KR102019664B1 - Cell sorting method and device using difference of moving speed - Google Patents

Abstract

The present invention relates to a cell sorting method using the difference of moving speed, and an apparatus for performing the same, the method comprising a step of distinguishing a growth region in which cells grow and a non-growth region in which cells cannot grow, and keeping the non-growth region closed; a step of attaching and growing cells in the growth region; and a step of opening the non-growth region so that cells grown in the growth region move to the non-growth region and distinguishing the cells located in the growth region and the cells moved to the non-growth region by the difference in the moving speed according to the cell characteristics.

Description

Cell sorting method using a difference in moving speed, a device for performing the same {Cell sorting method and device using difference of moving speed}

The present invention relates to a cell sorting method using a moving speed difference, an apparatus for performing the same, and more particularly, a method for identifying the migration of cells by controlling the area of the cell attachment surface, and distinguishing between fast and slow cells, It relates to a device for doing this.

In general, cell mobility is observed by growing cells and observing migration. Cell migration is important in regenerative medicine and tissue engineering because it can help tissue regeneration or cancer cell metastasis modeling and diagnosis.

In order to observe the movement of the cells, the cells are attached to a specific surface and grown. As growth progresses, cell migration occurs. It is known that the movement of the cells changes depending on the roughness of the attachment surface, whether or not the coating is applied, and the wettability. In particular, it is known that the movement speed is different depending on the attachment surface area to which the cells are attached.

However, the conventional method for observing cell mobility is to observe cell migration under the same conditions, which is not suitable for the observation of cells whose cell characteristics change.

For example, cancer cells exhibit the characteristics of population migrating cells, but when transformed into metastatic cancer cells, they exhibit the characteristics of individual migration.

Collective and individual migration following the epithelial-mesenchymal transition, Ian Y.Wong, Sarah Javaid, Elisabeth A.Wong, Sinem Perk, Daniel A. Haber, Mehmet Toner and Daniel Irimia1, nature materials 17 August 2014.

SUMMARY OF THE INVENTION The present invention has been made in an effort to provide a method and apparatus for performing the method capable of observing, distinguishing and separating the movement characteristics of modified cells by varying the area of the cell adhesion surface.

In particular, it is an object of the present invention to provide a method for separating cancer cells and metastatic cancer cells, apparatus for performing the method.

Cell division method using the movement speed difference according to an aspect of the present invention for solving the above problems, distinguishes the growth region in which the cells grow and the non-growth region in which the cells cannot grow, and closed the non-growth region And the step of attaching and growing the cells to the growth region, and opening the non-growth region to move the cells grown in the growth region to the non-growth region, depending on the difference in the movement speed according to the cell characteristics. And separating and separating the cells moved into the non-growth region and the cells located in the growth region.

According to one embodiment of the invention, the cells are attached to the attachment surface including perforations having a diameter of nanometer size, it is possible to control the attachment area.

According to one embodiment of the present invention, the narrower the adhesion area, the deeper the difference in movement speed according to the characteristics of the cells moving to the non-growth area.

According to one embodiment of the present invention, after the division, cells of the population movement characteristic may remain in the growth region, and cells of the individual movement characteristics may be located in the non-growth region.

In addition, the cell sorting apparatus according to another aspect of the present invention, the case for providing an internal space, the insert partition wall is inserted into the inside of the case to divide the internal space, attached to the inner surface of the case, the diameter is nanometer Including a plurality of perforations as a unit, it may include a substrate that can control the area to which cells can be attached.

According to an embodiment of the present invention, the case and the insert partition wall may be made of a material to which cells are not attached.

According to an embodiment of the present invention, the insert partition wall may be removed after the cells are grown in the case to provide a space for the cells to move.

According to one embodiment of the present invention, a cell having a high speed of movement is located in a space where the insert partition is removed and opened, and a cell having a low speed of movement is located at its original position to distinguish and separate cells.

Cell division method using the movement speed difference of the present invention, the apparatus for performing the same, by adjusting the adhesion area of the cell to increase the movement speed difference of the cells having different characteristics according to the attachment area, the cell by the difference in the movement speed It can be easily distinguished and separated.

1 is a flow chart of a cell sorting method using a moving speed difference according to a preferred embodiment of the present invention.
2 is an explanatory diagram to help understanding of the present invention.
3 is an exploded perspective view of a cell sorting apparatus according to a preferred embodiment of the present invention.
4 is a coupling state diagram of FIG. 3.
5 is a conceptual diagram and a micrograph of a substrate applied to the present invention.
6 shows the results of migration of individual mobile cells and population mobile cells.

Hereinafter, with reference to the accompanying drawings, a cell sorting method using the movement speed difference of the present invention, an apparatus for performing the same will be described in detail.

Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art, and the embodiments described below may be modified in various other forms, and The scope is not limited to the following embodiments. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the inventive concept to those skilled in the art.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a", "an" and "the" may include the plural forms as well, unless the context clearly indicates otherwise. Also, as used herein, "comprise" and / or "comprising" specifies the presence of the mentioned shapes, numbers, steps, actions, members, elements and / or groups of these. It is not intended to exclude the presence or the addition of one or more other shapes, numbers, acts, members, elements and / or groups. As used herein, the term "and / or" includes any and all combinations of one or more of the listed items.

Although the terms first, second, etc. are used herein to describe various members, regions, and / or portions, it is obvious that these members, components, regions, layers, and / or portions are not limited by these terms. . These terms do not imply any particular order, up or down, or superiority, and are only used to distinguish one member, region or region from another member, region or region. Accordingly, the first member, region, or region described below may refer to the second member, region, or region without departing from the teachings of the present invention.

Hereinafter, embodiments of the present invention will be described with reference to the drawings schematically showing embodiments of the present invention. In the drawings, for example, variations in the shape shown may be expected, depending on manufacturing techniques and / or tolerances. Accordingly, embodiments of the present invention should not be construed as limited to the specific shapes of the regions shown herein, but should include, for example, changes in shape resulting from manufacturing.

1 is a flow chart of a cell sorting method using a moving speed difference according to a preferred embodiment of the present invention.

Referring to FIG. 1, a step of partitioning a cell adhesion surface and a space where cells are not attached (S10), a step of attaching cells to the cell adhesion surface (S20), and growth of the attached cells do not attach the cells. Growing cells in the region excluding the space (S30), opening the space (S40), and check the movement speed by allowing the grown cells to move to the open space, and distinguish the cells according to the movement speed And separating (S50).

Hereinafter, the configuration and operation of the cell sorting method using the movement speed difference according to the preferred embodiment of the present invention configured as described above will be described in more detail.

2 is an explanatory diagram to help understanding of the present invention.

The present invention needs to distinguish between a region where cells grow and a region where cells do not grow.

In FIG. 2, the non-growing region 2 is secured in the middle of the growing region 1. The non-growing region (2) is blocked by the insert barrier during growth of the cells in the growth region (1), and the cells are moved to the non-growing region (2) by removing the insert barrier when the cells are completely grown in the growth region (1). You can do that.

First, in step S10 it partitions the cell attachment surface and the cell is not attached as shown in FIG. The cell attachment surface is located in the growth region 1, and the space where the cells are not attached is the non-growth region 2, and the space is blocked by the insert partition wall.

The cell attachment surface may be a surface of a nanostructure. Compared to the planar structure, the nano structure limits the area to which cells are attached, so that differences in movement speed may occur depending on the type of cells.

In other words, the cell attachment surface, which is the periphery of the growth region 1, is a nanostructure, which causes a difference in movement speed according to the type of cells.

Then, in step S20 to attach the cells to the cell attachment surface of the growth region (1). At this time, the cell adhesion surface may provide a nanostructured surface to control the area of the cell adhesion surface.

If the area of the cell attachment surface is reduced by using the nanostructure, the moving speed increases for the individual moving cells, and the moving speed for the group moving cells decreases.

Then, the attached cells are grown as in step S30 to fill all of the growth region (1).

Next, the insert growth barrier 2 located in the non-growth region 2 is removed in step S40 to open the non-growth region 2.

The opening of the non-growth region 2 provides a space for the cells grown in the growth region 1 to move.

Then, after a predetermined time elapses as in step S50, the degree of cell migration is measured and cells are classified. At this time, the cells can be sufficiently moved, and after 30-60 hours after opening the non-growth area (2) so that the cells with slow movement and the cells with high movement speed can be clearly distinguished, the degree of movement of the cells is measured and the cells are measured. Separate.

For example, in the case of individual mobile cells, cells moving to the non-growing region (2) at a high speed on the surface of the nanostructure, and in the case of population moving cells moving at a slow speed, the cells filling the non-growing region (2) have individual migration characteristics. It is a cell having, and the cells remaining in the growth region (1) can be separated and separated as a population transfer cell.

As described above, the present invention distinguishes between the growth region 1 and the non-growth region 2, and adjusts the adhesion area of the cells to clarify the difference in the moving speed according to the characteristics of the cells, thereby increasing the non-growing region in the growth region 1. In (2), cells can be distinguished and separated by the difference in migration speed.

In the above description, the non-growth region 2 is provided in the middle of the growth region 1, but any method may be applied as long as the non-growth region 2 and the growth region 1 are distinguished from each other.

For example, in FIG. 2, the growth region 1 may be located on both the left and right sides of the non-growth region 2, but only the region located on the left side of the non-growth region 2 is defined as the growth region 1. In addition, the region located on the right side may be defined as a non-growth region so that the cells grown in the growth region 1 move to the non-growth region 2, and the cells may be separated and separated by the difference in movement speed. .

The cell attachment surface may include a plurality of perforations to control its area, and the area of the cell adhesion surface may be the sum of the growth region 1 and the non-growth region 2. The division of the growth zone (1) and the non-growth zone (2) can divide a boundary by dividing a part of the cell attachment surface, and the means for dividing the cell by pressing a part of the cell attachment surface to adhere to the growth zone (1) Can be used irrespective of the constitution so long as the cells are grown in an isolated state and the means for dividing the compartment is removed so that the cells attached to the growth region 1 can move to the non-growth region 2.

For example, the means for dividing the compartment may be a polyhedron, a cylindrical structure such as a cube with an open top and bottom.

Hereinafter, a separator for applying to the cell sorting method of the present invention will be described in detail.

Figure 3 is an exploded perspective view of a cell sorting apparatus according to a preferred embodiment of the present invention, Figure 4 is a binding state diagram of FIG.

3 and 4, the case 10 defining the growth region 1, the insert partition wall 20 inserted into the center portion of the case 10, the case 10 and the insert partition wall, respectively. And a cover 40 attached to the lower side of the nanostructure 20 and positioned in the growth region 1, and a cover 40 for pressing and fixing the insert barrier 20.

The shape of the case 10 is a structure that provides a hexahedral space with the upper and lower portions open, and the insert partition wall 20 is inserted into an inner central portion of the case 10. Although the case 10 is illustrated as providing a growth region 1 of a hexahedron, the case 10 may be manufactured in various structures such as a circle and a polygon.

A guide 11 into which the insert partition wall 20 is inserted is provided at an inner central portion of the case 10.

The substrate 30 is a material to which cells can be attached, and the case 10 and the insert partition wall 20 are made of materials to which cells cannot be attached.

Specifically, the substrate 30 may use a polycarbonate (PC), and the case 10 and the insert partition wall 20 may use polydimethylsiloxane (PDMS).

The substrate 30 has a nanostructured surface, which may be transferred by a hot embossing process.

When adhering the case 10 to the upper portion of the substrate 30, an adhesive means such as a double-sided tape may be used.

After adhering the case 10 to the upper portion of the substrate 30, the insert partition 20 is inserted into the guide 11 to be coupled to the inside of the case 10. The growth region 1 is divided by the insert partition 20, and the non-growth region 2 is defined between the divided growth regions 1.

The height of the insert partition wall 20 is slightly higher than the height of the case 10, and then presses the insert partition wall 20 with the cover 40 to apply pressure to the substrate 30, the insert partition wall 20 is Before removal, cells are not allowed to migrate to the lower substrate 30 of the insert barrier 30.

5 shows a conceptual diagram and a micrograph of the substrate 30.

The perforations of 100 to 500nm are arranged regularly and regularly on the plate of PC material to form nanostructures, and the area to which cells are attached can be controlled by the formation of nanostructures.

As described above, the smaller the adhesion area, the higher the movement speed of individual mobile cells, and the lower the movement speed of the population mobile cells.

6 shows the results of migration of individual mobile cells and population mobile cells.

Fibroblast cells with individual migration characteristics show faster migration rates as the adhesion area decreases, and epithelial cells with population migration characteristics show slower migration rates as the attachment area decreases.

The insert partition wall 20 is inserted into the case 10 to partition the surface to which the cell is described with reference to FIG. 1 and the space where the cell is not attached, that is, the growth region 1 and the non-growth region 2. (Step S10).

The cells are attached to the substrate 30 (step S20) and grown (S30).

When the growth region 1, which is a space inside the case 10, is filled with cells by cell growth, the cover 40 and the insert partition wall 20 are removed to open the non-growth region 2 (S40). ).

The space is generated by the opening of the non-growth region 2 and the grown cells move to the open non-growth region 2.

After the set time has elapsed, the non-growing area 2 is filled with fast moving cells, and is separated and separated from the slow moving cells (S50).

<Example>

Typically, when a histological examination is performed to identify cancer cells, it is possible to identify a form in which cells such as fibroblasts are mixed with cancer cells.

The tissue for examination is attached to the substrate 30 located at the bottom of the inner space of the case 10 to grow cells.

When the cells are filled in the space formed by the case 10 and the insert partition wall 20, the insert partition wall 20 is removed.

At this time, the insert partition wall 20 is removed to move the cells into the open space, fibroblasts having individual movement characteristics are moved at a relatively faster speed. On the contrary, the cancer cells having the mass migration characteristics are slow in movement, and thus, the fibroblasts are located in the space where the insert partition 20 is present, and the cancer cells remain in their original positions to separate the cancer cells.

It will be apparent to those skilled in the art that the present invention is not limited to the above embodiments and may be variously modified and modified without departing from the technical spirit of the present invention. will be.

10: Case 20: Insert bulkhead
30: substrate 40: cover

Claims (9)

Dividing the growth region in which cells grow and the non-growth region to which cells are not attached on a substrate including perforations having a diameter of 100 to 500 nm, and separating the growth region and the non-growth region using an insert partition wall;
Attaching and growing cells to the growth region; And
By removing the insert partition wall, the cells grown in the growth zone and the non-growth zone are moved to the non-growth zone, but the cells and the growth zone are moved to the non-growth zone due to the difference in the movement speed according to the cell characteristics. Cell sorting method using a moving speed difference comprising the step of separating into cells located. delete The method of claim 1,
The cell separation method using the moving speed difference, characterized in that the difference in the moving speed according to the characteristics of the cell moving to the non-growing area as the area attached to the growth region decreases. The method of claim 3,
The cell sorting method using the moving speed difference, characterized in that the cells of the group movement characteristics remain in the growth region after the division, the cells of individual movement characteristics are located in the non-growth region. delete delete A case providing an inner space;
An insert partition wall inserted into the case to divide an internal space;
Attached to the bottom of the case, including a plurality of perforations having a diameter of 100 to 500nm, includes a substrate that can adjust the area to which cells can be attached,
The cells are attached to one side of the substrate divided by the insert partition wall and grown. When the growth is completed, the insert partition wall is removed to move a part of the cells grown to another area of the substrate where the cells are not grown. A cell separator, comprising dividing cells into two parts. The method of claim 7, wherein
The case and the insert partition wall is a cell separator, characterized in that the material is not attached to the cell. delete

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