KR20230095008A - Microfluidic device having improved mesh structure - Google Patents

Abstract

A microfluidic device providing a space for cell culture according to the present invention includes a mesh unit disposed in at least a portion of the space for cell culture, and the mesh unit extends along a three-dimensional coordinate axis and a plurality of fins forming a fluid channel; and an opening formed between the plurality of pins to communicate the inside and outside of the fluid channel with each other.

Description

Microfluidic device having a mesh structure {MICROFLUIDIC DEVICE HAVING IMPROVED MESH STRUCTURE}

The present invention relates to a microfluidic device for simulating the function or response of a three-dimensional microbiological tissue.

In order to predict efficacy and side effects in the human body, preclinical trials are followed prior to clinical trials. Currently, preclinical experiments are conducted through animal experiments and basic cell experiments, which have problems such as animal ethics and reliability of results. An organ-simulating chip, one of the attempts to overcome these problems, is a microenvironment composed of organs of the human body and cultured cells on a microchip, which can simulate the structure and function of an actual organ. These attempts present a biological microenvironment highly similar to human tissue and provide significant results accordingly.

A PDMS (polydimethylsiloxane) chip, which is widely used as a long-term simulated chip model, is fabricated through a soft lithography method used in semiconductor processing technology. In the PDMS chip, a fluid is patterned in a flow path composed of microstructures at the level of tens or hundreds of micrometers in a hydrophobic environment. When the surface of the PDMS material has a hydrophobic environment, the fluid can be patterned without leaking between the microstructures by pushing the fluid with an appropriate pressure at a contact angle of 90 degrees or more. Fibrin gel, collagen, and Matrigel, which are mainly used hydrogels, can induce a phase change from a liquid phase to a solid phase depending on conditions, respectively, so that the shape can be maintained in the patterned space later. However, PDMS chips are not suitable for mass production due to complicated selective etching or bonding processes during the manufacturing process.

On the other hand, a chip using 3D bioprinting is manufactured by directly 3D printing gel to make a shape. This 3D bio-printing chip can pattern a structure similar to that in an actual living body by selecting locations for each cell type. In addition, it can be patterned in various forms depending on the conditions of various factors such as the type of gel, viscosity, output temperature, and cell concentration. However, the process of creating and removing bio-printing-based 3D structures is complicated and difficult to mass-produce.

One object of the present invention is to provide a microfluidic device having a mesh structure in which fluid patterning can be easily performed.

Another object of the present invention is to provide a microfluidic device configured to allow additional fluid or cells to be injected after fluid loading is performed.

Another object of the present invention is to provide a microfluidic device in which gas is easily discharged and bubbles are not generated when fluid loading is performed.

However, the technical problem to be achieved by the present embodiment is not limited to the technical problems described above, and other technical problems may exist.

In order to solve the above problems, the present application is a microfluidic device that provides a space for cell culture, a fluid channel including a first channel surrounding the center and a second channel disposed in the center, a plurality of fluid channels forming the fluid channel A pin, a plurality of openings formed between the plurality of pins to communicate the inside and outside of the fluid channel with each other, a second injection part formed to communicate the external space of the fluid channel with the second channel, and A microfluidic device having a gas discharge passage formed so that the first channel communicates with the outside along a predetermined direction so as not to form a closed loop, and the second channel communicates with the outside along the predetermined direction. provides

The problem solving means of the present application is not limited thereto, and it should be construed as including all solutions within the range that a person skilled in the art can understand or predict.

The microfluidic device according to the present invention is formed to have a mesh structure in which an inner space is divided by a plurality of pins and an inner space is opened by openings, so that the process of receiving fluid and selectively patterning can be performed conveniently and quickly. can Complex structures can be simulated and complex reactions identified by simple structures and patterning methods.

In addition, the microfluidic device according to the present invention has a first channel and a second channel, and various materials can be easily injected. In particular, when fluid loading is performed, gas in the fluid channel can be easily discharged to prevent generation of bubbles.

1 shows a microfluidic device according to an embodiment of the present disclosure.
2 shows a microfluidic device according to another embodiment of the present application.
3 is a perspective view of one embodiment of the present application.
4 is a front view of one embodiment of the present application.
5 is a rear view of one embodiment of the present application.
Figure 6 is another rear view of one embodiment of the present application, a view for showing a fluid channel.
7 is a diagram illustrating governing equations in a micro-mesh structure according to an embodiment of the present disclosure.
8 is a diagram illustrating experimental values of CBP according to the size and shape of a mesh structure according to an embodiment of the present disclosure.
9 is a diagram illustrating an example of 3D fluid patterning through pipette injection according to an embodiment of the present disclosure.
10 is a diagram illustrating an example of fluid patterning using dipping according to an embodiment of the present disclosure.
11 is a diagram illustrating an example of fluid patterning through dipping and draining according to an embodiment of the present disclosure.
12 is a diagram illustrating an example of co-culture of HUVEC and Lung Fibroblast for simulating perfused microvessels according to an embodiment of the present disclosure.
13 is a diagram illustrating an example of co-culture of cancer cells and blood vessel cells through dipping and draining according to an embodiment of the present disclosure.
14 shows a general process of loading fluid into the fluid channel of the present invention, and loading steps with a time difference.
15 illustrates forming a vascular network within the microfluidic device of the present disclosure according to an embodiment of the present disclosure.
Figure 16 shows the culture of tumor spheroids in the microfluidic device of the present application according to an embodiment of the present application.
Figure 17 shows the analysis of cluster tumor size using the microfluidic device of the present application according to an embodiment of the present application.
Figure 18 is a measurement of cell viability (viability) for each cluster size in each condition according to an embodiment of the present application.

Hereinafter, embodiments of the present application will be described in detail so that those skilled in the art can easily practice with reference to the accompanying drawings. However, the present disclosure may be implemented in many different forms and is not limited to the embodiments described herein. And in order to clearly describe the present application in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the present specification, when a member is said to be located “on” another member, this includes not only a case where a member is in contact with another member, but also a case where another member exists between the two members.

Throughout the present specification, when a part "includes" a certain component, it means that it may further include other components without excluding other components unless otherwise stated.

As used throughout this specification, the terms "about," "substantially," and the like are used at or approximating the value when manufacturing and material tolerances inherent in the stated meaning are given, and do not convey the understanding of this application. Accurate or absolute figures are used to help prevent exploitation by unscrupulous infringers of the disclosed disclosure. The term "step of (doing)" or "step of" as used throughout the present specification does not mean "step for".

Throughout this specification, the term "combination(s) of these" included in the expression of the Markush form means one or more mixtures or combinations selected from the group consisting of the components described in the expression of the Markush form, It means including one or more selected from the group consisting of the above components.

Throughout this specification, reference to "A and/or B" means "A or B, or A and B".

Hereinafter, embodiments and embodiments of the present disclosure will be described in detail with reference to the accompanying drawings. However, the disclosure may not be limited to these embodiments and examples and drawings.

1 to 6 show one embodiment of the present invention from various angles.

In the microfluidic device 100 according to one embodiment of the present invention, in the microfluidic device 100 providing a space for cell culture, a first channel 121a surrounding the center and a second channel disposed in the center A fluid channel 121 including (121b), a plurality of pins 122 forming the fluid channel 121, and a plurality of pins 122 so that the inside and outside of the fluid channel 121 communicate with each other. A plurality of openings 123 formed therebetween, a second injection part 124b formed to communicate the external space of the fluid channel 121 and the second channel 121b with each other, and the first channel 121a A gas discharge passage portion 125 formed to communicate with the outside along a predetermined direction so as not to form a closed loop, and the second channel 121b to communicate with the outside along the predetermined direction, A microfluidic device 100 is provided.

The plurality of fins 122 include a first fin 122a surrounding the outer circumferential side of the first channel 121a, a second fin 122b formed on the upper portion of the first channel, and the first channel 121a. ) may be provided with a third fin (122c) formed on the inner circumference of the upper portion.

The second fins 122b may be spaced apart from each other to form at least one of the openings 123 .

The second pin 122b may connect the first pin 122a and the third pin 122c.

The third pin 122c may form the second injection part 124b, and the second channel 121b may be formed under the second injection part 124b.

The height of the first fin 122a may be higher than the heights of the second fin 122b and the third fin 122c.

The height of the second pin 122b may be the same as that of the third pin 122c.

The distance at which the plurality of pins 122 are spaced apart from each other may be set to induce the first fluid to flow through the first channel 121a but to suppress the flow to the second channel 121b.

The area of each of the openings 123 may be set to induce the first fluid to flow through the first channel 121a but to suppress the flow to the second channel 121b.

A first injection part 124a configured to communicate an external space of the fluid channel 121 and the first channel 121a may be further provided.

The gas discharge passage part 125 is formed to extend downward from the lower surface of the pin adjacent to the second injection part 124b, and the rail part 126 prevents the first channel 121a from forming a closed loop. can include

The height of the rail part 126 may be smaller than the height of the first channel 121a or the second channel 121b.

The microfluidic device 100 according to the present invention may be formed in a mesh structure through a plurality of pins 122 and a plurality of openings 123, and may be manufactured on a scale of several hundred μm to several tens of millimeters using 3D printing. can Specifically, the size and shape of the micro-mesh optimized according to the height of the fluid to be patterned may be designed as a three-dimensional shape. Furthermore, there is an advantage of a high degree of freedom, such as being able to fabricate a twisted structure, which was impossible in other biochips.

As a method capable of patterning the hydrogel inside the microfluidic device 100 according to the present invention, there is a method of directly dispensing fluid into the mesh structure using a pipette, syringe, or the like. In particular, a higher degree of freedom in patterning is possible by directly injecting a fluid into a mesh at a desired location rather than having a separately determined injection unit.

The microfluidic device 100 according to the present invention may be directly immersed in a fluid to pattern the fluid in the mesh structure. Even if the fluid is accommodated in a large reservoir in advance and the entire microfluidic device 100 according to the present invention is immersed in the fluid, it is possible to selectively pattern only a specific portion by adjusting the mesh size and immersion height. there is. In addition, a draining process may be added to perform selective patterning. Specifically, the device may be rotated to increase the hydraulic pressure acting on the fluid inside the device so that the fluid is drained except for the fluid at a desired location.

Many existing bioprinting technologies directly print hydrogel with one nozzle, or print a plastic frame or water-soluble sacrificial layer that can guide the hydrogel with one of the two nozzles, and then insert the cells with the other nozzle. It is a method of printing a hydrogel that is available. When a hydrogel is directly printed with one nozzle, there is a limit to the resolution because most of the hydrogel's material properties (eg, hardness, Young's modulus) are not good. When bio-printing is performed through two nozzles, since guide frame production and hydrogel printing are performed at the same time, there are limitations in the process and the printing speed is significantly reduced. In the present invention, the guide frame can be mass-produced in advance through a general digital light processing (DLP) method and then simply immersed in a fluid or injected, so that it can easily proceed from production to cell patterning.

On the other hand, PDMS, a representative material previously used as a biochip, has a fatal disadvantage of adsorbing hydrophobic small molecules, making it difficult to derive accurate results according to dosage, which is one of the ultimate goals of biomimetic experiments, screening for new drug development It has no value as a platform. In addition, PDMS has a limited production yield, so it is difficult to derive results from a large number of experimental samples. According to the present invention, fluid patterning can be performed using both hydrophilic and hydrophobic materials.

In addition, mass production is possible using 3D printing technology that can produce the same shape as the design in a short time, and efficient design is possible due to structural diversity compared to injection molding. Specifically, biocompatibility can be secured through parylene coating, and fatal disadvantages of existing biochips are compensated for in that they are free from adsorption problems of hydrophobic small molecules, and at the same time, mass production is possible, achieving the goal of biochip can do.

Existing devices have a structure in which the upper part is closed except for a space for supplying a culture medium or a region for coupling with an external system. Alternatively, in the microfluidic device 100 according to the present invention, tissues and cell spheres may be introduced by designing holes in the main cell culture area. In addition to additional tissue introduction, the open structure can be exposed to oxygen supply and the external environment, enabling mimicking the environment for the differentiation of specific cells. In addition, the annular structure allows the fluid transfer process to be uniform in all directions compared to the linear structure, and the transfer of biochemical substances can be achieved more effectively when it is composed of biological elements.

The microfluidic device 100 according to the present invention can control flow by forming a contact angle of fluid on a hydrophilic or hydrophobic surface. That is, when the fluid randomly spreads from the surface by capillary force and encounters a structure that serves as a guide, the fluid can be transported along the way. This method does not require complicated experimental capabilities, so the results performed by any experimenter can appear similar, and furthermore, there is an advantage in that similar results can be quickly obtained with very high efficiency if an electric pipette or an automated device is involved. .

The microfluidic device 100 according to the present invention is not only loaded with a pipette but also easily injected with a bioprinter, so that existing bioink can be easily used and has high efficiency. The inlet can be widened to be easily compatible with the bioprinter, and the fluid can be designed to naturally follow the structure no matter where it is injected.

According to the present invention, the hole channel formed by primary patterning can be co-cultured by inserting another cell or implementing a vascularized spheroid by inserting a cancer cell spheroid. can build Unlike other platforms, it is possible to utilize the entire height of 1 mm to realize a shape that covers the entire mass of cancer cells. Since the central hole can be filled with a small volume of 4 to 5 μl, a person directly inserts cancer cell mass, vascular endothelial growth factor, or different cells into the central empty hole at the desired concentration for precise co-culture. Secondary patterning can be performed by injection.

In addition, the present invention is provided with a gas discharge passage (125). When the gas discharge passage 125 does not exist, there is only an inlet for loading the fluid into the second channel 121b, so when injecting the fluid using a fluid loading device such as a pipette, different testers may use different methods depending on the skill and skill of the experimenter. results can emerge. After the fluid is accommodated in the first channel 121a surrounding the second channel 121b, the fluid is loaded into the second channel 121b. At this time, the gas existing in the space to accommodate the fluid in the second channel 121b Since there is no space to be discharged other than the inlet, air bubbles may occur as the fluid and gas are mixed. In order to solve this problem, the present application is provided with a gas discharge passage (125). Through the above additional configuration, the generation of air bubbles can be prevented.

In addition, in order to maximize the effect of the present invention, a rail unit 126 was additionally provided. 5 and 6, the rail unit 126 may be formed at the boundary between the first channel 121a and the second channel 121b, the first channel 121a and the gas passage, and the rail unit 126 ) The height may be formed smaller than the height of each fluid channel. Accordingly, the fluid in the first channel 121a may not invade the second channel 121b and the gas discharge passage 125 .

The above principle is the same as that of the mesh structure described in the first embodiment. Specifically, the addition of the rail portion 126 may have an effect of reducing the radius of curvature of the fluid, which increases the Laplace pressure so that the fluid flows through the first channel 121a and the second channel 121b. It can be induced to maintain the interface without invading between the. The rail part 126 is preferably formed so as to be connected to each other at the part where the first channel 121a and the second channel 121b, and the first channel 121a and the gas passage part contact each other, and are located at the upper part based on the contact surface. It is desirable to form

Hereinafter, specific embodiments of the microfluidic device 100 according to the present invention will be described.

Example 1. Description of the mesh structure principle of the present application

In one embodiment of the present invention, based on the phenomenon of liquid being trapped inside the mesh structure, a micro-mesh channel was implemented in three dimensions. A liquid retains its shape below a certain limiting pressure due to the surface tension between the liquid and solid surfaces in the microstructure. The micromesh structure is for controlling microfluids in a 3D channel, and was produced using a photocurable polymer resin with a DLP-type 3D printer. The thickness of the smallest rectangular frame of the micromesh was 200 μm, and the hole size of the micromesh varied from 400 μm to 800 μm.

7 is a diagram explaining governing equations within a micro mesh structure.

When filling with fluid into the mesh structure, a pressure less than the capillary burst pressure (CBP) must be applied to the mesh to prevent the fluid from bursting out. The pressure applied to the fluid is determined by the height of the fluid itself, and the resulting critical height is used to design the overall structure size. In addition, it was confirmed that burst pressure mainly appeared depending on the shape of the mesh hole.

Referring to FIG. 7 , just before the fluid bursts out of the mesh, in the case of a micropore or structure, the fluid has a convex shape toward the outside of the hole, that is, toward the air, due to attraction with the solid. At this time, the pressure of the fluid is in equilibrium with the pressure applied to the fluid and follows the Young-Laplace equation. In the equation the equilibrium pressure of a fluid is predicted according to the curvature of both radii in the direction perpendicular to the fluid at a given point. For example, if the shape of the micromesh is circular, since the radius of curvature in the vertical direction is constant regardless of the point to be considered, it is possible to predict at what pressure the fluid will burst according to the mesh size of a specific size, as shown in the equation of FIG. 7 .

8 is a diagram showing experimental values of capillary burst pressure (CBP) according to the size and shape of the mesh structure. As shown in the graph on the left of FIG. 8, as a result of experiments by slowly increasing the hydrostatic pressure in the micro mesh platform, when the mesh size increased from 400 μm to 800 μm, CBP showed a similar trend in both expected and measured values. show what you have This means that when fabricating taller structures, smaller mesh structures should be used.

If the 3D shape of a hole or gap is not circular, it is difficult to predict the fluid shape immediately before bursting. Therefore, in order to predict how much pressure is maintained in the shape of the micromesh, a shape factor was defined and the value was approximated through experiments. The value of the shape factor when the shape of the mesh is not a circle but a polygon was experimentally determined. The graph on the right of FIG. 8 is a graph comparing the calculated value when the mesh shape is circular and the CBP in a polygonal mesh structure ranging from an actual triangle to a hexagon. The shape coefficient values have the same values as those in the table of FIG. 8 and decrease as the variable increases (triangle->hexagon). In various application studies, the predicted value of the pressure can be calculated when the mesh is made rectangular, and the shape factor can be experimentally measured when the structure is made with a general shape mesh.

As calculated in FIG. 8 , it is theoretically possible to hold a fluid up to 4 cm in height within a platform with a mesh of about 400 μm. Taking advantage of the advantage of designing with a 3D printer, various types of microfluidic channels 121 were fabricated. 9 is a diagram illustrating an embodiment of 3D fluid patterning through pipette injection. As shown in FIG. 9, a microchannel with a width of 5 mm and a length of 2 cm was designed and manufactured, and a cube with a width of 5 mm and a cross cell with a width of 5 mm can be connected up and down so that the overall shape is a square. A 3D microchannel with a micromesh size of 400 μm and a frame thickness of 200 μm was designed to form two separate three-dimensional twisted channels and successfully demonstrated that the fluid was patterned. Because they are all open channels, they can be filled with fluid by injection methods such as pipettes, syringes, and tubes.

FIG. 10 is a diagram showing an example of fluid patterning using dipping, and FIG. 11 is a diagram showing an example of fluid patterning using dipping and draining. As shown in FIG. 10, when a platform with mesh sizes of 400, 600, and 800 μm and three different channel structures is designed on one plane and gradually submerged in liquid, the pressure applied to the channel gradually increases, selectively to be patterned. The advantage of filling the channels by dipping is that the size of the mesh can be changed to selectively fill the fluid in an orderly manner without the need for other external pumps or valves. This method can be applied not only to the microfluidic device 100 for 3D cell culture, but also to an experimental tool that can easily check biochemical microfluidic reactions.

When fluid is filled in a platform using a micromesh, the micromesh simultaneously serves as an inlet through which the fluid can enter and a virtual wall that prevents the fluid from bursting. The liquid is trapped within the platform when the hydrostatic pressure is less than the surface tension of the mesh pores, and bursts and drains when the hydrostatic pressure increases and reaches the critical surface tension of the mesh pores. The hydrostatic pressure is determined by the total height of the device.

In Figure 11, it is designed to convert the gravitational height into geometric asymmetry by rotating the platform. The length, width and height of this platform are 40 mm, 1.4 mm and 17 mm, respectively, and the mesh size of letters S, N and U is 400 μm and the mesh size of the background is 1000 μm. When it is immersed in liquid, the entire channel is filled with liquid, and even after the device is lifted from the liquid reservoir, the liquid is still trapped in the entire channel because the hydrostatic pressure at the 16 mm height does not exceed the CBP of the 1000 μm mesh. However, rotating the device 90 degrees changes the height and length of the device. Since the hydrostatic pressure of 40 mm is greater than the CBP of 1000 μm mesh and less than that of 400 μm mesh, the liquid in the characters remains still and the liquid in the background bursts. This dipping, spinning, and ejection process allows the liquid to be patterned in a defined area much faster than injecting with a pipette or syringe. The bottom picture of Figure 11 shows the formation of one microliter droplet array (24) inside the channel by applying this patterning method, which can be applied to further multi-cell co-culture or organoid research.

12 is a view showing an example of co-culture of HUVEC and Lung Fibroblast for simulating perfused microvessels. To mimic the microvascular structure within the micromesh-based platform, a channel with a mesh hole size of 400 μm and a frame width and thickness of 200 μm was fabricated. Thereafter, parylene was coated to a height of about 2 to 3 μm for biocompatibility and to reduce the toxicity of the resin using an uncured 3D printer. For observation, the bottom surface was bonded with PC (polycarbonate) film coated with silicon without mesh. The platform consisted of three microchannels, each 1.5 cm long and 5 mm wide. First, Human Umbilical Vein Endothelial Cell (HUVEC) and fibrin gel were injected into the middle channel. A mixture of Lung Fibroblast (LF) and fibrin gel was injected into the remaining two channels among the three channels. After curing by injecting HUVEC into the middle channel and LF into both channels, they were cultured for 7 days. In the fluorescence microscope image, it was confirmed that relatively large capillaries ranging from 50 μm to 300 μm were obtained.

13 is a diagram illustrating an example of co-culture of cancer cells and blood vessel cells through dipping and draining. In an embodiment of the present invention, a 3D patterning technique was developed in which a microfluidic platform was continuously immersed in a mixture of fibrin gel and cells. The patterning method consists of two dipping processes and one draining process in separate hydrogels. In FIG. 13 the central patterning area of the platform is 1 mm x 1 mm x 1 mm and the side patterning areas are 5 mm x 3 mm x 3 mm in length, width and height. The mesh size of the central patterning area is 400 μm thick and the mesh size of the side patterning area is 1000 μm. Two types of hydrogels were prepared in separate wells of a 24-well plate to reconstitute the tumor microenvironment.

The first hydrogel was composed of fibrin gel and glioblastoma cells (U87MG), and the second hydrogel was composed of fibrin gel, human brain microvascular endothelial cells (HBMEC), and LF. A small amount of thrombin was added to the hydrogels immediately before the platform was immersed in each hydrogel. When the first hydrogel is immersed and drained, only the hydrogel in the lateral patterning area is drained while the hydrogel in the central patterning area is trapped within the platform by the difference in CBP. Then, after the fibrin gel is solidified, the platform is immersed in a second hydrogel to selectively pattern U87MG in the center and vascular cells in the lateral region.

Figure 13 is the result of observation after 7 days of culturing the cells in the above manner. Nuclei of all cells were stained blue with DAPI, and HBMEC were stained green with CD31. As a result of confocal microscopy, it was confirmed that a vascular network was selectively formed on the lateral part of the platform, and it was confirmed that U87MG was well cultured, as the nuclei of cells other than vascular cells were stained in the central part.

Example 2. Improvement of co-cultivation success rate through mesh structure and gas discharge passage 125

As described above, the present invention can solve the bubble trap problem in which bubbles are generated by providing the configuration of the gas discharge passage 125 and greatly improve the patterning speed of the second channel 121b.

When using one embodiment of the present application, when the hydrogel is loaded into the second channel (121b), it is not the same timing as the hydrogel of the first channel (121a), but it is patterned well without problems even if it is loaded after a later time. confirmed that In particular, even if the hydrogel is patterned in the first channel 121a, the entire chip media is immersed in it, and the above media is removed after a few days, and then a new hydrogel is patterned in the second channel 121b, the patterning is easy and the first channel 121a ) and the second channel 121b, it was confirmed that the interaction is also actively made. This means that when co-cultivating blood vessels and tumor cells having different cell growth rates, the blood vessel cells can be loaded first to establish a blood vessel network, and then the tumor cells can be injected at a desired timing.

A and B of FIG. 14 show a process of loading fluid into the fluid channel 121 of the present application. When comparing the device with and without the gas discharge passage 125 as in the present application, the patterning of the first channel 121a (Part A) is possible at the same speed, but the patterning of the second channel of the present invention is possible. (121b) (Part B) patterning is a phenomenon in which bubbles are stagnant at the boundary between the first channel 121a (Part A) and the second channel 121b (Part B), and the gas discharge passage 125 does not exist. Compared to the patterning of non-devices, the improvement was over 95%.

In addition, the present application is able to set a time difference between patterning the first channel 121a and patterning the second channel 121b by providing the gas discharge passage 125 . That is, a co-culture method is possible in which the first channel 121a is first loaded, cells therein are grown for a certain period of time, and then the second channel 121b is loaded. In C of FIG. 14, the second channel 121b (Part B) is not loaded at the same time as the first channel 121a (Part A), but the media is first loaded, and then all of the media is sucked in and then the hydrogel is loaded. Indicates the process of reloading. According to this, it can be used as a co-culture platform between cells with different cell growth rates. In particular, when establishing a tumor-microenvironment, a vascular network is first formed and then the tumor is patterned to perform co-culture.

According to one embodiment, the width of the gas discharge passage 125 may be 1 mm, and the size of the second injection part 124b of the second channel 121b may be 1.5 mm. At this time, the second channel 121b can accommodate up to 5 μl of hydrogel.

Example 3. Vascular experiment and tumor cluster co-culture experiment method through a new patterning method within the platform

FIG. 15 is a view showing the formation of a vascular network within the microfluidic device 100 of the present application, confirming the interaction between the first channel 121a (Part A) and the second channel 121b (Part B). . 15A is a schematic diagram of angiogenesis, and the results can be seen in FIG. 15B. In the first channel 121a (Part A), a hydrogel containing lung fibroblasts was inserted, and in the second channel 121b (Part B), endothelial cells (EC) were placed. Angiogenesis was simulated by inserting it to confirm the interaction between each channel. 15C and D show schematic diagrams of vasculogenesis or actual experimental results, respectively. Unlike the previous experiment, when the hydrogel containing lung fibroblasts and vascular endothelial cells was loaded in both the first channel 121a (Part A) and the second channel 121b (Part B), the first channel 121a ( It was confirmed that new/formed blood vessels in Part A) and the second channel 121b (Part B) met each other to establish a vascular network.

A and B of FIG. 16 show schematic diagrams and results of culturing with tumor spheroids, respectively. 16 C and D are a schematic diagram of cluster co-culture and a photograph of fluorescence imaging by putting cancer cells in an actual cluster form, respectively. In the first channel 121a (Part A), a hydrogel containing lung fibroblasts and vascular endothelial cells was included to build blood vessels, and after 3 days, tumor cells were placed in the hydrogel in the second channel 121b (Part B). included and loaded.

In order to utilize the large surface area of the second channel 121b (Part B), it can be seen that clusters of various sizes grow in one microfluidic device 100 when cancer cells are inserted and then grown. Previously, drug screening was performed by cultivating clusters of one size in one well in a 96-well plate, whereas clusters of various sizes can be obtained in one well by utilizing the present invention. In particular, various data can be easily obtained through a single well during drug screening.

Example 4. Introduction of a platform and analysis method capable of analyzing cluster tumors of various sizes at once

In order to show the efficiency of drug screening of tumor clusters grown in the microfluidic device 100 according to the present invention, the size of all clusters was analyzed (see A in FIG. 17).

166T is the name of the carcinoma, and the anticancer drug used for drug screening is FOX (5-Fu + Oxaliplatin). B in FIG. 17 shows all clusters in the second channel 121b (Part B) organized by size for each case. C in FIG. 17 is an enlarged view of the lower part of B, and the red line is the average cluster size. As can be seen in the graph, it can be seen that the control group has a larger average cluster size than the drugs FOX 50, 100, and 200, and overall, absolutely large clusters are also distributed in the control group and FOX50. can confirm that 17D are representative fluorescence images of each condition.

There are clusters of various sizes in each condition, and the graphs in FIG. 18 measure cell viability for each cluster size. Control grows well compared to other conditions to the extent that the overall size comes out up to 30000, and it can be seen that the cell viability is as high as about 80% or more for each size. However, looking at the FOX conditions, it can be seen that the overall cluster size is reduced and the cell survival rate according to the size is greatly reduced.

Through these results, in the case of the newly improved microfluidic device 100, co-culture is very easy unlike before, and thus has a great advantage in drug screening. In addition, it shows that it is a platform capable of meaningful analysis according to control and drug concentration through actual drug screening, showing a further improvement.

100: microfluidic element 110: casing
120: mesh unit 121: fluid channel
121a: first channel 121b: second channel
122: fin 122a: first fin
122b: second pin 122c: third pin
123: opening 124a: first injection part
124b: second injection part 125: gas discharge passage part
126: rail part

Claims (12)

In the microfluidic device providing a space for cell culture,
a fluid channel including a first channel surrounding the center and a second channel disposed in the center;
a plurality of pins forming the fluid channel;
a plurality of openings formed between the plurality of pins to communicate the inside and outside of the fluid channel with each other;
a second injection part formed to communicate an external space of the fluid channel with the second channel; and
A microfluidic device having a gas discharge passage formed so that the first channel communicates with the outside along a predetermined direction so as not to form a closed loop, and the second channel communicates with the outside along the predetermined direction. . According to claim 1,
The plurality of pins,
a first fin surrounding an outer circumferential side of the first channel;
a second fin formed above the first channel; and
A microfluidic device comprising a third fin formed on an upper portion of the inner circumference of the first channel. According to claim 2,
The second fins are spaced apart from each other to form at least one of the openings, the microfluidic device. According to claim 2,
The second pin connects the first pin and the third pin, the microfluidic device. According to claim 2,
The third pin forms the second injection part, and the second channel is formed under the second injection part. According to claim 2,
The height of the first fin is higher than the heights of the second fin and the third fin, the microfluidic device. According to claim 2,
The height of the second fin is the same as the height of the third fin, the microfluidic device. According to claim 1,
The distance at which the plurality of pins are spaced apart from each other is set to induce the first fluid to flow through the first channel but to suppress the flow to the second channel. According to claim 1,
The microfluidic device, characterized in that the area of each of the openings is set to induce the first fluid to flow through the first channel while suppressing the flow to the second channel. According to claim 1,
The microfluidic device further comprising a first injection part formed to communicate an external space of the fluid channel and the first channel to each other. According to any one of claims 1 to 10,
The gas discharge passage portion includes a rail portion extending downward from a lower surface of a pin adjacent to the second injection portion to prevent the first channel from forming a closed loop. According to claim 11,
The height of the rail portion is smaller than the height of the first channel or the second channel, the microfluidic device.

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