KR20220165668A - Microfluidic chip compiring tumor spheroid - Google Patents

Abstract

The present invention relates to a microfluidic device, a cell culture method in the microfluidic device, and a drug screening method. The microfluidic device mimics a structure and a function of 2D-3D connective tissue or a 3D spheroid and blood vessels to provide an environment suitable for a tumor spheroid, and thus can be used as a model for the development of targeted therapeutic agents for cancer and new drugs by replacing animal models in vitro.

Description

Microfluidic chip containing tumor spheroid {MICROFLUIDIC CHIP COMPIRING TUMOR SPHEROID}

The present invention provides an environment suitable for tumor spheroids by mimicking the structure and function of 2D-3D connective tissue or 3D spheroids and blood vessels, replacing in vitro animal models for cancer target therapy and new drug development. It relates to a microfluidic device that can be used as a model, a cell culture method in the microfluidic device, and a drug screening method.

Organ-on-a-chip, which integrates organ tissues in the form of a chip, has been developed to solve ethical problems caused by the use of experimental animal models to test the efficacy and side effects of drugs and problems caused by heterogeneous differences. It became. Organ chip is a technology that implements desired functions by culturing cells constituting a specific organ on a small chip to mimic the shape and physiological characteristics of the organ. Using this, it is possible to study in detail the cell behavior of a specific organ and the mechanism of physical and chemical reactions in the microenvironment, and can be used as a model for new drug development and toxicity evaluation.

In the development of organ chips, the cell culture model using the existing two-dimensional (2D) cell culture uses a petri dish to culture cells in two dimensions on the bottom of the dish, so many cell types It has a problem of not being able to explain tissue-specific differentiated functions or accurately predicting tissue function and drug activity in vivo.

In particular, the geometric limitations of these two-dimensional cell culture models have the possibility of changing the profile of tumor cell formation and tissue behavior and gene expression, and there is no continuous supply of nutrients and oxygen, or circulation of waste products. There is an increasing need for 3D cell culture technology that well simulates the spatial structure and biochemical complexity of tissues and vascular structure that can be cultured continuously through perfusion.

In addition, since the 3D model using spheroids mimics the in vivo situation well, it is possible to implement directional growth and the complexity of intercellular connections in in vitro experiments, and to study tissue functions on a molecular basis. Compared to two-dimensional cell culture models, it is advantageous to capture signaling pathways, metabolism, cell stress, and drug responsiveness of various disease states.

On the other hand, microfluidics technology developed along with semiconductor processing technology has enabled mass production of structures with micro-scale specifications with high accuracy. In particular, photolithography and soft lithography technologies have made it possible to easily produce a microchannel structure through stamping using a semi-permanent mold, and through this, there are attempts to simulate microstructures such as blood vessels of the human body or cell layers of organs in vitro.

Existing research has fabricated a chip composed of polydimethylsiloxane (PDMS) microchannels and a porous membrane using photolithography or soft lithography technology, and implemented the surrounding environment structure of tumor cells through cell and spheroid culture inside the chip. was carried out in the form of However, microfluidic organ chips made of PDMS have limitations in that they are difficult to apply to the actual pharmaceutical industry due to the disadvantage that drugs or proteins are often lost due to adsorption on the PDMS surface.

Therefore, the present inventors have improved the problems of existing microfluidic organ chips using PDMS, and realized perfusion through 3D culture of endothelial cells and tumor spheroids in the channel to form a vascular network, and a microfluidic device similar to the inside of the human body. was developed to complete the present invention.

The present invention solves problems caused by ethical problems and heterogeneous differences according to the use of experimental animal models, and forms a vascular network through 3-dimensional culture of endothelial cells and tumor spheroids in a channel to form a 2D-3D connective tissue or 3D connective tissue. An object of the present invention is to provide a microfluidic device that simulates the structure and function of dimensional spheroids and blood vessels.

The present invention is an insert comprising a first central channel; a base including a second central channel, a third side channel, and a fourth side channel; and a porous membrane positioned between the insert and the base.

The present invention is an insert comprising a first central channel; a base including a second central channel, a third side channel, and a fourth side channel; And it relates to a cell culture method in a microfluidic device comprising a porous membrane positioned between the insert and the base,

assembling a porous membrane positioned between an insert including a first central channel and a base including a second central channel, a third side channel, and a fourth side channel;

assembling the insert to a base including a second central channel, a third side channel, and a fourth side channel;

injecting a second cell into the second central channel;

injecting a first cell into the first central channel; and

Provided is a cell culture method in a microfluidic device comprising the step of perfusing a first fluid into the second central channel, the third side channel, and the fourth side channel.

The present invention provides a method for screening a drug capable of inhibiting or treating tumors using the microfluidic device.

The microfluidic device of the present invention includes tumor spheroids in a channel and endothelial cells for vascular mimics, so that it can simulate major structures and mass transfer phenomena around various tumor cells in vivo and can be used for anticancer drug efficacy evaluation.

In addition, the microfluidic device of the present invention enables precise quantification of the nanoparticle distribution around tumor spheroids, and since the insert is detachable from the base, the tumor spheroids included in the microfluidic device are selectively separated and analyzed without damage. available for

In addition, the microfluidic device of the present invention is capable of 3-dimensional culture of tumor spheroids, realizing the spatial structure and biochemical complexity of cells in vivo, thereby better mimicking the structure and function of tissue barriers.

1 and 2 are cross-sectional views of a microfluidic device.
3 is a perspective view of a microfluidic device.
4 and 5 are exploded and combined views of the microfluidic device.
6 is a cross-sectional view of a microfluidic device including cells.
7 and 8 are cross-sectional views showing a configuration and a method for attaching and detaching an insert and a base.
9 is a cross-sectional view showing fluid flow in the base of the microfluidic device.
10 and 11 are perspective views of the insert.
12 and 13 are plan views of the base.
14 is a bottom view of the base.
15 and 16 are perspective and cross-sectional views showing a configuration and a method for attaching and detaching an insert.
17 and 18 are a-a' cross-sectional views and enlarged cross-sectional views of a microfluidic device in which an insert and a base are attached.
19 and 20 are front and bottom views of a microfluidic device in which an insert and a base are attached.
21 is a perspective view illustrating fluid flow in a microchannel included in an insert.
22 is a cross-sectional view showing fluid flow in microchannels included in the base.
23 and 24 are perspective views of the cover.
25 shows the form in which human vascular endothelial cells (RFP-HUVEC) passing through a porous membrane generate blood vessels and move from the upper end to the lower end of a three-dimensional tumor spheroid cultured inside a three-dimensional fibrin gel (Fibrin ECM) (Scale bar = 200 μm).

Hereinafter, with reference to the accompanying drawings, embodiments and examples of the present disclosure will be described in detail so that those skilled in the art can easily practice the present invention. However, the present application may be implemented in various forms and is not limited to the embodiments and examples described herein.

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.

The present invention is an insert comprising a first central channel; a base including a second central channel, a third side channel, and a fourth side channel; and a porous membrane disposed between the insert and the base, wherein the second central channel has a lower channel height than the first central channel.

As used herein, the term "microfluidic device" refers to a device that includes microchannels, etc. provided to allow fluid to flow on a substrate made of various materials including plastic, glass, metal, or silicon including organic polymeric materials. it means.

As used herein, the term "microchannel" refers to a microscopic sized channel having a dimension of millimeters, micrometers, or nanometers through which a fluid can flow.

As used herein, the term "insert" may refer to an object of a shape that can be inserted into a groove formed in another object, but is not limited thereto.

In one embodiment, the insert may be detachable from the base, and the base may include a groove into which the insert may be inserted. Preferably, the insert includes a first fastening head, and the base includes a second fastening head, so that the first fastening head and the second fastening head are hooked together so that the insert and the base are snap-fit ( snap fit) may be detachable. In this case, in order to prevent leakage between the first central channel and the second central channel, the porous membrane may receive a strong pressure between the insert and the base to serve as a gasket.

As used herein, the term “fastening head” may be made of a material having elasticity such as plastic, rubber, or silicone, and may be elastically deformed to achieve snap-fit attachment or detachment, but is not limited thereto.

As used herein, the term "snap-fit" refers to a fastening method in which fastening force is formed by the interaction of grooves that are engaged with each other without additional parts or fastening mechanisms.

In one embodiment, the first central channel and the second central channel may include an inlet and an outlet capable of inducing and controlling perfusion of the liquid medium. A method of inducing and controlling the perfusion may include, but is not limited to, a method using a hydrostatic pressure difference between the inlet and the outlet, and a method of connecting an external pump to the inlet and outlet.

In one embodiment, the base may include a first fluid reservoir and a second fluid reservoir. In addition, the base may include a second central channel, a third side channel, and a fourth side channel connecting the first fluid reservoir and the second fluid reservoir. Preferably, the height of the third side channel and the fourth side channel may be lower than that of the first central channel.

In one embodiment, the second central channel may be formed horizontally with the direction of the first central channel.

In one embodiment, the third side channel and the fourth side channel may be formed perpendicular to a direction of the first central channel.

In one embodiment, the microfluidic device may be made of plastic, glass, metal or silicon, but is not limited thereto. Preferably, it can be made of plastic using injection molding technology.

In one embodiment, the first central channel may include vascular endothelial cells. Preferably, it may be a human umbilical vein endothelial cell (HUVEC).

As used herein, the term "cell" refers to biological cells, including plant cells, animal cells (eg, mammalian cells), bacterial cells and fungal cells, and the like.

As used herein, the term “tissue vascular endothelial cells” refers to cells that play a role in maintaining a specific structure of a tissue and protecting a tissue from external stimuli. In addition, it refers to a cell that selectively permeates substances and maintains the homeostasis of a substance concentration in a tissue by using a strong binding force between tissue barrier cells or an extracellular matrix. Tissue vascular endothelial cells include epithelial tissue cells and mesenchymal cells.

In one embodiment, the second central channel may include tumor cells. In addition, a hydrogel may be additionally included.

The tumor cells are melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, It may be lung cancer, pancreatic cancer, gastric cancer, kidney cancer, colon cancer, hematopoietic tumor or metastatic cancer cells thereof, but is not limited thereto.

The hydrogel may be one or more selected from the group consisting of collagen, laminin, hyaluronic acid, minerals, fibrin, fibronectin, elastin, peptides, polyethylene glycol, and alginate. Preferably, the hydrogel may be collagen gel, fibrin gel, laminin gel, matrigel, animal-derived tumor basement membrane extract gel, tissue decellularization extracellular matrix gel, peptide gel, polyethylene glycol gel, or alginate gel, but is not limited thereto. . More preferably, it is laminin gel or matrigel.

As used herein, the term "hydrogel" is a hydrophilic polymer crosslinked by cohesive forces such as covalent bonds, hydrogen bonds, van der waals bonds, or physical bonds, and is a three-dimensional polymer that can swell by containing a large amount of water in an aqueous solution. It means a material having a polymer network structure.

In one embodiment, by separating the insert from the base, one or more cells selected from the group consisting of cells attached to the porous membrane and cells included in the second central channel may be separated, respectively, and the separated Cell analysis such as genetic analysis may be performed using the cells.

In one embodiment, after the insert is separated from the base, a necessary treatment, such as drug application, is applied to the first central channel, the insert is assembled to the base again to observe the cell response.

Assembling a porous membrane positioned between an insert including a first central channel and a base including a second central channel, a third side channel, and a fourth side channel;

assembling the insert to a base including a second central channel, a third side channel, and a fourth side channel;

injecting a second cell into the second central channel;

injecting a first cell into the first central channel; and

Provided is a cell culture method in a microfluidic device comprising the step of perfusing a first fluid into the second central channel, the third side channel, and the fourth side channel.

In one embodiment, the first central channel may include an inlet and an outlet capable of inducing and controlling perfusion of the liquid medium, and the first cells may be injected through the inlet of the first central channel.

In one embodiment, the second central channel may include an inlet and an outlet capable of inducing and controlling perfusion of the liquid medicine, and the second cells may be injected through the inlet of the second central channel.

In one embodiment, the first cell is a vascular endothelial cell; It may preferably be a human umbilical vein endothelial cell, and the second cell is a tumor cell; Preferably melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer, lung cancer , It may be selected from the group consisting of pancreatic cancer, gastric cancer, renal cancer, colon cancer, hematopoietic tumors, and metastatic cancer cells thereof.

The present invention is an insert comprising a first central channel; a base including a second central channel, a third side channel, a fourth side channel, a first fluid reservoir, and a second fluid reservoir; and a porous membrane positioned between the insert and the base, wherein the insert is detachable from the base. and perfusing through a fourth side channel; And it provides a blood vessel simulating method comprising the step of sequentially reaching the second fluid reservoir.

In one embodiment, the base may include the second central channel, the third side channel, the fourth side channel, the first fluid reservoir, and the second fluid reservoir.

The microfluidic device of the present invention will be described in more detail through the following drawings. The present invention may include a microfluidic device embodied in the form of FIGS. 1 to 24, but is not limited thereto. In particular, although specific embodiments of the microfluidic device of the present invention are shown in FIGS. 10 to 24, the present invention is not limited to the specific embodiments, and various modifications can be made by those skilled in the art to which the present invention belongs. Including what can be done.

A microfluidic device according to an embodiment of the present invention includes an insert 100, a base 200, and a porous membrane 300 positioned between the insert 100 and the base 200.

Referring to FIG. 1 , the insert 100 includes a first central channel 110 . In one embodiment, the bottom portion 400 may be attached to the lower end of the base 200 .

2 and 3, the base 200 of the microfluidic device may include a first fluid reservoir 240 and a second fluid reservoir 250, the first fluid reservoir 240 and the second fluid reservoir 240. It may include a second central channel 210, a third side channel 220, and a fourth side channel 230 connecting the reservoir 250.

4 and 5, the porous membrane 300 of the microfluidic device exists between the first central channel 110 and the second central channel 210, and the second central channel 210 is the first central channel 210. It may be formed in the direction of the channel 110 and horizontally.

Referring to FIG. 6 , the first central channel 110 of the microfluidic device may include first cells 111, and the second central channel 210 may include second cells 211. . In one embodiment, the first cells 111 may be attached to the upper surface of the porous membrane 300 and cultured, and the second cells 211 may be 3D cultured inside the second central channel 210. can

7 to 9, 18, and 19, the base 200 of the microfluidic device includes an insert groove 201 into which an insert 100 can be inserted, and the insert 100 includes a base 200 ) may be detachable from. In addition, the insert 100 includes a first fastening head 101, and the base 200 includes a second fastening head 202, so that the first fastening head 101 and the second fastening head ( 202) may be hooked to each other so that the insert 100 and the base 200 are detachable with a snap fit.

Referring to FIGS. 10 and 11 , the insert 100 of the microfluidic device may include a first central channel inlet 112 and a first central channel outlet 113 . The first central channel inlet 112 and the first central channel outlet 113 may be connected to the first central channel 110 in the form of a tube, and an electrode is inserted through the tube at a predetermined position and depth to obtain a TEER value. Precise measurements may be possible.

12 to 20 and 22, the base 200 of the microfluidic device may have a structure in which one or more inserts 100 are detachable.

Referring to FIGS. 23 and 24 , the microfluidic device may include a cover 500 .

Referring to FIG. 21 , the fluid is injected through the first central channel inlet 112 included in the insert 100 of the microfluidic device, and passes through the first central channel 110 toward the first central channel outlet 113. fluid can be perfused.

9 and 22, by injecting a fluid into the first fluid reservoir 240 included in the base 200 of the microfluidic device, a third side channel 220, a second central channel 210, The fluid may be sequentially passed through the 4 side channels 230 and the second fluid reservoir 250 .

[Example 1]

Fabrication of a tumor microenvironmental device combining vascular mimic structure and 3D cancer tissue cell culture

Using the assembled microfluidic device, a tumor microenvironment (TME) simulating microfluidic device combining a blood vessel-mimicking structure and three-dimensional tumor spheroid culture was fabricated in the following way.

10 μl of fibronectin was injected into the first and second central channels so that the cells could be evenly attached to the microfluidic device. After incubation for 1 hour, 10 μl of human lung fibroblasts (NHLF) at a concentration of 10 ⁶ cells/ml was injected into the second central channel and cultured for 2 hours to evenly adhere to the fibroblasts. Three-dimensional tumor spheroids with a size of 300 to 400 μm embedded in 15 μl of fibrin gel (Fibrin ECM) were injected into the second central channel and cultured for 30 minutes to harden the fibrin gel. 10 μl of human vascular endothelial cells (RFP-HUVEC) expressing RFP at a concentration of 15 x 10 ⁶ cells/ml were injected into the first central channel. In a state in which the culture medium was filled in the first fluid reservoir connected to the third lateral channel, the second central channel, and the fourth lateral channel, the vascular endothelial cells were cultured for 15 hours or more so that the vascular endothelial cells could be evenly adhered to the surface of the porous membrane. Three-dimensional tumor spheroids and vascular mimic structures were imaged using a confocal microscope (Zeiss LSM 780).

As shown in FIG. 25, RFP-expressing human vascular endothelial cells (RFP-HUVEC) passing through a porous membrane generate blood vessels, and in the direction from the upper end to the lower end of the three-dimensional tumor spheroid cultured inside the three-dimensional fibrin gel (Fibrin ECM). The moving shape was confirmed.

100: insert
101: first fastening head
110: first central channel
111 first cell
112: first central channel inlet
113: first central channel exit
200: base
201: insert insertion groove
202: second fastening head
210: second central channel
211 second cell
212: second central channel inlet
213: second central channel exit
220: third side channel
230: fourth side channel
240 first fluid reservoir
250: second fluid reservoir
300: porous membrane
400: bottom
500: cover

Claims (21)

an insert including a first central channel;
a base including a second central channel, a third side channel, and a fourth side channel; and
A porous membrane positioned between the insert and the base,
The second central channel is characterized in that the height of the channel is lower than the first central channel, microfluidic device.
The microfluidic device according to claim 1, wherein the insert is detachable from the base.
3. The method of claim 2, wherein the insert comprises a first fastening head;
The base includes a second fastening head,
The microfluidic device, characterized in that the first fastening head and the second fastening head are hooked to each other so that the insert and the base are attached and detached with a snap fit.
The microfluidic device according to claim 2, wherein the base includes a groove into which the insert can be inserted.
The microfluidic device according to claim 1, wherein the first central channel and the second central channel include an inlet and an outlet capable of inducing and controlling perfusion of the liquid medium.
The microfluidic device of claim 1 , wherein the base further comprises a first fluid reservoir and a second fluid reservoir.
The microfluidic device of claim 6, wherein the base includes a second central channel, a third side channel, and a fourth side channel connecting the first fluid reservoir and the second fluid reservoir.
The microfluidic device according to claim 1, wherein the second central channel is formed in a direction parallel to that of the first central channel.
The microfluidic device according to claim 1, wherein the third side channel and the fourth side channel are formed perpendicular to a direction of the first central channel.
The microfluidic device according to claim 1, wherein the microfluidic device is made of plastic, glass, metal or silicon.
The microfluidic device according to claim 1, wherein the first central channel comprises vascular endothelial cells.
The microfluidic device according to claim 11, wherein the vascular endothelial cells are human umbilical vein endothelial cells (HUVECs).
The microfluidic device according to claim 1, wherein the second central channel comprises tumor cells.
The method of claim 13, wherein the tumor cell is melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, A microfluidic device, characterized in that at least one selected from the group consisting of leukemia, lymphoma, liver cancer, lung cancer, pancreatic cancer, gastric cancer, kidney cancer, colon cancer, hematopoietic tumors, and metastatic cancer cells thereof.
14. The microfluidic device of claim 13, wherein the second central channel additionally comprises a hydrogel.
The microfluidic device according to claim 15, wherein the hydrogel is at least one selected from the group consisting of collagen, laminin, hyaluronic acid, minerals, fibrin, fibronectin, elastin, peptides, polyethylene glycol, and alginate.
According to claim 1, wherein the insert is separated from the base, characterized in that one or more cells selected from the group consisting of cells attached to the porous membrane and cells included in the second central channel can be separated, respectively. To do, microfluidic device.
assembling a porous membrane positioned between an insert including a first central channel and a base including a second central channel, a third side channel, and a fourth side channel;
assembling the insert to a base including a second central channel, a third side channel, and a fourth side channel;
injecting a second cell into the second central channel;
injecting a first cell into the first central channel; and
Perfusing a first fluid through the third side channel, the second central channel, and the fourth side channel
Including, the cell culture method in the microfluidic device.
19. The method of claim 18, wherein the first central channel includes an inlet and an outlet capable of inducing and controlling perfusion of the liquid medium,
The cell culture method in a microfluidic device, characterized in that the first cell is injected through the first central channel inlet.
19. The method of claim 18, wherein the second central channel includes an inlet and an outlet capable of inducing and controlling perfusion of the liquid medium,
The cell culture method in a microfluidic device, characterized in that the second cell is injected through the second central channel inlet.
The method of claim 18, wherein the first cell is a human umbilical vein endothelial cell (HUVEC);
The second cell is melanoma, squamous cell carcinoma, breast cancer, head and neck cancer, thyroid cancer, soft tissue sarcoma, osteosarcoma, testicular cancer, prostate cancer, ovarian cancer, bladder cancer, skin cancer, brain cancer, angiosarcoma, mast cell tumor, leukemia, lymphoma, liver cancer , Lung cancer, pancreatic cancer, gastric cancer, kidney cancer, colorectal cancer, hematopoietic tumors and metastatic cancer cells thereof, characterized in that selected from the group consisting of, a cell culture method in a microfluidic device.

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