Microfluidic chip and method for producing spheroid or organoid in vitro model using the same

Abstract

To provide a method for forming a spheroid or organoid in vitro model, which improves analysis convenience and enables formation of various co-culture models.SOLUTION: A microfluidic chip includes: a cell inlet 11 into which a fluid containing cells is injected and a cell channel 12 connected thereto; and an ECM (extracellular matrix) inlet 21 into which a fluid containing an ECM component is injected and an ECM channel 22 connected thereto. The cell channel is located adjacent to a part of one side of the ECM channel, a micro barrier 40 having a lower height than the channels is located between them, and the micro barrier has a shape in which a plurality of concave patterns 41, curved in a direction of the ECM channel, are arranged at regular intervals. The present invention also provides a method of forming a spheroid or organoid in vitro model using the microfluidic chip.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microfluidic chip using a micro partition wall having a concave pattern shape and a method for forming a spheroid or organoid in vitro model using the same.

Background Art

[0002] Numerous drugs have been developed and evaluated for the treatment of human diseases, and research on in vitro human tissue and organ simulation models for drug development has been underway. Among the models, there are spheroids and organoids formed three-dimensionally by collecting cells, which have the advantage of being able to provide more complex and various microenvironments than two-dimensionally cultured cells. However, general spheroids and organoids have the problem that it is difficult to smoothly supply oxygen and nutrients because there are no perfusable three-dimensional blood vessels, and it is difficult to form an in vivo environment within the model.

[0003] To overcome this limitation, a model in which spheroids and organoids are co-cultured with a blood vessel network in a microfluidic chip has been developed. However, conventional co-culture models generally have the drawback that the process of injecting spheroids and organoids into the chip is quite complicated and difficult. Moreover, when culturing spheroids and organoids in a conventional microfluidic chip, there is a limitation in that it is difficult to adjust their numbers and positions.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The object of the present invention is to provide a spheroid or organoid microfluidic chip, which improves analytical convenience in the formation of spheroid or organoid in vitro models and enables the formation of various co-culture models, and which includes a cell injection port into which a fluid containing cells is injected and a cell channel connected thereto; and an ECM injection port into which a fluid containing ECM (extracellular matrix) components is injected and an ECM channel connected thereto; wherein the cell channel is located adjacent to a portion of one side of the ECM channel, and a micropartition wall having a height lower than that of the channel is located between the cell channel and the micropartition wall has a shape in which a plurality of recessed patterns curved toward the ECM channel are arranged at regular intervals.

[0006] However, the technical problems that the present invention aims to solve are not limited to the above problems, and other problems not mentioned will be clearly understood by those skilled in the art from the following description. [Means for solving the problem]

[0007] The present invention provides a spheroid or organoid microfluidic chip comprising a cell inlet into which a fluid containing cells is injected and a cell channel connected thereto; and an ECM inlet into which a fluid containing ECM (extracellular matrix) components is injected and an ECM channel connected thereto; wherein the cell channel is located adjacent to a portion of one side of the ECM channel, and a micropartition wall having a height lower than that of the channel is located between the cell channel and the micropartition wall has a shape in which a plurality of recessed patterns curved toward the ECM channel are arranged at regular intervals.

[0008] The cells and the ECM components may interact with each other between the cell channel and the ECM channel where the micro-diaphragm is not located.

[0009] The height of the micro-partition wall may be 1 / 5 to 1 / 2 of the height of the channel.

[0010] The concave patterns in the micro-partitions may be connected to inclined chamfers at both ends.

[0011] The diameter of the recessed pattern in the micro-partition wall may be 1 / 5 to 1 / 3 of the maximum width of the ECM channel.

[0012] The interval between the recessed patterns in the micro-partition wall may be 100 μm to 5,000 μm.

[0013] The cell may be a cancer cell or a neuronal cell.

[0014] The device further includes a second cell channel located adjacent to a portion of the other side of the ECM channel.

[0015] A second micro-partition having a height lower than that of the channels is positioned between the second cell channel and the ECM channel, and the second micro-partition may have a shape in which a plurality of second concave patterns curved toward the ECM channel are arranged at regular intervals.

[0016] The second cells may be of the same type as or different from the cells, and may be one or more co-cultured cells selected from the group consisting of cancer cells, neural cells (and vascular endothelial cells).

[0017] In one embodiment of the present invention, there is provided a method for forming an in vitro spheroid or organoid model, comprising: (a) injecting a fluid containing cells into a cell injection port of the microfluidic chip; and (b) allowing the injected cells to form or culture spheroids or organoids at recessed pattern positions arranged at regular intervals within the cell channel. [Effects of the Invention]

[0018] In the microfluidic chip according to the present invention, a micro partition wall having a height lower than that of the channels is located between the cell channel and the ECM channel, and the micro partition wall has a shape in which a plurality of concave patterns curved in the ECM channel direction are arranged at regular intervals. Thereby, spheroids or organoids can be captured and cultured through the concave patterns, and the interaction between the spheroids or organoids and the ECM components can be observed between the cell channel and the ECM channel where the micro partition wall is not located. Further, spheroids or organoids can be captured and cultured through the concave patterns.

[0019] Thereby, when forming or culturing spheroids and organoids, there is an advantage that it is easy to adjust the number and position thereof, the reliability of analysis data can be improved, and it is sufficient to analyze only at a determined position, so that the convenience of analysis can be enhanced.

[0020] Therefore, the spheroids and organoids cultured according to the present invention can be utilized as an animal substitute model for screening various drugs.

Brief Description of Drawings

[0021] [Fig. 1] It is a diagram schematically showing a microfluidic chip according to an embodiment of the present invention and a method for forming an in vitro model of spheroids or organoids using the same. [Fig. 2] It is a diagram showing a design change for optimizing the diameter of the concave pattern and the maximum width of the ECM channel in the microfluidic chip according to an embodiment of the present invention. [Fig. 3A] and [Fig. 3B] It is a diagram schematically showing a microfluidic chip provided with an ECM channel having a concave pattern on one side according to the first embodiment example of the present invention. [Fig. 3C] and [Fig. 3C] It is a diagram in which pancreatic cancer cells are applied as cells and vascular endothelial cells are applied as the second cells to the microfluidic chip and co-cultured for 7 to 9 days, and as a result, pancreatic cancer cell spheroids are observed. [Figure 3D] This is a diagram showing the capture position and number of groups of peripheral nerve cell spheroids as a result of applying peripheral nerve cells as cells and pancreatic cancer cells as second cells to the microfluidic chip and co-culturing them for 8 days. [Figure 4A] This is a diagram schematically showing a microfluidic chip provided with an ECM channel having a concave pattern on both sides according to the second embodiment of the present invention. [Figure 4B] This is a diagram showing lung cancer cell organoids as a result of applying all lung cancer cells as cells and second cells to the microfluidic chip and culturing them for 7 days. [Figure 5A] This is a diagram schematically showing a microfluidic chip provided with an ECM channel and a second ECM channel according to the third embodiment of the present invention. [Figure 5B] to [Figure 5D] This is a diagram showing pancreatic cancer cell spheroids as a result of applying pancreatic cancer cells as cells and vascular endothelial cells as second cells to the microfluidic chip and co-culturing them for 7 to 9 days. [Figure 5E] and [Figure 5F] This is a diagram showing peripheral nerve cell spheroids as a result of applying peripheral nerve cells as cells and vascular endothelial cells as second cells to the microfluidic chip and co-culturing them for 11 days.

Best Mode for Carrying Out the Invention

[0022] When culturing spheroids and organoids in a conventional microfluidic chip, there was a limit in that it was difficult to adjust their number and position. To overcome this, a microfluidic chip using a micro partition with a concave pattern shape was designed, and the height of the micro partition and the diameter of the concave pattern were optimized to complete the present invention.

[0023] Hereinafter, the present invention will be described in detail.

[0024] <Microfluidic Chip and Method for Forming an Ex Vivo Model of Spheroids or Organoids Using the Same>

[0025] The present invention provides a spheroid or organoid microfluidic chip, comprising: a cell injection port into which a fluid containing cells is injected and a cell channel connected thereto; and an ECM injection port into which a fluid containing an ECM (extracellular matrix) component is injected and an ECM channel connected thereto. The cell channel is located adjacent to a part of one side surface of the ECM channel, and a micro partition wall having a height lower than that of the channel is located therebetween. The micro partition wall has a shape in which a plurality of concave patterns curved in the direction of the ECM channel are arranged at regular intervals.

[0026] The present invention also provides a method for forming a spheroid or organoid in vitro model, comprising: (a) injecting a fluid containing cells into the cell injection port of the microfluidic chip; and (b) forming or culturing spheroids or organoids at the positions of the concave patterns where the injected cells are arranged at regular intervals in the cell channel.

[0027] Hereinafter, with reference to the accompanying drawings, embodiments of the present invention will be described in detail so that those having ordinary knowledge in the technical field to which the present invention pertains can easily implement them. The present invention can be implemented in various different forms and is not limited to the embodiments described herein.

[0028] To clearly explain the present invention, parts not related to the description are omitted, and the same reference numerals are given to the same or similar components throughout the specification.

[0029] In the following, when any configuration is formed on the "upper (or lower)" or "inside (or outside)" of the substrate, it not only means that any configuration is formed in contact with the upper (or lower) or inside (or outside) of the substrate, but also is not limited by the fact that no other configuration is included between the substrate and any configuration formed on the upper (or lower) or inside (or outside) of the substrate.

[0030] Referring to Figures 1 and 3A, a microfluidic chip according to one embodiment of the present invention includes a cell inlet 11 into which a fluid containing cells is injected and a cell channel 12 connected thereto; and an ECM inlet 21 into which a fluid containing ECM (extracellular matrix) components is injected and an ECM channel 22 connected thereto.

[0031] In addition, the cell channel 12 is located adjacent to a portion of one side of the ECM channel 22, and a micro-partition 40 having a height lower than that of the channel is located between them, and the micro-partition 40 is characterized in that it has a shape in which multiple concave patterns 41 curved toward the ECM channel 22 are arranged at regular intervals.

[0032] Specifically, the cells and the ECM components directly contact and interact with each other between the cell channel 12 and the ECM channel 22, where the micro-partition 40 is not located. Therefore, the height h of the micro-partition 40 may be 1 / 5 to 1 / 2 of the channel height H, preferably 1 / 5 to 2 / 5, but is not limited to this. The micro-partition 40 serves to support the ECM components so that the fluid containing the ECM components does not leak out of the ECM channel after injection and before gelation. If the height h of the micro-partition 40 is too low compared to the channel height H, the fluid containing the ECM components may leak out of the ECM channel before gelation after injection. On the other hand, if the height h of the micro-partition 40 is too high compared to the channel height H, the contact area between the channels is small, making interaction difficult.

[0033] Further, the diameter D of the concave pattern 41 in the micro partition wall 40 may be 1 / 5 to 1 / 3 of the maximum width W of the ECM channel 22, preferably 1 / 5 to 3 / 10, but is not limited thereto. The diameter D of the concave pattern 41 means the diameter when the concave pattern 41 is a semi-circle, and means the average of the major axis and the minor axis when the concave pattern 41 is a semi-ellipse. The maximum width W of the ECM channel 22 means the longest width in the distance between the other side surface of the ECM channel 22 and the micro partition wall 40 (that is, the width in the distance between the other side surface of the ECM channel 22 and the micro partition wall 40 where the concave pattern 41 or the inclined surface 42 is not formed). At this time, if the diameter D of the concave pattern 41 in the micro partition wall 40 is too large, there will be problems that the fluid injected under excessive pressure leaks out of the ECM channel or the gel filing of the fluid containing the ECM component cannot be smoothly performed. On the other hand, if the diameter D of the concave pattern 41 in the micro partition wall 40 is too small, it is not effective for capturing the spheroid or organoid.

[0034] The interval between the concave patterns 41 in the micro partition wall 40 may be 100 μm to 5,000 μm, preferably 300 μm to 3,000 μm, more preferably 600 μm to 3,000 μm, but is not limited thereto. Such an interval between the concave patterns 41 is larger than the diameter D of the concave pattern 41 in the micro partition wall 40 and can be controlled in consideration of the analysis amount of the spheroid or organoid.

[0035] At this time, the concave pattern 41 in the micro partition wall 40 can be connected to inclined surfaces (chamfers) 42 at both ends respectively. The inclined surfaces 42 are designed to make it easier to capture spheroids or organoids, and the inclined surfaces 42 may be inclined toward the concave pattern 41. The inclined surfaces 42 can be inclined so as to reduce the shear stress exerted by the fluid on the cell body and minimize the influence exerted during the capture of spheroids or organoids. Also, the inclined surfaces 42 are effective for gel filing of the fluid containing ECM components. Specifically, the inclined surface (chamfer) 42 may be inclined to the maximum extent up to the radius (or depth) (D / 2) of the concave pattern 41, may be 10° or more, and is preferably 10° to 40°, but is not limited thereto.

[0036] In the fluid containing the cells, the cells may be cells for culturing into spheroids or organoids, or may already be in the form of spheroids or organoids. For example, the cells may be cancer cells or nerve cells. In a specific embodiment of the present invention, pancreatic cancer cells, lung cancer cells or peripheral nerve cells were used. Also, the fluid may be various media for culturing cells, and in a specific embodiment of the present invention, a DMEM and EGM-2 mixed medium, a PNS medium or an LTM medium was used.

[0037] In addition, the fluid containing the ECM component may be a fluid containing at least one selected from the group consisting of alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyethylene glycol diacrylate (PEGDA), gelatin, Matrigel, poly(L-lactic acid) (PLLA), carboxymethyl cellulose, SAP, PHEMA-MMA, dextran, and chitosan. A fluid containing collagen, fibrin, or Matrigel is preferred, but not limited thereto. At this time, considering the viscosity of the fluid containing the ECM component, it may be a fluid containing collagen at 6 mg / mL or less, a fluid containing fibrin at 10 mg / mL or less, or a fluid containing Matrigel at 7 mg / mL or less.

[0038] Furthermore, the microfluidic chip according to an embodiment of the present invention further includes a second cell channel 32 located adjacent to a part of the other side surface of the ECM channel 22, and the second cell channel 32 can be connected to a second cell inlet 31 into which a fluid containing second cells is injected. Also, a second micro partition wall (not shown) having a height lower than that of the channel may be located between the second cell channel 32 and the ECM channel 22. Thereby, the second cells and the ECM component directly abut and interact with each other between the second cell channel 32 and the ECM channel 22 where the second micro partition wall (not shown) is not located. Similarly, the height h of the second micro partition wall (not shown) may be 1 / 5 to 1 / 2 of the channel height H, preferably 1 / 5 to 2 / 5, but not limited thereto.

[0039] Referring to FIG. 4A, the second micro partition wall (not shown) may have a shape in which a plurality of second concave patterns curved in the direction of the ECM channel are arranged at regular intervals. The diameter D' of the second concave pattern in the second micro partition wall (not shown) may be 1 / 5 to 1 / 3 of the maximum width W of the ECM channel 22, preferably 1 / 5 to 3 / 10, but is not limited thereto. The diameter D' of the second concave pattern means the diameter when the second concave pattern is a semi-circle, and means the average of the major axis and the minor axis when the second concave pattern is a semi-ellipse. Also, the maximum width W of the ECM channel 22 means the longest width in the distance between one side surface of the ECM channel 22 and the second micro partition wall (not shown) (that is, the width in the distance between one side surface of the ECM channel 22 and the second concave pattern or the second micro partition wall (not shown) where no inclined surface is formed). At this time, if the diameter D' of the second concave pattern in the second micro partition wall (not shown) is too large, there will be problems that the fluid injected under excessive pressure leaks out of the ECM channel or the gel filing of the fluid containing the ECM component cannot be performed smoothly. On the other hand, if the diameter D' of the second concave pattern in the second micro partition wall (not shown) is too small, it is not effective for capturing spheroids or organoids.

[0040] The interval between the second concave patterns in the second micro partition wall (not shown) may be 100 μm to 5,000 μm, preferably 300 μm to 3,000 μm, more preferably 600 μm to 3,000 μm, but is not limited thereto. Such an interval between the second concave patterns is larger than the diameter D' of the second concave pattern in the second micro partition wall (not shown) and can be controlled in consideration of the analysis amount of spheroids or organoids.

[0041] At this time, the second concave pattern in the second micro partition wall (not shown) can be connected to second inclined surfaces (chamfers) at both ends respectively. The second inclined surfaces are designed to facilitate the capture of spheroids or organoids, and the second inclined surfaces can be inclined toward the second concave pattern. The inclination of the second inclined surfaces can be set so as to reduce the shear stress exerted by the fluid on the cell body and minimize the influence exerted during the capture of spheroids or organoids. Also, the inclined surface 42 is effective for gel filing of the fluid containing the ECM component. Specifically, the second inclined surface may be inclined to the maximum extent up to the radius (or depth) (D' / 2) of the second concave pattern, may be 10° or more, and is preferably 10° to 40°, but is not limited thereto.

[0042] In the fluid containing the second cells, the second cells are cells that can be co-cultured with the cells, and may be of the same or different types as the cells. For example, the second cells may be one or more co-culture cells selected from the group consisting of cancer cells, nerve cells, and vascular endothelial cells. When the second cells are vascular endothelial cells, a vascular layer can be formed inside the second cell channel 32 along the direction of the other side surface of the ECM channel 22. In a specific embodiment of the present invention, vascular endothelial cells, pancreatic cancer cells, or lung cancer cells were used. Also, the fluid may be various media for culturing cells, and in a specific embodiment of the present invention, RPMI and EGM-2 mixed medium, PNS medium, or LTM medium was used.

[0043] Referring to FIG. 5A, a microfluidic chip according to another embodiment of the present invention includes a cell injection port 11 into which a fluid containing cells is injected and a cell channel 12 connected thereto; and an ECM injection port 21 into which a fluid containing an ECM (extracellular matrix) component is injected and an ECM channel 22 connected thereto.

[0044] Further, the cell channel 12 is located adjacent to a part of one side surface of the ECM channel 22, and a micro partition wall 40 having a height lower than that of the channel is located therebetween. The micro partition wall 40 has a shape in which a plurality of concave patterns 41 curved in the direction of the ECM channel 22 are arranged at regular intervals.

[0045] Since the cell injection port 11, the cell channel 12, the ECM injection port 21, the ECM channel 22, and the micro partition wall 40 have been described above, redundant explanations are omitted.

[0046] Furthermore, the microfluidic chip according to an embodiment of the present invention further includes a second ECM injection port 23 into which a fluid containing a second ECM component and, optionally, a fluid containing a second cell are injected, and a second ECM channel 24 connected thereto. A part of one side surface of the second ECM channel 24 may be located adjacent to a part of the other side surface of the ECM channel 22, and a second micro partition wall (not shown) having a height lower than that of the channel may be located therebetween.

[0047] In addition, the microfluidic chip according to an embodiment of the present invention may further include a second cell channel 32 located adjacent to a part of the other side surface of the second ECM channel 24. The second cell channel 32 can be optionally connected to a second cell injection port 31 into which a fluid containing a second cell is injected. Also, a third micro partition wall (not shown) having a height lower than that of the channel may be located between the second cell channel 32 and the second ECM channel 24.

[0048] Similarly, the height h' or h'' of the second or third micro partition wall (not shown) may be 1 / 5 to 1 / 2 of the channel height H, preferably 1 / 5 to 2 / 5, but is not limited thereto.

[0049] The fluid containing the second ECM component may be the same as or different from the fluid containing the first ECM component, and may be a fluid containing at least one selected from the group consisting of alginate, collagen, peptide, fibrin, hyaluronic acid, agarose, polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyethylene glycol diacrylate (PEGDA), gelatin, Matrigel, poly(L-lactic acid) (PLLA), carboxymethyl cellulose, SAP, PHEMA-MMA, dextran, and chitosan. A fluid containing collagen, fibrin, or Matrigel is preferred, but not limited thereto. At this time, considering the viscosity of the fluid containing the ECM component, it may be a fluid containing collagen at 6 mg / mL or less, a fluid containing fibrin at 10 mg / mL or less, or a fluid containing Matrigel at 7 mg / mL or less.

[0050] Also, in the fluid containing the second cell, the second cell is a cell that can be co-cultured with the cell and may be of the same or different type as the cell. For example, the second cell may be one or more co-cultured cells selected from the group consisting of cancer cells, nerve cells, and vascular endothelial cells. When the second cell is a vascular endothelial cell, it can penetrate into the second ECM channel 24 and form a vascular network by forming a reticular structure. In a specific embodiment of the present invention, vascular endothelial cells, pancreatic cancer cells, or lung cancer cells were used. Also, the fluid may be various media for culturing cells. In a specific embodiment of the present invention, RPMI and EGM-2 mixed medium, PNS medium, or LTM medium was used.

[0051] As described above, in the microfluidic chip according to the present invention, a micro partition wall having a height lower than that of the channels is located between the cell channel and the ECM channel, and the micro partition wall has a shape in which a plurality of concave patterns curved in the ECM channel direction are arranged at regular intervals. Thereby, spheroids or organoids are captured and cultured through the concave patterns, and the interaction between the spheroids or organoids and the ECM components can be observed between the cell channel and the ECM channel where the micro partition wall is not located. Also, spheroids or organoids can be captured and cultured through the concave patterns.

[0052] Thereby, when forming or culturing spheroids and organoids, there is an advantage that it is easy to adjust their number and position, the reliability of analysis data can be improved, and the convenience of analysis can be enhanced by analyzing only at the determined positions. In particular, when capturing spheroids and organoids, the number and position of the captured spheroids and organoids can be specified from the center of the concave pattern through the control of gravity and the flow of fluid.

[0053] Therefore, the spheroids and organoids cultured according to the present invention can be utilized as an animal substitute model for screening various drugs. Hereinafter, preferred examples are presented to assist in understanding the present invention. However, the following examples are provided to more easily understand the present invention, and the content of the present invention is not limited by the following examples.

[0054] Example Example 1: Optimal Design of a Microfluidic Chip Equipped with an ECM Channel 22 A spheroid / organoid microfluidic chip is designed, which consists of a cell injection port 11 into which a fluid containing cells is injected and a cell channel 12 connected thereto; an ECM injection port 21 into which a fluid containing ECM (extracellular matrix) components is injected and an ECM channel 22 connected thereto; and a second cell injection port 31 into which a fluid containing second cells is injected and a second cell channel 32 connected thereto.

[0055] At this time, the cell channel 12 is located adjacent to a part of one side surface of the ECM channel 22, and a micro partition wall (h: 200 μm) 40 with a height lower than that of the channel (H: 500 μm) is located therebetween. The micro partition wall 40 has a shape in which a plurality of concave patterns 41 curved in the ECM channel direction are arranged at regular intervals (at an interval of 1,600 μm with respect to the center point in the diameter D of the concave pattern 41). In particular, it is characterized in that it is connected to inclined surfaces (chamfers) 42 formed at an angle of 30° at both ends of the concave pattern 41. On the other hand, between the cell channel 12 and the ECM channel 22 where the micro partition wall is not located, the cells and ECM components are in direct contact and can interact with each other.

[0056] On the other hand, the second cell channel 32 is located adjacent to a part of the other side surface of the ECM channel 22, and a second micro partition wall (h': 200 μm) (not shown) with a height lower than that of the channel (H': 500 μm) is located therebetween. On the other hand, between the second cell channel 32 and the ECM channel 22 where the second micro partition wall is not located, the second cells and ECM components are in direct contact and can interact with each other.

[0057] If necessary, the second micro partition wall has a shape in which a plurality of second concave patterns curved in the ECM channel direction are arranged at regular intervals (at an interval of 1,600 μm with respect to the center point in the diameter D of the second concave pattern). In particular, it is characterized in that it is connected to inclined surfaces (chamfers) formed at an angle of 30° at both ends of the second concave pattern.

[0058] Furthermore, design changes were made to optimize the diameter of the recessed pattern 41. As shown in Table 1 below, spheroid / organoid microfluidic chips were designed by adjusting the diameter D of the recessed pattern 41 and the maximum width W of the ECM channel 22 in various ways. A fluid containing ECM components was then injected into the ECM injection port 21, and it was observed whether the injected fluid gelled without leaking out of the ECM channel.

[0059] As a result, as can be seen from Table 1 and Figure 2 below, when the diameter D of the recessed pattern 41 is 1 / 5 to 1 / 3 of the maximum width W of the ECM channel 22, as in the first, second, and fourth design proposals, it was confirmed that gelation of the fluid containing collagen type 1 (2 mg / mL) was maintained without leakage from the ECM channel. In particular, when the diameter D of the recessed pattern 41 is 1 / 5 to 3 / 10 of the maximum width W of the ECM channel 22, as in the first and second design proposals, it was confirmed that this is more preferable for maintaining gelation. On the other hand, when the diameter D of the recessed pattern 41 is 1 / 2 or more of the maximum width W of the ECM channel 22, as in the third design proposal, it was confirmed that a problem of the injected fluid leaking out of the ECM channel occurred.

[0060] [Table 1]

[0061] Example 2: Optimal design of a microfluidic chip equipped with an ECM channel 22 and a second ECM channel 24

[0062] We designed a spheroid / organoid microfluidic chip consisting of a cell inlet 11 through which a fluid containing cells is injected and a cell channel 12 connected to it; an ECM inlet 21 through which a fluid containing ECM (extracellular matrix) is injected and an ECM channel 22 connected to it; a second ECM inlet 23 through which a fluid containing a second ECM component is injected and a second ECM channel 24 connected to it; and a second cell inlet 31 through which a fluid containing a second cell is injected and a second cell channel 32 connected to it.

[0063] At this time, the cell channel 12 is located adjacent to a part of one side surface of the ECM channel 22, and a micro partition wall (h: 200 μm) 40 with a height lower than that of the channel (H: 500 μm) is located therebetween. The micro partition wall 40 has a shape in which a plurality of concave patterns 41 curved in the ECM channel direction are arranged at regular intervals. In particular, it is characterized in that the micro partition wall 40 is connected to inclined surfaces 42 at both ends of the concave pattern 41, respectively. On the other hand, the diameter D of the concave pattern 41 and the maximum width W of the ECM channel 22 were 300 μm and 1,000 μm, respectively.

[0064] On the other hand, a part of one side surface of the second ECM channel 24 is located adjacent to a part of the other side surface of the ECM channel 22, and a second micro partition wall (h': 200 μm) (not shown) with a height lower than that of the channel (H': 500 μm) is located therebetween. In addition, the second cell channel 32 is located adjacent to a part of the other side surface of the second ECM channel 24, and a third micro partition wall (h'': 200 μm) (not shown) with a height lower than that of the channel (H': 500 μm) is located therebetween.

[0065] Example 3: Cultivation using a microfluidic chip provided with an ECM channel 22 having a concave pattern on one side surface

[0066] (1) Referring to Example 1, a spheroid / organoid microfluidic chip was designed by optimizing the diameter of the concave pattern 41 on one side surface. At this time, the micro partition wall 40 has a shape in which the concave pattern is arranged, and the second micro partition wall (not shown) has a shape in which no pattern is separately formed (Fig. 3A).

[0067] (2) Then, a fluid containing collagen type 1 (2 mg / mL) was injected into the ECM injection port 21 to form a vascular layer. Next, a fluid (RPMI and EGM-21:1 mixed medium) containing vascular endothelial cells (HUVEC) (2,000,000 cells / mL) was injected into the second cell injection port 31 to form a vascular layer. Meanwhile, a fluid (DMEM and EGM-21:1 mixed medium) containing a total of five spheroids made of approximately 1,000 pancreatic cancer cells (PANC1) was injected into the cell injection port 11. The pancreatic cancer cell spheroids were trapped in the recessed pattern 41 and co-cultured for 9 days, as shown in Figure 3B.

[0068] (3) On the other hand, pancreatic cancer cell spheroids were captured and co-cultured for 7 days in the same manner as in (2), except that a fluid containing fibrin (10 mg / mL) was injected into the ECM injection port 21 and a fluid (RPMI and EGM-21:1 mixed medium) containing vascular endothelial cells (HUVECs) (4,000,000 cells / mL) was injected into the second cell injection port 31. This is shown in Figure 3C.

[0069] (4) On the other hand, peripheral nerve cell spheroids were captured and co-cultured for 8 days in the same manner as in (2), except that a fluid (PNS medium) containing pancreatic cancer cells (2,000,000 cells / mL) was injected into the second cell injection port 31, and a fluid (PNS medium) containing groups of 0, 1, 3, or 5 spheroids made of approximately 1,000 peripheral nerve cells was injected into the cell injection port 11. This is shown in Figure 3D.

[0070] As shown in Figure 3D, it was confirmed that the position from the center of the concave pattern where the peripheral neuron spheroids were captured could be specified depending on the number of groups of injected peripheral neuron spheroids, and that the number of groups of peripheral neuron spheroids captured and cultured within the concave pattern could be adjusted.

[0071] Example 4: Culture using a microfluidic chip equipped with ECM channels 22 having concave patterns on both sides

[0072] (1) A spheroid / organoid microfluidic chip was designed with the diameter of the concave patterns on both sides optimized, as shown in Example 1. The chip features a second concave pattern arranged on both the micropartition 40 and the second micropartition (not shown) (FIG. 4A).

[0073] (2) After that, a fluid containing collagen type 1 (2 mg / mL) was injected into the ECM injection port 21, followed by gelation. Next, a fluid (LTM medium) containing a total of three groups of organoids made from approximately 1,000 patient-derived lung cancer cells was injected into the cell injection port 11 and the second cell injection port 31, respectively, and the organoids were captured in the concave pattern 41 and the second concave pattern, respectively, and cultured for 7 days, as shown in Figure 4B.

[0074] Example 5: Culture using a microfluidic chip equipped with an ECM channel 22 and a second ECM channel 24

[0075] (1) Referring to Example 2, a spheroid / organoid microfluidic chip was designed in which the diameter of the recessed pattern 41 was optimized (Figure 5A).

[0076] (2) Then, a fluid containing collagen type 1 (2 mg / mL) was injected into the ECM injection port 21 and gelled. Fibrin (10 mg / mL) was injected into the second ECM injection port 23, and a fluid (RPMI and EGM-21:1 mixed medium) containing vascular endothelial cells (HUVEC) (4,000,000 cells / mL) was injected into the second cell injection port 31, followed by gelling, forming a vascular network. Meanwhile, a total of five spheroids consisting of approximately 1,000 pancreatic cancer cells (PANC1) were injected into the cell injection port 11, and optionally, a fluid (DMEM and EGM-21:1 mixed medium) containing EGF (50 ng / mL) was injected into the recessed pattern 41. The pancreatic cancer cell spheroids were then co-cultured for 7 to 9 days, as shown in Figures 5B to 5D, respectively.

[0077] As shown in Figures 5B-5D, after 7 to 9 days of co-culture, pancreatic cancer cell spheroids were observed to be trapped and uniformly cultured within the curved recessed patterns toward the ECM channel. In particular, the addition of EGF induced the migration of pancreatic cancer cells, and active invasion was observed.

[0078] (3) Meanwhile, peripheral nerve cell spheroids were captured in the same manner as in (2), except that a fluid (RPMI and EGM-21:1 mixed medium) containing vascular endothelial cells (HUVEC) (4,000,000 cells / mL) was injected into the second cell injection port 31, and a fluid (PNS medium) containing a total of three groups of spheroids made of approximately 1,000 peripheral nerve cells was injected into the cell injection port 11, and the spheroids were cultured for 11 days, as shown in Figures 5E and 5F.

[0079] As shown in Figures 5E and 5F, after 11 days of co-culture, the peripheral neuron spheroids were captured within the concave pattern curved toward the ECM channel, and the axons of the peripheral neurons were uniformly cultured while extending toward the ECM channel. Sensory neurons were identified through BRN3A (green signal) and peripheral neurons were identified through TRPV1 (red signal).

[0080] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical spirit or essential characteristics of the present invention. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting.

Claims

1. A cell injection port into which a fluid containing cells is injected and a cell channel connected thereto; and An ECM injection port into which a fluid containing an ECM (extracellular matrix) component is injected and an ECM channel connected thereto; comprising The cell channel is located adjacent to a part of one side surface of the ECM channel, and a micro partition wall having a height lower than that of the channel is located therebetween. The micro partition wall is characterized in that a plurality of concave patterns curved in the ECM channel direction are arranged at regular intervals. A spheroid or organoid microfluidic chip.

2. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the cells and the ECM components interact between the cell channel and the ECM channel where the micro partition wall is not located. The spheroid or organoid microfluidic chip according to claim 1.

3. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the height of the micro partition wall is 1 / 5 to 1 / 2 of the channel height. The spheroid or organoid microfluidic chip according to claim 1.

4. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the concave patterns in the micro partition wall are each connected to an inclined surface (chamfer) at both ends. The spheroid or organoid microfluidic chip according to claim 1.

5. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the diameter of the concave patterns in the micro partition wall is 1 / 5 to 1 / 3 of the maximum width of the ECM channel. The spheroid or organoid microfluidic chip according to claim 1.

6. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the interval between the concave patterns in the micro partition wall is 100 μm to 5,000 μm. The spheroid or organoid microfluidic chip according to claim 1.

7. The spheroid or organoid microfluidic chip according to claim 1, characterized in that the cells are cancer cells or nerve cells. The spheroid or organoid microfluidic chip according to claim 1.

8. The spheroid or organoid microfluidic chip according to claim 1, further comprising a second cell channel located adjacent to a part of the other side surface of the ECM channel. The spheroid or organoid microfluidic chip according to claim 1.

9. A second micro partition wall having a height lower than that of the channel is located between the second cell channel and the ECM channel. The second micro partition wall is characterized in that a plurality of second concave patterns curved in the ECM channel direction are arranged at regular intervals. The spheroid or organoid microfluidic chip according to claim 8.

10. The second cell is of the same or different type as the cell, and is one or more co-cultured cells selected from the group consisting of cancer cells, nerve cells, and vascular endothelial cells, characterized in that The spheroid or organoid microfluidic chip according to claim 8.

11. (a) a step of injecting a fluid containing cells into the cell injection port of the microfluidic chip according to any one of claims 1 to 10; and (b) a step of forming or culturing spheroids or organoids at positions of concave patterns arranged at regular intervals in the cell channel by the injected cells; characterized by including A method for forming an in vitro model of spheroids or organoids.

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