KR20160000029A - Mehtod for inducing differentiation of mesenchymal stromal cells using light with red wavelength - Google Patents

Abstract

The present invention relates to a method for forming spheroid from mesenchymal stem cells derived from fat and differentiating the formed spheroid into vascular endothelial cells, by irradiating light in the red wavelength range for the mesenchymal stem cells derived from fat by using a LED light source. The method of the present invention can differentiate and induce the stem cells to the vascular endothelial cells, without using a differentiation-inducing material, and can be broadly used for developing drug for treating vascular diseases in an economical manner.

Description

{METHOD FOR INDUCING DIFFERENTIATION OF MESENCHYMAL STOMAL CELLS USING LIGHT WITH RED WAVELENGTH}

The present invention relates to a method for inducing the differentiation of mesenchymal stem cells using light of a red wavelength band. More specifically, the present invention relates to a method for inducing differentiation of mesenchymal stem cells using light of a red wavelength band, To form spleoids, and to differentiate the formed spheroids into vascular endothelial cells.

In general, differentiation refers to the process by which cells in the early stages become characteristic of each tissue, a representative example of which is seen in the development of animals. In other words, 'embryogenesis' must occur in order for a single cell called embryo, which is made by combining sperm and egg, to be made into various tissue cells such as bone, heart and skin. Stem cells that have this differentiation ability are the cells of the pre-differentiation stage of each cell that constitutes the tissue. It is capable of infinite proliferation in the undifferentiated state, and has the potential to be differentiated into various tissue cells by a specific differentiation stimulus ≪ / RTI >

Recently, it has been reported that the adipose tissue contains an undifferentiated cell group that is excellent in self-renewal ability and can be differentiated into various cells such as liver, bone, cartilage, fat, blood vessels, heart, nervous system, etc. Cucin et al., BBRC 301: 1016, 2003; Miranville et al., Circulation 110: 349, 2004; S. Gronthos et al., J. Cell Physiol. 189: 54, 2001; MJ Seo et al., BBRC 328: 258, 2005). As adipose-derived stem cells (ASCs), which are multipotential stem cells derived from adipose tissue, can be extracted in a large amount compared to mesenchymal stem cells, the acquisition process is easy and safe, And it has advantages in terms of tissue accessibility, stability, efficacy, and economics, and is attracting attention as a new source of multipotential stem cells.

Human adipose stem cells (BBRC 330: 142, 2005), human adipose stem cells capable of differentiating into osteogenic and adipocytes (Ying Cao et al., BBRC 332: 370, 2005), human adipose stem cells capable of differentiating into neurons (Kristine M. Safford et al., BBRC 294: 371, 2005), adipocyte stem cell differentiating into adipocytes (Rei Ogawa et al., BBRC 319: (Hani A. Awad et al., Biomaterials 25: 3211, 2004), which can differentiate into osteogenic and cartilage-forming cells (Rei Ogawa et al., BBRC 313: 871, (Juri Fujimura et al., BBRC 333: 116, 2005) capable of differentiating into neurons, and adipose stem cells (US Patent No. 6,777, 231) capable of differentiating into bone cells, chondrocytes, neurons or muscle cells have.

In addition, it has been reported that the adipose stem cells can be induced to differentiate into vascular endothelial cells in vivo and in vitro. For example, adipocyte stem cells can be differentiated into vascular endothelial cells by culturing in a culture vessel coated with matrigel, a special extracellular matrix component isolated from sarcoma mice, using a medium supplemented with various growth factors (Cao Y, et al., Biochem. Biophys. Res. Commun. 332: 370-379, 2005). After forming adipocytes (spheroids) by culturing adipose stem cells in serum-free medium, (Fig. 1). In this study, we have shown that Flk-1-expressing vascular endothelial cells can be obtained by culturing for one week at 37 ° C. However, these methods require more than three weeks for differentiation, and contamination may occur due to long-term cell culture. In order to differentiate into specific cells, special culture medium such as growth factor must be used. It is difficult to do so. In addition, conventionally, in order to induce mesenchymal stem cells into vascular cells, they have been cultured in a culture medium or a culture medium containing a vascular cell inducing component, and there is no equipment for promoting the differentiation of vascular cells.

Under these circumstances, the present inventors have made extensive efforts to develop a method for differentiating adipose-derived mesenchymal stem cells into vascular endothelial cells more effectively. As a result, it has been found that when adipose-derived mesenchymal stem cells are cultured in a spheroid form, It was found that the stem cells can be effectively differentiated into vascular endothelial cells, and the present invention has been completed.

One object of the present invention is to provide a method for differentiating adipose-derived mesenchymal stem cells into vascular endothelial cells, comprising culturing spleens of adipose-derived mesenchymal stem cells while irradiating light with a red wavelength band.

The present inventors paid attention to a method of irradiating light using an LED light source while performing various studies in order to develop a method that can more effectively differentiate adipose stem cells into endothelial cells. That is, when the stem cells are cultured and the LED light source is used to irradiate the light of the red wavelength band, the proliferation and mobility of the stem cells are enhanced to promote the formation of spoloids, and the formed spoloids are continuously irradiated with the red wavelength band light , Expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) capable of promoting differentiation into vascular endothelial cells are promoted in stem cells forming the above spheroids, Alternatively, without the genetic manipulation, the differentiation of the spheroids into vascular endothelial cells can be promoted.

As a result of studying the conditions for improving the differentiation efficiency of differentiating the adipocyte stem cells into vascular endothelial cells by irradiating light of the red wavelength band, it was found that light having a wavelength of 600 to 700 nm and a power of 5 to 30 mW / The amount of energy is 3 to 18 J / cm 2), and the culture medium of adipose-derived mesenchymal stem cells is subjected to a surface treatment with a polymer imparting hydrophobicity to the cell culture container, or a hydrophobic cell culture container (non-tissue culture- treated well plate, and it was confirmed that it is preferable to inoculate adipose stem cells into the culture vessel in an amount of 1 x 10 ⁵ cells or more in each well of a 24-well plate.

In order to accomplish the above object, the present invention provides a method for differentiating adipose stem cells into vascular endothelial cells by irradiating red stem cells with red wavelength band light.

Specifically, the method for differentiating adipocyte stem cells provided by the present invention into vascular endothelial cells comprises culturing adipocyte stem cells in a culture vessel having a hydrophobic surface and culturing the adipocyte stem cell with a power of 5 to 15 mW / To the adipose stem cells.

The term "mesenchymal stromal cells (MSCs)" of the present invention refers to mesenchymal stromal cells (MSCs) having excellent self-renewal ability and capable of differentiating into various cells such as liver, bone, cartilage, fat, blood vessel, Stem cells.

In the present invention, adipose-derived stem cells (ASCs) were used as the mesenchymal stem cells. Since the adipose-derived mesenchymal stem cells can be obtained from adipose tissue that can be extracted in large quantities in comparison with other kinds of mesenchymal stem cells, the process is easy and safe, It is advantageous in terms of tissue accessibility, stability, effectiveness, and economy.

The method of differentiating the adipose-derived mesenchymal stem cells into vascular endothelial cells according to the present invention essentially includes irradiating the adipose-derived mesenchymal stem cells with light of a red wavelength band. Promoting the proliferation and migration of stem cells to promote the formation of a celloid known as a spheroid or a 3D cell mass (3DCM), and when the light is continuously irradiated onto the spheroid, The expression and secretion of various growth factors (FGF, VEGF, HGF, etc.) capable of promoting the differentiation of stem cells into the vascular endothelial cells are promoted, and the accumulation or accumulation of the spheroids And the growth factor activates the differentiation signal transduction involved in stem cell differentiation, so that the concentration of the spheroid into the vascular endothelial cells It is possible to promote anger.

At this time, the light is not particularly limited as long as it exhibits an effect of promoting the differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, but it is preferable to use light generated from an LED (light emitting diode) Preferably, light generated from the LED and having a wavelength of 600 to 700 nm and a power of 5 to 30 mW / cm 2 can be used, most preferably a light generated from the LED and having a wavelength of 660 nm and a power of 10 mW / Can be used.

In addition, the light irradiation condition is not particularly limited as long as it is an effect of promoting differentiation of adipose derived mesenchymal stem cells into vascular endothelial cells, but preferably an interval of 1 to 5 cm from adipose derived mesenchymal stem cells A method of irradiating the LED light source for 10 to 15 minutes per day may be used.

In addition, in the culture solution or culture matrix for culturing the adipose stem cells, a substance inducing differentiation of stem cells into vascular endothelial cells is treated or light is irradiated at a red wavelength band without genetic modification to form spleen formation and angiogenesis factor By inducing expression, stem cells can be differentiated into vascular endothelial cells. In this case, the culture solution to be used is not particularly limited as long as it is a medium commonly used for culture and / or differentiation of adipose stem cells. Preferably, the medium is a medium containing serum such as DMEM (Dulbeco's modified eagle medium) or Ham's F12 Can be used. For example, in an embodiment of the present invention, a medium supplemented with fetal bovine serum (FBS) was used in DMEM / F12 medium mixed with DMEM and Ham's F12 at a ratio of 1: 1. However, it is also possible to carry out the method of the present invention using a medium containing no serum.

In addition, in order to promote the differentiation of the adipose derived mesenchymal stem cells, the adipose-derived mesenchymal stem cells may be inoculated into a culture container surface-treated with a hydrophobic polymer or a culture container made of the polymer . The hydrophobicity-imparting polymer is not particularly limited, but is preferably selected from the group consisting of polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE) Poly (L-lactic acid) (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA) as polypropylene (PP), polytetrafluoroethylene (PTFE) ), Poly (caprolactone) (PCL), poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives or copolymers thereof (poly (lactic acid-co-glycolic acid) ), Poly (L-lactic acid-co-caprolactone) (PLCL), poly (glycolic acid-co-caprolactone) (PGCL) and the like.

Finally, in order to easily form spheroids from the adipose-derived mesenchymal stem cells, the adipose-derived mesenchymal stem cells are preferably inoculated at a density of 1 × 10 ⁵ to 2 × 10 ⁶ cells / cm 2. When inoculated at a density of less than 1 × 10 ⁵ cells / cm 2, the size of the spheroid is small and the deviation is severe. In the case of inoculation at a density of 2 × 10 ⁶ cells / cm 2 or more, Lt; / RTI > However, when inoculated at a density of 1 x 10 < ⁵ > to 2 x 10 < ⁶ > cells / cm2 or more, sprooids having a diameter of about 1 mm detectable by the naked eye can be formed.

The vascular endothelial cells differentiated by the above method can be used for the treatment of various vascular diseases or wounds. The vascular diseases include, but are not limited to, cardiovascular diseases, cerebrovascular diseases, ischemic diseases, More preferably, it may be a disease such as arteriosclerosis, stable and unstable angina pectoris, peripheral cardiovascular disease, hypertension, heart failure, peripheral circulatory disorder, myocardial infarction, stroke, transient and ischemic seizure, subarachnoid hemorrhage.

The method of the present invention can induce the differentiation of stem cells into vascular endothelial cells without using a differentiation inducing substance, and thus it can be widely used for development of a more cost effective vascular disease therapeutic agent.

Fig. 1 is a micrograph showing the results of verifying stem cells isolated from human adipose tissue. Fig.
2 is a photograph showing the LED illuminating device.
FIG. 3 is a graph showing the growth rate change of the adipose-derived mesenchymal stem from LED by irradiation.
FIG. 4A is a photograph showing the result of culturing adipose derived mesenchymal stem cells according to the presence or absence of ECM protein coated on a culture container. FIG.
FIG. 4B is a photograph showing the morphological changes of adipose derived mesenchymal stem cells over time during inoculation of adipose-derived mesenchymal stem cells into NTCP and irradiation of light from an LED light source.
FIG. 5 is a photograph and a graph showing the results of measurement of changes in the expression level of angiogenesis-related proteins according to whether irradiation of LEDs or formation of spoiloids is formed.
6 is a photograph showing the result of immunofluorescence staining for an adipose tissue-derived mesenchymal stem cell irradiated with an LED.
The photograph on the right side of FIG. 6 is a photograph showing the result of Western blot analysis to determine whether CD31 is expressed depending on whether the LED is irradiated or not and whether spleoid is formed.
FIG. 7 is a graph showing FACS analysis results of two-dimensional cultured stem cells irradiated with LEDs and stem cells formed with spheroids.
FIG. 8 is a photograph and a graph showing the results of comparing the therapeutic effects of spleen-formed stem cells by irradiating LEDs on an animal model of lower limb ischemic mice, wherein A indicates the time when 1, 7, 14 and 21 days And B is a graph showing the percentage of damage of the lower limb in the whole mouse after 21 days, and C is a graph showing the percentage of damage of the lower limb at 1, 7, 14 and 21 days after injection And D is a graph showing the proportion of mice treated with lower limb ischemia in all the mice after 21 days have elapsed.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Origin of human fat Intermediate lobe  Isolation of stem cells

The pluripotent stem cells were isolated from human subcutaneous adipose tissue, and they were observed with a phase contrast microscope (Nikon), DAPI staining was performed, or flow cytometry was performed. CD29, CD90, and CD105 were used as surface antigens for the identification of mesenchymal stem cells, and CD34, CD31, KDR (Flk1, ) And? -SMA, their expression patterns were analyzed using flow cytometry (FIG. 1).

Fig. 1 is a micrograph showing the results of verifying stem cells isolated from human adipose tissue. Fig. As shown in Fig. 1, it was confirmed that the cells isolated from human subcutaneous adipose tissue were adipose-derived mesenchymal stem cells having mesenchymal stem cell phenotype.

Example  2: LED  Origin by provocation Intermediate lobe  Stem cell Growth rate  change

Water-soluble Tetrazolium Salts (WST) assay (Dojindo laboratories) was performed to observe changes in the growth rate of adipose derived mesenchymal stem cells. Approximately, stem cells of 2 x 10 ³ cells were inoculated in a 96-well plate and irradiated with light of 0 to 6 J / cm 2 using an LED irradiation apparatus capable of irradiating light at 660 nm. After a certain time had elapsed, the culture medium was removed, 20 의 of WST solution and 180 의 of fresh culture solution were added, reacted at 37 캜 for 4 hours, and the absorbance was measured at OD450 (Figs. 2 and 3).

Fig. 2 is a photograph showing the LED irradiating apparatus, and Fig. 3 is a graph showing a change in the growth rate of the sebum-derived mesenchymal stem, which is caused by LED irradiation. As shown in FIG. 3, as the irradiation time of light increased, the proliferation of stem cells was accelerated. As the amount of energy contained in the irradiated light was increased, the proliferation level of stem cells was increased.

In particular, stem cell proliferation was accelerated when light of 0.5 J / cm 2 or more was irradiated, and the proliferation level of stem cells was proportional to the amount of energy contained in the light until irradiation of 3 J / cm 2 However, when irradiated with light of 6 J / cm 2, it was confirmed that the proliferation level of stem cells was rather reduced.

Therefore, in order to promote the proliferation of stem cells, it is preferable to irradiate light of 0.5 to 3 J / cm 2.

Example  3: Intermediate lobe  Stem cell Spherical body ( spheroid : 3 DCM , 3D cell mass ) formation

To investigate the correlation between the adherent activity of adipose derived mesenchymal stem cells on the surface of the culture vessel and the formation of spoloids, 24-well plates (NTCP, Polystyrene) was inoculated with 4 x 10 ⁴ cells / cm 2 adipose-derived mesenchymal stem cells per well and cultured in DMEM / F12 medium containing 10% FBS for 3 days. After the culture was terminated, the formation of spheroids of adipose derived mesenchymal stem cells on each cell adhesion surface was observed (Fig. 4A).

FIG. 4A is a photograph showing the result of culturing adipose derived mesenchymal stem cells according to the presence or absence of ECM protein coated on a culture container. FIG. As shown in FIG. 4, sponge of adipose-derived mesenchymal stem cells was formed with a visually detectable size in the NTCP in which the surface was hydrophobic and cell adhesion was weakly induced, and the spheroid had a diameter of about 1 mm or more . On the contrary, in NTCP coated with fibronectin, in which cell adhesion is strongly induced, it was confirmed that adipose derived mesenchymal stem cells were two-dimensionally adhered to the surface of the plate in a single layer, and cell aggregate was not formed.

From the above results, the formation of spheroids of adipose derived mesenchymal stem cells is influenced by the adhesion activity to the surface of the culture container used. In order to form spleen of a detectable size visually, initially, cell adhesion is weakly induced It is preferable to use a culture vessel having a hydrophobic surface such as polystyrene-based NTCP that allows cells to be desorbed and proliferated in a suspended state with an increase in cell density over time.

Further, in order to examine the incubation time of the adipose-derived mesenchymal stem cells required to form a sprooid having a detectable size in the NTCP, the adipose-derived mesenchymal stem cells obtained in Example 1 were suspended in 5% FBS-containing DMEM / F12 medium was incubated at a concentration of 0.5 x 10 < ⁴ > to 1 x 10 < ⁵ > cells / cm < 2 > for 10 minutes each day for 3 days while irradiating light from an LED light source (Fig.

FIG. 4B is a photograph showing the morphological changes of adipose derived mesenchymal stem cells over time during inoculation of adipose-derived mesenchymal stem cells into NTCP and irradiation of light from an LED light source. As shown in FIG. 4B, it was confirmed that spleoids of a size that can be detected with the naked eye were formed at the end of three days

Example  4: LED  Changes in the production of angiogenic differentiation factors of adipose-derived stem cells by irradiation

Two-dimensional cultured stem cells (control), two-dimensional cultured stem cells with low-level light therapy (LLLT), and spheroids with spheroids without LED illumination To compare the expression levels of angiogenesis-related proteins in the respective stem cells obtained from L-spheroids, human angiogenesis array kit (R & D Systems, Ltd., Abingdon, UK) The expression levels of angiogenesis-related proteins were measured from the respective stem cells (Fig. 5). The detection signals were measured using an image reader LAS-3000 (Kodak, Rochester, NY), and then the detection signals were measured using a MultiGauge 4.0 software (Kodak).

FIG. 5 is a photograph and a graph showing the results of measurement of changes in the expression level of angiogenesis-related proteins according to whether irradiation of LEDs or formation of spoiloids is formed. 5, expression levels of angiogenesis-related proteins were increased in spleen-formed stem cells rather than two-dimensional cultured stem cells. Among the spleen-formed stem cells, the expression level of spleen- And the level of expression of angiogenesis-related proteins was increased in the stem cells in which the sphere was formed.

Example  5: Spearoid Immunostaining  analysis

The adipose tissue-derived mesenchymal stem cells isolated in Example 1 were inoculated into a 24-well NTCP (Non-Treated Cell Culture Plate) at a concentration of 1 × 10 ⁵ cells / / RTI > The formed spheroids were recovered, fixed at -70 ° C using an OCT compound, cut into a thickness of 4 쨉 m using a microtome, and then the sections were immobilized on a slide glass and immunostained.

As another method, the recovered spheroids were physically broken using a syringe, placed on a slide glass for 4 hours, the slide glass was washed several times with PBS, and immersed in 4% paraformaldehyde solution After fixing for 30 minutes at room temperature, the cells were washed again with PBS and immunostained.

The immunological staining was carried out by immersing the slide glass prepared above in PBS containing primary antibody against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, washing three times with PBS , Followed by treatment with a secondary antibody capable of binding to the primary antibody and reaction in a dark room for 1 hour. After the reaction was completed, the slide glass was washed three times with PBS, mounted, and analyzed by fluorescence microscopy (left side of FIG. 6).

6 is a photograph showing the result of immunofluorescence staining for an adipose tissue-derived mesenchymal stem cell irradiated with an LED. As shown in the photograph on the left side of FIG. 6, the sulforoids formed from the adipose-derived mesenchymal stem cells by the LED irradiation showed positive responses to CD29, CD34, Flk1 and CD31. CD29 is a surface antigen specifically expressed in mesenchymal stem cells and epithelial cells, and CD34, KDR (Flk1) and CD31 are surface antigens specifically expressed in vascular endothelial cells.

Thus, it was found that the spheroids formed by culturing the adipose-derived mesenchymal stem cells in a culture vessel having a hydrophobic surface irradiated with light of a red wavelength band were composed of vascular endothelial cells differentiated from adipose-derived mesenchymal stem cells .

On the other hand, when a two-dimensional cultured stem cell group (control), a group (LLLT) irradiated with LED on a two-dimensional cultured stem cell, a stem cell group (spheroid) Western blot analysis was performed on each of the stem cells obtained from the stem cell group (spheroid + LLLT) to determine the expression of CD31 (right picture in FIG. 6).

The photograph on the right side of FIG. 6 is a photograph showing the result of Western blot analysis to determine whether CD31 is expressed depending on whether the LED is irradiated or not and whether spleoid is formed. As shown in the right picture of FIG. 6, CD31 was expressed only in the spleen-formed stem cells, and among the stem cells formed with the sprooids, LEDs were irradiated rather than the spleen- CD31 < / RTI >

Example  6: LED  Vascular differentiation of adipose-derived stem cells by irradiation FACS

FACS analysis of CD31, Cd34, and KDR was performed on each stem cell obtained from a group (ASC + LLLT) irradiated with two-dimensional cultured stem cells (ASC + LLLT) and LEDs and obtained from a sprooid-formed stem cell group Respectively.

The stem cells of each group were collected and physically disrupted using a syringe, placed on a slide glass for 4 hours, and the slide glass was washed several times with PBS. The slide glass was immersed in 4% paraformaldehyde solution Immobilized at room temperature for 30 minutes, washed again with PBS, and immunostained for CD31, Cd34 and KDR, surface antigens specifically expressed in vascular endothelial cells.

The immunological staining was carried out by immersing the slide glass prepared above in PBS containing primary antibody against various differentiation markers (CD29, CD34, KDR (Flk1) and CD31), reacting overnight, washing three times with PBS , Followed by treatment with a secondary antibody capable of binding to the primary antibody and reaction in a dark room for 1 hour. After the reaction was terminated, the slide glass was washed 3 times with PBS, mounted and analyzed by FACS (FIG. 7).

FIG. 7 is a graph showing FACS analysis results of two-dimensional cultured stem cells irradiated with LEDs and stem cells formed with spheroids. As shown in FIG. 7, it was confirmed that the spleroid formed by the LED irradiation showed positive responses to the surface antigens CD29, CD34, KDR (Flk1) and CD31 that are specifically expressed in the vascular endothelial cells.

Therefore, it can be seen that the irradiated speroids exhibit the characteristics of vascular endothelial cells differentiated from adipose derived mesenchymal stem cells.

Example  7: Ischemia  Mouse on animal model Speroid  Check the treatment effect after transplantation

First, a 5-week-old BALB / c nude mouse (20 g body weight; Narabio, Seoul, Korea) was used to ligate the femoral aorta with 5-0 silk suture.

The ligation site of the prepared dorsal ischemic mouse animal model was injected with PBS (control), 1.5 × 10 ⁶ cells of two-dimensional cultured stem cells (ASC), LED (LLLT) or 1.5 × 10 ⁶ to examine the LED cell number of injected stem cells, the spheroid is formed, and (L-spheroid) 21 il sayuk then exterior damage level of not (no loss, to necrosis and recovered) for the skin and blood stream ( cutaneous blood flow) was photographed or measured (Fig. 8). At this time, the skin blood flow photograph was obtained by placing a mouse on a 37 ° C hot plate, scanning and photographing a laser dot color image.

FIG. 8 is a photograph and a graph showing the results of comparing the therapeutic effects of spleen-formed stem cells by irradiating LEDs on an animal model of lower limb ischemic mice, wherein A indicates the time when 1, 7, 14 and 21 days And B is a graph showing the percentage of damage of the lower limb in the whole mouse after 21 days, and C is a graph showing the percentage of damage of the lower limb at 1, 7, 14 and 21 days after injection And D is a graph showing the proportion of mice treated with lower limb ischemia in all the mice after 21 days have elapsed. As shown in FIG. 8, it can be seen that the use of stem cells formed with the steroids by irradiating the LEDs can effectively treat vascular lesions generated in the lower limb ischemic mice.

Claims (6)

Derived mesenchymal stem cells are inoculated into a culture vessel having a hydrophobic surface and incubated with light of 5 to 30 mW / cm 2 and 600 to 700 nm in the state of forming the adipose-derived mesenchymal stem cells or spoloids Derived mesenchymal stem cells, comprising the step of irradiating the mesenchymal stem cells with adipose-derived mesenchymal stem cells into vascular endothelial cells.
The method according to claim 1,
Wherein the hydrophobic culture container is a culture container surface-treated with a polymer imparting hydrophobicity or a culture container made of the polymer.
The method according to claim 1,
The hydrophobic polymer may be selected from the group consisting of polystyrene, polymethyl methacrylate (PMMA), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluor (PLLA), poly (D, L-lactic acid) (PDLLA), poly (glycolic acid) (PGA), poly (caprolactone) (PCL ), Poly (hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, derivatives thereof, and copolymers thereof.
The method according to claim 1,
Wherein the inoculation density of adipose derived mesenchymal stem cells is 1 x 10 ⁵ to 2 x 10 ⁶ cells / cm 2.
The method according to claim 1,
Wherein the light is irradiated for 10-15 minutes per day using an LED light source positioned at an interval of 1-5 cm from adipose-derived mesenchymal stem cells.
The method according to claim 1,
Wherein the adipose-derived mesenchymal stem cells are differentiated into vascular endothelial cells after spore formation by LED irradiation.

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