KR102250589B1 - Nanofiber hybrid membrane for culturing stem cells, medium and method for culturing stem cells using the same - Google Patents

Abstract

The present invention relates to a nanofiber hybrid membrane for stem cell culture and a stem cell culture method using the same, comprising a nanofiber membrane formed by stacking nanofibers having a fiber diameter of less than 1㎛, wherein the nanofiber membrane is located in the stem cell One side to become; And the other surface on which the supporting cells are located, wherein the nanofiber membrane is provided with a plurality of three-dimensional micropores for supplying nutrients required for the stem cells to grow from the supporting cells to the stem cells.

Description

Nanofiber hybrid membrane for culturing stem cells, stem cell culture device using the same, and method for culturing stem cells {Nanofiber hybrid membrane for culturing stem cells, medium and method for culturing stem cells using the same}

The present invention relates to a nanofiber hybrid membrane for stem cell culture, and more particularly, a nanofiber membrane having a structure most similar to the extracellular matrix (ECM) of the human body is used as a separation membrane between stem cells and support cells. The present invention relates to a nanofiber hybrid membrane for culturing stem cells, which allows easy attachment of stem cells and obtains high-quality stem cells in an appropriate culture environment, a stem cell incubator using the same, and a method for culturing stem cells.

In recent years, while biotechnology continues to develop, researches have been attempted to restore its function by regenerating tissues and organs using stem cells.

Stem cells are referred to as cells that can differentiate into various tissues that make up the human body, and stem cells to restore tissues and organs damaged or damaged by accidents or diseases are divided into embryonic stem cells and adult stem cells. do.

Embryonic stem cells have the risk of developing tumors and are difficult to use for therapeutic purposes due to ethical and legal issues, but adult stem cells are cultivated stem cells extracted from adipose tissue or bone marrow, and are ethical and legal issues. It is free to solve the problem of embryonic stem cells.

Currently, the main research directions for stem cell cultivation can be divided into a technique of obtaining stem cells by co-culturing stem cells with feeder cells, and a method of culturing only stem cells without feeder cells. Ultimately, it is advantageous to proceed to the side without supporting cells, but at the present stage, there is a problem such as having to replace the expensive culture medium every day when there are no supporting cells. More recently, research is underway to cultivate pluripotent stem cells.

On the other hand, it is an important task to mass-produce undifferentiated stem cells without contamination in stem cell research. In the case of co-cultivation of feeder cells and stem cells, it is necessary to remove mouse-derived feeder cells when applying stem cells to clinical trials. There is this.

Therefore, in culturing stem cells, a technology that separates feeder cells and stem cells so that only nutrients can be transferred from the feeder cells to the stem cells is being developed. For example, Korean Patent No. 10-0744445 discloses a feeder cell. A method of culturing human embryonic stem cells using a medium containing a porous membrane attached to the bottom surface is disclosed, and Korean Patent No. 10-0832592 discloses a polymer having a plurality of pores for feeder cells and stem cells. A method of culturing stem cells in which they are separated by a membrane and divided into two spaces for cultivation is disclosed.

In such prior art literature, the porous membrane used for culturing stem cells is manufactured by a phase separation technique and has a structure that is heterogeneous from the stem cells, so it takes a long time to attach the stem cells to the porous membrane, and it is a rather unsuitable culture environment. There is a problem that cells cannot be obtained.

Accordingly, as a result of repeating research to solve the problems of the prior art documents, the present inventors developed a nanofiber hybrid membrane formed by stacking nanofibers with a diameter of less than 1㎛ similar to the extracellular matrix structure formed of collagen fibrils and fibers. The present invention can be used to eliminate or significantly reduce the problems of prior art documents.

Korean Patent Publication No. 10-0744445 Korean Registered Patent Publication No. 10-0832592

An object of the present invention is a nanofiber hybrid membrane for stem cell culture that can shorten the time for attaching stem cells to the nanofiber membrane and obtain high-quality stem cells in an appropriate culture environment, and a stem cell incubator and stem cell culture using the same There is a way to provide.

Another object of the present invention is a nanofiber hybrid membrane for stem cell culture capable of efficiently co-culturing stem cells and support cells by applying a nanofiber membrane as a separation membrane between stem cells and support cells, and a stem cell culture device and stem using the same It is to provide a cell culture method.

Another object of the present invention is to provide a nanofiber hybrid membrane for culturing stem cells that can easily observe the culture state of stem cells with the naked eye, a stem cell incubator and a method for culturing stem cells using the same.

In order to achieve the above object, the present invention is made of a nanofiber membrane formed by stacking nanofibers having a fiber diameter of less than 1㎛, the nanofiber membrane is one side on which the stem cells are located; And the other side on which the supporting cells are located; wherein the nanofiber membrane includes a stem in which a plurality of three-dimensional micropores for supplying nutrients necessary for the stem cells to grow from the supporting cells to the stem cells are connected from the surface to the rear surface It provides a nanofiber membrane for cell culture.

In addition, the present invention provides a nanofiber membrane layer composed of nanofibers with a fiber diameter of less than 1 μm and a nonwoven or woven layer having a fiber diameter of 10 μm to 100 μm in the form of 2 layers and 3 layers. It provides a method of manufacturing a nanofiber hybrid membrane for stem cell culture in a form capable of smoothly supplying nutrients necessary for the growth of stem cells by improving mechanical properties and handling properties compared to a single nanofiber membrane.

In addition, the present invention provides a nanofiber hybrid membrane formed by hybridizing a nanofiber membrane having a plurality of three-dimensional micropores formed by stacking nanofibers with a fiber diameter of less than 1 μm and a nonwoven or woven fabric having a plurality of pores. Interposing between the stem cells and the support cells; And culturing the stem cells by supplying nutrients from the support cells to the stem cells through the pores of the nanofiber hybrid membrane and the nonwoven or woven fabric, and culturing the stem cells. It provides a cultivation method of.
Moreover, the present invention, the culture vessel; A nanofiber membrane inserted into the culture vessel and having a plurality of three-dimensional micropores by stacking nanofibers having a fiber diameter of less than 1 μm; Support cells attached to one surface of the nanofiber membrane; And stem cells attached to the other surface of the nanofiber membrane, wherein the nanofiber membrane provides a stem cell incubator separating a space in which the stem cells are cultured and a space in which the support cells are cultured.

As described above, in the present invention, stem cells can be quickly and smoothly attached to the nanofiber membrane having a structure most similar to the extracellular matrix (ECM) of the human body, thereby shortening the cultivation time of stem cells. In addition, there is an advantage in that only high-quality stem cells can be obtained by creating a suitable culture environment.

In addition, in the present invention, by applying the nanofiber membrane as a separation membrane between stem cells and support cells, only nutrients necessary for stem cells to grow are passed through a number of three-dimensional micropores formed in the nanofiber membrane, and the size is relatively larger than the pore size. By preventing impurities from passing through, there is an advantage that stem cells with minimal impurities can be produced in large quantities.

In addition, in the present invention, there is an advantage that it is possible to efficiently co-culture the stem cells and the support cells by dividing them into two spaces by separating the support cells and the stem cells by a nanofiber membrane having an open pore structure.

In addition, in the present invention, by hybridizing nanofibers and nonwovens or woven fabrics, there is an effect of simultaneously improving the weak mechanical properties and handling properties of the nanofiber membrane, and allowing support cells and stem cells to be easily attached to the nanofiber hybrid surface.

In addition, in the present invention, by culturing stem cells by applying a nanofiber membrane laminated with transparent nanofibers, there is an advantage in that the culture state of the stem cells can be easily observed with the naked eye.

1 is a schematic cross-sectional view for explaining a state in which stem cells and support cells are coupled to a nanofiber hybrid membrane for stem cell culture according to the present invention,
2 is a schematic view showing a partial view for explaining the nanofiber membrane for culturing stem cells according to the present invention,
3(a) is a flowchart of a method of culturing stem cells using a nanofiber membrane for stem cell culture according to the present invention, and FIG. 3(b) is a flowchart of a method of manufacturing a nanofiber membrane for stem cell culture according to the present invention. It is a flow chart.
4A to 4D are schematic cross-sectional views for explaining the structure of the nanofiber and nanofiber hybrid membrane for stem cell culture according to the present invention,
5 (a) and (b) are photographs of the surface and cross-section of a scanning electron micrograph of PVdF nanofibers prepared according to an embodiment of the present invention,
6 is a PMI graph showing the pore distribution of PVdF nanofibers prepared according to an embodiment of the present invention,
7 (a) and (b) are scanning electron micrographs of the surface and cross-section of PAN nanofibers prepared according to an embodiment of the present invention.
Figures 8 (a) and (b) are diagrams showing the surface and contact angle measurement of the plasma-treated PVdF nanofibers prepared according to an embodiment of the present invention,
9(a)-(c) are scanning electron micrographs of a cross section of a nanofiber hybrid membrane for stem cell culture prepared according to an embodiment of the present invention.
10 is a graph of counting adipocyte-derived and bone-derived stem cells grown using a nanofiber hybrid membrane prepared according to an embodiment of the present invention.

The terms or words used in the specification and claims should not be construed as being limited to their usual or dictionary meanings, and the inventor may appropriately define the concept of terms in order to describe his own invention in the best way. It should be interpreted as a meaning and concept consistent with the technical idea of the present invention based on the principle that there is.

Accordingly, the embodiments described in the present specification and the configurations shown in the drawings are only the most preferred embodiment of the present invention, and do not represent all the technical spirit of the present invention. It should be understood that there may be equivalents and variations.

Hereinafter, the present invention will be described in more detail with reference to the drawings.

1 is a schematic cross-sectional view illustrating a state in which stem cells and support cells are coupled to a nanofiber hybrid membrane for stem cell culture according to the present invention, and FIG. 2 is a schematic cross-sectional view illustrating a nanofiber membrane for stem cell culture according to the present invention. It is a part of the electron micrograph shown to, Figure 3 (a) is a flow chart of a method for culturing stem cells using a nanofiber membrane for culturing stem cells according to the present invention, Figure 3 (b) is a stem according to the present invention It is a flow chart of a method of manufacturing a nanofiber membrane for cell culture.

The nanofiber membrane for stem cell culture according to the present invention uses a nanofiber membrane 100 having a plurality of three-dimensional micropores 101 by stacking nanofibers having a fiber diameter of less than 1 μm, and the nanofiber membrane 100 ) Is interposed between the stem cells 110 and the support cells 120 and inserted into a culture vessel (not shown) to co-culture the stem cells 110 and the support cells 120.

In this case, as shown in FIG. 1, stem cells 110 may be attached to one surface of the nanofiber membrane 100, and support cells 120 may be attached to the other surface of the nanofiber membrane 100. Such stem cells 110 and support cells 120 are cultured and attached to the nanofiber membrane 100.

Here, a plurality of three-dimensional micropores 101 provided in the nanofiber membrane 100 supply nutrients necessary for the stem cells 110 to grow from the support cells 120 to the stem cells 110.

In addition, the stem cells 110 and the support cells 120 may be mounted on a separate medium and inserted into a culture vessel.

Here, when the stem cells 110 and the support cells 120 are cultured in the same medium, the serum may induce natural differentiation of stem cells. In the present invention, the nanofibers for stem cell culture are used as the nanofiber membrane 100. By implementing a membrane, the space in which the stem cells 110 are cultivated and the space in which the support cells 120 are cultivated are separated by the nanofiber membrane 100, thereby preventing the natural differentiation of stem cells from occurring.

For reference, stem cells are cells that can differentiate into various tissues that make up the human body, and support cells are cells that provide nutrients necessary for stem cells to grow and inhibit the differentiation of stem cells into specific cells. to be.

When the stem cell culture method of the present invention is described using the stem cell culture nanofiber membrane, as shown in FIG. 3(a), first, nanofibers having a diameter of less than 1 μm are stacked to form a number of three-dimensional micropores. A nanofiber membrane for culturing stem cells made of a nanofiber membrane having a is interposed between the stem cells and the supporting cells (S200). Here, as an example method of performing the'S200' process, stem cells are cultured and attached to one side of the nanofiber membrane, and support cells are cultured and attached to the other side of the nanofiber membrane, so that between the stem cells and the supporting cells. The nanofiber membrane is interposed.

Thereafter, the stem cells are cultured (S210) by supplying nutrients from the support cells to the stem cells through the plurality of three-dimensional micropores of the nanofiber membrane.

In addition, in another method of culturing stem cells of the present invention, a nanofiber membrane having a plurality of three-dimensional micropores formed by stacking nanofibers with a fiber diameter of less than 1㎛ and a nonwoven fabric or a woven fabric having a plurality of pores are hybridized. Interposing the formed nanofiber hybrid membrane between the stem cells and the supporting cells; And culturing the stem cells by supplying nutrients from the support cells to the stem cells through the nanofiber hybrid membrane and the pores of the nonwoven or woven fabric.

Meanwhile, the above-described nanofiber membrane 100 is in the form of a membrane having a plurality of three-dimensional micropores 112 in which nanofibers 111 composed of a fiber diameter of less than 1 μm are stacked, as shown in FIG. It has a sheet shape in a state.

As described above, in the present invention, by applying the nanofiber membrane as a separation membrane between the stem cells and the supporting cells, nutrients necessary for the stem cells to grow through a plurality of three-dimensional micropores formed in the nanofiber membrane are smoothly transferred from the supporting cells to the stem cells. However, impurities having a size relatively larger than the pore size cannot pass through a large number of three-dimensional micropores, so there is an advantage that only stem cells can be produced in large quantities while minimizing impurities in culturing stem cells.

In addition, in the present invention, the diameter and structure of the nanofiber membrane has a structure most similar to the extracellular matrix (ECM) of the human body composed of collagen fibers or collagen fibrils. Therefore, it is possible to shorten the cultivation time of stem cells by rapidly and smoothly attaching the stem cells to the nanofiber membrane by using nanofibers similar to the ECM structure, which is the extracellular matrix structure of humans. There is an advantage that can be obtained.

In addition, in the present invention, there is an advantage that it is possible to efficiently co-culture the stem cells and the support cells by dividing them into two spaces by separating the support cells and the stem cells by a nanofiber membrane having an open pore structure.

The nanofiber membrane 100 applied to the present invention is a single type of polymer or a mixed spinning solution obtained by mixing at least two types of polymers and dissolving in a solvent, or dissolving different polymers in a solvent, respectively. It can be obtained by cross-spinning through a spinning nozzle.

The nanofiber membrane 100 dissolves a single or mixed polymer in a solvent to prepare a spinning solution, and then electrospins the spinning solution to form a porous nanofiber membrane made of ultrafine nanofibers, and heats at a temperature below the melting point of the polymer. It is formed by pressing and adjusting the pore size and the thickness of the membrane.

It is preferable that the nanofiber membrane 100 is set to a thickness of 10 to 50 μm. In addition, it is preferable that the fiber diameter of the nanofiber membrane 100 is 50 to 600 nm, and the pore size is 0.1 to 0.5 μm.

Referring to FIG. 3, in the nanofiber membrane for stem cell culture according to the present invention, first, a spinning solution is prepared by dissolving single or two or more types of fiber-forming polymer materials in an organic solvent (S100). Thereafter, the spinning solution is electrospun to form a nanofiber membrane for culturing stem cells in which nanofibers are stacked (S110).

Subsequently, the nanofiber membrane is molded by thermal fusion and then cut into a predetermined size (S120). The cut nanofiber membrane is used after sterilization treatment before stem cell culture.

In addition, after the'S110' or'S120' process, a process of performing a surface hydrophilization treatment by plasma treatment on the surface of the nanofiber membrane may be further performed.

Here, in the present invention, the forming and cutting process and the plasma treatment process may be selectively performed.

The hybridization process is not limited to special methods such as hot plate calendering, hot melt bonding, ultrasonic bonding, laminating, etc., and any method capable of complexing a nanofiber layer with an existing material may be used.

In this way, when a nanofiber membrane and a nonwoven or woven fabric are hybridized and used as a nanofiber hybrid membrane for stem cell culture, the mechanical properties and handling properties can be improved compared to the membrane of the nanofiber alone, and the surface irregularities and pore structures are also Compared to the nanofiber membrane alone, the micro-nano structure makes it easier to settle the stem cells.

4A to 4D are schematic cross-sectional views for explaining the structure of the nanofiber membrane for stem cell culture according to the present invention.

Referring to FIGS. 4A to 4D, the nanofiber membrane for stem cell culture according to the present invention may be implemented in the structure of FIG. 4A consisting of the nanofiber membrane 100 alone.

In addition, in the present invention, the nanofiber membrane 100 is a two-layer structure of FIG. 4B hybridized to a nonwoven fabric or a woven fabric 200, or a nanofiber membrane 100 is interposed between a pair of nonwoven fabrics 210 and 220. It can be implemented in a hybridized three-layer structure of FIG. 4C. In addition, the non-woven fabric 200 may be interposed between a pair of nanofiber membranes 100a and 100b, and may be implemented as a three-layer structure of FIG. 4D hybridized.

Here, the non-woven fabric or woven fabric (200, 210, 220) applied to the two-layer structure and the three-layer structure is any one of rayon, PP, PE, PET, nylon, cellulose, PVDF, collagen, PU, PLA, PLGA, and PCL. It may be selected, and it is preferable if sufficient thermal bonding with the nanofiber membrane 100 is possible without being dissolved, deformed, or discolored during stem cell culture. The hybridization between the non-woven fabric or the woven fabric 200, 210, 220 and the nanofiber membrane 100 is preferably thermally bonded without an adhesive. At this time, in a two-layer structure, the thickness of the nonwoven fabric 200 is preferably 10 to 100 μm, and the thickness of the nanofiber membrane 100 is 5 to 20 μm, and in the three-layer structure, the thickness of the nanofiber membrane 100 is 1 to 10 It is preferable that the thickness is as thin as possible in µm.

In addition, as shown in FIG. 4A, the structure consisting of the nanofiber membrane 100 alone can be hydrophilicized on the surface of the nanofiber by plasma treatment, thereby facilitating the attachment of stem cells to the surface of the nanofiber.

Here, when the nanofiber membrane for stem cell culture is opaque, it is difficult to visually check the stem cell culture during stem cell culture, and thus there is a disadvantage in that it is observed with a microscope or staining method. However, in the present invention, the nanofiber membrane 100 Nanofibers can be implemented as PMMA, PC, PU, PCL, PLA, PLGA alone nanofibers. In this case, the nanofiber membrane 100 becomes translucent, so that the culture state of stem cells can be easily observed with the naked eye. There is this.

In addition, the nanofiber membrane 100 applied to the present invention may use a synthetic polymer or a natural polymer as a material capable of electrospinning as a usable polymer material, and the synthetic polymer or natural polymer may be used alone or in combination. There is no particular limitation as long as it is a polymer material capable of forming nanofibers by electrospinning.

Example 1

As a fiber-molding polymer, polyvinyliden fluoride (PVdF) is mixed and dissolved in a mixed solvent of DMAc (dimethylacetamide)/acetone (mixing ratio is 7/3 wt.%) so that it is 15 wt.%. A spinning solution was prepared. The spinning solution thus prepared is transferred to the spinning nozzle pack under a spinning condition with an applied voltage of 25 kV, a distance between the spinning nozzle and the current collector, 20 cm, discharge volume per hole 0.05 cc/g·hole·min, temperature 30°C, and 60% relative humidity. Electrospinning was performed. The PVdF nanofiber membrane obtained under the above conditions was subjected to thermocompression using a roller heated to 140°C.

Figures 5 (a) and (b) show scanning electron micrographs of the surface and cross section of the PVdF nanofiber membrane obtained under the above conditions, and Figure 6 shows the results of a PMI (Capillary Flow Porometer) showing the average pore distribution. As shown in the picture, most of the fiber diameters were less than 1 μm and averaged 400 nm. The thickness of the PVdF nanofibers obtained under the above conditions was about 30 μm, and the average pore size was about 0.3 μm.

Example 2

A spinning solution was prepared by mixing and dissolving polyacrylonitrile (PAN) as a fiber-molding polymer in a DMAc (dimethylacetamide) solvent to 12 wt.%. The thus prepared spinning solution was subjected to electrospinning and thermocompression bonding in the same manner as in the above example to obtain a membrane made of PAN nanofibers having an average fiber diameter of about 300 nm, an average pore size of 0.45 μm, and a membrane thickness of 20 μm. 7 shows a scanning electron micrograph of the surface and cross section of the PAN nanofiber.

Example 3

The PVdF nanofiber membrane prepared in Example 1 was surface-modified using a plasma cleaner. As the gas used at this time, argon (Ar) was supplied at 100 sccm and the surface was modified at 400W for 60 sec.

8(a) and (b) show the surface structure of the plasma-treated PVdF nanofiber membrane and the result of the contact angle with water. According to the plasma treatment, the contact angle of water decreased, and the contact angle of the surface of the PVdF nanofibers was modified to be hydrophilic from 110° to 75° by the plasma treatment.

Example 4

The nanofibers obtained in Example 1 were thermally bonded using a PET nonwoven fabric having a weight of 30 gsm at a temperature of 160° C. of a hot plate by the method b-d of FIG. 4 to prepare a nanofiber hybrid membrane. 9 shows a scanning electron microscope photograph of the morphology of the cross section of the nanofiber hybrid membrane prepared by the above method.

Example 5

As feeder cells, mitomycin (Mitomycin C, Sigma, Cat.No.M-4287) was added at a concentration of 10 ug/ml for 1 hour 30 to use mouse fetal fibroblasts extracted and cultured from mice (CF1, C57BL6). After treatment for a minute ^{, inoculate at a density of 4×10 4} /㎠, and then inoculate and culture a piece of embryonic stem cells (HSF6) the next day. As a medium composition, 3.069g/l sodium bicarbonate (Sigma, USA, Cat.No.S5761), 2 mM L-glutamine (Sigma, Cat. No.S8540), 1% penicillin (50U/ml) (Sigma, Cat.No.P4687)/streptomysin (50ug/ml) (Sigma, USA, Cat.No.S1277), 20% Knock-Out serum replacement (SR) ; Invitrogen BRL, Cat. No. 10828-028), 4 ng/ml Basic Fibroblasts Growth Factor (bFGF; Invitrogen BRL, Cat. No. 13256-029). Incubated in 37°C, 5% CO ₂ incubator. The medium was exchanged daily and used as a control.

Example 6

In order to use the nanofiber hybrid membrane as a separation membrane for stem cell culture, the nanofiber hybrid membrane prepared in Examples 1 and 4 was used using a commercially available 6 well culture dish.

Mouse fetal fibroblasts, which are feeder cells, were treated with mycomycin-c, a growth inhibitor, for 2 hours to inhibit proliferation, ^{and then inoculated at a density of 4 × 10 4} /㎠, and after 24 hours, a nanofiber hybrid membrane After adding the embryonic stem cell medium to the top and confirming that the medium is equilibrated between the upper and lower portions, stem cells (Adipose stem cells, bone marrow stem cells) were inoculated on the membrane, and the stem cells were cultured in the same manner as in Example 5. I did. To observe the growth of stem cells, colonies stained through alkaline phosphatase staining were counted as undifferentiated, and colonies that were not stained were counted as differentiated colonies. Alkaline phosphatase staining was performed by mixing a solution of NBT/BCIP (Roche, Germany, Cat.No. 1 681 451) in a pH of 9.5 Tris-Cl at a ratio of 99:1 to confirm color development, and a 400 times microscope observation was performed. . In FIG. 10, values indicated by counting fat and bone-derived stem cells are shown.

In the above, the present invention has been illustrated and described by way of example and description of specific preferred embodiments, but the present invention is not limited to the above-described embodiments, and common knowledge in the technical field to which the present invention pertains within the scope not departing from the spirit of the present invention. Various changes and modifications will be possible by those who have.

100,100a,100b: nanofiber membrane 101,112: fine pores
110: stem cell 111: nanofiber
120: support cells 200, 210, 220: non-woven fabric or woven fabric

Claims (14)

delete delete delete delete delete delete delete delete delete delete delete After dissolving two or more different polymers in each solvent, cross-spinning is carried out through different spinning nozzles, and nanofibers with a fiber diameter of less than 1㎛ are stacked to consist of two or more kinds of components having a number of three-dimensional micropores. Obtaining a multi-component nanofiber membrane;
Obtaining a multi-component nanofiber hybrid membrane by laminating the multi-component nanofiber membrane with a nonwoven fabric having a plurality of pores, or interposing a pair of nonwoven fabrics to form a composite;
Interposing the multi-component nanofiber hybrid membrane between stem cells and support cells; And
Culturing the stem cells by supplying nutrients from the support cells to the stem cells through a plurality of three-dimensional micropores of the multi-component nanofiber hybrid membrane; Culture method. The method of claim 12,
A method of culturing stem cells using a multi-component nano-fiber hybrid membrane, characterized in that the surface irregularities of the multi-component nano-fiber hybrid membrane form a micro-nano structure. The method of claim 12,
The method of culturing stem cells using a multi-component nano-fiber hybrid membrane, characterized in that the hybridization of the multi-component nano-fiber membrane and the non-woven fabric is performed by thermal bonding.

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