Microfluidic device, its manufacturing method, and toxicity evaluation method using the same

Abstract

A microfluidic device is provided, comprising: a fluid bed into which a cell culture medium or plasma is injected, a concentration gradient of the injected plasma is formed, and a cell suspension in which cells are diluted is introduced; and a spheroid forming layer in which a plurality of spheroids are formed from the cells of the cell suspension.

Description

Technology Field

[0001] The present application relates to a microfluidic device, a method for manufacturing the same, and a method for evaluating toxicity using the same. More specifically, it relates to a microfluidic device capable of rapidly and accurately determining the degree of toxicity according to the roundness of spheroids at various concentrations of plasma, a method for manufacturing the same, and a method for evaluating toxicity using the same. Background Technology

[0003] Colistin, also known as polymyxin E, may be an antibiotic against multidrug-resistant Gram-negative infections, including pneumonia.

[0004] However, colistin can have kidney toxicity as a major side effect.

[0005] As a result, when colistin is used, kidney function may decline, and in particular, when colistin is used in patients with pre-existing kidney problems or the elderly, kidney function may worsen.

[0006] Accordingly, evaluation and / or monitoring of kidney function may be required when using colistin.

[0007] Conventionally, to evaluate kidney function, blood could be collected from a patient and biological markers in the plasma contained in the collected blood could be measured, or urine tests or diagnostic imaging tests could be performed.

[0008] For example, Korean Patent Publication No. 10-2021-01332 discloses a method for evaluating kidney function of a subject, wherein the method comprises the steps of administering a first plurality of nanoparticles having a first dose to a subject, collecting a urine sample and / or blood sample from the subject after a first period following the administration, characterizing the nanoparticles in the urine sample and / or blood sample through a measurement process to obtain characteristic parameters, and evaluating kidney function by comparing the characteristic parameters with characteristic parameters of a control group measured for a control group having normal kidney function.

[0009] As another example, Korean Registered Patent Publication No. 10-1342035 discloses a method for confirming gene expression levels upon treatment with amphotericin B or cisplatin, comprising the steps of: treating isolated human normal kidney cells with a test compound; isolating RNA from experimental group cells treated with the test compound in the step of treating the test compound and control cells not treated with the test compound; synthesizing the RNA of the experimental group and control cells in the step of isolating RNA into cDNA while labeling the experimental group and control cells with different fluorescent substances; hybridizing the cDNA labeled with different fluorescent substances in the step of labeling fluorescent substances with the DNA microarray chip of claim 1; analyzing the DNA microarray chip that reacted in the hybridization step; and confirming the degree of gene expression integrated in the DNA microarray chip of claim 1 by comparing it with the control group in the data analyzed in the step of analyzing the microarray chip.

[0010] However, plasma collection for evaluating kidney function may be limited depending on the patient's biological condition, and there may be limits to the amount of plasma collected.

[0011] In addition, conventional two-dimensional cell culture may not be able to reproduce the complex three-dimensional microenvironment found in biological tissues.

[0012] As a result, there may be limitations in understanding cell behavior and drug responses with conventional two-dimensional cell culture.

[0013] Accordingly, there is a need for a platform and / or evaluation method that can assess the renal function of patients administered colistin using even a small amount of plasma. The problem to be solved

[0015] The technical problem that this application aims to solve is to provide a microfluidic device capable of rapidly and accurately determining the degree of toxicity according to the roundness of spheroids at various concentrations of plasma, a method for manufacturing the same, and a method for evaluating toxicity using the same.

[0016] Another technical problem that this application aims to solve is to provide a microfluidic device capable of determining the timing of drug toxicity onset based on patient-specific characteristics, for example, the individual patient's metabolism and the presence or absence of underlying diseases, a method for manufacturing the same, and a method for evaluating toxicity using the same.

[0017] Another technical problem that the present application aims to solve is to provide a microfluidic device capable of predicting the onset of nephrotoxicity in advance, a method for manufacturing the same, and a method for evaluating toxicity using the same.

[0018] Another technical problem that this application aims to solve is to provide a microfluidic device with improved sensitivity to small amounts of drugs, a method for manufacturing the same, and a method for evaluating toxicity using the same.

[0019] Another technical problem that the present application aims to solve is to provide a microfluidic device capable of evaluating the renal function of a patient administered colistin by a non-invasive method, a method for manufacturing the same, and a method for evaluating toxicity using the same.

[0020] The technical problems that this application aims to solve are not limited to those described above. means of solving the problem

[0022] To solve the above technical problem, the present application provides a microfluidic device.

[0023] According to one embodiment, the microfluidic device may include a fluid bed into which a cell culture medium or plasma is injected, a concentration gradient of the injected plasma is formed, and a cell suspension in which cells are diluted is introduced, and a spheroid forming layer in which a plurality of spheroids are formed from the cells of the cell suspension.

[0024] According to one embodiment, the fluidized bed comprises an injector having an injector port into which the cell culture medium or the plasma is injected, a concentration gradient forming part having a branched flow path forming a concentration gradient of the plasma injected through the injector port, and a cell inlet having a plurality of cell inlets into which the cell suspension in which the cells are diluted is introduced, and the spheroid forming layer may include a plurality of spheroid forming spaces that are in communication with the plurality of cell inlets and in which a plurality of spheroids are formed from the cells of the cell suspension.

[0025] According to one embodiment, the spheroid forming space may be curved downward in the direction opposite to the cell inlet.

[0026] According to one embodiment, the concentration gradient forming part can form a concentration gradient of the plasma corresponding to the number of terminal ends of the branched flow path.

[0027] According to one embodiment, the microfluidic device may further include a storage layer that is in communication with the inlet and includes a storage space in which the cell culture medium or plasma is stored before being injected into the inlet.

[0028] According to one embodiment, the fluid bed may further include a connecting channel connecting the inlet and the branched channel, and a discharge section including a discharge port through which the cell culture medium or the plasma is discharged.

[0029] According to one embodiment, the diameter of the outlet may be larger than the diameter of the inlet.

[0031] To solve the above technical problem, the present application provides a toxicity evaluation method.

[0032] According to one embodiment, the toxicity evaluation method may include the steps of: preparing a cell suspension by diluting cells; introducing the cell suspension into a microfluidic device; providing a cell culture medium to the cell suspension and forming a plurality of spheroids from the cells; providing plasma having a concentration gradient to the plurality of spheroids; and measuring the roundness of the plurality of spheroids to which the plasma has been provided, and evaluating toxicity according to the roundness.

[0033] According to one embodiment, the determination of toxicity of the plasma is performed within 2 days, and the time at which toxicity manifests can be determined for each patient, and the determination of the time at which toxicity manifests can be earlier than the time at which toxicity actually manifests in the patient.

[0034] According to one embodiment, the toxicity evaluation method may further include the step of forming a coating layer to reduce the adhesion of the cell to the microfluidic device.

[0035] According to one embodiment, the cell comprises kidney cells, and the cell suspension is 1 x 10 6 >10 cells / mL to 1 x 10⁶ 7 It can have a concentration of less than cells / mL.

[0036] According to one embodiment, the plasma comprises the plasma of a patient to whom colistin has been administered, and the colistin may have a concentration of 50 μg / mL or more to less than 100 μg / mL.

[0038] To solve the above technical problem, the present application provides a method for manufacturing a microfluidic device.

[0039] According to one embodiment, the method for manufacturing the microfluidic device may include the steps of: providing a first polymer material to a mold for forming an injection part including an injector for injecting a cell culture medium or plasma, a concentration gradient forming part including a branched flow path for forming a concentration gradient of the plasma injected through the injector, and a cell inlet part including a plurality of cell inlets for introducing a cell suspension in which cells are diluted, and forming a fluid layer; providing the first polymer material to a mold for forming a plurality of spheroid forming spaces in which a plurality of spheroids are formed from the cells of the cell suspension, and forming a spheroid forming layer; and stacking the fluid layer on the spheroid forming layer so that the plurality of cell inlets and the plurality of spheroid forming spaces are in communication.

[0040] According to one embodiment, the method for manufacturing the microfluidic device may further include the steps of: forming a storage layer by laser processing a second polymer material different from the first polymer material to form a storage space in which the cell culture medium or plasma is stored before being injected into the injector; and stacking the storage layer on the fluid bed so that the storage space and the injector are in communication.

[0041] According to one embodiment, the fluid layer and the storage layer can be laminated through the first polymer material. Effects of the invention

[0043] According to an embodiment of the present application, a microfluidic device may be provided comprising: a fluid bed into which a cell culture medium or plasma is injected, a concentration gradient of the injected plasma is formed, and a cell suspension in which cells are diluted is introduced; and a spheroid forming layer in which a plurality of spheroids are formed from the cells of the cell suspension.

[0044] According to an embodiment of the present application, the microfluidic device may include: a spheroid forming layer comprising a plurality of spheroid forming spaces in which a plurality of spheroids are formed from the cells of the cell suspension; an injection part comprising an injector into which the cell culture medium or the plasma is injected; a concentration gradient forming part comprising a branched flow path that forms a concentration gradient of the plasma injected through the injector; a fluidized bed comprising a cell inlet part comprising a plurality of cell inlets into which the cell suspension diluted with the cells is introduced; and a storage layer comprising a storage space in which the cell culture medium or the plasma is stored before being injected through the injector.

[0045] In a microfluidic device according to an embodiment of the present application, when the plasma to which the drug has been administered is provided to the spheroid, the roundness of the spheroid may be modified depending on whether or not there is toxicity. More specifically, if toxicity is manifested in the plasma to which the drug has been administered, the roundness of the spheroid may be reduced. In other words, if toxicity is manifested in the plasma to which the drug has been administered, the roundness of the spheroid may be compromised.

[0046] As a result, the microfluidic device according to the embodiment of the present application may have the advantage that staining and / or assay required in the toxicity evaluation of conventional cells can be omitted, because the toxicity of the plasma can be evaluated according to the roundness of the plurality of spheroids provided.

[0047] In addition, according to an embodiment of the present application, the microfluidic device can form a concentration gradient of the plasma corresponding to the number of terminals of the branched flow path. More specifically, the concentration gradient forming part can form n concentration gradients of the plasma when the branched flow path has m layers and n terminals.

[0048] Accordingly, the microfluidic device according to the embodiment of the present application has the technical effect of determining the degree of toxicity for various concentrations of the plasma. Furthermore, according to the present application, since the toxicity evaluation according to various concentrations of the plasma is performed as a single process rather than separately, there is also a simple and rapid technical effect. Brief explanation of the drawing

[0050] FIG. 1 is a drawing for explaining a microfluidic device according to an embodiment of the present application. FIG. 2 is a drawing for explaining steps S110 to S130 of a toxicity evaluation method according to an embodiment of the present application. FIG. 3 is a drawing for explaining steps S140 to S160 of a toxicity evaluation method according to an embodiment of the present application. FIG. 4 is a drawing for explaining steps S110 to S160 of a toxicity evaluation method according to an embodiment of the present application in more detail. FIG. 5 is a drawing for explaining steps S210 and S220 of a method for manufacturing a microfluidic device according to an embodiment of the present application. FIG. 6 is a drawing for explaining steps S230 to S250 of a method for manufacturing a microfluidic device according to an embodiment of the present application. FIG. 7 is a drawing for explaining step S210 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail. FIG. 8 is a drawing for explaining step S220 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail. FIG. 9 is a drawing for explaining step S230 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail. FIG. 10 is a diagram illustrating the concentration gradient of plasma according to an experimental example of the present application. Figure 11 is a photograph of a spheroid formed according to the concentration of a cell suspension according to an experimental example of the present application. FIG. 12 is a graph showing the area and roundness of spheroids formed according to the concentration of the cell suspension in an experimental example of the present application. FIG. 13 is a graph showing the diameter and roundness of spheroids formed according to the concentration of the cell suspension in an experimental example of the present application. Figure 14 is a photograph showing the toxicity analysis using a microfluidic device manufactured according to the experimental example of the present application. FIG. 15 is a graph showing the roundness and amount of colistin when toxicity is analyzed using a microfluidic device manufactured according to the experimental example of the present application. Figure 16 is a photograph showing the analysis of toxicity using a conventional two-dimensional cell culture microfluidic chip. Figure 17 is a graph showing the roundness and amount of colistin when toxicity is analyzed using a conventional two-dimensional cell culture microfluidic chip. FIG. 18 is a graph showing the viability of kidney cells using a microfluidic device manufactured according to the experimental example of the present application. Figure 19 is a graph showing the viability of kidney cells measured using a conventional two-dimensional cell culture microfluidic chip. FIG. 20 is a graph showing the spheroid survival rate of a first patient who developed nephrotoxicity at an early stage using a microfluidic device manufactured according to the experimental example of the present application. FIG. 21 is a graph showing the spheroid survival rate of a second patient who developed nephrotoxicity at a late stage using a microfluidic device manufactured according to the experimental example of the present application. FIG. 22 is a graph showing the spheroid survival rate of a third patient who did not exhibit nephrotoxicity using a microfluidic device manufactured according to the experimental example of the present application. Specific details for implementing the invention

[0051] Hereinafter, preferred embodiments of the present application will be described in detail with reference to the attached drawings. However, the technical concept of the present application is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the concept of the present application is sufficiently conveyed to those skilled in the art.

[0052] In this specification, when a component is described as being on another component, it means that it may be formed directly on the other component or that a third component may be interposed between them. Additionally, in the drawings, the thicknesses of the films and regions are exaggerated for the effective illustration of the technical content.

[0053] Additionally, although terms such as first, second, third, etc., have been used to describe various components in the various embodiments of this specification, these components should not be limited by such terms. These terms are used merely to distinguish one component from another. Accordingly, what is referred to as the first component in one embodiment may be referred to as the second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiment. Furthermore, in this specification, "and / or" is used to mean including at least one of the components listed before and after it.

[0054] In the specification, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "include" or "have" are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof. Additionally, in this specification, "connection" is used to include both indirectly connecting multiple components and directly connecting them.

[0055] Furthermore, in describing the present application below, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the application, such detailed description will be omitted.

[0057] FIG. 1 is a drawing for explaining a microfluidic device according to an embodiment of the present application.

[0058] Referring to FIG. 1, the microfluidic device (1000) may include a fluid bed (200) into which a cell culture medium or plasma (30) is injected and a concentration gradient of the injected plasma (30) is formed and a cell suspension in which cells are diluted is introduced, a spheroid forming layer (100) in which a plurality of spheroids (10) are formed from the cells of the cell suspension, and a storage layer (300) in which the cell culture medium or plasma (30) before injection is stored.

[0059] According to one embodiment, the spheroid forming layer (100) may include a plurality of spheroid forming spaces (111: 111a~111e) in which a plurality of spheroids (10) are formed from the cells of the cell suspension, as shown in FIG. 1 and FIG. 4.

[0060] According to one embodiment, the fluid bed (200) may include, as shown in FIGS. 1 and 4, an injection section (210) including an injection port (211: 211a, 211b) into which the cell culture medium or the plasma (30) is injected, a concentration gradient forming section (220) including a branched flow path (221: 221a~221l) that forms a concentration gradient of the plasma (30) injected through the injection port (211), and a cell inlet section (230) including a plurality of cell inlets (231) into which the cell suspension in which the cells are diluted is introduced.

[0061] According to one embodiment, the storage layer (300) may include a storage space (311: 311a, 311b) in which the cell culture medium or the plasma (30) is stored before being injected into the injection port (211), as shown in FIGS. 1 and FIGS. 4.

[0062] According to one embodiment, the microfluidic device (1000) may include a structure in which the fluid layer (200) is stacked on the spheroid forming layer (100) and the storage layer (300) is stacked on the fluid layer (200). In other words, the microfluidic device (1000) may include a structure in which the spheroid forming layer (100), the fluid layer (200), and the storage layer (300) are stacked sequentially.

[0063] According to one embodiment, a plurality of the spheroid forming spaces (111) of the spheroid forming layer (100) and a plurality of the cell inlets (231) of the fluid layer (200) may be connected.

[0064] Accordingly, the cell suspension introduced through the plurality of cell inlets (231) of the fluidized bed (200) can be provided to the plurality of spheroid forming spaces (111) of the spheroid forming layer (100).

[0065] As a result, in the plurality of spheroid forming spaces (111), a plurality of spheroids (10) can be formed from the cells of the cell suspension.

[0066] According to one embodiment, a plurality of the spheroid-forming spaces (111) and a plurality of the cell inlets (231) may be formed in equal numbers. For example, a plurality of the spheroid-forming spaces (111) and a plurality of the cell inlets (231) may each be formed at the ends of the branched flow channels (221) that form the concentration gradient of the plasma (30). More specifically, for example, if the number of ends of the branched flow channels (221) is 5 (221h to 221l), 10 of the spheroid-forming spaces (111) and 10 of the cell inlets (231) may be formed per end of the branched flow channels (221). In other words, the microfluidic device (1000) may include a total of 50 of the spheroid-forming spaces (111) and a total of 50 of the cell inlets (231).

[0067] According to one embodiment, the spheroid forming space (111) may be curved downward in the opposite direction of the cell inlet (231), as shown in FIGS. 1 and 4.

[0068] As a result, when the cell suspension is provided to the downwardly curved spheroid forming space (111), the spheroid (10) having roundness along the downwardly curved shape of the spheroid forming space (111) can be formed.

[0069] Accordingly, there is a technical effect in that toxicity can be easily evaluated according to the variation of the roundness of the spheroid (10) having the roundness. More specifically, the plasma (30) may be plasma to which a drug (dr) has been administered. For example, the plasma (30) may be plasma to which colistin has been administered. However, it is not limited thereto.

[0070] According to one embodiment, when the plasma (30) to which the drug (dr) has been administered is provided to the spheroid (10), the roundness of the spheroid (10) may be modified depending on whether there is toxicity. More specifically, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be reduced. In other words, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be compromised.

[0071] As a result, according to the present invention, since the toxicity of the plasma (30) can be evaluated according to the roundness of the plurality of spheroids (10) provided, there may be an advantage that the staining and / or assay required in the toxicity evaluation of conventional cells can be omitted.

[0072] According to one embodiment, the inlet (211) of the fluid bed (200) and the storage space (311) of the storage bed (300) may be connected, and the inlet (211) and the branched flow path (221) may be connected through a connecting flow path (251).

[0073] Accordingly, the cell culture medium or plasma (30) stored in the storage space (311) of the storage layer (300) can be injected into the injector (211) of the fluid layer (200), and in the case of the plasma (30), a concentration gradient can be formed as it flows through the branched flow path (221).

[0074] As a result, the plasma (30) having a concentration gradient can be provided to the spheroid (10).

[0075] According to one embodiment, the concentration gradient forming part (220) can form a concentration gradient of the plasma (30) corresponding to the number of terminals of the branched flow path (221). More specifically, the concentration gradient forming part (220) can form n concentration gradients of the plasma (30) when the branched flow path (221) has m layers and n terminals. For example, referring to FIGS. 1 and FIGS. 4, when the branched flow path (221) has 3 layers and 5 terminals (221h to 221l), the concentration gradient of the plasma (30) can be formed in 5 ways. However, it is not limited thereto.

[0076] According to one embodiment, the storage space (311) and the injection port (211) may each be two (311a, 311b, 211a, 211b). The cell culture medium stored in one of the storage spaces (311a) may be introduced through one of the injection ports (211a) which is connected to one of the storage spaces (311a). The plasma (30) stored in another storage space (311b) may be introduced through another injection port (211b) which is connected to the other storage space (311b). At this time, if the branched Euro (221) has three layers and five ends (221h to 221l), the concentration gradient of the plasma (30) can be formed in five ways, namely, from one of the storage spaces (311a) where the cell culture medium is stored to the other of the storage spaces (311b) where the plasma (30) is stored, at 0%, 12.5%, 50%, 87.5%, and 100%.

[0077] Accordingly, according to the present application, there is a technical effect of determining the degree of toxicity for various concentrations of the plasma (30). Furthermore, according to the present application, since the toxicity evaluation according to various concentrations of the plasma (30) is performed in a single process rather than separately, there is also a simple and rapid technical effect.

[0078] According to one embodiment, the fluid bed (200) may further include a discharge section (240) comprising a discharge port (241: 241a~241e) through which the cell culture medium or the plasma (30) is discharged, as shown in FIGS. 1 and FIGS. 4.

[0079] According to one embodiment, as illustrated in FIG. 1, in the fluid bed (200), the diameter (d2) of the outlet (241) may be larger than the diameter (d1) of the inlet (211). For example, the diameter (d1) of the inlet (211) may be 2 mm and the diameter (d2) of the outlet (241) may be 3 mm. However, it is not limited thereto.

[0080] As a result, the cell culture medium or plasma (30) injected through the injection port (211) can be easily discharged through the outlet port (241) after the reaction.

[0082] According to the embodiments of the present application described above, the microfluidic device (1000) may include: a spheroid forming layer (100) comprising a plurality of spheroid forming spaces (111) in which a plurality of spheroids (10) are formed from the cells of the cell suspension; an injection section (210) comprising an injection port (211) into which the cell culture medium or the plasma (30) is injected; a concentration gradient forming section (220) comprising a branched flow path (221) that forms a concentration gradient of the plasma (30) injected through the injection port (211); a fluid layer (200) comprising a cell inlet section (230) comprising a plurality of cell inlets (231) into which the cell suspension diluted with the cells is introduced; and a storage layer (300) comprising a storage space (311) in which the cell culture medium or the plasma (30) is stored before being injected through the injection port (211). there is.

[0083] In a microfluidic device (1000) according to an embodiment of the present application, when the plasma (30) to which the drug (dr) has been administered is provided to the spheroid (10), the roundness of the spheroid (10) may be modified depending on whether there is toxicity. More specifically, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be reduced. In other words, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be compromised.

[0084] As a result, the microfluidic device (1000) according to the embodiment of the present application may have the advantage that staining and / or assay required in the toxicity evaluation of conventional cells can be omitted, because the toxicity of the plasma (30) can be evaluated according to the roundness of the plurality of spheroids (10) provided.

[0085] Additionally, according to an embodiment of the present application, the microfluidic device (1000) can form a concentration gradient of the plasma (30) corresponding to the number of terminals of the branched flow path (221). More specifically, the concentration gradient forming part (220) can form n concentration gradients of the plasma (30) when the branched flow path (221) has m layers and n terminals.

[0086] Accordingly, the microfluidic device (1000) according to the embodiment of the present application has the technical effect of determining the degree of toxicity for various concentrations of the plasma (30). Furthermore, according to the present application, the toxicity evaluation according to various concentrations of the plasma (30) is not performed separately but is carried out as a single process, so there is also a simple and rapid technical effect.

[0088] FIG. 2 is a drawing for explaining steps S110 to S130 of a toxicity evaluation method according to an embodiment of the present application, FIG. 3 is a drawing for explaining steps S140 to S160 of a toxicity evaluation method according to an embodiment of the present application, and FIG. 4 is a drawing for explaining steps S110 to S160 of a toxicity evaluation method according to an embodiment of the present application in more detail.

[0089] Referring to FIGS. 2 and FIGS. 4, a coating layer for lowering the adhesion of the cell can be formed on the microfluidic device (1000) (S110).

[0090] According to one embodiment, prior to the formation of the coating layer, alcohol may be supplied into the microfluidic device (1000) through the storage space (311), the injection port (211), or the cell inlet (231). More specifically, for example, 70% ethanol may be supplied into the microfluidic device (1000) through the storage space (311), the injection port (211), or the cell inlet (231). However, it is not limited thereto.

[0091] As a result, the inside of the microfluidic device (1000) can be cleaned.

[0092] According to one embodiment, after the interior of the microfluidic device (1000) is washed with the alcohol, phosphate buffered saline (PBS) may be supplied multiple times into the microfluidic device (1000) through the storage space (311), the inlet (211), or the cell inlet (231). More specifically, for example, the phosphate buffered saline (PBS) may be supplied three times into the microfluidic device (1000). However, it is not limited thereto.

[0093] As a result, the interior of the microfluidic device (1000) can be additionally cleaned.

[0094] According to one embodiment, after the interior of the microfluidic device (1000) is further washed with the phosphate-buffered physiological saline (PBS), bovine serum albumin (BSA) may be supplied into the microfluidic device (1000) through the storage space (311), the inlet (211), or the cell inlet (231). More specifically, for example, 3% bovine serum albumin (BSA) may be supplied into the microfluidic device (1000) at room temperature through the storage space (311), the inlet (211), or the cell inlet (231). However, it is not limited thereto.

[0095] As a result, the coating layer can be formed inside the microfluidic device (1000).

[0096] Accordingly, the adhesion force of the cell inside the microfluidic device (1000) can be reduced.

[0097] As a result, when the cell suspension in which the cell is diluted is introduced into the microfluidic device (1000) in which the cell's adhesion force is lowered, the cell can be easily transferred to the spheroid forming space (111) of the spheroid forming layer (100).

[0098] Accordingly, the spheroid (10) can be easily formed in the spheroid forming space (111).

[0099] According to one embodiment, the formation of the coating layer may be performed for a predetermined period of time. More specifically, for example, the formation of the coating layer may be performed for one hour. However, it is not limited thereto.

[0100] According to one embodiment, phosphate-buffered saline (PBS) may be additionally provided multiple times inside the microfluidic device (1000) on which the coating layer is formed. More specifically, for example, the phosphate-buffered saline (PBS) may be additionally provided three times inside the microfluidic device (1000). However, it is not limited thereto.

[0101] As a result, the interior of the microfluidic device (1000) can be additionally cleaned.

[0102] Referring further to FIGS. 2 and FIGS. 4, the cell can be diluted to prepare the cell suspension (S120).

[0103] According to one embodiment, the concentration of the cell suspension prepared by diluting the cell can be controlled.

[0104] As a result, the roundness and / or size of the spheroid (10) formed can be controlled.

[0105] For example, the cell suspension is 1 x 10 6 >10 cells / mL to 1 x 10⁶ 7It may be diluted to have a concentration of less than cells / mL. More specifically, for example, the cell suspension is 5 x 10 6 It can be diluted to have a concentration of cells / mL.

[0106] As a result, the shape and size of the spheroid (10) formed from the cell may be suitable for toxicity evaluation. More specifically, for example, 5 x 10 6 The spheroid (10) formed by providing the cell suspension having a concentration of cells / mL can have a roundness improved by more than 50 compared to day 1 on a basis of 100 after 3 days, and can have maximum roundness after 4 days. Also, 5 X 10 6 The spheroid (10) formed by providing the cell suspension having a concentration of cells / mL is 1 X 10 6 When the above cell suspension having a concentration of cells / mL is provided and 1 X 10 7 The cell suspension having a concentration of cells / mL may have a larger area and / or diameter while maintaining the roundness than when provided.

[0107] Accordingly, the spheroid (10) having roundness and area and / or diameter suitable for toxicity evaluation can be formed, and toxicity evaluation using the spheroid (10) can be accurate and rapid.

[0108] Referring further to FIGS. 2 and FIGS. 4, the cell suspension can be introduced into the microfluidic device (1000) (S130).

[0109] According to one embodiment, the cell suspension may be introduced into a plurality of spheroid forming spaces (111) through a plurality of cell inlets (231, see FIG. 1).

[0110] According to one embodiment, the cells included in the cell suspension may be kidney cells. However, it is not limited thereto.

[0111] Referring further to FIGS. 3 and 4, the cell culture medium is provided to the cell suspension and a plurality of spheroids (10) can be formed from the cells (S140).

[0112] According to one embodiment, while a plurality of spheroids (10) are being formed in a plurality of spheroid forming spaces (111), the cell culture medium may be continuously injected through the injection port (211, see FIG. 1). For example, the cell culture medium may be injected at a ratio of 100 times the amount of the cell suspension. More specifically, for example, 1 μL of the cell suspension and 100 μL of the cell culture medium may be injected. However, it is not limited thereto.

[0113] As a result, not only does the interior of the microfluidic device (1000) not dry out, but the cell culture medium can also be continuously supplied to the plurality of spheroid forming spaces (111).

[0114] Accordingly, a plurality of the spheroids (10) can be easily formed in the spheroid forming space (111). More specifically, when the cells of the cell suspension are provided in the spheroid forming space (111) and the cell culture medium is provided, a plurality of the cells can aggregate in the spheroid forming space (111) to form a single spheroid (10).

[0115] According to one embodiment, the spheroid forming space (111) may be curved downward in the opposite direction of the cell inlet (231).

[0116] As a result, the longer the time the spheroid (100) is formed in the downwardly curved spheroid forming space (111), the greater the roundness of the spheroid (100) along the downwardly curved shape of the spheroid forming space (111).

[0117] Furthermore, the longer the time during which the spheroid (100) is formed in the spheroid forming space (111), the harder the spheroid (100) can become.

[0118] Referring further to FIGS. 3 and 4, the plasma (30) having a concentration gradient can be provided to a plurality of the spheroids (10) (S150).

[0119] According to one embodiment, when the cell culture medium is injected through one of the injection ports (211a), the plasma (30) may be introduced through the other injection port (211b).

[0120] According to one embodiment, the plasma (30) may be the plasma (30) of a patient to whom the drug (dr) has been administered. For example, the drug (dr) may be colistin. More specifically, for example, the drug (dr) may be colistin having a concentration of 50 μg / mL or more and less than 100 μg / mL. However, it is not limited thereto.

[0121] According to an embodiment of the present application, the microfluidic device (1000) may have excellent sensitivity to a small amount of drug (dr). For example, the microfluidic device (1000) may detect toxicity at a concentration of 50 μg / mL or more and less than 100 μg / mL of the drug (dr), i.e., colistin.

[0122] In contrast, the sensitivity of conventional toxicity determination methods using two-dimensional cell culture could be reduced. More specifically, in conventional toxicity determination methods using two-dimensional cell culture, a high drug (dr) concentration of 100 μg / mL or higher could be required.

[0123] However, according to an embodiment of the present application, the microfluidic device (1000) has excellent sensitivity, so toxicity can be clearly determined even with the drug (dr) having a concentration of 50 μg / mL or more to less than 100 μg / mL.

[0124] According to one embodiment, the plasma (30) of the patient to whom the colistin has been administered may be obtained by a centrifugation method after blood is collected from the patient. Additionally, the obtained plasma may be stored at a sub-zero temperature before being provided to the microfluidic device (1000). For example, the plasma may be stored at -100°C or higher and -60°C or lower. More specifically, for example, the plasma may be stored at -80°C. However, it is not limited thereto.

[0125] According to one embodiment, in order to provide the plasma (30) having a concentration gradient to a plurality of the spheroids (10), the plasma (30) may be introduced through the storage space (311) of the storage layer (300) and, more specifically, through another injector (211b) of the fluid layer (200), and may flow into the branched flow path (221).

[0126] As a result, the above plasma (30) may have a concentration gradient.

[0127] According to one embodiment, the plasma (30) may have a concentration gradient formed in a number corresponding to the number of terminals of the branched flow path (221). More specifically, when the branched flow path (221) has m layers and n terminals, the concentration gradient of the plasma (30) may be formed in n ways. For example, referring to FIG. 4, when the branched flow path (221) has 3 layers and 5 (221h to 221l) terminals, the concentration gradient of the plasma (30) may be formed in 5 ways. However, it is not limited thereto.

[0128] Accordingly, according to the present application, there is a technical effect of determining the degree of toxicity for various concentrations of the plasma (30). Furthermore, according to the present application, since the toxicity evaluation according to various concentrations of the plasma (30) is performed as a single process rather than separately, there is also a simple and rapid technical effect. For example, the determination of toxicity of the plasma (30) can be performed quickly within 2 days.

[0129] Furthermore, according to an embodiment of the present application, since the degree of toxicity can be determined for various concentrations of the plasma (30), the timing of the onset of toxicity can be determined for each patient. For example, in the case of a patient in whom toxicity develops relatively early, the roundness may be altered in the spheroid (10) provided with the plasma (30) at a relatively low concentration, for example, 12.5%. In another example, in the case of a patient in whom toxicity develops relatively late, the roundness may be altered in the spheroid (10) provided with the plasma (30) at a relatively high concentration, for example, 87.5%. Or, in another example, in the case of a patient in whom toxicity does not develop, the roundness of the spheroid (10) may not be altered at any concentration of the plasma (30) in which the concentration gradient is formed.

[0130] In other words, according to an embodiment of the present application, the microfluidic device (1000) can determine the toxicity by reflecting the patient's characteristics, for example, the patient's individual metabolism and / or underlying disease.

[0131] Accordingly, according to an embodiment of the present application, a customized treatment strategy optimized for various patients can be provided through a single microfluidic device (1000).

[0132] Referring further to FIGS. 3 and 4, the roundness of a plurality of spheroids (10) provided with the plasma (30) is measured, and toxicity can be evaluated according to the roundness (S160).

[0133] According to one embodiment, when the plasma (30) to which the drug (dr) has been administered is provided to the spheroid (10), the roundness of the spheroid (10) may be modified depending on whether there is toxicity. More specifically, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be reduced. In other words, if toxicity is manifested in the plasma (30) to which the drug (dr) has been administered, the roundness of the spheroid (10) may be compromised.

[0134] As a result, according to the present invention, since the toxicity of the plasma (30) can be evaluated according to the roundness of the plurality of spheroids (10) provided, there may be an advantage that the staining and / or assay required in the toxicity evaluation of conventional cells can be omitted.

[0136] According to the embodiments of the present application described above, the toxicity evaluation method may include the steps of: forming a coating layer to reduce the adhesion of the cell to the microfluidic device (1000) (S110); diluting the cell to prepare the cell suspension (S120); introducing the cell suspension into the microfluidic device (1000) (S130); providing the cell culture medium to the cell suspension and forming a plurality of spheroids (10) from the cell (S140); providing the plasma (30) having a concentration gradient to the plurality of spheroids (10) (S150); and measuring the roundness of the plurality of spheroids (10) to which the plasma (30) has been provided, and evaluating toxicity according to the roundness (S160).

[0137] According to the toxicity evaluation method according to an embodiment of the present application, since the coating layer is formed inside the microfluidic device (1000), the adhesion force of the cell inside the microfluidic device (1000) can be lowered.

[0138] As a result, when the cell suspension in which the cell is diluted is introduced into the microfluidic device (1000) in which the cell's adhesion force is lowered, the cell can be easily transferred to the spheroid forming space (111) of the spheroid forming layer (100).

[0139] Accordingly, the spheroid (10) can be easily formed in the spheroid forming space (111).

[0140] In addition, according to the toxicity evaluation method according to the embodiment of the present application, the concentration of the cell suspension prepared by diluting the cells can be controlled.

[0141] As a result, the roundness and / or size of the spheroid (10) formed can be controlled.

[0142] Accordingly, the spheroid (10) having roundness and area and / or diameter suitable for toxicity evaluation can be formed, and toxicity evaluation using the spheroid (10) can be accurate and rapid.

[0143] In addition, according to the toxicity evaluation method according to an embodiment of the present application, while a plurality of spheroids (10) are formed in a plurality of spheroid forming spaces (111), the cell culture medium can be continuously injected through the injection port (211, see FIG. 1).

[0144] As a result, not only does the interior of the microfluidic device (1000) not dry out, but the cell culture medium can also be continuously supplied to the plurality of spheroid forming spaces (111).

[0145] Accordingly, a plurality of the spheroids (10) can be easily formed in the spheroid forming space (111). More specifically, when the cells of the cell suspension are provided in the spheroid forming space (111) and the cell culture medium is provided, a plurality of the cells can aggregate in the spheroid forming space (111) to form a single spheroid (10).

[0146] In addition, according to the toxicity evaluation method according to the embodiment of the present application, there is a technical effect of being able to determine the degree of toxicity for various concentrations of the plasma (30). Furthermore, according to the present application, since the toxicity evaluation according to various concentrations of the plasma (30) is performed as a single process rather than separately, there is also a simple and rapid technical effect. For example, the determination of toxicity of the plasma (30) can be performed quickly within 2 days.

[0147] Furthermore, according to an embodiment of the present application, since the degree of toxicity can be determined for each different concentration of the plasma (30), the time at which toxicity manifests for each patient can be determined. More specifically, according to an embodiment of the present application, the toxicity can be determined by reflecting the characteristics of the patient, for example, the patient's individual metabolism and / or underlying disease.

[0148] Accordingly, according to an embodiment of the present application, a customized treatment strategy optimized for various patients can be provided through a single microfluidic device (1000).

[0150] FIG. 5 is a drawing for explaining steps S210 and S220 of a method for manufacturing a microfluidic device according to an embodiment of the present application, FIG. 6 is a drawing for explaining steps S230 to S250 of a method for manufacturing a microfluidic device according to an embodiment of the present application, FIG. 7 is a drawing for explaining step S210 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail, FIG. 8 is a drawing for explaining step S220 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail, and FIG. 9 is a drawing for explaining step S230 of a method for manufacturing a microfluidic device according to an embodiment of the present application in more detail.

[0151] Referring to FIGS. 5 and 7, a first polymer material (50) is provided in a mold (500a) for forming the injection part (210) including the injection port (211) into which the cell culture medium or the plasma (30) is injected, the concentration gradient forming part (220) including the branched flow path (221) that forms a concentration gradient of the plasma (30) injected through the injection port (211), and the cell inlet part (230) including a plurality of cell inlet ports (231) into which the cell suspension diluted with the cell is introduced, and the fluid bed (200) can be formed (S210). The first polymer material (50) may be, for example, polydimethylsiloxane (PDMS). However, it is not limited thereto.

[0152] According to one embodiment, the mold (500a) may include a pattern (510a) for forming the injection port (211), the branched flow path (221), and the cell inlet (231) (S211).

[0153] According to one embodiment, the first polymer material (50) may be provided to the mold (500a) so as to cover the pattern (510a) of the mold (500a) (S212).

[0154] According to one embodiment, the first polymer material (50) provided to cover the pattern (510a) can be cured (cr) (S213).

[0155] According to one embodiment, the area formed by the pattern (510a) in the first polymer material (50) that has been cured (cr) is cut (ct), and the fluid layer (200) can be formed (S214).

[0156] Referring further to FIGS. 5 and 8, the first polymer material (50) is provided in a mold (500b) for forming a plurality of spheroid forming spaces (111) in which a plurality of spheroids (10) are formed from the cells of the cell suspension, and the spheroid forming layer (100) can be formed (S220). The first polymer material (50) may be, for example, polydimethylsiloxane (PDMS). However, it is not limited thereto.

[0157] According to one embodiment, the mold (500b) may include a pattern (510b) for forming a plurality of spheroid forming spaces (111) (S221).

[0158] According to one embodiment, the pattern (510b) may be formed so that the depth and / or width of the spheroid forming space (111) is adjusted. For example, the pattern (510b) may be formed so that the spheroid forming space (111) has a depth of 200 μm and / or a width of 400 μm. However, it is not limited thereto.

[0159] As a result, the size of the spheroid (10) formed in the spheroid forming space (111) can be controlled according to the depth and / or width of the spheroid forming space (111).

[0160] According to one embodiment, the first polymer material (50) may be provided to the mold (500b) so as to cover the pattern (510b) of the mold (500b) (S222).

[0161] According to one embodiment, the first polymer material (50) provided to cover the pattern (510b) can be cured (cr) (S223).

[0162] According to one embodiment, the outline of the first polymer material (50) that has been cured (cr) can be cut so that a plurality of spheroid forming spaces (111) formed in the first polymer material (50) that has been cured (cr) and a plurality of cell inlets (231) of the fluidized bed (200) are connected (S224).

[0163] According to one embodiment, the first polymer material (50) may be additionally provided in an area including a cylindrical shape (110) formed by the pattern (510b) in the first polymer material (50) in which the outline is cut (ct) and cured (cr) (S225).

[0164] According to one embodiment, when the area including the cylindrical shape (110) to which the first polymer material (50) is additionally provided is scraped with a scraper (sc) and the fluid layer (200) is stacked, a micro-well shape that is downwardly curved in the opposite direction of the cell inlet (231) may be formed (S226). The scraper (sc) may be, for example, a slide glass. However, it is not limited thereto.

[0165] According to one embodiment, the scratched area is hardened to form the spheroid forming space (111), and the spheroid forming layer (100) including the spheroid forming space (111) can be formed (S227).

[0166] Referring further to FIGS. 6 and FIGS. 9, the fluid layer (200) can be laminated on the spheroid forming layer (100) so that a plurality of the cell inlets (231) and a plurality of the spheroid forming spaces (111) are in communication (S230).

[0167] According to one embodiment, before the fluid layer (200) is laminated on the spheroid forming layer (100), the opposing surfaces of the spheroid forming layer (100) and the fluid layer (200) may each be surface treated (S231). For example, the surface treatment may be performed using oxygen plasma. However, it is not limited thereto.

[0168] As a result, the opposing surfaces of the spheroid forming layer (100) and the fluid layer (200) can each be activated.

[0169] According to one embodiment, the fluid layer (200) may be laminated on the surface-treated spheroid forming layer (100) (S232).

[0170] Since the opposing surfaces of the spheroid forming layer (100) and the fluid layer (200) are each activated, the stacking of the fluid layer (200) on the spheroid forming layer (100) can be easily done.

[0171] Referring further to FIG. 6, a second polymer material different from the first polymer material (50) is laser-processed to form the storage layer (300), which includes the storage space (311) in which the cell culture medium or plasma (30) is stored before being injected into the injection port (211) (S240). The second polymer material may be, for example, acrylic. However, it is not limited thereto.

[0172] Referring further to FIG. 6, the storage layer (300) can be stacked on the fluid layer (200) so that the storage space (311) and the injection port (211) are in communication (S250).

[0173] According to one embodiment, the fluid layer (200) and the storage layer (300) can be laminated through the first polymer material (50).

[0174] As a result, the fluid layer (200) and the storage layer (300) can be easily stacked through the first polymer material (50), which is the forming material of the fluid layer (200).

[0176] Specific experimental examples and characteristic evaluation results of the present application are described below.

[0178] FIG. 10 is a diagram illustrating the concentration gradient of plasma according to an experimental example of the present application.

[0179] Referring to FIG. 10, according to an experimental example of the present application, phosphate buffered saline (PBS) was provided in one of the storage spaces (311a), and a fluorescent substance was provided in place of the plasma (30) in the other storage space (311b).

[0180] Through FIG. 10, it can be seen that when the fluorescent material is provided to another storage space (311b), the fluorescent material flows through the branched channels (221: 221a~221l), and a concentration gradient is formed.

[0181] Thus, when the plasma (30) is provided to another storage space (311b), it can be predicted that a concentration gradient may be formed as the plasma (30) flows into the branched flow path (221l).

[0182] In the experimental example of the present application, the concentration gradient of the fluorescent substance was five, namely, 0%, 12.5%, 50%, 87.5%, and 100% from one of the storage spaces (311a) where the acid-buffered physiological saline (PBS) is stored to the other of the storage spaces (311b) where the plasma (30) is stored.

[0183] Through this, it can be seen that the concentration gradient of the plasma (30) can be formed in five ways, namely, from one storage space (311a) where the acid-buffered physiological saline (PBS) is stored to the other storage space (311b) where the plasma (30) is stored, at 0%, 12.5%, 50%, 87.5%, and 100%.

[0185] FIG. 11 is a photograph of a spheroid formed according to the concentration of a cell suspension according to an experimental example of the present application, FIG. 12 is a graph showing the area and roundness of a spheroid formed according to the concentration of a cell suspension according to an experimental example of the present application, and FIG. 13 is a graph showing the diameter and roundness of a spheroid formed according to the concentration of a cell suspension according to an experimental example of the present application.

[0186] Referring to FIGS. 11 to 13, 5 X 10 6 It can be seen that the spheroid (10) formed by providing the cell suspension having a concentration of cells / mL can have a roundness improved by more than 50 compared to 1 day based on 100 after 3 days, and can have a maximum roundness after 4 days.

[0187] Also, 5 X 10 6 The spheroid (10) formed by providing the cell suspension having a concentration of cells / mL is 1 X 10 6 When the above cell suspension having a concentration of cells / mL is provided and 1 X 10 7 It can be seen that the cell suspension having a concentration of cells / mL can have a larger area and / or diameter while maintaining the roundness compared to when the cell suspension is provided.

[0188] Also, 5 X 10 6 The spheroid (10) formed by providing the cell suspension having a concentration of cells / mL is 1 X 10 6It can be seen that when the above cell suspension having a concentration of cells / mL is provided, the formed spheroid has a diameter of 120 μm, which is larger than the diameter of 50 μm. Meanwhile, 1 X 10 7 It can be seen that when the cell suspension having a concentration of cells / mL is provided, the formed spheroids collapse, making it difficult to measure the diameter.

[0190] FIG. 14 is a photograph showing toxicity analysis using a microfluidic device manufactured according to the experimental example of the present application, FIG. 15 is a graph showing the amount of roundness and colistin when toxicity is analyzed using a microfluidic device manufactured according to the experimental example of the present application, FIG. 16 is a photograph showing toxicity analysis using a conventional two-dimensional cell culture microfluidic chip, and FIG. 17 is a graph showing the amount of roundness and colistin when toxicity is analyzed using a conventional two-dimensional cell culture microfluidic chip.

[0191] In particular, referring to FIG. 15, it can be seen that toxicity determination using a microfluidic device (1000) manufactured according to an experimental example of the present application is performed within 2 days.

[0192] Referring to FIGS. 14 to 17, it can be seen that the microfluidic device (1000) manufactured according to the experimental example of the present application reacts more sensitively at a lower concentration of colistin than a conventional two-dimensional cell culture microfluidic chip.

[0193] In addition, while a significant decrease in viability was observed at a concentration of 100 μg / mL or higher in conventional two-dimensional cell culture microfluidic chips, it can be seen that a significant decrease in viability was observed at a concentration of 50 μg / mL or higher in the microfluidic device (1000) manufactured according to the experimental example of the present application.

[0194] Thus, it can be seen that the microfluidic device (1000) manufactured according to the experimental example of the present application has the advantage of mimicking the sensitive interaction between cells in a three-dimensional structure while simultaneously responding sensitively to minute environmental changes.

[0195] Accordingly, when a microfluidic device (1000) manufactured according to the experimental example of the present application is used, it can be proven that toxicity can be effectively detected through high sensitivity even at low concentrations of colistin, thereby enabling accurate toxicity assessment.

[0197] FIG. 18 is a graph showing the viability of kidney cells using a microfluidic device manufactured according to an experimental example of the present application, and FIG. 19 is a graph showing the viability of kidney cells using a conventional two-dimensional cell culture microfluidic chip.

[0198] Referring to FIGS. 18 and 19, plasma from a healthy person, to whom colistin has not been administered, was simultaneously injected into a microfluidic device (1000) manufactured according to an experimental example of the present application and a conventional two-dimensional cell culture microfluidic device, and the occurrence of nephrotoxicity in the two environments was compared.

[0199] As shown in FIG. 18, in the microfluidic device (1000) manufactured according to the experimental example of the present application, it can be seen that no nephrotoxicity was observed in any lane within 2 days.

[0200] On the other hand, as shown in Fig. 19, in a conventional two-dimensional cell culture microfluidic chip, it can be seen that a significant decrease in survival rate was observed when the human plasma concentration was 50% or higher, even without the inclusion of colistin.

[0201] Accordingly, when using conventional two-dimensional cell culture microfluidic chips, it was not possible to distinguish between nephrotoxicity induced by a patient's plasma and nephrotoxicity induced by a healthy donor's plasma, so it may be difficult to determine whether the expressed renal toxicity was caused by the influence of a specific substance or by the toxicity of the plasma itself.

[0202] On the other hand, when using a microfluidic device (1000) manufactured according to the experimental example of the present application, since no renal toxicity was observed in the plasma of a healthy person, the factor of renal damage caused by plasma can be excluded, and there may be an advantage in being able to clearly evaluate whether renal toxicity is induced after colistin administration.

[0203] Accordingly, according to the present invention, plasma from a healthy person can be used as a control group that does not induce renal toxicity, and renal toxicity can be evaluated using the patient's plasma and an in vitro system.

[0205] FIG. 20 is a graph showing the spheroid survival rate of a first patient who developed nephrotoxicity at an early stage using a microfluidic device manufactured according to an experimental example of the present application, FIG. 21 is a graph showing the spheroid survival rate of a second patient who developed nephrotoxicity at a late stage using a microfluidic device manufactured according to an experimental example of the present application, and FIG. 22 is a graph showing the spheroid survival rate of a third patient who did not develop nephrotoxicity using a microfluidic device manufactured according to an experimental example of the present application.

[0206] Referring to FIGS. 20 to 22, toxicity determination using a microfluidic device (1000) manufactured according to the experimental example of the present application is performed within 2 days, and the time at which toxicity manifests can be determined for each patient, and the determination of the time at which toxicity manifests can be earlier than the time at which toxicity actually manifests for the patient.

[0207] Specifically, referring to FIG. 20, plasma from a first patient administered colistin was collected using a microfluidic device (1000) manufactured according to the experimental example of the present application to check for nephrotoxicity, and it was found that nephrotoxicity was observed in the third lane or higher after 2 days.

[0208] Referring to FIG. 21, plasma from a second patient administered colistin was collected using a microfluidic device (1000) manufactured according to the experimental example of the present application to check for nephrotoxicity, and it was found that nephrotoxicity was observed in the fifth lane or higher after 2 days. In the case of the second patient, nephrotoxicity was not present at the time of plasma collection, but substantially nephrotoxicity was present 5 days after the plasma was collected from the second patient. In other words, according to the experimental example of the present application, the onset of nephrotoxicity was predicted 3 days prior to the substantial onset of nephrotoxicity in the second patient.

[0209] Thus, according to the present application, it can be demonstrated that there is a technical effect of being able to predict the onset of nephrotoxicity in advance before the patient's nephrotoxicity substantially manifests.

[0210] Referring to FIG. 22, plasma from a third patient administered colistin was collected using a microfluidic device (1000) manufactured according to the experimental example of the present application to check for nephrotoxicity, and it was found that no nephrotoxicity was observed in all lanes.

[0211] Thus, by using the microfluidic device (1000) manufactured according to the experimental example of the present application, it can be seen in a simple manner that the first patient develops nephrotoxicity at a relatively early stage, the second patient develops nephrotoxicity at a relatively late stage, and the third patient does not develop nephrotoxicity.

[0212] That is, according to the embodiments of the present application, since the degree of toxicity can be determined for each different concentration of plasma, the time at which toxicity manifests can be determined for each patient.

[0213] In other words, according to an embodiment of the present application, the microfluidic device (1000) can determine the toxicity by reflecting the patient's characteristics, for example, the patient's individual metabolism and / or underlying disease.

[0215] Although the present application has been described in detail using preferred embodiments, the scope of the present application is not limited to specific embodiments and should be interpreted by the appended claims. Furthermore, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the present application. Explanation of the symbols

[0217] 10: Spheroid 30: Plasma 100: Spheroid cambium 111: Spheroid formation space 200: Fluidized bed 210: Injection part 211: Inlet 220: Concentration gradient forming part 221: Divided Euro 230: Cell inflow section 231: Cell inflow point 240: Discharge section 241: Outlet 251: Connecting Euro 300: Storage layer 311: Storage space

Claims

Claim 1 A fluid bed into which a cell culture medium or plasma is injected, a concentration gradient of the injected plasma is formed, and a cell suspension in which cells are diluted is introduced; a spheroid forming layer in which a plurality of spheroids are formed from the cells of the cell suspension; and a storage layer comprising a storage space for storing the cell culture medium or the plasma, wherein the fluid bed and the storage layer are sequentially stacked on the spheroid forming layer, and the cell suspension comprises 1 X 10 6 >10 cells / mL to 1 x 10⁶ 7 A microfluidic device comprising having a concentration of less than cells / mL. Claim 2 A microfluidic device according to claim 1, wherein the fluid bed comprises: an injection section including an inlet into which the cell culture medium or the plasma is injected; a concentration gradient forming section including a branched flow path forming a concentration gradient of the plasma injected through the inlet; and a cell inlet section including a plurality of cell inlets into which the cell suspension in which the cells are diluted is introduced, and the spheroid forming layer comprises a plurality of spheroid forming spaces that are in communication with the plurality of cell inlets and in which a plurality of spheroids are formed from the cells of the cell suspension. Claim 3 A microfluidic device according to claim 2, wherein the spheroid forming space comprises being curved downward in the direction opposite to the cell inlet. Claim 4 A microfluidic device according to claim 2, wherein the concentration gradient forming part forms a concentration gradient of the plasma corresponding to the number of terminal ends of the branched flow path, the plasma comprises the plasma of a patient administered colistin, and the colistin comprises having a concentration of 50 μg / mL or more to less than 100 μg / mL. Claim 5 A microfluidic device according to claim 2, wherein the storage space of the storage layer is in communication with the inlet and includes storing the cell culture medium or plasma before being injected into the inlet. Claim 6 A microfluidic device according to claim 2, wherein the fluid bed further comprises a connecting channel connecting the inlet and the branched channel; and a discharge section including an outlet through which the cell culture medium or the plasma is discharged. Claim 7 A microfluidic device according to claim 6, wherein the diameter of the outlet is larger than the diameter of the inlet. Claim 8 The method comprises the steps of: preparing a cell suspension by diluting cells; introducing the cell suspension into a microfluidic device; providing a cell culture medium to the cell suspension and forming a plurality of spheroids from the cells; providing plasma having a concentration gradient to the plurality of spheroids; and measuring the roundness of the plurality of spheroids provided with the plasma and evaluating toxicity according to the roundness, wherein the cell suspension is 1 X 10 6 >10 cells / mL to 1 x 10⁶ 7 A toxicity assessment method comprising having a concentration of less than cells / mL. Claim 9 A toxicity assessment method according to claim 8, wherein the determination of toxicity of the plasma is performed within 2 days, and includes determining the time at which toxicity manifests for each patient, and the determination of the time at which toxicity manifests includes preceding the time at which toxicity substantially manifests for the patient. Claim 10 A toxicity evaluation method according to claim 8, further comprising the step of forming a coating layer on the microfluidic device to reduce the adhesion of the cell. Claim 11 In claim 8, the cell comprises kidney cells, and the cell suspension is 5 x 10 6 A toxicity evaluation method comprising having a concentration of cells / mL, wherein the diameter of the spheroid is maximum at the said concentration, and the roundness is maximum after 4 days at the said concentration. Claim 12 A toxicity evaluation method according to claim 8, wherein the plasma comprises plasma from a patient administered colistin, and the colistin comprises having a concentration of 50 μg / mL or more to less than 100 μg / mL. Claim 13 The method comprises the steps of: providing a first polymer material to a mold for forming an injection section including an injector for injecting cell culture medium or plasma, a concentration gradient forming section including a branched flow path for forming a concentration gradient of the plasma injected through the injector, and a cell inlet section including a plurality of cell inlets for introducing a cell suspension in which cells are diluted, and forming a fluid layer; providing the first polymer material to a mold for forming a plurality of spheroid forming spaces in which a plurality of spheroids are formed from the cells of the cell suspension, and forming a spheroid forming layer; stacking the fluid layer on the spheroid forming layer so that the plurality of cell inlets and the plurality of spheroid forming spaces are in communication with each other; processing a second polymer material different from the first polymer material with a laser to form a storage layer including a storage space for storing the cell culture medium or plasma before being injected through the injector; and stacking the storage layer on the fluid layer so that the storage space and the injector are in communication with each other, wherein the cell suspension is 1 X 10 6 >10 cells / mL to 1 x 10⁶ 7 A method for manufacturing a microfluidic device comprising having a concentration of less than cells / mL. Claim 14 A method for manufacturing a microfluidic device according to claim 13, wherein the fluid layer and the storage layer are laminated through the first polymer material, the first polymer material is polydimethylsiloxane, and the second polymer material is acrylic. Claim 15 A method for manufacturing a microfluidic device according to claim 13, wherein the plasma comprises plasma from a patient to whom colistin has been administered, and the colistin comprises having a concentration of 50 μg / mL or more to less than 100 μg / mL.

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