WO2022145743A1 - Method for producing three-dimensional blood-brain barrier structure of blood-brain barrier organ-on-a-chip using reverse rapid liquid printing, and blood-brain barrier organ-on-a-chip comprising same - Google Patents

Abstract

The present invention relates to a blood-brain barrier organ-on-a-chip using reverse rapid liquid printing. A method for producing a blood-brain barrier organ-on-a-chip using reverse rapid liquid printing, according to the present invention, enables the mimicking of an actual three-dimensional blood-brain barrier, and a blood-brain barrier organ-on-a-chip produced thereby can be used as an artificial organ model which can be an alternative to animal testing.

Description

Method for manufacturing a three-dimensional blood-brain barrier structure using reverse liquid rapid printing and blood-brain barrier organ-on-a-chip comprising the same

The present invention relates to a method for manufacturing a three-dimensional blood-brain barrier organ-on-a-chip using reverse liquid rapid printing, and to a blood-brain barrier organ-on-a-chip comprising the structure manufactured therefrom.

The blood-brain barrier (BBB) is a strong biological barrier that selectively permeates only blood substances essential for maintaining brain function to maintain normal brain function and protect the brain from external substances. In the process of developing new drugs, the central nervous system drugs cannot be delivered to target cells due to the blood-brain barrier (BBB), so the focus has been on the strategy of passing drugs or drug carriers through the blood-brain barrier (BBB). A model that can accurately predict the interaction of (BBB) is essential. By grafting organ-on-a-chip technology for accurate modeling of the biological blood-brain barrier (BBB), it is possible to artificially fabricate micro-scale structures to create a more accurate tissue microenvironment. R&D was in progress.

The currently used hydrogel interface technology is a method of realizing a three-dimensional co-culture environment by forming a two-dimensional surface using a hydrogel and attaching cerebrovascular cells to the hydrogel surface. The function of the blood-brain barrier can be verified by monitoring mass transfer in the cell wall formed by the attachment of cells. However, the existing two-dimensional blood-brain barrier (BBB) chip is only partially exposed to the culture medium after drug injection due to the limitation of two-dimensional cross-sectional contact, and changes in cells are concentrated around the contact surface. In addition, it has limitations as a research platform because it is difficult to simulate the complex functions and structures of the blood-brain barrier (BBB), such as directional blood flow and three-dimensional interactions between cells.

Accordingly, in order to overcome the problems of the conventional two-dimensional blood-brain barrier (BBB) chip model, a 3D blood-brain barrier (BBB) chip that implements a 3D blood vessel shape in a chip is being developed. Such a 3D blood-brain barrier (BBB) chip implements an environment similar to the human body through blood vessels implemented in three dimensions and can similarly simulate the tissue characteristics of cells. In addition, by implementing the shear stress applied to the blood-brain barrier (BBB), physical stimuli in vivo can be simulated, and various cells constituting the blood-brain barrier (BBB) are simultaneously cultured to enable intercellular interactions. Thus, it has the advantage of being able to reproduce a function similar to the biological blood-brain barrier (BBB).

Recently, technology development for realizing a 3D blood-brain barrier (BBB) chip in a three-dimensional environment by grafting 3D printing technology is being actively developed. 3D printing is a technology that builds three-dimensional objects by stacking materials in a layer-by-layer manner based on three-dimensional design data. 3D printing does not require a separate mold for product production, As products can be produced as-is, customized small-volume production, which was difficult with the existing manufacturing method, is possible.

However, the current 3D printing technology has a slower production speed compared to existing additive manufacturing technologies such as injection molding, casting, and milling, so it is suitable for small component production, but has a limitation in that it is difficult to manufacture large-sized products. In addition, the available materials are limited and the quality of the finished product is lower than that of industrial materials, making it difficult to apply in practice.

In order to solve the above problems, various 3D printing methods have been developed. Fused Deposition Modeling (FDM) method is a method of manufacturing by laminating after melting filaments. It can print products with strong durability and strength, but the output speed is slow and the quality of the finished product is low because the surface is not smooth. Poly Jet method is a method of manufacturing by jetting a photocurable liquid material and curing the material with a UV lamp. Although it is possible to produce a smooth and detailed product, the material is limited and the durability is low. The Stereo Lithography Apparatus (SLA) method is a method of curing a liquid material using a laser, and it is very good to realize the shape of a sharp edge, but it is expensive to maintain and it is difficult to remove the support required in the printing process. The Selective Laser Sintering (SLS) method is a method of manufacturing powder material by scanning a Co2 laser, and various materials can be used, but it is difficult to use because the laser irradiation direction must be carefully set depending on the material. The Digital Light Processing (DLP) method directly projects the output image from the beam projector and outputs it. It has excellent precision, but the usable materials and output size are limited. Furthermore, since the 3D printing method can output polymer only through a nozzle in the air, and the photocurable UV irradiation method can output only layer by layer, it is difficult to implement a three-dimensional 3D structure.

Therefore, there is an urgent need for a new microfluidic device manufacturing technology capable of realizing a three-dimensional microchannel.

The present invention has been devised to solve the problems of the prior art as described above, and an object of the present invention is to fabricate a three-dimensional blood-brain barrier structure of a blood-brain barrier organ-on-a-chip based on reverse rapid liquid printing (RRLP) technology. We want to provide a way to do it.

Another object of the present invention is to provide a novel blood-brain barrier organ-on-a-chip capable of accurately realizing a 3D blood-brain barrier existing in a living body without animal testing.

The present invention provides a method for manufacturing a three-dimensional blood-brain barrier structure of a blood-brain barrier organ-on-a-chip using reverse liquid rapid printing, comprising the following steps.

(a) preparing a first gel solution containing a biocompatible polymer and a second gel solution containing an anionic polymer;

(b) positioning a nozzle for discharging so that the first gel solution is printed inside the second gel solution, and reverse rapid liquid printing of the first gel solution into a bath filled with the second gel solution printing) to form a microchannel by discharging;

(c) storing the microchannel-formed bath prepared in step (b) at a low temperature of 1 to 10° C. and solidifying it to prepare a three-dimensional structure having microchannels;

(d) curing the three-dimensional structure by reacting it with a crosslinking agent; and

(e) removing the first gel solution from the cured three-dimensional structure.

The biocompatible polymer is collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, Matrigel, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL) , polylactic acid (PLA), polyglycolic acid (PGA), poly[(lactic-co-(glycolic acid))(PLGA), poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), polydioxanone (PDO), poly[(L-lactide)-co-(caprolactone)], poly(ester urethane) (PEUU), poly[(L-lactide)-co-(D -lactide)], poly[ethylene-co-(vinyl alcohol)](PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline ( PAN) and mixtures thereof may be any one selected from the group consisting of.

The anionic polymer may be any one or more selected from the group consisting of hyaluronic acid, alginate, pectin, carrageenan, chondroitin sulfate and dextran sulfate.

The second gel solution may further include a natural polymer forming a hydrogel.

The natural polymer may be any one selected from the group consisting of collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, MatrIgel, and mixtures thereof.

The second gel solution is any one or more human-derived brain selected from the group consisting of neurons, neural stem cells, microglia, astrocytes, and pericytes. It may further include tissue cells.

The diameter size of the microchannel may be controlled by adjusting the flow rate of the first gel solution and the discharge speed of the nozzle in step (b).

When the flow rate of the first gel solution in step (b) is 0.05 to 0.1 ml/min, the ejection speed of the nozzle may be 0.3 to 0.5 mm/s.

In step (c), the crosslinking agent may be any one or more selected from the group consisting of calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄ ), and calcium carbonate (CaCO ₃ ).

After step (e), (f) injecting cerebrovascular endothelial cells through the microchannel of the three-dimensional blood-brain barrier structure to form a cerebrovascular endothelial cell membrane on the inner wall of the microchannel; may further include.

Before the step (f), (f') injecting Matrigel through the microchannel of the three-dimensional blood-brain barrier structure, attaching the Matrigel to the inner wall of the microchannel of the three-dimensional blood-brain barrier structure; It may further include.

The present invention includes: a body including a first plate and a second plate joined to the first plate, wherein a recessed groove is formed on an inner surface of the first plate and an inner surface of the second plate; a chamber formed in the body by the recessed groove by bonding the first plate and the second plate; a three-dimensional blood-brain barrier structure existing inside the chamber and including at least one microchannel in the hydrogel; The first plate includes a plurality of through-holes for supplying or discharging fluid, and the second plate has guide channels recessed to a predetermined depth so that each of the through-holes and the microchannel of the blood-brain barrier structure communicate with each other. It provides a brain-vascular barrier organ-on-a-chip, characterized in that it is configured.

The hydrogel of the three-dimensional blood-brain barrier structure may include brain tissue cells, and a cerebrovascular endothelial cell membrane may be formed on the inner wall of the microchannel to mimic the human brain blood vessel barrier (BBB).

The features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings. Prior to this, the terms or words used in the present specification and claims are conventional and should not be interpreted in a dictionary meaning, and the concept of the term is appropriately defined in order for the inventor to best describe his invention. It should be interpreted as meaning and concept consistent with the technical idea of the present invention based on the principle that it can be done.

The three-dimensional blood-brain barrier structure manufacturing method of the blood-brain barrier organ on a chip using reverse liquid rapid printing according to the present invention can not only simulate the actual three-dimensional blood-brain barrier, but also a test method that can replace the current animal experiment can be used as

In addition, the manufactured blood-brain barrier organ-on-a-chip can delicately fabricate the blood-brain barrier (BBB) into a three-dimensional structure using a small amount of material. Therefore, the present invention is expected to be usefully used for three-dimensional culture of cells.

1 is an exemplary view showing a manufacturing process of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention (the symbols in FIG. 1 are as follows. 1: first gel solution, 2: second gel) Solution, 3: Nozzle of the printing device and the direction of movement of the nozzle, 4: Bath, 5: Temperature control device).

2 is a diagram illustrating phase separation between a first gel solution and a second gel solution.

3 is an apparatus for printing microfluidic channels using a reverse rapid liquid printing method.

4 shows preferred conditions of the 3-axis micro stage, drive motor, and motor controller in reverse rapid liquid printing.

5 is a perspective view and assembly diagram of a blood-brain barrier organ-on-a-chip according to an embodiment of the present invention. Vascular barrier structure, 111,112,113,114: micro channel, 200: body, 210: first plate, 211,212: first and second inlet, 213,214: first, second outlet, 215: depression, 220: second plate, 221,222,223,224: Guide channel, 225: recessed groove, 230: chamber).

6 is a plan view of the main body 200 of the blood-brain barrier organ-on-a-chip according to an embodiment of the present invention. 210: first plate, 211,212: first, second inlet, 213,214: first, second outlet, 215: recessed groove, 220: second plate, 221,222,223,224: guide channel, 225: recessed groove, 226: fastening ring, 230: chamber).

7 is a perspective view and a cross-sectional view of a body of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention. : first plate, 211: first inlet, 214: first outlet, 220: second plate, 221,224: guide channel, 230: chamber).

8 is a perspective view of a three-dimensional blood-brain barrier structure 100 according to an embodiment of the present invention, and the bottom view of FIG. 8 is a three-dimensional blood-brain barrier structure 100' according to another embodiment of the present invention. A perspective view (the symbols in Fig. 8 indicate the following: 100, 100': three-dimensional blood-brain barrier structure, 110, 110': hydrogel, 111,111', 112, 112', 113, 113', 114, 114':micro channel).

9 is a cross-sectional view of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention (the symbols in FIG. 9 are as follows. 100: 3-dimensional blood-brain barrier structure, 110: hydrogel, 111: microchannel, 111a: cerebrovascular endothelial cell membrane, 111b: brain tissue cells).

10 is a photograph taken with an optical microscope after a colored liquid is injected into the three-dimensional blood-brain barrier structure prepared in Example 1. FIG.

11 is a photograph of fluorescence images of living cells and dead cells in a three-dimensional blood-brain barrier structure. Figure 11a is a photograph taken of a fluorescence image of green living cells labeled with calcein AM, Figure 11b is a photograph taken of a fluorescence image of a red dead cell labeled with PI, Figure 11c is a photograph of living cells and dead cells It is a photograph taken together with a fluorescence image of

12 is an image showing the cell viability in a three-dimensional blood-brain barrier structure.

13A is a photograph of a three-dimensional blood-brain barrier structure immediately after injection of a cerebrovascular endothelial cell (hCMEC/D3) suspension into a microchannel.

13B is a photograph of a three-dimensional blood-brain barrier structure after injecting a cerebrovascular endothelial cell (hCMEC/D3) suspension into a microchannel and culturing for 30 minutes.

FIG. 13c is a photograph of a three-dimensional blood-brain barrier structure after cerebrovascular endothelial cell (hCMEC/D3) suspension was injected into a microchannel, cultured for 30 minutes, and removed.

The objects, specific advantages and novel features of the present invention will become more apparent from the following detailed description and preferred embodiments taken in conjunction with the accompanying drawings. In the present specification, in adding reference numbers to the components of each drawing, it should be noted that only the same components are given the same number as possible even though they are indicated on different drawings. Also, terms such as first, second, etc. may be used to describe various components, but the components should not be limited by the terms. The above terms are used only for the purpose of distinguishing one component from another. In addition, in describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description thereof will be omitted.

One aspect of the present invention relates to a method for manufacturing a three-dimensional blood-brain barrier structure of a blood-brain barrier organ-on-a-chip using reverse liquid rapid printing, comprising the following steps.

(a) preparing a first gel solution containing a biocompatible polymer and a second gel solution containing an anionic polymer;

(b) positioning a nozzle for discharging so that the first gel solution is printed inside the second gel solution, and reverse rapid liquid printing of the first gel solution into a bath filled with the second gel solution printing) using the method of discharging;

(c) forming a three-dimensional structure in which microchannels are formed by impacting the solution discharged in step (b) at a low temperature of 4°C;

(d) curing the three-dimensional structure having microchannels formed in step (c) by reacting it with a CaCl2 solution; and

(e) comprising the step of processing the cured three-dimensional structure of step (d).

1 is an exemplary view showing a manufacturing process of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention (the symbols in FIG. 1 are as follows. 1: first gel solution, 2: second gel) Solution, 3: Nozzle of the printing device and the direction of movement of the nozzle, 4: Bath, 5: Temperature control device).

First, a first gel solution containing a water-soluble polymer is prepared (step (a)).

The first gel solution may include a biocompatible polymer, and the biocompatible polymer is not particularly limited as long as it is not crosslinked by a divalent cation crosslinking agent, and preferably, the biocompatible polymer is collagen or gelatin. , chitosan, fibrin, cellulose, dextran, agar, pullulan, Matrigel, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL), polylactic acid (PLA), polyglycolic acid ( PGA), poly[(lactic-co-(glycolic acid)) (PLGA), poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), polydioxanone (PDO), Poly[(L-lactide)-co-(caprolactone)], poly(ester urethane) (PEUU), poly[(L-lactide)-co-(D-lactide)], poly[ethylene-co -(vinyl alcohol)] (PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline (PAN) and mixtures thereof It may be any one, a copolymer of the two or more biocompatible polymers, or a mixture thereof.

More preferably, the biocompatible polymer may be any one selected from the group consisting of polyethylene glycol (PEG), polyethylene oxide (PEO), and mixtures thereof, and most preferably, the biocompatible polymer is polyethylene glycol (PEG), PEO (polyethylene oxide) is preferably a mixture. Most preferably, the first gel solution contains 1 to 2% by weight of PEG and 10 to 20% by weight of PEO.

If the content range of the water-soluble polymer included in the first gel solution is less than or exceeding the above range, the density may be greatly changed, so that phase separation of the first gel solution and the second gel solution does not occur.

The first gel solution may further include a solvent, and the solvent is not particularly limited as long as it is a solvent capable of dissolving the water-soluble polymer and ensuring biostability, and preferably distilled water or PBS buffer.

The second gel solution includes an anionic polymer, and preferably, the anionic polymer may be any one or more selected from the group consisting of hyaluronic acid, alginate, pectin, carrageenan, chondroitin sulfate and dextran sulfate, , most preferably alginate.

The second gel solution may further include a natural polymer forming a hydrogel in addition to the anionic polymer, wherein the natural polymer is collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, mart It may be any one selected from the group consisting of Ligel (Matrigel) and mixtures thereof. The second gel solution acts as a granule gel serving as a support.

The second gel solution is preferably a mixture of gelatin and alginate, and the second gel solution contains 5 to 10 wt% of gelatin and 1 to 5 wt% of alginate Most preferred. When the gelatin is less than the ratio, a problem occurs in that mechanical strength is lowered, and when the alginate is used in less than the ratio, the crosslinking rate of the formed hydrogel is lowered, thereby causing a problem in that it is difficult to maintain the shape at room temperature.

The second gel solution may further include a solvent, and the solvent is not particularly limited as long as it is a solvent capable of dissolving the water-soluble polymer and ensuring biostability, and preferably distilled water or PBS buffer, and most Preferably, the same solvent as that of the second gel solution may be used.

The second gel solution is any one or more human-derived brain selected from the group consisting of neurons, neural stem cells, microglia, astrocytes, and pericytes. It may further include tissue cells. The cells can be obtained and used from human brain tissue.

The cells may be used alone or a mixture of two or more cells may be used, and a spheroid form or an organoid form may be used, and the microchannel after being sufficiently cultured in a culture medium before being mixed with the second gel solution or encapsulated in a hydrogel structure It can be cultured by perfusion of the medium.

The neuron refers to a nerve cell as a unit constituting the nervous system, the neural stem cell is a cell having the ability to differentiate into a nervous system cell capable of self-renewal, and the microglia are glial cells of the central nervous system derived from the mesoderm, microglia As a cell, it acts as a phagocyte responsible for transport, destruction, and removal of substances in tissues, and has the function of supporting the tissues of the central nervous system as well as supplying substances necessary for nerve cells and creating a suitable environment. The astrocytes are one of the cells constituting the glial supporting the nervous tissue, also called astrocytes or astrocytes, have small cell bodies and protrusions that split in various directions, and serve to help structure and metabolism of neurons. In the perivascular cells, the capillary wall consists only of flat endothelial cells, and there is no clear smooth muscle layer or connective tissue layer surrounding the perivascular cells, and connective tissue cells appear sporadically in the basement membrane layer of the endothelial cells, which are called perivascular cells, It is known to be edible.

Since the human-derived brain tissue cells are cells existing in the brain tissue and exist three-dimensionally in the brain, the second gel is placed so that these brain tissue cells are placed on the outer wall of the microchannel of the three-dimensional structure so that the present invention can imitate it. mixed into the solution. Since the second gel solution contains an anionic polymer or natural polymer that can be implemented similarly to the extracellular matrix, human brain tissue cells can survive well and maintain their intrinsic activity.

Next, a nozzle for discharging is positioned so that the first gel solution is printed inside the second gel solution, and the first gel solution is reversed rapid liquid printing in a bath filled with the second gel solution. ) to form a microchannel by discharging it (step (b)).

The first gel solution and the second gel solution are separated from each other without mixing due to the density difference, and through this characteristic, a hydrogel complex composed of two different layers can be obtained ( FIG. 2 ).

The temperature condition of step (b) is not particularly limited as long as the second gel solution maintains a liquid state in a temperature range, but may be preferably 20 to 50 °C, more preferably 20 to 40 °C, more More preferably, it may be 30 to 40 °C.

3 is an apparatus for printing microfluidic channels using a reverse rapid liquid printing method.

For reverse rapid liquid printing, a 3-axis micro stage capable of implementing 3D printing, a driving motor, a motor controller, a micro nozzle, a syringe pump, and a temperature control means may be included. The micro-nozzle preferably has a diameter capable of making a channel of 10 to 1500 μm. The syringe pump may move the gel solution to the nozzle. In order to prevent curing of the first and second gel solutions and maintain them in a liquid state, a temperature control means is provided, and a hot plate may be used as the temperature control means. Preferred conditions of the three-axis microstage, the driving motor, and the motor controller are shown in FIG. 4 .

Using the above-described characteristics, a microchannel having a desired structure is printed by discharging the first gel solution into the second gel solution using a reverse rapid liquid printing method. Specifically, by positioning the nozzle for discharging so that the first gel solution is printed inside the second gel solution, the first gel solution is reversed rapid liquid in a bath filled with the second gel solution. printing) method, by printing the microchannel with a pattern designed by moving it by the desired shape and length of the microchannel, it is possible to form a three-dimensional three-dimensional structure.

In this case, the thickness of the microchannel may be adjusted by adjusting the flow rate and velocity of the first gel solution supplied during the printing. The discharge speed may also be referred to as a printing speed. Table 1 shows the flow rate (flow rate) of the supplied first gel solution, the ejection speed (velocity) of the nozzle, and the width (㎛) of the microchannel manufactured through the flow rate (velocity). As the flow rate of the supplied first gel solution increases, the thickness of the microchannel increases, and it can be seen that the thickness of the microchannel decreases as the discharge speed of the nozzle increases. A flow rate of the first gel solution supplied during the printing may be appropriately selected according to a desired thickness of the microchannel. Specifically, in order to obtain a microchannel having a width of 100 to 1500 μm, preferably, the flow rate of the first gel solution may be 0.05 to 0.1 ml/min. The nozzle velocity (Velocity) may also be appropriately selected according to the desired thickness of the microchannel. Specifically, in order to obtain a microchannel having a width of 100 to 1500 μm, it may be preferably 0.1 to 0.5 mm/s, and more preferably 0.3 to 0.5 mm/s. However, considering the width of the blood-brain barrier, it is preferable that the width of the microchannel is 100 to 500 μm. It is most preferable to satisfy 0.5 mm/s.

The second gel solution in the bath may be filled to a height of 2 to 10 mm, but is not limited thereto.

Flow rate (ml/min)	Velocity (mm/s)	Microchannel width (μm)
0.005	0.1	0
0.03	0.3	0
	0.4	0
	0.5	0
0.05	0.1	900
	0.2	760
	0.3	250
	0.4	180
	0.5	100
0.07	0.1	1000
	0.2	880
	0.3	400
	0.4	230
	0.5	150
0.1	0.1	1200
	0.2	920
	0.3	500
	0.4	300
	0.5	210

Next, the microchannel-formed bath prepared in step (b) is stored at a low temperature of 1 to 10° C. and solidified to prepare a microchannel-formed three-dimensional structure (step (c)). Since the hydrogel formed from the first gel solution is solidified under the conditions of 1 to 10 ° C, it is stored at 1 to 10 ° C. low temperature in order to fix the three-dimensional micro channel structure in the microchannel formed bath prepared in step (b). to solidify.

The low temperature may be in a temperature range of 1 to 10 °C, but is not limited thereto, preferably 1 to 5 °C, and most preferably 4 °C.

The three-dimensional structure in which the microchannel formed in step (c) is formed may be a blood-brain barrier (BBB, blood-brain barrier) structure. As the discharged first gel solution permeates into the second gel solution, the microchannels in the form of three-dimensional three-dimensional fibrous structures are formed by rapidly diffusing while phase-separating without being mixed with each other.

Next, the three-dimensional structure having microchannels formed in step (c) is cured by reacting it with a crosslinking agent (step (d)).

The crosslinking agent may be a divalent cation aqueous solution, and the divalent cation aqueous solution may be at least one selected from the group consisting of calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄ ), and calcium carbonate (CaCO ₃ ), limited thereto. it's not going to be

In step (d), only the three-dimensional structure excluding the microchannel may be cured. For the curing, physical cross-linking as well as chemical cross-linking can be used, but for stable curing, a cross-linking method using a cross-linking agent is practical. In addition, the crosslinking time and degree of crosslinking can be controlled by adjusting the ratio of the crosslinking agent. Step (d) may be performed at 20 to 30 °C.

Finally, the first gel solution is removed from the cured three-dimensional structure of step (d) (step (e)). Since the first gel solution printed with the microchannel exists in a liquid state at room temperature, it can be removed by perfusing a phosphate buffer solution (PBS) through the three-dimensional structure.

Specifically, the first gel solution was removed by connecting a silicone tube to both ends of the three-dimensional structure and perfusion with phosphate buffer solution (PBS) at room temperature. The first gel solution was removed from the three-dimensional structure.

Through the above-described process, a microchannel of an empty space through which a fluid can flow is formed in the three-dimensional structure, and the fluid can be injected into the microchannel through both end portions of the three-dimensional structure.

It may further include processing the cured three-dimensional structure before the step (e). The cured three-dimensional structure may be processed into a desired shape to make a block of a required size. The processing method can be used without particular limitation as long as it is a method widely known in the art.

After step (e), washing with tertiary distilled water may be further included. This is to remove phosphate buffer solution (PBS) and other unreacted residual substances as solids after drying.

After the step (e), (f) injecting cerebrovascular endothelial cells through the microchannel of the three-dimensional blood-brain barrier structure may further include the step of forming a cerebrovascular endothelial cell membrane on the inner wall of the microchannel.

In the present invention, as the brain endothelial cells, vascular endothelial cells isolated from human brain microvascular endothelial cells may be used.

Before the step (f), Matrigel is injected through the microchannel of the three-dimensional blood-brain barrier structure so that the cerebrovascular endothelial cells can be well attached and stabilized on the inner wall of the microchannel, It may further include the step of attaching the matrigel to the microchannel inner wall of the three-dimensional blood-brain barrier structure.

That is, the present inventors have confirmed that a complex blood-brain barrier structure and a blood-brain barrier model can be manufactured using the different hydrogel solutions through a reverse rapid liquid printing method. In addition, it was confirmed that a three-dimensional blood-brain barrier structure can be implemented by including brain tissue cells and cerebrovascular endothelial cells, respectively, on the outside and inner walls of the microchannel of the blood-brain barrier structure.

As another aspect of the present invention, the present invention provides a blood-brain barrier organ-on-a-chip.

Hereinafter, a preferred embodiment of the present invention will be described in detail with reference to the accompanying drawings. 5 is a perspective view and an assembly view of a blood-brain barrier organ-on-a-chip according to an embodiment of the present invention. 6 is a plan view of the blood-brain barrier organ-on-a-chip according to an embodiment of the present invention. 7 is a perspective view and a cross-sectional view of a body of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention. 8 is a perspective view of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention, and the bottom view of FIG. 8 is a perspective view of a three-dimensional blood-brain barrier structure according to another embodiment of the present invention. 9 is a cross-sectional view of a three-dimensional blood-brain barrier structure according to an embodiment of the present invention.

The present inventors have tried to develop an artificial tissue model having a microchannel network similar to the structure of the blood-brain barrier, and thus have completed the present invention. The brain-vascular barrier organ-on-a-chip of the present invention is a three-dimensional microstructure that mimics brain tissue, and is configured to observe the interaction between brain tissue cells and vascular endothelial cells in the blood-brain barrier (BBB). is implemented.

The blood-brain barrier (BBB) is a capillary structure of the brain, and unlike general capillaries, endothelial cells are tightly coupled, so small molecules can be delivered to the brain through active transport, but large molecules have low permeability. For this reason, in order to develop brain-related and central nervous system-related drugs, the ability to penetrate the blood-brain barrier is required first. The blood-brain barrier organ-on-a-chip of the present invention is an organ chip that mimics the capillary structure of the brain, and the interaction between cells in the blood-brain barrier (BBB) can be observed.

Specifically, the blood-brain barrier organ-on-a-chip 10 of the present invention is a three-dimensional structure 100 formed while forming a capillary structure like the blood-brain barrier without brain endothelial cells and brain tissue cells mixed with each other, artificial tissue It is maintained in a stable and fixed state within the chip. The blood-brain barrier organ-on-a-chip 10 of the present invention can be used for drug efficacy tests and other harmful substances tests in addition to cell mechanism studies by simulating a living tissue that is a multi-structured blood-brain barrier structure. The form of the blood-brain barrier organ-on-a-chip 10 is not limited as long as it has a three-dimensional shape simulating a living tissue, for example, a blood-brain tissue.

The blood-brain barrier organ-on-a-chip 10 according to an embodiment of the present invention includes a first plate 210 and a second plate 220 bonded to the first plate 210, the first a body 200 in which recessed grooves 215 and 225 are formed on the inner surface of the plate 210 and the inner surface of the second plate 220; a chamber 230 formed in the body 200 by the recessed grooves 215 and 225 by bonding the first plate 210 and the second plate 220 to each other; The three-dimensional blood-brain barrier structure 100 is present in the chamber 230 and includes at least one microchannel (111, 112, 113, 114) in the hydrogel 110; The first plate 210 includes a plurality of through- holes 211, 212, 213, and 214 for supplying or discharging fluid, and the second plate 220 has each of the through-holes and micro-organisms of the blood-brain barrier structure. Each of the guide channels 221 , 222 , 223 and 224 formed by being recessed to a predetermined depth so that the channels communicate with each other are configured.

At the lower end of the through holes 211 , 212 , 213 , and 214 of the first plate 210 , a channel extending to the recessed grooves 215 and 225 is formed in combination with the guide channel of the second plate 220 . A recessed channel recessed by the can be additionally formed.

The shape of the blood-brain barrier organ-on-a-chip 10 of the present invention is not limited as long as it has a three-dimensional structure, and may specifically be a rectangular parallelepiped shape. The size of the blood-brain barrier organ-on-a-chip 10 is not particularly limited as long as it can be manufactured according to the manufacturing method of the present invention, but preferably has a width of 10-500 mm, 10-450 mm, 10-400 mm, 10-350 mm, 10-300 mm, 10-250 mm, 10-200 mm, 10-150 mm, 10-100 mm, 15-100 mm, 20-100 mm, 25-100 mm, 30-100 mm, 40 to 100 mm, 45 to 100 mm, 50 to 100 mm, 55 to 100 mm, 60 to 100 mm, 65 to 100 mm or 65 to 80 mm. In addition, the length of the blood-brain barrier organ on a chip 10 is 10-500 mm, 10-450 mm, 10-400 mm, 10-350 mm, 10-300 mm, 10-250 mm, 10-200 mm, 10- 150 mm, 10-100 mm, 10-50 mm, 20-50 mm, 25-50 mm, 30-50 mm, 35-50 mm or 35-45 mm

The blood-brain barrier organ-on-a-chip 10 of the present invention comprises a first plate 210 , a second plate 220 , a chamber 230 formed by the first and second plates, and a three-dimensional brain provided in the chamber. It may include a blood vessel barrier structure 100 .

The first plate 210 and the second plate 220 are not particularly limited as long as they are transparent materials, and transparent ceramics including glass, polydimethylsiloxane (PDMS: PolyDiMethylSiloxane), ecoflex, polystyrene ( polystyrene, general purpose polystyrene, polymethylmethacrylate, polyethylene terephthalate, polyester methacrylate, polypropylene, polycarbonate, Polyurethane, High impact polystyrene, Acrylonitnle butadiene styrene, Polyester, Polyamides, Polyethylene, Polytetrafluoroethylene ), polyethylene tereketone (Polyetheretherketone), acrylics (acrylics), amorphous polymers (amorphous polymers) and may be any one selected from the group consisting of polyethylene terephthalate (polyethylene terephthalate), preferably polydimethylsiloxane (PDMS) : PolyDiMethylSiloxane), polystyrene, polyurethane, polycarbonate, acrylonitrile butadiene styrene, polyethylene terketone (Polyetheretherketone) and polyethylene (Polyethylene) selected from the group consisting of It can be any one.

The first plate 210 and the second plate 220 may be manufactured through mold injection molding.

5 , 6 and 7 , the first plate 210 and the second plate 220 are bonded to each other to form the body 200 .

A recessed groove 215 into which a three-dimensional blood-brain barrier structure can be inserted is provided on the inner surface of the first plate 210 , and the recessed groove 215 of the first plate 210 is provided in the second plate 220 . ) and a recessed groove 225 of the same size corresponding to that, as the first plate 210 and the second plate 220 are joined, the recessed groove 215 of the first plate 210 and the second The recessed grooves 225 of the two plates 220 are engaged with each other to form a chamber 230 having an empty space.

The three-dimensional blood-brain barrier structure 100 may be inserted and coupled to the inside of the chamber 230, and the shape and size of the chamber 230 is the three-dimensional blood-brain barrier structure 100 to prevent fluid leakage. ), which is exactly the same as

5, 6 and 7, the first plate 210 includes a plurality of through- holes 211, 212, 213, and 214 for supplying or discharging the fluid. The through hole is provided to correspond to the microchannels 111 , 112 , 113 and 114 of the three-dimensional blood-brain barrier structure 100 .

According to one embodiment of the present invention, the first plate 210 is provided with at least two inlet (211, 212) and at least two outlet (213, 214), for convenience of description, at least two inlet and outlet is divided into "first inlet 211", "second inlet 212", "first outlet 213", and "second outlet 214".

The guide channels 221 , 222 , 223 , and 224 are provided on the same plane of the second plate 220 , and for convenience of description, “first guide channel 221” and “second guide channel 222” , "third guide channel 223" and "fourth guide channel 224" will be divided and described.

The first and second inlets 211 and 212 and the first and second outlets 213 and 214 are interconnected by microchannels 111, 112, 113, and 114 of the three-dimensional blood-brain barrier structure, and the The first and second injection holes 211 and 212 and the microchannels 111, 112, 113 and 114 are connected by the first and second guide channels 221 and 222 provided in the second plate 220, The microchannels 111 , 112 , 113 and 114 and the first and second outlets 213 and 214 are connected by third and fourth guide channels 223 and 224 .

The guide channels 221 , 222 , 223 and 224 inject the fluid introduced from the first and second inlets 211 , 212 into the microchannel, or the fluid discharged from the microchannel 111 , 112 , 113 , and 114 . It is a passage connected to move to the first and second outlets 213 and 214 .

When the fluid is injected into the blood-brain barrier organ-on-a-chip of the present invention through the first inlet 211 and the second inlet 212 , the three-dimensional blood-brain barrier is passed through the first guide channel 221 and the second guide channel 222 . The fluid is supplied to the microchannels 111, 112, 113, and 114 of the structure, respectively, and the fluid that has passed through the microchannels 111, 112, 113, 114 passes through the third and fourth guide channels 223 and 224. It is emitted to the first and second outlets 213 and 214 and can simulate the microvascular structure flowing between brain tissues of the human body. For this reason, the present invention is a morphological/genetic change model that mimics the flow of blood flow due to the fluid supplied to the three-dimensional blood-brain barrier structure, and the molecular biological changes that may appear in each cell are closer to the human body. can be formed and observed.

At the lower end of the through holes 211 , 212 , 213 , and 214 of the first plate 210 , a channel extending to the recessed grooves 215 and 225 is formed in combination with the guide channel of the second plate 220 . A recessed channel recessed by the can be additionally formed.

A fastening ring ( 226) may be further included.

The fastening ring 226 has a structure in which the entrance is gradually narrowed in the direction of the microchannels 111, 112, 113, and 114, and is inserted into the microchannel and fastened to minimize leakage at the connection part.

The three-dimensional blood-brain barrier structure 100 is inserted and seated inside the chamber 230, and may include at least one or more microchannels 111, 112, 113, and 114 in the hydrogel 110. have.

The size of the three-dimensional blood-brain barrier structure 100 is not particularly limited as long as it can mimic the blood-brain barrier structure, and specifically, the size of the cross-section may be 10 to 100 mm × 10 to 50 mm, preferably It may be 50 mm × 30 mm.

In addition, the three-dimensional blood-brain barrier structure 100 is not particularly limited as long as it has a three-dimensional shape that mimics the structure of the blood-brain barrier, but specifically may be a rectangular parallelepiped shape. The three-dimensional blood-brain barrier structure 100 has a plurality of micro-channels 111, 112, 113, and 114 penetrating the three-dimensional blood-brain barrier structure 100, and the micro-channels 111, 112, 113 , 114) is not limited as long as it has a three-dimensional structure, and specifically, each of the microchannels 111, 112, 113, and 114 may be the same as or different from each other. In addition, the microchannels 111 , 112 , 113 , and 114 may have a cylindrical shape having an empty space so that a fluid can flow therein. In addition, there may be a membrane formed by attaching cerebrovascular endothelial tissue to the inner wall of the microchannels 111 , 112 , 113 and 114 .

In the present invention, 'attachment' means injecting a formulation containing cerebrovascular endothelial cells into the blood-brain barrier organ-on-a-chip 10, and the microchannels 111, 112, 113, 114) means attaching the cerebrovascular endothelial cells to the inner wall, the attachment may be culturing the cerebrovascular endothelial cells, and the culture may be performed for 5 to 60 minutes, specifically 5 to 50 minutes, 5 It may be carried out for 40 minutes, 10 to 40 minutes, 15 to 40 minutes, 20 to 40 minutes or 25 to 35 minutes, and the temperature conditions of the incubation are 10 to 45 ° C, 20 to 45 ° C, 25 to 45 ° C, 30 to 45 °C, 30 to 40 °C, or 32 to 35 °C.

The hydrogel 110 mimics the surrounding tissue of the blood-brain barrier, and may be composed of a hydrogel including brain tissue cells, and preferably the hydrogel is an anionic that can be cross-linked by a divalent cation material. It may be composed of a composite material including a polymer. Specifically, it may include any one or more selected from the group consisting of hyaluronic acid, alginate, pectin, carrageenan, chondroitin sulfate and dextran sulfate, but is not particularly limited thereto.

The hydrogel may further include a natural polymer capable of forming a hydrogel in addition to the anionic polymer, and the natural polymer is collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, mart It may be any one selected from the group consisting of Ligel (MatrIgel) and mixtures thereof.

Referring to FIG. 9 , the hydrogel 110 includes brain tissue cells 111b to be similar to the surrounding tissue of the blood-brain barrier, and the brain tissue cells include neurons, neural stem cells, and microscopic It may be any one or more selected from the group consisting of glial cells (microglia), astrocytes (astrocyte) and peripheral cells (pericyte).

The diameters of the microchannels 111, 112, 113, and 114 may be the same as or different from each other, and preferably, the diameter of each microchannel may be formed in a channel structure having a diameter of 1 to 1500 μm, more preferably 10 to 1500 μm, 10 to 1000 μm, 10 to 600 μm, and 10 to 500 μm.

The microchannels 111, 112, 113, and 114 are formed to penetrate the three-dimensional blood-brain barrier structure, and a membrane including cerebrovascular endothelial cells is formed on the inner wall of the microchannel.

8 is a view for explaining the micro-channel arrangement of the three-dimensional blood-brain barrier structure 100. Referring to the upper drawing of FIG. 8 , the microchannels 111 , 112 , 113 , and 114 according to the present invention may have a three-dimensional network structure similar to that of a brain microvessel penetrating the three-dimensional blood-brain barrier structure 100 . Referring to the lower drawing of FIG. 8 according to another embodiment of the present invention, the microchannels 111', 112', 113', and 114' according to the present invention have a cylindrical shape penetrating the three-dimensional blood-brain barrier structure 100'. can be a structure of

The blood-brain barrier organ-on-a-chip of the present invention can be applied to drug delivery experiments. For example, the drug delivery test apparatus may include an organ-on-a-chip of the blood-brain barrier of the present invention, a microfluidic valve, a screening apparatus, and the like.

The microfluidic valve is a valve that can selectively control the flow of fluid in a chamber of a three-dimensional environment, and can control the inflow and discharge of fluid to and from the blood-brain barrier organ-on-a-chip of the present invention. Here, the fluid may be a candidate material for treating cerebrovascular barrier disorders and central nervous system diseases.

The screening device can monitor the effect of a drug solution in real time using electrochemical measurement and liquid chromatography mass spectrometry (LC-MS).

A drug delivery experiment using such a drug delivery test device may be performed as follows. First, a candidate substance is injected into the brain-vascular barrier organ-on-a-chip using a microfluidic valve, the discharged solution is collected, and the concentration of the candidate substance can be analyzed using a screening device. When a drug delivery experiment is performed using the blood-brain barrier organ-on-a-chip of the present invention, it is possible to solve the ethical problem of animal experiments and conduct drug delivery experiments at low cost through the blood-brain barrier (BBB) having the same biological environment as the animal model. have.

The blood-brain barrier disorder is due to a central nervous system disease, but is not limited thereto. The central nervous system disease is any one disease selected from the group consisting of spinal cord injury, stroke, cerebral infarction, cerebral ischemia, Alzheimer's disease, and multiple sclerosis, but is not limited thereto.

The present invention is a new artificial organ model that can relatively accurately predict the interaction of cells related to brain-vascular barrier-related disorders and central nervous system diseases in vivo without performing animal tests, and can replace existing animal experiments.

Although the technical idea of the present invention has been specifically described according to the above preferred embodiments, it should be noted that the above-described embodiments are for the purpose of explanation and not for the limitation thereof. In addition, those skilled in the art will understand that various implementations are possible within the scope of the technical idea of the present invention.

Example 1. Preparation of an organ-on-a-chip for the blood-brain barrier

As an embodiment of the present invention, the following process was performed to manufacture the blood-brain barrier organ-on-a-chip.

1) Culture of brain tissue cells (human astrocytes, human brain vascular pericytes)

The day before incubation, poly-L-lysinen at a concentration of 2 μg/cm 2 was coated on a 75T Flask, the coated vessel was washed with sterile distilled water, 15 ml complete culture solution and 1 × 10 ⁶ cells were dispensed (seeding), Incubated at 37 °C. The culture medium was changed every 2-3 days, and the astrocyte layer was washed twice with DPBS after 12-14 days. Then, 5 ml of 0.05% trypsin-EDTA and 5 ml of DPBS were added to the flask and reacted in a 37 ℃ CO ₂ incubator. . Then, when the cells fall out of the flask, add TNS solution and transfer the detached cells to a 50 ml tube. Into the tube, 5 ml of a culture solution together with the cells was centrifuged at 180 x g for 5 minutes. After removing the supernatant, 10 ml of fresh culture solution was added to prepare a cell suspension having a concentration of 1 × 10 ⁶ cell/ml.

2) Manufacture of blood-brain barrier organ-on-a-chip

First, 10% by weight of gelatin, 2% by weight of alginate, human astrocytes suspension (1 × 10 ⁶ cell/ml), human brain vascular pericytes suspension (1 × 10 ⁶ cell/ml) and the remaining amount of distilled water to prepare a second gel solution, 17.5 wt% of polyethylene glycol, 1.095 wt% of polyethylene oxide and the remaining amount of distilled water A first gel solution was prepared by mixing.

The second gel solution was poured into a bath equipped with a temperature control chamber, and a device capable of printing microfluidic channels using the reverse liquid rapid printing method of FIG. 5 was installed. The nozzle of the printing device was inserted so that the first gel solution was printed inside the second gel solution. Then, the first gel solution was discharged from the nozzle (flow rate 0.07 ml/min, discharge rate 0.3 mm/s) to print microchannels having a diameter of 400 μm. At this time, the temperature of the bath was maintained at 37 °C, and the first gel solution was maintained at 25 °C. Then, the microchannel formed bath was stored at 4 °C to solidify. After the solidified block was brought out to room temperature, an aqueous solution of CaCl ₂ was added to harden it. In the curing step, the microchannel of the second gel solution was not cured. Then, distilled water or PBS was perfused into the microchannel to remove the first gel solution to prepare a three-dimensional blood-brain barrier structure. The three-dimensional blood-brain barrier structure was processed into a size of 50 mm × 30 mm (width × length) to prepare a three-dimensional blood-brain barrier structure in the form of a cuboid.

In order to confirm the formation of microchannels in the three-dimensional blood-brain barrier structure prepared above, a colored liquid (rhodamine, Trypan blue) was injected into the three-dimensional blood-brain barrier structure and photographed with an optical microscope. 10 is a photograph taken with an optical microscope after a colored liquid is injected into the three-dimensional blood-brain barrier structure prepared in Example 1. FIG.

As shown in FIG. 10 , it was confirmed that the colored liquid in the three-dimensional blood-brain barrier structure was successfully perfused into the microchannel, and it was confirmed that the microchannel was well formed in a desired shape without breaking, clogging, or leaking.

The prepared three-dimensional blood-brain barrier structure was stained with calcein AM and photographed using a confocal microscope (LSM 700, ZEISS). Through this, when astrocytes (human astrocytes) and human brain vascular pericytes were co-cultured inside a three-dimensional blood-brain barrier structure, it was attempted to obtain fluorescence images of living and dead cells.

11 is a photograph of fluorescence images of living and dead cells in a three-dimensional blood-brain barrier structure, FIG. 11a is a photograph of fluorescence images of green living cells labeled with calcein AM, and FIG. 11b is a PI It is a photograph taken of the fluorescence image of the red dead cells marked with , and FIG. 11c is a photograph taken together of the fluorescence images of the living cells and the dead cells.

12 is a photograph of a three-dimensional blood-brain barrier structure taken simultaneously in the x, y, and z-axis directions using a confocal microscope and reconstructed into a three-dimensional image. According to this, it can be confirmed that the cell viability is very high throughout the three-dimensional blood-brain barrier structure.

Example 2. Blood-brain barrier organ on a chip

A three-dimensional blood-brain barrier structure was prepared in the same manner as in Example 1. In the microchannel of the three-dimensional blood-brain barrier structure, a 3 mg/mL solution of Matrigel was injected instead of a culture solution to coat the inner wall of the microchannel with Matrigel, and the brain at a concentration of 1 × 10 ⁶ cell/ml A vascular endothelial cell (hCMEC/D3) suspension was injected. In order for the cells to adhere well to the inner wall of the microchannel, both ends of the microchannel were blocked and incubated for 30 minutes. At this time, immediately after the injection of the cell suspension, each of the three-dimensional blood-brain barrier structures that had undergone the process of incubation for 30 minutes were photographed using a microscope, and the photographed photographs are shown in FIGS. 13A to 13C .

13A is a photograph of a three-dimensional blood-brain barrier structure immediately after injecting a cerebrovascular endothelial cell (hCMEC/D3) suspension into a microchannel. The cerebrovascular endothelial cells do not adhere to the inner wall of the microchannel and maintain a circular state. it can be seen that

13b is a photograph of a three-dimensional blood-brain barrier structure after injecting a cerebrovascular endothelial cell (hCMEC/D3) suspension into a microchannel and culturing for 30 minutes. was confirmed.

13c is a photograph of a three-dimensional blood-brain barrier structure after injecting a cerebrovascular endothelial cell (hCMEC/D3) suspension into a microchannel, culturing for 30 minutes, and removing it. It was confirmed that it was growing and forming a single film.

Claims (13)

1. (a) preparing a first gel solution containing a biocompatible polymer and a second gel solution containing an anionic polymer;

   (b) positioning a nozzle for discharging so that the first gel solution is printed inside the second gel solution, and reverse rapid liquid printing of the first gel solution into a bath filled with the second gel solution printing) to form a microchannel by discharging;

   (c) storing the microchannel-formed bath prepared in step (b) at a low temperature of 1 to 10° C. and solidifying it to prepare a three-dimensional structure having microchannels;

   (d) curing the three-dimensional structure by reacting it with a crosslinking agent; and

   (e) removing the first gel solution from the cured three-dimensional structure; a method for manufacturing a three-dimensional blood-brain barrier structure of the brain blood barrier organ on a chip using reverse liquid rapid printing comprising a.
2. According to claim 1,

   The biocompatible polymer is collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, Matrigel, polyethylene glycol (PEG), polyethylene oxide (PEO), polycaprolactone (PCL) , polylactic acid (PLA), polyglycolic acid (PGA), poly[(lactic-co-(glycolic acid))(PLGA), poly[(3-hydroxybutyrate)-co-(3-hydroxyvalerate) (PHBV), polydioxanone (PDO), poly[(L-lactide)-co-(caprolactone)], poly(ester urethane) (PEUU), poly[(L-lactide)-co-(D -lactide)], poly[ethylene-co-(vinyl alcohol)](PVOH), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyaniline ( PAN) and a mixture thereof.
3. According to claim 1,

   The anionic polymer is hyaluronic acid, alginate (Alginate), pectin, carrageenan, chondroitin sulfate, and a three-dimensional blood-brain barrier structure of the brain blood vessel barrier organ on a chip, characterized in that any one or more selected from the group consisting of dextran sulfate manufacturing method.
4. The method of claim 1,

   The second gel solution further comprises a natural polymer that forms a hydrogel.
5. 5. The method of claim 4,

   The natural polymer is any one selected from the group consisting of collagen, gelatin, chitosan, fibrin, cellulose, dextran, agar, pullulan, MatrIgel, and mixtures thereof. A method for manufacturing a three-dimensional blood-brain barrier structure of an organ on a chip.
6. The method of claim 1,

   The second gel solution is any one or more human-derived brain selected from the group consisting of neurons, neural stem cells, microglia, astrocytes, and pericytes. A three-dimensional blood-brain barrier structure manufacturing method of the blood-brain barrier organ on a chip, characterized in that it further comprises tissue cells.
7. According to claim 1,

   In the step (b), the three-dimensional blood-brain barrier structure manufacturing method of the blood-brain barrier organ on a chip, characterized in that the diameter size of the microchannel is controlled by adjusting the flow rate of the first gel solution and the ejection speed of the nozzle.
8. According to claim 1,

   When the flow rate of the first gel solution in step (b) is 0.05 to 0.1 ml/min, the ejection speed of the nozzle is 0.3 to 0.5 mm/s A method for manufacturing a barrier structure.
9. According to claim 1,

   In step (c), the crosslinking agent is calcium chloride (CaCl ₂ ), calcium sulfate (CaSO ₄ ), and calcium carbonate (CaCO ₃ ) The three-dimensional brain of the blood-brain barrier organ on a chip, characterized in that at least one selected from the group consisting of A method for manufacturing a blood vessel barrier structure.
10. According to claim 1,

    After step (e), (f) injecting cerebrovascular endothelial cells through the microchannel of the three-dimensional blood-brain barrier structure to form a cerebrovascular endothelial cell membrane on the inner wall of the microchannel; characterized by further comprising A method for manufacturing a three-dimensional blood-brain barrier structure of an organ-on-a-chip.
11. 11. The method of claim 10,

    Before step (f),

    (f') injecting matrigel through the microchannel of the three-dimensional blood-brain barrier structure, and attaching the matrigel to the inner wall of the microchannel of the three-dimensional blood-brain barrier structure; characterized by further comprising A method for manufacturing a three-dimensional blood-brain barrier structure of an organ-on-a-chip.
12. a body including a first plate and a second plate joined to the first plate, wherein a recessed groove is formed on an inner surface of the first plate and an inner surface of the second plate;

    a chamber formed in the body by the recessed groove by bonding the first plate and the second plate;

    a three-dimensional blood-brain barrier structure existing inside the chamber and including at least one microchannel in the hydrogel;

    The first plate includes a plurality of through-holes for supplying or discharging fluid, and the second plate has guide channels recessed to a predetermined depth so that each of the through-holes and the microchannel of the blood-brain barrier structure communicate with each other. Blood-brain barrier organ-on-a-chip, characterized in that it consists of.
13. 13. The method of claim 12,

    The hydrogel of the three-dimensional blood-brain barrier structure includes brain tissue cells, and a cerebrovascular endothelial cell membrane is formed on the inner wall of the micro-channel to mimic the human brain blood-vessel barrier (BBB). on a chip.

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