KR20210147337A - Fusion protein for cell culture including cell growth factor motif and use thereof - Google Patents

Abstract

The present invention relates to a fusion protein for cell culture, and more particularly, to a fusion protein for cell culture comprising a cell growth factor motif and a mussel adhesion 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, that is, cell viability, stem cell ability and cell migration 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.

Description

Fusion protein for cell culture including cell growth factor motif and use thereof {Fusion protein for cell culture including cell growth factor motif and use thereof}

The present invention relates to a fusion protein for cell culture, and more particularly, to a fusion protein for cell culture comprising a cell growth factor 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. 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.

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.

Accordingly, the present inventors completed the present invention by preparing a fusion protein comprising a cell growth factor b-FGF (basic-fibroblast growth factor) motif and a mussel adhesion protein, and confirming the cellular activity and yield increase effect of the fusion protein. .

Accordingly, an object of the present invention is to provide a fusion protein for cell culture comprising a cell growth factor 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 composition for increasing stem cell activity, and a cell culture machine.

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.

In order to achieve the above object, the present invention provides a fusion protein for cell culture comprising a cell growth factor 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.

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 viability, stem cell ability and cell migration 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.

1 is a diagram briefly showing a cell growth factor motif-based fusion protein according to the present invention.
2 is a diagram showing the results of analyzing the effect of the cell growth factor motif-based fusion protein according to the present invention on the Akt-Erk pathway through Western blotting.
3 is a diagram showing the results of analyzing the cell viability of human cardiac progenitor stem cells cultured in the cell growth factor-based incubator according to the present invention through MTS analysis.
4 is a diagram showing the result of analyzing the stem cell capacity of human cardiac progenitor stem cells cultured in the cell growth factor-based culture medium according to the present invention through flow cytometry.
5 is a diagram showing the results of analyzing the cell migration ability of human cardiac progenitor stem cells cultured in the cell growth factor-based incubator according to the present invention through a wound healing assay.
6 is a diagram showing the results of analyzing the cell yield according to the number of passages of human heart progenitor stem cells cultured in the cell growth factor-based incubator according to the present invention.

Hereinafter, the present invention will be described in detail.

According to an aspect of the present invention, there is provided a fusion protein for cell culture comprising a cell growth factor motif and a mussel adhesion protein.

In an embodiment of the present invention, the cell growth factor is preferably b-FGF (basic-fibroblast growth factor).

In an embodiment of the present invention, the cell growth factor motif is preferably represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 6.

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 water, blood, fat, placenta, umbilical cord blood, brain, liver, pancreas, heart, skin, nerve and muscle. It is preferred, and more preferably, culturing the heart-derived adult stem cells, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a medium composition comprising the fusion protein for cell culture.

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.

According to another aspect of the present invention, the present invention provides a cell culture machine coated with the fusion protein for cell culture.

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.

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.

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 of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example 1. Preparation of cell growth factor motif-based fusion protein and incubator

1-1. Cell growth factor motif selection

Peptide motifs of cell growth factor b-FGF (basic-fiblast growth factor) were selected, and the selected b-FGF motifs are shown in Table 1.

motif amino acid sequence b-FGF motif 001 (SEQ ID NO: 1) ANRYL AMKED GRLLA S b-FGF motif 002 (SEQ ID NO: 2) ERGVV SIKGV b-FGF motif 003 (SEQ ID NO: 3) WYVAL KRTGQ YKLG b-FGF motif 004 (SEQ ID NO: 4) HFKDP KRLYC K b-FGF motif 005 (SEQ ID NO: 5) FLPMS AKS b-FGF motif 006 (SEQ ID NO: 6) KTGPG QKIL

1-2. Cell growth factor motif-based fusion protein production

1, by binding the cell growth factor motif selected in Example 1-1 to the end of mussel adhesive protein-151 (MAF-151) (SEQ ID NO: 7), cell growth Factor motif-based fusion proteins were constructed.

The prepared cell growth factor motif-based fusion proteins were named b-FGF1, b-FGF2, b-FGF3, b-FGF4, b-FGF5 and b-FGF6, respectively.

1-3. Manufacture of cell growth factor-based incubator

A cell growth factor-based incubator was prepared using the cell growth factor motif-based fusion protein prepared in Example 1-2. Specifically, the cell growth factor motif-based fusion protein was coated on the plate at concentrations of 0.001, 0.01, 0.1 and 1 mg/ml, respectively.

The prepared cell growth factor-based incubator was used for the experiments described below. 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'.

Example 2. Analysis of the effect of the cell growth factor motif fusion protein on the Akt-Erk pathway

The effect of the cell growth factor motif fusion protein selected in Example 1-1 on the Akt-Erk pathway related to the cell proliferation of human cardiac progenitor stem cells was analyzed. Specifically, a mixed medium was prepared by adding the cell growth factor motif fusion protein in a solution state to a final concentration of 6 μM in the culture medium. Human cardiac progenitor stem cells were cultured for 24 hours in the prepared mixed medium. Thereafter, the cultured cells were harvested, and a lysis buffer was added to extract and quantify the protein. 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 Akt, p-AKT, ERK, p-ERK and GAPDH. 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. 2 .

As shown in FIG. 2 , it was confirmed that phosphorylation of AKT and ERK related to cell proliferation was induced in human progenitor stem cells cultured in a medium containing the cell growth factor motif fusion proteins bFGF1 to bFGF6. In particular, it was confirmed that bFGF4, bFGF5 and bFGF6 further induced phosphorylation of AKT and Erk. In the following experiments, cell growth factor motif fusion proteins bFGF4, bFGF5 and bFGF6 were used.

Example 3. Cell viability analysis of human cardiac progenitor stem cells cultured in a cell growth factor-based incubator

Cell viability of human heart progenitor stem cells was analyzed in a cell growth factor-based incubator coated with the cell growth factor motif fusion proteins bFGF4, bFGF5 and bFGF6 selected in Example 2 above. Specifically, the 96-well plate was divided into 4 sections, and then each of the cell growth factor motif fusion proteins (bFGF4, bFGF5 and bFGF6) and the control were coated. Each well of the prepared 96-well plate was seeded with 7,000 human heart progenitor stem cells and then cultured. 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 measurements, and the results are shown in FIG. 3 .

As shown in FIG. 3 , it was confirmed that the cell growth factor-based incubator coated with bFGF4, bFGF5, and bFGF6dl, respectively, had stronger cell viability than the control group. In particular, the cell viability of the cell growth factor-based incubator coated with bFGF4 at a concentration of 0.1 mg/ml was the best.

In the experiment to be described later, a cell growth factor-based incubator coated with bFGF4, which had the best cell viability, at a concentration of 0.1 mg/ml was tested.

Example 4. Analysis of stem cell ability of human cardiac progenitor stem cells cultured in a cell growth factor-based incubator

Through flow cytometry, the number of c-kit-positive cells according to the number of passages of human heart progenitor stem cells cultured in a cell growth factor-based incubator was analyzed. The c-kit is a representative marker of cardiac progenitor stem cells. Specifically, a cell growth factor-based incubator coated with bFGF4 at a concentration of 0.1 mg/ml was prepared. Human cardiac progenitor stem cells were seeded in the cell growth factor-based incubator and subcultured. After subculture, the same amount of cells was removed and the cells were permeablized using eBioscience Foxp3/Transcription factor sataining buffer set. The cells were treated with an unlabeled primary c-kit antibody (Santa cruz) (1:50) and then reacted at 4°C for 30 minutes. The cells were then washed 3 times with flow cytometry buffer. A fluorescently labeled secondary antibody (647-mouse, invitrogen) (1:200) was added to the washed cells and reacted at 4° C. for 30 minutes. After the reaction, the cells were washed 3 times with flow cytometry buffer. The washed cells were resuspended in 100 μl of flow cytometry buffer and analyzed by flow cytometry. The flow cytometry results are shown in FIG. 4 .

As shown in FIG. 4 , it was confirmed that the number of c-kit-positive cells among human cardiac progenitor stem cells cultured in a cell growth factor-based incubator was at a level similar to that of the control group (Normal). The above result means that even long-term culture of human heart progenitor stem cells in a cell growth factor-based incubator does not affect the phenotype.

Example 5. Analysis of cell migration ability of human cardiac progenitor stem cells cultured in a cell growth factor-based incubator

^{To analyze the cell migration ability, 1x10 5} human heart progenitor stem cells were seeded into each well of a cell growth factor-based incubator coated with bFGF4 at a concentration of 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. The movement of cells was observed with an optical microscope 4 hours after scratching, and the results are shown in FIG. 5 .

As shown in FIG. 5 , it was confirmed that the scratch range of the human heart progenitor stem cells cultured in the cell growth factor-based incubator was significantly reduced, that is, the cells moved a lot compared to the control group (Normal).

Example 6. Analysis of the yield of human cardiac progenitor stem cells cultured in a cell growth factor-based incubator according to the number of passages

It is important to secure a large number of cells in order to use them as stem cell therapeutics. Accordingly, the yield of human heart progenitor stem cells cultured in a cell growth factor-based incubator was analyzed. Specifically, human cardiac progenitor stem cells were cultured in a cell growth factor-based incubator, and the number of cells was measured at each subculture. An accumulation curve and a cell yield per generation graph were prepared based on the measured cell number measurement data, and are shown in FIG. 6 .

As shown in FIG. 6 , it was confirmed that the cell yield of human heart progenitor stem cells subcultured in a cell growth factor-based culture medium increased as the number of passages increased, compared to the control group (Normal). The above result means that when a cell growth factor-based incubator is used, it is possible to secure a large amount of cells, and the cell growth factor-based incubator can be usefully used in the manufacture of a cell therapeutic agent.

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.

<110> Pusan National University Industry-University Cooperation Foundation <120> Fusion protein for cell culture including cell growth factor motif and use thereof <130> 1.410 <160> 7 <170> KoPatentIn 3.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 001 <400> 1 Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly Arg Leu Leu Ala Ser 1 5 10 15 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 002 <400> 2 Glu Arg Gly Val Val Ser Ile Lys Gly Val 1 5 10 <210> 3 <211> 14 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 003 <400> 3 Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly 1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 004 <400> 4 His Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys 1 5 10 <210> 5 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 005 <400> 5 Phe Leu Pro Met Ser Ala Lys Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> b-FGF motif 006 <400> 6 Lys Thr Gly Pro Gly Gln Lys Ile Leu 1 5 <210> 7 <211> 206 <212> PRT <213> Artificial Sequence <220> <223> mussel adhesive protein-151 <400> 7 Met Ala Ser Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro 1 5 10 15 Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr 20 25 30 Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr 35 40 45 Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Gly 50 55 60 Cys Ser Ser Glu Glu Tyr Lys Gly Gly Tyr Tyr Pro Gly Asn Ala Tyr 65 70 75 80 His Tyr His Ser Gly Gly Ser Tyr His Gly Ser Gly Tyr His Gly Gly 85 90 95 Tyr Lys Gly Lys Tyr Tyr Gly Lys Ala Lys Lys Tyr Tyr Tyr Lys Tyr 100 105 110 Lys Asn Ser Gly Lys Tyr Lys Tyr Leu Lys Lys Ala Arg Lys Tyr His 115 120 125 Arg Lys Gly Tyr Lys Lys Tyr Tyr Gly Gly Ser Ser Glu Phe Glu Phe 130 135 140 Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro 145 150 155 160 Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys 165 170 175 Pro Ser Tyr Pro Pro Thr Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr 180 185 190 Tyr Lys Ala Lys Pro Ser Tyr Pro Pro Thr Tyr Lys Lys Leu 195 200 205

Claims (10)

A fusion protein for cell culture comprising a cell growth factor motif and a mussel adhesion protein. According to claim 1,
The cell growth factor is b-FGF (basic-fibroblast growth factor), a cell culture fusion protein. According to claim 1,
The cell growth factor motif is a fusion protein for cell culture, which is represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 6. According to claim 1,
The cell culture is to culture the 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, cell culture fusion protein. A medium composition comprising the fusion protein for cell culture according to any one of claims 1 to 4. Claims 1 to 4, comprising the fusion protein for cell culture according to any one of claims 1 to 4, a composition for increasing the activity of stem cells. 7. The method of claim 6,
The stem cell activity is at least one selected from the group consisting of cell viability, stem cell ability and cell migration ability, a composition for increasing the activity of stem cells. According to any one of claims 1 to 4, the cell culture machine coated with the fusion protein for cell culture. A method for increasing the activity of stem cells comprising the step of culturing the fusion protein for cell culture according to any one of claims 1 to 4 and the stem cells in vitro. A method for mass production of stem cells comprising the step of culturing the fusion protein for cell culture according to any one of claims 1 to 4 and the stem cells in vitro.

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