Method for producing stem cell culture medium with increased growth factor content using a double crosslinked hydrogel scaffold and composition obtained therefrom

Abstract

The present invention relates to a method for producing a stem cell culture medium having an increased content of growth factors using a doubly crosslinked hydrogel scaffold, and a composition obtained therefrom. Specifically, mesenchymal stem cells three-dimensionally cultured using a doubly crosslinked hydrogel scaffold stably attach to the hydrogel scaffold for a long period of time and continuously secrete a large amount of growth factors, specifically HGF, thereby increasing the content in the mesenchymal stem cell culture medium. As a result, it has been confirmed that the mesenchymal stem cell culture medium promotes fibroblast formation and increases collagen secretion. The mesenchymal stem cell culture medium having an increased content of growth factors produced by the method of the present invention is more suitable for clinical use and can be usefully used for clinical purposes.

Description

[Technical field]

[0001] The present invention relates to a method for producing a stem cell culture medium having an increased content of growth factors using a doubly cross-linked hydrogel scaffold, and a composition obtained therefrom. More particularly, the present invention relates to a method for producing a mesenchymal stem cell culture medium having an increased content of growth factors, specifically, hepatocyte growth factor (HGF), through three-dimensional culture of mesenchymal stem cells using a doubly cross-linked hydrogel scaffold, and a composition obtained therefrom. [Background technology]

[0002] It has been discovered that there are several types of progenitor cells in human bone marrow, and among these, progenitor cells that show multi-differentiation potential 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. Mesenchymal stem cells have the ability to self-renew and can differentiate into osteoblasts, chondrocytes, myocytes, marrow stromal cells, tendon ligament fibroblasts, and adipocytes.

[0003] In addition, mesenchymal stem cells are known to secrete a variety of growth factors, cytokines, and extracellular matrix proteins during the culture process. The biologically active factors secreted by mesenchymal stem cells have functions such as suppressing immune responses, inhibiting fibrosis and apoptosis in wound tissue, inducing angiogenesis, and stimulating cell division. Therefore, stem cell culture medium can be used for clinical purposes such as pharmaceutical and cosmetic compositions to promote wound healing.

[0004] Among them, hepatocyte growth factor (HGF) is a representative growth factor secreted from mesenchymal stem cells and is known to act on epithelial cells, endothelial cells and hematopoietic progenitor cells. It mainly plays a role in regulating cell growth, motility and morphogenesis, and is known to play a very central role in wound healing and organ formation by participating in the angiogenesis process, tumor formation and tissue regeneration. The reason why HGF can affect cells in various organs throughout the body is because c-Met, the only receptor for HGF, is widely distributed in the body, and as a result, HGF has recently been in the spotlight as a therapeutic agent that can treat various types of diseases.

[0005] Bovine serum is commonly used to culture mesenchymal stem cells, but culture media containing bovine serum is not suitable for clinical use, and therefore it is desirable to exclude bovine serum from stem cell culture, since the use of animal-derived proteins such as bovine serum is restricted for clinical use of cell culture media.

[0006] Since bovine serum proteins are essential for the proliferation and maintenance of stem cells, a method is required that allows for the cultivation of mesenchymal stem cells in a serum-free medium while still obtaining a culture medium containing high concentrations of physiologically active substances.

[0007] Domestic Patent Publication No. 10-2009-0001163 discloses a method for producing a large amount of bFGF or TGF-β from mesenchymal stem cells by culturing mesenchymal stem cells obtained from human bone marrow, umbilical cord blood or placenta tissue in a serum medium, subculturing them in a serum-free medium, and subjecting the cultured stem cells to a physical stimulus such as hypoxic culture. Similarly, International Patent Publication No. WO2007 / 086637 discloses a method for producing a large amount of bFGF, VEGF or TGF-β from mesenchymal stem cells by culturing adult stem cells extracted from mammalian adipocytes in a serum medium, subculturing them in a serum-free medium, subjecting the cultured stem cells to a physical stimulus such as hypoxic culture, ultraviolet light irradiation, nutrient deprivation or mechanical friction, and adding vitamins A, B, C or D to the culture medium.

[0008] In other words, conventional methods simply culture stem cells in two dimensions, or culture them in two dimensions under low-oxygen conditions to increase the content of VEGF, etc., and when considering that it is difficult to maintain stem cells stably in serum-free medium, there is a problem of reduced physiological activity of the stem cell culture medium.

[0009] Domestic Patent No. 10-1639988 discloses a method for three-dimensionally culturing mesenchymal stem cells with a biocompatible scaffold. However, the fibrin glue used as the biocompatible scaffold has a disadvantage that it has weak structural stability and is gradually degraded during culturing with cells.

[0010] Here, the present inventors have improved the existing manufacturing method using a single-bonded hydrogel scaffold and made efforts to develop a manufacturing method of a stem cell culture medium containing a high concentration of bioactive substances. As a result, by manufacturing mesenchymal stem cells in the form of a cell-double crosslinked hydrogel scaffold and culturing them in three dimensions, the mesenchymal stem cells stably attached to the scaffold for a long time during culture and continuously secreted growth factors, and in particular, the HGF content of the stem cell culture medium obtained from this was dramatically increased, and the ability to promote fibroblast proliferation was improved. In addition, it was confirmed that this increases the collagen secretion ability of mesenchymal stem cells and fibroblasts. Therefore, the present invention was completed by determining that the mesenchymal stem cell culture medium manufactured by the manufacturing method of the present invention is more suitable for clinical use. Summary of the Invention [Problem to be solved by the invention]

[0011] In the conventional method, a method of three-dimensionally culturing stem cells using biocompatible hydrogel in serum-free medium was used to produce a culture solution containing active ingredients such as high concentrations of growth factors and cytokines secreted from mesenchymal stem cells. When using biocompatible hydrogels, biocompatible hydrogels with good cell adhesion (e.g., fibrin gel) have the disadvantage of being structurally weak and gradually degraded during cell culture, while structurally stable biocompatible hydrogels (e.g., alginate) have the disadvantage of low cell adhesion and reduced active ingredients secreted by stem cells.

[0012] The object of the present invention is to provide a method for producing a stem cell culture medium with increased growth factor content by combining a component that can structurally encapsulate cells without being decomposed during long-term three-dimensional culture and a component that has excellent cell attachment ability (has the property of being able to easily attach cells), and to provide a composition obtained therefrom using a doubly crosslinked hydrogel scaffold that has the advantages of these two types of supports. [Means for solving the problem]

[0013] In order to achieve the object of the present invention, (1) mixing an alginate solution and a fibrinogen solution to prepare an alginate-fibrinogen mixture; (2) adding the alginate-fibrinogen mixture of step (1) to mesenchymal stem cells and then suspending the mesenchymal stem cells; (3) adding a solution containing a divalent cation and a thrombin solution to the mesenchymal stem cell suspension of step (2) to form a mesenchymal stem cell-doubly crosslinked hydrogel; and (4) After forming the mesenchymal stem cell-doubly crosslinked hydrogel in step (3), the mesenchymal stem cell is three-dimensionally cultured in a serum-free medium supplemented with at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor) to obtain a mesenchymal stem cell culture solution.

[0014] The present invention provides a method for producing a mesenchymal stem cell culture medium in which the content of growth factors, specifically, hepatocyte growth factor (HGF), is significantly increased.

[0015] The present invention also provides a mesenchymal stem cell culture medium in which the content of growth factors, specifically HGF, produced by the above-mentioned method is significantly increased. Effect of the Invention

[0016] The inventors performed 3D culture of mesenchymal stem cells using a double-crosslinked hydrogel scaffold that has the advantages of two types of hydrogels, one that can structurally encapsulate cells without being degraded during long-term 3D culture, and the other that can easily attach cells. As a result, the shape of the hydrogel scaffold is well maintained even after long-term culture, and the mesenchymal stem cells inside it are stably attached for a long time, and a large amount of growth factors, specifically HGF, are continuously secreted, which significantly increases the content of the mesenchymal stem cell culture medium compared to the mesenchymal stem cell culture medium produced with the conventional single-bonded hydrogel scaffold. In addition, the total amount of HGF secreted during the culture period was also significantly increased. As a result, the ability to promote fibroblast proliferation was improved, and collagen secretion of mesenchymal stem cells and fibroblasts was increased.

[0017] Therefore, the mesenchymal stem cell culture medium with a significantly increased content of growth factors, specifically HGF, prepared by the method of the present invention is more suitable for clinical use and can be usefully used for clinical purposes. [Brief description of the drawings]

[0018] [Figure 1a]FIG. 1a shows the relative content of HGF (FIG. 1a), VEGF (FIG. 1b), and EGF (FIG. 1c) in mesenchymal stem cell culture medium collected on days 3, 6, and 9 after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to one embodiment of the present invention. [Figure 1b] FIG. 1a shows the relative content of HGF (FIG. 1a), VEGF (FIG. 1b), and EGF (FIG. 1c) in mesenchymal stem cell culture medium collected on days 3, 6, and 9 after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to one embodiment of the present invention. [Figure 1c] FIG. 1a shows the relative content of HGF (FIG. 1a), VEGF (FIG. 1b), and EGF (FIG. 1c) in mesenchymal stem cell culture medium collected on days 3, 6, and 9 after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to one embodiment of the present invention. [Diagram 2] FIG. 1 shows the percentage difference in the total HGF content in mesenchymal stem cell culture medium collected on days 3, 6, 9, and 12 after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-linked hydrogel scaffold according to one embodiment of the present invention. [Diagram 3] FIG. 13 is a diagram showing the fibroblast proliferation ability after culturing fibroblasts by adding mesenchymal stem cell culture medium recovered after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to an embodiment of the present invention in a volumetric amount. [Figure 4]FIG. 1 shows the collagen content in mesenchymal stem cell culture medium collected after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to one embodiment of the present invention. [Diagram 5] FIG. 1 shows the amount of collagen secreted by fibroblasts after culturing by adding mesenchymal stem cell culture medium recovered after three-dimensional culture using a dual-crosslinked hydrogel scaffold or a single-bonded hydrogel scaffold according to an embodiment of the present invention to fibroblasts in different volumes and culturing the cells. Best Mode for Carrying Out the Invention

[0019] The terms used in the present invention are defined as follows.

[0020] As used herein, "mesenchymal stem cells" are defined as cells that are capable of self-renewal, have pluripotency, and express CD73 + , CD90 + , CD105 + , CD14 - , CD20 - , CD34 - , CD45 - The term "cells" refers to cells expressing a cell phenotype that has been previously described and may be isolated from, but is not limited to, fat, bone marrow, umbilical cord blood, liver, muscle, etc.

[0021] As used herein, the term "stem cell culture medium" refers to a cell culture supernatant in which stem cells are cultured. The stem cell culture medium contains various physiologically active substances secreted from cells during the stem cell culture process.

[0022] The present invention will now be described in more detail.

[0023] The present invention (1) mixing an alginate solution and a fibrinogen solution to prepare an alginate-fibrinogen mixture; (2) adding the alginate-fibrinogen mixture of step (1) to mesenchymal stem cells and then suspending the mesenchymal stem cells; (3) adding a solution containing a divalent cation and a thrombin solution to the mesenchymal stem cell suspension of step (2) to form a mesenchymal stem cell-doubly crosslinked hydrogel; and (4) After forming the mesenchymal stem cell-doubly crosslinked hydrogel in step (3), the method includes three-dimensionally culturing the mesenchymal stem cell in a serum-free medium supplemented with at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor) to obtain a mesenchymal stem cell culture solution.

[0024] The present invention provides a method for producing a mesenchymal stem cell culture medium having an increased content of growth factors.

[0025] In the method according to the present invention, in step (1), the alginate solution may be mixed at 4 to 40 mg / ml, specifically 5 to 30 mg / ml, more specifically 15 to 25 mg / ml, and the fibrinogen solution may be mixed at 1 to 40 mg / ml, specifically 5 to 30 mg / ml, more specifically 10 to 20 mg / ml, but is not limited thereto.

[0026] In addition, the alginate solution and the fibrinogen solution may be mixed in a weight ratio of 1:10 to 10:1, but is not limited thereto.

[0027] In the method according to the present invention, in step (2), mesenchymal stem cells can be isolated from fat, bone marrow, umbilical cord blood, liver or muscle, more specifically, but not limited to, fat. In particular, fat-derived stem cells have the advantage of being relatively easy to access and easy to collect, and can be obtained in large quantities from one individual.

[0028] In addition, the mesenchymal stem cells may be subcultured in a stromal medium and a proliferation medium, and more specifically, may be cultured in the following stages: a) culturing mesenchymal stem cells in a stromal medium and then in a proliferation medium; and b) subculturing said cells in a growth medium.

[0029] The growth medium is preferably a stromal medium supplemented with at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor).

[0030] In the above culture method, in step b), the cells are preferably cultured in a growth medium for one or more passages, more preferably for two or more passages, but is not limited thereto.

[0031] Furthermore, it is preferable that the proliferation medium is a matrix medium to which one or more of bFGF (basic fibroblast growth factor) or EGF (epidermal growth factor) is added. Specifically, it is preferable that the bFGF is added at 0.1 to 100 ng / ml and the EGF is added at 0.1 to 100 ng / ml, it is more preferable that the bFGF is added at 0.3 to 10 ng / ml and the EGF is added at 1 to 10 ng / ml, and it is even more preferable that the bFGF is added at 0.5 to 5 ng / ml and the EGF is added at 3 to 7 ng / ml, but this is not limited thereto.

[0032] In the method according to the present invention, the divalent cation in step (3) is Ca 2+ , Mg 2+ , Sr 2+ or Ba 2+ Examples of the solution containing the divalent cation include, but are not limited to, calcium chloride (C a Cl2), calcium lactate (C6H 10 O6Ca?5H2O), magnesium chloride (MgC l2 ), strondium chloride (SrC l2 ) or barium chloride (BaC l2 ), but are not limited to these.

[0033] In addition, in step (3), the solution containing a divalent cation may be added at a concentration of, but is not limited to, 10 to 300 mM, specifically 50 to 200 mM, more specifically 80 to 120 mM, and even more specifically 90 to 110 mM.

[0034] In the method according to the present invention, the thrombin solution may be added in step (3) at a concentration of, but not limited to, 1 to 25 U / ml, specifically 3 to 20 U / ml, even more specifically 4 to 15 U / ml, and even more specifically 5 to 10 U / ml.

[0035] In the method according to the present invention, a doubly crosslinked hydrogel scaffold having the advantages of two types of supports is formed by using a hydrogel component capable of structurally encapsulating cells without being decomposed during long-term three-dimensional culture through steps (1) to (3) and a hydrogel component having properties to which cells can easily attach, and mesenchymal stem cells can stably exist within the doubly crosslinked hydrogel.

[0036] In the method according to the present invention, it is preferable to add at least one of bFGF and EGF to the serum-free medium in step (4). Specifically, it is preferable to add 0.1-100 ng / ml of the bFGF and 0.1-100 ng / ml of the EGF, it is more preferable to add 0.3-10 ng / ml of the bFGF and 1-10 ng / ml of the EGF, and it is even more preferable to add 0.5-5 ng / ml of the bFGF and 3-7 ng / ml of the EGF, but the present invention is not limited thereto.

[0037] In the method according to the present invention, in the step (4), it is preferable to obtain the culture medium three or more times, specifically at 2-4 day intervals, more specifically at 3 day intervals, while changing the medium. In particular, it is preferable to obtain the culture medium three or more times in order to obtain a large amount of stem cell culture medium from a limited number of cells and to increase the content of growth factors contained in the culture medium.

[0038] In the method according to the present invention, growth factors, specifically HGF, are stably secreted from mesenchymal stem cells in the doubly crosslinked hydrogel scaffold during the culture period through steps (1) to (4), and the HGF content in the obtained mesenchymal stem cell culture medium is significantly increased. As a result, the obtained mesenchymal stem cell culture medium has an improved ability to promote fibroblast proliferation and increases the amount of collagen secreted by fibroblasts and stem cells. Specifically, the obtained mesenchymal stem cell culture medium can contain 700 to 900 ng / ml of collagen.

[0039] The culture of mesenchymal stem cells according to the present invention can be carried out according to the following process, and the medium used for cell culture is not limited thereto.

[0040] (1) Cultivation of mesenchymal stem cells

[0041] The mesenchymal stem cells obtained from the tissue were suspended in a stromal medium and cultured at 10,000 to 40,000 cells / cm. 2 The cells are inoculated into a culture vessel at a concentration of 10%, and then cultured in a substrate medium of DMEM or DMEM / F12 (Dulbecco's Modified Eagle Medium / Ham's F-12 Nutrient Broth) containing 10% bovine serum for approximately 24 hours.

[0042] (2) Cultivation in expansion media

[0043] After removing the substrate medium, the cells are cultured in a proliferation medium to proliferate the adherent cells. The proliferation medium is DMEM or DMEM / F12 containing 10% bovine serum, 0.1-100ng / ml of EGF (epidermal growth factor) and / or 0.1-100ng / ml of bFGF (basic fibroblast growth factor), which acts to rapidly proliferate adherent mesenchymal stem cells and greatly increase the cell mass in a short period of time.

[0044] (3) Subculture

[0045] When the cells fill 80-90% 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, the cells are diluted 1:3-1:4 and cultured in a new culture vessel with the growth medium. This method allows additional subculture.

[0046] (4) Three-dimensional culture using double-crosslinked hydrogel scaffolds

[0047] The cultured cells are washed with PBS at least three times to remove FBS, and an alginate-fibrinogen mixture prepared by mixing an alginate solution and a fibrinogen solution is added, after which the cells are suspended. The suspension is transferred to a plate, a bar, or a culture bag, and a solution containing a divalent cation, such as a calcium chloride solution and a thrombin solution, is uniformly applied to form a cell-doubly crosslinked hydrogel scaffold. The cell-doubly crosslinked hydrogel scaffold is cultured in DMEM or DMEM / F12 containing 0.1-100ng / ml of EGF (epidermal growth factor) and / or 0.1-100ng / ml of bFGF (basic fibroblast growth factor).

[0048] The present invention also provides a mesenchymal stem cell culture medium having an increased content of growth factors, which is prepared by the method of the present invention.

[0049] In order to obtain a mesenchymal stem cell culture medium with an increased content of growth factors, the inventors performed three-dimensional culture of mesenchymal stem cells using a double-crosslinked hydrogel scaffold that has the advantages of two types of hydrogels: a hydrogel component that can structurally encapsulate cells without being decomposed during long-term three-dimensional culture, and a hydrogel component that has the property of allowing cells to attach well. As a result, the shape of the hydrogel scaffold is well maintained even during long-term culture, and the mesenchymal stem cells inside it are stably attached for a long time, and the content of the mesenchymal stem cell culture medium is increased by continuously secreting a large amount of growth factors, specifically HGF. In addition, it was confirmed that the HGF content in the mesenchymal stem cell culture medium was significantly increased, improving the ability to promote fibroblast proliferation and increasing collagen secretion from mesenchymal stem cells and fibroblasts.

[0050] Therefore, the mesenchymal stem cell culture medium with increased growth factor content prepared by the method of the present invention is more clinically suitable and can be usefully used for clinical purposes.

[0051] The present invention will be described in detail below with reference to examples and experimental examples.

[0052] However, the following examples and experimental examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

[0053] Example 1. Method for culturing mesenchymal stem cells derived from human adipose tissue

[0054] Adipose tissue is typically obtained by, but not limited to, liposuction.

[0055] Adipose stem cells were isolated from adipose tissue obtained by liposuction as follows: The adipose tissue was washed 3-4 times with the same volume of KRB solution to remove blood. The same volume of collagenase solution as the adipose tissue was added and reacted in a 37°C water bath. This was transferred to a centrifuge tube and centrifuged at 20°C and 1,200 rpm for 10 minutes. The upper layer, the adipose layer, was removed, and the lower layer, the collagenase solution, was carefully separated without shaking. After adding a substrate medium and suspending, it was centrifuged at 20°C and 1,200 rpm for 5 minutes. At this time, the stromal vascular fraction sank to the bottom, and the upper layer was removed.

[0056] The stromal-vascular fraction was suspended in stromal medium, inoculated into a culture vessel, and cultured for 24 hours in a 37℃, 5% CO2 incubator. After removing the culture medium, the cells were washed with phosphate buffer solution and then cultured in stromal medium (DMEM / F12 medium containing 10% FBS) or in a proliferation medium containing basic fibroblast growth factor (bFGF) 1ng / ml, epidermal growth factor (EGF) 5ng / ml, or fibroblast growth factor (bFGF) 1ng / ml and epidermal growth factor (EGF) 5ng / ml. When the adipose stem cells grew to about 80-90% of the culture vessel, they were treated with trypsin to separate them into single cells and harvested. The obtained cells were diluted 1:3-1:4 with proliferation medium and subcultured.

[0057] Example 2. Method for three-dimensional culture of human adipose-derived mesenchymal stem cells on a double-crosslinked hydrogel scaffold

[0058] To prepare the dual-crosslinked hydrogel scaffolds, alginate solution was mixed with fibrinogen solution at a final concentration of 20 mg / ml and 15 mg / ml, respectively. This alginate-fibrinogen mixture was mixed thoroughly before use.

[0059] The stem cells obtained as single cells by trypsinization in Example 1 were washed three times with DMEM / F12 (without phenol red) medium to remove FBS. 7The alginate-fibrinogen mixture was added to the cells at a cell concentration of 4.0 × 10 cells / mL, and the cells were suspended. The cells were mixed thoroughly using a pipette, and then sprinkled onto a plate, barbecue, or culture bag. A solution of 100 mM calcium chloride (CaCl2) and 6.25 U / mL thrombin solution was applied to the cell-hydrogel mixture to form a cell-doubly crosslinked hydrogel scaffold. After 30 minutes, when the hydrogel scaffold had completely solidified, DMEM / F12 (without phenol red) medium containing 1 ng / mL basic fibroblast growth factor (bFGF) and 5 ng / mL epidermal growth factor (EGF) was added at a concentration of 4.0 × 10 cells / mL. 7 The cell culture medium was collected after 72 hours, and fresh medium was added to each well. The cell culture medium was collected 2 to 4 times and stored in a refrigerator.

[0060] Comparative Example 1. Method for three-dimensional culture of human adipose-derived mesenchymal stem cells on a single-bonded hydrogel scaffold

[0061] The stem cells obtained as single cells by trypsinization in Example 1 were washed three times with DMEM / F12 (without phenol red) medium to remove FBS. 7 Alginate solution was added to suspend the cells at a concentration of 4.0 × 10 cells / mL. The cells were mixed thoroughly using a pipette and then dispersed from a barbell or culture bag. 100 mM calcium chloride (CaCl2) solution was evenly applied to the cell-hydrogel mixture to form a cell-hydrogel scaffold. After 30 minutes, when the cell-hydrogel scaffold had completely solidified, DMEM / F12 (without phenol red) medium containing 1 ng / mL basic fibroblast growth factor (bFGF) and 5 ng / mL epidermal growth factor (EGF) was added at a concentration of 4.0 × 10 cells / mL. 7 The cell culture medium was collected after 72 hours, and fresh medium was added to each well. The cell culture medium was collected 2 to 4 times and stored in a refrigerator.

[0062] Experimental example 1. Analysis of growth factors contained in mesenchymal stem cell culture medium

[0063] The growth factors contained in the mesenchymal stem cell culture medium collected on days 3, 6, 9, and 12 of three-dimensional culture in Example 2 were analyzed using enzyme-linked immunosorbent assay (ELISA, R&D systems).

[0064] Specifically, 100 μL of mesenchymal stem cell culture medium recovered from Example 2 or Comparative Example 1 was added to each well of a 96-well plate and reacted at room temperature for 2 hours. If necessary, the culture medium was diluted 1:10 or 1:100 before addition. After washing four times with a washing solution, 100 μL of detection antibody was added and reacted at room temperature for 2 hours. After washing four times again with a washing solution, 100 μL of streptavidin-bound horseradish-peroxidase solution was added and reacted at room temperature for 20 minutes, and a substrate solution (tetramethyl benzidine and H2O2) was added to develop color. 2NH2SO4 solution was added to terminate the color reaction, and the absorbance at 450 nm was measured.

[0065] As a result, as shown in Figures 1a to 1c, it was confirmed that the mesenchymal stem cell culture medium (Dual scaffold) recovered after three-dimensional culture using the double cross-linked hydrogel scaffold of Example 2 contained about 5 times more HGF than the mesenchymal stem cell culture medium (Single scaffold) recovered after three-dimensional culture using the single bonded hydrogel scaffold of Comparative Example 1. On the other hand, it was confirmed that there was no significant difference in the VEGF and EGF contents between the mesenchymal stem cell culture medium (Dual scaffold) recovered after three-dimensional culture using the double cross-linked hydrogel scaffold of Example 2 and the mesenchymal stem cell culture medium (Single scaffold) recovered after three-dimensional culture using the single bonded hydrogel scaffold of Comparative Example 1.

[0066] In addition, as shown in FIG. 2, it was confirmed that the mesenchymal stem cell culture medium recovered after three-dimensional culture using the doubly cross-linked hydrogel scaffold of Example 2 contained approximately 3.5 times more HGF than the mesenchymal stem cell culture medium recovered after three-dimensional culture using the single-bonded hydrogel scaffold of Comparative Example 1.

[0067] Experimental example 2. Confirmation of fibroblast proliferation ability with the addition of mesenchymal stem cell culture medium

[0068] The proliferation ability of the fibroblasts in Example 2, which were three-dimensionally cultured and collected on the 3rd, 6th, and 9th days, to which the mixture of mesenchymal stem cell culture medium had been added, was confirmed.

[0069] Specifically, fibroblasts (Normal Human Dermal Fibroblast) were placed in a 24-well plate at 5.0 × 10 per well. 4 After 24 hours of incubation, the supernatant was removed, and serum-free FBM medium was added and incubated for 6 hours. After that, the supernatant was removed, and the mesenchymal stem cell culture solution recovered in Example 2 was diluted to 5%, 10%, and 20%, or the mesenchymal stem cell culture solution recovered in Comparative Example 1 was diluted to 20% and 50%, and added to each well. After 24 hours of incubation, the supernatant was removed, and 350 μL of serum-free medium containing tetrazolium salt was added to each well to develop color. After 1 to 3 hours of reaction, the absorbance at 450 nm was measured. Fibroblasts cultured in serum-free FBM medium were used as a negative control, and fibroblasts cultured in FGM medium, which is a medium containing 0.1% insulin, 0.1% rhFGF-B, and 2% FBS, which are ingredients that allow fibroblasts to grow optimally, were used as a positive control.

[0070] As a result, as shown in FIG. 3, it was confirmed that the mesenchymal stem cell culture medium recovered after three-dimensional culture using the doubly cross-linked hydrogel scaffold of Example 2 had a more excellent ability to promote fibroblast proliferation than the mesenchymal stem cell culture medium recovered after three-dimensional culture using the single-bonded hydrogel scaffold of Comparative Example 1.

[0071] Experimental Example 3. Analysis of collagen content in mesenchymal stem cell culture medium and fibroblasts treated with it

[0072] The collagen content in the mesenchymal stem cell culture medium collected after 72 hours of three-dimensional culture in Example 2 and the collagen secreted from the fibroblasts treated therewith were confirmed.

[0073] Specifically, fibroblasts were plated in a 24-well plate at 5 × 10 cells per well. 4 After inoculation at 100 cells / well, the cells were cultured for 24 hours. The supernatant was removed, and the mesenchymal stem cell culture medium recovered from Example 2 or the mesenchymal stem cell culture medium recovered from Comparative Example 1 was diluted to 20% or 50% in serum-free medium, respectively, and added to each well. The supernatant obtained after culturing for 24 hours was used for analysis.

[0074] Collagen analysis was performed using Procollagen type 1 peptide EIAKit (Takara), and both the original mesenchymal stem cell culture medium and the fibroblast supernatant obtained by treating the mesenchymal stem cell culture medium were analyzed.

[0075] As a result, as shown in FIG. 4, it was confirmed that the mesenchymal stem cell culture medium recovered after three-dimensional culture using the doubly cross-linked hydrogel scaffold of Example 2 had an increased collagen content compared to the mesenchymal stem cell culture medium recovered after three-dimensional culture using the single-bonded hydrogel scaffold of Comparative Example 1.

[0076] In addition, as shown in FIG. 5, it was confirmed that the amount of collagen secreted by fibroblasts treated with mesenchymal stem cell culture medium recovered after three-dimensional culture using the doubly cross-linked hydrogel scaffold of Example 2 was increased compared to the amount of collagen secreted by fibroblasts treated with the mesenchymal stem cell culture medium recovered after three-dimensional culture using the single-bonded hydrogel scaffold of Comparative Example 1. [Industrial Applicability]

[0077] The mesenchymal stem cell culture medium with increased growth factor content prepared by the method of the present invention is more suitable for clinical use and can be usefully used for clinical purposes.

Claims

1. mixing an alginate solution and a fibrinogen solution to prepare an alginate-fibrinogen mixture; adding the alginate-fibrinogen mixture of step (1) to mesenchymal stem cells and then suspending the mesenchymal stem cells; Adding a divalent cation-containing solution and a thrombin solution to the mesenchymal stem cell suspension of step (2) to form a mesenchymal stem cell-doubly crosslinked hydrogel; and After forming the mesenchymal stem cell-double crosslinked hydrogel in the step (3), the mesenchymal stem cells are three-dimensionally cultured in a serum-free medium containing at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor) to obtain a mesenchymal stem cell culture solution. A method for producing a mesenchymal stem cell culture medium with increased growth factor content.

2. 2. The method according to claim 1, wherein the alginate solution is mixed at 4 to 40 mg / ml in step (1).

3. 2. The method according to claim 1, wherein the alginate solution is mixed at 1 to 40 mg / ml in step (1).

4. 2. The method according to claim 1, wherein in step (1), the alginate solution and the fibrinogen solution are mixed in a weight ratio of 1:10 to 10:

1.

5. 2. The method of claim 1, wherein in step (2), the mesenchymal stem cells are isolated from any one selected from the group consisting of adipose tissue, bone marrow, umbilical cord blood, liver, and muscle.

6. In the step (2), the mesenchymal stem cells are a) culturing mesenchymal stem cells in a stromal medium and culturing them in a proliferation medium; and b) mesenchymal stem cells cultured by subculturing the cells in a growth medium; The method according to claim 1, wherein the growth medium is a stromal medium supplemented with at least one of bFGF (basic fibroblast growth factor) and EGF (epidermal growth factor).

7. The method according to claim 6, wherein the culture is performed for at least two passages in step b).

8. 7. The method according to claim 1, wherein the bFGF is added at 0.1 to 100 ng / ml and the EGF is added at 0.1 to 100 ng / ml.

9. In step (3), the divalent cation is Ca ２＋ , Mg ２＋ , Sr ２＋ , Ba ２＋ The method according to claim 1, characterized in that the method is any one selected from the group consisting of:

10. 2. The method of claim 1, wherein in step (3), a solution containing divalent cations is added at 10 to 300 mM.

11. 2. The method of claim 1, wherein the thrombin solution is added in an amount of 1 to 25 U / ml in step (3).

12. The method of claim 1, wherein in the step (4), the three-dimensional culture is performed by replacing the medium every 2 to 4 days to obtain a stem cell culture solution three or more times.

13. The method according to claim 1, wherein the growth factor is HGF.

14. 2. The method of claim 1, wherein the mesenchymal stem cell culture medium with increased growth factor content contains 700 to 900 ng / ml of collagen.

15. 15. A mesenchymal stem cell culture medium having an increased content of growth factors, produced by the method according to any one of claims 1 to 14.

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