Medium circulation-type biomimetic microphysiological system

Abstract

The present invention provides a medium circulation-type biomimetic microphysiological system for implementing, according to blood vessels, the movement of nutrients, the movement of signal transmission substances, and the like. Therefore, a material to be tested can be evaluated on a chip without a separate test animal. The medium circulation-type biomimetic microphysiological system (10) comprises: a material channel (100) for providing a passage through which a material injected from the outside can be discharged to the outside of the medium circulation-type biomimetic microphysiological system (10) after moving from the inside of the medium circulation-type biomimetic microphysiological system (10); a culture chamber (200) for providing a space enabling a three-dimensional cell culture; an inlet channel (300), which connects the material channel (100) and the culture chamber (200) so as to provide a passage through which a part of the material moving along the material channel (100) can be diffused or perfused to the culture chamber (200); and a micropillar array (400) which is positioned in the inlet channel (300) and which has a plurality of microposts spaced apart from each other.

Description

A biomimetic microphysiological system with a circulation system

[0001] The technology described below is for 3D cell culture of cells, tissues, and organoids (stem cell-based organoids, differentiated cell-based multicellular spheroids).

[0002] Microfluidics-based technologies deal with small volumes of fluid. Microfluidics are used in diverse fields, including biotechnology, medicine, the environment, and physics. Microfluidics-based technologies are being applied to 3D microphysiological systems (MPS).

[0003] A 3D microphysiological system is a device that cultures various organ cells on a chip. A 3D microphysiological system simulates the movement and absorption of nutrients, substances, and signaling molecules on the chip. Through this, a 3D microphysiological system can embody various physiological activities, functions, and structures within the body through a chip, and can be used to model various diseases and evaluate substances and mechanisms of action. A media-circulating biomimetic microphysiological system is one type of 3D microphysiological system. It is a chip that co-cultures blood vessels and organ analogues on a chip to create a microenvironment similar to the body, and then simulates the movement of nutrients and signaling molecules through the blood vessels.

[0004] [Prior Art Literature]

[0005] [Patent Document]

[0006] Korean Patent Publication No. 2022-0165668

[0007] One aspect is to provide a biomimetic microphysiological system with a circulation-type medium.

[0008] Another aspect is to provide a multi-medium circulation biomimetic microphysiological system for evaluating interactions between multiple organoids.

[0009] Another aspect is to provide a method for screening substances using a biomimetic microphysiological system with a circulation type medium.

[0010] Another aspect is to provide a method for fabricating a biomimetic microphysiological system with a circulating medium.

[0011] A medium-circulating biomimetic microphysiological system comprises: a material channel that provides a passageway so that a material injected from the outside can move into the medium-circulating biomimetic microphysiological system and then be discharged to the outside; a culture chamber that provides a space for culturing three-dimensional cells, tissues, and organ-like organisms; an inflow channel that connects the material channel and the culture chamber and provides a passageway so that a portion of the material moving along the material channel can diffuse or perfuse into the culture chamber; and a micropillar array positioned in the inflow channel and having a plurality of microposts spaced apart from each other.

[0012] The technology described below enables the use of a culture platform capable of nutrient supply, waste discharge, and material transfer and circulation for the cultivation of fully formed organoids.

[0013] The technology described below enables the use of a culture platform that can reflect the tumor microenvironment. The tumor microenvironment is the surrounding environment surrounding the cancer, including blood vessels, fibroblasts, immune cells, and the extracellular matrix.

[0014] The technology described below can be used to create a culture platform that can replace animal testing as a preclinical testing model for new drug development. The technology described below can also accurately predict substance responses. The technology described below can be utilized in disease modeling and in vitro evaluation / testing methods for new drug development.

[0015] Figure 1 is an overall overview of the technology described below.

[0016] Figure 2 is a perspective view of a biomimetic microphysiological system (10) for circulation according to an embodiment.

[0017] Figure 3 is a plan view of a biomimetic microphysiological system (10) of a circulation type according to an embodiment.

[0018] Figure 4 is a perspective view of a biomimetic microphysiological system (10) for circulation according to an embodiment when viewed from above.

[0019] Figure 5 is a bottom view of a biomimetic microphysiological system (10) for circulation according to an embodiment.

[0020] Figure 6 is a bottom perspective view of a biomimetic microphysiological system (10) for circulation according to an embodiment.

[0021] Figure 7 is a front view of a biomimetic microphysiological system (10) for circulation according to an embodiment.

[0022] Figure 8 is a cross-sectional view of a biomimetic microphysiological system (10) of a circulation type according to an embodiment.

[0023] Figure 9 is an enlarged drawing of part A and the process of moving a substance in a media circulation type biomimetic microphysiological system (10) according to an embodiment.

[0024] Figure 10 is an example of an inlet section (110) and an outlet section (120) of a biomimetic microphysiological system (10) according to an embodiment.

[0025] Figure 11 is an example of a culture chamber (200) of a culture medium circulation type biomimetic microphysiological system (10).

[0026] Figure 12 is a perspective view of a multi-medium circulation biomimetic microphysiological system (20) for evaluating interactions between multiple organ-like substances according to an embodiment.

[0027] Figure 13 is a plan view of a multi-medium circulation type biomimetic microphysiological system (20) for evaluating interactions between multiple organ-like substances according to an embodiment.

[0028] Figure 14 shows the manufacturing process of a biomimetic microphysiological system (10) for circulation according to an embodiment.

[0029] Hereinafter, embodiments are described in detail with reference to exemplary drawings. When designating components in each drawing, it should be noted that, where possible, identical components are given identical reference numbers, even if they appear in different drawings. Furthermore, when describing embodiments, detailed descriptions of related known configurations or functions will be omitted if they are deemed to hinder understanding of the embodiments.

[0030] The present inventors have developed a media-circulating biomimetic microphysiological system (or vascular organ chip) having a micropillar structure and an open culture chamber (Fig. 1). The present inventors have also developed a method for screening substances using the media-circulating biomimetic microphysiological system having the developed micropillar structure and an open culture chamber. The present inventors have also developed a method for fabricating a media-circulating biomimetic microphysiological system having a micropillar structure. The media-circulating biomimetic microphysiological system developed by the present inventors can be used to determine the effects of experimental substances on organoids and tumor microenvironments without animal testing.

[0031] The present invention is described in detail below.

[0032] One aspect of the present invention provides a medium circulation type biomimetic microphysiological system (10).

[0033] FIGS. 2 to 9 illustrate an embodiment of a media-circulating biomimetic microphysiological system (10). The shape of the media-circulating biomimetic microphysiological system (10) described in FIGS. 2 to 9 is expressed similarly to a polygon or a circle, but is not limited thereto, and the shape of the media-circulating biomimetic system (10) may have various shapes other than those expressed in FIGS. 2 to 9. For example, the media-circulating biomimetic microphysiological system (10) may have various shapes such as a triangle, a sphere, an ellipse, and a rectangular parallelepiped.

[0034] FIG. 2 is a perspective view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 3 is a plan view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 4 is a perspective view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment when viewed from above. FIG. 5 is a bottom view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 6 is a bottom perspective view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 7 is a front view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 8 is a cross-sectional view of a medium-circulating biomimetic microphysiological system (10) according to an embodiment. FIG. 9 is a drawing showing a process of moving a substance in a medium-circulating biomimetic microphysiological system (10) according to an embodiment and an enlarged view of part A. Figure 10 is an example of an inlet section (110) and an outlet section (120) of a biomimetic microphysiological system (10) according to an embodiment.

[0035] A medium-circulating biomimetic microphysiological system (10) includes a material channel (100) that provides a passageway so that a material injected from the outside can move inside the medium-circulating biomimetic microphysiological system (10) and then be discharged to the outside of the medium-circulating biomimetic microphysiological system (10); a culture chamber (200) that provides a space for three-dimensional co-culture; an inflow channel (300) that connects the material channel (100) and the culture chamber (200) and provides a passageway so that a portion of the material moving along the material channel (100) can diffuse or perfuse into the culture chamber (200); and a micro-pillar array (400) positioned in the inflow channel (300) and having a plurality of microposts spaced apart from each other. Furthermore, a fixing member (500) may be attached to the lower portion of the medium-circulating biomimetic microphysiological system (10).

[0036] In one embodiment of the present invention, the material channel (100) may be a channel that receives materials necessary for cell culture or experimental materials from the outside, supplies them into the culture chamber (200), and discharges the remaining materials to the outside.

[0037] In one embodiment of the present invention, a material channel (100) includes an inlet portion (110) for receiving a material from outside a media-circulating biomimetic microphysiological system (10); an outlet portion (120) for discharging the injected material to the outside of the media-circulating biomimetic microphysiological system (10); and a supply path portion (130) for connecting the inlet portion (110) and the outlet portion (120) to provide a passage so that the material received by the inlet portion (110) can move to the outlet portion (120).

[0038] In one embodiment of the present invention, the inlet portion (110) can serve as a passage for injecting a substance from outside the media-circulating biomimetic microphysiological system (10). That is, by injecting a substance into the inlet portion (110), the experimenter can supply the substance into the media-circulating biomimetic microphysiological system (10).

[0039] The inlet (110) may have a shape that allows for the injection of a substance from the outside. In one embodiment, the inlet (110) may have a shape that is open at the top during the experiment. That is, the experimenter can supply a substance into the culture-circulating biomimetic microphysiological system (10) through the open top of the inlet (110).

[0040] The inlet portion (110) may have a shape capable of storing a substance injected from the outside. For example, the inlet portion (110) may include a shape similar to a reservoir. In one embodiment, the inlet portion (110) may have a shape in which the upper cross-sectional area is wider than the lower cross-sectional area connected to the supply path portion (130), as shown in FIG. 10, so as to have a structure capable of storing a substance injected from the outside in the upper portion. The same may be true for the outlet portion (120).

[0041] One end of the inlet section (110) can be connected to the supply passage section (130). The material injected into the inlet section (110) can be moved to the supply passage section (130).

[0042] The inlet portion (110) may be single. Alternatively, the inlet portion (110) may be plural. When there are plural inlets (110), the inlets (110) can form a concentration gradient of the combined solution. That is, a concentration gradient can be formed by injecting substances with different concentrations into each inlet portion (110). For example, when there are two inlets (110), a concentration gradient can be formed by injecting a substance with a low concentration into one portion and a substance with a high concentration into the other portion.

[0043] In one embodiment of the present invention, the outlet portion (120) can serve as a passage for discharging a substance injected into the medium-circulating biomimetic microphysiological system (10) to the outside of the medium-circulating biomimetic microphysiological system (10). That is, the experimenter can discharging a substance inside the medium-circulating biomimetic microphysiological system (10) to the outside of the medium-circulating biomimetic microphysiological system (10) through the outlet portion (120).

[0044] The outlet section (120) may have a shape that discharges substances within the media circulation type biomimetic microphysiological system (10) to the outside. In one embodiment, the outlet section (120) may have a shape that is open at the top during an experiment. When a substance is moved to the outlet section (120) through the supply path section (130) connected to the lower side of the outlet section (120), the substance may be discharged to the outside through the upper side of the outlet section (120).

[0045] The outlet portion (120) may have a shape that is symmetrical with respect to the center of the cross-section. In one embodiment, the outlet portion (120) may have a cylindrical shape.

[0046] One end of the outlet section (120) can be connected to the supply path section (130). The material can be moved to the outlet section (120) through the supply path section (130).

[0047] In one embodiment of the present invention, the supply path (130) may serve as a passage that allows a substance injected into the inlet part (110) to be discharged through the outlet part (120). The substance injected from the inlet part (110) may serve to allow a portion of the substance to flow into the culture chamber (200) through the inlet channel (300) while moving along the supply path (130). That is, while the substance moves through the supply path (130), a portion of the substance may diffuse into the culture chamber (200) through the inlet part.

[0048] The supply path (130) may have a shape that surrounds the culture chamber (200). In one embodiment, when the culture chamber (200) has a cylindrical shape, the supply path (130) may have a circular shape so as to surround the outside of the cylindrical culture chamber (200).

[0049] One side of the supply path (130) may be connected to the inlet channel (300). In one embodiment, the inlet channel (300) may be positioned on the side based on the direction of movement of the material moving within the supply path (130).

[0050] The supply path section (130) may be single. Alternatively, the supply path sections (130) may be plural. A plurality of supply path sections (130) may be connected to a single inlet section (110). Alternatively, only a single supply path section (130) may be connected to a plurality of inlets (110). Alternatively, a plurality of supply paths may be connected to each of the plurality of inlets (110).

[0051] In one embodiment of the present invention, the culture chamber (200) may serve to provide a space into which a mixture of cells and substrates may be injected to enable three-dimensional cell culture. The culture chamber (200) may serve to provide a space into which a mixture of cells and substrates may be injected to enable three-dimensional co-culture. That is, an experimenter wishing to conduct a cell culture experiment may mix the cells and substrates to be cultured and then inject the mixture into the culture chamber (200).

[0052] The culture chamber (200) may have a shape that allows cell culture. In one embodiment, the culture chamber (200) may have a shape in which the upper part is open for cell experiments so that a mixture of cells and substrates can be injected, and the lower part is closed.

[0053] The culture chamber (200) can be injected with angiogenic cells and extracellular matrix (ECM). Organoids can be injected into the culture chamber (200). That is, organoids can be cultured within the culture chamber.

[0054] The culture chamber (200) may have a shape that is symmetrical with respect to the center of the cross-section. In one embodiment, the culture chamber (200) may have a simple cylindrical shape.

[0055] If the culture chamber (200) has a simple structure as shown in FIG. 11, problems may arise. Referring to FIG. 11(a), if the culture chamber (200) has a simple cylindrical structure, the extracellular matrix (ECM) injected into the culture chamber (200) may not be formed uniformly (horizontally). In this case, the injected extracellular matrix (ECM) may adhere to the surface of the culture chamber (200) due to surface energy, and the thickness of the extracellular matrix at the center of the culture chamber (200) may become thin. In particular, if the thickness of the extracellular matrix at the center of the culture chamber (200) becomes thinner compared to the injected extracellular matrix, the three-dimensional culture of organoids cultured in the culture chamber (200) may be inhibited. Therefore, the culture chamber (200) may have a structure for uniformly forming the injected extracellular matrix.

[0056] In one embodiment, the culture chamber (200) may have a structure of two stages (210 and 220) as shown in (b) of Fig. 11. The cross-sectional area of ​​the lower part (220) of the culture chamber (200) may be formed to be wider than the cross-sectional area of ​​the upper part (210). The thickness of the lower part (220) of the culture chamber (200) may be formed to be thinner than the thickness of the upper part (210). For example, the thickness of the lower part (220) may be 0 mm to 1 mm, and the thickness of the upper part (210) may be 5 mm or more.

[0057] In one embodiment, the culture chamber (200) may have a groove (230) formed on the inner wall surface as shown in (c) of Fig. 11. By forming the groove (230) on the inner wall surface, the injected extracellular matrix can be uniformly distributed.

[0058] One side of the culture chamber (200) may be connected to an inlet channel (300). As the material moves along the supply path (130), a portion of the material may be introduced into the culture chamber (200) through the inlet channel (300). In one embodiment, the culture chamber (200) may have a cylindrical shape. In this case, the inlet channel (300) may have a shape that surrounds the outside of the cylindrical culture chamber (200).

[0059] In one embodiment of the present invention, the inflow channel (300) may serve as a passage so that a portion of a substance moving along the supply path (130) may flow into the culture chamber (200). The culture chamber (200) and the supply path (130) may be connected through the inflow channel (300). The inflow channel (300) may have a structure that can function as a passage through which a substance can move, and does not necessarily need to have a shape like a pipe.

[0060] The inlet channel (300) may have a micropillar array (400) with a plurality of microposts spaced apart from each other.

[0061] In one embodiment of the present invention, the micro-pillar array (400) can prevent a mixture of cells and substrates in a culture chamber (200) from leaking into the supply channel (130) through the inflow channel (300) based on a plurality of micro-posts.

[0062] A micropillar array (400) can be used to control the fluid flow within a media-circulating biomimetic microphysiological system (10) using microposts. The micropillar array (400) can interfacially separate internal liquid and extracellular matrix based on a plurality of microposts and selectively allow substances contained in the material to pass through.

[0063] The micro-pillar array (400) may be positioned within the inflow channel (300). Alternatively, the micro-pillar array (400) may be positioned between the flow path (130) and the inflow channel (300). That is, it is sufficient for the micro-pillar array (400) to be positioned between the paths along which the material of the flow path (130) moves to the culture chamber (200).

[0064] The micropost may be a columnar structure with a length and height of tens to hundreds of micrometers (㎛). In one embodiment, the height of the micropost may be 50 ㎛ to 200 ㎛.

[0065] Microposts can be circular, obliquely oval, triangular, square or diamond shaped.

[0066] If the distance at which the microposts are spaced apart from each other is large, the extracellular matrix inside the culture chamber (200) may flow into the supply channel (130), which may block the supply channel (130). If the distance at which the microposts are spaced apart from each other is small, the material may not diffuse or perfuse well into the culture chamber (200) through the supply channel (130). Therefore, the distance at which the microposts are spaced apart from each other may be a distance at which the material can diffuse or perfuse well into the culture chamber (200) through the supply channel (130) without the extracellular matrix inside the culture chamber (200) flowing out into the supply channel (130).

[0067] To this end, the microposts can be designed in a mutually slanted circular shape to maximize the surface area of ​​the microposts, thereby widening the space between the upper and lower portions of the channel while minimizing the gap between them. The microposts can be arranged so that the length between the microposts is maximized relative to the transverse length of the microposts, thereby allowing the material to be transported to the culture chamber (200). The microposts can be spaced apart at a gap wider than the size of the microposts.

[0068] In one embodiment, if the microposts are circular, the microposts may be spaced apart at intervals greater than the diameter of the circle. For example, if the diameter of the circular microposts is 30 μm, the microposts may be spaced apart from each other at intervals of 50 μm.

[0069] In one embodiment, when the microposts are in the form of an oblique ellipse, the microposts may be spaced apart from each other by a wider interval than the length of the larger of the major and minor axes of the ellipse. Alternatively, when the microposts are in the form of an oblique ellipse, the microposts may be spaced apart from each other by a wider interval than the length of the major and minor axes of the ellipse, whichever is closest to the material channel (100). For example, when the major axis of an elliptical micropost is 50 μm in length and the minor axis is 30 μm in length, if the major axis is closer to the material channel (100), the microposts may be spaced apart from each other by a interval of 80 μm.

[0070] In one embodiment, if the microposts are triangular in shape, the spacing between the microposts may be greater than the length of one side of the triangle. Alternatively, if the microposts are triangular in shape, the spacing between the microposts may be greater than the length of one side of the triangle closest to the material channel (100). For example, if the length of one side of a micropost in the shape of an equilateral triangle is 30 μm, the microposts may be spaced apart from each other by a spacing of 50 μm.

[0071] In one embodiment, if the microposts are rectangular in shape, the spacing between the microposts may be greater than the length of one side of the rectangular shape. Alternatively, if the microposts are rectangular in shape, the spacing between the microposts may be greater than the length of one side of the rectangular shape to which the material channel (100) is closest. For example, if the length of one side of a rectangular micropost is 30 μm, the microposts may be spaced apart from each other by a spacing of 50 μm.

[0072] In one embodiment, when the microposts are in the shape of a rhombus, the spacing distance between the microposts may be greater than the length of one side of the microposts. For example, as shown in Fig. 9, the spacing distance (b) between the microposts may be greater than the length (a) of one side of the rhombus (a <b).

[0073] In one embodiment of the present invention, the fixing part (500) can be attached to the bottom of the media-circulating biomimetic microphysiological system (10) to fix the vascular organ chip. Alternatively, the fixing part (500) can serve as the bottom surface of the media-circulating biomimetic microphysiological system (10) when the media-circulating biomimetic microphysiological system (10) is manufactured by soft lithography, an injection molding method using plastic, etc., or an electroplating method using a plating plate after pattern processing on a silicon wafer and plating with copper, etc.

[0074] Another aspect of the present invention provides a multi-medium circulation biomimetic microphysiological system (20) for evaluating interactions between multiple organoids.

[0075] Figures 12 and 13 show an example of a multi-medium circulation type biomimetic microphysiological system (20) for evaluating interactions between multiple analogs. Figure 12 is a perspective view of a multi-medium circulation type biomimetic microphysiological system (20) for evaluating interactions between multiple analogs. Figure 13 is a plan view of a multi-medium circulation type biomimetic microphysiological system (20) for evaluating interactions between multiple analogs. The multi-medium circulation type biomimetic microphysiological system (20) for evaluating interactions between multiple analogs may have a structure in which a plurality of the aforementioned medium circulation type biomimetic microphysiological systems (10) are arranged on a single plate.

[0076] A multi-medium circulation type biomimetic microphysiological system (10) for evaluating interactions between various organoids includes a plurality of medium circulation type biomimetic microphysiological systems (10), and each of the plurality of medium circulation type biomimetic microphysiological systems (10) can be connected to each other by a material channel (100).

[0077] Different organoids can be cultured between multiple media-circulating biomimetic microphysiological systems (10).

[0078] As described below, organ-like bodies may refer to organ-specific cell aggregates manufactured by re-aggregating and recombining cells isolated from stem cells or organ-derived cells using a three-dimensional culture method. For example, a brain organ-like body may be cultured in a first media-circulating biomimetic microphysiological system (10), a heart organ-like body may be cultured in a second media-circulating biomimetic microphysiological system (10), and a liver organ-like body may be cultured in a third media-circulating biomimetic microphysiological system (10). The material channels (100) of the media-circulating biomimetic microphysiological systems (10) are connected to each other. Through this, a material secreted from an organ-like body on one media-circulating biomimetic microphysiological system (10) may affect another media-circulating biomimetic microphysiological system (10).

[0079] Another aspect of the present invention provides a material screening method using a media circulation type biomimetic microphysiological system (10).

[0080] A method for screening materials using a culture medium-circulating biomimetic microphysiological system (10) includes a first step of injecting angiogenic cells and an extracellular matrix (ECM) into a culture chamber (200); a second step of injecting materials necessary for cell culture and angiogenic cells through a material channel (100) to form blood vessels in the culture chamber (200); a third step of injecting an organ analog into the culture chamber (200); and a fourth step of injecting a test subject material through the material channel (100).

[0081] In one embodiment of the present invention, a step of coating the material channel (100) may be further included. For coating, extracellular matrix proteins such as fibronectin, collagen, and elastin may be used. By coating, cell adhesion, fluid flow, etc. can be improved.

[0082] In this specification, angiogenic cells may refer to cells that form blood vessels through interaction with angiogenic inducers, co-cultured cells, etc. contained in a cell culture medium. Angiogenic cells can form blood vessels through vasculogenesis or angiogenesis.

[0083] Angiogenic cells can be isolated from various body sites, such as human umbilical vein endothelial cells (HUVECs). Furthermore, cultured cells can be derived from various species other than humans, such as porcine, murine, and bovine, and can be selected based on the experimental objectives. Thus, the type of angiogenic cells can be appropriately selected by those skilled in the art, depending on the experimental objectives.

[0084] The angiogenic cells may be at least one of an endothelial cell, a stem cell-derived cell, and an endothelial progenitor cell. Furthermore, the angiogenic cells may be genetically mutated cells and / or transfected cells of the above cells. In one embodiment, the angiogenic cells may be human umbilical vein endothelial cells (HUVECs).

[0085] In one embodiment of the present invention, the extracellular matrix may play a role in creating a three-dimensional microenvironment of an organoid. Alternatively, the extracellular matrix may be tailored to a disease model.

[0086] The extracellular matrix may be at least one of a collagen gel, a fibrin gel, a Matrigel, a self-assembled peptide gel, a polyethyleneglycol gel, and an alginate gel.

[0087] In one embodiment of the present invention, the first step may include a step of further injecting co-culture cells.

[0088] In this specification, co-cultured cells may refer to cells that secrete biochemical substances necessary for blood vessel formation, such as angiogenic inducers, through interaction with angiogenic cells.

[0089] The co-cultured cells may be at least one of astrocytes, glial cells, mesothelial cells, fibroblasts, smooth muscle cells, cancer cells, pericytes, neuroglial cells, stem cells, stem-cell derived cells, and cells that interact with the vascular endothelium. In addition, the co-cultured cells may be genetically mutated cells and / or transfected cells of the above cells. If the blood vessel to be created is a cerebral blood vessel, the co-cultured cells are preferably astrocytes, glial cells, pericytes, or fibroblasts, and if the blood vessel to be created is a blood vessel other than a cerebral blood vessel, the co-cultured cells are preferably fibroblasts or smooth muscle cells. In addition, the type and combination of cultured cells and the culture method may be selected according to the purpose of the experiment. As an example, the co-culture cells may be normal human dermal fibroblasts (NHDF).

[0090] In this specification, the material required for cell culture may be a material including nutrients, growth factors, cytokines, or hormones required for culturing cells inside a medium-circulating biomimetic microphysiological system (10).

[0091] The material required for cell culture may include a material that stimulates angiogenic cells in the culture chamber (200) to create blood vessels.

[0092] Materials required for cell culture may include angiogenic proteins.

[0093] As used herein, an organ-like substance may refer to an organ-specific cell aggregate manufactured by re-aggregating and recombining cells isolated from stem cells or organ-derived cells using a three-dimensional culture method. Organ-like substances contain cells specific to the model organ, reproduce the characteristics of the organ, and are spatially organized in a manner similar to that of an actual organ. Organ-like substances may mimic the organs of living organisms to determine the effects of experimental substances on living organisms.

[0094] The organoid may include one or more organoids selected from the group consisting of human-derived stem cell-based organoids and differentiated cell-based multicellular spheroids.

[0095] Organoids can be in vitro-derived three-dimensional cell clusters derived from primary cells or stem cells that are capable of self-replication and self-organization and exhibit organ function.

[0096] Spheroids may be formed by allowing various cells that make up each organ of the body to take on a three-dimensional structure outside the body and mature their structure and function.

[0097] Organ analogs may include those mimicking organs of the human body. For example, the organ analog may include at least one of a liver analog, a brain analog, a kidney analog, a pancreas analog, a colon analog, a stomach cancer analog, and a tumor spheroid.

[0098] In one embodiment of the present invention, the third step may be a step of injecting organ-like bodies into the culture chamber (200) after blood vessels have been formed within the culture chamber. Simultaneous injection of organ-like bodies and vascular cells may inhibit blood vessel formation or prevent blood vessels from forming around the organ-like bodies. Therefore, to create precise three-dimensional culture conditions, blood vessels may be formed within the culture chamber (200) before the organ-like bodies are injected into the culture chamber (200).

[0099] In this specification, the experimental subject material may refer to a material to be tested to determine its effect on an organ-like substance within a culture chamber (200). The experimental subject material is injected into the medium-circulating biomimetic microphysiological system (10) and can be delivered to the organ-like substance through a blood vessel connected to the interior of the culture chamber (200). Through this, the effect (stability, toxicity, efficacy, etc.) of the experimental subject material on the organ-like substance can be evaluated.

[0100] The experimental subject material can be of any type and can include any substance intended for experimentation. In other words, any substance intended to investigate the effects of an experimenter on the microenvironment or organ analogue can be included as a test subject material. For example, the test subject material may include chemical substances, etc., or it may include drugs. The test subject material may include at least one of the following: chemotherapeutic agents, immunotherapeutic agents, metabolic anticancer agents, cell therapy agents, gene therapy agents, and anti-inflammatory agents. Alternatively, the test subject material may include living organisms such as cells or viruses.

[0101] In one embodiment of the present invention, a fifth step may be further included to confirm the effect of the experimental substance delivered to the organ-like body through the blood vessels formed in the culture chamber (200) after the fourth step.

[0102] Step 5 may be a step to determine one or more selected from the therapeutic effect of the disease, the usefulness of the substance, the safety of the substance, and the cytotoxicity of the substance.

[0103] In one embodiment of the present invention, the culture channel of the media-circulating biomimetic microphysiological system (10) may be multiple. Different concentrations of the test substance can be injected into the multiple culture channels. When different concentrations of the test substance are injected, a concentration gradient can be formed within the culture chamber (200).

[0104] Another aspect of the present invention provides a method for manufacturing a media-circulating biomimetic microphysiological system (10).

[0105] There are various methods for manufacturing a media-circulating biomimetic microphysiological system (10). For example, a method used to manufacture a microfluidic chip can be used to manufacture a media-circulating biomimetic microphysiological system (10). For example, a soft lithography method, an injection molding method, and an electroplating method that utilizes a plating plate by plating a silicon wafer after pattern processing can be used.

[0106] In one embodiment of the present invention, a method for manufacturing a medium-circulating biomimetic microphysiological system (10) includes a first step of patterning a supply channel (130), an inflow channel (300), and a micro-pillar (400) structure on a first material; a second step of curing a second material on the first material on which the supply channel (130), the inflow channel (300), and the micro-pillar array (400) structure are patterned; a third step of punching an inlet portion (110), an outlet portion (120), and a culture chamber (200) structure on the cured second material; and a fourth step of attaching a fixing portion to the bottom of the punched second material.

[0107] The first process may be a process of patterning a supply path (130), an inlet channel (300), and a micro-pillar array (400) structure on a first material coated with photoresist by irradiating the first material with ultraviolet rays. Alternatively, the first process may be a process of pattern processing and then plating the first material made of a silicon wafer.

[0108] In one embodiment of the present invention, after the second process, a process of cutting a part of the second material to adjust the size of the media circulation type biomimetic microphysiological system (10) may be further included.

[0109] In one embodiment of the present invention, the second material may be PDMS (Polydimethylsiloxane). Furthermore, the second material may be at least one of polycarbonate (PC), poly(methyl methacrylate) (PMMA), cyclic olefin copolymer (COC), polystyrene, polyethylene terephthalate (PET), polysulfones, polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-caprolactone) (PLCL), and poly(glycolic acid-co-caprolactone) (PGCL).

[0110] In one embodiment of the present invention, the fixing member may be made of a material made of glass. In one embodiment, the fourth process may include a process of firmly attaching the second material and the fixing member by treating the lower part of the second material made of PDMS and the fixing member made of glass with plasma.

[0111] In one embodiment of the present invention, a method for manufacturing a medium-circulating biomimetic microphysiological system (10) may include a process of patterning a supply channel portion (130), an inflow channel (300), and micro-pillars (400) on a silicon wafer, a process of forming a seed layer on the silicon wafer on which the supply channel portion (130), the inflow channel (300), and the micro-pillar (400) structures are patterned, a process of plating the surface of the seed layered silicon wafer, a process of forming a mold for injection molding by connecting a structure in which an inlet portion (110), an outlet portion (120), and a culture chamber (200) structures are formed on the plated silicon wafer, and a process of injecting a second material into the mold and injecting the mold. Furthermore, the method for manufacturing a medium-circulating biomimetic microphysiological system (10) may include a process of plasma treating the surface of an injected polymer and a film lamination process for packaging.

[0112] The above seed layering process may be a process of coating to induce surface electricity for nickel electroplating. The seed layering process may include a process of coating with copper to a thickness of 30 nm and a process of coating with titanium to a thickness of 10-50 nm. The nickel electroplating may include a process of plating nickel on the surface to a thickness of about 1 mm. After nickel electroplating, a CMP (Chemical Mechanical Polishing) process may be performed. Through a surface polishing process after nickel electroplating, silicon residue on the nickel-plated surface can be removed and the surface can be processed to be flat. The process of manufacturing a mold for injection molding may include a process of manufacturing a mold for injection molding by attaching a nickel-plated plate to a mold (43C).

[0113] [Drawing symbol explanation]

[0114] 10: A biomimetic microphysiological system with a circulation system

[0115] 20: Multi-medium circulation biomimetic microphysiological system for evaluating interactions between multiple organoids

[0116] 100: Material Channel

[0117] 110: Inlet

[0118] 120: Outlet section

[0119] 130: Supply Euro

[0120] 200: Incubation chamber

[0121] 210: Top of the culture chamber

[0122] 220: Bottom of the culture chamber

[0123] 230: Home

[0124] 300: Inflow channel

[0125] 400: Micropillar Array

[0126] 500: Fixed part

Claims

1. A material channel (100) that provides a passage so that a material injected from the outside can move inside the medium circulation type biomimetic microphysiological system (10) and then be discharged to the outside of the medium circulation type biomimetic microphysiological system (10); A culture chamber (200) that provides a space for 3D cell culture; An inflow channel (300) that connects the material channel (100) and the culture chamber (200) and provides a passage through which a portion of the material moving along the material channel (100) can diffuse or perfuse into the culture chamber (200); A medium-circulating biomimetic microphysiological system comprising a micropillar array (400) positioned in the above inflow channel (300) and having a plurality of microposts spaced apart from each other.

2. In paragraph 1, The above-mentioned media-circulating biomimetic microphysiological system is made of at least one of PDMS (Polydimethylsiloxane), PC (polycarbonate), PMMA (poly(methyl methacrylate), COC (cyclic olefin copolymer), polystyrene, polyethylene terephthalate (PET), polysulfones, polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D,L-lactic acid) (PDLLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-caprolactone) (PLCL), and poly(glycolic acid-co-caprolactone) (PGCL). A biomimetic microphysiological system with a circulating medium.

3. In the first paragraph, a medium circulation type biomimetic microphysiological system, wherein a fixing part (500) is attached to the lower part of the medium circulation type biomimetic microphysiological system.

4. In paragraph 1, A medium-circulating biomimetic microphysiological system, comprising: an inlet portion (110) for receiving a material from outside the medium-circulating biomimetic microphysiological system (10); an outlet portion (120) for discharging the injected material to the outside of the medium-circulating biomimetic microphysiological system (10); and a supply path portion (130) for connecting the inlet portion (110) and the outlet portion (120) to provide a passage so that the material received by the inlet portion (110) can move to the outlet portion (120).

5. In paragraph 1, A medium circulation type biomimetic microphysiological system, wherein the inlet portion (110) and the outlet portion (120) include a reservoir capable of containing a substance injected from the outside.

6. In paragraph 1, The above inlet portion (110) and the outlet portion (120) have a shape in which the upper cross-sectional area is wider than the lower cross-sectional area connected to the supply path portion (130), and have a structure capable of storing a substance injected from the outside in the upper portion, a media circulation type biomimetic microphysiological system.

7. In paragraph 1, The above culture chamber (200) is a medium circulation type biomimetic microphysiological system in which the cross-sectional area of ​​the lower part (220) is formed to be wider than the cross-sectional area of ​​the upper part (210) and the thickness of the lower part (220) is formed to be thinner than the thickness of the upper part (210).

8. In paragraph 1, The above culture chamber (200) is a medium circulation type biomimetic microphysiological system having a groove (230) formed on the inner wall.

9. In paragraph 1, The above micropost is a media-circulating biomimetic microphysiological system having a triangular, circular, oblique elliptical or rhombic shape.

10. In paragraph 9, A biomimetic microphysiological system for circulating media, wherein the microposts are spaced apart at intervals wider than the longer of the major and minor axes of the oval, when the microposts are obliquely oval-shaped.

11. It includes a plurality of media circulation type biomimetic microphysiological systems (10) having the structure of Article 1, A multi-medium circulation type biomimetic microphysiological system (10) for evaluating interactions between multiple organ-like organisms, each of which has a plurality of material channels (100) connected to each other.

12. In paragraph 11, The above multiple media circulation type biomimetic microphysiological systems (10) are multi-media circulation type biomimetic microphysiological systems for evaluating interactions between various organ analogs in which different organ analogs are cultured.

13. In the cell culture method using the medium circulation type biomimetic microphysiological system (10) of paragraph 1, Step 1: Injecting angiogenic cells and extracellular matrix (ECM) into the culture chamber (200); A second step of forming blood vessels in the culture chamber (200) by injecting materials necessary for cell culture and the angiogenic cells through the material channel (100); A third step of injecting a long-term analogue into the culture chamber (200); and A method for screening materials using a medium-circulating biomimetic microphysiological system (10), including a fourth step of injecting a material to be tested through the material channel (100).

14. In paragraph 13, A method for screening materials using a medium-circulating biomimetic microphysiological system, further comprising a step of coating the material channel (100) before the second step.

15. In paragraph 13, A method for screening a substance using a medium-circulating biomimetic microphysiological system, wherein the first step includes a step of further injecting co-cultured cells.

16. In paragraph 13, A method for screening a substance using a media-circulating biomimetic microphysiological system, wherein the organoid is one or more organoids selected from the group consisting of organoids and spheroids.

17. In paragraph 13, The third step is a method for screening materials using a medium-circulating biomimetic microphysiological system, in which the organ-like substance is injected into the culture chamber (200) after blood vessels are formed in the culture chamber (200).

18. In paragraph 13, A method for screening materials using a medium-circulating biomimetic microphysiological system, further comprising a fifth step of confirming the effect of the experimental material delivered to the organ-like body through the blood vessels formed in the culture chamber (200) after the fourth step.

19. A first process of patterning a supply path (130), an inflow channel (300), and a micro pillar (400) structure on the first material; A second process of curing a second material on a first material having a patterned supply path (130), inlet channel (300) and micro pillar array (400) structure; A third process of punching an inlet portion (110), an outlet portion (120), and a culture chamber (200) structure into the above-mentioned cured second material; and A method for manufacturing a media-circulating biomimetic microphysiological system, comprising a fourth process of attaching a fixing part to the lower part of the punched second material.

20. In paragraph 19, The above first process is a method for manufacturing a medium circulation type biomimetic microphysiological system, which is a process of patterning the supply path section (130), the inflow channel (300), and the micro-pillar array (400) structure on the first material by irradiating ultraviolet rays on the first material coated with photoresist.

21. In paragraph 19, The above first process is a method for manufacturing a media circulation type biomimetic microphysiological system, which is a process of pattern processing and then plating a first material made of a silicon wafer.

22. In paragraph 19, The second material is at least one of PDMS (Polydimethylsiloxane), PC (polycarbonate), PMMA (poly(methyl methacrylate), COC (cyclic olefin copolymer), polystyrene, polyethylene terephthalate (PET), polysulfones, polyvinyl chloride (PVC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), poly(L-lactic acid) (PLLA), poly(D, L-lactic acid) (PDLLA), poly(glycolic acid) (PGA), poly(caprolactone) (PCL), poly(hydroxyalkanoate), polydioxanone (PDS), polytrimethylene carbonate, poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-caprolactone) (PLCL), and poly(glycolic acid-co-caprolactone) (PGCL), a media-circulating biomimetic. A method for creating a microphysiological system.

23. In paragraph 19, A method for manufacturing a medium-circulating biomimetic microphysiological system, wherein the fourth process includes a process of firmly attaching the second material and the fixing part (500) by treating the lower part of the second material made of PDMS and the fixing part (500) made of glass with plasma.

24. A process of patterning a supply path (130), an inflow channel (300), and micropillars (400) on the first material. A process for manufacturing a mold for injection molding by connecting a structure in which an inlet portion (110), an outlet portion (120) and a culture chamber (200) structure are formed on a first material patterned with the above-mentioned supply path portion (130), inlet channel (300) and micro pillar (400) structures, and A method for manufacturing a media-circulating biomimetic microphysiological system, comprising a process of injecting a second material into the mold manufactured above and performing injection molding.

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