KR20200025246A - Method of activating ion channel of cell, Structure for ion channel activation of cell and Manufacturing method thereof - Google Patents

Abstract

The present invention relates to a structure for ion channel activation of a cell and a method for activating an ion channel of a cell, and more particularly, includes a bottom surface and a columnar structure disposed on the bottom surface, wherein the columnar structure is a cell. Immediately after seeding of the cells, the cells are arranged at contact intervals of two to three pillar-shaped structures and have a height at which the cells do not touch the bottom surface, thereby enabling to activate the ion channels of the cells, It relates to a preparation method and a method for activating an ion channel of a cell.

Description

Method for activating ion channel of cell, Structure for ion channel activation of cell and Manufacturing method

The present invention relates to a method of activating an ion channel of a seeded cell, a structure capable of activating an ion channel of a cell, and a method of manufacturing the same.

Cancer is a complex disease with genetics that has many possible causes ranging from carcinogens, diet and physical activity, UV exposure and possible infections. It is expected to provide a better understanding of cancer dynamics. Recently, there have been active pursuits of cellular responses to possible carcinogens. It is known that not only chemical interferences (carcinogens) but also the physical environment can determine cell fate. In this regard, technology that intentionally regulates cell traits is a key technology widely used in drug development, tissue engineering, and regenerative medicine. In drug screening, which develops a drug that intentionally makes cancer cells more aggressive and prevents metastasis, the development of specific tissues / organs through transformation and mass production of cells for therapeutic purposes are among the most studied and used technologies. do. In order to transform cells, cells are usually transformed by using organic small molecule drugs, but recently, by transforming cells by applying physical stimuli (pressure, temperature, environment, etc.) or controlling an extracellular matrix (ECM). A lot of research is being conducted.

Transformation using physical stimulation has a relatively limited effect on cells than stimulation by organic small molecule drugs, which provides an advantage of relatively reducing the side effects of drug-type stimulation. Recently, research on inducing transformation by incorporating cells into various nano / micro structures has been actively conducted with the development of nano / micro patterning technology.

Most of these studies, however, are lab-level and report only a few of the characteristic changes caused by nano / microstructure stimuli. In particular, the growth of various cells has been reported in the micro / nano pillar-shaped structures, and the correlation between the geometry / size of these structures and the transformation of the cells is only revealed at some level.

Accordingly, it is necessary to develop a structure that can induce the transformation of cells, in particular the activation of the ion channel in the desired form, and profile the physiological changes accordingly accordingly.

Korean Patent Publication No. 2014-0050514

It is an object of the present invention to provide a structure for inducing activation of ion channels of seeded cells.

It is an object of the present invention to provide a method for activating ion channels of seeded cells.

It is an object of the present invention to provide a method for preparing a construct for inducing activation of ion channels of seeded cells.

1. A bottom surface and a pillar-shaped structure disposed on the bottom surface, wherein the pillar-shaped structure, the spacing of the contact between the two or three of the pillar-shaped structure immediately after the seeding (seeding) of the cell Arranged to have a height that the cells do not reach the bottom surface, the ion channel activation structure of the cell.

2. The structure of 1 above, wherein the columnar structure has an interval in which the cell is in contact with two to three columnar structures until 10 hours or less immediately after seeding, the ion channel activation structure of the cell.

3. In the above 1, wherein the height of the columnar structure is greater than the longest height of the vertical cross section of the seeded cells, the structure for ion channel activation of the cell.

4. In the above 1, wherein the columnar structure has an aspect ratio of 5 or more, the structure for ion channel activation of the cell.

5. The method of 1 above, wherein the cell is at least one selected from cardiomyocytes or A549 cells, the ion channel activation structure of the cell.

6. preparing the cells; A pillar-shaped structure disposed on the bottom surface and on the bottom surface, wherein the pillar-shaped structure is arranged at intervals at which cells contact two to three pillar-shaped structures immediately after seeding of the cells; And seeding the cells in a structure for ion channel activation of cells, the cells having a height that does not touch the bottom surface.

7. The method of 6 above, wherein the cell is at least one selected from cardiomyocytes or A549 cells, ion channel activation method of cells.

8. The method according to the above 7, wherein the A549 cells have a period of increasing mobility after seeding in the ion channel activation structure of the cell, the ion channel activation method of the cell.

9. The method according to the above 7, wherein the cardiomyocytes are seeded in the structure for ion channel activation of the cell, the heart rate is increased, the ion channel activation method of the cell.

10. A pillar-shaped structure disposed on the bottom surface and the bottom surface, and immediately after seeding of the cells, the cells are arranged at contact intervals of two to three pillar-shaped structures, wherein the bottom surface A first step of preparing an intermediate mold including an elastic polymer in which a pillar-shaped structure having a height of which cells do not touch is engraved in an intaglio; A second step of imprinting the intermediate mold on a photocurable resin and irradiating light for 30 seconds to 5 minutes; And a third step of removing the intermediate template from the photocured resin.

11. The method of 10 above, wherein the first step includes a pillar-shaped structure disposed on the bottom surface and the bottom surface before, and the cells form two to three pillars immediately after seeding of the cells. Preparing a silicon master template by forming a pillar-shaped structure having a height that does not touch cells on the bottom surface and arranged at a contact interval of the structure of the silicon wafer to prepare a silicon master template; Manufacturing method.

12. The method according to the above 10, wherein the cell is at least one selected from cardiomyocytes or A549 cells, the method of manufacturing a structure for ion channel activation of cells.

The structure in which the micrometer-sized pillar-shaped structure of the present invention is regularly arranged may transform a cell's trait, and in particular, may activate an ion channel.

More specifically, in the case of A549 cells, chemotaxis is induced to increase the movement speed of the cells, and in the case of cardiomyocytes, the heart rate can be increased.

-go-go-Related　Gene), Kv4.3(Potassium voltage-gated channel subfamily D member 3), Kv7.1(Potassium voltage-gated channel), Kir2.1(inward-rectifier potassium ion channel) 및 Kir2.2(inward-rectifier potassium ion channel)를 비교예 1과 실시예 1을 배양 시간을 1주 및 2주로 나누어 비교 분석한 것이다.1 is a perspective view illustrating an example of a state in which cells are seeded in an ion channel activation structure of a cell including a columnar structure according to the present invention (100: bottom, 200: columnar structure).
Figure 2 is a view showing the definition of the dimensions associated with the columnar structure on the structure for ion channel activation of the cell according to the present invention.
Figure 3 shows the correlation between the cell and the structure of the columnar structure, (a) is Comparative Example 3, (b) Example 1 of the present invention, (c) is Comparative Example 2, (d) Comparative Example 1 is shown.
4 is a view showing that the height (H) of the columnar structure can exhibit the maximum effect when the height (H) of the cross-section of the cell is greater than or substantially equal to the longest diameter (C _H ).
FIG. 5 is a graph showing the maximum mobility in the ion channel activation construct in which cells contact only two to three pillar-shaped structures within 10 hours (initial state) after A549 cells are seeded.
Figure 6 shows the correlation between the height of the columnar structure and the movement speed of the A549 cells, comparing the structure of Comparative Example 1, Comparative Example 2, Example 1 (a) in the A549 cells according to the incubation time The movement speed is measured, (b) the wound area according to the culture time, (c) is the wound healing rate according to the culture time.
FIG. 7 shows (a) Control (6-Well plate, Falcon), (b) Comparative Example 1 (flat, structure without columnar structure), (c) Example 1 (diameter of columnar structure = 2 μm, the center-to-center distance of the columnar structure = 16 μm) of the cardiomyocytes cultured in the ion channel activation structure of the cells, the optical transmission picture of the general transmission type.
FIG. 8 is a graph showing that the beat rate of cardiomyocytes cultured in the ion channel activation construct of cells is increased by comparing Comparative Example 1 with Control (6-Well plate, Falcon), Example 1 (** p < 0.01, Tukey's test).
9 is a diagram confirming whether the ion channel in the cardiomyocytes is activated through protein expression. (a) is the cardiac marker, cTnT (cardiac Troponin T); Expression of NCX1 (Na ⁺ / Ca ²⁺ Exchange 1), a marker of automated beating, was confirmed. Comparative Example 1 and Example 1 is analyzed by dividing the culture time by 1 week and 2 weeks.
Figure 10 is also confirmed through the protein expression whether the ion channel is activated in the cardiomyocytes. (a) shows the expression levels of Nav1.5 (Sodium channel, voltage-dependent) and Cav1.2 (Calcium channel, voltage-dependent, L type, alpha 1C subunit) which are markers related to depolarization in the ion channel of the heart. Example 1 and Comparative Example 1 was compared and analyzed by dividing the culture time into 1 week and 2 weeks, (b) is the hERG (human Ether- ) markers associated with repolarization

-go-go -Related Gene), Potassisium voltage-gated channel subfamily D member 3 (Kv4.3), Potassisium voltage-gated channel (Kv7.1), inward-rectifier potassium ion channel (Kir2.1), and Kir2.2 (Inward-rectifier potassium ion channel) is a comparative analysis of Comparative Example 1 and Example 1 by dividing the culture time by 1 week and 2 weeks.

An embodiment of the present invention relates to a structure for activating a cell's ion channel, including a columnar structure, and has a geometry and size capable of maximizing activation of the cell's ion channel, thereby activating the cell's ion channel. It provides a structure for ion channel activation of the cells.

As used herein, the term "ion channel" refers to a membrane protein necessary for ions inside and outside the cell to circulate. The ion channel maintains the necessary ion concentration inside the cell, and controls the movement of ions to maintain the size of the cell. Neurons, which are the cells that make up the nervous system, are required to maintain the resting membrane potential, which is ready for signal transduction, and play a role in the cell's active state, ie, the neuron (action potential) that is intracellular neurotransmission. Basically, every cell has ion channels and is divided into two types: ionophoric protein and ion transporter.

As used herein, the term "space between pillar-shaped structures" means the distance between the centers of horizontal sections of adjacent different column-shaped structures. In figure 2 the definition of "gap between pillar-shaped structures" is schematically illustrated.

Hereinafter, embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, the following drawings attached to the present specification are intended to illustrate preferred embodiments of the present invention, and together with the contents of the present invention serves to further understand the technical spirit of the present invention, the present invention described in such drawings It should not be construed as limited to matters.

Figure 1 schematically shows an embodiment of the ion channel activation structure of the cell of the present invention.

Referring to FIG. 1, an embodiment of a structure according to the present invention includes a bottom surface 100 and a structure 200 having a columnar shape disposed on the bottom surface.

The columnar structure 200 serves to support and stimulate the seeded cells to induce transformation of the cells.

The pillar-shaped structure 200 according to an embodiment of the present invention is arranged at a contact interval of two to three pillar-shaped structures immediately after seeding of the cells, and the cells do not touch the bottom surface. May have a height. When the columnar structure 200 has the gap and the height, the ion channel of the seeded cell may be activated.

More specifically, the cells seeded in one embodiment of the structure according to the present invention are subjected to a large compressive stress in the lower part of the cell which is not supported by the columnar structure 200, in which case the ion channels of the cells are activated. It is thought to be. However, this is only a prediction and should not be construed as being limited thereto.

2 is a schematic illustration of various examples of seeded cells contacting the construct.

Referring to FIG. 2, (b) corresponds to an embodiment of the present invention, (a) is supported by more than three columnar structures, and (c) is the bottom surface 100. ) Is a case where cells touch, and (d) is a case of Comparative Example 1 without a columnar structure. As described above, the ion channel may be activated only in the case of (b).

3 schematically shows a method of measuring the spacing, horizontal cross-sectional diameter and height of the columnar structure 200.

The spacing between the pillar-shaped structures 200 is controlled so that the cells come into contact with two to three pillar-shaped structures 200 immediately after seeding, and the spacing may be adjusted according to the type or size of the cells. For example, in the case of A549 cells or cardiomyocytes, the interval of the pillar-shaped structure 200 may be 12 μm to 20 μm, preferably 14 μm to 18 μm, but is not limited thereto.

In one embodiment of the present invention, the columnar structure 200 may have an interval in which the cells are in contact with two to three columnar structures up to 10 hours or less immediately after seeding. When the pillar-shaped structure 200 has the gap, the degree of ion channel activation of the seeded cells may be further maximized.

In one embodiment of the present invention, the horizontal cross-sectional diameter of the columnar structure 200 should not be small enough that the structure is not firmly maintained due to the weight of the cell and does not bend or penetrate the cells, and the seeded cells May be a diameter that does not reach the top of the structure and is not sized to be located only at the top.

Therefore, the horizontal cross-sectional diameter may vary depending on the specific cell type, for example, may be 1 μm to 3 μm, more preferably 1.5 μm to 2.5 μm, but is not limited thereto.

At a diameter of less than 1 μm, columnar structures may penetrate the cell membrane or the structures may be easily bent due to the weight of the cells.

In addition, at diameters greater than 3 μm, the space between substantially adjacent pillars can be narrowed, making it difficult to show interactions between the cell and the bottom surface, and because of the large flat space on top of the pillars, the cells do not attach across multiple pillar structures. And can only be attached to one structure.

In one embodiment of the present invention, the height of the columnar structure 200 may be a height that the seeded cells do not reach the bottom surface (100). For example, it may be a height greater than the longest height of the vertical sections of the seeded cells. The cells seeded in the columnar structure 200 should have a height that does not touch the bottom surface 100 to activate the ion channels of the cells.

The specific value of the height of the columnar structure 200 may be adjusted according to the type of cells seeded. For example, in the case of A549 cells or cardiomyocytes, the height may be 10 to 16 μm, preferably 12 to 14 μm. However, it is not limited thereto. 4 schematically shows the relationship between the longest height of the vertical cross section of the cell and the height of the columnar structure 200.

In one embodiment of the present invention, the columnar structure 200 may have an aspect ratio of 5 or more.

Here, the aspect ratio means the ratio of the diameter and the height of the columnar structure 200. For example, a structure having an aspect ratio of 5 may have a height of 10 μm when the diameter is 2 μm in a ratio of 5 to a diameter of a columnar structure.

When the columnar structure 200 has the aspect ratio, the degree of ion channel activation of the seeded cells may be further maximized.

In one embodiment of the present invention, the material of the pillar-shaped structure 200 is not limited to organic materials, inorganic materials, etc., there is no cytotoxicity, the strength of the seed when the cells may be a material that is not bent by the cells. .

In addition, the material may be advantageous to be easily processed in a form that can support the culture and transparent to facilitate the observation and culture of the cells.

For example, the cured product of the transparent photocurable resin composition, PDMS, hardness, etc. of which the hardness is adjusted may be included, but is not limited thereto.

The bottom surface 100 supports the columnar structure 200, may be formed of the same material as the columnar structure 200, or may be integrated with the columnar structure 200.

In one embodiment of the invention, the cells may be at least one selected from cardiomyocytes or A549 cells.

When the cells are seeded and cultured in the above-described construct of the present invention, the ion channels of the cells may be activated. When the ion channel of the cell is activated, the heart rate can be increased in cardiomyocytes, and chemotaxis in A549 cells can increase cell mobility.

The present invention also provides a method for activating an ion channel of a cell.

One embodiment according to the ion channel activation method of the cell of the present invention, preparing a cell; And a columnar structure disposed on the bottom surface, wherein the columnar structure is arranged at intervals at which cells are in contact with two to three columnar structures immediately after seeding of the cells. And seeding the cells in a structure for ion channel activation of cells, the cells having a height that does not reach the bottom surface.

One embodiment according to the ion channel activation method of the cells of the present invention can be carried out by seeding and culturing the prepared cells in the above-described structure of the present invention.

Cells that can be applied to one embodiment of the ion channel activation method of the present invention is not particularly limited, for example, may be at least one selected from cardiomyocytes or A549 cells.

As described above, when the ion channel of the cell is activated, the heart rate may be increased in the case of cardiomyocytes, and the mobility of the cell may be increased in the case of A549 cells by inducing chemotaxis.

The present invention also provides a method for producing a structure for ion channel activation of a cell.

One embodiment of the method for producing a structure for ion channel activation of a cell of the present invention,

A pillar-shaped structure disposed on the bottom surface and the bottom surface, and immediately after seeding of the cells, the cells are arranged at contact intervals of two to three pillar-shaped structures, and the cells on the bottom surface A first step of preparing an intermediate mold including an elastic polymer in which a pillar-shaped structure having a height which does not reach is engraved in an intaglio; A second step of imprinting the intermediate mold on a photocurable resin and irradiating light for 30 seconds to 5 minutes; And a third step of removing the intermediate template from the photocured resin.

The intermediate mold may be solidified by applying heat or light to a raw material which is a liquid, and the resulting mold may be made of a material present as a solid at room temperature. The use of a liquid raw material allows solidification of the shape of the mast mold. In addition, the mold may be made of a material containing an elastic polymer to facilitate the step of removing the intermediate mold from the structure of the final column, which is the third step, so that the solidified structure has elasticity, The intermediate mold has almost no residue after the desorption, and the separated intermediate mold also maintains its original shape and can be used repeatedly. Examples of the elastomeric polymer include polydimethylsiloxane (PDMS), polystyrene-based polymer, polyolefin-based polymer, polyvinyl chloride-based polymer, polyurethane-based polymer, polyester-based polymer, polyamide-based polymer, collagen, hydrogel, solidifying carbon and silicone rubber And the like, but are not limited thereto.

Specifically, the first step includes a bottom surface and a columnar structure disposed on the bottom surface, and immediately after seeding of the cells, the cell contacts two to three of the columnar structures. The method may further include preparing a silicon master mold by forming a pillar-shaped structure having a height that does not touch cells on the bottom surface and arranged on the silicon wafer. Formation of the silicon master mold can be carried out using any of the fine patterning techniques known in the art without limitation. For example, it may be formed by photolithography and deep reactive ion etching (deep RIE) or wet etching. The method may further include providing hydrophobicity to the surface of the silicon master mold.

The provision of the hydrophobicity is a self-assembly formed by treating with octadecylsilane (OTS) or trichloroperfluorooctylsilane (trichloro (1H, 2H, 2H-perfluorooctyl) silane) which can hydrophobize the surface. This can be accomplished by functionalizing with a self-assembled monolayer (SAM). The hydrophobic treatment can facilitate the separation of the PDMS intermediate template formed from the master template later in the first step.

Preferably, the intermediate template prepared from the first step can be used repeatedly. The PDMS intermediate template of the present invention not only facilitates separation of the final polymer structure, but also exhibits no change in physical and / or chemical properties even after repeated use over 10 times, which is useful for mass production of cell culture substrates according to the present invention. Can be used. In particular, since it can manufacture once from an expensive silicone master mold and can use it repeatedly, there are also economic advantages.

Hereinafter, preferred examples are provided to help understanding of the present invention, but these examples are only for exemplifying the present invention, and do not limit the appended claims, but are within the scope and spirit of the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made to the present invention, and such modifications and changes belong to the appended claims.

Example  One

In a cell culture system including regularly arranged pillars, the size of the pillars was deliberately changed to determine the effect of the pillar size and spacing on the cells cultured on the system.

First, a master mold was prepared by patterning the photolithography and solution etching processes on an 8-inch silicon wafer. The master pillar structure was manufactured at the National Nanofab Center in Korea. In order to impart surface hydrophobicity, the prepared silicon pillar array was functionalized with self-assembled monolayers (SAM) of trichloroperfluorooctylsilane (trichloro (1H, 2H, 2H-perfluorooctyl) silane). . Hydrophobic SAM treatment is required for easy separation of polydimethylsiloxane (PDMS) intermediate molds. Thereafter, PDMS mixed with a 10: 1 curing agent was poured onto the SAM-functionalized Si master mold and cured at 80 ° C. for 4 hours to prepare an intermediate PDMS mold. An intermediate PDMS master template was introduced for convenient release of the final polymer structure, and the PDMS intermediate template can be used multiple times (without too much of degrading more than 10 times). However, there is an economical advantage over using the Si master directly.

Finally, a UV-curable resin (NOA 63 from Norland Products) was applied onto targetsubstrates (PET) to form a resin layer. The resin layer formed was pressurized with the PDMS intermediate mold prepared above to transfer the pillar structure form formed on the mold to the resin layer. Next, a UV lamp was irradiated for 1 minute to cure the UV-curable resin. Thereafter, the PDMS intermediate template was removed from the sample to obtain a structure including the final pillar structure formed of UV-curable resin on PET. The diameter of the horizontal cross section of the obtained columnar structure was 2 μm, the spacing between the columnar structures was 16 μm, and the height was 13 μm.

The structure including the pillar structure formed on the PET substrate was cut into a size that can be inserted into each well of a commercially available 6-well plate (Falcon) and inserted into the 6-well plate.

Comparative example  One

The structure was manufactured in the same manner as in Example 1 except that the structure 200 in the form of a pillar was not formed on the UV-curable resin layer.

Comparative example  2

The structure was manufactured in the same manner as in Example 1 except that the height of the columnar structure was 2 μm.

Comparative example  3

The structure was manufactured in the same manner as in Example 1 except that the spacing of the columnar structures was 5 μm.

Experimental Example

1.Evaluation of Cell Mobility According to Column Height of A549 Cells Grown in Cell Ion Channel Activation Construct

(1) Experiment Method

A549 cells (initial state) were seeded and cultured in the constructs of Example 1 and Comparative Examples. In Example 1 it was confirmed that the A549 cells are supported by two to three columnar structures (FIG. 5). Since the cell mobility according to the incubation time, the wound to the surface where the cell was grown to remove the cell, and then the time it takes for the cell to move to fill the empty space, the standard method of measuring the movement speed of the wound wound Wound area and wound healing rate were evaluated according to the healing method.

(2) experimental results

Within 10 hours after the seeding of the A549 cells (initial state), it was confirmed that the mobility of the ion channel activation constructs in which the cells were in contact with only two to three pillar-shaped structures was maximized (FIG. 5).

In addition, when the cell mobility according to the culture time is compared with the structures of Comparative Examples 1, 2 and Example 1, it was confirmed that the cells cultured in the structure of Example 1 shows the highest mobility until the culture time 30 hours (FIG. 6A).

In the case of the wound area was found to be the smallest in the order of Example 1, Comparative Example 1, Comparative Example 2 (Fig. 6b), the wound healing rate was confirmed to increase in the order of Example 1, Comparative Example 2, Comparative Example 1. Could be (FIG. 6C).

Taken together, it can be seen that the A549 cells seeded in Example 1 were activated with ion channels.

2. Grown in the structure for ion channel activation of cells cardiomyocyte  Assessment of cell heart rate increase

(1) Experiment Method

Cardiomyocytes were seeded into the constructs of Example 1 and Comparative Examples. In Example 1, the cardiomyocytes were confirmed to be supported by two to three columnar structures, and micrographs were taken to compare with Comparative Example 1 (FIG. 7).

Heart rate per minute of cardiomyocytes after seeding in Example 1 and Comparative Example 1 was measured, and the results are shown graphically in FIG. 8.

In addition, the gene expression level of the markers indicating that the ion channel was activated after seeding in Comparative Example 1 and Example 1 was measured, and the graphs are illustrated in FIGS. 9 and 10.

-go-go-Related　Gene), Kv4.3(Potassium voltage-gated channel subfamily D member 3), Kv7.1(Potassium voltage-gated channel), Kir2.1(inward-rectifier potassium ion channel) 및 Kir2.2(inward-rectifier potassium ion channel)이다.These markers are cardiac marker cTnT (cardiac Troponin T), hyperpolarization-activated Cyclic Nucleotide-gated channel 4 (HCN4), NCX1 (Na ⁺ / Ca ^{2 +} Exchange 1), a marker of automated beating associated with ion channels Nav1.5 (Sodium channel, voltage-dependent) and Cav1.2 (Calcium channel, voltage-dependent, L type, alpha 1C subunit) markers related to depolarization in ion channels, hERG (human Ether- ) markers related to repolarization

-go-go -Related Gene), Potassisium voltage-gated channel subfamily D member 3 (Kv4.3), Potassisium voltage-gated channel (Kv7.1), inward-rectifier potassium ion channel (Kir2.1), and Kir2.2 (inward-rectifier potassium ion channel).

(2) Experiment result

Referring to FIG. 8, it could be seen that the heart rate per minute of the cardiomyocytes seeded in Example 1 was higher than that of cardiomyocytes seeded in the comparative example 1.

Referring to FIG. 9, cTnT and HCN4 were found to express less seeded cells in Example 1 than in Comparative Example 1, and NCX1 expressed more seeded cells in Example 1 than in Comparative Example 1. Could know.

Referring to FIG. 10, Nav1.5 was expressed more in the seeded cells in Example 1 at 1w, but was expressed in the cells seeded in Comparative Example 1 at 2w.

In addition, Cav1.2 and Kir2.2 had more expression in the cells seeded in Comparative Example 1 than in Example 1.

In the case of hERG, the expression amount of cells seeded in Comparative Example 1 was higher in 1w, but the expression amount of cells seeded in Example 1 was higher in 2w.

In addition, in the case of Kv4.3, Kv7.1, Kir2.1, the expression amount was higher in Example 1.

Taken together, it can be seen that in the cardiomyocytes seeded in Example 1, ion channels related to the maturation of the cardiomyocytes such as NCX, hERG, Kir2.1 are more activated.

Claims (12)

A pillar-shaped structure disposed on the bottom surface and the bottom surface, wherein the pillar-shaped structure is arranged at contact intervals of two to three of the pillar-shaped structures immediately after seeding of the cells; Structure having an ion channel activation of the cell, the cell has a height that does not touch the bottom surface.
The structure of claim 1, wherein the columnar structure has a gap in which the cell is in contact with two to three columnar structures until 10 hours or less immediately after seeding.
The structure of claim 1, wherein the height of the columnar structure is greater than the longest height of the vertical cross section of the seeded cell.
The structure of claim 1, wherein the columnar structure has an aspect ratio of 5 or more.
The structure of claim 1, wherein the cell is at least one selected from cardiomyocytes or A549 cells.
Preparing cells;
A pillar-shaped structure disposed on the bottom surface and on the bottom surface, wherein the pillar-shaped structure is arranged at intervals at which cells contact two to three pillar-shaped structures immediately after seeding of the cells; Seeding the cells in a structure for ion channel activation of cells, the cells having a height that does not reach the bottom surface;
Ion channel activation method of a cell comprising a.
The method of claim 6, wherein the cell is at least one selected from cardiomyocytes or A549 cells.
The method of claim 7, wherein the A549 cell has a period of increasing mobility after seeding the ion channel activation construct of the cell.
The method according to claim 7, wherein the cardiomyocytes are seeded in the ion channel activation structure of the cell, the heart rate is increased, the ion channel activation method of the cell.
A pillar-shaped structure disposed on the bottom surface and the bottom surface, and immediately after seeding of the cells, the cells are arranged at contact intervals of two to three pillar-shaped structures, and the cells on the bottom surface A first step of preparing an intermediate mold including an elastic polymer patterned in an engraved structure of a columnar structure having a height that is not touched;
A second step of imprinting the intermediate mold on a photocurable resin and irradiating light for 30 seconds to 5 minutes; And
And a third step of removing the intermediate template from the photocured resin.
The structure of claim 10, wherein the first step includes a bottomed structure and a columnar structure disposed on the bottom surface, wherein the cells have two or three pillar-shaped structures immediately after seeding. Preparing a silicon master template by forming a pillar-shaped structure having a height in which the cells do not reach the bottom surface and contacting the bottom surface of the silicon wafer, thereby preparing a silicon master template; Way.
The method of claim 10, wherein the cell is at least one selected from cardiomyocytes or A549 cells.

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