KR20150111535A - Nanofiber hybrid membrane for culturing stem cells and method for culturing stem cells using the same - Google Patents

Abstract

The present invention relates to a nanofiber hybrid membrane for culturing stem cells and method for culturing stem cells using the same comprising: a one side consisting of a nanofiber membrane which is formed by laminating nanofibers having a fiber diameter of less than 1μm; and the other side where support cells are placed, wherein the a nanofiber membrane has a plurality of pores for supplying nutrients necessary to grow the stem cells from the support cells to the stem cells.

Description

TECHNICAL FIELD The present invention relates to a nanofiber hybrid membrane for culturing stem cells, and a method for culturing stem cells using the same.

The present invention relates to a nanofiber hybrid membrane for culturing a stem cell, and more particularly, to a nanofiber hybrid membrane having a structure most similar to an extracellular matrix (ECM) of a human body, To a method for culturing a stem cell using the same, and to a method for culturing a stem cell using the same, which can obtain high quality stem cells with a suitable culture environment.

Recently, as biotechnology continues to develop, research is underway to regenerate tissues and organs using stem cells to restore their functions.

Stem cells are called cells that can differentiate into various tissues that make up the human body. Stem cells for reconstructing damaged tissues and organs by accident or disease are divided into embryonic stem cells and adult stem cells do.

Embryonic stem cells have a risk of developing tumors and are difficult to be used for therapeutic purposes due to ethical and legal problems. However, adult stem cells are cultured stem cells extracted from adipose tissue or bone marrow, and ethical and legal problems And the problem of embryonic stem cells can be solved.

Currently, the main research direction of stem cell cultivation is divided into the technique of obtaining stem cells by co-culturing feeder cells and stem cells, and the method of culturing only stem cells without feeder cells (feeder cell free). Ultimately, it is advantageous to proceed to the absence of support cells, but in the present stage, there is a problem such that an expensive culture solution should be replaced every day in the absence of supporting cells. More recently, research is under way to cultivate pluripotent stem cells.

In the meantime, in the study of stem cells, mass production of undifferentiated stem cells without contamination is an important task. When co-culturing stem cells and supporting cells, disadvantages of removing mouse-derived stem cells when applying stem cells to clinical use .

Therefore, a technique has been developed in which stem cells are separated from support cells and stem cells to allow only nutrients to migrate from stem cells to stem cells. For example, in Korean Patent No. 10-0744445, feeder cells Discloses a method for culturing human embryonic stem cells using a medium containing a porous membrane attached to a bottom surface thereof. Korean Patent No. 10-0832592 discloses a method for culturing a feeder cell and a stem cell with a polymer having plural pores A method for culturing a stem cell in which the cells are separated into a membrane and cultured in two spaces is disclosed.

In this prior art document, the porous membrane used for culturing stem cells has a heterogeneous structure with the stem cells prepared by the phase separation technique. Therefore, it takes a long time to adhere the stem cells to the porous membrane, and the culture medium is somewhat inadequate. There is a problem that cells can not be obtained.

As a result, the inventors of the present invention have found that a nanofiber hybrid membrane formed by laminating nanofibers having a diameter of less than 1 μm, which is similar to the extracellular matrix structure formed of collagen fibrils and fibers, The present invention has been accomplished in order to solve the problems of the prior art documents in a remarkable manner.

Korean Patent Registration No. 10-0744445 Korean Patent Registration No. 10-0832592

An object of the present invention is to provide a nanofiber hybrid membrane for stem cell culture capable of shortening the time for adhering stem cells to a nanofiber membrane and obtaining high quality stem cells in a suitable culture environment and a method for culturing stem cells using the same There is.

Another object of the present invention is to provide a nanofiber hybrid membrane for culturing stem cells, which can efficiently culture co-cultured stem cells and supporting cells by applying a nanofiber membrane as a membrane between stem cells and supporting cells, and a method for culturing stem cells using the same .

It is another object of the present invention to provide a nanofiber hybrid membrane for culturing stem cells, which can easily observe the cultured state of stem cells by naked eyes, and a method for culturing stem cells using the same.

In order to achieve the above object, the present invention provides a nanofiber membrane formed by lamination of nanofibers having a fiber diameter of less than 1 탆, wherein the nanofiber membrane has one side on which stem cells are located; Wherein the nanofiber membrane comprises a plurality of three-dimensional micropores for feeding the nutrients necessary for the stem cells to grow from the supporting cells to the stem cells, A nanofiber membrane for cell culture is provided.

In addition, the present invention provides a non-woven fabric or a woven fabric layer having a fiber diameter of less than 1 占 퐉 and a non-woven fabric or a woven fabric layer having a fiber diameter of 10 占 퐉 to 100 占 퐉 in two layers, The present invention also provides a method for manufacturing a nanofiber hybrid membrane for culturing a stem cell in which the nutrients necessary for growth of stem cells can be smoothly improved by improving mechanical properties and handling properties of the nanofiber single membrane.

The present invention also provides a nanofiber hybrid membrane comprising a nanofiber membrane having a plurality of three-dimensional micropores formed by lamination of nanofibers having a fiber diameter of less than 1 μm and a nonwoven fabric or woven fabric having a plurality of pores hybridized with each other Intervening between stem cells and supporting cells; And culturing the stem cells by supplying nutrients from the supporting cells to the stem cells through the pores of the nanofiber hybrid membrane and the nonwoven fabric or the woven fabric, thereby culturing the stem cells using the nanofiber hybrid membrane for stem cell culture. .

As described above, in the present invention, the stem cell can be rapidly and smoothly adhered to the nanofiber membrane having the structure most similar to the extracellular matrix (ECM) of the human body, thereby shortening the culturing time of the stem cell , There is an advantage that only a high-quality stem cell can be obtained by generating a suitable culture environment.

In the present invention, the nanofiber membrane is used as a separation membrane between stem cells and supporting cells. Thus, only the nutrients required for the stem cells to grow are passed through a plurality of three-dimensional micropores formed on the nanofiber membrane, It is advantageous to mass-produce the stem cells having impurities minimized by preventing impurities from passing therethrough.

In addition, in the present invention, the support cells and the stem cells are separated into nanofiber membranes having an open pore structure, and are divided into two spaces, whereby the stem cells and support cells can be efficiently co-cultured.

In addition, in the present invention, by hybridizing the nanofibers with the nonwoven fabric or the woven fabric, it is possible to simultaneously improve the weak mechanical properties and handleability of the nanofiber membrane and to easily attach the supporting cells and the stem cells to the surface of the nanofiber hybrid.

In addition, in the present invention, culturing of stem cells by applying a nanofiber membrane laminated with transparent nanofibers makes it possible to easily observe the cultured state of the stem cells with the naked eye.

FIG. 1 is a schematic cross-sectional view illustrating a state where a stem cell and supporting cells are bound to a nanofiber hybrid membrane for stem cell culture according to the present invention,
FIG. 2 is a schematic view illustrating a nanofiber membrane for culturing stem cells according to the present invention,
FIG. 3 (a) is a flow chart of a method of culturing stem cells using a nanofiber membrane membrane for culturing stem cells according to the present invention, and FIG. 3 (b) FIG.
4A to 4D are schematic cross-sectional views illustrating a structure of a nanofiber / nanofiber hybrid membrane for culturing stem cells according to the present invention,
5 (a) and 5 (b) are photographs of the surface and cross-section of the PVdF nanofiber fabricated according to one embodiment of the present invention by scanning electron microscopy,
6 is a PMI graph showing the pore distribution of PVdF nanofibers produced according to one embodiment of the present invention,
7 (a) and 7 (b) are scanning electron micrographs of the surface and cross-section of PAN nanofibers produced according to one embodiment of the present invention.
8 (a) and 8 (b) are graphs showing the surface and contact angle measurement of the plasma-treated PVdF nanofiber prepared according to one embodiment of the present invention,
9 (a) - (c) are scanning electron micrographs of a section of a nanofiber hybrid membrane for culture of stem cells prepared according to an embodiment of the present invention.
FIG. 10 is a graph showing counts of bone-derived stem cells derived from fat cells grown using the nanofiber hybrid membrane prepared according to an embodiment of the present invention.

The terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary terms and the inventor may appropriately define the concept of the term in order to best describe its invention It should be construed as meaning and concept consistent with the technical idea of the present invention.

Therefore, the embodiments described in this specification and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not represent all the technical ideas of the present invention. Therefore, It is to be understood that equivalents and modifications are possible.

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

FIG. 1 is a schematic cross-sectional view illustrating a state where stem cells and support cells are bound to a nanofiber hybrid membrane for culturing stem cells according to the present invention. FIG. 2 is a schematic view illustrating a nanofiber membrane membrane for culturing stem cells according to the present invention FIG. 3 (a) is a flow chart of a method for culturing stem cells using a nanofiber membrane for stem cell culture according to the present invention, and FIG. 3 (b) FIG. 2 is a flow chart of a method of manufacturing a nanofiber membrane for cell culture. FIG.

The nanofiber membrane for culture of stem cells according to the present invention comprises a nanofiber membrane 100 having a plurality of three-dimensional micropores 101 laminated with nanofibers having a fiber diameter of less than 1 mu m, Is interposed between the stem cells 110 and the support cells 120 and inserted into a culture container (not shown) to co-culture the stem cells 110 and the support cells 120.

1, stem cells 110 are attached to one surface of the nanofiber membrane 100 and support cells 120 are attached to the other surface of the nanofiber membrane 100. The stem cells 110 and supporting cells 120 are attached to the nanofiber membrane 100 by being cultured.

The plurality of three-dimensional micropores 101 provided in the nanofiber membrane 100 supply the nutrients necessary for the stem cells 110 to the stem cells 110 from the supporting cells 120.

In addition, the stem cells 110 and the supporting cells 120 may be placed in a separate medium and inserted into a culture container.

Here, when the stem cells 110 and the support cells 120 are cultured in the same medium, serum may induce spontaneous differentiation of the stem cells. In the present invention, the nanofiber membrane 100 may contain nanofibers for culturing stem cells By implementing the membrane, the space in which the stem cells 110 are cultured and the space in which the support cells 120 are cultured is separated into the nanofiber membrane 100, thereby preventing the stem cells from spontaneous differentiation.

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

As shown in FIG. 3 (a), nanofibers having a diameter of less than 1 μm are laminated to form a plurality of three-dimensional micropores A nanofiber membrane membrane for culturing a stem cell is interposed between the stem cell and the supporting cell (S200). As an example of performing the 'S200' process, a stem cell is cultured on one side of a nanofiber membrane, adhered thereto, and a supporting cell is adhered to the other side of the nanofiber membrane to attach the stem cell. Interposing a nanofiber membrane.

Thereafter, nutrients are supplied to the stem cells from the supporting cells through a plurality of three-dimensional micro pores of the nanofiber membrane to cultivate the stem cells (S210).

In another method of culturing a stem cell of the present invention, a nanofiber membrane having a plurality of three-dimensional micropores formed by laminating nanofibers having a fiber diameter of less than 1 μm and a nonwoven fabric or woven fabric having a plurality of pores are hybridized Intervening 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 pores of the nanofiber hybrid membrane and nonwoven fabric or woven fabric.

2, 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 having a fiber diameter of less than 1 mu m are laminated, Shaped sheet shape.

As described above, in the present invention, by applying the nanofiber membrane as a membrane separating stem cells and supporting cells, nutrients necessary for the stem cells to grow through a plurality of three-dimensional micropores formed on the nanofiber membrane are transferred from the supporting cells to the stem cells Impurities having a size larger than the pore size can not pass through a plurality of three-dimensional micropores, and thus it is possible to produce a large number of stem cells while minimizing impurities in culturing the stem cells.

In addition, in the present invention, the diameter and structure of the nanofiber membrane are most similar to the extracellular matrix (ECM) of the human body, which is composed of collagen fiber or collagen fibril. Therefore, it is possible to rapidly and stably attach the stem cells to the nanofiber membrane using the nanofibers similar to the ECM structure of the human extracellular matrix structure, thereby shortening the culturing time of the stem cells. In addition, Can be obtained.

In addition, in the present invention, the support cells and the stem cells are separated into nanofiber membranes having an open pore structure, and the cells are divided into two spaces, so that the stem cells and support cells can be efficiently co-cultured.

The nanofiber membrane 100 applied to the present invention may be prepared by electrospinning a mixed spinning solution obtained by mixing a single kind of polymer or at least two kinds of polymers dissolved in a solvent or dissolving different polymers in a solvent, Can be obtained by cross-irradiation through a spinning nozzle.

The nanofiber membrane 100 may be prepared by dissolving a single or mixed polymer in a solvent to prepare a spinning solution, electrospinning the spinning solution to form a porous nanofiber membrane made of microfine nanofiber, And adjusting the pore size and the thickness of the membrane.

The nanofiber membrane 100 is preferably set to a thickness of 10 to 50 mu m. The nanofiber membrane 100 preferably has a fiber diameter of 50 to 600 mu m and a pore size of 0.1 to 0.5 mu m.

Referring to FIG. 3, the nanofiber membrane for culturing stem cells according to the present invention is prepared by dissolving single or at least two types of fibrous polymeric 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).

Next, the nanofiber membrane is formed by thermal fusion, and then cut to a predetermined size (S120). The cut nanofiber membrane is used by sterilization treatment before stem cell culture.

Further, after the step 'S110' or 'S120', the surface of the nanofiber membrane may be subjected to a surface hydrophilizing treatment by a plasma treatment.

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

The hybridization process is not limited to special methods such as hot plate calendering, hot melt bonding, ultrasonic bonding, and laminating, and it is sufficient that the nanofiber layer and the existing material can be combined with each other.

If the nanofiber membrane and the nonwoven fabric or the woven fabric are hybridized and used as a nanofiber hybrid membrane for stem cell culture, the mechanical properties and handleability of the nanofiber alone can be improved, and the surface irregularity and pore structure The micro-nano-structure of the nanofiber membrane alone is compared with that of the nanofiber membrane.

4A to 4D are schematic cross-sectional views illustrating the structure of a nanofiber membrane for culturing stem cells according to the present invention.

4A to 4D, the nanofiber membrane for culturing stem cells according to the present invention can be realized by the structure of FIG. 4A, which is made of the nanofiber membrane 100 alone.

4B in which the nanofiber membrane 100 is hybridized with the nonwoven fabric or the woven fabric 200 or a structure in which the nanofiber membrane 100 is interposed between the pair of nonwoven fabrics 210 and 220 Layered structure shown in FIG. 4C. Layer structure of FIG. 4D in which the nonwoven fabric 200 is interposed between the pair of nanofiber membranes 100a and 100b.

The nonwoven fabric or the woven fabric 200, 210 or 220 applied to the two-layer structure and the three-layer structure may be any one of rayon, PP, PE, PET, nylon, cellulose, PVDF, collagen, PU, PLA, PLGA, And it is preferable that sufficient thermal bonding with the nanofiber membrane 100 is possible without dissolving, deforming, or discoloring in the stem cell culture. The hybridization of the nonwoven fabric or the woven fabric (200, 210, 220) and the nanofiber membrane (100) is preferably thermally bonded without an adhesive. In this case, the thickness of the nonwoven fabric 200 (10 to 100 μm, the thickness of the nanofiber membrane 100) is preferably 5 to 20 μm, and the thickness of the nanofiber membrane 100 is 1 to 10 Lt; RTI ID = 0.0 > m, < / RTI >

In addition, as shown in FIG. 4A, the nanofiber membrane 100 alone can facilitate the attachment of stem cells to the surface of the nanofibers by surface-hydrophilizing the surface of the nanofibers by plasma treatment.

In the case where the nanofiber membrane for stem cell culture is opaque, it is difficult to visually confirm stem cell culture during stem cell culture, and there is a disadvantage in observing by microscopic observation or staining. However, in the present invention, In this case, since the nanofiber membrane 100 is translucent, it is possible to easily observe the cultured state of the stem cells with the naked eye. In this case, the nanofiber membrane 100 can be made translucent by PMMA, PC, PU, PCL, PLA or PLGA alone. .

The nanofiber membrane 100 according to the present invention may be a synthetic polymer or a natural polymer that can be electrospun as a usable polymer material. The synthetic polymer or natural polymer may be used singly or in combination, There is no particular limitation on a polymer substance capable of forming nanofibers by electrospinning.

Example 1

Polyvinylidene fluoride (PVdF), a fiber-forming polymer, was mixed and dissolved in a mixed solvent of DMAc (dimethylacetamide) / acetone (mixing ratio is 7/3 in wt.%) At 15 wt. A spinning solution was prepared. The spinning solution thus prepared was transferred to a spinning nozzle pack and spin-coated at a spinning temperature of 30 ° C and a relative humidity of 60% under the conditions of an applied voltage of 25 kV, a distance of 20 cm between the spinneret and the collector, a discharge rate per hole of 0.05 cc / Electrospinning was performed. The PVdF nanofiber membrane obtained under the above conditions was subjected to thermocompression bonding using a roller heated at 140 占 폚.

5 (a) and 5 (b) show SEM images of the surface and cross section of the PVdF nanofiber membrane obtained under the above conditions, and FIG. 6 shows the PMI (Capillary Flow Porometer) showing the average pore distribution. As shown in the photograph, the fiber diameter was mostly less than 1 탆, and the average diameter was 400 nm. The thickness of the PVdF nanofiber obtained under the above conditions was about 30 탆, and the average pore size was about 0.3 탆.

Example 2

Polyacrylonitrile (PAN) as a fiber-forming polymer was mixed and dissolved in DMAc (dimethylacetamide) to a concentration of 12 wt.% To prepare a spinning solution. The spinning solution thus prepared was electrospun and thermocompression-bonded 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 mu m, and a membrane thickness of 20 mu m. Fig. 7 shows a scanning electron micrograph of the surface and cross section of PAN nanofibers.

Example 3

The surface modification of the PVdF nanofiber membrane prepared in Example 1 was performed using a plasma cleaner. At this time, surface reforming was performed at 400 W for 60 seconds while supplying argon (Ar) at 100 sccm.

8 (a) and 8 (b) show the surface structure of the plasma-treated PVdF nanofiber membrane and the results of contact angle with water. The contact angle of water decreased with plasma treatment. The contact angle of PVdF nanofiber was modified by hydrophilization from 110 75 to 75 플라 by plasma treatment.

Example 4

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

Example 5

As feeder cells, mitomycin C (Sigma, Cat. No. M-4287) was inoculated at a concentration of 10 ug / ml for 1 hour 30 min to use mouse embryo fibroblasts that were firstly extracted and cultured from mice (CF1, C57BL6) Min, and inoculated at a density of 4 × 10 ⁴ / cm 2, and cultured the next day by inoculating a piece of embryonic stem cell (HSF6). For the medium composition, 3.069 g / l sodium bicarbonate (Sigma, USA, Cat. No. S5761), 2 mM L-glutamine (Sigma, Cat. No.5761) were added to MEM / F12 medium (GIBCO, USA, Cat. No. 12500-062). 20% Knock-Out serum replacement (Sigma, Cat. No. 46687) / streptomysin (50 ug / ml) (Sigma, USA, Cat No. S1277), 1% penicillin ; Invitrogen BRL, Cat. No. 10828-028) and 4 ng / ml Basic Fibroblasts Growth Factor (bFGF; Invitrogen BRL, Cat. No. 13256-029). And cultured at 37 ° C in a 5% CO2 incubator. The medium was changed daily and used as a control.

Example 6

To use the nanofiber hybrid membrane as a membrane for stem cell culture, the nanofiber hybrid membrane prepared by the above Examples 1 and 4 was carried out using a commercially available 6-well culture dish.

The mouse embryonic fibroblast, a feeder cell, was treated with mitomycin-C (growth inhibitor) for 2 hours to inhibit proliferation and was inoculated at a density of ⁴ 10 ⁴ / cm 2. After 24 hours, the nanofiber hybrid membrane (Adipose stem cell, Bone marrow stem cell) was inoculated on the membrane. After culturing the stem cells in the same manner as in Example 5, Respectively. The colonies were stained with alkaline phosphatase staining, and colonies with undifferentiated staining were counted with differentiation colonies. Alkaline phosphatase staining was performed with NBT / BCIP (Roche, Germany, Cat. No 1 681 451) solution was mixed and reacted at a ratio of 99: 1 to confirm color development, and a microscopic observation of 400 times was carried out. FIG. 10 shows the values obtained by counting the fat and bone-derived stem cells.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is clearly understood that the same is by way of illustration and example only and is not to be construed as limited to the embodiments set forth herein. Various changes and modifications may be made by those skilled in the art.

100, 100a, 100b: nanofiber membranes 101, 112: micropores
110: stem cell 111: nanofiber
120: supporting cells 200, 210, 220: nonwoven fabric or woven fabric

Claims (4)

And a nanofiber membrane formed by laminating nanofibers having a fiber diameter of less than 1 mu m,
Wherein the nanofiber membrane comprises one surface on which the stem cells are located; And a second surface on which the support cells are located,
Wherein the nanofiber membrane is provided with a plurality of three-dimensional micropores for supplying nutrients necessary for the stem cells to grow from the supporting cells to the stem cells. The nanofiber membrane for culturing stem cells according to claim 1, wherein the nanofiber membrane has a pore size of 0.1 to 0.5 탆 and a thickness of 10 to 50 탆. The hybrid nanofiber membrane for culturing stem cells according to claim 1, wherein the nanofiber membrane is laminated to a nonwoven fabric or a woven fabric or interposed between a pair of nonwoven fabrics or a nonwoven fabric or a woven fabric. A nanofiber hybrid membrane composed of a nanofiber membrane having a plurality of three-dimensional micropores formed by lamination of nanofibers having a fiber diameter of less than 1 탆 and a nonwoven fabric or a woven fabric having a plurality of pores hybridized with each other is sandwiched between stem cells and supporting cells ; And
And culturing the stem cells by feeding nutrients from the supporting cells to the stem cells through a plurality of three-dimensional micropores of the nanofiber hybrid membrane. Lt; / RTI >

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