Biomimetic chip, method for manufacturing same, and method for coating extracellular matrix by using same
Patent Information
- Application Number
- PCT/KR2025/099584
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Priority Date
- 2025-02-07
- Filing Date
- 2025-03-06
- Publication Date
- 2025-10-02
AI Technical Summary
Conventional microfluidic channels with curved structures in biomimetic chips experience non-uniform fluid flow, leading to uneven distribution of coating agents and cell adhesion, which complicates cell culture uniformity and simulation of the in vivo extracellular matrix environment.
A biomimetic chip design with linear microfluidic channels and a porous membrane, combined with oxygen plasma and silane coupling, ensures uniform fluid circulation and alignment of extracellular matrix fibers for enhanced cell attachment and barrier functionality.
The solution maintains uniform cell density and adhesion on the microchannel surface, simulates the in vivo environment effectively, and enhances cell culture stability by aligning the extracellular matrix fibers, thereby improving cell culture uniformity and barrier functionality.
Smart Images
Figure KR2025099584_02102025_PF_FP_ABST
Abstract
Description
Biomimetic chip, its manufacturing method, and extracellular matrix coating method using the same
[0001] The present invention relates to a biomimetic chip, a method for manufacturing the same, and a method for coating an extracellular matrix using the same.
[0002] Micro Physiological System (MPS) is a biomimetic chip that reproduces the structure and microenvironment of human organs in vitro by combining human-derived 3D cell culture technology and blood flow-simulating microfluidic technology. It is a technology that fixes cells in a microfluidic channel for a certain period of time and a certain space and observes cell growth, etc.
[0003] MPS is utilized in various fields such as disease mechanisms, new drug screening, and patient-tailored medical technology, and depending on the purpose of use, the microfluidic channels inside the chip are applied in various designs such as single, double, and multi-channel.
[0004] In MPS for applying cell circulation model, the structure of conventional microfluidic channel has a curved structure. When the flow of fluid is given to the microfluidic channel of this curved structure in order to simulate circulating cells, the flow of fluid is concentrated on the inside of the curve, which may cause a difference in the velocity of the fluid. Due to this difference in the velocity of the fluid in the curved area, the coating agent used when modifying the surface of the microfluidic channel accumulates in the curved area, making it difficult to uniformly treat the surface of the microfluidic channel, and there is a problem that it causes non-uniform adhesion of cells cultured on the surface of the microfluidic channel during cell culture in the future.
[0005] Meanwhile, an extracellular matrix can be coated on the microfluidic channel to fix cells in a certain space for a certain period of time and observe cell growth, etc.
[0006] Currently commercialized extracellular matrix coating methods mainly involve simple coatings using single proteins such as collagen, fibronectin, and laminin, or coatings using complex basement membrane proteins such as Matrigel. These existing coating methods, formed through simple immersion or adsorption, mostly form two-dimensional structures with random orientations, failing to effectively reflect the sophisticated structural characteristics of the in vivo basement membrane and making it difficult to simulate the complex in vivo extracellular matrix environment.
[0007] In the present invention, the biomimetic chip can easily apply cell culture and cell circulation models by introducing a linear microfluidic channel.
[0008] Additionally, a robust cell barrier similar to the in vivo environment can be implemented.
[0009] In order to achieve the above-described object, one embodiment of the present invention relates to a biomimetic chip including a substrate having a first groove formed on one surface, a cover part arranged to face the substrate and having a second groove formed on a surface facing the substrate, a porous membrane arranged between the substrate and the cover part to have an area overlapping at least the first groove and the second groove, and a plurality of openings having a first inlet, a first outlet, a second inlet, and a second outlet that penetrate the cover part in a thickness direction of the cover part or in a direction perpendicular to the thickness direction.
[0010] A biomimetic chip according to an embodiment of the present invention maintains a constant circulation speed of fluid inside a microfluidic channel to induce attachment at a uniform density on the surface of the microchannel during cell culture and circulation, and can be immediately and strongly attached through chemical bonding by combining using oxygen plasma and a silane coupling agent.
[0011] In addition, the extracellular matrix coating method according to an embodiment of the present invention can effectively implement the function of the basement membrane in the body by optimizing cell-matrix interaction by introducing an aligned fiber structure into a porous membrane, thereby strengthening the function of the cell barrier.
[0012] FIG. 1 is an exploded perspective view schematically illustrating an example of a biomimetic chip according to one embodiment of the present invention.
[0013] Figure 2 is a cross-sectional view schematically illustrating an example of the biomimetic chip of Figure 1.
[0014] FIG. 3 is a flowchart illustrating an example of a method for manufacturing a biomimetic chip according to another embodiment of the present invention.
[0015] Figure 4 is a process diagram schematically illustrating an example of a method for manufacturing a biomimetic chip of Figure 3.
[0016] Figure 5 is a graph showing the bonding strength of a silane coupling agent according to a solvent.
[0017] Figure 6 is a photograph showing the cell culture results of a biomimetic chip manufactured using the biomimetic chip manufacturing method of Figure 3.
[0018] Figure 7 is a photograph showing the experimental results of a biomimetic chip manufactured using the biomimetic chip manufacturing method of Figure 3.
[0019] Figure 8 is a flowchart illustrating an example of an extracellular matrix coating method according to another embodiment of the present invention.
[0020] Figure 9 is a schematic diagram schematically illustrating an example of the extracellular matrix coating method of Figure 8.
[0021] Figure 10 is a photograph showing the alignment of the extracellular matrix coated with the extracellular matrix coating method of Figure 8.
[0022] Figure 11 is a photograph of cells cultured on an extracellular matrix coated with the extracellular matrix coating method of Figure 8, obtained by fluorescence analysis.
[0023] Figure 12 is a graph evaluating the function of cells cultured on an extracellular matrix coated under various conditions.
[0024] Figure 13a is a photograph of a fluorescent analysis of cells cultured on an extracellular matrix coated under various conditions and co-cultured with a cancer cell line.
[0025] Figure 13b is a graph showing the relative quantification of the fluorescently labeled tight junction proteins in Figure 13a.
[0026] Figure 14a is a photograph of the metastasis of cancer cells analyzed by fluorescence when cells and cancer cell lines were co-cultured on an extracellular matrix coated under various conditions.
[0027] Figure 14b is a graph showing the number of fluorescently labeled cancer cells per volume in Figure 14a.
[0028] Figure 14c is a graph showing the permeability coefficient due to metastasis of cancer cells when co-cultured with cancer cell lines on an extracellular matrix coated with various conditions.
[0029] In order to achieve the above-described object, one embodiment of the present invention relates to a biomimetic chip including a substrate having a first groove formed on one surface, a cover part arranged to face the substrate and having a second groove formed on a surface facing the substrate, a porous membrane arranged between the substrate and the cover part to have an area overlapping at least the first groove and the second groove, and a plurality of openings having a first inlet, a first outlet, a second inlet, and a second outlet that penetrate the cover part in a thickness direction of the cover part or in a direction perpendicular to the thickness direction.
[0030] The first groove may be formed to be longer than the length of the second groove, the first inlet and the first outlet may correspond to an end of the first groove, and the second inlet and the second outlet may correspond to an end of the second groove.
[0031] The first groove has a length extending in one direction and a width intersecting the length, and the width may include a plurality of convex regions formed spaced apart from each other and having the same or greater width along the length.
[0032] The above convex regions may overlap the first inlet, the first outlet, the second inlet, and the second outlet.
[0033] Biofluid can be injected into the first groove and the second groove.
[0034] The biofluid may include a material selected from circulating cells, adherent cells, scaffolds, chemicals, and biomolecules.
[0035] The biofluid can be selectively exchanged through the porous membrane in an area where the first groove and the second groove overlap.
[0036] The above circulating and adherent cells can be cultured in a single layer or multilayer structure or can be cultured by circulating along the channels of the chip.
[0037] The scaffold may comprise a protein or hydrogel protein comprising an extracellular matrix.
[0038] The above chemicals may include cell culture media and additives.
[0039] The biomolecule may comprise a signaling molecule or a neurotransmitter.
[0040] Another embodiment of the present invention for achieving the above-described purpose discloses a method for manufacturing a biomimetic chip, including the steps of preparing a substrate, a cover part, and a porous membrane, the step of surface-treating the substrate and the cover part with plasma gas, the step of surface-treating the porous membrane with a silane coupling agent, and the step of bonding the surface-treated substrate, the cover part, and the porous membrane.
[0041] The plasma gas contains oxygen or air, and the oxygen or the air can modify the surface of the substrate and the cover part with hydroxyl groups.
[0042] The above silane coupling agent may contain water or ethanol as a solvent.
[0043] Another embodiment of the present invention for achieving the above-described purpose provides an extracellular matrix coating method comprising the steps of introducing a solution containing an extracellular matrix protein onto a substrate, forming a constant flow in the solution, coating the extracellular matrix protein so that it is directionally aligned in the direction of the flow, and introducing cells onto the extracellular matrix coating having the directionality.
[0044] In the above coating step, plasma and calcium chloride (CaCl2) can be further injected while forming the above constant flow.
[0045] Before describing in detail the preferred embodiments of the present invention below, it should be noted that the terms and words used in this specification and claims should not be interpreted as limited to their usual or dictionary meanings, but should be interpreted as meanings and concepts that are consistent with the technical spirit of the present invention.
[0046] The present invention is capable of various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the detailed description. The effects and features of the present invention, as well as the methods for achieving them, will become clearer with reference to the embodiments described in detail below, along with the drawings. However, the present invention is not limited to the embodiments disclosed below and can be implemented in various forms.
[0047] Throughout this specification, the terms first, second, etc. are used not in a limiting sense but for the purpose of distinguishing one component from another.
[0048] Throughout this specification, singular expressions include plural expressions unless the context clearly indicates otherwise.
[0049] Throughout this specification, terms such as “include” or “have” mean that a feature or component described in the specification is present, and do not preclude the possibility that one or more other features or components may be added.
[0050] Throughout this specification, when a part such as a film, region, component, etc. is said to be on or above another part, it includes not only the case where it is directly on top of the other part, but also the case where another film, region, component, etc. is interposed between them.
[0051] The identifiers used in each step are for convenience of explanation and do not indicate the order of the steps. The steps may be performed in a different order than stated, unless the context clearly dictates otherwise. In other words, the steps may be performed in the same order as stated, substantially simultaneously, or in the opposite order.
[0052] Hereinafter, embodiments of the present invention will be described. However, the scope of the present invention is not limited to the following preferred embodiments, and those skilled in the art can implement various modified forms of the contents described herein within the scope of the present invention.
[0053] For convenience of explanation, the sizes of components in the drawings may be exaggerated or reduced. For example, the sizes and thicknesses of each component shown in the drawings are arbitrarily indicated for convenience of explanation, and thus the present invention is not necessarily limited to what is shown.
[0054] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. When describing with reference to the drawings, identical or corresponding components are given the same reference numerals and redundant descriptions thereof will be omitted.
[0055] The present invention relates to a biomimetic chip, a method for manufacturing the same, and a method for coating an extracellular matrix using the same.
[0056] FIG. 1 is an exploded perspective view schematically illustrating an example of a biomimetic chip according to one embodiment of the present invention, and FIG. 2 is a cross-sectional view schematically illustrating an example of the biomimetic chip of FIG. 1.
[0057] Referring to FIGS. 1 and 2, a biomimetic chip (1) may include a substrate (10) having a first groove (11) formed on one surface, a cover portion (20) having a second groove (21) formed thereon and including a plurality of openings (12, 13, 22, 23) formed in a thickness direction intersecting the second groove (21), and a porous membrane (30) disposed between the substrate (10) and the cover portion (20) and covering at least a portion of the first groove (11) and the second groove (21).
[0058] The substrate (10) may have a first groove (11) formed in the center of one surface. For example, the first groove (11) may be formed in the center of the substrate (10) with a length in one direction and a width in a direction crossing the length. By forming the first groove (11) with a length and a width, a space for a fluid to flow may be defined in the first groove (11), and therefore, the first groove (11) may function as a microfluid channel or chamber, and may be connected to a first inlet (12) and a first outlet (13) described below to induce a flow of the first biofluid so that the first biofluid may circulate inside the first groove (11).
[0059] As an optional embodiment, the first groove (11) may be formed with a region having a width that is not constant and intersecting with the length, and having a larger width. In this case, a plurality of regions having a larger width may be formed, and may correspond to the first inlet (12) and the first outlet (13) described below.
[0060] Meanwhile, the substrate (10) may be a transparent substrate whose inside can be observed from the outside. For example, the substrate (10) may be made of any material that can be formed transparently, and as a specific example, the substrate (10) may include at least one selected from among polydimethylsiloxane (PDMS), polyethersulfone (PES), poly(3,4-ethylenedioxythiophene), poly(styrenesulfonate), polyimide, polyurethane, polyester, perfluoropolyether (PFPE), and polycarbonate.
[0061] The cover portion (20) can be positioned to face the substrate (10). The cover portion (20) includes a second groove (21). For example, the second groove (21) can be formed to have at least an area overlapping the first groove (11).
[0062] Meanwhile, the cover portion (20) may be a transparent substrate whose inside can be observed from the outside. For example, the cover portion (20) may be formed of any material that can be formed transparently, and as a specific example, the cover portion (20) may include at least one selected from among polydimethylsiloxane (PDMS), polyethersulfone (PES), poly(3,4-ethylenedioxythiophene), poly(styrenesulfonate), polyimide, polyurethane, polyester, perfluoropolyether (PFPE), and polycarbonate.
[0063] As a specific example, the cover portion (20) may have a second groove (21) formed on one surface of the cover portion (20) facing the substrate (10).
[0064] As an optional embodiment, the second groove (21) may be formed in the center or an area adjacent to one surface of the cover portion (20).
[0065] In addition, the first groove (11) and the second groove (21) may overlap at least in one area to form a tube shape. At this time, the second groove (21) may be formed to have a shorter length than the first groove (11) and may overlap in one area of the first groove (11). Specifically, the second groove (21) may overlap in the longitudinal direction of the first groove (11) on both sides along the center of the first groove (11), and both end portions of the second groove (21) may be located inside the both end portions of the first groove (11).
[0066] Meanwhile, a plurality of openings (12, 13, 22, 23) may be formed in a direction intersecting the second groove (21), for example, in a vertical thickness direction (Z) or a direction perpendicular to the thickness direction (Z), that is, in a direction horizontal to the second groove.
[0067] For example, the second groove (21) faces the first groove (11) and overlaps to form a tube shape, and is separated by a porous membrane (30) described later, so that each of the first groove (11) and the second groove (21) can function as a microfluidic channel or chamber. At this time, the depth of the microfluidic channel formed by the first groove (11) and the porous membrane (30) can be 150 µm to 250 µm, and the depth of the microfluidic channel formed by the second groove (11) and the porous membrane (30) can be 800 µm to 1200 µm.
[0068] The plurality of openings (12, 13, 22, 23) may each include a first inlet (12), a first outlet (13), a second inlet (22) and a second outlet (23).
[0069] For example, the first inlet (12) and the first outlet (13) may be arranged to be spaced apart from each other, and may be arranged at least spaced apart from the second groove (21) and on the outside of the second groove (21).
[0070] In addition, the first inlet (12) and the first outlet (13) may be formed to correspond to the first groove (11), for example, the first inlet (12) and the first outlet (13) may be formed to correspond to one end and the other end of the first groove (11), respectively.
[0071] It can be formed to correspond to the end of the first groove (11) formed on the substrate (10),
[0072] The second inlet (22) and the second outlet (23) can be arranged to be spaced apart from each other, and can be arranged at least spaced apart from the first inlet (12) and the first outlet (13) and on the inside of the first inlet (12) and the first outlet (13).
[0073] In addition, the second inlet (22) and the second outlet (23) may be formed to correspond to the second groove (21) of the cover part (20), and for example, the second inlet (22) and the second outlet (23) may be formed to correspond to one end and the other end of the second groove (21), respectively.
[0074] At this time, the length of the first groove (11) and the length of the second groove (21) may be formed to be equal to or different from each other, so that the first inlet (12) and the second inlet (22) and the first outlet (13) and the second outlet (23) may be formed to be spaced apart from each other.
[0075] The first inlet (12) and the first outlet (13) are formed to overlap with the first groove (11), for example, to correspond to both ends of the first groove (11), so as to be connected to the first groove (11) and form a flow path through which the fluid flows. At this time, the first inlet (12) and the first outlet (13) can be attached or detached to a tube connected to an external pump, so that the flow of the fluid circulating in the first groove (11) can be controlled at a desired speed through the pump.
[0076] The second inlet (22) and the second outlet (23) may be formed to overlap with the second groove (21), for example, to correspond to both ends of the second groove (21), and may be connected to the second groove (21) to form a flow path through which the fluid flows. At this time, the second inlet (22) and the second outlet (23) may be detachably connected to a tube connected to an external pump, so that the flow of the fluid circulating in the second groove (21) may be controlled at a desired speed through the pump.
[0077] That is, the first inlet (12), the first outlet (13), the second inlet (22) and the second outlet (23) can all be attached to and detached from the tube, and the tube is connected to an external pump, so that the flow of the fluid injected into the first groove (11) and the second groove (21) by the pump and then discharged can be controlled.
[0078] As an optional embodiment, the first groove (11) may include regions with different widths. For example, in FIG. 1, four regions with different widths of the first groove (11), specifically, four regions convex on both sides (hereinafter defined as "convex regions") with a large width, may be formed, and these four convex regions of the first groove (11) may correspond to and overlap multiple openings (12, 13, 22, 23), respectively.
[0079] Meanwhile, the first groove (11) and the openings (12, 13, 22, 23) can be formed perpendicular to each other, and the fluid flowing into the openings (12, 13, 22, 23) and the first groove (11) and the second groove (21) by the joint formed perpendicularly at a right angle can rapidly change in velocity and pressure. At this time, by forming a convex region overlapping with a plurality of openings (12, 13, 22, 23) in the first groove (11), a space is formed in the convex region, and the connection portion of the first groove (11) and the plurality of openings (12, 13, 22, 23) that are vertically connected at right angles has a streamlined shape, thereby preventing the flow direction of the fluid from changing abruptly, thereby preventing the flow velocity and hydraulic pressure of the fluid flowing in the first groove (11) and the second groove (21) from changing, and the shear stress caused by the fluid flowing in the first groove (11) and the second groove (21) can be maintained constant.
[0080] Meanwhile, the fluid flowing into the first groove (11) and the second groove (21) may include a biofluid. For example, the biofluid may be circulated within the first groove (11) by being injected into the interior of the first groove (11) through the first inlet (12) and discharged to the outside through the first outlet (13), and may be circulated within the second groove (21) by being injected into the interior of the second groove (21) through the second inlet (22) and discharged to the outside through the second outlet (23).
[0081] As an optional embodiment, the biofluid may be injected into the first groove (11) through the first inlet (12) and remain statically inside the first groove (11) without being discharged, and may be injected into the second groove (21) through the second inlet (22) and discharged to the outside through the second outlet (23) to circulate inside the second groove (21).
[0082] As an optional embodiment, the biofluid may be circulated inside the first groove (11) by being injected into the interior of the first groove (11) through the first inlet (12) and discharged to the outside through the first discharge port (13), and may be injected into the interior of the second groove (21) through the second inlet (22) and remain statically inside the second groove (21) without being discharged.
[0083] As an optional embodiment, the biofluid may be injected into the first groove (11) through the first inlet (12) and may exist in a static state inside the first groove (11) without being discharged, and may be injected into the second groove (21) through the second inlet (22) and may exist in a static state inside the second groove (21) without being discharged.
[0084] The biofluid may include, for example, a material selected from circulating and adherent cells, a scaffold, chemicals, and biomolecules, and specifically, the circulating and adherent cells may include all types of cells, such as endothelial cells, epithelial cells, stromal cells, and immune cells derived from animals including humans, the scaffold may include a protein or hydrogel containing constituent molecules of cells including an extracellular matrix, the chemicals may include a cell culture medium and additives and drugs added to the cell culture medium, and the biomolecules may include, but are not limited to, all molecules produced by an organism, such as a signaling substance or a neurotransmitter, and may include all types of materials that are generally available in the past, depending on the purpose of the experiment.
[0085] The porous membrane (30) is placed between the substrate (10) and the cover portion (20), and may be placed, for example, between the first groove (11) and the second groove (21).
[0086] As a specific example, the porous membrane (30) may be arranged to overlap the first groove (11) and the second groove (21), and may be arranged to cover at least one area of the space between the first groove (11) and the second groove (21).
[0087] For example, the porous membrane (30) may include, but is not limited to, a PET porous membrane, and may include various materials, and may include any material that can be conventionally used as a membrane (30).
[0088] Meanwhile, the porous membrane (30) is arranged between the first groove (11) and the second groove (21) in the area where the first groove (11) and the second groove (21) overlap, so that the biofluid injected into the first groove (11) can be selectively moved to the second groove (21), or conversely, the biofluid injected into the second groove (21) can be selectively moved to the first groove (11).
[0089] In an optional embodiment, the porous membrane (30) can selectively move biofluid containing circulating cancer cells injected into the second groove (21) to the first groove (11). In this case, endothelial cells cultured on the surface of the porous membrane (30) facing the second groove (21) can be infiltrated by circulating cancer cells injected into and moved into the second groove (21).
[0090] In the case of cancer cells, when co-cultured with endothelial cells, the intercellular bonds of the endothelial cells can be weakened, inducing condensation of the endothelial cells. This weakens the intercellular bonds of the endothelial cells, which can easily cause the collapse of the endothelial cell barrier cultured on the porous membrane (30). This collapse of the endothelial cell barrier causes disordered transendothelial migration (TEM) of cancer cells through the endothelial cells, making it difficult to maintain the membrane permeability of the porous membrane (30) on which the endothelial cells are cultured in an environment similar to that in vivo.
[0091] To address this, the present invention provides, as an optional embodiment, a porous membrane (30) that can be coated with an extracellular matrix oriented in one direction. For example, to coat the extracellular matrix oriented in one direction, a constant flow can be applied to a biofluid containing the extracellular matrix. By applying this constant flow, the extracellular matrix can be aligned and oriented in one direction, and a fiber-shaped extracellular matrix coating layer can be formed.
[0092] In an optional embodiment, when endothelial cells and cancer cells are co-cultured in the second groove (21), a biofluid containing an extracellular matrix may first be injected into the second groove (21) so that the extracellular matrix may be coated on the surface of the second groove (21) and the porous membrane (30) facing the second groove direction. At this time, a constant flow may be applied to the biofluid so that the extracellular matrix may be oriented and aligned in one direction, and a fiber-shaped extracellular matrix coating layer may be formed on the surface of the second groove (21) and the porous membrane (30) facing the second groove direction.
[0093] As above, endothelial cells can be introduced and aligned on a fiber coating layer in which the extracellular matrix is aligned.
[0094] Endothelial cells cultured in these aligned fiber structures can exhibit enhanced cell proliferation rates and increased expression of genes related to cell-to-cell adhesion, enabling them to function as excellent barriers without collapsing even when co-cultured with cancer cells.
[0095] As a result, the barrier of endothelial cells cultured on the aligned fiber coating layer can maintain structural stability even in a co-culture environment with cancer cells, which can provide a physiological advantage of restricting the disordered transendothelial migration (TEM) of cancer cells and maintaining membrane permeability.
[0096] Meanwhile, the first groove (11) and the second groove (21) of the biomimetic chip (1) can be separated by a porous membrane (30) to form a straight-line microfluidic channel or chamber, that is, a straight-line microfluidic channel or chamber including a separate inlet and outlet can be formed in each of the first groove (11) and the second groove (21), so that various treatments such as cells, scaffolds, and drugs can be performed in the microfluidic channels of each of the first groove (11) and the second groove (21) depending on the model to be applied.
[0097] As an optional embodiment, when applying a circulation model to a biomimetic chip (1), first, an extracellular matrix can be injected into the second groove (21) through the second inlet (22) of the biomimetic chip (1). The PLL (Poly-L-Lysine) coating agent and extracellular matrix protein, which are easy for cell attachment and are injected through the second inlet (22), can be discharged through the second outlet (23), thereby coating the second groove (21). After the second groove (21) is coated, human pulmonary vascular endothelial cells and a culture medium are injected and discharged again through the second inlet (22) and the second outlet (23), thereby forming a monolayer of human pulmonary vascular endothelial cells on the surface of the second groove (21), so that an in vivo blood vessel can be simulated in the second groove (21).
[0098] When a blood vessel is simulated in the second groove (21), cancer cells can be circulated in the second groove (21) through the second inlet (22) and the second outlet (23), and cancer cells (circulating tumor cells) circulating in the second groove (21) can attach onto the simulated endothelial cells. At this time, some cancer cells can move to the first groove (11) through the porous membrane (30) and infiltrate into the extracellular matrix formed in the first groove (11). At this time, in order to facilitate the distinction between endothelial cells and cancer cells, CellTracker TM Using this, pulmonary vascular endothelial cells can be labeled with Green CMFDA Dye, and circulating cancer cells can be labeled with Red CMFPX Dye.
[0099] Meanwhile, since the first groove (11) and the second groove (21) have a straight structure, when the flow rates of the first biofluid and the second biofluid are adjusted and injected into the microfluid channel to circulate, the extracellular matrix and coating agent can be uniformly coated throughout the microfluid channel or chamber, human pulmonary vascular cells can be uniformly cultured as a monolayer, and circulating cancer cells can be uniformly attached to endothelial cells.
[0100] FIG. 3 is a flowchart illustrating an example of a method for manufacturing a biomimetic chip according to another embodiment of the present invention.
[0101] Referring to FIG. 3, a method for manufacturing a biomimetic chip may include a step of preparing a substrate, a cover part, and a porous membrane (S100), a step of surface-treating the substrate and the cover part with plasma gas (S200), a step of surface-treating the porous membrane with a silane coupling agent (S300), and a step of bonding the surface-treated substrate, cover part, and porous membrane (S400).
[0102] In the step (S100) of preparing a substrate, a cover portion, and a porous membrane, the substrate (10) and the cover portion (20) can be manufactured using a mold having a shape of a first groove (11) and a second groove (21) by milling with a CNC machine, and can be prepared by a molding method in which liquid polydimethylsiloxane (PDMS) is poured into the mold to imitate the shape of the mold, and the cover portion (20) can be prepared by additionally forming a plurality of openings with a desired diameter using a puncher in the PDMS imitated in the mold.
[0103] A porous membrane can be prepared by manufacturing a PET thin film to correspond to the length of the second groove (21).
[0104] Figure 4 is a process diagram schematically illustrating an example of a method for manufacturing a biomimetic chip of Figure 3.
[0105] Referring to Fig. 4, in the step (S200) of surface-treating the substrate and the cover part with plasma gas, the substrate (10) and the cover part (20) may be surface-treated with oxygen plasma (O2plasma). For example, the PDMS forming the substrate (10) and the cover part (20) may have a surface that is CH3 - It is composed of CH3 and has hydrophobicity. When the surface is treated with oxygen plasma (O2plasma), the surface of PDMS becomes CH3. - In OH - It can be substituted with and become hydrophilic. At this time, oxygen plasma (O2plasma) can be treated for 1 to 3 minutes under the conditions of 400 mTorr to 500 mTorr and 80 W to 120 W.
[0106] In the step (S300) of surface-treating the porous membrane with a silane coupling agent, the porous membrane (30) may be surface-treated with a silane coupling agent, and the silane coupling agent may specifically include a (3-Aminopropyl)triethoxysilane (APTES) coupling agent. At this time, the APTES coupling agent may be prepared by adding 5 molar% of APTES to a solvent containing ethanol (Et-OH) or distilled water, and may be treated on the surface of the porous membrane (30) by heating to 70°C to 90°C.
[0107] The porous membrane (30) surface-treated with APTES in this way can have its surface modified with amino groups.
[0108] In the step (S400) of bonding the surface-treated substrate, cover part, and porous membrane, the substrate (10) and cover part (20) surface-treated with oxygen plasma (O2 plasma) have a surface with hydroxyl groups (OH - ) and the porous membrane (30) surface-treated with an APTES coupling agent has an amino group on its surface, so that the substrate (10) and the cover portion (20) can form a strong covalent bond with the porous membrane (30). Therefore, by placing the porous membrane (30) between the substrate (10) and the cover portion (20), the substrate (10), the cover portion (20), and the porous membrane (30) can be firmly attached to each other.
[0109] Meanwhile, the bonding strength between the substrate (10), the cover portion (20), and the porous membrane (30) may vary depending on the solvent used in the APTES coupling agent.
[0110] Figure 5 is a graph showing the bonding strength of a silane coupling agent according to a solvent.
[0111] Referring to FIG. 5, when distilled water (DW) is used as a solvent, the fluid pressure that the biomimetic chip can withstand without fluid leakage is approximately 23 psi, and when ethanol (Et-OH) is used as a solvent, the fluid pressure that the biomimetic chip can withstand without fluid leakage is approximately 35 psi. It can be confirmed that the bonding strength is approximately 1.5 times stronger when ethanol is used than when distilled water is used as a solvent.
[0112] Figure 6 is a photograph showing the cell culture results of a biomimetic chip manufactured using the biomimetic chip manufacturing method of Figure 3.
[0113] Referring to Figure 6, human pulmonary vascular endothelial cells cultured in a microfluidic channel were stained with VE-cadherin (endothelial specific adhesion molecule, White) and DAPI (nuclear DNA, Blue) markers, and then fluorescence analysis was performed to confirm that the human pulmonary vascular endothelial cells injected into the microfluidic channel formed a monolayer on the surface of the microfluidic channel, and that blood vessels in vivo were mimicked in the microfluidic channel.
[0114] Figure 7 is a photograph showing the experimental results of a biomimetic chip manufactured using the biomimetic chip manufacturing method of Figure 3.
[0115] Referring to Figure 7, (a) shows human pulmonary vascular endothelial cells cultured in the second groove, (b) shows circulating cancer cells circulating in the second groove, and (C) shows cancer cells attached to human pulmonary vascular endothelial cells formed in the second groove.
[0116] Referring to (a), (b), and (c), as a result of culturing pulmonary vascular endothelial cells using the biomimetic chip of the present invention, it can be confirmed that pulmonary vascular endothelial cells are cultured in a monolayer at a uniform density in a microchannel having a straight structure, and it can be confirmed that cancer cells are uniformly attached to the top of the endothelial cells without fluid leakage even when circulating cancer cells using tubing.
[0117] As a result, the biomimetic chip maintains a constant circulation rate of fluid within the microfluidic channel, thereby inducing attachment at a uniform density on the microchannel surface during cell culture and circulation, and can be immediately and strongly attached through chemical bonding by combining using oxygen plasma and a silane coupling agent.
[0118] Below, a method for coating an extracellular matrix using a biomimetic chip according to an embodiment of the present invention is described.
[0119] Figure 8 is a flowchart illustrating an example of an extracellular matrix coating method according to another embodiment of the present invention.
[0120] Referring to FIG. 8, the extracellular matrix coating method may include a step of introducing a solution containing an extracellular matrix protein onto a substrate (S1000), a step of forming a constant flow in the solution (S2000), a step of coating the extracellular matrix protein so that it is directionally aligned in the direction of the flow (S3000), and a step of introducing cells onto the extracellular matrix coating having the directionality (S4000).
[0121] In the step (S1000) of introducing a solution containing an extracellular matrix protein onto a substrate, the substrate may be a transparent substrate whose inside can be observed from the outside, and may include, for example, silicon, and as a specific example, may include polydimethylsiloxane (PDMS), and as a more specific example, may include a biomimetic chip according to an embodiment of the present invention.
[0122] In an optional embodiment, a first groove and a second groove may be positioned on the first groove and overlapping the first groove within the substrate, and a porous membrane may be positioned between the first groove and the second groove, such that a space may be defined by one side of the first groove and the porous membrane facing the first groove direction, and a space may be defined by the other side of the second groove and the porous membrane facing the second groove direction.
[0123] A solution containing the extracellular matrix can be injected into the space defined by the second groove and the other side of the porous membrane.
[0124] In the step (S2000) of forming a constant flow in the above solution, a constant shear stress can be applied to the solution injected into the space defined by the second groove and the other surface of the porous membrane by a pump located outside the substrate, so that a constant flow can be formed in the solution.
[0125] In the step (S3000) where the extracellular matrix protein is coated to be aligned directionally in the flow direction, the extracellular matrix contained in the solution in which a constant flow is formed in the space defined by the second groove and the other side of the porous membrane can be aligned in one direction in the space defined by the second groove and the other side of the porous membrane.
[0126] By sequentially injecting plasma and calcium chloride (CaCl2) into the extracellular matrix oriented in this manner, the extracellular matrix contained in the solution can be coated in the form of fibers on the second groove and the other side of the porous membrane.
[0127] Figure 9 is a schematic diagram schematically illustrating an example of the extracellular matrix coating method of Figure 8.
[0128] Referring to FIG. 9, the other side (310) of the porous membrane (300) can be coated with extracellular matrix proteins aligned in one direction.
[0129] A solution having a constant flow in the space (S) defined by the second groove (211) and the other side (310) of the porous membrane (300) may include, for example, fibronectin, which is an extracellular matrix, and when a solution containing fibronectin is injected with a constant flow into the space (S) defined by the second groove (211) and the other side (310) of the porous membrane (300), fibronectin may be arranged on the other side (310) of the membrane (300).
[0130] When fibronectin is placed on the other side (310) of the membrane (300), plasma containing fibrinogen is injected with a constant flow into the space (S) defined by the second groove (211) and the other side (310) of the porous membrane (300), so that fibrinogen can be placed on the fibronectin.
[0131] Next, when calcium chloride (CaCl2) is injected at a constant flow onto fibronectin and fibrinogen, factor XIIIa is activated, and fibronectin and fibrin are cross-linked and aligned, so that the other side (310) of the membrane (300) can be coated with an extracellular matrix in the form of fibrin fibers.
[0132] Figure 10 is a photograph showing the alignment of the extracellular matrix coated with the extracellular matrix coating method of Figure 8.
[0133] Referring to Figure 10, the orientation of the extracellular matrix under no-flow conditions (a) and flow conditions (b) can be confirmed.
[0134] As a result of treating the other side (310) of the porous membrane (300) with plasma at intervals of 1 hour, 3 hours, and 6 hours, in (a) a random orientation was observed, but in (b) it can be observed that the fibrin fibers are aligned along the direction of fluid flow.
[0135] In the step (S4000) of introducing cells onto the extracellular matrix coating having the above directionality, cells can be cultured onto the extracellular matrix coating having the directionality, and at this time, the cells can be cultured while being oriented along the extracellular matrix having the directionality.
[0136] Meanwhile, cells are not limited to a single type of cell, but various types of cells can be introduced and cultured on an extracellular matrix depending on the experimental purpose.
[0137] Figure 11 is a photograph of cells cultured on an extracellular matrix coated with the extracellular matrix coating method of Figure 8, obtained by fluorescence analysis.
[0138] Referring to Figure 11, human umbilical vein endothelial cells (HUVECs) cultured under coating conditions of static and flow conditions can be confirmed, which can be stained with markers of fibronectin (gray), fibrin (green), VE-cadherin (red), and DAPI (blue).
[0139] As a result of culturing vascular endothelial cells (HUVECs) on a substrate coated with fibrin fibers, it was confirmed that vascular endothelial cells (HUVECs) cultured on the coated substrate under no-flow conditions (Static) showed irregular cell arrangement, whereas vascular endothelial cells (HUVECs) cultured along the coated aligned fibers under flow conditions (Flow) were arranged in a directionally ordered manner.
[0140]
[0141] Example 1
[0142] After flowing a solution containing fibronectin with a constant flow into the microfluidic channel of a biomimetic chip, plasma and calcium chloride with a constant flow were sequentially flowed, thereby culturing vascular endothelial cells (HUVECs) on a membrane coated with fibrin fibers aligned with an orientation in the direction of flow.
[0143]
[0144] Example 2
[0145] By statically positioning a solution containing fibronectin, plasma, and calcium chloride in the microfluidic channels of a biomimetic chip, vascular endothelial cells (HUVECs) were cultured on a membrane coated with fibrin fibers.
[0146]
[0147] Comparative Example 1
[0148] By flowing a solution containing fibronectin through the microfluidic channels of the biomimetic chip, vascular endothelial cells (HUVECs) were cultured on the fibronectin-coated membrane.
[0149]
[0150]
[0151] Comparative Example 2
[0152] Vascular endothelial cells (HUVECs) were cultured on fibronectin-coated experimental well plates.
[0153]
[0154] Comparative Example 3
[0155] Endothelial cells (HUVECs) were cultured in uncoated experimental well plates.
[0156]
[0157] Figure 12 is a graph evaluating the function of cells cultured on an extracellular matrix coated under various conditions.
[0158] Referring to FIG. 12, graphs of cell proliferation rate (a), permeability (b), and endothelial cell junction marker (PECAM-1) and tight junction marker (CLDN-5, OCLN, ZO-1) analysis (c) of Examples 1 and 2 and Comparative Examples 1 to 3 can be confirmed.
[0159] Referring to graph (a) and graph (b), it can be confirmed that the vascular endothelial cells (HUVECs) cultured in Example 1 exhibited an excellent barrier function by showing the lowest permeability along with a high proliferation rate compared to Example 2 and Comparative Examples 1 to 3.
[0160] Referring to graph (c), it can be confirmed that the function of vascular endothelial cells (HUVECs) cultured in Example 1 was improved through analysis of gene expression related to cell-to-cell binding and adhesion.
[0161] Experimental Example 1
[0162] MDA-MB-231, a highly metastatic breast cancer cell line, was co-cultured with vascular endothelial cells (HUVECs) cultured in Example 1.
[0163]
[0164] Experimental Example 2
[0165] In Example 2, MDA-MB-231, a highly metastatic breast cancer cell line, was co-cultured with cultured vascular endothelial cells (HUVECs).
[0166]
[0167] Experimental Example 3
[0168] In Comparative Example 1, MDA-MB-231, a highly metastatic breast cancer cell line, was co-cultured with cultured vascular endothelial cells (HUVECs).
[0169]
[0170] Experimental Example 4
[0171] MCF-7, a low-metastatic breast cancer cell line, was co-cultured with vascular endothelial cells (HUVECs) cultured in Example 1.
[0172]
[0173] Experimental Example 5
[0174] MCF-7, a low-metastatic breast cancer cell line, was co-cultured with vascular endothelial cells (HUVECs) cultured in Example 2.
[0175]
[0176] Experimental Example 6
[0177] In Comparative Example 1, MCF-7, a low-metastatic breast cancer cell line, was co-cultured with cultured vascular endothelial cells (HUVECs).
[0178]
[0179] Figure 13a is a photograph of a fluorescence analysis of cells cultured on an extracellular matrix coated under various conditions and a co-cultured cancer cell line, and Figure 13b is a graph showing the relative quantification of the fluorescently labeled tight junction protein in Figure 13a.
[0180] Referring to Figures 13a and 13b, the barrier function of the endothelial cell layer can be evaluated using two breast cancer cell lines with different metastatic properties.
[0181] Experimental Examples 1 to 3 are co-cultures of highly metastatic MDA-MB-231, which has the property of migrating by destroying tight junctions of endothelial cells, with vascular endothelial cells (HUVECs) of Examples 1, 2, and Comparative Example 1, and Experimental Examples 4 to 6 are co-cultures of low-metastatic MCF-7, which has a superior binding force between cancer cells, with vascular endothelial cells (HUVECs) of Examples 1, 2, and Comparative Example 1.
[0182] Intercellular adhesion of endothelial cells was quantified by fluorescence using the tight junction protein (VE-Cadherin) as a marker.
[0183] As a result of the analysis, it can be confirmed that the vascular endothelial cells (HUVECs) cultured in Experimental Example 1 maintained the same amount of tight junction protein (VE-Cadherin) as in Example 1, which was not co-cultured with highly migratory MDA-MB-231, thereby maintaining the barrier. On the other hand, it can be confirmed that the vascular endothelial cells (HUVECs) cultured in Experimental Examples 2 and 3 had a reduced amount of tight junction protein (VE-Cadherin) compared to Example 2 and Comparative Example 1, which were not co-cultured with MDA-MB-231, respectively, thereby destroying the barrier.
[0184] In addition, it can be confirmed that Experimental Examples 4 to 6 maintain the same tight junction protein quantification as Examples 1, 2, and Comparative Example 3, which were not co-cultured with low-metastatic MCF-7, respectively, thereby maintaining the barrier.
[0185] That is, it was confirmed that the vascular endothelial cells (HUVECs) coated on the extracellular matrix aligned with anti-metastatic properties maintained the barrier without collapse when co-cultured with highly metastatic MDA-MB-231 or low-metastatic MCF-7.
[0186] Figure 14a is a photograph of a fluorescent analysis of cancer cell metastasis during co-culture of cells cultured on an extracellular matrix coated under various conditions and a cancer cell line, Figure 14b is a graph showing the number of cancer cells per volume indicated by fluorescent light in Figure 14a, and Figure 14c is a graph showing the permeability coefficient due to cancer cell metastasis during co-culture of cells cultured on an extracellular matrix coated under various conditions and a cancer cell line.
[0187] Referring to Fig. 14a, to facilitate cell differentiation, vascular endothelial cells (HUVECs) can be stained with Red CMFDA Dye and cancer cells can be stained with Green CMFPX Dye, and when co-cultured with highly metastatic MDA-MB-231, it can be confirmed that fewer metastatic cancer cells (green marker) are observed in Experimental Example 1 compared to Experimental Examples 2 and 3.
[0188] Additionally, it can be confirmed that in experimental examples 4 to 6, when co-cultured with low-metastatic MCF-7, metastatic cancer cells (green marker) are hardly observed.
[0189] Referring to Fig. 14b, MDA-MB-231, which has strong tight junction destruction characteristics, showed metastatic behavior in all of Experimental Examples 1 to 3, but it can be confirmed that the number of metastatic cancer cells confirmed in Experimental Example 1 was the lowest, and through this, it can be seen that the vascular endothelial cells of Experimental Example 1 showed the lowest cancer cell invasion rate compared to Experimental Examples 2 and 3. In other words, it can be seen that the barrier function of the vascular endothelial cells of Experimental Example 1 was improved compared to Experimental Examples 2 and 3.
[0190] The above results can also be confirmed by comparing the permeability coefficients of vascular endothelial cells.
[0191] Referring to Fig. 14c, Experimental Example 1 maintains 1, while Experimental Examples 2 and 3 exceed 1, indicating that the barrier function of the vascular endothelial cells of Experimental Example 1 is improved compared to Experimental Examples 2 and 3.
[0192] That is, it was confirmed that the vascular endothelial cells (HUVECs) coated on the extracellular matrix aligned with anti-metastatic properties maintained the barrier without collapse when co-cultured with highly metastatic MDA-MB-231 or low-metastatic MCF-7.
[0193] As a result, the extracellular matrix coating method according to the embodiment of the present invention can implement a robust cell barrier similar to an in vivo environment by coating an extracellular matrix aligned with anti-radiation properties in the form of fibers on a biomimetic chip.
[0194] In addition, it can be usefully utilized in the development of biomimetic chips and experimental research using them by providing various biological functions such as enhancing proliferation and gene expression of endothelial cells, regulating permeability, and maintaining endothelial cell barriers in cancer cell co-culture environments by complementing the physiological environment of the in vivo basement membrane in vitro.
[0195] While the present invention has been described with reference to the embodiments illustrated in the drawings, these are merely exemplary, and those skilled in the art will understand that various modifications and equivalent alternative embodiments are possible. Therefore, the true scope of technical protection of the present invention should be determined by the technical spirit of the appended claims.
[0196] The specific implementations described in the embodiments are exemplary embodiments and do not limit the scope of the embodiments in any way. For the sake of brevity of the specification, descriptions of conventional electronic components, control provision methods, software, and other functional aspects of the above provision methods may be omitted. In addition, the lines connecting or connecting members between components illustrated in the drawings are merely representative of functional connections and / or physical or circuit connections, and may be replaced or represented as various additional functional connections, physical connections, or circuit connections in an actual device. In addition, if there is no specific mention such as “essential,” “important,” etc., the component may not be absolutely necessary for the application of the present invention.
[0197] The use of the term "above" and similar referential terms in the specification of embodiments (especially in the claims) may refer to both the singular and the plural. Furthermore, if a range is described in the embodiments, the invention encompasses the application of individual values within the range (unless otherwise stated), and is equivalent to describing each individual value constituting the range in the detailed description. Finally, unless the order of steps constituting a method according to an embodiment is explicitly stated or otherwise stated to the contrary, the steps may be performed in any suitable order. The embodiments are not necessarily limited by the order in which the steps are described. The use of all examples or exemplary terms (e.g., "for example," etc.) in the embodiments is merely intended to describe the embodiments in detail, and the scope of the embodiments is not limited by the examples or exemplary terms, unless otherwise defined by the claims. Furthermore, those skilled in the art will recognize that various modifications, combinations, and variations may be made within the scope of the appended claims or their equivalents, depending on design conditions and factors.
Claims
1. A substrate having a first groove formed on one side; A cover portion positioned so as to face the substrate and having a second groove formed on a surface facing the substrate; A porous membrane arranged between the substrate and the cover portion so as to have an area overlapping at least the first groove and the second groove; and Including a plurality of openings having a first inlet, a first outlet, a second inlet, and a second outlet that penetrate in the thickness direction or in a direction perpendicular to the thickness direction in the cover portion, Biomimetic chips.
2. In paragraph 1, The first groove is formed to be equal to or longer than the length of the second groove, A biomimetic chip, wherein the first inlet and the first outlet correspond to the end of the first groove, and the second inlet and the second outlet correspond to the end of the second groove.
3. In paragraph 1, A biomimetic chip, wherein the first groove has a length extending in one direction and a width intersecting the length, and the width includes a plurality of convex regions formed spaced apart from each other with a larger width along the length.
4. In paragraph 3, A biomimetic chip, wherein the convex regions overlap with the first inlet, the first outlet, the second inlet, and the second outlet.
5. In paragraph 1, A biomimetic chip, wherein a biofluid is injected into the first groove and the second groove.
6. In paragraph 5, A biomimetic chip, wherein the biofluid comprises a material selected from circulating cells, adherent cells, scaffolds, chemicals, and biomolecules.
7. In paragraph 5, A biomimetic chip, wherein the biofluid is selectively exchanged through the porous membrane in an area where the first groove and the second groove overlap.
8. In paragraph 6, A biomimetic chip in which the above circulating and adherent cells are cultured in a single layer or multilayer structure or are cultured while circulating along the channels of the chip.
9. In paragraph 6, The above scaffold is a biomimetic chip comprising an extracellular matrix protein or a hydrogel protein.
10. In paragraph 6, The above chemical substance is a biomimetic chip including cell culture medium and additives.
11. In paragraph 6, A biomimetic chip, wherein the biomolecule comprises a signal transmitter or a neurotransmitter.
12. Step of preparing the substrate, cover and porous membrane; A step of surface-treating the substrate and the cover with plasma gas; A step of surface-treating the porous membrane with a silane coupling agent; and A method for manufacturing a biomimetic chip, comprising the step of bonding the surface-treated substrate, the cover part, and the porous membrane.
13. In paragraph 12, A method for manufacturing a biomimetic chip, wherein the plasma gas contains oxygen or air, and the oxygen or the air modifies the surface of the substrate and the cover part with hydroxyl groups.
14. In paragraph 12, A method for manufacturing a biomimetic chip, wherein the silane coupling agent comprises water or ethanol as a solvent.
15. A step of introducing a solution containing extracellular matrix proteins onto a substrate; A step of forming a constant flow in the above solution; A step of coating the extracellular matrix protein so that it is oriented and aligned in the flow direction; and An extracellular matrix coating method comprising a step of introducing cells onto the extracellular matrix coating having the above directionality.
16. In paragraph 15, An extracellular matrix coating method comprising further injecting plasma and calcium chloride (CaCl2) to form a constant flow during the coating step.