KR20200061036A - Apparatus for manufacturing 3D tumor model - Google Patents

Abstract

Provided in the present invention is an apparatus for preparing a 3D tumor model. According to an embodiment of the present invention, the apparatus for preparing a 3D tumor model comprises: a first inlet part, into which an oil phase solution is introduced; a second inlet part, into which a water phase solution containing cells related to a 3D tumor model to be prepared is introduced; a branched passage part, in which the oil phase solution separately flows and the oil phase solution flows in opposite directions at one end; a water phase solution passage part, in which the water phase solution flows and which is disposed to cross the branched passage part; an extension passage part which extends from a cross point between the branched passage part and the water phase solution passage part toward an opposite side of the water phase solution passage part and in which a cell droplet containing cells is formed by mixing the water phase solution and the oil phase solution; a liquid droplet passage part in which cell droplets flow; and an outlet part from which the cell droplets are discharged to the outside. At this time, the cell droplets containing cells are incubated for a certain period of time, after which the cells are aggregated and formed into a 3D tumor model and a flow rate of the water phase solution and the oil phase solution and a concentration of the cells contained in the water phase solution can be controlled so that a finally obtained 3D tumor model can have a desired size.

Description

Apparatus for manufacturing 3D tumor model

The present invention relates to a three-dimensional tumor model manufacturing apparatus, in particular, to a three-dimensional tumor model manufacturing apparatus for mass production and uniform size control of a three-dimensional tumor.

It is common to use a cell model to determine drug efficacy for a drug substance, and in the case of 3D cells, it acts as a factor that interferes with the delivery of the drug substance, so that the efficacy of drugs similar to tumors in the body can be verified. It has the advantage of being able to.

However, a typical cell has a property of growing in a two-dimensional form, and the two-dimensional cell thus grown has different physical and biological characteristics from the cell of the three-dimensional form, making it difficult to effectively verify drug efficacy for a drug substance. there is a problem.

Accordingly, a technique for culturing a cell in a three-dimensional form has been developed, and methods that are widely used to date include a hanging drop method and a micromolding method. On the other hand, SpheroFilm (iN CYTO), Perfecta3D (3D Biomatrix), etc. are commercially available as these cultivation products. There is a limit to producing a 3D tumor model.

Therefore, there is a need for a technique for manufacturing a 3D tumor model more effectively by solving the low productivity problem in manufacturing the 3D tumor model.

Republic of Korea Registered Patent No. 10-1544391

In order to solve the problems of the prior art as described above, one embodiment of the present invention can improve the production yield of the three-dimensional tumor model using droplets while simultaneously controlling the size of the three-dimensional tumor model. It is intended to provide an apparatus for manufacturing a dimensional tumor model.

According to an aspect of the present invention for solving the above problems, the oil inlet solution (oil phase solution) is introduced into the first inlet; A second inflow part into which an oil phase solution containing cells related to a 3D tumor model to be manufactured is introduced; A branch flow path part in which the oil phase solution is separated and flows, and at one end, the oil phase solution flows in opposite directions; A fluid flow passage portion through which the aqueous solution flows and intersects the branch flow path portion; An extended flow path portion extending from an intersection point of the branch flow path portion and the fluid flow path portion to an opposite side of the liquid flow path portion, wherein cell droplets containing the cells are formed by mixing the aqueous solution and the oil solution; A droplet flow path portion through which the cell droplet flows; And an outlet through which the cell droplets are discharged to the outside, and the cell droplets containing the cells are aggregated to form a three-dimensional tumor model after culturing for a predetermined time, and the size of the finally obtained three-dimensional tumor model is targeted. A three-dimensional tumor model manufacturing apparatus is provided in which the flow rate of the aqueous phase solution and the oil phase solution and the concentration of cells contained in the aqueous phase solution are controlled so as to have a size.

In one embodiment, the apparatus for manufacturing a 3D tumor model includes a first syringe pump that supplies the oily solution to the first inlet; A second syringe pump supplying the aqueous solution to the second inlet; And it may further include a control unit for controlling the first syringe pump and the second syringe pump to adjust the flow rate of the oil phase solution and the water phase solution.

In one embodiment, the flow rate of the oil phase solution is 120 ~ 300μl/min, the flow rate of the water phase solution is 40 ~ 100μl/min, and the flow rate ratio of the oil phase solution and the oil phase solution may be 1:3. .

In one embodiment, the concentration of cells contained in the aqueous solution may be 1.47 × 10 ⁶ ~ 29.4 × 10 ⁶ pieces / ml.

In one embodiment, the cell droplet may have a diameter of 50 μm or more and 450 μm or less.

In one embodiment, the 3D tumor model may have a diameter of 50-150㎛.

In one embodiment, the width of the extended flow path portion may be smaller than the width of the branch flow path portion, the fluid flow path portion, and the droplet flow path portion.

In one embodiment, the width of the droplet flow path portion may extend in a curved shape from the extension flow path portion.

In one embodiment, the cells may be cancer cells.

In one embodiment, the cell droplets can be cultured for 24 hours to form the 3D tumor model.

In one embodiment, the three-dimensional tumor model may be discharged to the outside of the cell droplets by centrifugation.

In one embodiment, the diameter (Y1; μm) of the cell droplet according to the flow rate (X1; μl/min) of the aqueous solution may be calculated by the following equation;

Y1 =-0.9695 × X1 + 471.61 (R ² = 0.9866).

In one embodiment, the number (Y2) of cells in the cell droplet according to the cell concentration (X2; dog/mL) may be calculated by the following equation;

Y2 = 3 × 10 ^-5 × X2-16.687 (R ² = 0.9873).

The apparatus for manufacturing a 3D tumor model according to an embodiment of the present invention facilitates the 3D tumor model by continuously forming droplets in which cells for forming a tumor model are formed by mixing the aqueous solution and the oil solution. Since it can be produced in large quantities at the same time as manufacturing, it is possible to improve the production yield of the three-dimensional tumor model.

In addition, the apparatus for manufacturing a 3D tumor model according to an embodiment of the present invention is formed by adjusting the flow rate of the aqueous solution and the oil solution, and the concentration of cells contained in the aqueous solution, and the size of the cell droplets and the aggregation of cells. Three-dimensional tumor models of various sizes can be produced uniformly.

1 is a perspective view showing a three-dimensional tumor model manufacturing apparatus according to an embodiment of the present invention,
FIG. 2 schematically illustrates a process in which droplets are formed in region A of FIG. 1,
3 is an enlarged view of region A of FIG. 1,
Figure 4 is a block diagram of a three-dimensional tumor model manufacturing apparatus according to an embodiment of the present invention,
Figure 5 is a graph showing the relationship between the droplet diameter and yield according to the flow rate of the aqueous solution and the oil solution,
Figure 6 is a graph showing the relationship between the number of cells in the droplet according to the cell concentration of the aqueous solution,
7 is an image of a cell droplet produced by a 3D tumor model manufacturing apparatus according to an embodiment of the present invention,
8 is an image of a three-dimensional tumor model formed by culturing the cell droplets of FIG. 7,
Figure 9 is a graph showing the relationship between the diameter of the three-dimensional tumor model according to the cell concentration of the aqueous solution,
10 is an image of a three-dimensional tumor model according to the cell concentration of the aqueous solution.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art to which the present invention pertains can easily practice. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. In the drawings, parts not related to the description are omitted in order to clearly describe the present invention, and the same reference numerals are attached to the same or similar elements throughout the specification.

Hereinafter, an apparatus for manufacturing a 3D tumor model according to an embodiment of the present invention will be described in more detail with reference to the drawings. 1 is a perspective view showing a three-dimensional tumor model manufacturing apparatus according to an embodiment of the present invention, Figure 2 is a diagram schematically showing the process of droplet formation in the region A of Figure 1, Figure 3 is a diagram of Figure 1 This is an enlarged view of area A.

Referring to FIG. 1, a 3D tumor model manufacturing apparatus 10 according to an embodiment of the present invention is a droplet-based microfluidic system, the body portion 100, the first inlet portion 110, and the first flow path portion ( 111), the first branch flow path 112, the second branch flow path 113, the second inflow section 120, the second flow path section 121, the third flow path section 131, the extended flow path section 132 ) And the outlet portion 130.

The body portion 100 forms the body of the 3D tumor model manufacturing apparatus 10 and includes a transparent material, and may have a thin rectangular chip shape as a whole.

The first inlet part 110 is formed at one end of the body part 100 and may be formed as an opening connected to the inside of the body part 100. In addition, the first inlet 110 may be introduced into the oil solution 200. Here, the oily material 200 may be, for example, a mixture of Pico Surf 2 of Dolomite-microfludicis company at a concentration of 5% on a Novec 7500 Engineered fluid of 3M company.

In the first flow path 111, the first quarter flow path 112, and the second quarter flow path 113, the oil solution 200 introduced from the first flow path 110 may flow. Here, the first flow path 111 extends from the first flow path 110 and may be formed in a zigzag shape.

The first branch flow path portion 112 and the second branch flow path portion 113 are branch flow path portions in which the oil phase solution 200 is separated and flows. That is, the first branch flow path portion 112 and the second branch flow path portion 113 may be arranged to branch from the first flow path portion 111 to both sides.

Here, the first branch flow path portion 112 and the second branch flow path portion 113 may be formed in a substantially rectangular shape. Therefore, the first quarter flow path 112 and the second quarter flow path 113 may be integrated again at opposite sides of the first inflow portion 110. That is, the first branch flow path 112 and the second branch flow path 113 may be connected to the second flow path 121 and the third flow path 131.

At this time, the oil solution 200 may be flowed so as to be mixed in opposite directions from one end on the opposite side of the first inlet part 110 after being branched along the first branch passage part 112 and the second branch passage part 113. have.

The second inlet 120 is disposed to be spaced apart from the first inlet 110 by a predetermined distance, and may be formed as an opening connected to the interior of the body 100 similar to the first inlet 110. Here, the second inflow portion 120 may be disposed inside the first branch flow path portion 112 and the second branch flow path portion 113. The aqueous solution 300 may be drawn into the second inflow part 120.

At this time, the aqueous solution 300 may include cells associated with a 3D tumor model to be manufactured, as shown in FIGS. 2 and 3. Here, the cells may be cancer cells for forming a 3D tumor model, but are not limited thereto. For example, the cells included in the aqueous solution 300 may be normal cells for comparison with tumors.

Here, the aqueous solution 300 may be a mixture of cancer cell lines in a single cell unit on a cell culture solution. For example, the cultured breast cancer cell line (MCF-7) may be a single cell unit mixture on a cell culture solution (Dullecco Modified Eagle Medium, Fatal Bonine Serum, L-Glutamine, Penicillin-Streptomycin).

The second flow path portion 121 is a fluid flow path portion through which the aqueous solution 300 flows, one end being connected to the second flow path portion 120, and the other end of the first flow path portion 112 and the second flow path portion It can be connected with (113).

In this case, the second flow path part 121 may be disposed to intersect the first flow path part 112 and the second branch flow path part 113 vertically.

The third flow path portion 131 is a droplet flow path portion through which the cell droplet 400 flows, one end of which is connected to the extended flow path portion 132, and the other end of which may be connected to the outlet portion 130. The third flow path part 131 may be formed to have a width larger than the width W1 of the second flow path part 121.

The extended flow path portion 132 has one end connected to the second flow path 121, the first branch flow path 112, and the second branch flow path 113, and the other end to be connected to the third flow path portion 131. have. That is, the extended flow path portion 132 may be formed to extend from the intersection of the branch flow path portions 112 and 113 and the fluid flow path portion 121 to the opposite side of the fluid flow path portion 121.

At this time, the second flow path portion 121 and the extended flow path portion 132 are substantially disposed on a straight line, and the first branch flow path portion 112 and the second branch flow path portion 113 are substantially disposed on a straight line. Can be. In addition, the first branch flow path portion 112 and the second branch flow path portion 113, the second flow path portion 121, and the extended flow path portion 132 may be connected in a direction perpendicular to each other. Therefore, the first branch flow path 112, the second branch flow path 113, the second flow path portion 121, and the extended flow path portion 132 may be connected in a cross-shaped intersection.

2 and 3, cell droplets 400 including cells 301 may be formed by mixing the aqueous phase solution 300 and the oil phase solution 200.

The outlet portion 130 is connected to the third flow path portion 131 so that the cell droplet 400 can be discharged to the outside. Here, the outlet portion 130 may be formed as an opening that is opened from the inside of the body portion 100 to the outside.

Here, the flow path parts 111, 112, 121, 131, and 132 may be formed of a polydimethylsiloxane (PDMS) polymer. At this time, in order to more easily form the cell droplets 400, hydrophobic treatment may be performed on the flow path portions of the 3D tumor model manufacturing apparatus 10.

For example, hydrophobic treatment is performed on the flow path portions of the 3D tumor model manufacturing apparatus 10 using Aquapel of Pittsburgh Glass Works, a glass water-repellent coating, and the PDMS polymer and the cells of the flow path portions are processed. BSA (Bovine Serum Albumin) may be processed to prevent abnormal adhesion.

As shown in FIG. 2, the width W3 of the extended flow path portion 132 is the width W1 of the second flow path portion 121 and the first branch flow path portion 112 and the second branch flow path portion 113. It may be smaller than the width (W2). In addition, the third flow path portion 131 may be formed such that its width extends in a curved shape from the extended flow path portion 132.

Thereby, when the water phase solution 300 and the oil phase solution 200 cross to form the cell droplet 400, the flow of the water phase solution 300 flowing into the third flow path part 131 flows into the oil phase solution 200. By easily breaking, the droplets can be effectively formed.

4 is a block diagram of a 3D tumor model manufacturing apparatus according to an embodiment of the present invention.

The 3D tumor model manufacturing apparatus 10 according to an embodiment of the present invention may further include a control unit 11, a first syringe pump 12, and a second syringe pump 13.

The control unit 11 may control the pumps of the first syringe pump 12 and the second syringe pump 13 to adjust the flow rates of the oil phase solution 200 and the aqueous phase solution 300.

The first syringe pump 12 may supply the oil solution 200 to the first inlet 110. Here, the first syringe pump 12 may be connected to a container in which the first inlet part 110 and the oil solution 200 are stored through a tube. The first syringe pump 12 may control the absolute flow rate of the oil phase solution 200.

The second syringe pump 13 may supply the aqueous solution 300 to the second inlet 120. Here, the second syringe pump 13 may be connected to a container in which the second inlet part 120 and the aqueous solution 300 are stored through a tube. The second syringe pump 13 may control the absolute flow rate of the aqueous solution 300.

Cells 301 may be injected into the three-dimensional tumor model manufacturing apparatus 10 as described above, thereby forming the cell droplets 400 by injecting the aqueous phase solution 300 and the oil phase solution 200 included therein.

That is, when the oil phase solution 200 is injected through the first inlet part 110, the oil phase solution 200 flows to the first flow passage part 111, and then the first quarter flow passage part 112 and the second quarter flow. Branched through the flow path portion 113.

At this time, when the aqueous solution 300 containing the cells 301 is injected through the second inlet part 120, the aqueous solution 300 flows to the second flow part 121.

As shown in FIG. 2, the oil phase solution 200 flowing through the first quarter flow path 112 and the second quarter flow path 113 is a water phase solution 300 flowing through the second flow path 121. ).

At this time, the oil phase solution 200 is mixed with the flow of the aqueous phase solution 300 in a vertical direction to cut the flow of the aqueous phase solution 300, thereby forming a cell droplet 400. The formed cell droplets 400 may flow to the outlet 130 through the third flow passage 131.

The cell droplets 400 thus generated may be cultured for 24 hours. Here, the cell culture medium may be included in the cell droplet 400. As an example, the resulting cell droplets 400 can be cultured in an environment of 37.5° C., 5% CO ₂ for 24 hours.

At this time, the cells 301 in the cell droplet 400 can be aggregated to generate a 3D tumor model. Thereafter, the three-dimensional tumor model can be separated from the cell droplet 400 by centrifugation.

On the other hand, while changing the flow rate of the aqueous solution 300 and the oil phase solution 200 and the concentration of the cells 301 contained in the aqueous solution 300, the production yield of the cell droplets and the cell droplet 400 and the three-dimensional tumor model The size of was observed through experiments.

First, a 3D tumor model manufacturing apparatus 10 as shown in FIGS. 1 and 2 was manufactured. Here, the thickness t of the body portion 100 is 150 µm, the width W3 of the extended flow path portion 132 which is the droplet bonding portion is 300 µm, and the width W1 of the first flow path 111 is The width W2 of the 600 µm, the first branch flow path 112 and the second branch flow path 113 was 400 µm.

The breast cancer cell line (MCF-7) was used as the cell. These cells were cultured in high-grade DMEM/F12 basal medium supplemented with 10% v/v fetal serum, 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine. Cells were separated using trypsin-EDTA (all cell reagents were purchased from Invitrogen Corporation (USA)).

In addition, five concentrations of MCF-7 (1×, 2.5×, 5×, 10×, and 20×10 ⁶ /mL) were used to determine the final diameter of the 3D tumor model. To form cell-encapsulated micro-droplets, the cell suspension was used as aqueous solution. The cell suspension contained cell culture medium and suspended cancer cells.

At this time, 5% diluted surfactant (Pico-Surf™, Dolomite Microfluidics, Royston, UK) was used in the fluorinated oil (Novec™ 7500, 3M, Maplewood, MN, USA) to form stable and uniform micro droplets. . Surfactants are chemical solutions containing fluorocarbon carrier oil.

In addition, 1H, 1H, 2H, 2H, -perfluoro (perfluoro)-1-octanol (Sigma-Aldrich, St. Louis, MO, USA) using the fragments were divided into 3D after incubation for 24 hours and cultured Tumor spheroids were recovered.

At this time, 1H, 1H, 2H, 2H,-perfluoro-1-octanol was added to the droplet solution and gently mixed with the oil phase solution. The supernatant containing the culture medium and the released spheroid were separated from the mixture solution by centrifugation. Conventional cell culture systems (MCO-18 AC, Panasonic Healthcare Co.) under magnetic stirring of the spheroid and culture medium to prevent the formation of two-dimensional cell monolayers as well as to minimize unexpected binding between the spheroids ., Ltd., Tokyo, Japan).

On the other hand, in order to generate MCF-7 cells encapsulated in droplets, cells suspended in the culture medium were used as an aqueous solution as shown in FIG. 3. At this time, the cells were encapsulated as droplets under various flow rates for the oil phase solution 200 and the water phase solution 300.

Here, the flow rate of the oil phase solution 200 is 20 to 100 μl/min, and the flow rate ratio between the aqueous phase solution 300 and the oil phase solution 200 is fixed to 1:3.

Figure 5 is a graph showing the relationship between the droplet diameter and yield according to the flow rate of the aqueous solution and the oil solution.

As shown in Fig. 5, in the slowest condition (20 µl/min) for the aqueous solution 300, the droplet diameter and production yield were found to be 451.5 ± 5.3 µm and 7 mm 2 (7 generated per second), respectively. On the other hand, at the fastest conditions (100 μl/min), droplet diameter and product yield were found to be 370.5 ± 4.6 μm and 57 mm 3, respectively.

At this time, the droplet diameter and production yield were inversely related. That is, the droplet diameter and the flow velocity of the water phase solution 300 and the oil phase solution 200 are inversely related, and the production yield and the flow rate of the water phase solution 300 and the oil phase solution 200 are proportional.

 Here, when the diameter of the droplet exceeds 450 µm, its shape is irregularly formed. This is due to the mechanical limitations of the experimental equipment, such as the slowest flow rate that is large in the droplet size and stable in the syringe pump.

Moreover, when the diameter of the droplets exceeds 500 μm, a phenomenon occurs in which the droplets do not maintain their shape and merge.

Therefore, the droplet diameter generated by the 3D tumor model manufacturing apparatus 10 is preferably 50 μm or more and 450 μm or less. At this time, the flow rate of the oil phase solution 200 is 120 ~ 300 μl/min, and the flow rate of the water phase solution 300 is preferably 40 ~ 100 μl/min. Here, the flow rate ratio of the oil phase solution 200 and the water phase solution 300 may be 1:3.

Next, at least 1000 droplets per minute (16.7 mm 3) were prepared with the largest droplet diameter set to maximize the number of encapsulated cells. At this time, the flow rates of the water phase solution 300 and the oil phase solution 200 were fixed at 40 and 120 μl/min, respectively.

Under the flow rate conditions above, cell encapsulated micro-droplets were generated.

At this time, MCF-7 samples were prepared at 5 concentrations (1x, 2.5x, 5x, 10x and 20x 10 ⁶ /ml) to determine the average number of cells encapsulated within each micro-droplet.

In each case, MCF-7 cells per liquid crystal were measured, as shown in FIG. 6. 6 is a graph showing the relationship between the number of cells in a droplet according to the cell concentration of the aqueous solution.

As shown in Fig. 6, the number of MCF-7 cells per droplet according to the concentration of the cells averaged 500.3±39.9 (20×10 ⁶ cells/mL), 282.2±14.3 (10×10 ⁶ cells/mL), 89.4± It was measured as 15.0 (5×10 ⁶ pieces/ml), 42.9±6.24 (2.5×10 ⁶ pieces/ml), and 17.5±3.81 (1×10 ⁶ pieces/ml).

Theoretically, the expected number of cells per droplet depending on the cell concentration can be calculated based on the cell solution volume of a single droplet. For a cell concentration of 20×10 ⁶ /ml and a droplet diameter of 400 μm, it was expected that approximately 670 cells (about 33.5 nl/droplet) were encapsulated in each droplet. In all cases, the number of encapsulated cells was reduced by 30-35%. This is due to cell sinking due to gravity resulting in non-homogeneous cell suspension and the cells sticking to the tube surface as well as disposable syringes. In this experiment, lost cells were observed with an average of 32.3%.

Therefore, the concentration of cells contained in the aqueous solution 300 is preferably 1.47 × 10 ⁶ ~ 29.4 × 10 ⁶ pieces/ml by compensating for the number of lost cells. At this time, the diameter of the three-dimensional tumor model is preferably 50 ~ 150㎛.

Cell aggregation was observed during the first hour as shown in the supporting material (SMovieclip1). Compact spheroid formation was observed 3 hours after droplet culture. The rotational motion of the compact spheroid was observed during the 24 hour incubation.

7 is an image of photographing cell droplets produced by a 3D tumor model manufacturing apparatus according to an embodiment of the present invention, and FIG. 8 is an image of 3D tumor model formed by culturing the cell droplets of FIG. 7, and FIG. 9 Is a graph showing the relationship between the diameter of the 3D tumor model according to the cell concentration of the aqueous solution, and FIG. 10 is an image of the 3D tumor model according to the cell concentration of the aqueous solution.

The morphological changes of MCF-7 in droplets are shown in FIGS. 7 and 8. In FIG. 7, cell droplets containing cells with initial concentration aggregate with each other by culture. In Fig. 8, a three-dimensional tumor model is formed in droplets by cell aggregation.

It was incubated for 24 hours while changing the initial cell-seed droplets and initial cell concentration. As shown in Figure 9, there were 17.5, 42.9, 89.4, 282.2 and 500.3 cells in 3D tumor spheroid models with diameters of 54.2, 73.4, 94.6, 123.2 and 148.3 μm, respectively (linear curve fitting, R ²⁾ = 0.9936).

In FIGS. 10A to 10E, droplets containing cells of initial concentrations (1x, 2.5x, 5x, 10x, and 20x 10 ⁶ cells/ml) were aggregated and the cells were aggregated after 24 hours of culture, resulting in a three-dimensional tumor model. It is formed. 9 and 10, the cell concentration and the size of the 3D tumor model show a proportional relationship.

Cell encapsulated droplets increased the diameter of the 3D spheroid model after 3 days. At 10×10 ⁶ cells/ml, single cells of the 3D tumor spheroid model began to separate after 36 hours because there was no accumulation of nutrients and gas exchange or metabolic waste in a confined space.

Further, for the same reason, the yield of spheroid formation of 20 x 10 ⁶ particles/ml was about 50%. Thus, it was found that the time limit for droplet culture was 24 hours for 10 x ⁶ cells/ml cells. Based on these results, one single MCF-7 cell requires 3.7 pL/h of culture medium to survive inside the droplet. Theoretically, 20×10 ⁶ pcs/ml require 44.7 nl of culture medium with respect to 450 μm diameter micro-droplets.

6 and 9, it can be seen that the final diameter of the 3D tumor model can be determined by the number of encapsulated cells in a single micro-droplet.

As a result, the size of the cell droplet 400 and the size of the three-dimensional tumor model formed by aggregation of the cells 301 are included in the flow rate of the aqueous solution 300 and the oil phase solution 200 and the aqueous phase solution 300 We have learned that it can be effectively adjusted according to the concentration of cells 301.

Here, based on the experimental results of FIG. 5, the diameter Y1 of the cell droplet 400 according to the flow rate X1 of the aqueous solution 300 may be calculated by the following equation.

Y1 =-0.9695 × X1 + 471.61 (R ² = 0.9866)

Here, the unit of the diameter Y1 of the cell droplet 400 is μm, and the unit of the flow rate X1 of the aqueous solution 300 is μl/min.

In addition, based on the experimental results of FIG. 9, the number (Y2) of cells in the cell droplet 400 according to the initial cell concentration (X2) may be calculated by the following equation.

Y2 = 3 × 10 ^-5 × X2-16.687 (R ² = 0.9873)

Here, the unit of cell concentration (X2) is dog/ml.

With such a configuration, the apparatus for manufacturing a 3D tumor model according to an embodiment of the present invention can easily manufacture a 3D tumor model and simultaneously manufacture a large amount, thereby improving the production yield of the 3D tumor model.

In addition, the present invention can uniformly produce a three-dimensional tumor model of various sizes formed by the size of the cell droplets to be produced and the aggregation of cells.

Although one embodiment of the present invention has been described above, the spirit of the present invention is not limited to the embodiments presented herein, and those skilled in the art to understand the spirit of the present invention may add elements within the scope of the same spirit. However, other embodiments may be easily proposed by changing, deleting, adding, or the like, but it will also be considered to fall within the scope of the present invention.

10: 3D tumor model manufacturing apparatus 100: body portion
110: first inflow section 111: first flow section
112: Euro portion of the first quarter 113: Euro portion of the second quarter
120: second inlet part 121: second flow part
130: outlet portion 131: the third flow path
132: extended flow path 200: paid solution
300: aqueous solution 301: cells
400: cell droplet 11: control
12: first syringe pump 13: second syringe pump

Claims (13)

A first inflow part into which an oil phase solution flows;
A second inflow part into which an oil phase solution containing cells related to a 3D tumor model to be manufactured is introduced;
A branch flow path part in which the oil phase solution is separated and flows, and at one end, the oil phase solution flows in opposite directions;
A fluid flow passage portion through which the aqueous solution flows and intersects the branch flow path portion;
An extended flow path portion extending from an intersection point of the branch flow path portion and the fluid flow path portion to an opposite side of the liquid flow path portion, wherein cell droplets containing the cells are formed by mixing the aqueous solution and the oil solution;
A droplet flow path portion through which the cell droplet flows; And
The cell droplets include an outlet that flows outward,
The cell droplets containing the cells are aggregated and formed into a three-dimensional tumor model after culturing for a predetermined time.
3D tumor model manufacturing apparatus in which the flow rate of the aqueous solution and the oil phase solution and the concentration of cells contained in the aqueous phase solution are controlled so that the size of the finally obtained 3D tumor model has a target size. According to claim 1,
A first syringe pump supplying the oily solution to the first inlet;
A second syringe pump supplying the aqueous solution to the second inlet; And
3D tumor model manufacturing apparatus further comprising a control unit for controlling the first syringe pump and the second syringe pump to control the flow rate of the oil phase solution and the water phase solution. According to claim 1,
The flow rate of the oil phase solution is 120 to 300 μl/min,
The flow rate of the aqueous solution is 40-100 μl/min,
A device for manufacturing a three-dimensional tumor model in which the flow rate ratio between the oil solution and the oil solution is 1:3. According to claim 1,
The concentration of cells contained in the aqueous solution is 1.47 × 10 ⁶ ~ 29.4 × 10 ⁶ /ml 3D tumor model manufacturing apparatus. According to claim 1,
The cell droplets are three-dimensional tumor model manufacturing apparatus having a diameter of 50㎛ to 450㎛. According to claim 1,
The three-dimensional tumor model is a three-dimensional tumor model manufacturing apparatus having a diameter of 50 ~ 150㎛. According to claim 1,
The width of the extended flow path portion is three-dimensional tumor model manufacturing apparatus smaller than the width of the branch flow path portion, the fluid flow path portion, and the droplet flow path portion. According to claim 1,
The droplet flow path portion is a three-dimensional tumor model manufacturing apparatus whose width extends in a curved shape from the extended flow path portion. According to claim 1,
The cell is a cancer cell 3D tumor model manufacturing apparatus. According to claim 1,
The cell droplets are cultured for 24 hours, and a 3D tumor model manufacturing apparatus in which the 3D tumor model is formed. According to claim 1,
The three-dimensional tumor model is a three-dimensional tumor model manufacturing apparatus that is discharged to the outside of the cell droplets by centrifugation. According to claim 1,
A device for manufacturing a three-dimensional tumor model in which the diameter (Y1; μm) of the cell droplet according to the flow rate (X1; μl/min) of the aqueous solution is calculated by the following equation.
Y1 =-0.9695 × X1 + 471.61 (R ² = 0.9866) According to claim 1,
The number of cells in the cell droplet (Y2) according to the cell concentration (X2; dogs/mL) is a three-dimensional tumor model manufacturing apparatus calculated by the following equation.
Y2 = 3 × 10 ^-5 × X2-16.687 (R ² = 0.9873)

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