. 10-2010-0008763 (Jan. 26, 2010), human adipose tissue pellets were cultured in DMEM medium containing FBS, NAC and ascorbic acid, and FBS, NAC, ascorbic acid, calcium , rEGF, insulin, and hydrocortisone. The stem cells adhered to the Keratinocyte-SFM medium were separated, and NAC, ascorbic acid, calcium, rEGF, insulin, Culturing in Keratinocyte-SFM medium containing hydrocortisone, collecting the medium, and obtaining a pure cell culture by centrifugation and filtration.

That is, in the conventional method, only two-dimensional culture is carried out under hypoxic condition in order to increase the content of VEGF or the like in a simple two-dimensional culture of stem cells. Considering that the stem cells are not stably maintained in serum-free medium The physiological activity of the stem cell culture solution is lowered. That is, there is a problem in that the content of VEGF and the like contained in the culture liquid is limited even if hypoxic condition is given in the two-dimensional culture, and the stem cell culture liquid can be obtained only once.

The present invention relates to a method for culturing mesenchymal stem cells stably in a serum-free medium and a method for producing a mesenchymal stem cell culture comprising the stem cell culture solution obtained therefrom in order to obtain a mesenchymal stem cell culture medium containing a high amount of cytokine and cell growth factor And to provide the above objects. The mesenchymal stem cell culture composition containing a large amount of the biologically active factor obtained according to the present invention can be used for wound healing, cosmetic compositions and the like.

In order to accomplish the above object, the present invention provides a method for producing a mesenchymal stem cell culture liquid comprising the steps of:

(a) culturing mesenchymal stem cells;

(b) collecting mesenchymal stem cells cultured in two or more passages and culturing the cells in a serum-free medium in a three-dimensional culture with a biocompatible scaffold; And

(c) collecting the culture medium.

Here, (a) the culture of mesenchymal stem cells is preferably subcultured after culturing in medium and growth medium, and (b) three-dimensional culture of mesenchymal stem cells is carried out by culturing at least three It is preferable to obtain a stem cell culture solution at least three times. The serum-free medium may be supplemented with at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor).

In addition, the biocompatible scaffold can be a natural and synthetic polymer as a cell scaffold having a surface that is adhered to the cell, such as alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, poly (alpha-hydroxy acid) Poly (vinyl alcohol), polyanhydride, and the like.

To achieve these and other advantages and in accordance with the purpose of the present invention, as embodied and broadly described herein, the present invention provides a mesenchymal stem cell culture composition comprising VEGF and EGF at a high concentration, comprising a large amount of growth factors and cytokines. Preferably, the mesenchymal stem cell culture composition of the present invention contains VEGF more than 4 times and EGF more than 17 times as compared with the conventional culture composition.

In addition, the present invention provides wound healing agents and cosmetic compositions comprising such a mesenchymal stem cell culture medium composition containing such a large amount of growth factors and cytokines.

The present invention relates to a method and a composition for producing a stem cell culture liquid containing a large amount of a physiologically active substance obtained by effectively culturing mesenchymal stem cells in a serum-free medium. Mesenchymal stem cells used for culturing can be obtained from bone marrow, umbilical cord blood, adipose tissue and the like. In particular, adipose-derived stem cells are relatively easy to access, easy to harvest, and capable of obtaining large quantities from a single individual.

In the present invention, a large amount of mesenchymal stem cells can be effectively obtained in a short period of time by culturing the mesenchymal stem cells using the basal medium and the growth medium as appropriate. In addition, in the method of the present invention, a three-dimensional culture is performed using a biocompatible scaffold in a serum-free medium in the course of obtaining a culture medium, and the cells can be stably maintained for a long period of time, so that a large amount of cell culture liquid can be produced. As described above, the stem cell culture solution produced according to the method of the present invention is characterized in that the content of growth factors such as VEGF and EGF is much higher than the culture solution produced by the conventional cell culture method of 2-dimensional culture.

Since the mesenchymal stem cells are obtained from human bone marrow, adipose tissue, umbilical cord blood and the like, the amount of mesenchymal stem cells obtained from these tissues is also very limited. In order to use the stem cell culture fluid for clinical use, it is preferable that a large amount of stem cell culture fluid is obtained from a limited number of cells and that the content of the physiologically active substance contained in the culture fluid is high.

Generally, in order to cultivate and maintain the mesenchymal stem cells, a bovine serum is required. In order to use the cell culture medium for clinical use, it is preferable that the bovine serum is not contained in the culture medium. It is difficult to maintain the cells stably if the cow-derived serum is not contained in the culture medium of mesenchymal stem cells.

The present invention provides a method for stably culturing mesenchymal stem cells in a serum-free medium containing no bovine serum. The culture solution obtained by this method contains a very high amount of growth factors as compared with the culture solution produced by the conventional method. For example, the content of VEGF (vascular endothelial growth factor) contained in the culture medium of the present invention obtained by the method of the present invention is about 4 times higher than that of the conventional culturing method, thereby providing a remarkably improved effect, It can be very useful.

According to the method of the present invention, when the stem cells are cultured in a three-dimensional manner together with a biocompatible scaffold such as fibrin glue, the cells can be cultured in a simple form of a bottle without using a plate commonly used for cell culture A very economical and large-capacity culture solution can be easily obtained. In addition, since the number of cells is limited according to the size of the cell culture surface in the two-dimensional culture, the amount of the physiologically active substance secreted from the stem cells is limited, whereas when the three-dimensional culture according to the present invention is performed, the number of cells can be adjusted It is possible to increase the concentration of the physiologically active substance in the culture liquid as needed.

Generally, when mesenchymal stem cells are cultured on a serum-free medium, they are not stably maintained and are eliminated from the cell culture surface and die. In the present invention, the mesenchymal stem cells are cultured together with the biocompatible scaffold, so that the mesenchymal stem cells having the property of attaching and growing can be stably maintained in the three-dimensional scaffold. When fibrin glue was used as an example of a biocompatibility scaffold, the mesenchymal stem cells showed at least 80% survival rate over 14 days and continuously secreted a higher amount of VEGF than 2-dimensional culture.

In addition, according to the method of the present invention, when the culture is performed by adding basic fibroblast growth factor (bFGF) and / or epidermal growth factor (EGF) to a serum-free medium, the stability of the cells and the content of secreted growth factors in the culture medium are increased .

As used herein, the term "mesenchy stem cells" refers to cells that are self-proliferating, multipotent and capable of expressing CD73 +, CD90 +, CD105 +, CD14-, CD20-, CD34-, And may be isolated from bone marrow, adipose tissue, umbilical cord blood, etc., but is not limited thereto.

In the present invention, the term "physiologically active substance" refers collectively to cytokines, cell growth factors, and the like that may affect the functions of cells or the body. Examples of physiologically active substances include VEGF (vascular endothelial growth factor), EGF (epidermal growth factor), HGF (hepatocyte growth factor), TGF-beta (Tumor growth factor-beta) But is not limited thereto.

"Stem cell culture fluid" refers to a cell culture supernatant in which stem cells have been cultured. Stem cell cultures contain various physiologically active substances secreted from cells during stem cell culture.

&Quot; Biocompatibility scaffold " refers to a scaffold that has affinity for a cell and is made of a material having a so-called " cell adhesive " surface and capable of adhering and culturing cells three-dimensionally. Examples of natural-derived supports include, but are not limited to, alginate, protein, collagen, fibrin, hyaluronic acid, cellulose, and the like. Examples of the synthetic polymer include poly (alpha-hydroxy acid) series, poly (vinyl alcohol), polyanhydride, and the like.

The present invention discloses a method of obtaining a stem cell culture solution from mesenchymal stem cells, and related methods have already been disclosed in various documents (Dermatol. Surg. 2008, 34: 1323-1326; J. Dermatol. Sci. 2007, 48: 15-24; Korean Patent Registration No. 10-0899329). The present invention improves the conventional method and suggests a method for mass-producing a stem cell culture liquid containing a clinically useful physiologically active substance at a high concentration in a more convenient manner. Specifically, in the conventional method, two-dimensional culture is performed under a hypoxic condition in order to perform a simple two-dimensional culture or increase the content of VEGF, etc. In contrast, in the present invention, a three-dimensional culture is performed with a biocompatible scaffold such as fibrin glue . According to the method of the present invention, when the mesenchymal stem cells are cultured in a serum-free medium, the content of human-derived growth factors, particularly VEGF and EGF, can be drastically increased by performing three-dimensional culture together with fibrin glue. In other words, even in the case of the two-dimensional culture, it is difficult to increase the content of VEGF and the like contained in the culture liquid even if hypoxic condition is given, and the stem cell culture liquid can be obtained only once. However, Since the growth factors are continuously secreted, the culture medium can be obtained at least three times while changing the culture medium once every three days. In addition, according to the three-dimensional culture method of the present invention, unlike the conventional plate culture, the culture can be performed in a bottle, so that large-scale production is possible.

In the present invention, mesenchymal stem cells can be obtained from bone marrow, adipose tissue, umbilical cord blood, and the like, but are not limited thereto.

Hereinafter, the present invention will be described in detail.

The culture of the mesenchymal stem cells according to the present invention can be carried out according to the following procedure, but the culture medium used for cell culture is not limited thereto.

(1) Culture of mesenchymal stem cells

The mesenchymal stem cells obtained from the tissue are suspended in a medium and cultured in a culture vessel at a concentration of 10,000 to 40,000 cells / cm 2. The substrate medium is cultured in DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) medium containing 10% bovine serum for about 24 hours.

(2) Culture in expansion media.

After the substrate medium is removed, the adherent cells are proliferated by culturing in a proliferation medium. The proliferation medium was DMEM or DMEM / F12 containing 10% fetal bovine serum, epidermal growth factor (EGF) at a concentration of 0.1 to 100 ng / ml and / or basic fibroblast growth factor (bFGF) at a concentration of 0.1 to 100 ng / Adherent mesenchymal stem cells are rapidly proliferating to increase the amount of cells in a short period of time.

(3) Passage culture

When the cells fill 80 to 90% of the bottom of the culture vessel, the growth medium is removed and the cells are removed from the culture vessel by trypsinization. For subculture, cells are diluted 1: 3 ~ 1: 4 and incubated with the growth medium in a new culture vessel. Additional subculture is possible in this way.

(4) incubation with a biocompatible scaffold

The cultured cells are washed with PBS 3 times or more to remove FBS, and cultured in a serum-free medium in the form attached to a biocompatible scaffold. Cell culture in a scaffold does not require a conventional cell culture container, and thus it is possible to cultivate large quantities in an aseptic bottle or culture bag, thereby enabling convenient cultivation at a lower cost.

According to the method of the present invention, the mesenchymal stem cells can be stably maintained for a long period of time by performing the three-dimensional culture using the biocompatible scaffold in the serum-free medium to produce a large amount of cell culture medium, The content of growth factors such as EGF can be dramatically increased. Unlike the conventional plate culture, the culture can be performed in a bottle, so that a simple, highly economical and large-capacity culture liquid can be easily obtained.

The stem cell culture composition obtained by the method of the present invention is more suitable for clinical use because the content of the cell growth factor is significantly increased as compared with the composition produced by the conventional method, There is excellence in the clinical applications involved.

1 shows the contents of EGF, VEGF, HGF and TGF β1 in the adipose stem cell culture obtained on the 3rd, 6th and 9th days after two-dimensional (2D) or three-dimensional (3D) culture of adipose derived mesenchymal stem cells Fig.
FIG. 2 is a graph showing the results of measurement of collagen synthesis amount of fibroblasts after culture of stem cell cultures obtained from three-dimensional cultures in a fibroblast culture medium.

Hereinafter, the present invention will be described in more detail by way of examples. However, these examples are merely examples of the present invention, and the scope of the present invention is not limited thereto.

Example  1: derived from human fat Intermediate lobe  How to culture stem cells

Fat tissue is usually obtained by liposuction, but is not limited thereto.

Adipose stem cells were isolated from adipose tissue obtained by liposuction as follows: Adipose tissue was washed 3 to 4 times with the same volume of KRB solution to remove blood. A volume of collagenase solution such as adipose tissue was added and reacted at 37 ° C in a water bath. This was transferred to a centrifuge tube and centrifuged at 1200 rpm for 10 minutes at 20 ° C. The upper layer of fat layer was removed and the lower layer of collagenase solution was carefully removed to avoid shaking. Substrate media were suspended and centrifuged at 20 ° C and 1200 rpm for 5 minutes. At this time, the lower layer was a stroma-vascular fraction, and the supernatant was removed.

The stroma-vascular fraction was suspended in a substrate medium, inoculated into a culture vessel, and cultured in a 5% CO ₂ incubator at 37 ° C for 24 hours. After removing the culture medium, the cells were washed with a phosphate buffer solution and incubated with substrate medium (DMEM / F12 medium containing 10% FBS) or substrate medium with 1 ng / ml basic fibroblast growth factor (bFGF) or epidermal growth factor (EGF) ng / ml, or 1 ng / ml of fibroblast growth factor (bFGF) and 5 ng / ml of epidermal growth factor (EGF). If the adipose stem cells were grown to about 80-90% of the culture vessel, trypsin treatment was performed to isolate them into single cells. The obtained cells were diluted to 1: 3 to 1: 4 with a growth medium and subcultured.

Example  2: human adipocyte stem cells were fibrin Glue  How to make three-dimensional culture

The stem cells obtained by single-cell treatment with trypsin were washed three times with DMEM / F12 (phenol red) medium to remove FBS. The cells were suspended by adding DMEM / F12 (without phenol red) solution containing 20% of thrombin solution so as to be 1.3 x 10 ⁷ cells / ml. Fibrinogen was diluted 1: 2 with DMEM / F12 (phenol red). The cell suspension and the fibrinogen diluent were mixed using a dual syringe and then sprayed into a bag or culture bag to form a fibrin clot. After 20-30 minutes, if the fibrin clot completely hardened, DMEM / F12 (without phenol red) containing 1 ng / ml basic fibroblast growth factor (bFGF) and 5 ng / ml epidermal growth factor (EGF) 10 < ⁷ > After 72 hours, the cell culture broth was recovered and the cell culture broth was recovered 2 times by adding fresh broth and refrigerated.

Example  3: Quantification of cell growth factor contained in stem cell culture fluid

Cell growth factors contained in stem cell cultures were analyzed by enzyme linked immunosorbent assay (R & D system). To each well of the 96-well plate was added 100 占 퐇 of the stem cell culture solution and reacted at room temperature for 2 hours. If necessary, the culture was diluted to 1: 10 to 1: 100. After washing 4 times with a washing solution, 100 μl of a detection antibody solution capable of recognizing the growth factors was added, followed by reaction for 2 hours. After washing four times with a washing solution, streptavidin-conjugated horseradish peroxidase solution was added in an amount of 100 μl. The mixture was reacted at room temperature for 20 minutes, and a substrate solution (H ₂ O ₂ and tetramethylbenzidine) was added to develop color. 2N H ₂ SO ₄ solution was added to stop the color reaction and the absorbance was measured at 450 nm.

Vascular endothelial growth factor (HGF), vascular endothelial growth factor (VEGF), and vascular endothelial growth factor (VEGF) in the stem cell culture obtained in the three-dimensional culture (3D) according to the present invention and in the stem cell culture obtained at 3 days, 6 days, The results of quantitation of TGF-beta 1 are shown in the following Table 1 and FIG.

Table 1 below shows the content of cell growth factors contained in the stem cell culture liquid after three-dimensional culture (3D) and two-dimensional culture (2D).

EGF VEGF HGF TGF-β1 pg / ml / 10 < ⁶ > cells Day 3 2D 1092.35 6234.50 13201.55 2290.40 3D 18339.38 22935.06 11638.45 1727.10 Day 6 2D 72.99 3446.95 10006.48 1355.79 3D 16162.13 16505.74 19368.41 1515.65 Day 9 2D 2332.40 1737.33 6439.56 474.78 3D 13249.88 7465.54 17960.75 1279.91

As shown in Table 1, when the stem cells were three-dimensionally cultured, the content of the cell growth factor in the stem cell culture fluid was greatly increased, and the concentration of EGF and VEGF was remarkably increased. EGF increased by 16.8, 221.4, and 5.7 times, respectively, and VEGF was increased by 3.7, 4.8, and 4.3 times, respectively, in the culture medium on days 3, 6, and 9. The content of HGF in the culture obtained from the three-dimensional culture was slightly lower than that of the two-dimensional culture on the third day, but increased by 1.9 times and 2.8 times on the sixth and ninth days, respectively. TGF-β1 was also found to be 1.1 times higher on the 6th day and 2.7 times higher on the 9th day in the culture medium obtained by the three-dimensional culture method.

1 shows the contents of EGF, VEGF, HGF and TGF β1 in the adipose stem cell culture obtained on the 3rd, 6th and 9th days after two-dimensional (2D) or three-dimensional (3D) culture of adipose derived mesenchymal stem cells Fig. Fig. As shown in 1, VEGF content in the culture medium obtained in the present invention after the 3-dimensional culture (3D) culture methods according to 3, 6, 9 was respectively 22900, 16500, 7470 pg / ㎖ / 10 6 cells, And 6230, 3450 and 1740 pg / ml / 10 ⁶ cells, respectively, in the conventional culture method (2D).

Therefore, according to the method of the present invention, the effect of increasing the VEGF content in the culture medium obtained at 3, 6, and 9 days after stem cell culture is improved by about 4 times. Especially, the content of epidermal growth factor (EGF) in the culture medium of 3 days was increased about 17 times as compared with the conventional culture method.

Example  4: Stem cell culture medium containing fibroblast Collagen  Secretion measurement

Fibroblast (CCD-986sk) was inoculated on a 48-well plate at 5 × 10 ⁴ per well and cultured for 24 hours. After the medium was discarded and washed with PBS, the stem cell culture was diluted to 0.1%, 1%, and 10% with DMEM and added to each well. After culturing for 24 hours, the amount of collagen was measured by collecting the culture solution. Collagen analysis was performed using a Procollagen type I peptide EIA kit (Takara) and corrected for the amount of collagen contained in the stem cell culture fluid. Table 2 below shows the amount of fibroblast collagen synthesis depending on the stem cell culture fluid content.

Relative increase of collagen Control group 1.00 EGF 10 ng / mL 1.31 3D culture solution 0.1% 1.63 1.0% 2.03 10.0% 3.45

As shown in Table 2, when 0.1%, 1.0%, and 10.0% of the stem cell cultures obtained from the three-dimensional culture were added to the serum-free DMEM medium, collagen synthesis amounts were increased by 1.63, 2.03, and 3.45 times, respectively . When compared with 10 ng / ㎖ of EGF treated with the positive control group, the effect of 0.1% treatment of stem cell culture was more excellent.

FIG. 2 is a graph showing the results of measurement of collagen synthesis amount of fibroblasts after culture of stem cell cultures obtained from three-dimensional cultures in a fibroblast culture medium.

As shown in Table 2 and FIG. 2, since the stem cell culture composition obtained according to the method of the present invention increases the collagen synthesis of fibroblasts, it can be used in medicines and cosmetic compositions intended for wound healing.

Claims (9)

A method for producing a mesenchymal stem cell culture fluid,
(a) culturing mesenchymal stem cells in a serum-derived medium, culturing the medium in a serum-proliferating medium by adding at least one of bFGF or EGF,
(b) culturing the cells obtained from the growth medium in two or more passages in the growth medium,
(c) mixing the suspension of the cultured mesenchymal stem cells with a biocompatible scaffold material suspension, and spraying the mixed suspension to a culture container to form a three-dimensional structure;
(d) a three-dimensional culture step of obtaining a stem cell culture solution at least three times in a serum-free medium supplemented with at least one of bFGF or EGF after the curing is completely cured, and
and (e) collecting the cultured stem cell culture fluid. The method for producing a mesenchymal stem cell culture medium containing a high concentration of growth factor. delete delete delete The method according to claim 1, wherein the biocompatible scaffold material is any one of natural or synthetic high molecular weight growth factor-containing mesenchymal stem cells. delete delete delete delete

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