KR101473617B1 - Electrospun fish collagen/synthetic polymer hybrid nanofiber scaffolds, method thereof and use thereof - Google Patents

Abstract

The present invention provides a method for producing a nanofiber support and a fish collagen/synthetic polymer composite nanofiber support produced through the method, wherein the method comprises a first step of dissolving a synthetic polymer in an organic solvent; a second step of producing a fish collagen solution by dissolving fish collagen in water; a third step of adding and mixing the synthetic polymer solution produced from the first step in the fish collagen solution produced from the second step; and a fourth step of producing a nanofiber support by electrospinning the mixture produced from the third step. The composite nanofiber support can exhibit higher mechanical stability than existing nanofiber supports including synthetic polymer ingredients and constitutes a cell culturing environment similar to that of a living body, wherein three-dimensional cell culturing efficiency such as cell adhesion, cellular viability, and cell distributing efficiency is remarkably improved, and an excellent biomimetic function is exhibited so that the same is appropriate as a three-dimensional cell-culturing support. Accordingly, a three-dimensional cell-culturing method using the fish collagen/synthetic polymer nanofiber support of the present invention is appropriate as a three-dimensional cell-culturing model which can be used in testing drug sensitivity and studying the formation, growth, spread, and death of cancer cells. The same can be used in studying the development of a biological tissue which can recover or replace a part or all of bones, cartilages, blood vessels, bladder, skin, and tissues such as muscles.

Description

TECHNICAL FIELD [0001] The present invention relates to a fish collagen / synthetic polymer nanofiber scaffold, a method of producing the same,

The present invention relates to a nanofiber support prepared by electrospinning in a mixed solution containing, as an effective component, hydrophilic natural polymers such as fish collagen and a hydrophobic synthetic polymer, a method for producing the nanofiber support, and a use thereof.

Tissue engineering has recently attracted attention as a fusion medicine which is intended to be applied for the purpose of clinical treatment by maintaining, enhancing, restoring or replacing biological functions using cells, engineering materials and appropriate physiological / biochemical factors. It is a field.

The most important technique of this tissue engineering is to attach the cells separated from the biotissue to a porous scaffold made of a biodegradable polymer and to transplant them in vivo or to cultivate them for a certain period of time in vitro to produce new biotissues. Is used in various fields such as restoring or replacing a part or whole of tissues such as bones, cartilage, blood vessels, bladder, skin, muscles, etc., so as to develop artificial tissues / organs For the development of tissue engineering, development of bio-environmental simulation technology as well as biomaterials, cells, and biologically active factors is essential.

Cell culture methods so far have been based on culturing cells on a two-dimensional surface consisting of polystyrene or glass substrates to support the growth of adherent cells. However, since the monolayer cell culture method cultured on such a two-dimensional surface can not accurately reflect the biotissue environment of the three-dimensionally grown cells attached to the extracellular matrix, There are many differences. As a result, the two-dimensional cell culture and the three-dimensional cell culture show general morphological differences, and the expression of the receptor, the transcriptional regulation of the gene, the cell migration and the apoptosis through the normal two-dimensional cell culture, Is very different from the phenomenon that occurs in actual biotissue environment.

In addition, conventional two-dimensional cell culture can not consider different kinds of intercellular interactions because one kind of the same cell is cultured. Although some attempts have been made to solve this problem partially through 2D co-culture, there have been many differences from the cellular environment in living tissues.

In order to solve the problems in the two-dimensional cell culture, it is required to research and develop a three-dimensional cell culture method considering the spatial organization of the cells. In order to solve the problem, As a method for constructing a living cell culture environment, research and development have been carried out to form a three-dimensional cell culture environment by culturing cells in a porous biocompatible scaffold with holes, Studies on the production method of scaffolds using polymer and natural polymers and the precise cell culture method according to the tissue to be studied have been carried out.

However, to date, there has been no known method of producing scaffolds most suitable for regenerating living tissue.

Korean Patent No. 10-1130239

In order to solve the above-mentioned problems, the present invention relates to a method for producing a hydrophilic natural polymer, which comprises mixing a fish collagen, a hydrophilic natural polymer, and a hydrophobic synthetic polymer, such as poly (e-caprolactone); (Hereinafter, referred to as " PCL "), and to provide a nanofiber support by the above production method, and to provide a high-efficiency / high-function biomimetic three-dimensional cell culture model using the same .

The present invention relates to a method for preparing a synthetic polymer, comprising: a first step of dissolving a synthetic polymer in an organic solvent; A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution; A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously; And a fourth step of preparing a nanofiber support by electrospunning the mixed solution prepared in the third step.

The present invention also relates to a method for producing a synthetic polymer, comprising: a first step of dissolving a synthetic polymer in an organic solvent; A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution; A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously; A fourth step of electrospinning the mixed solution prepared in the third step to produce a nanofiber support; And a step of culturing the cells on the nanofiber support prepared in the fourth step.

The fish collagen / synthetic polymer nanofiber support according to the present invention is a composite nanofiber support prepared by electrospinning a solution prepared by adding fish collagen, a natural polymer, to a conventional synthetic polymer material, Can exhibit higher mechanical stability than the support. As a result of cell culture using the fish collagen / synthetic polymer nanofiber scaffold of the present invention, the fish collagen / synthetic polymer nanofiber scaffold of the present invention can provide a cell culture environment very similar to a living body, and can improve cell adhesion, cell viability, And 3 - dimensional cell culturing efficiency as well as excellent biomimetic function. Further, since the concentration of the solvent can be easily controlled, a suitable three-dimensional environment can be provided according to the cell to be cultured.

Therefore, the three-dimensional cell culture method using the fish collagen / synthetic polymer nanofiber scaffold of the present invention is suitable as a three-dimensional cell culture model that can be used for drug sensitivity test, formation of cancer cells, growth, metastasis and death, Cartilage, blood vessels, bladder, skin, muscles, etc., for the development and replacement of living tissue.

FIG. 1 is a photograph (× 400) of a fish collagen / PCL nanofiber support of the present invention analyzed by a confocal laser scanning microscope after rhodamine staining to confirm the structure of the fish collagen / PCL nanofiber support and the degree of mixing of fish collagen and PCL.
Figure 2 shows the cell adhesion of PCL nanofiber scaffold and Fc 0.1 wt% -Sc 20 wt%, Fc 1 wt% -Sc 20 wt% and Fc 2 wt% -Sc 20 wt% fish collagen / PCL nanofiber scaffolds to fibroblast actin staining Confocal laser scanning microscope photographs confirmed that the green is F-actin and the blue color is DAPI (x400).
3 is S _c 50 wt% fish collagen stock solution of F _c 2% by weight prepared by using, F _c 3% by weight, F _c 4 wt%, F _c 5% by weight and the F _c of 10% by weight fish collagen / A micrograph of the microstructure of the PCL nanofiber support.
4 is S _c 70 wt% fish collagen stock solution of F _c 2% by weight prepared by using, F _c 3% by weight, F _c 4 wt%, F _c 5% by weight and the F _c of 10% by weight fish collagen / A micrograph of the microstructure of the PCL nanofiber support.
5 is S _c 20% by weight, S _c 30% by weight, S _c 40% by weight, and S _c 50% by weight of the fish collagen stock solution of F _c 2% fish collagen / PCL nanofiber weight of the support prepared using It is a scanning electron microscope photograph showing the microstructure.
Figure 6 is a scanning electron micrograph showing the microstructure of the F _c 4 weight% fish collagen / PCL support prepared using fish collagen stock solution of 40% by weight, S _c and S _c 50% by weight.
7 is a PCL nanofiber support _{(F c 0%; PCL 100} %) and then three-dimensionally with one days the person non-small cell lung cancer state (NCI-H1703) cultured in, using a fiber-actin staining in the PCL nanofiber support (A), 3-D reconstructed photograph (B), and enlarged image (C) photographs of a 3-dimensional distribution profile of cells under a confocal laser scanning microscope - Actin, blue is DAPI (x400).
8 is F _c 2% by weight -S _c 30 wt% fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after culture, F _c 2% by weight -S _c Three-dimensional distribution of cells in a 30 wt% fish collagen / PCL nanofiber scaffold using fiber-based actin staining. Sequential photographs (A) and three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
9 is F _c 2 -S wt% _c 50% by weight fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after culture, F _c 2% by weight -S _c Three-dimensional distribution characteristics of cells in a 50% by weight fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method are shown in a sequential photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 10 is F _c 2% by weight -S _c 70 wt% fish collagen / PCL nanofibers after support three-dimensionally the person 1 days of non-small cell lung cancer state (NCI-H1703) cultured in, F _c 2% by weight -S _c Three-dimensional distribution of cells in a 70 wt% fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method. A continuous photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 11 is F _c 5% by weight -S _c 50% by weight fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after the culturing, 5% by weight of F _c -S _c Three-dimensional distribution characteristics of cells in a 50% by weight fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method are shown in a sequential photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 12 is F _c 5% by weight -S _c 70 wt% fish collagen / PCL nanofibers for a day at the support From the primary non-small cell lung cancer (NCI-H1703) were cultured in three dimensions, F _c 5% by weight -S _c Three-dimensional distribution of cells in a 70 wt% fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method. A continuous photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 13 F _c 10% by weight -S _c 50% by weight fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after incubation, the fibrous support in the PCL nanofiber (A), 3-D reconstructed photograph (B), and enlarged image (C) of a 3-dimensional distribution profile of cells using an actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope , Green is F-actin, and blue is DAPI (x400).
Figure 14 F _c 10% by weight -S _c 70% by weight fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after incubation, _c F 10% by weight -S _c Three-dimensional distribution of cells in a 70 wt% fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method. A continuous photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 15 F _c 15% by weight -S _c 70% by weight fish collagen / PCL nanofiber support the three-dimensional human non-small cell lung cancer state (NCI-H1703) in the enemy one days after incubation, 15 wt% F _c -S _c Three-dimensional distribution of cells in a 70 wt% fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method. A continuous photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Figure 16 F _c 20 wt% 70 wt% _c -S fish collagen / PCL nanofibers for a day at the support From the primary non-small cell lung cancer (NCI-H1703) were cultured in three dimensions, F _c 20% by weight -S _c Three-dimensional distribution of cells in a 70 wt% fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method. A continuous photograph (A) and a three-dimensional In the reconstructed photograph (B) and the enlarged image (C), green is F-actin and blue is DAPI (× 400).
Using a fiber-actin staining after the user three-dimensionally with one days the man prostate cancer cell lines (DU-145) cultured in, PCL nanofiber support; Fig. 17 is a PCL nanofiber support _{(PCL 100% F c 0%} ) (A), 3-D reconstructed photograph (B), and enlarged image (C) photographs of a 3-dimensional distribution profile of cells observed with a Z-stack of 1 μm intervals under a confocal laser scanning microscope. Actin, and blue is DAPI (x400).
Figure 18 is F _c 2% by weight -S _c 30 wt% fish collagen / PCL nano-fibers in a support person prostate cancer cell lines (DU-145) in a three-dimensional manner one days after incubation, _c F _c -S 2% by weight (A), three-dimensional reconstruction (X-ray) observation of the three-dimensional distribution characteristics of cells in a 30 wt% fish collagen / PCL nanofiber support using a fibrous actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 19 is F _c 2% by weight -S _c 50 wt% fish collagen / PCL nanofiber support three-dimensionally the person 1 days prostate cancer cell lines (DU-145) were cultured in, F _c 2% by weight _c -S (A), 3-D reconstruction (A), and 3-D reconstruction of a 50% by weight fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method and observing the three- Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 20 is F _c 2% by weight -S _c 70 wt% fish collagen / PCL nanofibers for a day at the support person prostate cancer cell lines (DU-145) were cultured in three dimensions, F _c 2% by weight _c -S (A), three-dimensional reconstruction (X-ray) observation of the three-dimensional distribution characteristics of cells in a 70 wt% fish collagen / PC nanofiber support using a fibrous actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 21 is F _c 5% -S _c 50% by weight fish collagen / PCL nanofiber support the three-dimensional human prostate cancer cell lines (DU-145) from the enemy one days after the incubation, fiber-actin in the PCL nanofiber support weight (A), 3-D reconstructed photograph (B), and enlarged image (C) photographs of a 3-dimensional distribution pattern of cells using a staining method with a Z-stack of 1 μm intervals under a confocal laser scanning microscope, Green is F-actin, and blue is DAPI (× 400).
Figure 22 is F _c 5% by weight -S _c 70 wt% fish collagen / PCL nanofiber support three-dimensionally the person 1 days prostate cancer cell lines (DU-145) were cultured in, F _c 5% by weight of _c -S (A), three-dimensional reconstruction (X-ray) observation of the three-dimensional distribution characteristics of cells in a 70 wt% fish collagen / PCL nanofiber support using a fiber-type actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 23 is F _c 10 wt% 50 wt% _c -S fish collagen / PCL nanofibers after three-dimensionally the person 1 days prostate cancer cell lines (DU-145) culture in a support, _c F 10% by weight _c -S (A), 3-D reconstruction (A), and 3-D reconstruction of a 50% by weight fish collagen / PCL nanofiber scaffold using a fiber-type actin staining method and observing the three- Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 24 F _c 10 wt% 70 wt% _c -S fish collagen / PCL nano-fibers in a support person prostate cancer cell lines (DU-145) in three dimensions after 1 days incubation, the fiber-actin in the PCL nanofiber support (A), 3-D reconstructed photograph (B), and enlarged image (C) photographs of a 3-dimensional distribution pattern of cells using a staining method with a Z-stack of 1 μm intervals under a confocal laser scanning microscope, Green is F-actin, and blue is DAPI (× 400).
Figure 25 F _c 15 wt% 70 wt% _c -S fish collagen / PCL nanofibers after three-dimensionally the person 1 days prostate cancer cell lines (DU-145) culture in a support, _c F 15% by weight _c -S (A), three-dimensional reconstruction (X-ray) observation of the three-dimensional distribution characteristics of cells in a 70 wt% fish collagen / PCL nanofiber support using a fiber-type actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
Figure 26 F _c 20 wt% 70 wt% _c -S fish collagen / PCL nanofibers after three-dimensionally the person 1 days prostate cancer cell lines (DU-145) culture in a support, _c F 20% by weight _c -S (A), three-dimensional reconstruction (X-ray) observation of the three-dimensional distribution characteristics of cells in a 70 wt% fish collagen / PCL nanofiber support using a fiber-type actin staining method with a Z-stack at 1 μm intervals under a confocal laser scanning microscope Photograph (B) and enlarged image (C) Photographs show that green is F-actin and blue is DAPI (× 400).
27 is a PCL nanofiber support _{(F c 0%; PCL 100} %) 3 -dimensionally by one days the mouse B cell lymphoma cell line (A20) in a culture and then, using a fiber-actin staining in the PCL nanofiber support cells (A), a three-dimensional reconstruction photograph (B), and an enlarged image (C) photograph of a three-dimensional distribution characteristic of a F-Actin And the blue color is DAPI (x400).
Figure 28 is F _c 2% by weight -S _c 30 wt% fish collagen / PCL nanofiber After the support 3-dimensionally by one days the mouse B cell lymphoma cell line (A20) in culture, F _c 2% by weight _c 30 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
29 is F _c -S 2% _c 50% by weight fish one days for the collagen / PCL mouse in nanofiber support B cell lymphoma cell line (A20) in three dimensions, the weight after incubation, _c F 2 wt% -S _c 50 (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 30 is F _c -S 2% _c 70% by weight of fish collagen / PCL nanofiber the mouse B cell lymphoma cell line (A20) in a three-dimensional manner in the support 1 days after incubation, fiber-actin staining in the PCL nanofiber support weight (A), 3-D reconstructed photograph (B), and enlarged image (C) photographs of a 3-dimensional distribution profile of cells using a Z-stack at 1 μm intervals under a confocal laser scanning microscope. Is F-actin, and blue is DAPI (x400).
Figure 31 is F _c 5% by weight -S _c 50 wt% fish collagen / PCL nanofibers after the support 1 days the mouse B cell lymphoma cell line (A20) three-dimensionally cultured at, F _c 5 _c 50 wt% -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 32 is F _c 5% and then 70% by weight of fish 1 _c -S days and collagen / PCL mouse in nanofiber support B cell lymphoma cell line (A20) in three dimensions by weight of the culture, F _c 5% by weight _c 70 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 33 F _c 15% by weight -S _c 70% by weight fish collagen / PCL nanofiber support 3D mouse B cell lymphoma cell line (A20) from the enemy one days after culture, F _c 15 _c 70% by weight -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 34 is F _c 20 wt% 70 wt% _c -S fish 1 days and the collagen / PCL mouse in nanofiber support B cell lymphoma cell line (A20) were cultured in three dimensions, F _c 20 _c 70% by weight -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
35 is a PCL nanofiber support _{(F c 0%; PCL 100} %) 3 -dimensionally by one days the mouse T-cell lymphoma cell line (EL4) in a culture and then, using a fiber-actin staining in the PCL nanofiber support cells (A), a three-dimensional reconstruction photograph (B), and an enlarged image (C) photograph of a three-dimensional distribution characteristic of a F-Actin And the blue color is DAPI (x400).
36 is F _c 2% by weight -S _c 30 wt% fish collagen / PCL nanofiber After the support 3-dimensionally by one days the mouse B cell lymphoma cell line (A20) in culture, F _c 2% by weight _c 30 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 37 is F _c 2 -S wt% _c 50% by weight fish one days for the collagen / PCL mouse in nanofiber support B cell lymphoma cell line (A20) were cultured in three dimensions, and then, F _c 2% by weight _c 50 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 38 is F _c 2% by weight -S _c 70 wt% fish collagen / PCL nanofibers after the support 1 days the mouse B cell lymphoma cell line (A20) three-dimensionally cultured at, F _c 2% by weight _c 70 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 39 is F _c 5% by weight -S _c 50 wt% fish collagen / PCL nanofibers after the support 1 days the mouse B cell lymphoma cell line (A20) three-dimensionally cultured at, F _c 5 _c 50 wt% -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
Figure 40 is F _c 5% and then 70% by weight of fish 1 _c -S days and collagen / PCL mouse in nanofiber support B cell lymphoma cell line (A20) in three dimensions by weight of the culture, F _c 5% by weight _c 70 -S (A), 3-D reconstruction photographs of 3-dimensional distribution characteristics of cells in a weight% fish collagen / PCL nanofiber support using fiber-type actin staining with a 1-μm spacing Z-stack under confocal laser scanning microscopy (B) and an enlarged image (C), green is F-actin and blue is DAPI (x400).
41 is a result of analyzing the effects on the survival of the PCL nanofiber substrate and F _c 2% by weight -S _c 50 wt% fish collagen / PCL during cell culture in the nanofibers of the nanofiber support each support is a mouse B-cell lymphoma cells , And cultured for 2 days after inoculation with mouse B cell lymphoma cells, and confirmed by live confinement laser microscopy (× 400).
42 is a result of analyzing the effects on the survival of the PCL nanofiber substrate and F _c 2% by weight -S _c 50 wt% fish collagen / PCL during cell culture in the nanofibers of the nanofiber support each support is a mouse B-cell lymphoma cells , And cultured for 6 days after inoculation with mouse B-cell lymphoma cells, and confirmed by Live-Dead cells with a confocal laser scanning microscope (× 400).
To FIG. 43 to evaluate the proliferation ability of the drug to reach cancer cells in a 3D environment, F _c 2% by weight of the present invention -S _c 50 wt% fish collagen / PCL mouse using nano fiber support T 3d of lymphoma cells The concentration of doxorubicin, a standard chemotherapeutic agent for lymphoma, was measured at 1 μM concentration and the degree of anticancer drug diffusion was confirmed by confocal laser scanning microscopy.
44 is a conventional two-dimensional environment of the present invention and F _c -S 4% _c 50% by weight of fish collagen / PCL one lung cancer cells cultured in the three-dimensional environment with a nanofiber support (NCI-H1703, A) by weight and prostate (DU-145, B) was determined by measuring cell survival rate.
45 is a conventional two-dimensional environment in malignant and F _c -S 4% by weight _c 50 wt% fish collagen / PCL each human prostate cancer (DU-145) cultured in a 3D environment with nanofibers support of the present invention And the expression level of Hes 1 and Notch genes was confirmed by RT-PCR.
46 is malicious in a conventional two-dimensional environment, and F _c -S 4% by weight _c 50 wt% fish collagen / PCL each human prostate cancer (DU-145) cultured in a 3D environment with nanofibers support of the present invention The expression levels of snail, slug, endothelin-1, CD44, VEGF, and HIF-1α genes, which are closely related to the prognosis, were determined by RT-PCR.
47 is a flow cytometric analysis results of measurement for the present invention the F _c 2% by weight 50% by weight _c -S CSCs tumor-forming ability and multipotential of collagen of the fish / PCL nanofiber support 3D cultured mouse T cell lymphoma cell line in.

The present invention relates to a method for preparing a synthetic polymer, comprising: a first step of dissolving a synthetic polymer in an organic solvent; A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution; A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously; And a fourth step of preparing a nanofiber support by electrospunning the mixed solution prepared in the third step.

The synthetic polymer solution of the first step may be prepared by dissolving 8 to 10 parts by weight of a synthetic polymer in 100 parts by weight of an organic solvent. More preferably 8.8% by weight of a synthetic polymer solution, but is not limited thereto.

If the addition range of the synthetic polymer is out of the range, electrospinning may not be performed or it may be difficult to produce homogeneous nanofibers during electrospinning.

The synthetic polymer may be selected from the group consisting of polycaprolactone (poly (e-caprolactone); PCL], poly lactic acid; PLA], polyglycolic acid; PGA], poly (hydroxy valerate); PHV], poly (lactic-co-glycolic acid) [poly (lactic-co-glycolic acid)]; PLGA], poly-hydroxy butyrate (PHB), poly (hydroxy butyrate-co-valerate) PHBV], polyanhydride, polyorthoester, poly (vinyl alchol); (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), poly-N-isopropylacrylamide, diol / And may include at least one member selected from the group consisting of polyester and polyester-amide / polyester-urethane. More preferably, the synthetic polymer is selected from the group consisting of polycaprolactone (poly (e-caprolactone); PCL] may be used, but the present invention is not limited thereto.

The polycaprolactone [poly (e-caprolactone); PCL] is a synthetic polymer with a semi-crystalline powder form and a low melting point. It has a melting characteristic compared to other polymers. Also, poly (e-caprolactone); PCL] is a non-toxic and biocompatible material that exhibits a very slow degradation rate and is widely used as a material for scaffolds for tissue engineering.

The organic solvent may be selected from the group consisting of chloroform, hexafluoroisopropane, tetrahydrofuran, dimethylformamide, dichloromethane, acetone, dioxane, trifluoroethane, and mixed solvents thereof.

The fish collagen aqueous solution of the second step may dissolve fish collagen in an amount of 10 to 70 parts by weight based on 100 parts by weight of water.

If the addition amount of the fish collagen is out of the range, the fish collagen does not dissolve homogeneously in water, the fish collagen-PCL mixture can not be homogeneously prepared, or homogeneous nanofibers can not be produced.

The collagen may be collagen extracted from marine organisms, more preferably fish collagen, but is not limited thereto.

Collagen is a fiber-like protein, a natural polymer that is the main component of the outer cell matrix in the connective tissue. This collagen plays a role in regulating cell attachment and proliferation, supporting tissue or organs, maintaining the body shape surrounding the body tissues, and regenerating tissues. It is used for sponges, fibers, gels, solutions, And can be processed in various forms such as a tubular material. Fish collagen is a natural high molecular substance extracted from marine fish scales. Collagen extracted from these marine organisms is easier to lower molecular peptide than collagen extracted from mammals, and has excellent absorption of human body. In addition, the amino acid composition of collagen extracted from marine organisms is different from that of human collagen, and can be easily distinguished by special antibodies.

However, it is difficult to control the mechanical strength and decomposition rate of collagen at a very low level, and it is difficult to gel by using alone collagen.

The water is the most common solvent in nature and is more economical than organic solvents and does not show toxicity. The present invention is the first to demonstrate that water can be used in a solvent that constitutes a mixed solution used in the manufacture of nanofibers using electrospinning. More specifically, it is possible to prepare a mixed solution for electrospinning by dissolving a natural polymer using water which is not toxic, instead of an organic solvent exhibiting toxicity to a cell. In addition to the fish collagen, other kinds of water And can be applied to the manufacture of nanofibers by electrospinning using natural polymer components.

In the third step, the synthetic polymer solution of the first step may be added so that the synthetic polymer may be contained in an amount of 6.3 to 8.5 parts by weight based on 100 parts by weight of the mixed solution. More specifically, % Poly (e-caprolactone); PCL] solution may be added in an amount of 71.4 to 97.1 parts by weight based on 100 parts by weight of the mixed solution. However, the present invention is not limited thereto. In addition, the fish collagen aqueous solution prepared in the second step may be added in an amount of 2 to 20 parts by weight based on 100 parts by weight of the mixed solution. However, the present invention is not limited thereto.

If the addition amount of the synthetic polymer solution and the aqueous fish collagen solution is out of the range of the third step, the electrospinning may not be properly performed or it may be difficult to produce homogeneous nanofibers during electrospinning.

In addition, water may be added in an amount of 0.9 to 10 parts by weight based on 100 parts by weight of the mixed solution of the third step, but is not limited thereto.

The present invention also relates to a method for producing a synthetic polymer, comprising: a first step of dissolving a synthetic polymer in an organic solvent; A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution; A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously; A fourth step of electrospinning the mixed solution prepared in the third step to produce a nanofiber support; And a step of culturing the cells on the nanofiber support prepared in the fourth step.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> Nanofibrous support preparation and mixing analysis

1-1. PCL  Manufacture of nanofiber support

Polycaprolactone, average Mn 80,000, Sigma (St. Louis, Mo., USA); Hereinafter referred to as PCL) was added chloroform and uniformly dissolved by using a magnetic stirrer for 1 hour to finally prepare a 8.8 wt% PCL solution and then electrospun. In the case of electrospinning, the needle was connected to a wire mesh type collector (0.5 mm in diameter, 0.5 mm in diameter) under the conditions of 27 gauge, a scattering distance of 8 cm, a voltage of 11 kV, a fluid velocity of 0.5 mL / Mesh 1x1 mm), and the spinning time was 60 to 120 minutes, preferably about 90 minutes. Thereafter, a PCL nanofiber support was prepared by drying in a 60 ° C oven for 10 seconds to remove the residual solvent contained in the mat.

1-2. Fish Collagen / PCL  Manufacture of nanofiber support

Chloroform was added to Polycaprolacton (average Mn 80,000, Sigma; hereinafter referred to as 'PCL') and dissolved homogeneously for 1 hour using a magnetic stirrer to finally prepare a 8.8% by weight PCL solution. In order to prepare fish collagen stock solution, fish collagen (Fish collagen, Geltech, Busan, Korea) was added to distilled water and mixed in a test tube mixer (Vortex, Genie-2, 60 Hz, Scientific Industries, Bohemia, NY, USA) to prepare 20 wt% ( _C 20 wt%) fish collagen aqueous solution [20 wt% fish collagen stock solution; 20 wt% stock (S) solution; 20% by weight of S _C ; S _C 20%).

Then, S _C 20 wt% fish collagen stock solution was added to the 8.8 wt% PCL solution so that the final concentration of fish collagen was 0.1, 1 and 2 wt%, and the mixture was homogeneously mixed for 3 hours using a magnetic stirrer to produce fish collagen / PCL mixed solution was prepared.

The 0.1, 1 and 2 wt% fish collagen / PCL mixed solution prepared by the above method was electrospun by using an electrospinning apparatus. In the case of electrospinning, the needle was connected to a wire mesh type collector (0.5 mm in diameter, 0.5 mm in diameter) under the conditions of 27 gauge, a scattering distance of 8 cm, a voltage of 9 kV, a fluid velocity of 0.4 mL / hr, a temperature of 21 to 22 ° C. and a humidity of 40 to 45% Mesh 1x1 mm). The electrospinning time was 60 to 90 minutes, preferably about 70 minutes.

1-3. Rhodamine  Used Fish Collagen / PCL  Mixing analysis of nanofiber support

To confirm the mixing of fish collagen and PCL, 0.01 g of Rhodamine B (Sigma) was added to an aqueous solution of S _C 20% by weight fish collagen and dissolved homogeneously using a test tube mixer. Thereafter, an aqueous S _C 20 wt% fish collagen solution containing rhodamine B was added to 8.8 wt% PCL solution so that the final concentration of fish collagen was 1 wt%, and the mixture was stirred to prepare a rhodamine-1% fish collagen / PCL mixed solution .

A nanofiber support was prepared by electrospinning the Rhodamine-1 wt% fish collagen / PCL solution prepared above in the electrospinning conditions of Example 1-2. The prepared rhodamine-1 wt% fish collagen / PCL nanofiber support was placed on a slide glass, sealed with a cover glass, and then mixed using a confocal laser scanning microscope.

As a result, as shown in Fig. 1, it was confirmed that the nanofibers constituting the support were well stained with rhodamine.

From the above results, it was confirmed that the aqueous solution of fish collagen was homogeneously mixed with PCL.

< Example  2> PCL  And Fish Collagen / PCL  Of nanofiber support Cell adhesion  analysis

2-1. PCL  And Fish Collagen / PCL  Cell culture using nanofiber support

Thymic deep cortex or cortical reticular epithelial cell (CREC), a type of normal mouse thymic epithelial cell (TEC) (Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, USA) and 10% (v / v) fetal bovine serum was Gibco, cultured and maintained under Invitrogen) containing Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA) for 37 ℃, 5% CO ₂ atmosphere conditions, using;, Invitrogen) and 100 ㎍ / mL streptomycin (streptomycin . The culture medium was replaced with new one every 2-3 days.

In Example 1, the PCL nanofiber support and prepared as 0.1, 1, and 2 wt.% Fish collagen final concentration of fish collagen / PCL nanofibers into a support for each 96-well plate cell CREC to 5 × 10 ⁴ cells per well And cultured for 6 hours.

2-2. Fibrous actin  Of nanofiber support using staining Cell adhesion  analysis

In order to confirm the cell adhesion of the nanofiber support, the supernatant on which the cells were cultured was washed twice with phosphate buffered saline (hereinafter referred to as &quot; PBS &quot;) and fixed with 4% paraformaldehyde fixative at room temperature And fixed for 20 minutes. Then, the cells were washed twice with PBS, and then they were infiltrated into 0.1% Triton-X 100 solution at room temperature for 5 minutes, followed by washing twice with PBS.

To remove nonspecific staining, the cells were treated with 0.2% bovine serum albumin (BSA) solution at room temperature for 20 minutes and then washed twice with PBS.

After that, actin-detecting Phalloidin-FITC (Promega, Madison, Wis., USA) was diluted 1: 150 in 0.2% BSA solution and reacted at room temperature for 40 minutes. Tris- buffered saline TBS "). The supernatant was stained with DAPI (Vector Lab, Inc., Burlingame, Calif., USA) for nuclear staining of the cells, Using a laser confocal microscope, the adhesion of cells to the supporter was confirmed.

As a result, as shown in Fig. 2, the 1 wt% fish collagen-PCL nanofiber support exhibited similar adhesion as the PCL nanofiber support, and the 1 and 2 wt% fish collagen support significantly increased cell adhesion force than the PCL nanofiber support , And the higher the concentration of fish collagen, the better the cell culture efficiency of the support. Therefore, through the following examples, conditions optimized for three-dimensional culture of cells at a concentration of 2 wt% or more of fish collagen were confirmed.

< Example  3> Optimized Fish Collagen  Concentration analysis

3-1. A concentration of at least 2% by weight Fish Collagen / PCL  Manufacture of nanofiber support

By the addition of fish collagen in distilled water to a concentration of fish collagen stock solution (concentration of fish collagen stock solution, S c) S c 10 wt%, S _c 20 wt%, S _c 30 wt%, S _c 40% by weight, S _c 50%, S _c 60% by weight, S _c 70% by weight, S _c 80% by weight, S _c 90 were prepared in wt.%, and S _c 100% by weight, S _c 10% this, respectively, S _c 20%, S _c 30%, S _c 40%, S _c 50%, S _c 60%, S _c 70%, S _c 80%, S _c 90% and S _c 100%. However, since 100% of S _c can not contain PCL at all, it is excluded from the selection condition (× in Table 1), and the concentration of S _c 80% or more is excluded from the selection condition because the viscosity can not be added because the viscosity is too high (X mark in Table 1). In the case of S _c S _c of more than 50% high-concentration 60% is not a 50% or 70% S _c and S _c significant differences were also negative 60% S _c.

Therefore, the fish collagen storage solution to be added to the mixed solution was prepared as S _c 10%, S _c 20%, S _c 30%, S _c 40%, S _c 50% and S _c 70%.

Chloroform was added to PCL (Polycaprolacton, average Mn 80,000, Sigma) and dissolved uniformly for 1 hour using a magnetic stirrer to finally prepare a 8.8 wt% PCL solution.

The final concentration of fish collagen in the fish collagen solution (F _C ) was adjusted to 2 wt% of F _C by adding fish collagen solution prepared at each concentration to 8.8 wt% PCL solution, F _C 3 wt%, F _C 4 wt.%, F _C 5 wt%, F _C 10 wt.%, F _C 15 wt.%, F _C 20 wt.%, F _C 30 wt.%, F _C 40% by weight, and F _C was prepared a 50 wt.% of Fish collagen / PCL blend solution, each F _C 2% this, F _C 3%, F _C 4%, F _C 5%, F _C 10%, F _C 15%, F _C 20% , F _C 30%, F _C 40% and F _C 50%. Referring to Table 1, the nanofiber support was not homogeneously produced or radiated when it contained more than 1.1 mL of water based on the total volume of the mixed solution of 10 mL (number in parentheses in italics). Therefore, it was confirmed that the nanofiber support was successfully prepared when the amount of water contained was 0.09 to 1 mL based on the total volume of the mixed solution of 10 mL (the numerical portion indicated by the bold in Table 1).

Finally, the mixed solution prepared according to the amount of water in Table 1, the amount of fish collagen in Table 2, and the amount of the 8.8 wt% PCL synthesized polymer solution in Table 3 based on 10 mL of the mixed solution was electrospun to obtain a nanofiber support . Electrospinning was carried out using a wire mesh type collector (0.5 mm in diameter, 0.5 mm in diameter) under conditions of 27 gauge needle, 8 cm radiation distance, 9 kV voltage, 0.4 mL / hr fluid velocity, Mesh 1x1 mm). The electrospinning time was 60 to 90 minutes, preferably about 70 minutes.

Mixed solution 10 mL  The content of water contained in the reference ( mL )
In mixture Of fish collagen  Final concentration ( F _c ,%) 2 3 4 5 10 15 20 30 40 50 100

Fish Collagen  Storage solution concentration
( S _C ,%)

10 (1.8) (2.7) (3.6) (4.5) × × × × × × × 20 0.8 (1.2) (1.6) (2) (4) × × × × × × 30 0.47 0.7 0.93 (1.17) (2.33) (3.5) (4.67) × × × × 40 0.3 0.45 0.6 0.75 (1.5) (2.25) (3) (4.5) × × × 50 0.2 0.3 0.4 0.5 One (1.5) (2) (3) (4) × × 60 0.13 0.2 0.27 0.33 0.67 One (1.33) (2) (2.67) (3.33) × 70 0.09 0.13 0.17 0.21 0.43 0.64 0.86 (1.29) (1.71) (2.14) × 80 × × × × × × × × × × × 90 × × × × × × × × × × × 100 × × × × × × × × × × ×

Mixed solution 10 mL  The content of fish collagen included in the standard ( mL )
In mixture Of fish collagen  Final concentration ( F _c ,%) 2 3 4 5 10 15 20 30 40 50 100

Fish Collagen  Storage solution concentration
( S _C ,%)
10 × × × × × × × × × × × 20 0.2 × × × × × × × × × × 30 0.2 0.3 0.4 × × × × × × × × 40 0.2 0.3 0.4 0.5 × × × × × × × 50 0.2 0.3 0.4 0.5 1.0 × × × × × × 60 0.2 0.3 0.4 0.5 1.0 1.5 × × × × × 70 0.2 0.3 0.4 0.5 1.0 1.5 2.0 × × × × 80 × × × × × × × × × × × 90 × × × × × × × × × × × 100 × × × × × × × × × × ×

Mixed solution 10 mL  The content of 8.8 wt% PCL solution included in the standard ( mL )

In mixture Of fish collagen  Final concentration ( F _c ,%) 2 3 4 5 10 15 20 30 40 50 100

Fish Collagen  Storage solution concentration
( S _C ,%)


10 × × × × × × × × × × × 20 9.00 × × × × × × × × × × 30 9.33 9.00 8.67 × × × × × × × × 40 9.50 9.25 9.00 8.75 × × × × × × × 50 9.67 9.40 9.20 9.00 8.00 × × × × × × 60 9.67 9.50 9.33 9.17 8.33 7.50 × × × × × 70 9.71 9.57 9.43 9.29 8.57 7.86 7.14 × × × × 80 × × × × × × × × × × × 90 × × × × × × × × × × × 100 × × × × × × × × × × ×

3-2. Fish Collagen  Depending on concentration Fish Collagen / PCL  The structure of the nanofiber support and diameter  analysis

In order to confirm the surface structure of the PCL nanofiber support prepared in Example 1-1 and the fish collagen / PCL nanofiber support prepared in Example 3-1 using a scanning electron microscope (SEM) Each nanofiber support was fixed with 2.5% glutaraldehyde fixative solution for 24 hours (4 ℃) and then fixed with 1% osmium tetroxide at room temperature for 1 hour. Fixed support samples were washed 2-3 times with 0.1 M phosphate buffer (pH 7.4) and dehydrated through an ethanol series. Each of the dehydrated nanofiber support samples was frozen at -80 ° C for 3 hours, lyophilized for 12 hours using a vacuum freeze drier (FDS series, ilShinBioBase), and immersed in an ion sputter (E-1010, Hitachi , Tokyo, Japan), and the surface structure of the nanofiber support was analyzed with a scanning electron microscope (Hitachi) under an accelerating voltage of 20 kV. F _c 2% -S _c 50%, F _c 3% -S _c 50%, F _c 4% -S _c 50%, F _c 5% -S _c 50%, F _c 10% -S _c 50% F _c 3% -S _c 70%, F _c 5% -S _c 70%, F _c 10% -S _c 70%, F _c 15% -S _c 70%, F _c 20% -S _c 70% The fish collagen / PCL nanofiber support with F _c 2% -S _c 20%, F _c 2% -S _c 30%, F _c 2% -S _c 40% and F _c 4% -S _c 40% Diameter and nano / microfibre distribution were measured at double magnification.

As a result, as shown in FIGS. 3, 4, 5 and 6, the fibers constituting the fish collagen / PCL nanofiber support were composed of nanofibers having a nanometer diameter and micro fibers having a micrometer diameter, Fiber. Therefore, even if a mixed solution of a PCL solution dissolved in an organic solvent such as chloroform and a fish collagen solution dissolved in water is electrospun with a polymer solution made of a mixed solvent of an organic solvent and water, a nano / micro hybrid fiber It was confirmed that the constructed fish collagen / PCL mat could be successfully manufactured.

In addition, F _c 2% with 50% S _c stock _{solution, F c 3%, F c} 4%, F c 5% , and F _c 10% results confirming the microstructure of the support of the final fish collagen concentration of a scanning electron microscope, Fig. 3, it was confirmed that the size of pores between the nanofibers decreased as the final collagen concentration of the support increased. Therefore, it was confirmed that as the final collagen concentration of the support increases, the average diameter of the nanofibers decreases.

The microstructure of the supporter prepared with the final fish collagen concentration was confirmed by scanning electron microscope using the S _c 70% stock solution, F _c 2%, F _c 3%, F _c 4%, F _c 5% and F _c 10% It was confirmed that as the final collagen concentration of the support increases, the size of the pores between the nanofibers decreases. Especially S _c 50%, and S _c a 70% stock solution in a support made using F _c of more than 5% higher concentration of fish collagen / PCL nanofiber support in because generally smaller in size and the diameter of the nanofibers of the cavity 3 of the cell It is considered that it is difficult to provide an ideal environment for the dimensional distribution. Therefore, the microstructure of the support was observed according to the concentration of the stock solution at F _c 2% and F _c 4%.

As a result Referring to FIG. 5, F _c mean diameter of 2% nanofibers were _{1.47 ± 0.6 μm (S c 20} %), 1.31 ± 0.7 μm (S c 30%), 1.28 ± 0.7 μm (S c 40% ) And 1.01 ± 0.4 μm (S _c 50%) respectively. Also referring to Figure 6, F _c mean diameter of 4% of nanofibers was respectively _{1.53 ± 1.0 μm (S c 40} %) and _{0.96 ± 0.5 μm (S c 50} %). 5 and 6, it was confirmed that as the concentration of F _c 2% and F _c 4% stock solution increases, the pore size becomes smaller.

The result from the support of the F _c is less than 5% increasing the concentration of the stock solution was found that the average pore diameter and the reduction in the nanofiber.

< Example  4> Fish Collagen / PCL  Nano / Micro hybrid  In the fiber support Fish Collagen  Analysis of three-dimensional culture characteristics of solid cancer cells according to concentration

4-1. Cell and cell culture

Human non-small cell lung cancer cells (NCI-H1703) were purchased from the American Type Culture Collection (ATCC, Manassas, Va., USA) and used as human prostate cancer cells -145) were purchased from Korean Cell Line Bank. NCI-H1703 and DU-145 cell lines were treated with 10% (v / v) fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (HyClone, Logan, UT, USA) containing streptomycin (Gibco, Invitrogen) at 37 ° C under atmospheric conditions containing 5% CO ₂ . The culture medium was replaced with new one every 2-3 days.

4-2. Fish Collagen / PCL  Three-dimensional cell culture using nanofiber support

In order to compare the three - dimensional culture characteristics of fish collagen / PCL nanofiber scaffolds prepared according to the concentration of fish collagen, experimental groups were set as follows.

Fish collagen / PCL mixture concentration of the fish collagen stock solution used in manufacturing (S _c) and F _c 0% according to the concentration (F _c) of the final fish collagen in the fish collagen / PCL mixed solution used for the electrospinning (PCL 100% ), F _c 2% -S _c 30%, F _c 2% -S _c 50%, F _c 2% -S _c 70%, F _c 5% -S _c 50%, F _c 5% -S _c 70 %, F _c 10% _c 50% -S, -S F _c 10% _c 70%, _c F 15% 70% _c -S, -S F _c 20% _c 70% by dividing the 10 types of the group The fish collagen / PCL nanofiber support was prepared according to the method of manufacturing the fish collagen / PCL nanofiber support of Example 1, and then the fish collagen / PCL nanofiber support was placed in a 96-well plate and the concentration of the collagen / PCL nanofiber support was adjusted to ⁵ × 10 ⁴ cells / Human non-small cell lung cancer (NCI-H1703) and human prostate cancer (DU-145) cells were inoculated and cultured, respectively.

4-3. Actin  Person using dye Solid rock  Analysis of three-dimensional distribution of cells

F-actin staining of human non-small cell lung cancer (NCI-H1703) and prostate cancer cell line (DU-145), which were cultured three-dimensionally for 1 day using fish collagen / PCL nanofiber scaffolds, Respectively.

First, the cell-cultured fish collagen / PCL composite nanofiber support was fixed with 4% paraformaldehyde fixative at room temperature for 20 minutes. It was then washed twice with PBS and infiltrated into 0.1% triton-x 100 solution at room temperature for 5 minutes and then washed twice with PBS. To remove nonspecific staining, the cells were treated with 0.2% bovine serum albumin (BSA) solution at room temperature for 20 minutes and then washed twice with PBS. After that, actin-detecting Phalloidin-FITC (Promega, Madison, Wis., USA) was diluted 1: 150 in 0.2% BSA solution and reacted for 40 minutes. Tris- buffered saline ), Placed on a slide, stained with DAPI (Vector Lab, Inc., Burlingame, CA, USA) for nuclear staining of cells, covered with a cover glass, The three-dimensional distribution of the cells in each nanofiber support was confirmed with a microscope (FV1000-IX81, Olympus, Japan).

The human non-small cell lung cancer state (NCI-H1703) cells, a 3-D as a result of incubation 7 and human prostate cancer cell lines (DU-145) cells; As a result, PCL nanofiber support _{(PCL 100% F c 0%} ) (NCI-H1703) from the surface of the nanofiber supporter to a depth of about 55 [mu] m, and human prostate cancer cell line (DU-145) The cells were found to reach a depth of about 28 μm.

From the above results, it can be confirmed that the pores between the nanofibers in the PCL nanofiber support (F _c 0%) are relatively wide, and the cells reach a considerably deep region. However, referring to FIG. 7C and FIG. 17C in which each cross section at 1 占 퐉 intervals was observed, human non-small cell lung cancer (NCI-H1703) cells and human prostate cancer cell line (DU-145) It is observed that it takes a form of a round shape, which is a form of cells that are not attached to the general spreading. In addition, there was almost no observed growth of the cells, which are related to the survival, proliferation and interaction of the cells, and most of the cells were scattered and isolated.

On the other hand, as a result of three-dimensional culture of human non-small cell lung cancer (NCI-H1703) cultured in fish collagen / PCL nanofiber support, FIGS. 8, 9 and 10 and human prostate cancer cell line (DU- Referring to Figure 18, 19 and 20, F _c 2% for -S _c 30% fish collagen / PCL nanofiber support, one lung cancer cells was confirmed that entered to a depth of about 42 from the surface of the nanofiber support ㎛ (Fig. 8), confirming that the human prostate cancer cells enter the depth of about 58 mu m from the surface of the nanofiber support (Fig. 18). In addition, F _c 2% -S _c 50% fish collagen / PCL nanofibers in the support cells are human lung cancer went into to a depth of about 36 ㎛ from the surface of the nanofibers, the support (9), human prostate cancer cell lines (DU-145) Lt; RTI ID = 0.0 &gt; 100 &lt; / RTI &gt; F _c 2% -S _c 70% fish collagen / PCL human lung cancer cells if the nanofibers are went into the support to a depth of about 36 ㎛ from the surface of the nanofibers, the support (10), human prostate cancer cell lines (DU-145) Were found to have reached a depth of about 100 mu m or more (Fig. 20).

From the above results, _{F c 2% -S c 30%} , F c 2% -S c 50% _c 2 and F _c -S% 70% fish collagen / PCL nanofiber substrates included to nano cells can lead to extremely deep portion The pores between the fibers were found to be sufficiently wide. 18C, 19C and 20C, which are the result of three-dimensional culture of human prostate cancer cell line (DU-145), which is a result of three-dimensional culture of human non-small cell lung cancer cell line (NCI-H1703) , F _c -S 2% _c 30%, _c F _c -S 2% and 50% F _c 2% -S _c 70% fish collagen / PCL most of the cell culture in the nanofiber support to cell survival, proliferation, and presence cells showed the appearance growing attached to each other, 7C or Fig. Unlike the cell culture properties of the PCL nanofiber support (F _c 0%) of 17C cells were dispersed by itself as the morphological characteristics that reflect the interaction I have not found anything at all.

Thus, F _c 2% -S _c 30%, F _c 2% -S _c 50% and F _c 2% -S _c 70% fish collagen / PCL nanofiber scaffold provide an ideal three dimensional cell growth environment, 3-dimensional cell culture efficiency.

And also 5% -S F _c 50% _c and F _c 5% -S _c 70% fish collagen / PCL nanofiber support a person non-small cell lung cancer state (NCI-H1703) 3D as a result of 11 and 12 culture From Referring to prostate cancer cell lines (DU-145) 3-D as a result of 21 and 22 _{incubation, F c 5% -S c 50} % fish collagen / PCL nanofiber in support of human lung cancer cells nanofiber support (Fig. 11), and the human prostate cancer cell lines (DU-145) were found to reach a depth of about 38 탆 (Fig. 21). In addition, in the F _c 5% -S _c 70% fish collagen / PCL nanofiber scaffold, it was found that human lung cancer cells enter the depth of about 24 μm from the surface of the nanofiber scaffold (FIG. 12) 145) were found to have reached a depth of about 20 μm (FIG. 22).

From the above results, F _c 5% -S _c 50%, and F _c 5% -S _c 70% fish collagen / PCL nanofiber support F _c -S 2% _c 30% _c 2 and F _c -S% 50% Fish It was confirmed that the pores between nanofibers were narrower than the collagen / PCL nanofiber support, so that the cells were relatively shallow. However, as a result of checking each cross section at 1 탆 intervals, the cells were evenly distributed as in the cells of FIGS. 8C and 9C. In addition, _c and F _c 5% -S and 50% F _c 5% -S _c 70% fish collagen / PCL nanofiber person in support of non-small cell lung cancer state (NCI-H1703) 3D as a result of FIG. 11C and 12C incubation Referring to FIGS. 21C and 22C as a result of three-dimensional culture of human prostate cancer cell line (DU-145), most of the cells showed adhesion and growth for cell survival, proliferation and interaction, Unlike the cell culture characteristics of the PCL nanofiber support (F _c 0%), no single cells were found to be scattered. Therefore, it was confirmed that F _c 5% -S _c 50% and F _c 5% -S _c 70% fish collagen / PCL nanofiber scaffold provide an ideal three-dimensional cell growth environment, thereby exhibiting a high three-dimensional cell culture efficiency .

And also F _c 10% _c 50% -S and 10% F _c -S _c 70% fish collagen / PCL nanofiber support person non-small cell lung cancer state (NCI-H1703) 3D as a result of 13 and 14 in culture From Referring to prostate cancer cell lines (DU-145) a three-dimensional culture which result in Fig. 23 and _{24, F c 10% -S c} 50% fish collagen / PCL nanofiber in support of human lung cancer cells nanofiber support (Fig. 13), and the human prostate cancer cell line (DU-145) reached a depth of about 22 탆 (Fig. 23). In addition, F _c 10% _c 70% fish collagen -S / PCL nanofiber support the human lung cancer cells was found to be entered to a depth of about 26 ㎛ from the surface of the nanofibers, the support (14), human prostate cancer cell lines (DU- 145) reached a depth of about 22 탆 (Fig. 24).

From the above results, F _c 10% _c 50% -S and 10% F _c -S _c 70% fish collagen / PCL nanofiber support F _c -S 2% _c 30% _c 2 and F _c -S% 50% Fish It was confirmed that the pores between the nanofibers were narrower than the collagen / PCL nanofiber support, and the cells were relatively shallow. However, as a result of checking each cross section at 1 탆 intervals, the cells were evenly distributed as in the cells of FIGS. 8C and 9C. In addition, F _c 10% _c 50% -S and 10% F _c -S _c 70% fish collagen / PCL nanofiber person non-small cell lung cancer in a main support (NCI-H1703) for 3D culture is also a result of the 13C and 14C 23C and 24C, which are three-dimensional cultures of human prostate cancer cell line (DU-145), showed that most of the cells adhered to each other for cell survival, proliferation and interaction, Unlike the cell culture characteristics of the PCL nanofiber support (F _c 0%) of 7C, no single cells were found scattered. Therefore, it was confirmed that F _c 10% -S _c 50% and F _c 10% -S _c 70% fish collagen / PCL nanofiber scaffold provide an ideal three-dimensional cell growth environment, thereby exhibiting high three-dimensional cell culture efficiency .

And also F _c 15% _c 70% -S and 20% F _c -S _c 70% fish collagen / PCL nanofiber support a person non-small cell lung cancer state (NCI-H1703) a three-dimensional culture which results in Figures 15 and 16 From Referring to prostate cancer cell lines (DU-145) a three-dimensional culture as a result of 25 and _{26, F c 15% -S c} 70% fish collagen / PCL nanofiber in support of human lung cancer cells nanofiber support (Fig. 15), and the human non-small cell lung cancer germinate reached a depth of about 20 mu m (Fig. 25). In addition, F _c 20% _c 70% fish collagen -S / PCL nanofiber in support man lung cancer cells was found to be entered to a depth of about 26 ㎛ from the surface of the nanofibers, the support (16), one to about 24 non-small cell lung cancer Mu] m (Fig. 26).

From the above results, F _c 15% _c 70% -S and 20% F _c -S _c 70% fish collagen / PCL nanofiber support F _c -S 2% _c 30% _c 2 and F _c -S% 50% Fish It was confirmed that the pores between the nanofibers were narrower than the collagen / PCL nanofiber support, and the cells were relatively shallow. However, as a result of checking each cross section at 1 탆 intervals, the cells were evenly distributed as in the cells of FIGS. 8C and 9C. In addition, F _c 15% _c 70% -S and 20% F _c -S _c 70% fish collagen / PCL nanofiber support the three-dimensional culture of human non-small cell lung cancer state (NCI-H1703) the result of Fig. 15C and 16C and Referring to FIGS. 25C and 26C, which are three-dimensional cultures of human prostate cancer cell line (DU-145), most of the cells showed adhesion and growth for cell survival, proliferation and interaction, and FIG. 7C of PCL cell state that is present in scattered cells alone, unlike the culture properties of the nanofiber support (F _c 0%) it was not found at all. Therefore, it was confirmed that F _c 15% -S _c 70% and F _c 20% -S _c 70% fish collagen / PCL nanofiber scaffold provide an ideal three-dimensional cell growth environment, thereby exhibiting high three-dimensional cell culture efficiency .

From the result _{F c 2%, F c 5} %, F c 10%, F c 15% , and F _c 20% fish collagen / concentration (F _c) of the final fish collagen in PCL mixture is F _c fish more than 2% Collagen-containing fish collagen / PCL nanofiber scaffolds have been demonstrated to be effective for three-dimensional culture of human non-small cell lung cancer (NCI-H1703) and human prostate cancer cell lines (DU-145). In particular, the fish collagen / PCL nanofiber support among F _c -S 2% _c 30% _c to F 2% 3-dimensional culture of -S _c 50% fish collagen / PCL nanofibers, the support is a cell that contains the F _c above 2% It is confirmed that it can be usefully used.

< Example  5> Fish Collagen / PCL  On a nanofiber support Fish Collagen  Analysis of three-dimensional culture characteristics of blood cancer cells according to concentration

5-1. Cell and cell culture

Mouse T cell lymphoma cells (EL4) and mouse B cell lymphoma cells (A20) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). EL4 cell lines were cultured in RPMI 1640 supplemented with 10% v / v fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / ml penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (Gibco, Invitrogen ) Containing Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA) containing 5% CO ₂ at 37 ° C. The A20 cell line was treated with 10% v / v fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (Gibco, Invitrogen ) Were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) containing 5% CO ₂ Containing atmospheric conditions. The culture medium was replaced with new one every 2-3 days.

5-2. Fish Collagen / PCL  Three-dimensional cell culture using nanofiber support

In order to compare the three - dimensional culture characteristics of fish collagen / PCL nanofiber scaffolds prepared according to the concentration of fish collagen, experimental groups were set as follows.

Fish collagen / PCL mixture concentration of the fish collagen stock solution used in manufacturing (S _c) and F _c 0% according to the concentration (F _c) of the final fish collagen in the fish collagen / PCL mixed solution used for the electrospinning (PCL 100% ), F _c 2% -S _c 30%, F _c 2% -S _c 50%, F _c 2% -S _c 70%, F _c 5% -S _c 50%, F _c 5% -S _c 70 %, F _c 10% _c 50% -S, -S F _c 10% _c 70%, _c F 15% 70% _c -S, -S F _c 20% _c 70% by dividing the 10 types of the group Following the method of example 1 Fish collagen / PCL nanofiber of the support, fish collagen / PCL nanofibers into the fish collagen / PCL nanofiber substrate after producing the support in a 96-well plate, with 4x10 ⁵ cells / well density Mouse T cell lymphoma cell line (EL4) and mouse B cell lymphoma cell line (A20) were inoculated and cultured, respectively.

5-3. Actin  Analysis of three-dimensional distribution of mouse blood cancer cells using staining

F-actin staining of mouse B cell lymphoma cell line (A20) and mouse T cell lymphoma cell line (EL4) cultured three-dimensionally for 1 day and 3 days using fish collagen / PCL nanofiber scaffold For the first time, the cultured fish collagen / PCL nanofiber support was fixed with 4% paraformaldehyde fixative at room temperature for 20 min. Then, the cells were washed twice with PBS, followed by permeation to 0.1% triton-X 100 solution for 5 minutes at room temperature, followed by washing twice with PBS. To remove nonspecific staining, the cells were treated with 0.2% bovine serum albumin (BSA) solution at room temperature for 20 minutes and then washed twice with PBS. After that, actin-detecting Phalloidin-FITC (Promega, Madison, Wis., USA) was diluted 1: 150 in 0.2% BSA solution and reacted for 40 minutes. Tris- buffered saline ), Placed on a slide, stained with DAPI (Vector Lab, Inc., Burlingame, CA, USA) for nuclear staining of cells, covered with a cover glass, And confirmed with a microscope (FV1000-IX81, Olympus, Japan).

As a result, PCL nanofiber support _{(F c 0%; PCL 100} %) to the mouse B cell lymphoma cell line (A20) 3-D as a result of the culture 27 and mouse T cell lymphoma cell line (EL4) cells in the three-dimensional culture which 36, it was confirmed that the mouse B cell lymphoma cell line (A20) was introduced to the depth of about 40 mu m from the surface of the nanofiber support, and the mouse T cell lymphoma cell line (EL4) And it was confirmed that it entered depth.

From the above results, it can be confirmed that the pores between the nanofibers in the PCL nanofiber support (F _c 0%) are relatively wide, and the cells reach a considerably deep region.

However, referring to FIG. 27C and FIG. 36C showing each cross section at 1 占 퐉 intervals, the number of cells in which the mouse B cell lymphoma cell line (A20) cells and mouse T cell lymphoma cell line (EL4) cells were distributed was considerably small, F-Actin was not homogeneously distributed along the cell membrane, and expression was greatly reduced or accumulated irregularly in the local region. In addition, the cell size was smaller than that of normal cells and showed very irregular size and shape.

From the above results, it was confirmed that the PCL nanofiber support does not provide an environment suitable for three-dimensional culture of blood cancer cells.

On the other hand, as a result of three-dimensional culture of mouse B cell lymphoma cell line (A20) in the fish collagen / PCL nanofiber scaffold and three-dimensional culture of the mouse T cell lymphoma cell line (EL4) Referring to 38 and 39, in the case of the F _c 2% -S _c 30% fish collagen / PCL nanofiber support, a mouse B-cell lymphoma cells was confirmed that entered to a depth of about 66 ㎛ from the surface of the nanofibers, the support ( 28), it was confirmed that the mouse T cell lymphoma cells reached a depth of about 60 mu m from the surface of the nanofiber support (Fig. 37). In the F _c 2% -S _c 50% fish collagen / PCL nanofiber support mouse B cell lymphoma cell went into the depth of about 70 ㎛ from the surface of the nanofibers, the support (29), the mouse T-cell lymphoma cells were about To a depth of 80 [mu] m (Fig. 39). For the F _c 2% -S _c 70% fish collagen / PCL nanofiber support mouse B cell lymphoma cell went into the depth of about 110 ㎛ from the surface of the nanofibers, the support (30), the mouse T-cell lymphoma cells were about And it was found that it reached the depth of 42 탆 (FIG. 39).

From the above results, _{F c 2% -S c 30%} , F c 2% -S c 50% _c 2 and F _c -S% 70% fish collagen / PCL nanofiber substrates included to nano cells can lead to extremely deep portion This is because not only the size of the pores between the fibers is appropriate but also the physical properties of the nanofibers provide an environment suitable for cell migration and maintenance. Also, when each cross section of 1 ㎛ spacing was observed, the cells were evenly distributed at a sufficiently high density over a considerable thickness. Also, note the mouse B-cell lymphoma cells, the results of a three-dimensional culture Figure 28C and 29C and the mouse T-cell lymphoma cells, the three-dimensional FIG. 37C and 38C as a result of the _{culture, F c 2% -S c 30} % , and F _c Most of the cells of the 2% -S _c 50% nanofiber scaffold were significantly different from the cell culture characteristics of the PCL nanofiber support (F _c 0%) of FIG. 27C or FIG. 36C, - Actin was homogeneously distributed along the cell membrane and was very well represented in the round shape of the normal cell cortex. In addition, the cell size was similar to that of normal cells, but also showed regular size and shape.

Therefore, it was confirmed that F _c 2% -S _c 30% and F _c 2% -S _c 50% fish collagen / PCL nanofiber scaffold provide an ideal three-dimensional cell growth environment, thereby exhibiting high three-dimensional cell culture efficiency .

On the other hand, F _c -S 2% _c 70% fish collagen / PCL For nanofiber support 30C and FIG. Referring to FIG. 39C, cell distribution, but the size, shape and F- actin staining characteristics of the cells are substantially similar to the normal cells Were lower than those of F _c 2% -S _c 30% and F _c 2% -S _c 50% fish collagen / PCL nanofiber scaffolds. In addition, _{F c 5% -S c 50%} ( Fig. _{31), F c 5% -S} c 70% ( Fig. _{32), F c 10% -S} c 70% ( Fig. _{33), F c 15% -S} c 70% (Fig. 34), F _c 20% _c 70% -S (FIG. 35) In the case of fish collagen / PCL nanofiber support mouse B-cell lymphoma cells to about 46 ㎛ respectively, 36 ㎛, 26 ㎛, 34 ㎛ and 24 Mu m depth.

From the above results, _{F c 5% -S c 50%} , F c 5% -S c 70%, F c 10% -S c 70%, F c 15% -S c 70%, F c 20% -S c 70% fish collagen / PCL nanofiber substrates included because F _c 2% -S _c 30% _c 2 and F _c -S% of a narrow gap than 50% fish collagen / PCL nanofiber support that the cells are relatively shallow, the distribution . However, the cells in the enlarged cross-section of Figs. 31C, 32C, 33C, 34C and 35C are distributed evenly as in the cells of F _c 2% -S _c 30% and F _c 2% -S _c 50% fish collagen / PCL nanofiber scaffolds , And the size, shape and F-actin staining characteristics of the cells were almost similar to normal cells.

_{However, F c 2% -S c 70} % cell density, such as fish collagen / PCL nanofiber support is significantly lower.

Mouse T-cell lymphoma cells were grown in F _c 5% -S _c 50% (FIG. 38) and F _c 5% -S _c 70% (FIG. 39) fish collagen / PCL nanofiber scaffolds to depths of 30 μm and 20 μm, respectively However, in the other F _c 10% -S _c 70%, F _c 15% -S _c 70%, F _c 20% -S _c 70% fish collagen / PCL nanofiber scaffolds, And the number of cells was also very low.

These results support the F _c 10% or higher concentration of from (F _c 10% _c 50% -S, -S F _c 10% _c 70%, _c F 15% 70% _c -S, -S F _c 20% _c 70 %) Did not provide a good three-dimensional cell growth environment.

Therefore, the concentration (F _c ) of the final fish collagen in a mixture of F _c 2%, F _c 5%, F _c 10%, F _c 15% and F _c 20% fish collagen / PCL through the above- the F _c F _c 2% to about 5%, and the fish collagen is a fish collagen / PCL nanofiber support comprises providing a suitable culture environment the three-dimensional cell cultures, more preferably F _c 2 _c -S% 30% To F _c 2% -S _c 50% fish collagen / PCL nanofiber scaffold was found to be a suitable support for three-dimensional cell culture.

< Example  6> Fish Collagen / PCL  Three-dimensionally cultured cells in a nanofiber support Survival  Measure

6-1. Cell and cell culture

Mouse B cell lymphoma cells (A20) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). The A20 cell line was treated with 10% v / v fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (Gibco, Invitrogen ) Were cultured in RPMI-1640 medium (HyClone, Logan, UT, USA) containing 5% CO ₂ Containing atmospheric conditions. The culture was replaced with new one every 1-2 days.

6-2. Fish Collagen / PCL  Three-dimensional cell culture using nanofiber support

In order to measure the viability of three-dimensionally cultured cells in fish collagen / PCL nanofiber scaffolds, the concentration (S _c ) of fish collagen stock solution used in the production of fish collagen / PCL mixture and the concentration of fish collagen / PCL the classification of F _c 0% of the support (PCL100%) and F _c 2% -S _c 50% according to the concentration (F _c) of the final fish collagen was used in. The method of producing the fish collagen / PCL nanofiber support was carried out by the method of manufacturing the fish collagen / PCL nanofibers of Example 1 above. Each nanofiber support was placed in a 96-well plate and A20 cells were inoculated at a concentration of 2 x ¹⁰ cells / well and cultured.

6-3. Live / Dead  dyeing

Live / Dead staining was performed to confirm the effect of the supporter on the survival of A20 cells in the culture of the fish collagen / PCL nanofiber support according to the present invention.

First, A20 cells were inoculated in a fish collagen / PCL composite nanofiber support as in Example 6-2 and cultured for 2 days and 6 days. Each nanofiber support was washed once with PBS, diluted with 2 μL of calcein AM and 0.5 μL of EthD-1 (LIVE / DEAD Viability Kit, Molecular Probes) in 1 ml of PBS, Stained in the dark and analyzed with a confocal laser scanning microscope (FV1000-IX81, Olympus, Japan).

As a result, the survival rate of A20 cells cultured on the fish collagen / PCL nanofiber support according to the present invention was much higher than that of the PCL nanofiber support, as shown in FIG. 42 on the second day of culture and FIG. 43 on the sixth day of culture.

From the above results, it was confirmed that the fish collagen / PCL nanofiber support provides a proper growth environment for three-dimensional culture of A20 cells.

< Example  7> Fish Collagen / PCL  In a three-dimensional cultured cancer cell using a nanofiber support Korean - Bibo model conformity analysis

7-1. Cell and cell culture

Human non-small cell lung cancer cells (NCI-H1703) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and used for chemotherapy-sensitive chemotherapy , And human prostate cancer cell (DU-145) were purchased from Korean Cell Line Bank. NCI-H1703 cell line and DU-145 cell line were treated with 10% (v / v) fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) hygromycin (streptomycin; Gibco, Invitrogen) containing RPMI-1640 medium 37 ℃ using (HyClone, Logan, UT, USA ), 5% CO 2 Containing atmospheric conditions. Mouse T cell lymphoma cells (EL4) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). EL4 cell lines were cultured in RPMI 1640 supplemented with 10% v / v fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / ml penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (Gibco, Invitrogen ) Containing Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA) containing 5% CO ₂ at 37 ° C. The culture was replaced with new one every 1-2 days.

7-2. Fish Collagen / PCL  For cancer cells cultured three-dimensionally on a nanofiber support Drug spreading ability  Measure

To determine the diffusion capacity of the drug to reach cancer cells in a 3D environment using nano fiber _{support, F c 2% -S c 50} % fish collagen / PCL] After the fabrication of nano-fiber substrate placed in a 96-well plate EL4 cells Were inoculated at a concentration of 1 x ¹⁰ cells / well and cultured for 3 days.

Then, doxorubicin (Cell Signaling Technology, Danvers, MA, USA) was treated at a concentration of 1 μM for 12 hours and 24 hours, and the fish collagen / PCL nanofiber support was washed with PBS. The nuclei were stained with DAPI solution, The distribution of doxorubicin was confirmed by confocal laser microscopy (FV1000-IX81, Olympus, Tokyo, Japan). The results are expressed as mean ± standard deviation (SD) for each assay. Statistical analysis was performed using a two-tailed t-test, and statistical significance was evaluated based on P <0.05.

As a result, as shown in FIG. 44, it was confirmed that doxorubicin was diffused into most regions where EL4 cells, which are mouse T lymphoma cells, were distributed in the supporter.

From the above results, it was confirmed that the three-dimensional cancer cell culture model using the fish collagen / PCL nanofiber support according to the present invention can be useful for screening for susceptibility testing of various drugs such as anticancer drugs.

7-3. Fish Collagen / PCL  Measurement of anticancer drug susceptibility to cancer cells cultured three-dimensionally on a nanofiber support

To confirm the suitability of a 3 - D cancer cell culture model using fish collagen / PCL nanofiber scaffolds, we analyzed the sensitivity of 3 - D cancer cell culture model using fish collagen / PCL nanofiber scaffolds.

First, in the same way as in Example 1 F _c 4% -S _c produced a 50% fish collagen / PCL nanofiber support and placed in a 96-well plate the NCI-H1703, and DU-145 cells, respectively, 5x10 ⁴ cells / well And then cultured.

NCI-H1703 and DU-145 cells cultured three-dimensionally on the nanofiber scaffold and NCI-H1703 and DU-145 cells cultured in the usual two-dimensional culture conditions were supplemented with 3 [mu] M doxorubicin (doxorubicin, 5927; Cell Signaling Technology , Danvers, MA, USA) and 3 μM paclitaxel (T7402, Sigma) for 24 and 48 hours, respectively. The absorbance was measured at 450 nm using a microplate reader using WST- Were measured. The results are expressed as mean ± standard deviation (SD) for each assay. Statistical analysis was performed using a two-tailed t-test, and statistical significance was evaluated based on P <0.05.

As a result, referring to FIG. 45A, when doxorubicin was treated with 3 μM for 24 hours in human non-small cell lung cancer (NCI-H1703), the survival rate was 55.1% (P <0.01) , And the cell survival rate was 94.5% (P <0.01) in the three - dimensional environment as compared with the control group. The survival rate was 40.8% (P <0.01) compared with the control group in the 2-dimensional environment after 48 hours, and the cell survival rate was 95.7% in the 3-dimensional environment compared with the control group.

In addition, when paclitaxel was treated with 3 μM of the prostate cancer cell line (DU-145), the survival rate was 50.2% (P <0.05) in the two-dimensional environment as shown in FIG. 45B , And 93.5% (P <0.01) in the 3-D environment compared to the control group. In the 2-D environment, the survival rate was 14.6% (P <0.01) In the 3D environment, cell viability was 64.2% (P <0.05) compared to the control group.

Based on the above results, it can be shown that the three-dimensional environment can effectively exhibit drug resistance to anticancer agents rather than the two-dimensional environment. Thus, the three-dimensional cancer cell culture model using fish collagen / PCL nanofiber scaffolds can provide a drug resistance- And it can be used for testing.

7-4. Fish Collagen / PCL  Three-dimensional cancer cell culture on nanofiber scaffolds Malignancy  analysis

Fish collagen / PCL nanofibers in the three-dimensional tumor culture model using a support for malignant function analysis, the method of Example 1, the F _c 4 _c -S% 50% fish collagen / PCL nanofibers support 96 made in the same Well plate and DU-145 cells were inoculated at a concentration of 5 x 10 ⁴ cells / well. After 2 days of incubation, DU-145 cells were cultured in the same condition for 2 days. Semi-quantitative reverse transcription-polymerase chain reaction analysis (RT-PCR) was performed.

First, total RNA was isolated from the cultured cells according to the manufacturer's instructions using trizol reagent (Ambion, Invitrogen) in each of the cultured DU-145 cells.

(DT) 12-18 oligo (dT) 12-18] Oligo (dT) primer, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 _{mM MgCl 2, 40 mM DTT,} 0.5 mM deoxynucleotide triphosphate (dNTP) mixture, 10 units RNase inhibitor , and 200 unit MMLV (Moloney murine leukemia virus ) reverse transcriptase polymerase; buffer containing (reverse transcriptase Promega, Madison, WI , USA) Single-strand cDNA was synthesized from 1 μg of total RNA using 25 μL of the solution.

To confirm the PCR reaction for gene amplification, the primers of Table 4 were used. The PCR reaction of human Hes1 and Notch-1, -2, -3, and 4 was performed using 10 pmol of the primer and 1 μl cDNA sample, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl ₂ , 0.1% Triton X Initial denaturation was performed at 94 ° C for 5 minutes using 25 μL of a buffer solution containing 100 μM dNTP, -100 μM, 0.2 mM dNTP and 5 units Taq DNA polymerase (Promega) 45 sec at 60 ° C and 45 sec at 72 ° C for 35 cycles and CD44, Snail and GAPDH were performed at 94 ° C for 30 sec, at 57 ° C for 45 sec and at 72 ° C for 45 sec Was performed in 30 cycles. In the case of human slug, HIF-1α, VEGF, and endothelin-1, initial denaturation was performed at 94 ° C. for 5 minutes, followed by 30 seconds at 94 ° C., 45 seconds at 60 ° C., and 45 seconds at 72 ° C. Cycle. After the PCR reaction, the reaction product samples produced by 2% agarose gel electrophoresis were stained with EtBr (Ethidium bromide) and analyzed under UV light illumination.

As a result, the three-dimensional culture of human prostate cancer cells (DU-145) using two-dimensional or fish collagen / PCL nanofiber scaffolds as shown in FIG. 46 was performed and Hes 1 and Notch-1, -2 , -3, and 4 were confirmed by RT-PCR analysis. As a result, expression of both Hes 1 and Notch-1, -2, -3, and 4 was increased in cancer cells cultured in a 3-dimensional environment than 2-dimensional culture Respectively.

From the above results, it was confirmed that the cancer cells cultured in the three-dimensional environment were increased in malignancy unlike the two-dimensional environment. In the three-dimensional cancer cell culture model using the fish collagen / PCL nanofiber support according to the present invention, ) Environment is well simulated.

47, the malignancies of cancer cells, particularly the markers snail and slug, which are closely related to the epithelial-mesenchymal transition (EMT), VEGF, the vascular endothelial growth factor of cancer cells , VEGF secretion factor HIF-1α, CD44, an important marker of cancer stem cell, and expression of endothelin-1, which plays a key role in tumor growth and metastasis, In the cultured cancer cells.

From the above results, in the case of three-dimensional cancer cell culture based on fish collagen / PCL nanofiber support, the culture environment of cancer cells such as growth, progression, metastasis and malignancy of cancer can exhibit characteristics very similar to living body, 3-dimensional cell culture method using the nanofiber scaffold of the present invention, in view of providing an in-vivo environment that could not be provided by the method, can be applied not only to research on cancer cell, growth, metastasis and malignancy of cancer cells, And the like.

Gene name SEQ ID NO: Primer sequence Human Hes 1
One Forward: 5'-CCTGTCATCCCCGTCTACAC-3 ' 2 Reverse direction: 5'-CACATGGAGTCCGCCGTAA-3 ' Human Notch-1
3 Forward: 5'-TACAAGTGCGACTGTGACCC-3 ' 4 Reverse direction: 5'-CACACGTAGCCACTGGTCAT-3 ' Human Notch-2
5 Forward: 5'-CAACCGCAATGGAGGCTATG-3 ' 6 Reverse: 5'-GCGAAGGCACAATCATCAATGTT-3 ' Human Notch-3
7 Forward: 5'-TGGCGACCTCACTTACGACT-3 ' 8 Reverse: 5'-CACTGGCAGTTATAGGTGTTGAC-3 ' Human Notch-4
9 Forward: 5'-CCTGGCTCCTTCAACTGCC-3 ' 10 Reverse direction: 5'-GCAAGTAGGTCCAGACAGGT-3 ' Human Snail
11 Forward: 5'-AGACCCACTCAGATGTCAA-3 ' 12 Reverse: 5'-CATAGTTAGTCACACCTCGT-3 ' Human Slug
13 Forward: 5'-GGTCAAGAAGCATTTCAAC-3 ' 14 Reverse direction: 5'-GGTAATGTGTGGGTCCGA-3 ' Human VEGF
15 Forward: 5'-CGAAACCATGAACTTTCTGC-3 ' 16 Reverse direction: 5'-CCTCAGTGGTCACACACTCC-3 ' Human HIF-1α
17 Forward: 5'-GGCGCGAACGACAAGAAAA-3 ' 18 Reverse: 5'-GTGGCAACTGATGAGCAAGC-3 ' Human CD44
19 Forward: 5'-CTCCAGTGAAAGGAGCAGCA-3 ' 20 Reverse direction: 5'-AGCAGGGATTCTGTCTGTGC-3 ' Human Endothelin-1 21 Forward: 5'-TGCTCCTGCTCTTCCCTGATGGATAAAGAGTGTGTC-3 ' 22 Reverse direction: 5'-GGTCACATAACGCTCTCTGGAGGGCTT-3 ' Human GAPDH
23 Forward: 5'-TGGAGAAACCTGCCAAGTATG-3 ' 24 Reverse direction: 5'-TTGTCATACCAGGAAATGAGC-3 '

< Example  8> Fish Collagen / PCL  In a three-dimensional cultured cancer cell using a nanofiber support Korean - Bibomosa Amniotic stem cell induction / Growth and Cancer Cell Differentiation Model Suitability Analysis

8-1. Cell and cell culture

Mouse T cell lymphoma cells (EL4) were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

EL4 cell lines were cultured in RPMI medium supplemented with 10% (v / v) fetal bovine serum (FBS; Gibco, Invitrogen), 100 IU / mL penicillin (Gibco, Invitrogen) and 100 ug / mL streptomycin (Gibco, Invitrogen ) Containing Dulbecco's Modified Eagle Medium (DMEM, HyClone, Logan, UT, USA) containing 5% CO ₂ at 37 ° C. The culture medium was replaced with new one every 2-3 days.

8-2. High processing Flow cell  Analyzer ( flow cytometer ) analysis

Fish collagen / PCL nm in the three-dimensional tumor culture model using a fiber-forming ability and the support as CSCs same manner as in Example 1, in order to analyze the tumor cells of the multipotential F _c 2% 50% _c -S] After the fabrication of nanofibers support The fabricated nanofiber support was placed in a 96-well plate and EL4 cells were inoculated at a concentration of 2 x ¹⁰ cells / well and cultured for 2 days.

The cultured cells were washed twice with Hank's Balanced Salt Solution (HBSS, GibcoLife Technologies) and resuspended in 3 mL of FACS buffer (0.1% BSA, 0.1% sodium chloride, 500 mL of HBSS) The cells were pre-incubated with anti-FcγRII / III (2.4G2, hybridoma supernatant) to avoid nonspecific binding with antibodies after resuspension of the cells.

CD4 antibody (FITC-CD4 or PE-CD4) conjugated with FITC or PE, the primary fluorescent-labeled primary anti-mouse monoclonal antibody, in HBSS containing 2% , APC-conjugated anti-sCD-1 antibody (APC-CD8), c-kit-conjugated anti-c-kit antibody -sca-1) at a concentration of 5-10 μg / mL for 30 minutes under ice-cooling. Background fluorescence was determined through cells that were reacted with isotype-matched nonreactive antibodies.

After the cells were washed twice with HBSS, the cells were resuspended in 500 μL of FACS buffer, and the expression of cell surface molecules was analyzed by Dot-plot method using a flow cytometer. The obtained results were analyzed using FlowJo software (Tree Star, Ashland, OR, USA).

As a result, the proportion of c-kit ⁺ sca-1 ⁺ cells corresponding to cancer stem cells among the EL4 cells cultured for 2 days under the 2D environment was 0.97% The proportion of c-kit ⁺ sca-1 ⁺ C-stem cells in one EL4 cell increased to 4.45%.

From the above results, it was confirmed that, in the case of three-dimensional cancer cell culture based on fish collagen / PCL nanofiber support, the culture environment of cancer cells related to the formation and growth of cancer stem cells is similar to a living body, 3-dimensional cell culture method using the fish collagen / PCL nanofiber support of the present invention is useful for research on cancer stem cell and anti-cancer drug resistance mechanism and development of highly efficient chemotherapy Can be used as a culture model.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Pusan National University Industry-University Cooperation Foundation <120> Electrospun fish collagen synthetic polymer hybrid nanofiber          scaffolds, method thereof and use thereof <130> ADP-2014-0163 <160> 24 <170> Kopatentin 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 cctgtcatcc ccgtctacac 20 <210> 2 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 cacatggagt ccgccgtaa 19 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 tacaagtgcg actgtgaccc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 cacacgtagc cactggtcat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 caaccgcaat ggaggctatg 20 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gcgaaggcac aatcatcaat gtt 23 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 tggcgacctc acttacgact 20 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 cactggcagt tataggtgtt gac 23 <210> 9 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 cctggctcct tcaactgcc 19 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 gcaagtaggt ccagacaggt 20 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agacccactc agatgtcaa 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 catagttagt cacacctcgt 20 <210> 13 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 ggtcaagaag catttcaac 19 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 ggtaatgtgt gggtccga 18 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 cgaaaccatg aactttctgc 20 <210> 16 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cctcagtggt cacacactcc 20 <210> 17 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 ggcgcgaacg acaagaaaa 19 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 gtggcaactg atgagcaagc 20 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ctccagtgaa aggagcagca 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 agcagggatt ctgtctgtgc 20 <210> 21 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 tgctcctgct cttccctgat ggataaagag tgtgtc 36 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 22 ggtcacataa cgctctctgg agggctt 27 <210> 23 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 23 tggagaaacc tgccaagtat g 21 <210> 24 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 24 ttgtcatacc aggaaatgag c 21

Claims (9)

A first step of dissolving the synthetic polymer in an organic solvent;
A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution;
A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously; And
And a fourth step of preparing a nanofiber support by electrospinning the mixed solution prepared in the third step. [Claim 2] The method according to claim 1, wherein the synthetic polymer solution in the first step is prepared by dissolving 8 to 10 parts by weight of a synthetic polymer in 100 parts by weight of an organic solvent. The method according to claim 1 or 2, wherein the synthetic polymer is selected from the group consisting of poly (e-caprolactone); PCL], poly lactic acid; PLA], polyglycolic acid; PGA], poly (hydroxy valerate); PHV], poly (lactic-co-glycolic acid) PLGA, poly-hydroxy butyrate (PHB), poly (hydroxy butyrate-co -valerate); PHBV], polyanhydride, polyorthoester, poly (vinyl alchol); (PVA), polyethylene glycol (PEG), polyurethane, polyacrylic acid (PAA), poly-N-isopropylacrylamide, diol / A method of producing a composite nanofiber support comprising the steps of: preparing a composite nanofiber support comprising at least one selected from the group consisting of polyester and polyester-amide / polyester-urethane. The method according to claim 1 or 2, wherein the organic solvent is selected from the group consisting of chloroform, hexafluoroisopropane, tetrahydrofuran, dimethylformamide, dichloromethane, acetone, dioxane, trifluoroethane, &Lt; RTI ID = 0.0 & [Claim 2] The method according to claim 1, wherein the fish collagen aqueous solution in the second step is prepared by dissolving fish collagen in an amount of 10 to 70 parts by weight based on 100 parts by weight of water. [6] The method of claim 1, wherein the mixed solution of the third step comprises a synthetic polymer solution so that the synthetic polymer may be contained in an amount of 6.3 to 8.5 parts by weight based on 100 parts by weight of the mixed solution. [2] The method of claim 1, wherein the mixed solution of the third step comprises a fish collagen aqueous solution so that the fish collagen is contained in an amount of 2 to 20 parts by weight based on 100 parts by weight of the mixed solution. [Claim 2] The method according to claim 1, wherein the mixed solution in the third step is added in an amount of 0.9 to 10 parts by weight based on 100 parts by weight of the mixed solution. A first step of dissolving the synthetic polymer in an organic solvent;
A second step of dissolving fish collagen in water to prepare a fish collagen aqueous solution;
A third step of adding the aqueous fish collagen solution prepared in the second step to the synthetic polymer solution prepared in the first step and mixing them homogeneously;
A fourth step of electrospinning the mixed solution prepared in the third step to produce a nanofiber support; And
And culturing the cells on the nanofiber support prepared in the fourth step.

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