KR101959208B1 - Droplet based biochip, a method for producing a tumor, and a method for deciding a metastasis tumor - Google Patents

Abstract

A droplet-based biochip, a method of producing a tumor using the same, and a method of determining a metastasis of a tumor produced by the method, the droplet-based biochip includes a body portion, a first inlet portion, a first flow portion, a second inlet portion, A third flow path portion and an outflow portion. An oil phase flows into the first inlet portion toward the inside of the body portion. The oil channel flows through the first flow path portion. The second inflow portion is spaced apart from the first inflow portion, and the water phase material flows into the inside of the body portion. And the aqueous phase flows in the second flow path portion. And the third flow path portion flows through the cell droplet. The outlet portion is formed at an end of the third flow path portion, and the cell droplet flows out of the body portion.

Description

TECHNICAL FIELD [0001] The present invention relates to a droplet-based biochip, a tumor production method using the same, and a method for determining a metastasis of a tumor produced by the method. [0002] DROPLET BASED BIOCHIP, A METHOD FOR PRODUCING A TUMOR, AND METHOD FOR DECIDING A METASTASIS TUMOR [

The present invention relates to a droplet-based biochip, a method of producing a tumor using the same, and a method of determining the metastasis of the tumor. More particularly, the present invention relates to a droplet-based biochip for mass production of a 3-dimensional tumor, The present invention relates to a method for producing a tumor and a method for determining the metastasis of a tumor to determine the metastasis of the tumor produced through the tumor production method.

It is common to use a cell model to determine the drug efficacy of a new drug substance. In particular, in the case of a 3-dimensional cell, the drug acts as a factor that interferes with the delivery of the drug substance, .

However, a general cell has a characteristic of growing in a two-dimensional form, and the cells of the two-dimensional form thus grown have physical and biological characteristics different from those of the three-dimensional form, There is a difficult problem.

Accordingly, techniques for culturing three-dimensional cells have been developed, and many methods such as hanging drop, micromolding, etc. have been used so far. On the other hand, SpheroFilm (iN CYTO) and Perfecta3D (3D Biomatrix) are commercially available as the current culture products, but the limited number of 361 wells (SpheroFilm), 96 or 384 wells (Perfecta3D) There is a limit to producing 3D tumor models.

Therefore, there is a need for a technique for producing a three-dimensional tumor model more effectively by solving the problem of low productivity in the production of such a three-dimensional tumor model.

Korean Patent No. 10-1544391 Korean Patent No. 10-1637937

SUMMARY OF THE INVENTION Accordingly, it is an object of the present invention to provide a three-dimensional tumor model using droplets, The present invention relates to a droplet-based biochip capable of mass production with high productivity.

Another object of the present invention is to provide a tumor production method using the droplet-based biochip.

It is still another object of the present invention to provide a method for determining the metastasis of a tumor produced by the tumor production method.

The droplet-based biochip according to one embodiment of the present invention includes a body portion, a first inlet portion, a first flow portion, a second inlet portion, a second flow portion, a third flow portion, and an outlet portion do. An oil phase flows into the first inlet portion toward the inside of the body portion. The oil channel flows through the first flow path portion. The second inflow portion is spaced apart from the first inflow portion, and the water phase material flows into the inside of the body portion. And the aqueous phase flows in the second flow path portion. And the third flow path portion flows through the cell droplet. The outlet portion is formed at an end of the third flow path portion, and the cell droplet flows out of the body portion.

In one embodiment, the apparatus may further include first and second branch flow path portions branched from the first flow path portion, and the oil phase material flows separately from each other.

In one embodiment, the ends of the first and second branch channel portions are connected to the ends of the second channel portion, and the liquid material and the oil phase material are mixed with each other to form the cell liquid droplet.

In one embodiment, the oil phase material may flow in opposite directions from the ends of the first and second branch flow paths.

In one embodiment, the water-based material may comprise cells.

In order to realize the object of the present invention described above, an oily material is produced in a tumor production method according to an embodiment. Thereby preparing a water-based material. And preprocesses the biochip in which a plurality of flow paths are formed. The oily material and the water-based material are respectively injected into the bio-chip to produce a cell droplet. The resulting cell droplet is cultured to complete a tumor model.

In one embodiment, in the step of preparing the water-based material, the cancer cell line can be prepared by mixing the cell culture liquid on a single cell basis.

In one embodiment, the step of pretreating the biochip may include a step of subjecting the inner walls of the channels of the biochip to hydrophobic treatment, and a step of treating the inner wall of the channels of the biochip with bovine serum albumin (BSA).

In one embodiment, in the step of generating the cell droplet, the oily material flowing through the first flow path portion of the flow path portions and the water phase material flowing through the second flow path portion of the flow path portions are mixed with each other, .

In one embodiment, in the step of generating the cell droplet, the flow velocity of each of the oily material and the water-based material, or the flow velocity ratio of the oily material and the water-based material is controlled in consideration of the size and productivity of the cell droplet .

In one embodiment, the flow rates of each of the oily material and the water-based material may be 120 [mu] l / min and 80 [mu] l / min.

In one embodiment, the step of completing the tumor model, the cells can be droplets 37.5 ℃, 24 hours at 5% CO ₂ concentration of the culture environment.

In the method for determining the metastasis of a tumor according to an embodiment for realizing the object of the present invention described above, an oil substance is prepared. Thereby preparing a water-based material. And preprocesses the biochip in which a plurality of flow paths are formed. The oily material and the water-based material are respectively injected into the bio-chip to produce a cell droplet. The resulting cell droplet is cultured to complete a tumor model. To determine whether the completed tumor model is a single tumor or a single cell.

In one embodiment, if the tumor model is a single tumor, it is determined to be a non-metastatic tumor, and if the tumor model is a single cell, it can be determined to be a metastatic tumor.

According to the embodiments of the present invention, the biochip is designed so that the oily material and the water-based material flow through separate channels and can be mixed with each other to form droplets, thereby effectively forming droplets containing cells therein, This makes it easy to complete a 3D tumor model.

Particularly, it is possible to control the size and productivity of the cell droplet by controlling the flow rate or the flow rate of the oily material and the water-based material during the formation of the cell droplet for the three-dimensional tumor model, You can complete the model.

In addition, since the cell droplet can be continuously produced through mixing of the continuous water-based material and the oily material, and a three-dimensional tumor model can be produced only through culturing for 24 hours, a relatively large amount of three-dimensional tumor can be produced Do.

In addition, it is possible to judge whether metastasis is metastatic or non-metastatic based on the cell unit status of the tumor model without using the conventional protein or gene test method, and thus it is possible to judge the metastasis of the tumor relatively easily.

1 is a perspective view illustrating a droplet-based biochip according to an embodiment of the present invention.
FIG. 2 is an enlarged view of the 'A' region of FIG. 1.
FIG. 3 is a flowchart illustrating a tumor production method using the droplet-based biochip of FIG. 1 and a method of determining the metastasis of a tumor produced by the tumor production method.
FIG. 4A is an image of the cytolysy produced through the tumor production method of FIG. 3, and FIG. 4B is an enlarged view of the 'B' region of FIG. 4A.
FIG. 5 is an image of a tumor model cultured through the tumor production method of FIG. 3; FIG.
6A and 6B are images showing non-metastatic tumors and enlarged views thereof.
7A and 7B are images showing metastatic tumors and enlarged views thereof.

While the present invention has been described in connection with what is presently considered to be the most practical and preferred embodiment, it is to be understood that the invention is not limited to the disclosed embodiments. It is to be understood, however, that the invention is not intended to be limited to the particular forms disclosed, but on the contrary, is intended to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the invention. Like reference numerals are used for like elements in describing each drawing. The terms first, second, etc. may be used to describe various components, but the components should not be limited by the terms.

The terms are used only for the purpose of distinguishing one component from another. The terminology used in this application is used only to describe a specific embodiment and is not intended to limit the invention. The singular expressions include plural expressions unless the context clearly dictates otherwise.

In the present application, the term "comprises" or "comprising ", etc. is intended to specify that there is a stated feature, figure, step, operation, component, But do not preclude the presence or addition of one or more other features, integers, steps, operations, components, parts, or combinations thereof.

Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in commonly used dictionaries are to be interpreted as having a meaning consistent with the contextual meaning of the related art and are to be interpreted as either ideal or overly formal in the sense of the present application Do not.

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a perspective view illustrating a droplet-based biochip according to an embodiment of the present invention. FIG. 2 is an enlarged view of the 'A' region of FIG. 1.

1 and 2, the droplet-based biochip 10 according to the present embodiment includes a body 100, a first inlet 110, a first flow passage 111, a first branch flow passage 112 The second flow path portion 113, the second inflow portion 120, the second flow path portion 121, the third flow path portion 131, and the outflow portion 130.

The body part 100 forms a body of the bio-chip 10 and includes a transparent material, and has a thin chip shape as a whole.

The first inlet 110 is formed at one end of the body 100 and is formed as an opening connected to the inside of the body 100. Thus, the oil phase 200 is introduced through the first inlet 110.

The first flow path portion 111 starts from the first inflow portion 110 to form a zigzag flow path as shown in FIG. 1, and then the first branch path portion 112 and the second branch path portion Quot;

The first and second branching flow path portions 112 and 113 branched in this manner are branched and extended along both outer sides of the second inlet portion 120 and the second flow path portion 121, And is connected to the second flow path portion 121 and the third flow path portion 131.

The second inlet 120 is spaced apart from the first inlet 110 by a predetermined distance and is formed as an opening connected to the inside of the body 100 in the same manner as the first inlet 110 . Thus, the aqueous phase 300 is introduced through the second inlet 120.

In this case, the water-based material 300 includes the cells 301 as shown in FIG. 2, in which case the cells may be cancer cells.

1, one end of the second flow path portion 121 is connected to the second inflow portion 120. One end of the second flow path portion 121 is connected to the first and second branch flow path portions 112 and 113, And the other end is connected to the first and second branch flow path portions 112 and 113 and the third flow path portion 131, respectively.

One end of the third flow path portion 131 is connected to the first and second branch flow path portions 112 and 113 and the second flow path portion 121 and the other end of the third flow path portion 131 is connected to the outflow portion 130 do.

The outflow part 130 is connected to the third flow path part 131 and is formed as an opening part through which the cell liquid droplet flows out from the inside of the body part 100.

Through the structure of the droplet-based biochip 10 as described above, the water-based material 300 including the cells 301 and the oily material 200 are formed as the cell droplets 400.

That is, the oil material 200 flowing through the first inlet 110 flows through the first oil passage 111 and then flows through the first and second branch passage portions 112 and 113 So that they flow from each other.

Meanwhile, the water-based material 300 including the cells 301 flowing through the second inlet 120 flows through the second flow passage 121.

2, the oily material 200 flowing through the first and second branching flow path portions 112 and 113 flows through the second flow path portion 121, The cell droplet 400 is mixed with the water-based material 300 including the first channel portion 301 and the cell droplet 400 is flowed through the third channel portion 131.

In this case, the first and second branch flow paths 112 and 113 are connected to face each other, and the second flow path 121 is perpendicular to the first and second branch flow paths 112 and 113 Direction. The third flow path portion 131 is connected to the second flow path portion 121 in the same direction.

Thus, the first and second branch flow path portions 112 and 113 and the second and third flow path portions 121 and 131 cross each other in a direction perpendicular to each other to form a cross shape.

Accordingly, the oily material 200 is formed into the cell droplet 400 by mixing with the water-based material 300 containing the cells 301 provided in the vertical direction after flowing in the direction opposite to each other.

In this case, since the oily material 200 and the water-based material 300 are vertically mixed with each other, mixing into the cell droplet is easier, and the oily material 300 The cell liquid droplet 400 is guided by the third flow path portion 131 extending in the same direction as the extending direction of the second flow path portion 121 which is the flow direction of the water phase material 300, Lt; / RTI >

By controlling the flow rates of the oily material 200 and the water based material 300 and the flow velocity ratio of the oily material 200 and the water based material 300 to be described later, Or the cell number in the cell droplet 400 or the productivity of the cell droplet 400 can be controlled.

FIG. 3 is a flowchart illustrating a tumor production method using the droplet-based biochip of FIG. 1 and a method of determining the metastasis of a tumor produced by the tumor production method. FIG. 4A is an image of the cytolysy produced through the tumor production method of FIG. 3, and FIG. 4B is an enlarged view of the 'B' region of FIG. 4A. FIG. 5 is an image of a tumor model cultured through the tumor production method of FIG. 3; FIG.

Referring to FIG. 3, in the method for producing tumor according to the present embodiment, an oil material 200 is prepared (step S10).

The oily material 200 can be prepared, for example, by blending Pico Surf 2 from Dolomite-microfludicis with a concentration of 5% on 3M Novec 7500 Engineered fluid.

Thereafter, in the tumor production method, the water-based material 300 is prepared (step S20).

For example, the breast cancer cell line (MCF-7) is cultured in a cell culture medium (Dulleco Modified Eagle Medium, Fatal Bonine Serum, or the like) in a cell culture medium. L-Glutamine, Penicillin-Streptomycin). In this case, the initial concentration may be, for example, 10 ⁷ cells / ml.

Thereafter, in the tumor production method, the biochip 10 is pretreated (step S30).

1 and 2, the biochip 10 has a plurality of flow paths formed in the body 100, and therefore, the flow path of the biochip 10 is not limited to the above- Perform preprocessing on the flow paths.

The pretreatment of the flow path portions is performed to facilitate the formation of the cell droplets 400, and hydrophobic treatment is performed on the flow path portions inside the biochip 10.

For example, the hydrophobic treatment is performed on the flow path portions of the biochip 10 using Aquapel of Pittsburgh Glass Works, a glass water-repellent coating agent, BSA (Bovine Serum Albumin) can be treated to prevent abnormal adhesion of cells with PDMS polymer.

Thereafter, in the tumor production method, the oily material 200 and the water-based material 300 are injected into the bio-chip 10 to generate the cell droplet 400 (step S40).

The oily material 200 is injected through the first inlet 110 and flows through the first and second branch passage portions 112 and 113 after passing through the first passage portion 111 , The water-based material 300 is injected through the second inlet portion 120 including the cell 301 and flows through the second flow passage portion 121.

Thus, the water-based material 300 including the oily material 200 and the cells 301 at the ends of the first and second branching flow paths 112 and 113 and the end of the second flow- The cell droplet 400 flows through the third flow path portion 131 and then flows out to the outside through the third outflow portion 130. [

In this case, the structure and the flow mechanism of the channel portions of the biochip 10 are described in detail with reference to FIG. 1 and FIG. 2, and a duplicate description will be omitted.

Meanwhile, when the cell droplet 400 is formed, various cell droplets can be formed under various control conditions.

In this case, in consideration of the size of the cell droplet 400, the number of cells 301 included in the cell droplet 400, the productivity of the cell droplet 400, and the like, 300, or the flow rate ratio of the oily material 300 and the water-based material 300 can be controlled.

For example, in the case of the cell droplet 400 shown in FIGS. 4A and 4B, the flow rate of each of the oil material and the water-based material is formed by injecting at 120 μl / min and 80 μl / min, The yield of the cell droplet can be formed at a rate of 30 Hz per second, and the average size of the cell droplet can be formed at 508 탆.

In this case, an average of about 250 breast cancer cells per one cell droplet shown may be included.

Thereafter, in the tumor production method, the generated cell droplet is cultured to complete a tumor model (step S50).

5, the generated cell droplet 400 is maintained at 37.5 DEG C in a 5% CO ₂ concentration environment, for example, as shown in FIG. 5. In this case, the cell culture liquid is contained in the cell droplet. More specifically, As a result of culturing for 24 hours, a three-dimensional tumor model with an average diameter of 220 mu m is formed.

The thus formed three-dimensional tumor model is completed by discharging it out of the cell droplet 400 through centrifugation.

6A and 6B are images showing non-metastatic tumors and enlarged views thereof. 7A and 7B are images showing metastatic tumors and enlarged views thereof.

Meanwhile, as shown in FIG. 3, the method of determining tumor metastasis according to the present embodiment is the same as the tumor production method from the step of producing the oily substance (step S10) to the step of completing the tumor model (step S50) Do.

Thereafter, in the method of determining the metastasis of the tumor, it is further determined whether the completed tumor model is a single tumor or a single cell (step S60).

In the case of cancer cells, they have the characteristic of forming a three-dimensional single tumor, but in the case of metastatic cancer cells, they do not form a single tumor but maintain a single cell unit.

Using the characteristics of these metastatic cancer cells, in the method of determining the metastasis of the tumor, it is determined that the completed 3D tumor model is a non-metastatic tumor if the 3D tumor is a single tumor and if the completed 3D tumor model is a single cell, It can be judged to be a tumor.

In other words, in the case of non-metastatic tumors, even at very low density of several to several dozen, they form one single tumor, and the boundary of the cell membrane disappears, the formation of tight junction proteins, generation of an extra cellular matrix.

Referring to FIGS. 6A and 6B, considering the geometrical characteristics of the completed 3D tumor model in the cell droplet 400, it is determined that the 3D tumor model is a non-metastatic tumor 411).

7A and 7B, considering the shape characteristic of the completed 3D tumor model in the cell droplet 400, since it has a shape of a single cell rather than a single tumor, the 3D tumor The model is judged as metastatic tumor 412.

However, when the cell droplet 400 is cultured to complete a tumor model, a single tumor is formed in the case of the non-metastatic tumor 411 due to the limited nutritional state of the culture liquid in the cell droplet 400, In the case of a longer incubation time (for example, 48 to 72 hours), since a single tumor may be separated into single cells again and exhibit metastasis, it is preferable that the culture is performed by optimizing the incubation time to about 24 hours, .

According to the embodiments of the present invention as described above, the biochip is designed so that the oily material and the water-based material flow through separate channels and are mixed with each other to form droplets, thereby effectively forming droplets containing the cells And thus, a three-dimensional tumor model can be easily completed.

Particularly, it is possible to control the size and productivity of the cell droplet by controlling the flow rate or the flow rate of the oily material and the water-based material during the formation of the cell droplet for the three-dimensional tumor model, You can complete the model.

In addition, since the cell droplet can be continuously produced through mixing of the continuous water-based material and the oily material, and a three-dimensional tumor model can be produced only through culturing for 24 hours, a relatively large amount of three-dimensional tumor can be produced Do.

In addition, it is possible to judge whether metastasis is metastatic or non-metastatic based on the cell unit status of the tumor model without using the conventional protein or gene test method, and thus it is possible to judge the metastasis of the tumor relatively easily.

It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the present invention as defined by the following claims. It can be understood that it is possible.

The droplet-based biochip according to the present invention, the method for producing tumor using the same, and the method for determining the metastasis of tumor produced therewith have industrial applicability that can be used for producing a three-dimensional tumor model.

10: Biochip 100: Body part
110: first inlet portion 111: first flow portion
112: first branch flow portion 113: second branch flow portion
120: second inlet portion 121: second flow portion
130: outlet portion 131: third flow path portion
200: Oily material 300: Water-based material
301: cell 400: cell droplet
410: tumor 411: non-metastatic tumor
412: metastatic tumor

Claims (14)

delete delete delete delete delete Producing an oily material;
Preparing a water-based material by mixing a cancer cell line on a cell culture liquid in a single cell unit;
Treating the biochip in which a plurality of flow paths are formed;
Injecting the oily material through a first inlet of the bio-chip, injecting a water-based material mixed with the cancer cell line through a second inlet of the bio-chip to continuously produce cell droplets;
Culturing the resulting cell droplet for 24 hours to complete a tumor model; And
Determining that the completed tumor model is a non-metastatic tumor if the single tumor is a tumor and determining that the tumor model is a metastatic tumor if the single tumor is a single tumor. delete 7. The method of claim 6, wherein the step of pre-
Hydrophobic processing an inner wall of the channel portions of the biochip; And
Treating bovine serum albumin (BSA) of the inner wall of the channel portions of the biochip. 7. The method according to claim 6, wherein in the step of generating the cell droplet,
Wherein the oily material flowing through the first flow path portion of the flow path portions and the water phase material flowing through the second flow path portion of the flow path portions are mixed with each other to generate a cell droplet. 10. The method according to claim 9, wherein in the step of generating the cell droplet,
Wherein the flow rate of each of the oily substance and the water-based substance, or the flow rate ratio of the oily substance and the water-based substance is controlled in consideration of the size and productivity of the cell droplet. 11. The method of claim 10,
Wherein the flow rates of the oily substance and the water-based substance are 120 l / min and 80 l / min, respectively. 7. The method of claim 6, wherein in completing the tumor model,
Method of determining metastatic tumors which comprises culturing said cell in liquid droplets 37.5 ℃, 5% CO ₂ concentration of the environment.


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