KR20110083191A

KR20110083191A - Cell separator using magnet

Info

Publication number: KR20110083191A
Application number: KR1020100003286A
Authority: KR
Inventors: 김영호
Original assignee: (주) 셀모코리아; 김영호
Priority date: 2010-01-14
Filing date: 2010-01-14
Publication date: 2011-07-20
Also published as: KR101160188B1

Abstract

PURPOSE: A cell sorting apparatus using a magnet is provided to apply the magnetic force and to simply and efficiently isolate cells. CONSTITUTION: A cell sorting apparatus comprises an upper housing(1), an upper socket(2), a lower socket(3), a lower socket cover(4), a magnet part(5), and a lower housing(6). The upper housing comprises an upper socket connection part which is protruded in a cylindrical shape and an upper socket connection latch which is protruded in a central direction. The upper socket has a side of cylindrical shape with an open front side; a back side which is connected with the side and has an upper socket hole for injecting cell lysate liquid; and a first upper/lower fixing part for fixing the lower socket.

Description

Cell separator using magnetic force {CELL SEPARATOR USING MAGNET}

The present invention relates to a cell separator using magnetic force, and more particularly, to a cell separator for separating a required cell by applying a magnetic force to a cell separation solution in which specific cells are mixed.

In US Pat. No. 6,602,422 (micro column system), a cell mixture in which magnetic beads are attached as a magnetic carrier in a column in which small metal beads are stacked is placed in a gravity direction while putting a cell mixture in a direction of gravity. When magnetic force is applied from the side, specific cells are adsorbed in the process of moving to the pores between the magnetized iron balls, and the remaining cells other than the specific cells flow down, where the specific cells attached to the iron balls are separated. After removing the applied magnetic force, the aqueous solution is collected by flowing a buffer solution. However, in the case of separating cells of various sizes using the columnar cell separation device, the size and size of adsorption of specific cells to be separated may be because the size of the pores generated between the iron balls may interfere with the movement of the cells. Depending on the characteristics and the size of the magnetic beads, the size of the metal beads used must be replaced each time. In addition, the cells mixed in the cell mixture may make it difficult to collect the specific cells separated while directly sticking to the surface of the metal ball. Furthermore, in order to eliminate the clogging of the pores between the metal balls as the cells aggregate, there is also a hassle to periodically homogenize using a pipette or the like during the separation process.

On the other hand, in US Patent No. 5,602,042 (method and device for magnetic separation of bioparticles from a mixture), a separation means having a magnet and a flat plate is applied while being vertically and rotationally immersed in a liquid mixture filled in a sealed container. The magnetic force causes the magnetic beads attached bioparticles to be attached to and separated from the plate. However, in the case of separating using the vessel-type cell separation device, other bioparticles adhered to the housing side () of the separation means in the process of removing the separation means from the mixed solution to the outside also gather in a flat plate and the separation efficiency is very high. Will be lowered. In addition, the separation of the liquid in a sealed container filled with a large amount of sample is expensive, and the separation means for implementing the vertical and rotational movement mechanism is complicated, the exposed portion such as the housing of the separation means Direct contact with the mixed solution increases the possibility of contamination of the mixed solution.

From this, in Korean Patent Application No. 2004-25421 (method and method for cell separation using water droplet-type cell suspension) proposed by the present inventors, a cell mixture containing specific cells with magnetic beads is used as a drop-shaped cell. It is produced as a mixed solution, and the magnetic force is applied thereto to distinguish the specific cells in the upper part and the remaining cells other than the specific cells in the lower part, and then additionally supply the buffer aqueous solution to the cell mixture in the form of water droplets separately to the other specific cells in the lower part. Simple cells and simple processes were realized by collecting only specific cells while remaining cells were completely separated by gravity. However, when the cells are separated using the droplet mixture, the apparatus and process for forming the cellular mixture in the form of droplets are complicated.

In addition, since the lower part of the droplet-shaped cell mixture is exposed to air, the remaining cells other than the specific cells in the inner surface are shaken even by the flow of fine ambient air, and further, the remaining cells other than the specific cells to be separated are removed. In order to inject a buffer aqueous solution in order to some of the remaining cells other than the specific cells in the lower back there is a problem that the separation efficiency is lowered to move toward the upper specific cells.

On the other hand, if the remaining cells other than the specific cells are attached to the surface where the specific cells are collected during the separation process, the specific cells may be adversely affected in the subsequent experiments to be performed using the collected specific cells. It is necessary to increase the purity of maximally.

In the case of the Republic of Korea Patent Application No. 2006-7005874 proposed by the present inventors devised to solve the problems of the existing invention, the cell mixture layer by adjusting the interval between the upper plate and the lower plate containing the cell mixture located facing the bottom However, the present invention provides a cell separation apparatus and method for easily and efficiently separating cells through width control and disassembly while applying magnetic force from the upper plate portion of the cell mixture layer, but the present invention is not specifically implemented as a cell fluid separation apparatus. There has been a disadvantage that has not been demonstrated whether an effective separation to the extent practicable is possible.

The present invention has been made to solve the above problems, the magnet portion 5 which is located adjacent to the lower socket (3) in the cell separation solution located between the upper socket (2) and the lower socket (3) By applying the magnetic force of the upper socket (2) and the lower socket (3) through the separation of cells can be easily and efficiently separated, the purpose of providing a cell separator implemented in a specific form.

The present invention is to achieve the above object, is provided with a test tube holder 13 which is a hole for inserting a test tube 11, the cylindrical side of the rear is open and the front surface connected to the side (11) 12, the upper socket connection portion 14 protruding in a cylindrical shape from the front portion to the rear side in the inner side of the front surface 12 and the upper socket connection latch protruding in the center direction of the circle at the end of the upper socket connection portion 14; An upper housing 1 provided with 15; The front side has a cylindrical side 21, an upper socket hole 23 into which the cell separation solution is injected, and a rear side 22 connected to the side 21, and a lower portion formed outside the rear side 22. The upper socket suction plate 27 having a donut shape around the upper socket hole 23 by adsorbing the first upper and lower fixing parts 24 and the separating liquid for fixing the socket 3, the upper socket An upper socket (2) having an upper housing connecting portion (25) for engaging with the upper housing (1) by coupling with the upper socket connecting latch (15) formed inside the rear surface (2) of (2); A cylindrical side surface 31 having a rear opening, a lower socket hole 33 into which a cell separation solution is injected, and a front surface 32 connected to the side surface 31 and the outer side formed on the front surface 32 are provided. The lower upper and lower fixing section 24 and the second upper and lower fixing section 34 corresponding to the lower socket adsorption portion 36 for adsorbing the separation liquid from the side, the lower portion formed inside the front surface 32 of the lower socket 3 A lower socket 3 having a lower housing connecting portion 35 for connecting with the housing 6; A lower socket cover (4) having a circular plate formed therein for separating a liquid adsorption plate (41) for adsorbing the separation liquid injected on one side thereof and sealing the lower socket hole (33); Magnets for separating positive particles or negative particles present in the separation liquid, which are combined with the lower housing 6 below and in contact with the opposite side of the lower socket 3 of one side of the lower socket cover 4. Part 5; And a lower housing (6) having a magnet portion accommodating portion (63) for accommodating the magnet portion (5) and a lower socket fixing support portion (64) in a cylindrical shape that is open to the front. .

In addition, a lower socket cover adsorption protrusion 42 is formed to protrude from the separation liquid adsorption plate 41 of the lower socket cover 4 so that a proper solution of the separation solution is stably adsorbed on the separation liquid adsorption plate 41. It is characterized by being.

In addition, it is formed to correspond to the side 11 of the upper housing 1 and the side 61 of the lower housing 6, the step of the height of the height of the contact between the upper housing 1 and the lower housing 6 When the upper housing (1) and the lower housing (6) is coupled to the upper housing (1) or lower housing (6) in a predetermined direction by twisting the upper housing to be separated from each other by the step of the height ( 1) Departure surface and the lower housing 6 characterized in that the departing surface (16,66) is formed.

In addition, a lower socket aligning groove 37 for arranging the lower socket 3 is formed on the side surface 31 of the lower socket 3 and has a lower shape corresponding to the lower socket aligning groove 37. The socket fixing protrusion 65 is characterized in that formed in the corresponding position of the lower housing (6).

In addition, the first upper and lower fixing part 24 and the second upper and lower fixing part 34 are characterized in that the fixing pin and the fixing hole, or the fixing hole and the fixing pin, respectively.

According to the present invention, the magnetic separation force of the magnet part 5 positioned adjacent to the lower socket 3 is applied to the cell separation liquid located between the upper socket 2 and the lower socket 3, thereby allowing the upper socket 2 to be applied. By separating the lower socket (3) and the cell can be easily and efficiently separated, it is possible to provide a cell separator implemented in a specific form.

1 is an exploded perspective view of one embodiment of a cell separator of the present invention.
2 is a perspective view of the upper housing 1 of the present invention.
3 is a perspective view of the upper socket 2 of the present invention.
4 is a perspective view of the lower socket 3 of the present invention.
Figure 5 is a perspective view of the lower socket cover 4 of the present invention.
6 is a perspective view of the magnet part 5 of the present invention.
7 is a perspective view of the lower housing 6 of the present invention.
8 is a view showing a mechanism in which the upper housing 1 and the lower housing 6 are coupled and disengaged in one embodiment of the present invention.
9 to 15 are diagrams showing the method of using the cell separator of the present invention step by step.
Figure 16 and Figure 17 is a photograph showing the form of the cell separator in the cell separator in each step when separating the cells using the cell separator according to the present invention.
18 is a diagram schematically showing a mechanism for separating cell separation liquid using the cell separator of the present invention.

The present invention is provided with a cylindrical side surface 11 having a rear opening, a test tube holder 13 which is a hole into which a test tube can be inserted, and a front surface 12 connected to the side surface 11 and the front surface 12. The upper housing is provided with an upper socket connecting portion 14 protruding in a cylindrical shape from the front portion to the rear side and an upper socket connecting latch 15 protruding in the center direction of the circle at the end of the upper socket connecting portion 14. (One); The front side has a cylindrical side 21, an upper socket hole 23 into which the cell separation solution is injected, and a rear side 22 connected to the side 21, and a lower portion formed outside the rear side 22. The upper socket suction plate 27 having a donut shape around the upper socket hole 23 by adsorbing the first upper and lower fixing parts 24 and the separating liquid for fixing the socket 3, the upper socket An upper socket (2) having an upper housing connecting portion (25) for engaging with the upper housing (1) by coupling with the upper socket connecting latch (15) formed inside the rear surface (2) of (2); A cylindrical side surface 31 having a rear opening, a lower socket hole 33 into which a cell separation solution is injected, and a front surface 32 connected to the side surface 31 and the outer side formed on the front surface 32 are provided. The lower upper and lower fixing section 24 and the second upper and lower fixing section 34 corresponding to the lower socket adsorption portion 36 for adsorbing the separation liquid from the side, the lower portion formed inside the front surface 32 of the lower socket 3 A lower socket 3 having a lower housing connecting portion 35 for connecting with the housing 6; A lower socket cover (4) having a circular plate formed therein for separating a liquid adsorption plate (41) for adsorbing the separation liquid injected on one side thereof and sealing the lower socket hole (33); Magnets for separating positive particles or negative particles present in the separation liquid, which are combined with the lower housing 6 below and in contact with the opposite side of the lower socket 3 of one side of the lower socket cover 4. Part 5; And a lower housing (6) having a magnet portion accommodating portion (63) for accommodating the magnet portion (5) and a lower socket fixing support portion (64) in a cylindrical shape that is open to the front. .

Hereinafter, the present invention will be described in detail with the accompanying drawings.

1 is an exploded perspective view of an embodiment of a cell separator of the present invention.

The components of the present invention are listed in order, and include an upper housing (1), an upper socket (2), a lower socket (3), a lower socket cover (4), a magnet part (5), and a lower housing (6). .

2 is a perspective view of the upper housing 1. The upper housing 1 has a cylindrical shape with a rear opening, and a test tube holder 13 is provided on the front surface 12. The test tube holder 13 is a part for inserting a test tube (not shown) for containing the separated cell separation solution during the operation of the cell separator according to the present invention into the cell separator of the present invention, a test having a normal diameter of a circle According to the shape of the tube consists of a circular hole. Inside the open cylinder of the upper housing 1, the remaining components except for the lower housing 6 described below are inserted.

An upper socket connecting portion 14 is provided inside the front housing 12 of the upper housing 1. In this embodiment, an upper portion of a cylindrical shape having a diameter smaller than the diameter of the upper housing 1 is provided inside the front housing 12. The socket connecting portion 14 is provided to protrude to the rear side. The upper socket connection portion 14 is formed with an upper socket connection latch 15 as a specific configuration means for coupling the upper housing 1 with the upper socket 2 described below, which has a circular cross section. It is connected to the upper socket connecting portion 14, it is made of a small protrusion formed in the direction of the center of the circle. The protrusion-type upper socket connection latch 15 is fixed to the upper housing connecting portion 25, which is a corresponding portion of the upper socket 2 to be described below to securely couple the upper housing 1 and the upper socket 2 to each other. It plays a role.

3 shows a perspective view of the upper socket 2. The right direction of FIG. 3 is a part connected to the upper housing 1, and the left direction of FIG. 3 is a part connected to the lower socket 3 below, which will be referred to as front and rear, respectively, for convenience of description.

The upper socket 2 is formed of a cylindrical side surface 21 having an open front and a rear surface 22 having an upper socket hole 23 into which a cell separation solution is injected.

On the outside of the rear surface 22 is formed a first upper and lower fixing part 24 for fixing the lower socket 3 to be described below and an upper socket adsorption plate 27 for adsorbing the injected separation liquid. In addition, the inside of the rear side 22 of the upper socket 2 is provided with an upper housing connecting portion 25 for coupling with the upper housing connecting latch 15 and the upper housing 1, Figure 3 As can be seen in the right figure of the upper housing connecting portion 25 is protruded in the form of a cross-section in the direction of the front side from the rear to the front side from the rear side 22 of the upper socket 2, for connecting the upper socket A plurality of grooves 26 are formed to allow the clasp 15 to be inserted.

4 shows a perspective view of the lower socket 3. The left direction of FIG. 4 is a portion connected to the upper socket 2, and the right direction of FIG. 4 is a portion connected to the lower housing 4, which is referred to herein as a front and a rear for convenience of description.

The lower socket 3 has a cylindrical side surface 31 having an open rear side, and a front surface 32 connected to the side surface 31, and a front surface 32 having a lower socket hole 33 into which a cell separation solution is injected. )

A second upper and lower fixing part 34 is formed at a shape and a position corresponding to the first upper and lower fixing parts 24 of the upper socket 2 on the outside of the front surface 32. The first upper and lower fixing parts 24 and the second upper and lower fixing parts 34 may be implemented in the form of fixing pins (fixing holes) and fixing holes (fixing pins) corresponding to each other.

In addition, the lower socket adsorption portion 36 for adsorbing the separation liquid from the side is formed in the form of a band of the edge portion of the circle of the lower socket hole 33, the lower socket ( Inside the front face 32 of 3), a lower housing connecting portion 35 for connecting with the lower housing 6 is provided from the front to the rear side.

The lower housing connecting portion 35 protrudes from the front to the rear of the lower socket 3 in the front direction, and a cross section protrudes in the shape of a circle along the outer surface of the lower socket hole 33. have.

In addition, it is preferable that a lower socket aligning groove 37 is formed on the side surface 31 of the lower socket 3 to accurately position the lower socket 3.

5 is a perspective view of the lower socket cover 4.

The lower socket cover 4 is in the form of a circular plate, one side of the circular plate functions as a separation liquid adsorption plate 41, in the present embodiment, the lower socket hole formed in the lower socket 3 ( 33) and has a circular shape and size to cover completely. When combined with the lower socket 3, the circular separation solution adsorption plate 41 completely blocks the lower socket hole 33 and serves to adsorb the injected cell separation solution.

In addition, the separation solution adsorption plate 41 may be provided with one or more lower socket cover adsorption protrusions 42 may help to adsorb the separation solution so that the separation of the cell separation solution is made stable.

6 is a perspective view of the magnet portion 5.

The magnet part 5 serves to separate positive particles or negative particles existing in the injected cell separation liquid by using magnetic force, and the magnet part accommodating part 63 provided in the lower housing 6 described below. Is accommodated in, is positioned to be in contact with the rear side of the lower socket cover (4). The magnetic force applied to the magnet part 5 can overcome the magnitude of gravity acting on the specific cells in order to move the specific cells of the cell separation solution contained in the upper socket adsorption plate 27 to the separation solution adsorption plate 41. Maintain more than enough size.

7 is a perspective view of the lower housing 6.

The lower housing 6 has a cylindrical shape that is open to the front, and has a magnet accommodating portion 63 for accommodating the magnet portion 5 in the middle of the rear side 62 of the lower housing 6. From the front side. In addition, the lower socket (3) is accurately coupled to the lower socket fixing support portion 64 and the lower socket aligning groove 37 for fixing the lower socket 3 in contact with the inner surface of the lower socket (3) A lower socket fixing protrusion 65 for positioning is provided.

8 is a view illustrating a mechanism in which the upper housing 1 and the lower housing 6 are coupled to and separated from each other according to an embodiment of the present invention.

The release surfaces 16 and 66 formed on the side surfaces of the upper housing 1 and the lower housing 6 are in contact with each other when the upper housing 1 and the lower housing 6 are coupled to each other. Instead of forming the release surfaces 16 and 66 of the upper housing 1 and the lower housing 6 in a straight line, the upper housing 1 and the upper housing 1 and the case When the lower housing 6 is separated, when the upper housing 1 or the lower housing 6 is twisted in a predetermined direction (clockwise or counterclockwise), it may be easily separated from each other by the height difference.

Next, a method of separating the cell separation solution using the cell separator of the present invention will be described.

9 is a view showing a form in which the lower housing 6, the lower socket 3 and the upper socket 2 are coupled.

An assembly in which the upper socket 2 and the lower socket 3 are coupled to the front surface of the lower housing 6 to which the magnet part (not shown) is coupled is coupled through a portion of the lower socket 3.

The upper housing 1 is coupled to the assembly of FIG. 9. In the coupling, the grooves of the detaching surfaces 16 and 66 of the upper housing 1 and the lower housing 6 fit together well (FIG. 10).

Cell separation solution is injected into the conjugate of FIG. 10 using a pipette. The injection of the cell separation solution is made through the test tube holder 13, the upper socket hole 23, and the lower socket hole 33 of the upper housing 1 and the combination of the upper socket 2 and the lower socket 3. Inject well to inside (Figure 11).

After injecting the cell separation solution into the conjugate, the test tube is bound to the test tube holder of the upper housing (1).

Invert the cell separator of FIG. 12. After inverting, left for 10 to 15 minutes to separate the cell separation solution (Fig. 13).

When it is determined that the cells have been separated after a certain time, while the test tube is coupled, the upper housing 1 is turned in the direction of the disengagement surface of the upper housing 1 and the lower housing 6 as shown in the arrow direction of FIG. 14. Through this process, the fixing pins (not shown) of the upper and lower sockets 2 and 3, which are coupled therein, are broken and separated vertically (FIG. 14).

In each of the upper and lower housings 1 and 6, the positive and negative cells are separated into the upper and lower sockets 2 and 3 by the magnetic force of the magnet unit 5, respectively. . The cell separation solution separated in the upper housing 1 is collected in a test tube so that it can be used (FIG. 15).

16 and 17 are photographs showing the form of the cell separation liquid in the cell separator in each step when separating the cells using the cell separator according to the present invention.

(a) is a photograph immediately after the cell separation solution is injected between the upper socket 2 and the lower socket 3.

(b) is a state in which the assembly is located at right angles to the direction of gravity in the process of inverting the assembly, even when the separation liquid is not collapsed even when it is vertical as shown in the photograph.

(c) ~ (e) is a photograph when the separation between the layers occurs, as the separation interval is gradually widened, gravity, the lower socket adsorption portion 36 and the separating liquid adsorption plate 41 of the lower socket cover ) Shows the state of being separated into the upper and lower parts by the suction force and magnetic force by the magnet portion (5).

18 is a diagram schematically showing a mechanism for separating cell separation liquid using the cell separator of the present invention.

In the first picture, the magnet 5 having positive magnetic force is in contact with the lower socket cover 4 covering the lower socket hole in the assembly where the upper and lower sockets 2 and 3 are coupled. The cell separation solution injected through the upper and lower socket holes is filled in the assembly, and the cell separation solution is mixed with the positive particles and the negative particles before separation.

In the second figure, when the magnet part 5 and the assembly 2,3 are inverted by 180 °, the magnet part 5 is positioned upward, attracting negative particles in the cell separation solution, and the positive particles are opposite to the magnet. They are gathered downward to move away from wealth. When the first upper and lower fixing parts 24 are broken, the combination of the upper socket 2 and the lower socket 3 is separated, and the cell separation solution is separated from the suction socket 41 and the lower socket cover suction protrusion 42 of the lower socket cover. ) And the lower socket adsorption portion 36 starts to separate by the suction force and gravity.

In the third picture, the separation between the upper and lower cell separation liquids is completely completed while the upper and lower sockets 2 and 3 are spaced apart. Negative particles are collected in the cell separation liquid on the lower socket 3 side adjacent to the magnet part 5, and cell separation liquid containing the positive particles is collected on the opposite side of the upper socket 2, and the upper socket 2 The cell separation solution collected at the) side may be dropped downward by gravity through the upper socket hole 23. Each isolated cell particle is collected in a test tube or pipette and used for the required application.

1: upper housing 2: upper socket
3: lower socket 4: lower socket cover
5: magnet part 6: lower housing
23: upper socket hole 27: upper socket suction plate
33: lower socket hole 41: separation liquid adsorption plate

Claims

A cylindrical side 11 having an open rear, a test tube holder 13 which is a hole into which a test tube can be inserted, and a front side 12 connected to the side 11 and an inner side of the front side 12 are provided. An upper housing (1) provided with an upper socket connection portion (14) protruding in a cylindrical shape from the front portion to the rear side and an upper socket connection latch (15) protruding in the center direction of the circle at the end of the upper socket connection portion (14). ;
The front side has a cylindrical side 21, an upper socket hole 23 into which the cell separation solution is injected, and a rear side 22 connected to the side 21, and a lower portion formed outside the rear side 22. The upper socket suction plate 27 having a donut shape around the upper socket hole 23 by adsorbing the first upper and lower fixing parts 24 and the separating liquid for fixing the socket 3, the upper socket An upper socket (2) having an upper housing connecting portion (25) for engaging with the upper housing (1) by coupling with the upper socket connecting latch (15) formed inside the rear surface (2) of (2);
A cylindrical side surface 31 having a rear opening, a lower socket hole 33 into which a cell separation solution is injected, and a front surface 32 connected to the side surface 31 and the outer side formed on the front surface 32 are provided. The lower upper and lower fixing section 24 and the second upper and lower fixing section 34 corresponding to the lower socket adsorption portion 36 for adsorbing the separation liquid from the side, the lower portion formed inside the front surface 32 of the lower socket 3 A lower socket 3 having a lower housing connecting portion 35 for connecting with the housing 6;
A lower socket cover (4) having a circular plate formed therein for separating a liquid adsorption plate (41) for adsorbing the separation liquid injected on one side thereof and sealing the lower socket hole (33);
Magnets for separating positive particles or negative particles present in the separation liquid, which are combined with the lower housing 6 below and in contact with the opposite side of the lower socket 3 of one side of the lower socket cover 4. Part 5; And
A lower housing 6 having a cylindrical shape open to the front and having a magnet part accommodating part 63 for accommodating the magnet part 5 and a lower socket fixing support part 64.
Cell separator comprising a.

The method of claim 1,
The lower socket cover adsorption protrusion 42 is formed to protrude from the separating liquid adsorption plate 41 of the lower socket cover 4 so that a proper solution of the separation liquid is stably adsorbed to the separation liquid adsorption plate 41. Cell separator.

The method of claim 1,
It is formed to correspond to the side 11 of the upper housing 1 and the side 61 of the lower housing 6, there is a step of the height of the contact between the upper housing 1 and the lower housing (6). When the upper housing 1 and the lower housing 6 are coupled to each other so that the upper housing 1 or the lower housing 6 is twisted in a predetermined direction, the upper housing 1 is separated from each other by the height difference. Separation surface and the lower housing 6 The cell separator, characterized in that the separation surface (16,66) is formed.

The method of claim 1,
A lower socket aligning groove 37 for arranging the lower socket 3 is formed on the side surface 31 of the lower socket 3, and the lower socket fixing of a shape corresponding to the lower socket aligning groove 37 is performed. Cell separator characterized in that the molten protrusion (65) is formed at a corresponding position of the lower housing (6).

The method of claim 1,
The first upper and lower fixing part 24 and the second upper and lower fixing part 34 are fixed pins and fixing holes, or a cell separator, characterized in that the fixing holes and fixing pins, respectively.