KR102460414B1 - Light blocking porous nanofiber membrane and apparatus for cell culture comprising the same - Google Patents

Abstract

An object of the present invention is to provide a cell culture apparatus comprising a porous nanofiber membrane having an excellent light-shielding effect, a method for manufacturing a porous nanofiber membrane having an excellent light-shielding effect, and a porous nanofiber membrane having an excellent light-shielding effect. According to the present invention, a porous nanofiber membrane having an excellent light-shielding effect is provided, and it is possible to provide a cell culture device suitable for cell invasion analysis using the porous nanofiber membrane of the present invention. Therefore, it is expected that the porous nanofiber membrane of the present invention and a cell culture device including the same can be widely applied in related fields such as pathological observation using a cell invasion assay in the future.

Description

A porous nanofiber membrane exhibiting a light-shielding effect and a cell culture device comprising the same {LIGHT BLOCKING POROUS NANOFIBER MEMBRANE AND APPARATUS FOR CELL CULTURE COMPRISING THE SAME}

The present invention relates to a porous nanofiber membrane, a method for producing the same, and a cell culture apparatus including the same, and more particularly, to a porous nanofiber membrane having excellent light-shielding effect, a method for producing the same, and a cell culture apparatus including the same.

The porous nanofiber membrane is a membrane formed by entangled or arranging a plurality of nanofibers in one direction, and may be typically manufactured through an electrospinning process. In this case, in the electrospinning process, a viscous polymer solution may be used as a spinning solution to be spun in the form of nanofibers having a nano diameter by using an electrical repulsive force. In particular, since the electrospinning process can use a polymer material having various properties such as durability, chemical resistance, biocompatibility, and biodegradability, it is possible to manufacture a porous nanofiber membrane having various properties according to process parameters.

The porous nanofiber membrane produced through such electrospinning not only has a large surface area to volume ratio, but also has a structural similarity to the extracellular matrix in vivo, so it can be widely used in the medical field, biotechnology field, etc. have.

On the other hand, cell invasion assay is an assay that evaluates the movement of cells through membranes by biological or environmental signals. can At this time, a cell culture device such as a cell culture insert is used, and the cell culture insert is a cell culture construct inserted into a plate having an insert, and is carried out by movement, diffusion, uptake, metabolization and It is widely used in secretion investigations, etc. Korean Patent No. 10-1367855 and Korean Patent No. 10-2068465 disclose a cell culture apparatus including a membrane in which a plurality of through-holes are uniformly formed and a membrane having an appropriate permeability.

Conventional cell invasion assays culture cells tagged with immunofluorescent proteins, give biological signals, and then react to the cells to penetrate and migrate through the membrane through an inverted fluorescence microscope. Analyze.

On the other hand, when a membrane having permeability is used, there is a problem in that fluorescence exhibited by non-invasive cells is photographed together. For nanofibers having a light-shielding effect, a method of forming carbon black on the surface of a general polymer-based nanofiber through a carbonization process is widely used. However, there is a problem that the process time is long and the cost is high because a process for stabilizing the furnace for a long time is required in the carbonization process. In addition, in the case of carbonized nanofibers, there is cytotoxicity, and there is a inconvenience of requiring additional treatment such as hydrogel coating during cell culture.

Accordingly, if a porous nanofiber membrane is developed that solves the problems of the conventional membrane, has light blocking power, and is easy to use for cell invasion assays, it is expected to be widely applied in related fields.

The present invention is to provide a porous nanofiber membrane having an excellent light-shielding effect.

Another aspect of the present invention is to provide a method for manufacturing a porous nanofiber membrane having an excellent light-shielding effect.

Another aspect of the present invention is to provide a cell culture apparatus comprising a porous nanofiber membrane having an excellent light-shielding effect.

According to one aspect of the present invention, there is provided a porous nanofiber membrane, which exhibits a light-shielding effect, produced by forming a network of nanofibers made of a polymer including a light-shielding material.

According to another aspect of the present invention, a cell culture apparatus is provided, including the porous nanofiber membrane and endothelium.

According to another aspect of the present invention, preparing a polymer solution by dissolving a light-shielding material and a polymer in a solvent; and using the polymer solution as a spinning solution to form a porous nanofiber membrane through electrospinning.

According to the present invention, a porous nanofiber membrane having an excellent light-shielding effect is provided, and it is possible to provide a cell culture device suitable for cell invasion analysis using the porous nanofiber membrane of the present invention. Therefore, it is expected that the porous nanofiber membrane of the present invention and a cell culture device including the same can be widely applied in related fields such as pathological observation using a cell invasion assay in the future.

1 shows the observation (about 50 times) of the structure of the nanofiber prepared by the method for manufacturing a porous nanofiber membrane of the present invention with an optical microscope, and FIG. 1 (a) contains 2.5% by weight of carbon black 1 (b) is prepared by electrospinning a polymer solution containing 5% by weight of carbon black, FIG. 1 (c) is a polymer containing 7.5% by weight of carbon black It is a photograph of the nanofiber structure prepared by electrospinning the solution.
2 is a graph showing the measurement of the light-shielding effect (Transmittance) having a wavelength of 300 to 700 nm according to the electrospinning time of the porous nanofiber membrane prepared according to Example 1. From this, it can be confirmed that it has a light blocking effect of 90% or more.
3 is a graph showing the measurement of the average pore size (pore size) according to the electrospinning time of the porous nanofiber membrane prepared according to Example 1. It can be seen that all of them have a pore size of 3 to 6 μm.
Figure 4 shows the number of cells that invaded after 8, 16, and 24 hours according to the cell invasion assay according to Experimental Example 3 after culturing human umbilical vein endothelial cells (HUVECs) for 24 hours. .
5 is a photograph of the fluorescence of cells that invaded according to the cell invasion assay of Experimental Example 3 under the membrane using an inverted fluorescence microscope. It can be confirmed that no fluorescence is observed when the cells do not migrate (0h).
6 shows an electron micrograph of the porous nanofiber membrane according to Example 1.
7 shows a cell culture insert comprising a porous nanofiber membrane.

Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. However, the embodiments of the present invention may be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below.

According to the present invention, there is provided a porous nanofiber membrane having an excellent light-shielding effect.

More specifically, the porous nanofiber membrane of the present invention is manufactured by forming a network of nanofibers made of a polymer including a light blocking material.

The light-shielding material of the present invention may be at least one selected from the group consisting of carbon black, iron oxide dye, squid ink dye, ink, charcoal and charcoal, and may preferably be carbon black, but is not limited thereto, and has a light-shielding effect It can be used without limitation if it corresponds to a pigment that can have

The nanofibers of the present invention may be prepared from a polymer solution including the light-shielding material and the polymer, and in this case, when the porous nanofiber membrane of the present invention is for cell culture, the polymer is, for example, polycaprolactone (PCL), polyurethane (PU), polyvinyl alcohol (PVA), gelatin, silk fibroin, polylactic acid (PLLA), polylactate-co-glycolate (PLGA), polyglycolic acid (PGA), polyethylene oxide (PEO), polyethylene glycol ( PEG), polycarbonate (PC), and polyacrylonitrile (PAN) may be at least one selected from the group consisting of, preferably nanofibers prepared with a polymer solution containing polycaprolactone (PCL). , In this case, the polymer is not limited thereto, and may be changed according to its purpose and use.

In the nanofiber of the present invention, since the light-shielding material is homogenized in the nanofiber, it is possible to obtain the effect of significantly lower cytotoxicity compared to the case where the surface is coated with the light-shielding material. On the other hand, when the process of coating the outer surface of the nanofiber with a light-shielding material, there are also disadvantages that the process time is long and the cost is high.

The light-shielding material is preferably contained in an amount of 1 to 10% by weight, more preferably in an amount of 2.5 to 7.5% by weight, and when it is less than 1% by weight, there is a problem in that sufficient light-shielding power cannot be obtained, and when it exceeds 10% by weight There is a problem in that the light-shielding material is agglomerated and the homogenized electrospinning process is not performed. As the content of the light-shielding material increases within the content range, the viscosity of the polymer solution may increase, and thus the diameter of the nanofiber to be manufactured may increase.

The nanofibers of the porous nanofiber membrane of the present invention are for mimicking the structure of collagen fibrils constituting the extracellular matrix, and may have an average diameter of 300 to 3000 nm, as long as they can mimic the structure of collagen fibrils, However, the present invention is not limited thereto.

The average pore size of the membrane is preferably 1 to 10 μm, more preferably 3 to 6 μm, and when it is less than 1 μm, there is a problem that cells cannot penetrate the membrane, and when it exceeds 10 μm, the size of the pores becomes large. Therefore, when culturing endothelial cells, the penetration rate of endothelial cells is increased, so it is difficult to observe the phenomenon at sufficient time intervals.

The average thickness of the membrane is preferably 10 to 500 μm, more preferably 30 to 150 μm, and when it is less than 10 μm, there is a problem that it can be easily damaged during cell culture due to insufficient mechanical rigidity of the membrane, and when it exceeds 500 μm There is a problem that the infiltration phenomenon cannot be observed at all because the cell penetration phenomenon is excessively inhibited.

The membrane of the present invention has a light blocking power of 90% or more at a wavelength of 300 to 700 nm, and can be used to measure the fluorescence exhibited by cells. It has intrinsic excitation wavelength and emission wavelength. For example, DiI dye, which is a fluorescent molecule, is excited at a wavelength of 488 nm and emits fluorescence at a wavelength of 565 nm.

The method for manufacturing the porous nanofiber membrane of the present invention is not particularly limited, but may be formed by electrospinning.

More specifically, the method for producing a porous nanofiber membrane of the present invention comprises the steps of dissolving a light-shielding material and a polymer in a solvent to prepare a polymer solution; and forming a porous nanofiber membrane through electrospinning using the polymer solution as a spinning solution.

The electrospinning is preferably performed by applying a voltage of 1 to 25 kV, and more preferably, is performed by applying a voltage of 15 to 20 kV. When the voltage is less than 1 kV, the nanofiber diameter increases to a micro level. And, when it exceeds 25 kV, there is a problem in that DC current flows due to dielectric breakdown.

In addition, in order to perform a stable electrospinning process, the flow rate of the polymer solution was adjusted to 0.5 to 2.0 ml/hr, a temperature of 0 to 35° C., and a humidity of 40 to 60%. In this case, humidity means relative humidity.

A suitable solvent for the preparation of the porous nanofiber membrane is at least one selected from the group consisting of chloroform, methanol, ethanol, camphorsulfonic acid, formic acid, dimethylformamide, distilled water and hexafluoroisopropanol or a mixed solvent thereof, wherein chloroform and alcohol are A mixed solvent having a volume ratio of 2:1 to 4:1 is preferable, and more preferably, chloroform and methanol have a volume ratio of 3:1. In the case of using such a mixed solvent of chloroform and alcohol, there is an advantage in electrospinning because the solubility is high and the dielectric constant is large.

The porous nanofiber membrane obtained by the present invention may be used for cell culture, and since the porous nanofiber membrane of the present invention has an excellent light-shielding effect, the porous nanofiber membrane of the present invention is used in a cell culture device suitable for cell invasion analysis. provision becomes possible. The cell culture apparatus including the porous nanofiber membrane of the present invention as described above includes, for example, a cell culture insert, a microfluidic chip, and the like, but is not limited thereto.

When the porous nanofiber membrane of the present invention is applied to a cell culture-related use, in this case, the cells are human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBECs). -5i), but is not limited thereto, and cells having a cell size equal to or greater than the pore size of the porous nanofiber membrane of the present invention are preferred.

According to the present invention, a cell culture apparatus comprising the porous nanofiber membrane and endothelium of the present invention may be provided, wherein the endothelial cells are human umbilical vein endothelial cells (HUVECs) Or it may be a human brain microvascular endothelial cell (HBEC-5i).

The cell culture device may be manufactured by applying a voltage between the electrospinning device and the cell culture device, for example, an electrolyte solution applied on a cell culture insert or a microfluidic chip, and stacking the spun nanofibers on the cell culture device. In this case, the spinning conditions are as described above in relation to the porous nanofiber membrane.

Therefore, it is expected that the porous nanofiber membrane of the present invention and a cell culture device including the same can be widely applied in related fields such as pathological observation using a cell invasion assay in the future.

Hereinafter, the present invention will be described in more detail through specific examples. The following examples are only examples to help the understanding of the present invention, and the scope of the present invention is not limited thereto.

Example

1. Preparation of Porous Nanofiber Membrane

Example 1

0.28 g of carbon black was dissolved in 10 g of a solvent in which chloroform and methanol were mixed in a volume ratio of 3:1 to homogenize. Thereafter, 0.77 g of polycaprolactone was dissolved to prepare a polymer solution. Using the polymer solution as a spinning solution, nanofibers prepared through electrospinning were laminated on a cell culture device to prepare a porous nanofiber membrane.

Electrospinning system (HV power supply; NanoNC HV30, Syringe pump; NanoNC EP100) was used for the electrospinning machine. The release rate of the spinning solution during spinning was adjusted to 0.7 ml/h. The voltage was adjusted to 18 kV. Since it is important to maintain an appropriate temperature and humidity so that the solvent is completely volatilized before the fibers are accumulated on the cell culture device, the temperature was set to about 25° C. and the humidity was set to about 50%.

It was confirmed that the nanofibers obtained here had an average diameter of 773.30 nm. Figure 1 (a) shows an optical micrograph of the fiber produced by the spinning conditions as described above.

Example 2

A polymer solution was prepared by dissolving 0.57 g of carbon black in 10 g of a solvent in which chloroform and methanol were mixed in a volume ratio of 3:1, followed by dissolving 0.80 g of polycaprolactone, and using the polymer solution as a spinning solution. A porous nanofiber membrane was prepared by stacking the nanofibers prepared through electrospinning under the same conditions as in Example 1, except that.

It was confirmed that the nanofibers obtained here had an average diameter of 1867.71 nm. Figure 1 (b) shows an optical micrograph of the fiber produced by the spinning conditions as described above.

Example 3

A polymer solution was prepared by dissolving 0.88 g of carbon black in 10 g of a solvent in which chloroform and methanol were mixed in a volume ratio of 3:1, followed by dissolving 0.82 g of polycaprolactone, and the polymer solution was used as a spinning solution. A porous nanofiber membrane was prepared by stacking the nanofibers prepared through electrospinning under the same conditions as in Example 1, except that.

It was confirmed that the nanofibers obtained here had an average diameter of 2163.91 nm. Figure 1 (c) shows an optical micrograph of the fiber produced by the spinning conditions as described above.

2. Measurement of light blocking rate and pore size according to electrospinning time

Experimental Example 1

In order to confirm the light blocking rate according to the electrospinning time of the porous nanofiber membrane prepared in Example 1, the light transmittance was measured at a wavelength of 300 to 700 nm after electrospinning for 30, 60, and 90 seconds. indicated. The light transmittance was measured through spectral scanning in a wavelength range of 300 to 700 nm using a commercial microplate reader.

As shown in Figure 2 below, when performed for 30, 60, and 90 seconds, each 98.9%. The light blocking rates of 98.9% and 98.4% were confirmed.

Experimental Example 2

In order to confirm the pore size according to the electrospinning time of the porous nanofiber membrane prepared in Example 1, electrospinning was performed for 30, 60, and 90 seconds, and then the pore size was measured and shown in FIG. 3 . After photographing the fabricated nanofiber structure at 1500 times magnification with an electron microscope, the nanofiber pore size was measured through image processing.

As shown in FIG. 3 below, it was confirmed that all of them had a pore size of 3 to 6 μm when performed for 30, 60, and 90 seconds.

3. Cell Culture and Cell Invasion Assay Experiments

Experimental Example 3

A porous nanofiber membrane having an average pore size of 4.70 μm and an average thickness of 42.87 μm was laminated on the cell culture insert while preparing the nanofibers under the conditions set in 1 above.

Human retinal pigment epithelial cells (ARPE-19) were seeded in the cell culture plate at 1.9x10 ⁵ /well and then cultured for 24 hours. In the insert, human umbilical vein endothelial cells (HUVECs) were seeded on a porous nanofiber membrane at 1x10 ⁵ /well and then cultured for 24 hours. After 24 hours, a plate with ARPE-19 was treated with 5 μM of A2E, irradiated with ultraviolet light for 30 seconds, and then combined with an insert containing human umbilical vein endothelial cells, and 8, 16, and 24 hours later, under an inverted fluorescence microscope. was used to measure the number of human umbilical vein endothelial cells that invaded under the insert. The measured number of cells is shown in FIG. 4 .

After tagging the cells using DiI dye, excitation with 488 nm fluorescence light and emitted fluorescence images were taken. The photographed fluorescence image is shown in FIG. 5 .

As shown in FIG. 4 below, it was confirmed that the invading cells increased as time passed. In addition, as shown in FIG. 5 , when no cell invasion was made (0h), fluorescence did not appear.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible within the scope without departing from the technical spirit of the present invention described in the claims. It will be apparent to those of ordinary skill in the art.

Claims (18)

Carbon black, iron oxide dye, squid ink dye, ink, charcoal, and nanofibers made of a polymer comprising a light-shielding material selected from the group consisting of charcoal are formed by forming a network, and have an average thickness of 10 to 500 μm. A porous nanofiber membrane for cell culture that exhibits a light-shielding effect.
delete According to claim 1,
The nanofiber is a porous nanofiber membrane for cell culture, which is produced through electrospinning of a polymer spinning solution containing a light-shielding material.
According to claim 1,
The light-shielding material is characterized in that contained in 2.5 to 7.5% by weight based on the total weight of the porous nanofiber membrane, porous nanofiber membrane for cell culture.
According to claim 1,
The polymer is polycaprolactone (PCL), polyurethane (PU), polyvinyl alcohol (PVA), gelatin, silk fibroin, polylactic acid (PLLA), polylactate-co-glycolate (PLGA), polyglycolic acid ( PGA), polyethylene oxide (PEO), polyethylene glycol (PEG), polycarbonate (PC) and at least one selected from the group consisting of polyacrylonitrile (PAN), a porous nanofiber membrane for cell culture.
According to claim 1,
The nanofiber has an average diameter of 300 to 3000nm, a porous nanofiber membrane for cell culture.
According to claim 1,
The membrane is a porous nanofiber membrane for cell culture, having a light blocking power of 90% or more at a wavelength of 300 to 700 nm.
According to claim 1,
The membrane is a porous nanofiber membrane for cell culture, having an average pore size of 3 to 6 μm.
delete A cell culture device comprising a porous nanofiber membrane and endothelium for cell culture according to any one of claims 1 to 8.
11. The method of claim 10,
The endothelial cells are human umbilical vein endothelial cells (HUVECs) or human brain microvascular endothelial cells (HBEC-5i), a cell culture device.
11. The method of claim 10,
The cell culture apparatus is a cell culture insert or a microfluidic chip, characterized in that the cell culture apparatus.
preparing a polymer solution by dissolving a light blocking material and a polymer in a solvent; and
forming a porous nanofiber membrane for cell culture with an average thickness of 10 to 500 μm through electrospinning for 15 to 120 seconds using the polymer solution as a spinning solution;
A method for producing a porous nanofiber membrane for cell culture, comprising, exhibiting a light-shielding effect.
14. The method of claim 13,
The light-shielding material is at least one selected from the group consisting of carbon black, iron oxide dye, squid ink dye, ink, charcoal and charcoal.
14. The method of claim 13,
The method for producing a porous nanofiber membrane for cell culture, wherein the flow rate of the polymer solution during the electrospinning is 0.5 to 2.0ml/hr, a temperature of 0 to 35°C, and a humidity of 40 to 60%.
14. The method of claim 13,
The electrospinning is performed by applying a voltage of 1 to 25 kV, a method for producing a porous nanofiber membrane for cell culture.
14. The method of claim 13,
The solvent is at least one selected from the group consisting of chloroform, methanol, ethanol, camphorsulfonic acid, formic acid, dimethylformamide, distilled water and hexafluoroisopropanol or a mixed solvent thereof, characterized in that the porous nanofiber membrane for cell culture manufacturing method.
14. The method of claim 13,
The solvent is a method for producing a porous nanofiber membrane for cell culture, characterized in that it is a mixed solvent of chloroform and alcohol in a volume ratio of 2:1 to 4:1.

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