KR101539453B1 - Cell tissue homogenizer and cell tissue homogenizing or extracellular matrix extracting method using the same - Google Patents

Abstract

The present invention relates to a cell homogenizer connected to a syringe and including a cutting member, and a method of homogenizing a cell tissue using the same or a method of extracting an extracellular matrix.
According to one embodiment of the present invention, homogenization of cell tissue by connecting a syringe to a cell homogenizer containing a cutting member is inexpensive because it does not require additional power such as electricity, etc., and the homogenization can be achieved by simply operating the piston of the syringe It is convenient and possible, and ③ there is no need to prepare additional equipment, so time is short, and ④ homogenization is performed inside the enclosed device from outside, so that the possibility of contamination by foreign substances is low.
In addition, according to another embodiment of the present invention, the cell homogenizer can be bound and separated, so that it is easy to clean the equipment or replace the auxiliary substrate such as the cutting member.

Description

[0001] The present invention relates to a cell homogenizer and cell tissue homogenizing or extracellular matrix extracting method using same,

The present invention relates to a cell homogenizer including a cutting member and connected to a syringe, and a method of homogenizing a cell tissue using the same, or a method of extracting an extracellular matrix.

Extracts that can be separated from cell tissue can be used for various purposes, and especially extracellular matrix, which can be obtained from adipose tissue, is used in various fields such as treatment and molding .

However, the cell tissue is generally composed of various constituents combined with each other. In the case of adipose tissue, since it is composed of connective tissue fibroblasts, reticulum cells, micro-fat droplets and fat globes, In order to separate the desired substance from the cell tissue of the cell, tissue must first be homogenized by cutting, grinding, or the like.

Particularly, as a method of homogenizing cell tissue for separating extracellular matrix from cell tissue, conventionally, cell tissue is minced by a cutting tool such as a surgical blade, and a medical grinder The grinding method was used. In particular, a medical device named 'Filler Geller' was mainly used as a medical pulverizer for pulverizing cell tissue including adipose tissue (see FIG. 1).

However, there are the following problems in the method of directly directing the cell tissue.

First, since the cell tissue is directly exposed to the outside of a human hand, there is a high possibility of contamination by foreign substances.

Second, it is inconvenient and time-consuming because a person directly applies force to the cell tissue with a cutting tool.

On the other hand, a method of pulverizing cell tissue with a medical pulverizer is a method of pulverizing pulverized cells directly in contact with a cell tissue contained in a container. However, the possibility of contamination with foreign substances is lower than the direct pulverization method.

First, since the price of the electric pulverizer is expensive, it has a disadvantage that it is expensive.

Second, since medical mills are generally bulky and heavy, it takes a long time to install and prepare them.

Third, although the possibility of contamination by foreign substances is low, the crushing operation is not performed in a closed space, and there is still possibility of contamination from the outside because the crusher is exposed to the outside before and after the crushing operation.

Therefore, there has been a demand for a cell tissue homogenization technique capable of solving the above problems.

Published Patent No. 2012-0082298

It is an object of the present invention to solve the problems of the prior art and to provide a cell tissue homogenizer which is inexpensive, convenient, time-consuming, A cell tissue homogenization method or an extracellular matrix extraction method using the same.

In addition, when it is made of a cell homogenizer capable of binding and separating, it is desirable to clean equipment or to replace an auxiliary substrate or the like.

In order to solve the problems and problems of the prior art, the present invention provides a cell homogenizer in which both ends are coupled to the injection part 17 of the syringe 16 to connect the two syringes 16, Specifically, the connector body 11 has a passage 12 formed therein. A fastening portion 13 located at both end portions of the connection port main body 11 and connected to the injection portion 17 of the syringe 16 to thereby connect the inside of the syringe 16 and the passage 12; And the at least one cutting member 14 installed inside the passage 12. The sample including the cell tissue injected into the syringe 16 is moved through the passage 12 of the connecting tube body 11 And the cell tissue is homogenized by being crushed by a cutting member (14) provided inside the channel (12).

In addition, two or more cutting members 14 may be provided so that a plurality of the cutting members 14 are arranged to intersect each other at 90 degrees, and may be positioned perpendicular to the moving direction of the sample. The passage 12 has a cross- .

On the other hand, the cell tissue may be a cell tissue including adipose tissue.

According to another embodiment of the present invention, the connector main body 11 includes: a female connector 22 having a cavity 21 formed therein; (24) formed with a small cavity (23) therein and capable of being fitted into the cavity of the female connector (22); And at least one fixing groove 25 is provided on the inner circumferential surface of the passage 12 so that the cutting member 14 can be bound and separated to the fixing groove 25, The cutting member engaging portion 26 including the cutting member 14 is inserted into the small cavity 23 of the connecting hole 24 so that the cutting member engaging portion 26 including the cutting member 14 can be inserted into the small hole 23 And the male connecting port 24 into which the cutting member engaging portion 26 is inserted can be inserted into and bound to the cavity 21 of the female connector 22.

The o-ring 27 may be coupled to the outer circumferential surface of the male connection port 24 so that the o-ring 27 may be coupled to the male- The connector 24 can be inserted into the cavity 21 of the female connector 22 to be engaged.

In the meantime, the present invention includes a coupling step of coupling the cell-tissue homogenizer coupling part 13 and the injection part 17 of the two syringes 16 according to the embodiment described above, respectively; And a sample containing the cell tissue injected into the syringe 16 are crushed by the cutting member 14 provided inside the passage 12 while moving through the passage 12 of the connecting tube body 11 to homogenize the cell tissue Wherein the pulverizing step is repeated at least once or more in order to cut the cell tissue to a smaller size and homogenize the cell tissue.

The present invention also provides an extracellular matrix extraction method further comprising the step of separating the extracellular matrix by centrifuging the homogenized cell tissue by the above-described cell tissue homogenization method.

According to one embodiment of the present invention, homogenization of cell tissue by connecting a syringe to a cell homogenizer containing a cutting member is not economical because it does not use a complicated device requiring additional power such as electricity , ② It is convenient because homogenization is possible by simply operating the piston of the syringe. ③ Because there is no need to prepare additional equipments, it takes less time. ④ Because homogenization is performed inside the enclosed device from the outside, Effect can be obtained.

In addition, according to another embodiment of the present invention, when the cell homogenizer is manufactured as a separate type rather than an integral type, each of the accessory components can be individually bound and separated so that the equipment is washed or the accessory base material such as a cutting member is replaced It has an advantage that it is easy.

1 shows the appearance of 'Filler Geller' as a medical fat tissue grinder as a prior art.
Figure 2 is a schematic representation of the structure of a cell homogenizer coupled to a syringe 16, in accordance with an embodiment of the present invention.
Figs. 3 and 4 schematically show that the connector body 11 is designed to be engageable and detachable, according to an embodiment of the present invention.
Fig. 5 schematically shows another embodiment of the connector body 11 which can be bound and separated.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. Prior to the description, terms and words used in the present specification and claims should not be construed as limited to ordinary or dictionary meanings and should be construed in accordance with the technical concept of the present invention.

Throughout this specification, when a member is " on " another member, this includes not only when the member is in contact with another member, but also when there is another member between the two members.

Throughout this specification, when an element is referred to as "including" an element, it is understood that it may include other elements as well, without departing from the other elements unless specifically stated otherwise.

FIG. 2 schematically illustrates the structure of a cell homogenizer connected to a syringe according to an embodiment of the present invention.

The cell tissue homogenizer has a fastening part 13 at both ends of the connecting rod main body 11 and a passage 12 formed inside the connecting rod main body 11. Also, at least one or more cutting members 14 or filters 15 may be included in the interior of the passageway 12.

The connection port body 11 may have various shapes such as a cylindrical shape or a rectangular parallelepiped shape, and the passage 12 therein preferably has a shape of a tube having a circular section, but is not limited thereto. The cross-sectional area of the passage 12 in the radial direction is preferably constant, but is not limited thereto.

The fastening portion 13 is engaged with the injection portion 17 of the syringe 16 and serves to connect the inside of the syringe 16 with the passage 12. The sample injected into one syringe 16 can enter the passage 12 in the connecting port body 11 through the fastening portion 13 and pass through the passage 12 to the inside of the other syringe 16 Can also enter.

The fastening portion 13 and the injection portion 17 can be coupled in various ways and are not limited thereto. For example, male thread may be formed on the outer circumferential surface of the fastening portion 13, female thread groove may be formed on the inner circumferential surface of the injection portion 17, So that they can be engaged with each other in a screw-engaged manner.

The cutting member 14 may be any member capable of cutting an object, such as a surgical cutting member, a knife including a razor blade, and the shape is not limited thereto.

Only one cutting member 14 may be provided in the passage 12, and a plurality of the cutting members 14 may be disposed parallel or intersect each other. The cutting member 14 is preferably positioned perpendicular to the direction in which the injected sample moves, and the blade of the cutting member 14 is preferably disposed in the direction of movement of the sample, but is not limited thereto.

The cutting member 14 may be a blade provided with both ends thereof.

The filter 15 may be installed in front of or behind the cutting member 14 and can perform the function of separating the crushed tissue by the cutting member 14 according to the pore size of the filter.

The two syringes 16 are connected to the fastening portion 13 and the piston 18 of the syringe 16 or one or both syringes 16 is separated to inject a sample including cell tissue into the syringe 16. When one piston 18 is pressed, the sample in the syringe 16 is moved toward the other syringe 16 by the pressure.

The cell tissue contained in the sample is cut while being moved in contact with the cutting member 14 and pressed against the opposite piston 18 which has been relaxed by the pressure, the cell tissue included in the sample moves again in the opposite direction, ).

As the above process is repeated, the cell tissue is pulverized to a smaller size, and the cell tissue can be homogenized. In addition, the above process may further include a step of separating the extracellular matrix by centrifuging the homogenized cell tissue.

Further, when a filter is further included in front of or behind the cutting member 14, the cell tissue broken by the cutting member 14 is separated in size by the filter. That is, only a desired portion of the cell tissue that has been pulverized through the filter can be obtained.

Meanwhile, the cell tissue used in the present invention may be a cell tissue including adipose tissue, but is not limited thereto.

The cell homogenizer may be fabricated as an integral unit, and may be used in a disposable or semi-permanent manner, but is not limited thereto.

Fig. 3 and Fig. 4 schematically show the connector main body 11 designed so as to be able to be bound and detached, according to another embodiment of the present invention.

The connector main body 11 may include a female connector 22 having a cavity 21 formed therein, a male connector 24 having a small cavity 23 formed therein, and a cut member engaging portion 26.

The cutting member fastening portion 26 is inserted into the small cavity 23 of the male coupling hole 24 and the male coupling hole 24 including the cutting member fastening portion 26 is inserted into the cavity 21 of the female coupling hole 22 The connector main body 11 can be fastened in such a manner that it can be separated in the reverse order of the fastening manner, so that it is easy to clean the inside.

On the other hand, the cutting member fastening portion 26 is provided with a passage 12 through which a sample containing cell tissue moves. At least one or more fastening grooves 25 are provided on the inner circumferential surface of the passage 12. The cutting member 14 can be bound to and separated from the fixing groove 25 and can be used for various purposes such as separating and cleaning the cutter member 14, reassembling it, or replacing it with another one.

The connector body 11 may further include an O-ring 27. The O-ring 27 seals the sample including the cell tissue moving in the passage 12 from the outside and is connected to the outer circumferential surface of the male connection port 24 and has the male connection port 24 Are inserted into the cavity 21 of the female connector 22.

The material of the O-ring 27 is preferably rubber, but not limited thereto.

The O-ring coupling groove 24 may be formed on the outer circumferential surface of the male coupling hole 24, but the present invention is not limited thereto.

5 is a perspective view showing another embodiment of the connector main body 11 in which the male thread 31 is formed on the outer circumferential surface of the male threaded portion 24 and the male thread 31 is formed on the inner peripheral surface of the hollow 21 of the female threaded portion 22 And the female thread groove 32 is formed.

Therefore, when the male coupling hole 24 and the female coupling hole 22 are coupled, the male thread 31 and the female screw groove 32 are engaged with each other, so that a firm coupling can be maintained.

The cell homogenizer capable of binding and separating the connector body 11 can be used semi-permanently, but can also be fabricated and used in a disposable manner, but is not limited thereto.

<Cell structure according to the number of cutting members Homogenization  Performance evaluation>

The following experiment was carried out to investigate the homogenizing ability of cell tissue according to the number of blades contained in the cell homogenizer.

The homogenization performance according to the number of cutting members was evaluated by measuring the number of pressures of the syringe piston required for homogenizing the fatty tissue to a certain level or more. A constant level is defined as the level at which the homogenized adipose tissue can be completely separated into free oil, extracellular matrix and water when centrifuged at 12000 rpm for 15 minutes with a SIGMA 6-16KS centrifuge .

The syringe attached to both ends of the present invention used in this experiment was a 50 cc BD luer-lock syringe and 30 cc of adipose tissue was used as the cell tissue.

As shown in FIG. 5, the cell homogenizer is a cell homogenizer capable of binding and separating by male threads and female thread grooves. The inner diameter of the inner passage is 4.3 mm, the outer diameter of the connecting body is 18 mm, 0.3 mm, and the size of the fastening portion was M16x1 TAB.

First, three types of cell homogenates were prepared as follows.

Experimental Example 1: A cell tissue homogenizer in which a cutting member having a blade thickness of 0.3 mm was placed.

Experimental Example 2: A cell tissue homogenizer in which two cutting members having a blade thickness of 0.3 mm were arranged so as to intersect each other at an internal angle of 90 degrees.

Experimental Example 3: A cell tissue homogenizer in which three cutting members each having a blade thickness of 0.3 mm were arranged so as to intersect each other with an internal angle of 60 DEG.

The syringe was connected to both ends of each cell homogenizer, the piston of one syringe was separated, the fat tissue was injected into the syringe, and the piston was again connected to the syringe. Then, the pistons of the two syringes were alternately repeatedly pressed to homogenize by crushing the fat tissue.

In the case of Experimental Example 1, the pressing operation was required about 50 to 70 times. In Experimental Example 2, the pressing operation was required about 20 to 30 times. In Experimental Example 3, It was difficult to obtain a homogenized fat tissue at a certain level.

From the above experiment, it was found that the use of two cutting members (Experiment 2) is the most efficient.

<Cell structure according to the arrangement form of the cutting member Homogenization  Performance evaluation>

In order to evaluate the homogenization performance according to the arrangement form of the cutting member included in the cell homogenizer according to an embodiment of the present invention, the following experiment was conducted.

As in the previous experiment, the syringe used in this experiment was a 50 cc BD luer-lock syringe and 30 cc of adipose tissue was used as the cell tissue. The experimental results are as follows.

The cell tissue homogenizer used was the same cell homogenizer as in Example 1, except that the number of the cutting members was fixed at two and only their arrangement was changed as follows.

Experimental Example 4: A cell-tissue homogenizer in which two cutting members having a blade thickness of 0.3 mm were arranged parallel to each other (0 deg.

Experimental Example 5: A cell tissue homogenizer in which two cutting members having a blade thickness of 0.3 mm were arranged so as to cross each other at an internal angle of 45 degrees.

EXPERIMENTAL EXAMPLE 6 A cell tissue homogenizer in which two cutting members having a blade thickness of 0.3 mm were arranged so as to intersect each other with an internal angle of 90 degrees.

The syringe is connected to both ends of each cell homogenizer, the piston of one syringe is separated, the fat tissue is injected into the syringe, and the piston is again connected to the syringe. Then, as the pistons of the two syringes were alternately and repeatedly pressed, it was observed whether or not the fat tissue was smoothly pulverized.

In the case of Experimental Example 4, a mass of untreated cell tissue was observed after about 30 times of pressing operation, and such a cell mass interferes with the homogenization process by blocking the internal passage of the cell homogenizer or the injection portion of the syringe.

In the case of Experimental Example 5, some unfractured cell tissues were partially observed after about 20 times of pressing operation, but only uniformly cut cell tissue particles were observed after about 30 times of pressing operation. In Experimental Example 6, Even after 20 pressing operations, only uniformly cut cell tissue particles could be identified.

It can be seen from Example 2 that the cutting members used in the cell homogenizer of the present invention are more efficient to be arranged in a cross shape, especially a cross shape, than when arranged in parallel.

<Cell structure according to the cross-sectional area change of the channel Homogenization  Performance evaluation>

In order to evaluate the homogenization performance according to the variation of the cross-sectional area in the radial direction of the channel of the cell homogenizer according to an embodiment of the present invention, the following experiment was conducted.

As in previous experiments, the syringe used in this experiment was a 50 cc BD luer-lock syringe and 30 cc of adipose tissue was used as the cell tissue. The experimental results are as follows.

The cell homogenizer was prepared so that two cutting members were installed so as to have an internal angle of 90 degrees with each other and the same cell homogenizer used in Example 1 or Example 2 was used except that the inner diameter of the passage was changed as follows Respectively.

Experimental Example 7: A cell homogenizer having an inner diameter of 3.9 mm at one end and an increasing diameter at the other end and an inner diameter of 4.7 mm at the other end.

EXPERIMENTAL EXAMPLE 8 A cell homogenizer having an inner diameter of 3.9 mm at both ends and an inner diameter of 4.7 mm at the center with an increasingly wider passage.

Experimental Example 9: Cellular homogenizer having a constant inner diameter of 4.3 mm in the entire channel.

The syringe was connected to both ends of each cell tissue homogenizer, the piston of one syringe was separated, the fat tissue was injected into the syringe, and the piston was again connected to the syringe. Then, the pistons of the two syringes were alternately repeatedly pressed to check whether or not the fat tissue was smoothly crushed.

In Experimental Example 7 and Experimental Example 8, a bottle-neck phenomenon occurs at a portion where the cross-sectional area is small, so that it is difficult for the cell tissue to move from one syringe to another syringe, And the homogenization performance was decreased. However, in the case of Experimental Example 9, this problem did not occur, and uniformly cut cell tissue particles could be obtained.

Therefore, through the above experiment, it was found that the cross - sectional area of passage is the most efficient when constant.

Table 1 below summarizes the homogenization performance of the cell homogenizer of the present invention based on the results of comparative experiments of Experiments 1 to 9 confirmed in Examples 1 to 3 as described above.

Experiment order Number of cutting members Cutting Member Placement Passage inner diameter Homogenization performance evaluation One One - 4.3 mm schedule medium 2 2 Cross at 90 ° 4.3 mm schedule Prize 3 3 Cross at 60 ˚ 4.3 mm schedule Ha 4 2 parallel 4.3 mm schedule Ha 5 2 Cross at 45 ° 4.3 mm schedule medium 6 2 Cross at 90 ° 4.3 mm schedule Prize 7 2 Cross at 90 ° 3.9 mm to 4.7 mm medium 8 2 Cross at 90 ° 3.9 mm to 4.7 mm medium 9 2 Cross at 90 ° 4.3 mm schedule Prize

11: connector body 22: female connector
12: passage 23: small cavity
13: fastening part 24:
14: cutting member 25: fixing groove
15: filter 26: cutting member fastening portion
16: Syringe 27: O-ring
17: injection part 28: O-ring fastening groove
18: Piston 31: Male threads
21: cavity 32: female screw groove

Claims (9)

Each of which is connected to an injection part 17 of a syringe 16 to connect two syringes 16,
(11) having a passage (12) formed therein;
A fastening portion 13 located at both end portions of the connector main body 11 and connected to the injection portion 17 of the syringe 16 to thereby connect the inside of the syringe 16 with the passage 12; And
(12) provided in the passage (12) And a cutting member (14)
The cutting members 14 are arranged so that a plurality of the cutting members 14 cross each other at an angle of 90 占 and are positioned perpendicular to the moving direction of the sample,
The passage 12 has a constant cross-sectional area in the radial direction,
A sample containing the cell tissue injected into the syringe 16 is crushed by the cutting member 14 provided inside the passage 12 while moving through the passage 12 of the connecting pipe body 11, Lt; RTI ID = 0.0 &gt; homogenized. &Lt; / RTI &gt; delete delete delete The method according to claim 1,
Wherein said cell tissue comprises adipose tissue. Each of which is connected to an injection part 17 of a syringe 16 to connect two syringes 16,
(11) having a passage (12) formed therein;
A fastening portion 13 located at both end portions of the connector main body 11 and connected to the injection portion 17 of the syringe 16 to thereby connect the inside of the syringe 16 with the passage 12; And
And at least one cutting member (14) installed inside the passage (12)
The connector main body (11)
A female connector (22) having a cavity (21) formed therein;
A water hole (24) in which a small cavity (23) is formed and which can be fitted into the cavity of the female connector (22); And
And at least one fixing groove 25 is provided on the inner circumferential surface of the passage 12 so that the cutting member 14 can be bound and separated to the fixing groove 25 , And a cutting member engaging portion (26) that can be fitted into the small cavity (23) of the male connector (24)
The cutting member engaging portion 26 including the cutting member 14 is inserted into the small cavity 23 of the male coupling hole 24,
The male connection port 24 into which the cutting member coupling portion 26 is inserted is inserted and bound to the cavity 21 of the female coupling port 22,
A sample containing the cell tissue injected into the syringe 16 is crushed by the cutting member 14 provided inside the passage 12 while moving through the passage 12 of the connecting pipe body 11, Lt; RTI ID = 0.0 &gt; homogenized. &Lt; / RTI &gt; The method according to claim 6,
The o-ring 27 is coupled to the outer circumferential surface of the male connection port 24 so that the o-ring 27 is connected to the male connection port 24 Is inserted and bound to the cavity (21) of the female connector (22). A coupling step of coupling the coupling part (13) of the cell homogenizer according to any one of claims 1 and 5 to the injection part (17) of the two syringes (16) respectively; And
A sample containing the cell tissue injected into the syringe 16 is crushed by the cutting member 14 provided inside the passage 12 while moving through the passage 12 of the connecting pipe body 11, And a homogenizing step,
Characterized in that said crushing step is repeated at least once more in order to cut and homogenize said cell tissue to a smaller size. The method for extracting extracellular matrix according to claim 8, further comprising centrifuging the homogenized cell tissue to separate the extracellular matrix.

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