WO2021241782A1 - Fusion protein for cell culture containing extracellular matrix motif and use thereof - Google Patents
Fusion protein for cell culture containing extracellular matrix motif and use thereof Download PDFInfo
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- WO2021241782A1 WO2021241782A1 PCT/KR2020/006921 KR2020006921W WO2021241782A1 WO 2021241782 A1 WO2021241782 A1 WO 2021241782A1 KR 2020006921 W KR2020006921 W KR 2020006921W WO 2021241782 A1 WO2021241782 A1 WO 2021241782A1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M1/00—Apparatus for enzymology or microbiology
- C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
Definitions
- the present invention relates to a fusion protein for cell culture, and more particularly, to a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
- Cardiovascular disease is one of the diseases with the highest mortality rate worldwide.
- Existing main treatment methods for this are medical treatment centered on drug administration and interventional treatment such as percutaneous cardiovascular intervention/coronary artery bypass graft to clear blocked blood vessels. A transplant is required.
- cardiovascular disease is still considered the number one cause of death worldwide, and it is reported that about 30% of acute myocardial infarction develops into chronic heart failure. Therefore, for a wide range of cardiovascular diseases that cannot be treated with current medical technology, stem cell therapy has been proposed as an alternative treatment to secure blood flow to the ischemic tissue and reduce tissue damage by promoting myocardial regeneration from the tissue around the ischemic site.
- stem cell therapy has been proposed as an alternative treatment to secure blood flow to the ischemic tissue and reduce tissue damage by promoting myocardial regeneration from the tissue around the ischemic site.
- a number of methods for promoting the proliferation and development of cells in the heart by directly or administering a myocardial regeneration promoting factor or its gene are in progress. is in need.
- cardiac progenitor cells can be differentiated into three cell types, such as cardiomyocytes, smooth muscle cells, and vascular endothelial cells, and are known to be involved in the regeneration of damaged heart tissue because of their ability to self-renew.
- adult stem cells are used for clinical application. have.
- stem cell amplification and biological activity enhancement is a key factor for the commercialization of cell therapy. Accordingly, it is necessary to have a system capable of supplying functionally superior stem cells.
- research to enhance cell function for the practical use of stem cell therapeutics is being actively conducted.
- the present inventors have completed the present invention by preparing a fusion protein comprising the extracellular matrix fibronectin and vitronectin motif and mussel adhesion protein, and confirming the increase in cell activity and yield of the fusion protein through experiments.
- a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
- Another object of the present invention is to provide a medium composition comprising the fusion protein for cell culture, a cell culture machine, and a composition for increasing stem cell activity.
- Another object of the present invention is to provide a method for increasing the activity of stem cells, comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- Another object of the present invention is to provide a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- the present invention provides a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
- the present invention also provides a medium composition comprising the fusion protein for cell culture.
- the present invention also provides a composition for increasing the activity of stem cells comprising the fusion protein for cell culture.
- the present invention also provides a cell culture device coated with the fusion protein for cell culture.
- the present invention also provides a method for increasing the activity of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- the present invention also provides a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- the fusion protein for cell culture according to the present invention not only increases the biological activity of cultured cells, that is, cell adhesion ability, cell migration ability, cell senescence inhibiting ability and colony formation ability, but also significantly improves the yield of cells. Therefore, the fusion protein for cell culture according to the present invention can be used in various ways in the field of cell culture and the field of cell therapy.
- FIG. 1 is a diagram schematically illustrating an ECM peptide motif-based fusion protein according to the present invention.
- FIG. 2 is a diagram showing the results of analyzing the cytotoxicity of the ECM-based incubator according to the present invention through MTS analysis.
- FIG. 3 is a diagram showing the results of morphological analysis of human cardiac progenitor stem cells cultured in an ECM-based incubator according to the present invention.
- FIG. 4 is a diagram showing the results of analyzing the expression of cell markers in human cardiac progenitor stem cells cultured in the ECM-based incubator according to the present invention through flow cytometry.
- FIG. 5 is a diagram showing the number of cells initially attached to the ECM-based incubator according to the present invention through MTS analysis.
- FIG. 6 is a diagram showing the results of analyzing the number of cells attached to the ECM-based incubator according to the present invention through fluorescence imaging.
- FIG. 7 is a diagram showing the results of analyzing the expression of cell adhesion-related factors in human cardiac progenitor stem cells cultured in an ECM-based culture medium according to the present invention through Western blotting.
- FIG. 8 is a diagram showing the results of analyzing the cell migration ability of human cardiac progenitor stem cells cultured in the ECM-based incubator according to the present invention through a wound healing assay.
- FIG. 9 is a diagram showing the results of morphological analysis of human cardiac progenitor stem cells subcultured in an ECM-based culture medium according to the present invention.
- FIG. 10 is a diagram showing the results of analyzing the cellular senescence of human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention through SA- ⁇ -gal analysis.
- FIG. 11 is a diagram showing the results of analyzing the expression of cellular senescence markers in human cardiac progenitor stem cells subcultured in an ECM-based culture medium according to the present invention through Western blotting.
- FIG. 12 is a diagram showing the results of confirming the doubling time according to the number of passages of human heart progenitor stem cells subcultured in the ECM-based incubator according to the present invention.
- FIG. 13 is a diagram showing the results of analyzing the colony-forming ability of human heart progenitor stem cells subcultured in an ECM-based culture medium according to the present invention.
- FIG. 14 is a diagram showing the results of analyzing the expression of stem cell ability-related mRNA in human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention through qPT-PCR.
- FIG. 15 is a diagram showing the results of analyzing the cell yield according to the number of passages of human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention.
- a fusion protein for cell culture comprising an extracellular matrix (ECM) motif and a mussel adhesion protein.
- ECM extracellular matrix
- the extracellular matrix refers to the structure of a cell formed by various substances secreted by the cell to the outside of the cell.
- the extracellular matrix functions to protect and support cells by filling the space between cells and tissues.
- the extracellular matrix plays an important role in cell development and differentiation mechanisms through cell-to-cell biochemical signal transduction.
- the extracellular matrix may be at least one selected from the group consisting of fibronectin, vitronectin, laminin, gelatin and collagen, and preferably fibronectin or vitronectin.
- the extracellular matrix motif is preferably represented by the amino acid sequence of SEQ ID NO: 1 or 2.
- the scope of the extracellular matrix motif includes functional equivalents of the peptides represented by the amino acid sequence of SEQ ID NO: 1 or 2 and salts thereof.
- the "functional equivalent” means at least 80% or more, preferably 90%, more preferably 95% or more sequence homology (ie, identity) to the peptide of SEQ ID NO: 1 or 2 as a result of addition, substitution or deletion of amino acids
- 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% refers to a peptide having substantially the same physiological activity as the peptide of SEQ ID NO: 1 or 2, including those having sequence homology.
- Sequence homology and identity herein are defined as the percentage of amino acid residues of a candidate sequence with respect to the amino acid sequence of SEQ ID NO: 1 or 2 after aligning the amino acid sequence of SEQ ID NO: 1 or 2 with the candidate sequence and introducing gaps. do. If necessary, conservative substitutions as part of sequence identity are not considered in order to obtain the maximum percentage sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the amino acid sequence of SEQ ID NO: 1 or 2 are not to be construed as sequences affecting sequence identity or homology.
- the mussel adhesive protein may be mussel adhesive protein-151 (MAP-151).
- MAP-151 mussel adhesive protein-151
- the 6-repeat structure of the 10-repeat amino acids of mussel adhesive protein-1 was modified with N- and C of mussel adhesive protein-5.
- the mussel adhesion protein-151 has advantages in that it has high productivity in the E. coli expression system and can be isolated and purified using acetic acid.
- the mussel adhesive protein is preferably represented by the amino acid sequence of SEQ ID NO: 5.
- the cell culture is culturing adult stem cells derived from one or more selected from the group consisting of bone marrow, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, nerve and muscle. It is preferred, but not limited thereto.
- the fusion protein for cell culture is preferably represented by the amino acid sequence of SEQ ID NO: 3 or 4.
- the fusion protein for cell culture of the present invention can be variously used in the field of cell culture and the field of stem cell therapeutics.
- the present invention provides a medium composition comprising the fusion protein for cell culture.
- the medium means a culture medium that can support the growth and survival of stem cells in in vitro culture conditions, and includes all of the usual medium used in the art suitable for the culture of stem cells.
- the medium and culture conditions can be selected according to the type of cell.
- the medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source and a trace element component.
- Examples of the cell culture minimal medium include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, MEM (Minimal essential Medium), GMEM (Glasgow's) Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but is not limited thereto.
- the present invention provides a composition for increasing the activity of stem cells comprising the fusion protein for cell culture.
- the activity of the stem cells is preferably at least one selected from the group consisting of cell adhesion ability, cell migration ability, cell senescence inhibitory ability and colony formation ability, but is not limited thereto.
- the present invention provides a cell culture machine coated with the fusion protein for cell culture.
- the cell culture device may be in the form of a cell culture plate, a Petri dish, a culture flask, a chamber slide, a chamber or a tube, but is not limited thereto.
- the cell culture device may be one or more materials selected from the group consisting of glass, polystyrene, polycaprolactone and polypropylene, but any material suitable for cell attachment may be used without limitation.
- the present invention provides a method for increasing the activity of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- the stem cells are preferably adult stem cells derived from one or more selected from the group consisting of bone marrow, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, nerve and muscle. and, more preferably, cardiac-derived adult stem cells, but is not limited thereto.
- the activity of the stem cells may be one or more selected from the group consisting of cell adhesion ability, cell migration ability, cell senescence inhibitory ability and colony formation ability, but is not limited thereto.
- the present invention provides a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
- stem cells In general, when subculturing cells, cell growth and division are stopped or significantly delayed due to cell senescence.
- the mass production method of stem cells according to the present invention by increasing the biological activity of the stem cells, even if the number of passages increases, the yield of stem cells can be maintained or increased. That is, it means that it is possible to secure a large amount of stem cells through the method according to the present invention.
- Example 1 ECM (extracellular matrix) peptide motif-based fusion protein production and ECM-based incubator production
- Peptide motifs were selected from fibronectin and vitronectin, which are extracellular matrix (ECM).
- the selected fibronectin-based motif is represented by the amino acid sequence of SEQ ID NO: 1
- the vitronectin-based motif is represented by the amino acid sequence of SEQ ID NO: 2.
- the prepared ECM peptide motif-based fusion proteins were named Fibro-P (Fibronectin-MAP) and Vitro-P (Vitronectin-MAP), respectively.
- the Fibro-P is represented by the amino acid sequence of SEQ ID NO: 3
- Vitro-P is represented by the amino acid sequence of SEQ ID NO: 4.
- An ECM-based incubator was prepared using the ECM peptide motif-based fusion protein prepared in Example 1-2. Specifically, the fusion proteins prepared in Example 1-2 were coated on the plate at concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 and 4.8 mg/ml, respectively. The prepared ECM-based incubator was used for the experiments described below.
- the cytotoxicity of the ECM-based culture medium prepared in Example 1 was confirmed through MTS analysis. Specifically, 5,000 C-kit-positive human cardiac progenitor cells (CSC) were seeded in an ECM incubator (96-well plate) and then cultured. The medium used for the culture was F-12 Ham's Medium (Hyclone, #SH30026.01, GE healthcare, Chicago IL, USA), 10% FBS (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin/streptomycin , 2 mM glutathione (sigma-Aldrich, St.
- F-12 Ham's Medium Hyclone, #SH30026.01, GE healthcare, Chicago IL, USA
- FBS Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA
- penicillin/streptomycin 2 mM glutathione
- the cells were cultured in an incubator of 5% CO 2 and 37 ⁇ C conditions, and the medium was replaced and subcultured every 2 days.
- the counting medium was prepared by mixing the CCK (Cell Counting Kit) solution and the culture medium of the WST-based cell viability/toxicity evaluation kit in a volume ratio of 1:10. After 24 hours of cell culture, 100 ⁇ l of the counting medium was added to each well and reacted for 1 hour. After completion of the reaction, absorbance was measured at 450 nm using a microplate reader. Cell viability was derived based on the absorbance measurement, and cytotoxicity was confirmed therefrom. The results of the cytotoxicity analysis are shown in FIG. 2 .
- Example 2 Morphological analysis of human cardiac progenitor stem cells cultured in the ECM-based incubator prepared in Example 1 was performed. Specifically, C-kit-positive human cardiac progenitor stem cells were cultured under the same conditions as in Example 2. The cultured human cardiac progenitor stem cells were observed using an optical microscope, and the results are shown in FIG. 3 .
- Example 4 Cell marker expression analysis of human cardiac progenitor stem cells cultured in an ECM-based incubator
- Example 1 Expression of representative cell markers (CD34, CD45, CD133, CD90, CD29, CD44 and CD105) of human cardiac progenitor stem cells cultured in the ECM-based incubator of Example 1 was analyzed through flow cytometry. Specifically, human cardiac progenitor stem cells were seeded in ECM-based incubators Fibro-P and Vitro-P, respectively, and then cultured for 14 days. The cell culture was cultured in the same manner as in the culture conditions of Example 2. A cell dilution was prepared by diluting the cultured human progenitor stem cells in 100 ⁇ l of flow cytometry buffer (2 mM EDTA, 2% FBS in PBS solution).
- Antibodies for each cell marker were diluted in a ratio of 1:100 to 1:200, and then added to the cell dilution and reacted at 4°C for 30 minutes. After the reaction, the cells were washed 3 times with flow cytometry buffer and then centrifuged to obtain a cell pellet. The cell pellet was resuspended in 100 ⁇ l of flow cytometry buffer, and fluorescently labeled cells were analyzed using a flow cytometer. The flow cytometry results are shown in FIG. 4 .
- the cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P showed cell marker expression similar to that of the control group (Normal) after culture.
- the above result means that even when human heart progenitor stem cells are cultured for a long time in ECM-based incubators Fibro-P and Vitro-P, the phenotype is not significantly affected, that is, no transformation occurs in the cultured cells.
- Example 5 Analysis of cell adhesion ability of human cardiac progenitor stem cells cultured in an ECM-based incubator
- Human cardiac progenitor endothelial cells were seeded in ECM-based incubators (96 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 and 4.8 mg/ml). Before the cells were sufficiently attached, the supernatant was removed and the cells were washed with PBS. In the above case, only the rapidly attached cells at the beginning of seeding remain in the incubator. The cells remaining in the incubator were confirmed through the MTS analysis of Example 2, and the results are shown in FIG. 5 .
- Human cardiac progenitor endothelial cells were seeded in 1x10 5 ECM-based incubators (6 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05 and 0.1 mg/ml) and cultured for 4 hours. After incubation, the cells were washed twice with PBS. The washed cells were fluorescently stained using NucBlue Live Ready Probes Reagent. Fluorescently stained cells were imaged using a lion heart instrument. As a control in this example, the commercially available plate of Examples 1-3 and a plate coated with fibronectin at a concentration of 0.05 mg/ml were used. The results of fluorescence imaging are shown in FIG. 6 .
- the ECM-based incubator coated with Fibro-P and Vitro-P had a larger number of adherent cells compared to a commercially available plate (Normal).
- the ECM-based incubator coated with 0.1 mg/ml Fibro-P, 0.05 mg/ml Vitro-P, and 0.1 mg/ml Vitro-P had more adherent cells compared to the plate coated with fibronectin.
- Fak-Src a cell adhesion-related factor
- ECM-based culture medium ECM-based culture medium
- ECM-based incubators (6 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05 and 0.1 mg/ml) were prepared.
- Human cardiac progenitor stem cells were seeded and then cultured in the prepared ECM-based culture machine. After culture, the medium was removed, and protein lysis buffer was treated to extract and quantify intracellular proteins. Based on the results of protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the same amount of protein.
- SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis
- the electrophoresed protein was transferred to a PVDF membrane and blocked with 5% skim milk for 30 minutes.
- Primary antibodies (1:1000) for each protein were added to the blocked PVDF membrane and incubated overnight at 4°C.
- the primary antibodies are FAK (Santa Cruz), p-FAK (Cell Signaling), Src (Cell Signaling), p-Src (Santa Cruz), and b-actin (Santa Cruz).
- the HRP-conjugated secondary antibody was reacted at room temperature for 1 hour. After completion of the reaction, it was developed using an X-ray film and an Automatic X-ray Film Processor (JPI Healthcare). In addition, a graph was created based on the results of X-ray development using ImageJ software. Western blotting results are shown in FIG. 7 .
- Example 6 Analysis of cell migration ability of human cardiac progenitor stem cells cultured in an ECM-based incubator
- 1x10 5 human cardiac progenitor stem cells were seeded into each well of an ECM-based incubator coated with Fibro-P and Vitro-P at 0.1 mg/ml, and cultured for 2 days.
- the cells in the incubator were more than 90% full, the supernatant was removed and washed once with PBS. After that, PBS was added to the wells, and scratches were made using a yellow tip. Cell movement was observed with an optical microscope 3 and 6 hours after scratching, and the results are shown in FIG. 8 .
- Example 7 Analysis of senescence of human cardiac progenitor stem cells subcultured in an ECM-based culture medium
- C-kit-positive human cardiac progenitor stem cells were cultured for 24 days in the ECM-based incubator prepared in Example 1.
- the cell culture was cultured in the same manner as in the culture conditions of Example 2.
- the subcultured human cardiac progenitor stem cells were used for cellular senescence analysis to be described later.
- human progenitor stem cells passaged in an ECM-based incubator coated with Fibro-P and Vitro-P undergo cellular senescence according to the culture period and number of passages. It showed an increase in length and width.
- human progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P had significantly smaller length and width compared to the control group (Normal).
- Cell senescence of subcultured human cardiac progenitor stem cells was analyzed using the SA- ⁇ -gal assay kit (cell signaling). The cellular senescence assay was tested according to the manufacturer's manual. Experimental results were confirmed through microscopic imaging, and based on this, the number of aged cells, that is, ⁇ -gal positive cells (green) per cell group, was measured and graphed. Cell senescence analysis results are shown in FIG. 10 .
- human cardiac progenitor stem cells In the case of adult stem cells, when subcultured continuously, the doubling time of the cells increases due to aging. Accordingly, the doubling time according to the number of passages of human cardiac progenitor stem cells was confirmed. Specifically, human cardiac progenitor stem cells were cultured in an ECM-based incubator, and the time and number of cells were measured at each subculture. The doubling time was calculated based on the measured results, and the results are shown in FIG. 12 .
- Example 8 Stem cell ability analysis of human cardiac progenitor stem cells subcultured in an ECM-based culture medium
- Example 9 Analysis of the yield of human cardiac progenitor stem cells subcultured in an ECM-based incubator
- the present inventors prepared a fusion protein of an ECM motif and a mussel adhesion protein, and prepared an ECM-based incubator using the fusion protein. It was confirmed that the ECM-based incubator not only increased the cell adhesion, cell migration and colony forming ability of human cardiac progenitor stem cells, but also attenuated cellular senescence and significantly increased cell yield. This means that the ECM-based incubator increases the biological activity of stem cells, and can be used in various fields in cell culture and stem cell therapeutics.
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Abstract
The present invention relates to a fusion protein for cell culture, and more particularly to a fusion protein containing an extracellular matrix motif and a mussel adhesive protein. It was confirmed that the fusion protein for cell culture according to the present invention not only increases the biological activity of cultured cells, i.e., cell adhesion ability, cell migration ability, ability to inhibit cellular senescence, and colony formation ability, but also significantly improves cell yield. Therefore, the fusion protein for cell culture according to the present invention may be utilized in various ways in the fields of cell culture and cell therapeutic agents.
Description
본 발명은 세포배양용 융합 단백질에 관한 것으로, 보다 상세하게는 세포외기질 모티프 및 홍합 접착 단백질을 포함하는 세포배양용 융합 단백질에 관한 것이다.The present invention relates to a fusion protein for cell culture, and more particularly, to a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
심혈관질환은 세계적으로 가장 사망률이 높은 질환 중 하나이다. 이에 대한 기존의 주된 치료방법은 약물 투여 중심의 내과적 치료법과 막힌 혈관을 뚫어주는 경피적 심혈관 중재술/관상동맥 우회 이식술 등의 중재적 치료가 행해지며, 말기심부전과 같은 말기 심장질환에 이르렀을 경우 심장이식이 요구된다. 표준치료법을 통해 심혈관 질환으로 인한 사망률은 감소하였지만 여전히 심혈관 질환은 전 세계 사망원인 1위로 손꼽히고 있으며, 급성 심근경색의 약 30%는 만성 심부전으로 발전된다고 보고되어진다. 때문에 현재 의학기술로 치료가 불가능한 광범위한 심혈관 질환의 경우 대안 치료로 허혈 부위 주변의 조직으로부터 심근재생을 촉진하여 허혈조직의 혈류를 확보하고 조직의 손상을 줄이고자 하는 줄기세포 치료법이 제안되고 있다. 또 다른 치료전략으로써, 심근재생 촉진인자를 직접 또는 이의 유전자를 투여하여 심장 내 세포의 증식 및 발달을 촉진하는 방법이 다수 진행되고 있으나 미약한 효과 및 부작용 문제가 제기되어 실질적인 도입까지는 더 많은 연구가 필요한 실정이다.Cardiovascular disease is one of the diseases with the highest mortality rate worldwide. Existing main treatment methods for this are medical treatment centered on drug administration and interventional treatment such as percutaneous cardiovascular intervention/coronary artery bypass graft to clear blocked blood vessels. A transplant is required. Although mortality due to cardiovascular disease has decreased through standard treatment, cardiovascular disease is still considered the number one cause of death worldwide, and it is reported that about 30% of acute myocardial infarction develops into chronic heart failure. Therefore, for a wide range of cardiovascular diseases that cannot be treated with current medical technology, stem cell therapy has been proposed as an alternative treatment to secure blood flow to the ischemic tissue and reduce tissue damage by promoting myocardial regeneration from the tissue around the ischemic site. As another treatment strategy, a number of methods for promoting the proliferation and development of cells in the heart by directly or administering a myocardial regeneration promoting factor or its gene are in progress. is in need.
한편, 심장전구세포는 심근 세포, 평활근 세포, 혈관내피세포 등과 같은 세가지 세포유형으로 분화가 가능하며, 자가재생하는 능력을 가지고 있어 손상된 심장 조직의 재생에 관여한다고 알려져 있다. 현재 줄기세포를 이용한 혈관 신생 및 재생 분야에서는 임상적용을 위해 성체줄기세포를 이용하고 있는데 환자 자신의 몸에서 추출하여 필요한 조직에 자가 이식이 가능하며 배아줄기세포와 비교하여 윤리적 문제가 없다는 장점을 가지고 있다. 하지만 서구화된 식생활과 운동의 감소, 고령화 등으로 인한 환자유래 세포의 노화 및 기능 저하로 인하여 실용화 단계로 이어지는데 어려움이 있다. 때문에 줄기세포의 증폭 및 생물학적 활성 증진을 위한 연구는 세포 치료 실용화를 위한 핵심 요소이다. 이에, 기능적으로 보다 더 우수한 줄기세포를 수급할 수 있는 시스템을 갖추는 것이 필요하다. 이러한 요구에 최근 줄기세포 치료제 실용화를 위한 세포의 기능을 강화시키기 위한 연구가 활발히 진행되고 있다. On the other hand, cardiac progenitor cells can be differentiated into three cell types, such as cardiomyocytes, smooth muscle cells, and vascular endothelial cells, and are known to be involved in the regeneration of damaged heart tissue because of their ability to self-renew. Currently, in the field of angiogenesis and regeneration using stem cells, adult stem cells are used for clinical application. have. However, it is difficult to reach the commercialization stage due to the aging and functional deterioration of patient-derived cells due to westernized diet, reduced exercise, and aging. Therefore, research for stem cell amplification and biological activity enhancement is a key factor for the commercialization of cell therapy. Accordingly, it is necessary to have a system capable of supplying functionally superior stem cells. In response to this demand, research to enhance cell function for the practical use of stem cell therapeutics is being actively conducted.
이에 본 발명자들은 세포외기질인 피브로넥틴 및 비트로넥틴 모티프 및 홍합 접착 단백질을 포함하는 융합 단백질을 제조하고, 상기 융합 단백질의 세포 활성 및 수확량 증가를 실험을 통해 확인함으로써 본 발명을 완성하게 되었다.Accordingly, the present inventors have completed the present invention by preparing a fusion protein comprising the extracellular matrix fibronectin and vitronectin motif and mussel adhesion protein, and confirming the increase in cell activity and yield of the fusion protein through experiments.
따라서 본 발명의 목적은 세포외기질(extracellular matrix) 모티프 및 홍합 접착 단백질을 포함하는 세포배양용 융합 단백질을 제공하는 것이다.Accordingly, it is an object of the present invention to provide a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
본 발명의 다른 목적은 상기 세포배양용 융합 단백질을 포함하는 배지 조성물, 세포배양기 및 줄기세포 활성 증가용 조성물을 제공하는 것이다.Another object of the present invention is to provide a medium composition comprising the fusion protein for cell culture, a cell culture machine, and a composition for increasing stem cell activity.
본 발명의 또 다른 목적은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 활성을 증가시키는 방법을 제공하는 것이다.Another object of the present invention is to provide a method for increasing the activity of stem cells, comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
본 발명의 또 다른 목적은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 대량 생산 방법을 제공하는 것이다.Another object of the present invention is to provide a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
상기 목적을 달성하기 위하여, 본 발명은 세포외기질 모티프 및 홍합 접착 단백질을 포함하는 세포배양용 융합 단백질을 제공한다.In order to achieve the above object, the present invention provides a fusion protein for cell culture comprising an extracellular matrix motif and a mussel adhesion protein.
또한 본 발명은 상기 세포배양용 융합 단백질을 포함하는 배지 조성물을 제공한다.The present invention also provides a medium composition comprising the fusion protein for cell culture.
또한 본 발명은 상기 세포배양용 융합 단백질을 포함하는 줄기세포의 활성 증가용 조성물을 제공한다.The present invention also provides a composition for increasing the activity of stem cells comprising the fusion protein for cell culture.
또한 본 발명은 상기 세포배양용 융합 단백질이 코팅된 세포배양기를 제공한다.The present invention also provides a cell culture device coated with the fusion protein for cell culture.
또한 본 발명은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 활성을 증가시키는 방법을 제공한다.The present invention also provides a method for increasing the activity of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
또한 본 발명은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 대량 생산 방법을 제공한다.The present invention also provides a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
본 발명에 따른 세포배양용 융합 단백질은 배양된 세포의 생물학적 활성, 즉 세포 부착능, 세포 이동능, 세포 노화 억제능 및 콜로니 형성능을 증가시킬 뿐만 아니라, 세포의 수확량을 현저히 향상시킨다는 것을 확인하였다. 따라서 본 발명에 따른 세포배양용 융합 단백질은 세포배양 분야 및 세포 치료제 분야에서 다양하게 활용될 수 있다.It was confirmed that the fusion protein for cell culture according to the present invention not only increases the biological activity of cultured cells, that is, cell adhesion ability, cell migration ability, cell senescence inhibiting ability and colony formation ability, but also significantly improves the yield of cells. Therefore, the fusion protein for cell culture according to the present invention can be used in various ways in the field of cell culture and the field of cell therapy.
도 1은 본 발명에 따른 ECM 펩타이드 모티프 기반 융합 단백질을 간략히 나타낸 도이다.1 is a diagram schematically illustrating an ECM peptide motif-based fusion protein according to the present invention.
도 2는 MTS 분석을 통해 본 발명에 따른 ECM 기반 배양기의 세포독성을 분석한 결과를 나타낸 도이다.2 is a diagram showing the results of analyzing the cytotoxicity of the ECM-based incubator according to the present invention through MTS analysis.
도 3은 본 발명에 따른 ECM 기반 배양기로 배양된 인간심장전구줄기세포의 형태학적 분석 결과를 나타낸 도이다.3 is a diagram showing the results of morphological analysis of human cardiac progenitor stem cells cultured in an ECM-based incubator according to the present invention.
도 4는 유세포 분석을 통해 본 발명에 따른 ECM 기반 배양기로 배양된 인간심장전구줄기세포의 세포 표지자의 발현을 분석한 결과를 나타낸 도이다.4 is a diagram showing the results of analyzing the expression of cell markers in human cardiac progenitor stem cells cultured in the ECM-based incubator according to the present invention through flow cytometry.
도 5는 MTS 분석을 통해 본 발명에 따른 ECM 기반 배양기에 초기에 부착된 세포의 수를 나타낸 도이다.5 is a diagram showing the number of cells initially attached to the ECM-based incubator according to the present invention through MTS analysis.
도 6은 형광 이미징을 통해 본 발명에 따른 ECM 기반 배양기에 부착된 세포 수를 분석한 결과를 나타낸 도이다.6 is a diagram showing the results of analyzing the number of cells attached to the ECM-based incubator according to the present invention through fluorescence imaging.
도 7은 웨스턴 블롯팅을 통해 본 발명에 따른 ECM 기반 배양기에서 배양된 인간심장전구줄기세포의 세포 부착 관련 인자의 발현을 분석한 결과를 나타낸 도이다.7 is a diagram showing the results of analyzing the expression of cell adhesion-related factors in human cardiac progenitor stem cells cultured in an ECM-based culture medium according to the present invention through Western blotting.
도 8은 상처 회복 분석(wound healing assay)를 통해 본 발명에 따른 ECM 기반 배양기에서 배양된 인간심장전구줄기세포의 세포 이동능을 분석한 결과를 나타낸 도이다.8 is a diagram showing the results of analyzing the cell migration ability of human cardiac progenitor stem cells cultured in the ECM-based incubator according to the present invention through a wound healing assay.
도 9는 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 형태학적 분석 결과를 나타낸 도이다.9 is a diagram showing the results of morphological analysis of human cardiac progenitor stem cells subcultured in an ECM-based culture medium according to the present invention.
도 10은 SA-β-gal 분석을 통해 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 세포 노화를 분석한 결과를 나타낸 도이다.10 is a diagram showing the results of analyzing the cellular senescence of human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention through SA-β-gal analysis.
도 11은 웨스턴 블롯팅을 통해 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 세포 노화 마커의 발현을 분석한 결과를 나타낸 도이다.11 is a diagram showing the results of analyzing the expression of cellular senescence markers in human cardiac progenitor stem cells subcultured in an ECM-based culture medium according to the present invention through Western blotting.
도 12는 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구 줄기세포의 계대 수에 따른 배가 시간을 확인한 결과를 나타낸 도이다.12 is a diagram showing the results of confirming the doubling time according to the number of passages of human heart progenitor stem cells subcultured in the ECM-based incubator according to the present invention.
도 13은 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구 줄기세포의 콜로니 형성능을 분석한 결과를 나타낸 도이다.13 is a diagram showing the results of analyzing the colony-forming ability of human heart progenitor stem cells subcultured in an ECM-based culture medium according to the present invention.
도 14는 qPT-PCR을 통해 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 줄기세포능 관련 mRNA의 발현을 분석한 결과를 나타낸 도이다.14 is a diagram showing the results of analyzing the expression of stem cell ability-related mRNA in human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention through qPT-PCR.
도 15는 본 발명에 따른 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 계대 수에 따른 세포 수확량을 분석한 결과를 나타낸 도이다.15 is a diagram showing the results of analyzing the cell yield according to the number of passages of human cardiac progenitor stem cells subcultured in the ECM-based culture medium according to the present invention.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명의 양태에 따르면, 본 발명은 세포외기질(extracellular matrix, ECM) 모티프 및 홍합 접착 단백질을 포함하는 세포배양용 융합 단백질을 제공한다.According to an aspect of the present invention, there is provided a fusion protein for cell culture comprising an extracellular matrix (ECM) motif and a mussel adhesion protein.
본 발명에 있어서, 세포외기질은 세포가 세포의 외부로 분비한 여러 가지 물질이 형성하는 세포의 구조를 의미한다. 상기 세포외기질은 세포와 조직 사이의 공간을 채워줌으로써 세포를 보호 및 지지하는 기능을 한다. 또한 세포외기질은 세포 간 생화학적 신호 전달을 통해 세포의 발생 및 분화 메커니즘에서 중요한 역할을 한다.In the present invention, the extracellular matrix refers to the structure of a cell formed by various substances secreted by the cell to the outside of the cell. The extracellular matrix functions to protect and support cells by filling the space between cells and tissues. In addition, the extracellular matrix plays an important role in cell development and differentiation mechanisms through cell-to-cell biochemical signal transduction.
본 발명의 구체예에서, 상기 세포외기질은 피브로넥틴, 비트로넥틴, 라미닌, 젤라틴 및 콜라겐으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 바람직하게는 피브로넥틴 또는 비트로넥틴일 수 있다.In an embodiment of the present invention, the extracellular matrix may be at least one selected from the group consisting of fibronectin, vitronectin, laminin, gelatin and collagen, and preferably fibronectin or vitronectin.
본 발명의 구체예에서, 상기 세포외기질 모티프는 서열번호 1 또는 2의 아미노산 서열로 표시되는 것이 바람직하다. 또한 상기 세포외기질 모티프의 범위에는 서열번호 1 또는 2의 아미노산 서열로 표시되는 펩타이드의 기능적 동등물 및 그들의 염을 포함한다. 상기 "기능적 동등물"이란 아미노산의 부가, 치환 또는 결실의 결과 서열번호 1 또는 2의 펩타이드와 적어도 80％ 이상의, 바람직하게는 90％, 더욱 바람직하게는 95％이상의 서열 상동성(즉, 동일성)을 갖는 것으로 예를 들면, 80％, 81％, 82％, 83％, 84％, 85％, 86％, 87％, 88％, 89％, 90％, 91％, 92％, 93％, 94％, 95％, 96％, 97％, 98％, 99％, 100％의 서열 상동성을 갖는 것을 포함하며, 서열번호 1 또는 2의 펩타이드와 실질적으로 동질의 생리활성을 나타내는 펩타이드를 말한다. 본 명세서에서 서열 상동성 및 동질성은 서열번호 1 또는 2의 아미노산 서열과 후보 서열을 정렬하고 갭(gaps)을 도입한 후 서열번호 1 또는 2의 아미노산 서열에 대한 후보 서열의 아미노산 잔기의 백분율로서 정의된다. 필요한 경우, 최대 백분율 서열 동질성을 수득하기 위하여 서열 동질성의 부분으로서 보존적 치환은 고려하지 않는다. 서열번호 1 또는 2의 아미노산 서열의 N-말단, C-말단 또는 내부 신장, 결손 또는 삽입은 서열 동질성 또는 상동성에 영향을 주는 서열로서 해석되지 않는다.In an embodiment of the present invention, the extracellular matrix motif is preferably represented by the amino acid sequence of SEQ ID NO: 1 or 2. In addition, the scope of the extracellular matrix motif includes functional equivalents of the peptides represented by the amino acid sequence of SEQ ID NO: 1 or 2 and salts thereof. The "functional equivalent" means at least 80% or more, preferably 90%, more preferably 95% or more sequence homology (ie, identity) to the peptide of SEQ ID NO: 1 or 2 as a result of addition, substitution or deletion of amino acids For example, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99%, refers to a peptide having substantially the same physiological activity as the peptide of SEQ ID NO: 1 or 2, including those having sequence homology. Sequence homology and identity herein are defined as the percentage of amino acid residues of a candidate sequence with respect to the amino acid sequence of SEQ ID NO: 1 or 2 after aligning the amino acid sequence of SEQ ID NO: 1 or 2 with the candidate sequence and introducing gaps. do. If necessary, conservative substitutions as part of sequence identity are not considered in order to obtain the maximum percentage sequence identity. N-terminal, C-terminal or internal extensions, deletions or insertions of the amino acid sequence of SEQ ID NO: 1 or 2 are not to be construed as sequences affecting sequence identity or homology.
본 발명의 구체예에서, 상기 홍합 접착 단백질(mussel adhesive protein, MAP)은 홍합 접착 단백질-151(MAP-151)일 수 있다. 상기 홍합 접착 단백질-151은 발현 시스템에서 발현율이 낮다는 문제를 해결하기 위하여, 유전적 수준에서 홍합 접착 단백질-1의 10개 반복 아미노산의 6회 반복 구조를 홍합 접착 단백질-5의 N- 및 C- 말단에 연결하여 제조된 하이브리드 홍합 접착 단백질이다. 상기 홍합 접착 단백질-151은 대장균 발현 시스템에서 생산성이 높고, 아세트산을 이용하여 분리 및 정제할 수 있다는 장점이 있다.In an embodiment of the present invention, the mussel adhesive protein (MAP) may be mussel adhesive protein-151 (MAP-151). In order to solve the problem that the mussel adhesive protein-151 has a low expression rate in the expression system, at the genetic level, the 6-repeat structure of the 10-repeat amino acids of mussel adhesive protein-1 was modified with N- and C of mussel adhesive protein-5. - It is a hybrid mussel adhesive protein prepared by ligation at the end. The mussel adhesion protein-151 has advantages in that it has high productivity in the E. coli expression system and can be isolated and purified using acetic acid.
본 발명의 구체예에서, 상기 홍합 접착 단백질은 서열번호 5의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the mussel adhesive protein is preferably represented by the amino acid sequence of SEQ ID NO: 5.
본 발명의 구체예에서, 상기 세포배양은 골수, 혈액, 지방, 태반, 제대혈, 뇌, 간, 췌장, 심장, 피부, 신경 및 근육으로 이루어진 군에서 선택된 1 이상에서 유래된 성체줄기세포를 배양하는 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the cell culture is culturing adult stem cells derived from one or more selected from the group consisting of bone marrow, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, nerve and muscle. It is preferred, but not limited thereto.
본 발명의 구체예에서, 상기 세포배양용 융합 단백질은 서열번호 3 또는 4의 아미노산 서열로 표시되는 것이 바람직하다.In an embodiment of the present invention, the fusion protein for cell culture is preferably represented by the amino acid sequence of SEQ ID NO: 3 or 4.
본 발명의 실시예를 통해, 본 발명에 따른 세포배양용 융합 단백질이 코팅된 배양기로 성체줄기세포를 배양하는 경우 세포 부착능, 세포 이동능 및 콜로니 형성능을 현저히 증가시키고, 세포 노화를 감쇠시키며, 세포 수확량을 현저히 증가시키는 것을 확인하였다. 따라서 본 발명의 세포배양용 융합 단백질은 세포배양 분야 및 줄기세포 치료제 분야에서 다양하게 활용될 수 있다.Through the examples of the present invention, when culturing adult stem cells in an incubator coated with the fusion protein for cell culture according to the present invention, cell adhesion capacity, cell migration capacity and colony formation capacity are significantly increased, and cellular senescence is attenuated, It was confirmed that the cell yield was significantly increased. Therefore, the fusion protein for cell culture of the present invention can be variously used in the field of cell culture and the field of stem cell therapeutics.
본 발명의 다른 양태에 따르면, 본 발명은 상기 세포배양용 융합 단백질을 포함하는 배지 조성물을 제공한다.According to another aspect of the present invention, the present invention provides a medium composition comprising the fusion protein for cell culture.
본 발명에 있어서, 배지(media)는 체외배양 조건에서 줄기세포 성장 및 생존을 지지할 수 있게 하는 배양액을 의미하고, 줄기세포의 배양에 적절한 당 분야에서 사용되는 통상의 배지를 모두 포함한다. In the present invention, the medium (media) means a culture medium that can support the growth and survival of stem cells in in vitro culture conditions, and includes all of the usual medium used in the art suitable for the culture of stem cells.
또한 세포의 종류에 따라 배지와 배양 조건을 선택할 수 있다. 배양에 사용되는 배지는 바람직하게는 세포배양 최소 배지(cell culture minimum medium; CCMM)로, 일반적으로 탄소원, 질소원 및 미량원소 성분을 포함한다. 상기 세포배양 최소 배지의 예로는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal essential Medium), BME(Basal Medium Eagle), RPMI1640, F-10, F-12, MEM(Minimal essential Medium), GMEM(Glasgow's Minimal essential Medium), Iscove's Modified Dulbecco's Medium 등이 있으나, 이에 제한되지 않는다.In addition, the medium and culture conditions can be selected according to the type of cell. The medium used for the culture is preferably a cell culture minimum medium (CCMM), and generally contains a carbon source, a nitrogen source and a trace element component. Examples of the cell culture minimal medium include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, MEM (Minimal essential Medium), GMEM (Glasgow's) Minimal essential Medium), Iscove's Modified Dulbecco's Medium, etc., but is not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 세포배양용 융합 단백질을 포함하는 줄기세포의 활성 증가용 조성물를 제공한다.According to another aspect of the present invention, the present invention provides a composition for increasing the activity of stem cells comprising the fusion protein for cell culture.
본 발명의 구체예에서, 상기 줄기세포의 활성은 세포 부착능, 세포 이동능, 세포 노화 억제능 및 콜로니 형성능으로 이루어진 군에서 선택된 1 이상인 것이 바람직하나, 이에 제한되지 않는다.In an embodiment of the present invention, the activity of the stem cells is preferably at least one selected from the group consisting of cell adhesion ability, cell migration ability, cell senescence inhibitory ability and colony formation ability, but is not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 세포배양용 융합 단백질이 코팅된 세포배양기를 제공한다.According to another aspect of the present invention, the present invention provides a cell culture machine coated with the fusion protein for cell culture.
본 발명의 구체예에서, 세포배양기는 세포 배양 플레이트, 페트리 디쉬, 배양 플라스크, 챔버 슬라이드, 챔버 또는 튜브 형태일 수 있으며, 이에 제한되지 않는다.In an embodiment of the present invention, the cell culture device may be in the form of a cell culture plate, a Petri dish, a culture flask, a chamber slide, a chamber or a tube, but is not limited thereto.
또한 상기 세포배양기는 유리, 폴리스티렌, 폴리카프로락톤 및 폴리프로필렌으로 이루어진 군에서 선택된 1 이상의 소재일 수 있으나, 세포가 부착되기에 적합한 소재라면 제한없이 사용할 수 있다.In addition, the cell culture device may be one or more materials selected from the group consisting of glass, polystyrene, polycaprolactone and polypropylene, but any material suitable for cell attachment may be used without limitation.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 활성을 증가시키는 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for increasing the activity of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
본 발명의 구체예에서, 상기 줄기세포는 골수, 혈액, 지방, 태반, 제대혈, 뇌, 간, 췌장, 심장, 피부, 신경 및 근육으로 이루어진 군에서 선택된 1 이상에서 유래된 성체줄기세포인 것이 바람직하며, 더 바람직하게는 심장 유래 성체줄기세포이나, 이에 제한되지 않는다.In an embodiment of the present invention, the stem cells are preferably adult stem cells derived from one or more selected from the group consisting of bone marrow, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, nerve and muscle. and, more preferably, cardiac-derived adult stem cells, but is not limited thereto.
본 발명의 구체예에서, 상기 줄기세포의 활성은 세포 부착능, 세포 이동능, 세포 노화 억제능 및 콜로니 형성능으로 이루어진 군에서 선택된 1 이상일 수 있으나, 이에 제한되지 않는다.In an embodiment of the present invention, the activity of the stem cells may be one or more selected from the group consisting of cell adhesion ability, cell migration ability, cell senescence inhibitory ability and colony formation ability, but is not limited thereto.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는 줄기세포의 대량 생산 방법을 제공한다.According to another aspect of the present invention, the present invention provides a method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture and the stem cells in vitro.
일반적으로, 세포의 계대배양을 할 경우 세포의 노화(senescence)로 인해 세포 성장 및 분열이 정지되거나 유의성있게 지연된다. 본 발명에 따른 줄기세포의 대량 생산 방법은 줄기세포의 생물학적 활성을 증가시킴으로써, 계대 수가 증가하여도 줄기세포의 수확량이 유지 또는 증가될 수 있다. 즉, 본 발명에 따른 방법을 통해 줄기세포의 대량 확보가 가능하다는 것을 의미한다.In general, when subculturing cells, cell growth and division are stopped or significantly delayed due to cell senescence. The mass production method of stem cells according to the present invention by increasing the biological activity of the stem cells, even if the number of passages increases, the yield of stem cells can be maintained or increased. That is, it means that it is possible to secure a large amount of stem cells through the method according to the present invention.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. ECM(extracellular matrix) 펩타이드 모티프 기반 융합 단백질 제작 및 ECM 기반 배양기 제조Example 1. ECM (extracellular matrix) peptide motif-based fusion protein production and ECM-based incubator production
1-1. ECM 펩타이드 모티프 선별1-1. ECM peptide motif selection
ECM(extracellular matrix)인 피브로넥틴(fibronectin) 및 비트로넥틴(vitronectin)으로부터 펩타이드 모티프를 선별하였다. 선별된 피브로넥틴 기반 모티프는 서열번호 1의 아미노산 서열로 표시되며, 비트로넥틴 기반 모티프는 서열번호 2의 아미노산 서열로 표시된다.Peptide motifs were selected from fibronectin and vitronectin, which are extracellular matrix (ECM). The selected fibronectin-based motif is represented by the amino acid sequence of SEQ ID NO: 1, and the vitronectin-based motif is represented by the amino acid sequence of SEQ ID NO: 2.
1-2. ECM 펩타이드 모티프 기반 융합 단백질 제작1-2. ECM peptide motif-based fusion protein construction
도 1에 나타낸 바와 같이, 홍합 접착 단백질-151(mussel adhesive protein-151, MAP-151)(서열번호 5)의 말단에 상기 실시예 1-1에서 선별된 ECM 펩타이드 모티프를 결합시켜, ECM 펩타이드 모티프 기반 융합 단백질을 제작하였다. As shown in FIG. 1, by binding the ECM peptide motif selected in Example 1-1 to the end of mussel adhesive protein-151 (MAP-151) (SEQ ID NO: 5), the ECM peptide motif A base fusion protein was constructed.
제작된 ECM 펩타이드 모티프 기반 융합 단백질은 각각 Fibro-P(Fibronectin-MAP) 및 Vitro-P(Vitronectin-MAP)로 명명하였다. 상기 Fibro-P는 서열번호 3의 아미노산 서열로 표시되며, Vitro-P는 서열번호 4의 아미노산 서열로 표시된다.The prepared ECM peptide motif-based fusion proteins were named Fibro-P (Fibronectin-MAP) and Vitro-P (Vitronectin-MAP), respectively. The Fibro-P is represented by the amino acid sequence of SEQ ID NO: 3, and Vitro-P is represented by the amino acid sequence of SEQ ID NO: 4.
1-3. ECM 기반 배양기 제조1-3. ECM-based incubator manufacturing
상기 실시예 1-2에서 제작된 ECM 펩타이드 모티프 기반 융합 단백질을 이용하여 ECM 기반 배양기를 제조하였다. 구체적으로, 상기 실시예 1-2에서 제작된 융합 단백질을 각각 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 및 4.8 mg/ml의 농도로 플레이트에 코팅하였다. 제조된 ECM 기반 배양기는 후술된 실험에 사용하였다.An ECM-based incubator was prepared using the ECM peptide motif-based fusion protein prepared in Example 1-2. Specifically, the fusion proteins prepared in Example 1-2 were coated on the plate at concentrations of 0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 and 4.8 mg/ml, respectively. The prepared ECM-based incubator was used for the experiments described below.
또한 후술되는 실험의 대조군은 시판중인 배양 플레이트(Cell Culture Dish, SPL, Cat. 20100)를 이용하였고, ‘No treat’ 또는 ‘Normal’로 표시하였다.In addition, a commercially available culture plate (Cell Culture Dish, SPL, Cat. 20100) was used as a control group for the experiment to be described later, and was marked as 'No treat' or 'Normal'.
실시예 2. ECM 기반 배양기의 세포독성 분석Example 2. Cytotoxicity analysis of ECM-based incubator
상기 실시예 1에서 제조된 ECM 기반 배양기의 세포독성은 MTS 분석을 통해 확인하였다. 구체적으로, C-kit 양성 인간심장전구줄기세포(cardiac progenitor cell, CSC) 5,000개를 ECM 배양기(96웰 플레이트)에 시딩한 후 배양하였다. 상기 배양에 사용한 배지는 F-12 Ham's Medium(Hyclone, #SH30026.01, GE healthcare, Chicago IL, USA), 10% FBS(Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin/streptomycin, 2mM glutathione(sigma-Aldrich, St.Louis, CA, USA), 10ng/mL recombinant human basic fibroblast growth factor(rb-FGF; Peprotech, Rocky Hill, NJ, USA) 및 0.005 unit/mL human erythropoietin(hEPO; R&D system, Minneapolis, MN, USA)을 포함한다. 또한 상기 세포는 5% CO
2 및 37˚C 조건의 배양기에서 배양하였으며, 2일 마다 배지를 교체 및 계대배양하였다. 한편, WST-기반 세포 생존능/독성 평가 키트의 CCK(Cell Counting Kit) 용액 및 배양 배지를 1:10의 부피비로 혼합하여 카운팅 배지를 준비하였다. 세포 배양 24시간 후 각 웰에 상기 카운팅 배지 100 μl씩 첨가하여 1시간 동안 반응시켰다. 반응 종료 후 마이크로플레이트 리더기를 이용하여, 450 nm에서 흡광도를 측정하였다. 흡광도 측정값에 기초하여 세포 생존율을 도출하였고, 이로부터 세포 독성을 확인하였다. 세포독성 분석 결과는 도 2에 나타내었다.The cytotoxicity of the ECM-based culture medium prepared in Example 1 was confirmed through MTS analysis. Specifically, 5,000 C-kit-positive human cardiac progenitor cells (CSC) were seeded in an ECM incubator (96-well plate) and then cultured. The medium used for the culture was F-12 Ham's Medium (Hyclone, #SH30026.01, GE healthcare, Chicago IL, USA), 10% FBS (Gibco, Thermo Fisher Scientific, Carlsbad, CA, USA), 1% penicillin/streptomycin , 2 mM glutathione (sigma-Aldrich, St. Louis, CA, USA), 10 ng/mL recombinant human basic fibroblast growth factor (rb-FGF; Peprotech, Rocky Hill, NJ, USA) and 0.005 unit/mL human erythropoietin (hEPO; R&D system, Minneapolis, MN, USA). In addition, the cells were cultured in an incubator of 5% CO 2 and 37˚C conditions, and the medium was replaced and subcultured every 2 days. On the other hand, the counting medium was prepared by mixing the CCK (Cell Counting Kit) solution and the culture medium of the WST-based cell viability/toxicity evaluation kit in a volume ratio of 1:10. After 24 hours of cell culture, 100 μl of the counting medium was added to each well and reacted for 1 hour. After completion of the reaction, absorbance was measured at 450 nm using a microplate reader. Cell viability was derived based on the absorbance measurement, and cytotoxicity was confirmed therefrom. The results of the cytotoxicity analysis are shown in FIG. 2 .
도 2에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 배양된 C-kit 양성 심장전구줄기세포는 시판중인 배양기(즉, 대조군)에 비해 세포 생존율이 높은 것을 확인하였다. 상기 결과는 실시예 1에서 제조된 ECM 기반 배양기는 세포 독성이 매우 낮다는 것을 의미한다.As shown in FIG. 2 , it was confirmed that C-kit positive progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P had a higher cell viability compared to a commercially available incubator (ie, control). The above result means that the ECM-based incubator prepared in Example 1 has very low cytotoxicity.
실시예 3. ECM 기반 배양기로 배양된 인간심장전구줄기세포의 형태학적 분석Example 3. Morphological analysis of human cardiac progenitor stem cells cultured in an ECM-based incubator
상기 실시예 1에서 제조된 ECM 기반 배양기로 배양된 인간심장전구줄기세포의 형태학적 분석을 실시하였다. 구체적으로, 상기 실시예 2와 같은 조건으로 C-kit 양성 인간심장전구줄기세포를 배양하였다. 배양된 인간심장전구줄기세포를 광학현미경으로 세포를 관찰하였으며, 그 결과는 도 3에 나타내었다.Morphological analysis of human cardiac progenitor stem cells cultured in the ECM-based incubator prepared in Example 1 was performed. Specifically, C-kit-positive human cardiac progenitor stem cells were cultured under the same conditions as in Example 2. The cultured human cardiac progenitor stem cells were observed using an optical microscope, and the results are shown in FIG. 3 .
도 3에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 배양된 인간심장전구줄기세포는 대조군과 형태학적으로 유사한 것을 확인하였다.As shown in FIG. 3 , it was confirmed that human cardiac progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P were morphologically similar to the control group.
실시예 4. ECM 기반 배양기로 배양된 인간심장전구줄기세포의 세포 표지자 발현 분석Example 4. Cell marker expression analysis of human cardiac progenitor stem cells cultured in an ECM-based incubator
유세포 분석을 통해 상기 실시예 1의 ECM 기반 배양기로 배양된 인간심장전구줄기세포의 대표적인 세포 표지자(CD34, CD45, CD133, CD90, CD29, CD44 및 CD105)의 발현을 분석하였다. 구체적으로, ECM 기반 배양기 Fibro-P 및 Vitro-P에서 인간심장전구줄기세포를 각각 시딩한 후 14일 동안 배양하였다. 상기 세포배양은 실시예 2의 배양조건과 동일하게 배양하였다. 배양된 인간심장전구줄기세포를 유세포 분석 버퍼(2 mM EDTA, 2% FBS in PBS solultion) 100 μl에 희석하여 세포 희석액을 제조하였다. 각 세포 표지자에 대한 항체ff 1:100 내지 1:200의 비율로 희석한 후 상기 세포 희석액에 첨가하여 4℃에서 30분 동안 반응시켰다. 반응 후 유세포 분석 버퍼로 세포를 3회 세척한 후 원심분리하여 세포 펠릿을 얻었다. 상기 세포 펠릿을 유세포 분석 버퍼 100 μl에 재부유시켰으며, 유세포 분석기를 이용하여 형광으로 표지된 세포를 분석하였다. 유세포 분석 결과는 도 4에 나타내었다.Expression of representative cell markers (CD34, CD45, CD133, CD90, CD29, CD44 and CD105) of human cardiac progenitor stem cells cultured in the ECM-based incubator of Example 1 was analyzed through flow cytometry. Specifically, human cardiac progenitor stem cells were seeded in ECM-based incubators Fibro-P and Vitro-P, respectively, and then cultured for 14 days. The cell culture was cultured in the same manner as in the culture conditions of Example 2. A cell dilution was prepared by diluting the cultured human progenitor stem cells in 100 μl of flow cytometry buffer (2 mM EDTA, 2% FBS in PBS solution). Antibodies for each cell marker were diluted in a ratio of 1:100 to 1:200, and then added to the cell dilution and reacted at 4°C for 30 minutes. After the reaction, the cells were washed 3 times with flow cytometry buffer and then centrifuged to obtain a cell pellet. The cell pellet was resuspended in 100 μl of flow cytometry buffer, and fluorescently labeled cells were analyzed using a flow cytometer. The flow cytometry results are shown in FIG. 4 .
도 4에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기로 배양된 세포는 배양 후 세포 표지자의 발현이 대조군(Normal)과 유사하였다. 상기 결과는 ECM 기반 배양기 Fibro-P 및 Vitro-P에서 인간심장전구줄기세포를 장기배양하여도, 표현형에는 큰 영향을 받지 않는다는 것, 즉 배양된 세포에 변형이 일어나지 않는다는 것을 의미한다.As shown in FIG. 4 , the cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P showed cell marker expression similar to that of the control group (Normal) after culture. The above result means that even when human heart progenitor stem cells are cultured for a long time in ECM-based incubators Fibro-P and Vitro-P, the phenotype is not significantly affected, that is, no transformation occurs in the cultured cells.
실시예 5. ECM 기반 배양기로 배양된 인간심장전구줄기세포의 세포 부착능 분석Example 5. Analysis of cell adhesion ability of human cardiac progenitor stem cells cultured in an ECM-based incubator
5-1. 초기 부착세포 확인5-1. Identification of early adherent cells
Fibro-P 및 Vitro-P를 다양한 농도(0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 및 4.8 mg/ml)로 코팅한 ECM 기반 배양기(96웰)에 인간심장전구내피세포를 시딩하였다. 세포가 충분히 부착되기 전에 상층액을 제거하고, PBS로 세포를 세척하였다. 위의 경우 시딩 초기에 빠르게 부착된 세포만 배양기에 남게된다. 배양기에 남아있는 세포를 상기 실시예 2의 MTS 분석을 통해 확인하였으며, 그 결과는 도 5에 나타내었다.Human cardiac progenitor endothelial cells were seeded in ECM-based incubators (96 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05, 0.1, 0.2, 0.4, 0.8, 1.2, 2.4 and 4.8 mg/ml). Before the cells were sufficiently attached, the supernatant was removed and the cells were washed with PBS. In the above case, only the rapidly attached cells at the beginning of seeding remain in the incubator. The cells remaining in the incubator were confirmed through the MTS analysis of Example 2, and the results are shown in FIG. 5 .
도 5에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기는 초기에 부착된 세포수가 대조군(No treat)에 비해 현저히 많은 것을 확인하였다.As shown in FIG. 5 , it was confirmed that the number of initially adhered cells in the ECM-based incubator coated with Fibro-P and Vitro-P was significantly higher than that of the control group (No treat).
5-2. 형광 이미징5-2. fluorescence imaging
Fibro-P 및 Vitro-P를 다양한 농도(0.05 및 0.1 mg/ml)로 코팅한 ECM 기반 배양기(6웰)에 인간심장전구내피세포를 1x10
5개 시딩한 후 4시간 동안 배양하였다. 배양 후 세포를 PBS로 2회 세척하였다. 세척된 세포를 NucBlue Live Ready Probes Reagent를 이용하여 형광염색하였다. Lion heart 기기를 이용하여 형광염색된 세포를 이미징하였다. 본 실시예에서 대조군은 상기 실시예 1-3의 시판중인 플레이트 및 피브로넥틴을 0.05 mg/ml의 농도로 코팅한 플레이트를 이용하였다. 형광이미징 결과는 도 6에 나타내었다. Human cardiac progenitor endothelial cells were seeded in 1x10 5 ECM-based incubators (6 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05 and 0.1 mg/ml) and cultured for 4 hours. After incubation, the cells were washed twice with PBS. The washed cells were fluorescently stained using NucBlue Live Ready Probes Reagent. Fluorescently stained cells were imaged using a lion heart instrument. As a control in this example, the commercially available plate of Examples 1-3 and a plate coated with fibronectin at a concentration of 0.05 mg/ml were used. The results of fluorescence imaging are shown in FIG. 6 .
도 6에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기는 시판중인 플레이트(Normal)에 비해 부착된 세포의 수가 많은 것을 확인하였다. 특히, 0.1 mg/ml Fibro-P, 0.05 mg/ml Vitro-P 및 0.1 mg/ml Vitro-P로 코팅된 ECM 기반 배양기는 피브로넥틴으로 코팅한 플레이트에 비해 부착된 세포가 많은 것을 확인하였다.As shown in FIG. 6 , it was confirmed that the ECM-based incubator coated with Fibro-P and Vitro-P had a larger number of adherent cells compared to a commercially available plate (Normal). In particular, it was confirmed that the ECM-based incubator coated with 0.1 mg/ml Fibro-P, 0.05 mg/ml Vitro-P, and 0.1 mg/ml Vitro-P had more adherent cells compared to the plate coated with fibronectin.
5-3. 세포 부착 관련 인자의 발현 분석5-3. Expression analysis of cell adhesion-related factors
ECM 기반 배양기에서 배양된 인간심장전구줄기세포에서 세포 부착 관련인자인 Fak-Src의 발현 및 인산화 여부를 웨스턴 블롯팅을 통해 분석하였다. 구체적으로, Fibro-P 및 Vitro-P를 다양한 농도(0.05 및 0.1 mg/ml)로 코팅한 ECM 기반 배양기(6웰)를 준비하였다. 준비된 ECM 기반 배앙기에 인간심장전구줄기세포를 시딩한 후 배양하였다. 배양 후 배지를 제거하였고, 단백질 용해 버퍼를 처리하여 세포 내 단백질을 추출 및 정량하였다. 단백질 정량 결과를 토대로, 동량의 단백질을 이용하여 SDS-PAGE(sodium dodecyl sulfate-polyacrylamide gel electrophoresis)를 실시하였다. 그 후, 전기영동된 단백질을 PVDF 멤브레인에 옮겨 5% 스킴밀크로 30분 동안 블로킹(blocking)하였다. 블로킹된 PVDF 멤브레인에 각 단백질에 대한 1차 항체(1:1000)를 첨가하여 4℃에서 밤새도록 배양하였다. 상기 1차 항체는 FAK(Santa Cruz), p-FAK(Cell Signalling), Src(Cell Singnaling), p-Src(Santa Cruz) 및 b-actin(Santa Cruz)이다. 1차 항체와 반응시킨 후 HRP가 결합된 2차 항체를 상온에서 1시간 동안 반응시켰다. 반응 종료 후 X-ray 필름 및 Automatic X-ray Film Processor(JPI Healthcare)를 이용하여 현상하였다. 또한 이미지J 소프트웨어를 이용하여 X-Ray 현상 결과에 기초한 그래프를 작성하였다. 웨스턴 블롯팅 결과는 도 7에 나타내었다.Expression and phosphorylation of Fak-Src, a cell adhesion-related factor, in human cardiac progenitor stem cells cultured in an ECM-based culture medium were analyzed by Western blotting. Specifically, ECM-based incubators (6 wells) coated with Fibro-P and Vitro-P at various concentrations (0.05 and 0.1 mg/ml) were prepared. Human cardiac progenitor stem cells were seeded and then cultured in the prepared ECM-based culture machine. After culture, the medium was removed, and protein lysis buffer was treated to extract and quantify intracellular proteins. Based on the results of protein quantification, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using the same amount of protein. After that, the electrophoresed protein was transferred to a PVDF membrane and blocked with 5% skim milk for 30 minutes. Primary antibodies (1:1000) for each protein were added to the blocked PVDF membrane and incubated overnight at 4°C. The primary antibodies are FAK (Santa Cruz), p-FAK (Cell Signaling), Src (Cell Signaling), p-Src (Santa Cruz), and b-actin (Santa Cruz). After reacting with the primary antibody, the HRP-conjugated secondary antibody was reacted at room temperature for 1 hour. After completion of the reaction, it was developed using an X-ray film and an Automatic X-ray Film Processor (JPI Healthcare). In addition, a graph was created based on the results of X-ray development using ImageJ software. Western blotting results are shown in FIG. 7 .
도 7에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기로 배양된 인간심장전구줄기세포는 인산화된 FAK 및 Src의 발현이 대조군(Nermal)에 비해 현저히 높은 것을 확인하였다. 상기 결과는 ECM 기반 배양기로 배양된 세포의 세포 부착능이 증가하는 것은, 다양한 세포의 부착을 조절하는 대표적인 신호전달 경로인 Fak-Src 신호의 활성화에 의한 것임을 의미한다.As shown in FIG. 7 , it was confirmed that the expression of phosphorylated FAK and Src in human cardiac progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P was significantly higher than that of the control group (Nermal). The above result means that the increase in the cell adhesion capacity of cells cultured in an ECM-based incubator is due to the activation of the Fak-Src signal, which is a representative signaling pathway regulating the adhesion of various cells.
실시예 6. ECM 기반 배양기로 배양된 인간심장전구줄기세포의 세포 이동능 분석Example 6. Analysis of cell migration ability of human cardiac progenitor stem cells cultured in an ECM-based incubator
세포의 이동능을 분석하기 위하여, Fibro-P 및 Vitro-P가 0.1mg/ml로 코팅된 ECM 기반 배양기의 각 웰에 인간심장전구줄기세포 1x10
5개를 시딩한 후 2일 동안 배양하였다. 배양기에 세포가 90% 이상 찼을 때 상층액을 제거한 후 PBS로 1회 세척하였다. 그 후 웰에 PBS를 첨가하였고, 옐로우팁을 이용하여 스크래치를 냈다. 스크래치를 낸지 3 및 6시간 후 세포의 이동을 광학현미경으로 관찰하였으며, 그 결과는 도 8에 나타내었다. To analyze the cell mobility, 1x10 5 human cardiac progenitor stem cells were seeded into each well of an ECM-based incubator coated with Fibro-P and Vitro-P at 0.1 mg/ml, and cultured for 2 days. When the cells in the incubator were more than 90% full, the supernatant was removed and washed once with PBS. After that, PBS was added to the wells, and scratches were made using a yellow tip. Cell movement was observed with an optical microscope 3 and 6 hours after scratching, and the results are shown in FIG. 8 .
도 8에 나타낸 바와같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기로 배양된 인간심장전구줄기세포는 대조군(Normal)에 비해 스크래치 범위가 현저시 감소, 즉, 세포가 많이 이동한 것을 확인하였다.As shown in FIG. 8, human cardiac progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P significantly reduced the scratch range compared to the control (Normal), that is, the cells migrated a lot. Confirmed.
실시예 7. ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 세포의 노화 분석Example 7. Analysis of senescence of human cardiac progenitor stem cells subcultured in an ECM-based culture medium
실시예 1에서 제조된 ECM 기반 배양기로 C-kit 양성 인간심장전구줄기세포를 24일 동안 배양하였다. 상기 세포배양은 실시예 2의 배양조건과 동일하게 배양하였다. 계대배양된 인간심장전구줄기세포는 후술되는 세포 노화 분석에 사용하였다.C-kit-positive human cardiac progenitor stem cells were cultured for 24 days in the ECM-based incubator prepared in Example 1. The cell culture was cultured in the same manner as in the culture conditions of Example 2. The subcultured human cardiac progenitor stem cells were used for cellular senescence analysis to be described later.
7-1. 형태학적 분석7-1. Morphological analysis
계대배양된 인간심장전구줄기세포의 형태학적 분석을 실시하였다. 배양된 인간심장전구줄기세포를 광학현미경으로 세포를 관찰하였으며, 그 결과는 도 9에 나타내었다.Morphological analysis of subcultured human cardiac progenitor stem cells was performed. Cultured human cardiac progenitor stem cells were observed using an optical microscope, and the results are shown in FIG. 9 .
도 9에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 배양 기간 및 계대 수에 따라 세포 노화가 진행되며, 상기 세포 노화에 따라 세포의 길이 및 너비가 증가하는 양상을 보였다. 다만, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기로 배양된 인간심장전구줄기세포는 대조군(Normal)에 비해 세포의 길이 및 너비가 현저히 작은 것을 확인하였다. As shown in FIG. 9, human progenitor stem cells passaged in an ECM-based incubator coated with Fibro-P and Vitro-P undergo cellular senescence according to the culture period and number of passages. It showed an increase in length and width. However, it was confirmed that human progenitor stem cells cultured in an ECM-based incubator coated with Fibro-P and Vitro-P had significantly smaller length and width compared to the control group (Normal).
7-2. 세포 노화에 대한 방어능 분석7-2. Analysis of defense against cellular aging
SA-β-gal assay kit(cell signaling)를 이용하여 계대배양된 인간심장전구줄기세포의 세포 노화를 분석하였다. 상기 세포 노화 분석은 제조사의 매뉴얼에 따라 실험하였다. 실험 결과는 현미경 이미징을 통해 확인하였고, 이를 토대로 세포그룹 당 노화된 세포, 즉 β-gal 양성 세포(녹색)의 수를 측정하여 그래프화하였다. 세포 노화 분석 결과는 도 10에 나타내었다.Cell senescence of subcultured human cardiac progenitor stem cells was analyzed using the SA-β-gal assay kit (cell signaling). The cellular senescence assay was tested according to the manufacturer's manual. Experimental results were confirmed through microscopic imaging, and based on this, the number of aged cells, that is, β-gal positive cells (green) per cell group, was measured and graphed. Cell senescence analysis results are shown in FIG. 10 .
도 10에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 대조군(Normal)에 비해 노화된 세포가 유의하게 적은 것을 확인하였다. 이는 Fibro-P 및 Vitro-P가 세포 노화를 감쇠시킨다는 것을 의미한다.As shown in FIG. 10 , it was confirmed that the number of senescent cells was significantly less in the human heart progenitor stem cells subcultured in the ECM-based incubator coated with Fibro-P and Vitro-P compared to the control group (Normal). This means that Fibro-P and Vitro-P attenuate cellular senescence.
7-3. 세포 노화 마커의 발현 분석7-3. Expression analysis of cellular senescence markers
웨스턴 블롯팅을 통해 계대배양된 인간심장전구줄기세포의 세포 노화 마커 P53 및 P16의 발현을 분석하였다. 상기 웨스턴 블롯팅은 실시예 5-3의 방법으로 실시하였고, 1차 항체는 p16(Abcam) 및 p53(Abcam)을 사용하였다. 웨스턴 블롯팅 결과는 도 11에 나타내었다.Expression of cellular senescence markers P53 and P16 in subcultured human cardiac progenitor stem cells was analyzed by Western blotting. The Western blotting was carried out as in Example 5-3, and p16 (Abcam) and p53 (Abcam) were used as primary antibodies. Western blotting results are shown in FIG. 11 .
도 11에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 대조군(Normal)에 비해 세포 노화 마커의 발현이 낮은 것을 확인하였다.As shown in FIG. 11 , it was confirmed that human cardiac progenitor stem cells subcultured in an ECM-based incubator coated with Fibro-P and Vitro-P had lower expression of cellular senescence markers compared to the control group (Normal).
7-4. 계대 수에 따른 배가 시간 확인7-4. Check doubling time according to the number of passages
성체줄기세포의 경우 지속적으로 계대배양할 경우 노화로 인해 세포의 배가 시간(doubling time)이 증가한다. 이에, 인간심장전구줄기세포의 계대 수에 따른 배가 시간을 확인하였다. 구체적으로, 인간심장전구줄기세포를 ECM 기반 배양기에서 배양하였고, 계대배양할 때 마다 시간 및 세포 수를 측정하였다. 측정된 결과를 바탕으로 배가 시간을 계산하였으며, 그 결과는 도 12에 나타내었다.In the case of adult stem cells, when subcultured continuously, the doubling time of the cells increases due to aging. Accordingly, the doubling time according to the number of passages of human cardiac progenitor stem cells was confirmed. Specifically, human cardiac progenitor stem cells were cultured in an ECM-based incubator, and the time and number of cells were measured at each subculture. The doubling time was calculated based on the measured results, and the results are shown in FIG. 12 .
도 12에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 배가 시간이 대조군(Normal)에 비해 현저히 짧은 것을 확인하였다.As shown in FIG. 12 , it was confirmed that the doubling time of the human progenitor stem cells subcultured in the ECM-based incubator coated with Fibro-P and Vitro-P was significantly shorter than that of the control group (Normal).
상기 결과는 Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 대조군에 비해 유의적으로 세포 노화가 감쇠한다는 것을 의미한다.The above results mean that cellular senescence was significantly attenuated in human cardiac progenitor stem cells subcultured in an ECM-based culture medium coated with Fibro-P and Vitro-P compared to the control group.
실시예 8. ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포의 줄기세포능 분석Example 8. Stem cell ability analysis of human cardiac progenitor stem cells subcultured in an ECM-based culture medium
8-1. 콜로니 형성능8-1. Colony forming ability
심장줄기세포의 주요 특징 중 하나인 줄기세포능에 미치는 영향을 콜로니 형성능 분석을 통해 확인하였다. 구체적으로, 인간심장전구줄기세포를 ECM 기반 배양기에서 3회 계대배양한 후 세포를 수확하였다. 수확된 세포(계대 수 : 4)를 단일세포로 준비하였다. 상기 단일세포를 96웰 플레이트(1% 젤라틴 코팅)에 시딩하고, 7 내지 14일 동안 배양하였다. 배양 기간 동안 현미경을 통해 콜로니 형성 여부를 확인하였다. 실험 결과는 콜로니가 형성된 웰의 수를 측정하여 그래프로 나타내었다. 콜로니 형성능 분석 결과는 도 13에 나타내었다.The effect on stem cell ability, one of the main characteristics of cardiac stem cells, was confirmed through colony forming ability analysis. Specifically, human cardiac progenitor stem cells were subcultured three times in an ECM-based incubator, and then the cells were harvested. Harvested cells (passage number: 4) were prepared as single cells. The single cells were seeded in 96-well plates (1% gelatin coating) and cultured for 7 to 14 days. During the culture period, colony formation was confirmed through a microscope. Experimental results were displayed by measuring the number of wells in which colonies were formed. The results of the colony forming ability analysis are shown in FIG. 13 .
도 13에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 콜로니 형성능이 대조군에 비해 유의하게 높은 것을 확인하였다.As shown in FIG. 13 , it was confirmed that the colony-forming ability of human cardiac progenitor stem cells subcultured in an ECM-based incubator coated with Fibro-P and Vitro-P was significantly higher than that of the control group.
8-2. mRNA 발현 확인8-2. mRNA expression check
ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포에서 줄기세포능 관련 mRNA(c-kit, Klf4 및 Nanog)의 발현을 분석하였다. 구체적으로, 인간심장전구줄기세포를 ECM 기반 배양기에서 3회 계대배양한 후 세포 펠릿을 수득하였다. 수득된 세포 펠릿에 TRIzol을 처리하여 RNA를 분리하였고, PrimeScript 1st stand cDNA Synthesis Kit(Takara)를 이용하여 cDNA를 합성하였다. 합성된 cDNA, 프라이머(c-kit, Klf4, Nanog) 및 Faststart Essential DNA green master mix(Roche)를 이용하여 qRT-PCR을 수행하였다. 상기 qRT-PCR은 Lightcycle 96 real-time pcr system(Roche)을 이용하였다. 줄기세포능 관련 mRNA의 발현을 분석한 결과는 도 14에 나타내었다.Expression of stem cell ability-related mRNA (c-kit, Klf4 and Nanog) was analyzed in human cardiac progenitor stem cells subcultured in an ECM-based culture medium. Specifically, human cardiac progenitor stem cells were subcultured three times in an ECM-based incubator, and then a cell pellet was obtained. RNA was isolated by treating the obtained cell pellet with TRIzol, and cDNA was synthesized using PrimeScript 1st stand cDNA Synthesis Kit (Takara). qRT-PCR was performed using the synthesized cDNA, primers (c-kit, Klf4, Nanog) and Faststart Essential DNA green master mix (Roche). The qRT-PCR was performed using the Lightcycle 96 real-time PCR system (Roche). The results of analyzing the expression of stem cell ability-related mRNA are shown in FIG. 14 .
도 14에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 c-kit 및 Klf4의 발현이 대조군(Normal)에 비해 유의하게 높은 것을 확인하였다. 특히, Fibro-P로 코팅된 ECM 기반 배양기에서 계대 배양된 인간심장전구줄기세포는 Nanog의 발현도 현저히 높은 것을 확인하였다.As shown in FIG. 14 , it was confirmed that the expression of c-kit and Klf4 in the human progenitor stem cells subcultured in the ECM-based incubator coated with Fibro-P and Vitro-P was significantly higher than that of the control group (Normal). . In particular, it was confirmed that the expression of Nanog was also significantly higher in human cardiac progenitor stem cells subcultured in an ECM-based incubator coated with Fibro-P.
실시예 9. ECM 기반 배양기로 계대배양된 인간심장전구줄기세포의 수확량 분석Example 9. Analysis of the yield of human cardiac progenitor stem cells subcultured in an ECM-based incubator
줄기세포 치료제로 이용하기 위해서는 세포 수를 다량 확보하는 것이 중요하다. 이에, ECM 기반 배양기에서 배양된 인간심장전구줄기세포의 수확량을 분석하였다. 구체적으로, 상기 실시예 7-4에서 계대 수에 따른 배가 시간을 확인하기 위해 측정한 세포수 측정 데이터를 토대로, accumulation curve를 작성하였다. 작성된 accumulation curve는 도 15에 나타내었다.It is important to secure a large number of cells in order to use them as stem cell therapeutics. Accordingly, the yield of human cardiac progenitor stem cells cultured in an ECM-based culture medium was analyzed. Specifically, an accumulation curve was prepared based on the cell number measurement data measured to confirm the doubling time according to the number of passages in Example 7-4. The created accumulation curve is shown in FIG. 15 .
도 15에 나타낸 바와 같이, Fibro-P 및 Vitro-P로 코팅된 ECM 기반 배양기에서 계대배양된 인간심장전구줄기세포는 계대 수가 증가할수록 대조군(Normal)에 비해 세포 수확량이 통계적으로 유의하게 많은 것을 확인하였다. 상기 결과는 ECM 기반 배양기를 이용할 경우 세포의 대량 확보가 가능한바, 상기 ECM 기반 배양기는 세포 치료제 제조에 유용하게 활용될 수 있음을 의미한다.As shown in FIG. 15 , it was confirmed that the cell yield of human progenitor stem cells subcultured in an ECM-based incubator coated with Fibro-P and Vitro-P was statistically significantly higher than that of the control group (Normal) as the number of passages increased. did The above result means that a large amount of cells can be secured when using an ECM-based incubator, and the ECM-based incubator can be usefully used for manufacturing cell therapeutics.
종합적으로 본 발명자들은 ECM 모티프 및 홍합 접착 단백질의 융합단백질을 제조하고, 이를 이용하여 ECM 기반 배양기를 제조하였다. 상기 ECM 기반 배양기는 인간심장전구줄기세포의 세포 부착능, 세포 이동능 및 콜로니 형성능을 증가시킬 뿐만 아니라 세포 노화를 감쇠시키고, 세포 수확량을 현저히 증가시킨다는 것을 확인하였다. 이는 ECM 기반 배양기가 줄기세포의 생물학적 활성을 증가시킨다는 것을 의미하는 바, 세포배양 분야 및 줄기세포 치료제 분야에서 다양하게 활용될 수 있다.Overall, the present inventors prepared a fusion protein of an ECM motif and a mussel adhesion protein, and prepared an ECM-based incubator using the fusion protein. It was confirmed that the ECM-based incubator not only increased the cell adhesion, cell migration and colony forming ability of human cardiac progenitor stem cells, but also attenuated cellular senescence and significantly increased cell yield. This means that the ECM-based incubator increases the biological activity of stem cells, and can be used in various fields in cell culture and stem cell therapeutics.
이상, 본 발명내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적인 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의해 정의된다고 할 것이다.Above, specific parts of the present invention have been described in detail, for those of ordinary skill in the art, it is clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
Claims (11)
- 세포외기질(extracellular matrix, ECM) 모티프 및 홍합 접착 단백질을 포함하는 세포배양용 융합 단백질.A fusion protein for cell culture comprising an extracellular matrix (ECM) motif and a mussel adhesion protein.
- 제1항에 있어서,According to claim 1,상기 세포외기질은 피브로넥틴, 비트로넥틴, 라미닌, 젤라틴 및 콜라겐으로 이루어진 군에서 선택된 1종 이상인, 세포배양용 융합 단백질.The extracellular matrix is at least one selected from the group consisting of fibronectin, vitronectin, laminin, gelatin and collagen, a fusion protein for cell culture.
- 제1항에 있어서,According to claim 1,상기 세포외기질 모티프는 서열번호 1 또는 2의 아미노산 서열로 표시되는 것인, 세포배양용 융합 단백질.The extracellular matrix motif is represented by the amino acid sequence of SEQ ID NO: 1 or 2, a fusion protein for cell culture.
- 제1항에 있어서,According to claim 1,상기 세포배양용 융합 단백질은 서열번호 3 또는 4의 아미노산 서열로 표시되는 것인, 세포배양용 융합 단백질.The fusion protein for cell culture is represented by the amino acid sequence of SEQ ID NO: 3 or 4, the fusion protein for cell culture.
- 제1항에 있어서,According to claim 1,상기 세포배양은 골수, 혈액, 지방, 태반, 제대혈, 뇌, 간, 췌장, 심장, 피부, 신경 및 근육으로 이루어진 군에서 선택된 1 이상에서 유래된 성체줄기세포를 배양하는 것인, 세포배양용 융합 단백질.The cell culture is bone marrow, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, fusion for cell culture of culturing adult stem cells derived from one or more selected from the group consisting of nerves and muscles protein.
- 제1항 내지 제5항 중 어느 한 항에 따른 세포배양용 융합 단백질을 포함하는, 배지 조성물.A medium composition comprising the fusion protein for cell culture according to any one of claims 1 to 5.
- 제1항 내지 제5항 중 어느 한 항에 따른 세포배양용 융합 단백질을 포함하는, 줄기세포의 활성 증가용 조성물.A composition for increasing the activity of stem cells, comprising the fusion protein for cell culture according to any one of claims 1 to 5.
- 제7항에 있어서,8. The method of claim 7,상기 줄기세포의 활성은 세포 부착능, 세포 이동능, 세포 노화 억제능 및 콜로니 형성능으로 이루어진 군에서 선택된 1 이상인, 줄기세포의 활성 증가용 조성물.The stem cell activity is at least one selected from the group consisting of cell adhesion ability, cell migration ability, cell senescence inhibitory ability and colony formation ability, a composition for increasing the activity of stem cells.
- 제1항 내지 제5항 중 어느 한 항에 따른 세포배양용 융합 단백질이 코팅된, 세포배양기.According to any one of claims 1 to 5, the cell culture machine is coated with the fusion protein for cell culture.
- 제1항 내지 제5항 중 어느 한 항의 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는, 줄기세포의 활성을 증가시키는 방법.A method for increasing the activity of stem cells, comprising the step of culturing in vitro (in vitro) the fusion protein and stem cells for cell culture of any one of claims 1 to 5.
- 제1항 내지 제5항 중 어느 한 항의 세포배양용 융합 단백질 및 줄기세포를 시험관 내(in vitro)에서 배양하는 단계를 포함하는, 줄기세포의 대량 생산 방법.A method for mass production of stem cells, comprising the step of culturing in vitro (in vitro) the fusion protein and stem cells for cell culture of any one of claims 1 to 5.
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