KR101766385B1 - Cell culture container and method for fabricating the same - Google Patents

Abstract

A cell culture container according to an embodiment of the present invention includes a cell culture surface for adhering cells to proliferate and differentiate cells, wherein the cell culture container comprises at least one or more cells having a mesenchymal cell culture surface A cell culture section including a well, and a cover for protecting the cell culture section.

Description

TECHNICAL FIELD [0001] The present invention relates to a cell culture container,

The present invention relates to a container for cell culture and a method for producing the same.

Cell culture was performed on polystyrene plates, well plates and glass petri dishes, and plastic plates of various shapes were produced by injection or extrusion.

However, the cell culture container used in the field of biotechnology of the existing tissue engineering department is disadvantageous in that it differs from the in vivo structure. That is, the two-dimensional plane of the conventional cell culture container is different from the three-dimensional shape in the living body.

Therefore, in recent years, researches are being conducted to develop a cell culture container for providing a similar environment to a living body in cell culture. Among them, cell culture containers combined with nanofibers are being developed.

Cell culture vessels containing nanofibers are cultured on a two-dimensional plane because the nanofibers are integrated on a flat surface.

However, since most of the plants and animals in the body as well as the organs in the body are made of the three-dimensional structure, there is a problem in that the cell culture efficiency is deteriorated in the cell culture container having the two-dimensional planar structure.

SUMMARY OF THE INVENTION Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a cell culture container capable of increasing the culture efficiency of a cell by providing a three-dimensional structure similar to a structure in a living body, and a manufacturing method thereof.

In order to accomplish the above object, a cell culture container according to an embodiment of the present invention includes a cell culture surface for adhering cells to proliferate and differentiate cells, A cell culture section including at least one well having a culture surface, and a cover for protecting the cell culture section.

The mesenchymal cell culture surface may be composed of polymer fibers, and the polymer fibers may be entangled.

The diameter of the polymer fibers may be from several tens nanometers to several hundreds nanometers.

The polymer fibers may be selected from the group consisting of polycaprolactone, polyurethane, polyvinylidene fluoride (PVDF), polystyrene and biopolymers such as collagen, gelatin, chitosan ). ≪ / RTI >

The cover may be located on the other side of the mesenchymal cell culture surface, and the cover may be located apart from the well.

The culture surface may be curved.

The cover may have a recess shaped to engage the well.

The diameter of the well may be 100 [mu] m to 25 mm.

According to another aspect of the present invention, there is provided a method of manufacturing a cell culture container, comprising: preparing a container storing an electrolyte; disposing a plate having at least one opening in the container to form a droplet; Forming a cell culture section having a well having a cell culture surface by spinning the polymer fiber on the spinning liquid droplet; disposing a cover for accommodating the cell culture section in the cell culture section opposite to the droplet; separating the cell culture section from the droplet .

In the step of disposing the cover, the cover may be disposed apart from the well.

In the step of forming the cell culture section, the electrospinning may be applied at 5 kV to 30 kV.

By using the cell culture container according to one embodiment of the present invention, it is possible to provide a cell culture container having a cell culture surface having a three-dimensional structure similar to a structure in vivo.

Further, it is possible to provide a cell culture container capable of increasing cell orientation efficiency by providing a cell culture surface of a meshed structure.

In addition, it is possible to easily manufacture a cell culture container by using electrospinning, thereby reducing the time and cost for manufacturing a cell culture container.

1 is a schematic view showing a cell culture container according to an embodiment of the present invention.
2 is a cross-sectional view taken along the line II-II in FIG.
FIGS. 3 and 4 are cross-sectional views of a cell culture container according to another embodiment of the present invention, taken along the line II-II of FIG. 1. FIG.
FIG. 5 is a perspective view of an intermediate stage of forming a cell culture unit according to an embodiment of the present invention.
Fig. 6 is a perspective view in the next step of Fig. 5; Fig.
7 is a cross-sectional view cut along the line VII-VII in FIG.
8 is a cross-sectional view at the next step of Fig.
9 is a photograph of a cell culture section formed according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

In the drawings, the thickness is enlarged to clearly represent the layers and regions. Like parts are designated with like reference numerals throughout the specification. Whenever a portion of a layer, film, region, plate, or the like is referred to as being "on" another portion, it includes not only the case where it is "directly on" another portion, but also the case where there is another portion in between. Conversely, when a part is "directly over" another part, it means that there is no other part in the middle.

In order to clearly illustrate the present invention, parts not related to the description are omitted, and the same or similar components are denoted by the same reference numerals throughout the specification.

FIG. 1 is a schematic view showing a cell culture container according to an embodiment of the present invention, FIG. 2 is a cross-sectional view cut along a line II-II in FIG. 1, Sectional view taken along the line II-II of Fig. 1, and is a cross-sectional view of the cell culture container according to the present invention.

1 and 2, a cell culture container 1000 according to an embodiment of the present invention includes a cell culture unit 200 having at least one well 22 and a connection unit 24 connecting the wells, , And a cover (300) for supporting and protecting the cell culture unit (200).

The diameter of the wells 22 may be between 100 μm and 25 mm, respectively, and may be arranged to form a matrix. Also, the plurality of well arrangements is not limited to this, and may be arranged to have various intervals. The bottom surface of the well 22 may be concave to have a curved shape and may be semicircular with the side walls.

Further, the well 22 may have a flat bottom surface, as shown in FIG. 3, and only corner portions and corners where the side wall meets the bottom surface may have a curved shape.

The cross-sectional shape of the well 22 is not limited to this, and may be formed to have various shapes as required.

The cell culture unit 200 may be made of a polymer fiber. The polymer fiber may include at least one of a thermoplastic resin, a thermosetting resin, an elastomer and a biopolymer. Examples of the polymer fiber include polycaprolactone, polyurethane, polyvinylidene fluoride (PVDF) , Polystyrene and biopolymers such as collagen, gelatin, chitosan, and the like.

The well 22 may be a cell culture surface on which the cells are cultured. The cell culture surface is intended to artificially enhance the proliferation and differentiation efficiency of cells, and the cells to be cultured are adhered onto the cell culture surface to induce differentiation in a desired direction. The mesenchymal stem cells include bone marrow-derived stem cells, placenta-derived stem cells, and adipose-derived stem cells. The cell culture container according to this embodiment can improve the adhesion, proliferation and differentiation efficiency of such cells.

That is, in one embodiment of the present invention, by forming wells and providing a three-dimensional structure, the cells can be cultured and propagated in a structure similar to an in-vivo environment.

In addition, the cell culture container in one embodiment of the present invention is interwoven with the polymer fibers having a few micro or hundreds to several tens of nanometers in diameter to provide a meshed cell culture surface. Therefore, it is expected that the adhesion area of the cells is increased to improve the cell attachment efficiency, thereby increasing the proliferation and differentiation efficiency of the cells.

Referring again to FIG. 1, the cover 300 is for supporting and protecting the cell culture unit 200, and is disposed on the other side of the cell culture surface of the cell culture unit 200.

The cover 300 has a receiving space 33 in which the cell culture unit 200 is accommodated. The accommodation space 33 may be formed as one space so that all the wells 22 included in the cell culture unit 200 can be accommodated.

4, the cover 300 may have a separate accommodating space 35 corresponding to each well 22 included in the cell culture unit 200. As shown in Fig.

The individual receiving space 35 may have a concave shape along the shape of the well 22. That is, when the bottom surface of the well 22 is curved, the individual accommodation space 35 may have a curved surface that engages the bottom surface of the well 22.

Meanwhile, the cover 300 is installed apart from the well of the cell culture unit 200. When the cover 300 and the cell culture unit 200 are spaced apart from each other, a fluid can flow through the space formed between the cover 300 and the cell culture unit 200.

Therefore, when a fluid having nutrients to be supplied to the cells is flowed into the fluid passage, nutrients can be supplied to the cells attached to the cell culture surface through the cell culture surface of the well.

Hereinafter, a method of forming the cell culture portion of the cell culture container shown in Fig. 2 will be described in detail with reference to the drawings.

FIG. 5 is a perspective view of an intermediate stage of forming a cell culture unit according to an embodiment of the present invention, FIG. 6 is a perspective view at the next step in FIG. 5, and FIG. 7 is a cross- 8 is a cross-sectional view at the next step of Fig. 7. Fig.

First, as shown in Fig. 5, a container 500 filled with an electrolyte is prepared.

The electrolyte preferably has an electric conductivity of 1 mS / cm 2 or more and a relative permittivity of 80 or more. For example, potassium chloride may be used in distilled water in an amount of 0.01 mol% or more, preferably 3 mol%.

Thereafter, the plate 600 having the plurality of openings 66 is aligned so as to be inserted into the container 500.

Next, as shown in Figs. 6 and 7, the plate 600 having a plurality of openings 66 is arranged to be in contact with the electrolyte in the container. At this time, it is preferable that the upper surface of the plate 600 is arranged so as not to be immersed in the electrolyte.

As such, when the plate 600 is placed in contact with the electrolyte, the electrolyte is pressed by the plate and the pressurized electrolyte is pushed out through the opening of the plate. At this time, the electrolyte droplet 45 is formed in the opening portion 66 by the surface tension of the electrolyte.

The shape and size of the droplet 45 may vary depending on the shape and size of the plane of the opening 66. The planar shape of the opening 66 may be formed in various shapes such as a circular shape or a polygonal shape.

In addition, the size of the droplet 45 may vary depending on the amount of electrolyte and the pressure applied by the plate 600. That is, when the plate 600 presses the electrolyte with the same pressure, when the amount of the electrolyte is large, the amount of the electrolyte pushed out through the opening 66 is larger than when the amount of the electrolyte is relatively small. The size can be larger.

Thus, by controlling the shape and size of the opening 66 and the amount of the electrolyte, a droplet 45 having a desired size and shape can be obtained.

8, after the electric radiator 800 is prepared, a voltage is applied between the electrospinning device 800 and the electrolyte, and the polymer fibers are radiated onto the droplet 45 to form the cell culture unit 200 .

Electrospinning allows the polymeric material to be stored in the solution tank to have a suitable viscosity for electrospinning, and then to discharge the polymeric material through the spinning nozzle of the spinning unit. The polymer material is hardened after being discharged and then scattered to form a polymer fiber. The polymer fibers are laminated on the droplets, and the fibers are entangled to form a meshed structure.

The polymer material may be a polymer solution having a concentration of 5% to 25% by mixing polycaprolactone in a solvent in which chloroform and methanol are mixed in a mass ratio of 1: 1.

Alternatively, acetone and dimethylformamide may be mixed in a volume ratio of 3: 7 and then mixed with 25 wt% of polyvinylidene fluoride to form a polymer solution having a concentration of 25% to 30%.

In addition, polymer solutions can be prepared using polystyrene, polycarbonate, collagen / polycarbonate.

In this case, it is preferable that the release speed of the polymer fiber through the nozzle is controlled to be in the range of 0.01 ml / h to 3 ml / h so that the polymer fiber can be placed on the droplet while maintaining the shape of the droplet.

An electric field is applied between the spinning nozzle and the electrolyte. If the intensity of the applied electric field is too low, it is difficult to produce uniformly thick polymer fibers because the electric discharge composition is not continuously discharged. It is difficult to form a meshed structure because it can not be smoothly focused on the droplet. On the other hand, when the electric field intensity is too high, it is difficult to form a meshed structure having a normal shape because the polymer fibers are not accurately adhered to the droplet. Therefore, it is preferable that the intensity of the electric field applied between the spinneret and the electrolyte is 5 kV to 30 kV.

When formed with the above-described discharge speed and electric field, the diameter of the polymer fiber may be several tens to several hundred nanometers.

As described above, when the polymer material is spun using an electrospinning device, the polymer fibers in the form of fibers are stacked on the droplets along the shape of the droplets, and the polymer fibers are irregularly and continuously intertwined.

The polymer fibers stacked on the droplet 45 form the wells 22 of the cell culture section 200 and the polymer fibers stacked on the plate 600 between the droplets form the connection section 24 of the cell culture section.

Since the polymer fibers are stacked on the droplets to form the wells 22, the cell culture unit 200 having the wells 22 of a desired shape and size can be formed by adjusting the shape and size of the droplets.

Next, as shown in FIG. 2, a cover 300 is attached to support and protect the cell culture unit 200 to complete a cell culture container.

As described above, in the present invention, a cell culture container including a well having a cell culture surface of a net-like structure is formed using electrospinning, and a cell culture container having a three-dimensional cell culture surface can be easily manufactured by attaching a cover. Therefore, it is possible to reduce the time and cost for fabricating the cell culture container.

9 is a photograph of a cell culture section formed according to an embodiment of the present invention.

As shown in Fig. 9, it can be confirmed that the cell culture surface of the cell culture section is entangled with the polymer fibers to form the mesenchymal culture surface.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (13)

delete delete delete delete delete delete delete delete delete delete Preparing a container in which an electrolyte is stored,
Disposing a plate having at least one or more openings in the container to form droplets protruding from the plate through the openings;
Forming a cell culture section having a well curved along the outline of the droplet projected onto the plate by spinning the polymer fiber on the droplet by electrospinning
≪ / RTI > delete 12. The method of claim 11,
In the step of forming the cell culture section,
Wherein said electrospinning is applied at a frequency of 5 kV to 30 kV.

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