KR20190032009A - Composition of media for culture or differentiation of stem cells comprising spirulina - Google Patents

Abstract

The present invention relates to a culture medium composition for culture and differentiation of stem cells comprising spirulina. The composition of the present invention includes spirulina instead of fetal bovine serum while being excellent in an effect of culturing and differentiating the stem cells, and thus can be used as a serum-free medium composition for the culture and differentiation of the stem cells.

Description

TECHNICAL FIELD The present invention relates to a composition for stem cell culture and differentiation comprising spirulina,

The present invention relates to a culture medium for stem cell culture and differentiation comprising Spirulina.

Cell culture is the most fundamental and important experimental technique for research in medicine today, including molecular biology and immunology. In animal cell culture methods used up to now, serum must be supplied to the cell culture medium for growth. Fetal bovine serum (FBS) is the most widely used serum. In addition, various kinds of differentiation inducing factors have been added to the culture medium containing fetal bovine serum for inducing culture and differentiation of adult stem cells.

However, it is difficult to exclude the stability problem such as interspecies stain during development of a stem cell treatment agent for clinical application by using an animal-derived protein source including fetal bovine serum in the culture process of stem cells using the culture medium. In addition, the manufacturing process of fetal bovine serum is inevitably ethical problem because fetal fetus is extracted from the uterus of the pregnant mother and the serum is separated and it is inevitably caused by ethical problems in the international society including the FDA of the United States and the European Medicines Agency (EMEA) Has recommended the use of fetal calf serum and the development of alternative agents. Furthermore, the controversy over the risk of transmission of bovine spongiform encephalopathy caused by fetal bovine serum has not been solved.

As a result of studies on a cell culture medium replacing fetal bovine serum, Korean Patent Laid-Open No. 10-2013-0006123 developed a serum-free medium, MesemCult-XF, which can effectively proliferate amniotic stem cells, Team of Arati A. Inamdar has developed plasma and platelet lysates that are used as cell growth supplements to produce clinical grade mesenchymal stem cells for use as cell therapy. (Inamdar et al., Journal of regenerative medicine & tissue engineering 2013)

However, in the case of the serum-free medium developed so far, the applicable cell line is very limited, the adaptation process of the cell line for culturing is difficult, the cell division and growth rate is slow, and various hormones and growth There is a disadvantage that it is troublesome to the user because addition of an additional factor is required.

Accordingly, the present inventors have continued research on a new medium capable of replacing the existing stem cell culture medium, confirming the possibility of culturing and differentiation of stem cells in a medium containing spirulina, and completed the present invention Respectively.

Korean Patent Publication No. 10-2013-0006123

Inamdar et al. journal of regenerative medicine & tissue engineering 2013

It is an object of the present invention to provide a medium composition for culturing and differentiating stem cells comprising spirulina.

In one aspect of the present invention, the present invention provides a medium composition for culturing and differentiating stem cells comprising Spirulina.

The term " spirulina " in the present invention is a microorganism belonging to an extremely small alga that is vigorously growing in the water of a tropical alkaline lake such as Lake Chad in Africa and Lake Texco in Mexico. Spirulina cells are classified as blue-green algae because they contain a large amount of chlorophyll and picocyanin pigments. However, as the structure of cyanobacteria has recently been revealed, spirulina is classified as blue-green algae -green bacteria.

The spirulina contained in the medium composition of the present invention can be used in the form of a powder or an extract, but is not limited thereto. Specifically, the dry powder of Spirulina may be an extract of centrifuged liquid after disruption by enzymatic, ultrasonic, high-temperature and high-pressure treatment.

Recently, the necessity of the study on the alternative medium of fetal bovine serum used for the culture of stem cells has been continuously developed, so that the study on the serum - free medium has been actively carried out. However, in the case of serum-free medium, problems such as limitations on applied cell lines, difficulties in cell culture, and high cost are not solved, and thus, the present invention still has limitations that can not be applied to research. As a result of studying serum-free medium using marine biomaterials having no research, the present inventors have found for the first time that stem cell culture and differentiation medium exhibit excellent culture and differentiation effects even when spirulina is used instead of fetal bovine serum. Completed.

As used herein, the term " stem cell " refers to a cell capable of differentiating into various cells through suitable environment and stimulation, and having autoproliferative capacity, including adult stem cells, pluripotent stem cells, It may be an allogenic stem cell or embryonic stem cell.

In the present invention, the stem cells may be adult stem cells. Specifically, the types of stem cells in which culture and differentiation are promoted by the medium composition of the present invention include periodontal ligament stem cells (PDLSC) A stem cell derived from bone marrow-derived stem cells (DMSC), a bone marrow-derived stem cell (BMSC), and adipose-derived stem cells (ADSC) . More specifically, it may be periodontal ligament stem cells or adipose derived stem cells.

As used herein, the term " periodontal ligament stem cell (PDLSC) " is a stem cell present in the ligament tissue between a tooth and a gum bone. The term " periodontal ligament stem cell " It is one type of mesenchymal stem cells that can be obtained during. The periodontal ligament stem cells are hyperplastic cells capable of differentiating into bone formation, cementum formation, fat production, cartilage formation, neural formation and collagen-forming cells under specific environment.

In the present invention, the term " dental pulp stec cell (DPSC) " is a stem cell existing in a dimension which is a soft tissue inherent tissue in the center of teeth and may be derived from the dimensions of human teeth have.

The term " bone marrow-derived stem cell (BMSC) " in the present invention means a stem cell derived from a bone marrow, which is a tissue or organ that makes blood cells such as red blood cells, white blood cells and platelets.

The term " adipose-derived stem cell (ADSC) " in the present invention refers to stem cells isolated from adipose tissue capable of differentiating into most mesenchymal cells such as adipocytes, osteoblasts, chondroblasts and myofibroblasts And may be used in the same or equivalent meaning as lipid precursor cells, stromal cells, multipotent adipose-derived cells or adipose derived adult stem cells.

The term " medium " in the present invention means a medium capable of supporting the growth, survival and differentiation of stem cells in vitro, and means a culture medium suitable for culturing and differentiation of stem cells used in the field Include all media. Depending on the type of cell, the type of medium and the culture conditions can be selected at the technical level of the field. The medium to be used for culturing is specifically a cell culture minimum medium (CCMM), which generally includes a carbon source, a nitrogen source and a trace element. The cell culture minimum medium is, for example, DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, Medium, GMEM, and Iscove's Modified Dulbecco's Medium. In addition, the medium may include antibiotics such as penicillin, streptomycin, gentamicin, or a mixture of two or more thereof.

For the purpose of the present invention, which enables the cultivation and differentiation of stem cells using spirulina, there is no particular limitation on the type of medium and the culture method, and spirulina may be used in the stem cell medium. At this time, various applications such as using spirulina alone or in combination with one or more known culture and differentiation inducing substances are possible.

In the present invention, the culture medium composition of the present invention enables subculture of stem cells.

In the present invention, the term " passage " means to continuously cultivate cells to continuously cultivate cells, specifically stem cells, in a healthy state for a long period of time. do. Replacement of the culture container once or culturing the cell group separately is referred to as a passage. In the present invention, the passage can be used in combination with a generation.

In one embodiment of the present invention, when the culture medium composition of the present invention is used, the periodontal ligament stem cells, the dermal-derived stem cells, the bone marrow-derived stem cells and the fat-derived stem cells are cultured from the first generation to the 10th generation, And differentiation was possible.

In the present invention, the medium composition of the present invention can promote the ability to differentiate stem cells.

In the present invention, the term " differentiation ability " refers to the ability of a stem cell to be specialized in the structure and function of a cell such as adipocytes, osteoblasts, chondroblasts, myofibroblasts, muscle cells or neurons. May be the ability of the stem cells to differentiate. Specifically, the culture medium composition of the present invention can promote the activity of alkaline phosphatase (ALP) of stem cells.

The term " alkaline phosphatase (ALP) " in the present invention means an enzyme that releases inorganic phosphoric acid from an organic phosphoric acid ester used as an index of calcification.

The medium composition for promoting the bone differentiation ability may further include at least one selected from the group of bone formation inducers including? -Glycerophosphate, dexamethasone and ascorbic acid.

In the present invention, the culture medium composition may further include fetal bovine serum (FBS). 8: 2, 7: 3, 6: 4, 5: 5, 4: 6, 3: 7, 2: : 8, 1: 9, or 0:10. Specifically, the weight ratio between fetal bovine serum and spirulina (fetal bovine serum: spirulina) may be 9: 1 to 4: 6, more specifically 9: 1.

In one example, the weight ratio of fetal bovine serum to spirulina (fetal bovine serum: Spirulina) in the culture composition for culturing and differentiating periodontal ligament stem cells may be 9: 1 to 3: 7, specifically 9: 1 to 7: 3 < / RTI >

For example, the weight ratio of fetal bovine serum and spirulina (fetal bovine serum: Spirulina) in the culture composition for the culture and differentiation of stem stem cells from 9: 1 to 3: 7 can be in the range of 9: 1 to 6: 4 < / RTI >

For example, the weight ratio of fetal bovine serum and spirulina (fetal bovine serum: Spirulina) of the culture composition for culturing and differentiation of bone marrow-derived stem cells may be 9: 1 to 2: 8, and specifically 9: 1 .

For example, the weight ratio of fetal bovine serum and spirulina (fetal bovine serum: Spirulina) in the culture composition for culturing and differentiating adipose-derived stem cells may be 9: 1 to 4: 6, specifically 9: 1 to 7: 3 < / RTI >

The culture medium containing spirulina of the present invention is capable of stem cell culture and differentiation. Therefore, the culture medium composition of the present invention can be used as a culture medium for stem cell culture and differentiation, which can replace fetal bovine serum.

FIG. 1 is a graph showing cell viability of periodontal ligament stem cells (PDLSC) up to 10th generation according to the replacement ratio of Spirulina animal cell culture medium (SACCS).
2 is a graph showing the cell survival rate of the dental pulp stem cells (DPSC) up to 10th generation according to the replacement ratio of Spirulina animal cell culture medium (SACCS).
FIG. 3 is a graph showing cell survival rates of bone marrow-derived stem cells (BMSC) up to 10th generation according to substitution ratio of Spirulina animal cell culture medium (SACCS).
FIG. 4 is a graph showing cell survival rate of adipose-derived stem cells (ADSC) up to 10th generation according to substitution ratio of Spirulina animal cell culture medium (SACCS).
FIG. 5 is a diagram showing phenotypes up to 10th generation of periodontal ligament stem cells (PDLSC) according to replacement ratio of Spirulina animal cell culture medium (SACCS). FIG.
FIG. 6 is a diagram showing phenotypes up to 10th generation of stem cell (DPSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS).
7 is a view showing a phenotype up to generation 10 of bone marrow-derived stem cells (BMSC) according to substitution ratio of Spirulina animal cell culture medium (SACCS).
Fig. 8 is a diagram showing phenotypes up to 10th generation of adipose-derived stem cells (ADSC) according to substitution ratio of Spirulina animal cell culture medium (SACCS).
FIG. 9 is a graph showing cell survival rates of first, fifth, and tenth generation of periodontal ligament stem cells (PDLSC) according to replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substance.
FIG. 10 is a graph showing cell survival rates of first-generation, fifth-generation, and tenth generations of stem-derived stem cells (DPSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substance.
FIG. 11 is a graph showing cell survival rates of bone marrow-derived stem cells (BMSCs) according to substitution ratios of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substances in first, fifth, and tenth generations.
FIG. 12 is a graph showing cell survival rates in first-, fifth-, and tenth generations of adipose-derived stem cells (ADSC) according to replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation-inducing substance.
13 shows the activity of alkaline phosphatase (ALP) in the first, fifth, and tenth generations of periodontal ligament stem cells (PDLSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) .
14 shows the activity of alkaline phosphatase (ALP) in first-, fifth-, and tenth generations of stem-derived stem cells (DPSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) .
15 shows the activity of alkaline phosphatase (ALP) in the first, fifth, and tenth generations of bone marrow-derived stem cells (BMSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) .
16 shows the activity of alkaline phosphatase (ALP) in first-, fifth-, and tenth generations of adipose-derived stem cells (ADSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) .
FIG. 17 is a graph comparing the degrees of calcification in the first, fifth, and tenth generations of periodontal ligament stem cells (PDLSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing material.
FIG. 18 shows the calcification of stem cells in the first, fifth, and tenth generations of periodontal ligament stem cells (PDLSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substances .
FIG. 19 is a graph comparing the degree of calcification in the 1st, 5th and 10th generations of the stem cell (DPSC) according to the substitution rate of the Spirulina animal cell culture medium (SACCS) containing the bone differentiation inducing substance.
FIG. 20 shows the calcification of stem cells in first-, fifth-, and tenth generations of stem-derived stem cells (DPSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) .
FIG. 21 is a graph comparing the degree of calcification of bone marrow-derived stem cells (BMSCs) in the 1st, 5th and 10th generation according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing the bone differentiation inducing substance.
FIG. 22 shows the calcification of stem cells in the first, fifth, and tenth generations of bone marrow-derived stem cells (BMSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substance .
Figure 23 is a graph comparing the degree of calcification in first, fifth, and tenth generations of adipose derived stem cells (ADSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substances.
Figure 24 shows the calcification of stem cells in first, fifth, and tenth generations of adipose-derived stem cells (ADSC) according to the replacement ratio of Spirulina animal cell culture medium (SACCS) containing bone differentiation inducing substances .

Hereinafter, preferred embodiments of the present invention will be described in order to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited thereto.

Example  1: Stem Cell Survival Rate

To confirm the possibility of replacing fetal bovine serum (FBS), which is a representative medium composition in stem cell culture medium, a medium containing spirulina animal cell culture solution (Spaculina animal cell culture solution, SACCS) And cell viability was measured for each generation. The Spirulina animal cell culture fluid was supplied by Korea Ocean Research & Development Institute. The spirulina extract contained in the SACCS was prepared by disintegrating the dried powder of Spirulina with an enzyme, an ultrasonic wave and a high pressure treatment and then centrifuging.

The cell survival rate was measured using periodontal ligament stem cells (PDLSC) purchased from the Cell Biotech Co., Ltd., dental pulp stec cells (DPSC) purchased from Lonza, bone marrow derived stem cells (BMSCs) and adipose-derived stem cells (ADSCs).

The stem cell culture medium was prepared by varying the substitution ratio of SACCS in the FBS medium. Specifically, the ratio of FBS: SACCS was adjusted to 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: : 5, 4: 6, 3: 7, 2: 8, 1: 9 and 0:10. The four kinds of stem cells were collected at a rate of ³ × 10 ³ cells / well in a 96-well plate. After 48 hours, the cultures were completely removed and α-MEM cultures containing 10% cell counting kit-8 (CCK-8) reagent were treated and cultured in an incubator at 37 ° C and 5% CO ₂ for 1 hour. Cell viability was then measured at 450 nm. The measurement results are as follows:

<Periodontal ligament stem cells (PDLSC)>

The cell viability of periodontal ligament stem cells was measured in all ratios in the first generation (# 1) compared to the control (FBS: SACCS = 10: 0, F10: S0) It was confirmed that the ratio of F4: S6 in the 5th generation, F3: S7 in the 6th generation, F6: S4 in the 7th to 9th generation, and F7: S3 in the 10th generation can be substituted And Table 1).

PDLSC F10: S0 F9: S1 F8: S2 F7: S3 F6: S4 F5: S5 F4: S6 F3: S7 F2: S8 F1: S9 F0: S10 #One 100 108.6 111.7 113.5 105.8 106.2 111.8 116.6 115.5 117.0 101.7 #2 115.1 118.2 100.4 143.3 139.3 119.1 135.3 123.0 108.4 68.2 # 3 110.0 144.6 159.7 127.7 153.1 139.9 136.0 147.9 132.4 68.1 #4 103.4 104.8 117.9 116.3 107.5 107.1 102.9 99.2 97.7 79.7 # 5 110.3 102.9 124.8 104.1 107.5 106.7 92.9 85.8 85.8 56.3 # 6 114.7 104.4 113.0 111.6 101.5 106.0 106.7 91.8 82.5 47.3 # 7 98.3 124.8 119.3 125.3 123.5 105.7 98.0 91.2 84.7 X #8 106.1 106.0 119.5 114.1 104.6 99.5 97.9 94.9 89.4 X # 9 111.5 112.5 111.0 115.7 99.2 97.6 86.5 78.4 76.7 X # 10 109.6 100.2 108.0 86.4 83.1 82.6 83.2 63.5 60.2 X

< Stem-derived stem cells ( DPSC )>

As a result of measurement of the cell viability of the stem cells of the resected stem cells, the ratio of F2: S8 in the first generation (# 1) to the ratio of F2: S8 can be substituted for the control group (FBS: SACCS = 10: 0, F10: S0) S5, the ratio of F4: S6 in the third to eighth generations, and the ratio of F6: S4 in the ninth to tenth generations (FIG. 2 and Table 2).

DPSC F10: S0 F9: S1 F8: S2 F7: S3 F6: S4 F5: S5 F4: S6 F3: S7 F2: S8 F1: S9 F0: S10 #One 100 96.8 99.6 104.1 97.8 101.9 103.1 100.8 101.5 96.7 83.9 #2 103.0 115.1 106.9 115.8 107.7 89.2 82.2 81.8 89.4 83.9 # 3 109.8 93.7 90.2 107.0 88.5 112.3 89.4 86.5 89.2 74.3 #4 102.7 117.7 98.7 97.2 117.5 113.6 84.4 79.0 76.0 68.7 # 5 112.6 102.4 104.1 104.7 106.0 105.7 92.4 89.0 87.0 74.0 # 6 107.4 112.4 113.4 108.7 111.0 122.0 90.3 83.0 67.7 60.3 # 7 104.8 103.5 104.2 100.5 96.7 98.4 93.2 88.9 65.2 X #8 104.4 113.2 114.4 103.7 102.0 100.6 96.6 84.2 X X # 9 107.0 101.0 104.0 98.7 94.0 89.4 75.7 69.4 X X # 10 118.2 110.1 107.1 105.2 95.5 88.5 84.0 62.3 X X

&Lt; Bone marrow stem cell (BMSC) >

As a result of measurement of the cell viability of the bone marrow-derived stem cells by generations, it was possible to replace the ratio of F2: S8 in the first generation (# 1) to the third generation with respect to the control group (FBS: SACCS = 10: 0, F10: S0) It was confirmed that the ratio of F7: S3 in the 7th generation, the ratio of F8: S2 in the 8th generation, and the ratio of F9: S1 in the 9th to the 10th generation can be substituted (FIG. 3 and Table 3).

BMSC F10: S0 F9: S1 F8: S2 F7: S3 F6: S4 F5: S5 F4: S6 F3: S7 F2: S8 F1: S9 F0: S10 #One 100 98.6 101.0 100.4 99.9 97.8 96.1 94.3 92.3 85.1 75.7 #2 113.7 111.6 108.2 105.0 112.9 111.7 113.8 108.1 94.7 81.8 # 3 105.3 101.0 101.4 88.8 80.7 101.3 102.0 97.3 80.9 X #4 93.1 87.7 87.3 71.5 63.2 76.7 76.7 67.2 59.3 X # 5 105.5 102.8 105.2 88.6 89.2 81.3 75.4 71.0 65.5 X # 6 95.8 101.5 97.8 64.7 53.7 57.1 52.0 47.7 47.9 X # 7 104.3 107.4 98.9 65.7 52.0 55.4 50.7 49.2 47.2 X #8 117.7 105.0 92.5 X X X X X X X # 9 99.6 76.7 68.5 X X X X X X X # 10 100.3 79.9 83.0 X X X X X X X

< Adipose-derived stem cells ( ADSC )>

As a result of measuring the cell viability of the fat-derived stem cells by generation, the ratio of F4: S6 in the first generation (# 1) to the control group (FBS: SACCS = 10: 0, F10: S0) It was confirmed that the ratio of F3: S7, F4: S6 in the fourth generation, F8: S2 in the fifth generation, F5: S5 in the sixth generation, and F7: S3 in the seventh to tenth generation (Fig. 4 and Table 4).

ADSC F10: S0 F9: S1 F8: S2 F7: S3 F6: S4 F5: S5 F4: S6 F3: S7 F2: S8 F1: S9 F0: S10 #One 100 96.3 95.7 99.1 96.4 95.0 95.0 92.9 89.1 84.6 74.1 #2 87.7 84.3 81.9 74.2 73.3 83.5 87.1 78.4 67.2 62.7 # 3 102.6 126.0 100.5 97.9 98.5 109.4 100.5 97.6 88.6 79.2 #4 94.5 97.2 97.4 84.6 82.6 94.2 83.2 83.0 79.2 72.4 # 5 99.9 105.3 73.0 61.6 60.3 59.3 58.8 55.7 49.1 X # 6 107.1 113.6 102.7 98.3 98.3 80.5 81.2 86.6 59.5 X # 7 101.4 111.7 110.6 96.4 91.8 94.9 85.3 63.9 55.9 X #8 107.5 104.9 97.9 90.0 90.8 90.9 84.6 X X X # 9 101.3 102.8 96.3 95.2 94.3 92.6 93.9 X X X # 10 103.9 102.9 104.5 103.4 96.4 95.1 92.2 X X X

Example  2: Morphological phenotyping of stem cells

The morphological phenotype was observed and photographed (original magnification X100) using the microscope before stem cell viability measurement of Example 1. The results were as follows:

<Periodontal ligament stem cells (PDLSC)>

The morphological phenotype of periodontal ligament stem cells was observed. In all the ratios, spindle-shaped cell morphology appeared in all ratios in the first generation (# 1) 5 in the fifth generation, F3: S6 in the sixth generation, F6: S4 in the seventh and ninth generations, and F7: S3 in the ten generations (FIG. 5) . On the other hand, in the case of F0: S10, all the periodontal ligament stem cells from the 5th generation were changed from spindle-shaped cell form to circular form, As a result, it was confirmed that periodontal ligament stem cells can be replaced with SACCS up to 30% of the control group, which is equal to the cell survival rate.

<Dimension-derived stem cells (DPSC)>

As a result of observing morphological phenotypes of stem-derived stem cells from the generations, spindle-shaped cell morphology appeared at the ratio of F10: S10 to F2: S8 in the first generation (# 1) It was confirmed that the ratio of F5: S5 in the second generation, F4: S6 in the third to fifth generation, F4: S6 in the sixth and eighth generations, and F6: S4 in the ninth and tenth generations 6). On the other hand, in the case of F0: S10, all the periodontal ligament stem cells from the 6th generation were changed from spindle-shaped cell form to circular form, As a result, it was confirmed that periodontal ligament stem cells can be replaced with SACCS up to 40% of the control group, which is equal to the cell survival rate.

&Lt; Bone marrow stem cell (BMSC) >

The morphological phenotype of bone marrow-derived stem cells was observed. In the first generation (# 1) to the third generation, spindle-shaped cell morphology appeared at the ratio of F10: S10 to F2: S8, (F7: S3 in the fourth to seventh generation, F8: S2 in the eighth generation, and F9: S1 in the ninth and tenth generation) (Fig. 7). On the other hand, in the case of F0: S10, all the periodontal ligament stem cells were changed from spindle-shaped cell form to circular form from 2nd generation, As a result, it was confirmed that bone marrow-derived stem cells can be replaced with SACCS up to 10% of the control group, which is equal to the cell survival rate.

<Fat-derived stem cells (ADSC)>

The morphological phenotype of adipose-derived stem cells was observed. In the first generation (# 1), the spindle-shaped cell morphology appeared at the ratio of F10: S10 to F4: S6, The ratio of F3: S7 in the 2nd to 3rd generation, F4: S6 in the fourth generation, F8: S2 in the fifth generation, F5: S5 in the sixth generation, and F7: S3 in the seventh to tenth generation (Fig. 8). On the other hand, in the case of F0: S10, all the periodontal ligament stem cells were changed from spindle-shaped cell form to circular form from 3rd generation, As a result, it was confirmed that the fat-derived stem cells can be replaced with SACCS up to 30% of the control group in the same manner as the cell survival rate.

Example  3: Stem cell Bone vertebrae  maintain

Example 3-1: Stem cell survival rate in differentiation medium

In order to confirm the possibility of replacing fetal bovine serum (FBS), which is a typical medium composition, in the differentiation medium for stem cell differentiation, the differentiation medium after stem cell culture was treated to determine the cell survival rate Respectively.

Specifically, the ratio of FBS: SACCS was adjusted to 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: : 5, 4: 6, 3: 7, 2: 8, 1: 9 and 0:10. (PDMSC), dental pulp stent cells (DPSC), bone marrow-derived stem cells (BMSC), and adipose-derived stem cells (STEMs) cell, ADSC) were collected at ³ × 10 ³ cells / well in a 96-well plate. After 48 hours, the cell adhesion was confirmed, and the cell adhesion was confirmed and cultured in? -MEM culture medium (5% FBS: SACCS, 1% penicillin and streptomycin, 50 μg / ml ascorbic acid, 1 μM dexamethasone, Glycerophosphate). &Lt; / RTI &gt; After 48 hours, the differentiation medium was completely removed and the α-MEM culture medium containing 10% cell counting kit-8 (CCK-8) reagent was treated and incubated for 1 hour at 37 ° C. in a 5% CO ₂ incubator. Cell viability was then measured at 450 nm. The measurement results are as follows:

<Periodontal ligament stem cells (PDLSC)>

The cell viability of periodontal ligament stem cells was different from that of control (FBS: SACCS = 10: 0, F10: S0) in the first generation (# 1) It was confirmed that the generation can substitute for the ratio of F7: S3 (Fig. 9). As a result, it was confirmed that SACCS could be substituted for up to 30% of the control group.

<Dimension-derived stem cells (DPSC)>

As a result of measurement of the cell viability of the stem cells derived from the differentiated cells, the ratio of F4: S6 in the first generation (# 1) to the control group (FBS: SACCS = 10: 0, F10: S0) It was confirmed that the generation can substitute up to the ratio of F7: S3 (Fig. 10). As a result, it was confirmed that SACCS could be substituted for up to 30% of the control group.

&Lt; Bone marrow stem cell (BMSC) >

The cell viability of the bone marrow-derived stem cells in the differentiation medium was compared with that of the control (FBS: SACCS = 10: 0, F10: SO) to the ratio of F8: S2 in the first generation (# 1) Generation, it was confirmed that substitution was possible up to the ratio of F9: S1 (Fig. 11). As a result, it was confirmed that SACCS could be substituted for up to 10% of the control group.

<Fat-derived stem cells (ADSC)>

As a result of measuring the cell viability of the fat-derived stem cells in the differentiation medium, the ratio of F5: S5 in the first generation (# 1) to the control group (FBS: SACCS = 10: 0, F10: S0) It was confirmed that the generation can substitute for the ratio of F7: S3 (Fig. 12). As a result, it was confirmed that SACCS could be substituted for up to 10% of the control group.

Example 3-2: Enzyme activity related to bone differentiation

Alkaline phosphatase, or alkaline phosphatase (ALP), is an enzyme that releases inorganic phosphoric acid from organic phosphate esters and is known as an indicator of calcification. Thus, it has been shown that periodontal ligament stem cells (PDLSC), dental pulp stec cells (DPSC), bone marrow-derived stem cells (BMSC), and adipose- derived stem cells (ADSC), the activity level of alkaline phosphatase was confirmed.

Specifically, the ratio of FBS: SACCS was adjusted to 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: : 5, 4: 6, 3: 7, 2: 8, 1: 9 and 0:10. The four kinds of stem cells were collected at a rate of ³ × 10 ³ cells / well in a 96-well plate. When cells reached 80% cell density after 48 hours, cells were cultured in? -MEM medium (5% FBS: SACCS, 1% penicillin and streptomycin, bone morphogenetic inducer: 50 μg / ml ascorbic acid, 1 μM dexamethasone, and 3 mM [beta] -glycerophosphate). After incubation for 7 days at 37 ° C in a 5% CO ₂ incubator, the cells were disrupted using 1X RIPA buffer. The protein supernatant was taken by centrifugation at 13,000 rpm for 10 minutes. 200 μL of pNP solution (Sigma) was added to 50 μL of the supernatant, and reacted in an incubator at 37 ° C. and 5% CO _{2 for} 30 minutes. Then, the activity level of the alkaline phosphatase was measured at 450 nm. The measurement results are as follows:

<Periodontal ligament stem cells (PDLSC)>

As a result of measuring the activity of the final bone differentiation enzyme by generation of periodontal ligament stem cells, it was found that in the first generation (# 1), the activity of F3: S7 was 33% or more relative to that of the control group (FBS: SACCS = 10: 0, F10: , And it was confirmed that the activity of F7: S3 was increased by 12% or more in the 10th generation (Fig. 13). As a result, it was confirmed that SACCS could be substituted for up to 30% of the control group.

<Dimension-derived stem cells (DPSC)>

In the first generation (# 1), activity of 11% or more to the ratio of F7: S3 to control (FBS: SACCS = 10: 0, F10: S0) , And in the 10th generation, the activity was increased by 13% or more to the ratio of F8: S2 (Fig. 14). As a result, it was confirmed that SACCS could be substituted for up to 20% of the control group.

&Lt; Bone marrow stem cell (BMSC) >

In the first generation (# 1), the activity of the bone marrow-derived stem cells was determined to be 18% or more as much as that of the control (FBS: SACCS = 10: 0, F10: S0) , And it was confirmed that the activity was increased by 40% or more up to the ratio of F9: S1 in the 10th generation (Fig. 15). As a result, it was confirmed that SACCS could replace 10% of the control group.

<Fat-derived stem cells (ADSC)>

In the first generation (# 1) and 10th generation, the ratio of F7: S3 to the control group (FBS: SACCS = 10: 0, F10: S0) 18% or more (Fig. 16). As a result, it was confirmed that SACCS could be substituted for up to 30% of the control group.

Example 3-3: Confirmation of formation of calcium nodule

It is known that when stem cells are differentiated into bone cells after proliferation, a calcium module is formed. Therefore, when cultured with SACCS culture medium, periodontal ligament stem cells (PDLSC), dental pulp stec cells (DPSC), bone marrow-derived stem cells (BMSC) The level of calcium nodule was checked to determine the ability of the adipose-derived stem cell (ADSC) to differentiate.

Specifically, the ratio of FBS: SACCS was adjusted to 10: 0, 9: 1, 8: 2, 7: 3, 6: 4, 5: : 5, 4: 6, 3: 7, 2: 8, 1: 9 and 0:10. (PDMSC), dental pulp stent cells (DPSC), bone marrow-derived stem cells (BMSC), and adipose-derived stem cells (STEMs) cell, ADSC) were collected at a rate of 2.5 × 10 ⁴ cells / well in a 6-well plate. When cells reached 60% cell density after 48 hours, cells were cultured in? -MEM culture medium (5% FBS: SACCS, 1% penicillin and streptomycin, bone morphogenetic inducer: 50 μg / ml ascorbic acid, 1 μM dexamethasone, and 3 mM [beta] -glycerophosphate). After incubation for 14 days at 37 ° C in a 5% CO ₂ incubator, alizarin red solution was used for staining and calcification was confirmed by microscopy. The measurement results are as follows:

<Periodontal ligament stem cells (PDLSC)>

We found that the calcification of periodontal ligament stem cells was more than 35% up to the ratio of F5: S5 to the control group (FBS: SACCS = 10: 0, F10: S0) And in the 10th generation, the calcification was increased by 18% or more to the ratio of F7: S3 (FIGS. 17 and 18).

<Dimension-derived stem cells (DPSC)>

As a result of measuring the age-specific calcification of stem-derived stem cells, it was confirmed that the first generation (# 1) showed an increase of more than 15% in calcification by the ratio of F7: S3 to the control group (FBS: SACCS = 10: 0, F10: S0) , And in the 10th generation, active calcification was observed at a rate of 7% or more up to the ratio of F8: S2 (FIGS. 19 and 20).

&Lt; Bone marrow stem cell (BMSC) >

The bone marrow-derived stem cells were examined for age-specific calcification, and it was confirmed that the first generation (# 1) showed a calcification increase of 4% or more by the ratio of F8: S2 to the control group (FBS: SACCS = 10: 0, F10: S0) And in the 10th generation, the calcification was increased by 24% or more to the ratio of F9: S1 (FIGS. 21 and 22).

<Fat-derived stem cells (ADSC)>

We found that the calcification of adipose-derived stem cells showed more than 20% increase in F7: S3 ratio compared to the control group (FBS: SACCS = 10: 0, F10: S0) , And in the 10th generation, the calcification was increased by 15% or more to the ratio of F7: S3 (FIGS. 23 and 24).

Claims (10)

A medium composition for culturing and differentiating stem cells comprising spirulina. The stem cell according to claim 1, wherein the stem cell is a periodontal ligament stem cell (PDLSC), a dental pulp stec cell (DPSC), a bone marrow-derived stem cell (BMSC) And adipose-derived stem cells (ADSC). &Lt; / RTI &gt; The culture medium of claim 1, wherein the stem cells are periodontal ligament stem cells (PDLSC) and adipose-derived stem cells (ADSC). The culture medium according to claim 1, wherein the culture medium composition is capable of subculturing stem cells. 2. The medium composition according to claim 1, wherein the culture medium composition promotes the pluripotency of stem cells. 6. The composition of claim 5, wherein the differentiating ability is a bone differentiation ability. 7. The medium composition according to claim 6, wherein the culture medium composition promotes the activity of alkaline phosphatase (ALP). The culture medium composition according to claim 1, wherein the culture medium composition further comprises fetal bovine serum (FBS). 9. The media composition of claim 8, wherein the Spirulina is a 9: 1 to 4: 6 weight ratio (fetal calf serum: Spirulina) to fetal bovine serum (FBS). 9. The media composition of claim 8, wherein the Spirulina is a 9: 1 weight ratio (fetal bovine serum: Spirulina) to fetal bovine serum (FBS).

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