KR101870948B1

KR101870948B1 - Method of fabricating 3D biomimetic tissue scaffold

Info

Publication number: KR101870948B1
Application number: KR1020160019373A
Authority: KR
Inventors: 성종환; 심규영; 이동욱; 염지원; 최낙원; 김소현; 박성수
Original assignee: 홍익대학교 산학협력단; 한국과학기술연구원; 성균관대학교산학협력단
Priority date: 2016-02-18
Filing date: 2016-02-18
Publication date: 2018-06-26
Also published as: KR20170097838A

Abstract

A three-dimensional organ model structure manufacturing method and a three-dimensional organ model structure are disclosed, wherein the method of manufacturing a three-dimensional organ model structure comprises the steps of: fabricating a first unremarkable mold having a first indented shape; Forming a first relief mold having a first embossed shape corresponding to the first relief shape, preparing a second relief mold having a second relief shape corresponding to the first relief shape using the relief mold, And forming an organ model structure having a second relief shape corresponding to the second relief pattern using the second relief mold.

Description

TECHNICAL FIELD [0001] The present invention relates to a method of fabricating a 3D biomimetic tissue scaffold,

The present invention relates to a method for producing a structure in which living organs are three-dimensionally simulated, and a three-dimensional organs simulated structure produced thereby.

For food or drug development, previous clinical trials and clinical trials are the steps to predict and evaluate the hazard and efficacy of the ingredients. Although animal or human cells can be used to predict adverse effects and efficacy prior to animal testing and clinical testing, cell culture models are not predictive enough to eventually require animal testing and clinical trials, It takes a lot of development time.

Regarding the improvement of the cell culture model, it is reported that when cells are cultured in a cell culture scaffold similar to the tissue structure of a human body without culturing the cells in a general culture dish, cell activity and functionality are improved . Accordingly, there is an increasing need to develop a three-dimensional organs-like structure capable of culturing cells corresponding to a three-dimensional tissue structure.

The background technology of the present application is disclosed in Korean Patent Laid-Open Publication No. 2015-0126520 (Application No. 2014-0053585).

It is an object of the present invention to solve the problems of the prior art described above, and it is an object of the present invention to provide a method and apparatus for reproducing a tissue structure of a complex organs three-dimensionally, Dimensional long-term simulation structure and a three-dimensional long-term simulation structure that can accurately reproduce the same.

It should be understood, however, that the technical scope of the embodiments of the present invention is not limited to the above-described technical problems, and other technical problems may exist.

According to an aspect of the present invention, there is provided a method of manufacturing a three-dimensional organ model structure, including: (a) fabricating a first negative-type mold having a first indentation; (b) fabricating a positive embossed mold having a first relief shape corresponding to the first relief pattern using the first relief mold; (c) fabricating a second negative-tone mold having a second engraved shape corresponding to the first embossed shape using the positive mold; And (d) fabricating an organ model structure having a second relief shape corresponding to the second relief pattern using the second relief mold.

As a technical means for accomplishing the above technical object, the three-dimensional organ model structure according to one embodiment of the present invention is a three-dimensional organ model structure structure manufactured by the method of manufacturing a three-dimensional organ model structure according to one embodiment of the present invention .

The above-described task solution is merely exemplary and should not be construed as limiting the present disclosure. In addition to the exemplary embodiments described above, there may be additional embodiments in the drawings and the detailed description of the invention.

According to the above-mentioned task solution of the present invention, it is possible to reproduce the tissue structure of a complex organs three-dimensionally more accurately than in the prior art, and also to reproduce the above-mentioned accurate reproducibility through a cell-friendly material such as a hydrogel having soft properties Simulation) can be made possible. In this regard, it has been reported that the function of cells is improved by simply culturing the cells in a simple three-dimensional form. According to the task solution of the present invention, three-dimensional A long-term simulated structure can be provided, the physiological similarity can be further increased, and the function of the actual tissue can be reproduced with higher reproducibility.

In addition, according to the above-described task solution of the present invention, a three-dimensional organs-like structure capable of maintaining high compatibility with existing experimental techniques and experimental instruments can be manufactured. For example, an observable level of a three-dimensional organs mimetic structure can be produced by a general microscope used for cell observation.

In addition, according to the present invention, it is possible to use the screening platform for testing the efficacy and side effects of drugs, cosmetics, and food ingredients. For example, the three-dimensional organ model structure manufactured by the task solution of the present application can be utilized as an artificial field model for evaluating the water absorption rate of food ingredients. In addition, the three-dimensional organs simulated by the method of the present invention can be developed into an experimental model of a specific disease and utilized in medical research. For example, a three-dimensional organs-like structure produced by the task solution of the present invention can be manufactured into a model of intestinal inflammation disease and can be utilized in the development of therapeutic agents and the study of disease mechanisms. The three-dimensional organs simulated by the method of the present invention can be utilized for the development of customized therapeutic agents. For example, a disease model reflecting the genetic characteristics of an individual can be developed using a three-dimensional organ model structure manufactured by the task solution of the present invention, and thus an individual-optimized therapeutic agent and treatment method can be tested.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an overall flow diagram of a method for fabricating a three dimensional organ model structure according to one embodiment of the present application.
2 is a detailed flowchart of one implementation of step S110 of the method for fabricating a three-dimensional organ model structure according to one embodiment of the present application.
3 is a conceptual diagram for explaining an embodiment of step S110 of the method of manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
4 is a conceptual diagram for explaining an embodiment of step S120 of the method for fabricating a 3D long-term simulation structure according to an embodiment of the present invention.
5 is a detailed flowchart of a first embodiment of step S130 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
FIGS. 6 and 7 are conceptual diagrams for explaining a first embodiment of step S130 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
FIG. 8 is a detailed flowchart of a first embodiment of step S140 of the method for manufacturing a three-dimensional organ model structure according to one embodiment of the present application.
9 is a conceptual diagram for explaining a first embodiment of step S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
10 is a detailed flowchart of a second embodiment of the step S130 of the method for manufacturing a three-dimensional organ model structure according to the embodiment of the present application.
11 is a conceptual diagram for explaining a second embodiment of the step S130 of the method of manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
FIG. 12 is a schematic perspective view for explaining an example of a stamp unit used in a step S130 and a step S140 of the method of manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
13 is a detailed flowchart of a second embodiment of the step S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
FIGS. 14 and 15 are conceptual diagrams for explaining a second embodiment of step S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.
FIG. 16 is a conceptual diagram for explaining one function of the master unit of the stamp unit used in the steps S130 and S140 of the method of manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily carry out the present invention. It should be understood, however, that the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. In the drawings, the same reference numbers are used throughout the specification to refer to the same or like parts.

Throughout this specification, when a part is referred to as being "connected" to another part, it is not limited to a case where it is "directly connected" but also includes the case where it is "electrically connected" do.

It will be appreciated that throughout the specification it will be understood that when a member is located on another member "top", "top", "under", "bottom" But also the case where there is another member between the two members as well as the case where they are in contact with each other.

Throughout this specification, when an element is referred to as "including " an element, it is understood that the element may include other elements as well, without departing from the other elements unless specifically stated otherwise.

The present invention relates to a method for producing a three-dimensional synovial structure and a three-dimensional synovial structure produced by the method. More specifically, the present application can be used to produce cell culture supports (three-dimensional organs) that are similar to those of human organs using cell-friendly materials (such as hydrogels such as gelatin hydrogel, collagen, etc.) .

The present invention can be applied to a method of constructing a structure in which villus existing on the intestinal wall is three-dimensionally simulated, but the present invention is not limited thereto. For example, according to the present application, other organ structures other than the intestinal lining structure can be produced. In addition to the above-mentioned hydrogel or collagen, a method of manufacturing a three-dimensional organ model structure using another material (another kind of hydrogel) may also be included in the present invention.

In addition, in the present invention, in order to fabricate a hydrogel for cell culture with a desired three-dimensional microstructure (for example, a size of several tens of micrometers), a semiconductor fine processing, a replica molding, a stamping method, And the like can be used in combination. In other words, the above processes can be combined in various ways in consideration of the shape and precision of the tissue to be manufactured by the present invention, material properties, cost, time, and the like.

According to the present invention, unlike the conventional method in which only a simple microstructure such as a right-angled shape can be realized, a three-dimensional long-term structure having various curves and aspect ratios can be manufactured and provided in a micrometer- have.

Hereinafter, a method for manufacturing a three-dimensional organ model structure according to one embodiment of the present invention (hereinafter referred to as a " method for manufacturing a three-dimensional organ model structure ") will be described.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is an overall flow diagram of a method for fabricating a three dimensional organ model structure according to one embodiment of the present application.

Referring to FIG. 1, the method of manufacturing a three-dimensional organ model structure (S100) includes steps S110 to S140.

Step S110 is a step of manufacturing the first negative-type mold 1 in which the first engraved shape 11 is formed. Here, the first negative-type mold 1 may be an engraved SU-8 mold. Step S120 is a step of manufacturing the positive embossed mold 2 having the first embossed shape 21 corresponding to the first embossed shape 11 by using the first negative embossing mold 1. [ Step S130 is a step of manufacturing the second negative-tone mold 3 in which the second engraved shape 31 corresponding to the first relief shape 21 is formed by using the positive-relief mold 2. Step S140 is a step of manufacturing an organ model structure 4 formed with a second relief shape 41 corresponding to the second relief shape 31 using the second relief mold 3.

FIG. 2 is a detailed flow chart of one embodiment of the step S110 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention, FIG. 3 is a flowchart Fig. 8 is a conceptual diagram for explaining an embodiment. Fig.

Referring to FIG. 3 (c), the first indentation 11 may include a groove or a hole in the form of a three-dimensional solid having a width varying in depth. In step S110, And may be formed by a photolithography process. Specifically, in step S110, the three-dimensional solid shape may be formed by at least one of overexposure (first embodiment) and photorefractive (second embodiment) exposure.

Here, the three-dimensional solid shape is preferably understood as a concept opposite to a cylindrical shape having a constant width (diameter) along the depth direction. That is, the first indentation 11 may be formed in one of a cylindrical shape having a constant width along the depth direction and a three-dimensional shape having a width varying along the depth direction, or a combination of two or more thereof have.

Referring to FIGS. 2 and 3, step S110 of manufacturing the first negative-type mold 1 in which the first engraved shape 11 is formed may include steps S111 to S113.

Step S111 is a step of forming a photoresist layer 13 on the base member 12 (see Fig. 3 (a)).

In step S112, a photomask 14 having a pattern 14a corresponding to the first intaglio 11 is disposed on the photoresist layer 13 and exposed to expose the photoresist layer 13 to the pattern 14a ) (See Fig. 3 (b)).

In this case, step S112 is a first embodiment (and an exposure method) in which the three-dimensional solid shape is formed, wherein a groove or a hole-like shape 3 having a width varying in depth by overexposure to the photoresist layer 13 The cross-linking corresponding to the three-dimensional shape can be performed. Here, overexposure means that exposure is performed longer than a predetermined exposure time to expose an area wider than the original area, and as shown by a thin dotted line in (b) of FIG. 3, Lt; / RTI >

Specifically, the first embodiment (and the exposure method) of step S112 is a method of making concave structures (see FIGS. 3B and 3C) by overexposing UV. For example, SU-8 2150 is applied to a base member 12 such as a wafer, the spin coater speed is adjusted to 500 rpm for 10 seconds and 1000 rpm for 60 seconds, and spin coating ) To make the height of SU-8 about 600μm. Thereafter, the base member 12 coated with SU-8 is placed on a 65 ° C hot plate for 30 minutes and then placed on a 95 ° C hot plate for 2 hours to soft bake . When the soft bake process is completed, photolithography is performed using MA6 Aligner such as MA6 Aligner II (4 inch only). The conditions of MA6 Aligner may be soft contact, Gap 100 μm, exposure energy 350 Mj / cm2. When the UV is overexposure for 35 to 40 seconds, the SU-8 is over-crosslinked to form a concave structure (a structure corresponding to a groove or a hole-like three-dimensional solid shape varying in width depending on the depth) (See Fig. 3 (b)).

On the other hand, the step S112 is a second embodiment (exposure method using light refraction) in which the three-dimensional solid shape is formed. By arranging and exposing a diffuser for guiding light refraction on the photomask 14, It is possible to perform crosslinking corresponding to a three-dimensional solid shape of a groove or a hole shape whose width changes according to the depth.

Specifically, the second embodiment of the step S112 (the exposure method using light refraction) is a method using an optical diffuser. For example, SU-850 is applied to a base member 12 such as a wafer and the spin coater speed is set to 500 rpm for 5 seconds and 1000 rpm for 30 seconds. Then, spin coating ) To make the height of the SU-8 about 300 μm. Thereafter, the base member 12 coated with SU-8 is placed on a 65 ° C hot plate for 10 minutes, and then placed on a 95 ° C hot plate for 30 minutes to soft bake . When the soft bake process is completed, photolithography is performed using MA6 Aligner such as MA6 Aligner II (4 inch only). The conditions of MA6 Aligner may be soft contact, Gap 100μm, exposure energy 350Mj / cm2. When a diffuser (for example, an Opal glass diffuser) is placed on the photomask 14 and exposed to UV for 20 seconds, the SU-8 is recessed due to the refraction phenomenon (groove or hole type (Corresponding to a three-dimensional solid shape)) (see Fig. 3 (b)).

The distance between the photomask 14 and the photoresist layer 13 (SU-8 substrate) and the intensity of ultraviolet light are controlled to produce a refraction of light so that the photoresist layer 13 is gradually It is possible to realize a three-dimensional shape of an engraved shape having a curved cross-section rather than a columnar shape having a general vertical inner peripheral surface.

Subsequently, SU-8 may be laminated one or more times on the crosslinked SU-8, if necessary, by repeating the above-described method. For example, when the UV is irradiated for 20 seconds after removing a diffuser (for example, an Opal glass diffuser), the three-dimensional solid shape may be formed into a three-dimensional solid shape having a constant width depending on the depth . At this time, it is preferable to draw a reference line in the photomask 14 in order to align (align) the sputtered SU-8 with the further deposited SU-8.

As described above, in steps S111 and S112, a groove or a hole-like three-dimensional solid shape varying in width according to the depth and a three-dimensional shape having no width variation according to the depth are connected to form a first indentation shape . &Lt; / RTI >

Referring to FIGS. 2 and 3, after the photolithography process is performed according to the first embodiment or the second embodiment of the step S112, the photoresist layer 13 is developed to remove the non-crosslinked portion, It is possible to proceed to step S113 of forming the first negative-type mold 1 having the recess 11 (see Fig. 3 (c)).

Illustratively, in step S113, PEB (Post Exposure Bake) was performed at 65 ° C for 5 minutes and at 95 ° C for 10 minutes, and developed using SU-8 Developer to remove unbridged SU-8 (Negative first angle mold) of the concave structure (a structure corresponding to a groove or a hole-like three-dimensional solid shape whose width changes according to the depth) can be manufactured.

Through the steps S111 to S113, the first negative-type mold 1 having the first indentation 11 corresponding to the three-dimensional solid shape can be manufactured.

On the other hand, the step S110 performed by the photolithography process described above can be replaced as necessary with the step of forming the first negative-type mold 1 having the first indentation 11 by using a laser. The step S110 of fabricating the first negative-tone mold 1 using a laser will be described as an example. First, a uniform size hole is made in the PMMA sheet using a UV laser (such as a UV laser micromachining system Resonetics Maestro 1000). At this time, the intensity of the laser may be 50 mJ, the pulse rate may be 75 pps (pulse per second), and the pulse number may be 1100, in which case the hole may be formed at a depth of about 0.5 mm . Illustratively, the distance between each of the holes may be 0.2 mm and the density may be 25 / mm < 2 >. With the use of such a laser, the first intaglio mold 1 in the form of a PMMA negative-embossed mold having a desired size and shape of the plastic can be manufactured by controlling the intensity of the laser and the frequency of irradiation.

4 is a conceptual diagram for explaining an embodiment of step S120 of the method for fabricating a 3D long-term simulation structure according to an embodiment of the present invention.

4, in step S120, the positive mold 2 pours and hardens a silicon solution (for example, PDMS) on the first negative mold 1 (see FIG. 4 (a)), (See Fig. 4 (b)) from the mold 1 as shown in Fig.

4, the Sylgard 184 A and B solutions are mixed at a ratio of 10: 1, and the mixed PDMS (Sylgard solution) is applied to the first negative-tone mold 1 (for example, an engraved SU- 0.0 > PMMA < / RTI > mold). Next, the pressure in the vacuum chamber is controlled to allow the PDMS to penetrate into the first negative-tone mold 12, and then the first negative-tone mold 12 in which the PDMS is poured is cured at 60 ° C for 6 hours or more. Next, the solidified embossed PDMS mold is separated from the first negative embossing mold 12 to obtain the embossed mold 2 (see Fig. 4 (b)).

Hereinafter, the first embodiment of steps S130 and S140, and the second embodiment of steps S130 and S140 will be described. The first embodiment may be a method of dissolving and separating a second negative mold (intermediate mold) from the manufactured long term simulation structure, and the second embodiment may be a method of manufacturing the long term simulation structure through stamping Method).

FIG. 5 is a detailed flow chart of a first embodiment of the step S 130 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention, and FIGS. 6 and 7 are cross- And is a conceptual diagram for explaining the first embodiment of step S130.

5 to 7, step S130 of manufacturing the second negative-tone mold 3 in which the second engraved shape 31 corresponding to the first positive-angle shape 21 is formed by using the positive- As one implementation, it may include steps S131 to S133.

6A, the frame including the bottom frame 22, the middle frame 23, and the top frame 24 is assembled before the step S131, and the frame corresponding to the second negative-tone mold 3 The material is injected into the frame. More specifically, for example, prior to step S131, 3.5% Alginate (in DI water, w / v) is injected into the frame as shown in FIG. 6A.

Next, as shown in Fig. 6 (b), the bottom frame 22 is detached.

Referring to FIG. 7A, in step S131, the cover gasket 32 is arranged so that the hole 32a formed in the cover gasket 32 surrounds the first relief shape 21 of the relief mold 2 .

7 (a), the embossed mold 2 is seated on the embossed mold seating portion 25 formed by the material (for example, Alginate) injected into the frame, and the embossed mold 2 The cover gasket 32 can be disposed so as to surround the first embossed shape 21 of the gasket.

7B, in step S132, the hole 32a of the cover gasket 32 is filled with a material corresponding to the second negative-tone mold 3, and the membrane 33 and the perforated plate 34 are filled with the material corresponding to the second negative- And the material corresponding to the second negative-tone mold 3 is crosslinked by supplying the crosslinking solution to the perforated plate 34 so that the second negative-tone mold 3 is inserted into the hole 32a of the cover gasket 32 .

More specifically, for example, a polycarbonate membrane (8 μm / 25 mm) is covered on the second negative-tone mold 3, and then the aluminum (perforated plate) having the hole is covered. Next, a 60 mM CaCl2 solution is sufficiently sprayed on the cover gasket 32 to crosslink Alginate (the material corresponding to the second negative-tone mold). At this time, the upper gasket 35 as shown in FIG. 7 (b) can serve as a reservoir in which the crosslinking solution stays for a predetermined time.

For reference, alginate has been mainly described as a material corresponding to the second negative-type mold (which may be referred to as an intermediate mold), but the present invention is not limited thereto. That is, it is possible to use a variety of gels if the gel can be melted under a physiological condition that is not harmful to the cells as the second negative mold (intermediate mold). For example, it is also possible to cross-link with agarose instead of alginate, then melt the cross-linked agarose gel by raising the temperature. Alternatively, it is also possible to cross-link a hydrogel such as collagen or gelatin, and then selectively melt through an enzyme to remove it. As a material corresponding to the second negative angle mold, a material such as collagen, gelatin, and agarose may be used. In the case of using a material other than alginate, Depending on the material, the method of crosslinking and melting afterwards may be different.

As understood from the material description of the second negative-angle mold (middle mold), the main purpose of the second negative-angle mold is to have a soft material such as hydrogel when separating the object (long-term simulation structure) from the second negative- So that the object can be separated in a direction to maintain the shape of the designed tissue structure perfectly without being deformed or broken. That is, as described above, the second negative-angle mold may be provided with a material (for example, a hydrogel such as alginate) that can be melted and separated to maintain the structure of the object as it is.

Next, referring to FIG. 7C, step S133 is a step S133 of separating the cover gasket 32 from the positive mold 2 together with the second negative-tone mold 3. By separating the cover gasket 32 and the second negative-tone mold 3 from the positive mold 2 in this way, it is possible to manufacture the long-term simulation structure 4 by using this in the first embodiment of the step S140 to be described later.

FIG. 8 is a detailed flowchart of a first embodiment of step S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention. FIG. 1 is a conceptual diagram for explaining a first embodiment.

8 and 9, step S140 of fabricating the organ model structure 4 in which the second relief shape 41 corresponding to the second intaglio shape 31 is formed using the second negative mold 3 As a first embodiment, steps S141 and S142 may be included.

Step S141 is a step of injecting and crosslinking the material corresponding to the long term simulation structure 4 on the second negative-tone mold 3 provided in the hole of the cover gasket 32 (see Fig. 9 (a)).

Specifically, for example, prepare DMEM, 1N NaOH and 8.23% collagen solution on ice, add 500 μl of 8.23% collagen, 50 μl of DMEM and 20 μl of 1N NaOH to the E-tube, give. The mixed collagen is injected flatly into a second negative mold (alginate mold) using a pipette. The mold injected with collagen solution is crosslinked in an incubator at 37 ° C for 20 to 30 minutes. 0.1% glutaraldehyde (in PBS, v / v) was sprayed onto the crosslinked collagen structure, followed by crosslinking for 3 hours at 37 ° C in an incubator.

Step S142 is a step of dissolving and removing the second negative-tone mold 3 (see Fig. 9 (b)).

Specifically, for example, when a crosslinked collagen structure is treated in a solution of 50 mM EDTA (in DI water, w / v) for 10 hours or more, the second negative mold (intermediate mold) Only the collagen structure remains. This collagen structure is treated in 7% L-glutamic acid (in 1N HCl, w / v) for 6 hours or more and washed three times a day with PBS. The collagen structure remaining after dissolving and removing the second negative-tone mold 3 is an example of the final organ model structure 4.

FIG. 10 is a detailed flowchart of a second embodiment of the step S130 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention. FIG. 12 is a conceptual diagram for explaining a second embodiment of the present invention. FIG. 12 shows an example of the stamp unit used in the steps S130 and S140 of the method for fabricating a three-dimensional organ model structure according to an embodiment of the present invention. And is a schematic three-dimensional diagram for explaining.

Referring to FIGS. 10 and 11, step S130 may include steps S131a and S132a as a second embodiment. For reference, the first embodiment of the above-described step S130 is a method using the second negative mold (intermediate mold) combined with the cover gasket, while the second embodiment of the step S130 uses the second negative mold Intermediate mold) can be used.

Step S131a is a step of pouring and hardening the silicone solution on the relief mold 2 and separating the relief mold 2 from the relief mold 2 to manufacture the second relief mold 3 (see FIGS. 11A and 11B).

Step S131a may be a step of manufacturing an engraved PDMS mold using an embossed PDMS mold. Illustratively, referring to FIG. 11 (a), Sylgard 184 A and B solution are mixed at a ratio of 10: 1, and then the mixed PDMS (Sylgard solution) is poured into the embossing mold 2. Next, the pressure in the vacuum chamber is controlled so that the PDMS can penetrate into the positive mold 2, and then the positive mold 2 in which the PDMS is poured is cured at 60 DEG C for 6 hours or more. Next, referring to FIG. 11 (b), a solid intaglio PDMS mold can be separated from the positive mold 2 to obtain the second negative mold 3.

More specifically, a relief PDMS is placed in a vacuum chamber, a predetermined amount of trichlorosilane is dropped on the slide glass, and the glass is put into a vacuum chamber. And a predetermined time is waited while a predetermined vacuum or higher is maintained by the pressure pump. When trichlorosilane coating is applied to the embossed PDMS mold, the intaglio PDMS can be well separated even after the PDMS is poured and hardened on the embossed PDMS mold

Step S132a is a step S132a of attaching the second negative-tone mold 3 to the lower end of the stamp unit 36 (see Fig. 11 (c)).

Referring to FIG. 12, the stamp unit 36 used in the second embodiment of the step S130 is formed of a "Corning 3470 Transwell polyester membrane cell culture inserts" (described later) to minimize the difficulty of the molding process Corresponding to the well unit 37, hereinafter referred to as " Transwell 3470 "). For example, the stamp unit 36 may be provided by machining 17 mm x 17 mm x 18 mm duralumin to the designed dimensions. Next, referring to FIG. 11 (c), the second negative-tone mold 3 such as a relief PDMS mold may be cut to a predetermined size and fixed to the lower end of the stamp unit 36 with an adhesive material 36c.

The height and detailed configuration of the stamp unit 36 will be described later in more detail with reference to the second embodiment of the step S140.

FIG. 13 is a detailed flowchart of a second embodiment of step S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention. FIGS. 14 and 15 are views showing a method of manufacturing a three-dimensional organ model structure according to one embodiment of the present invention And is a conceptual diagram for explaining a second embodiment of the step S140. FIG. 16 is a conceptual diagram for explaining one function of the master unit of the stamp unit used in the steps S130 and S140 of the method for manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.

Referring to FIGS. 13 to 15, step S140 may include steps S141a to S143a as a second embodiment.

The step S141a is a step of filling the hollow portion of the well-shaped unit 37 (insert well) having the upper opening therein with a material corresponding to the organ model structure 4 (see Fig. 14 (a)).

Illustratively, a stamp unit 36 with a second negative-tone mold 3, such as a relief PDMS mold, is inserted into the hollow portion of the well-shaped unit 37 (Transwell 3470) (Transwell 3470), and the stamp unit 36 is separated from the well unit 37 (Transwell 3470) in accordance with the inserting position of the stamp unit 36 in the well unit 37 (Transwell 3470). At this time, the lubricating oil treatment may be performed on the second negative-tone mold 3 portion of the stamp unit 36. Next, a material corresponding to the organ model structure 4 (such as a collagen solution) is filled into the well-shaped unit 37 in accordance with the indicated height. At this time, the filling of the collagen solution can be carried out by a desired amount through injection using a pipette.

14, a permeable member 37b, such as a membrane, may be attached to the bottom of the well unit 37 (bottom surface of the hollow portion). As the well type unit 37, Transwell 3470 can be used as described above.

In addition, since the three-dimensional organ structure provided by the present invention can be said to be the main purpose of the cell culture, it is preferable that the present method for producing a three-dimensional organ structure (S100) is provided as a cell-friendly process in the first half thereof. For example, it is preferred that no organic solvents or high concentration of base or estimation conditions are applied to the cells. Accordingly, as a material corresponding to the organopolymer structure 4, a material having cell-friendly physical properties can be used. For example, a hydrogel (such as collagen) may be used as the material corresponding to the long term simulation structure 4.

In step S142a, a stamp unit 36 having a second negative-tone mold 3 attached thereto is inserted into the hollow portion of the well-shaped unit 37 to transfer the material corresponding to the long-term simulation structure 4 to the second negative-tone mold 3 Stamping step (see Fig. 14 (b)).

Step S142a may be performed in such a manner that the stamp unit 36 subjected to the surface treatment (lubrication) is slowly inserted after the injection of the material corresponding to the organ model structure 4 such as a collagen solution is completed. That is, before the step S142a is performed, surface treatment with lubricant may be performed as described above. Such surface treatment with lubricant is carried out by a pressure acting on the surface of the organ model structure 4 that has been in contact with the second negative mold during the second negative mold removal process for separating the stamp unit 36 from the organ model structure 4 The long term simulation structure 4 is prevented from being deteriorated in yield. Illustratively, Poloxamer 188 solution or silicon oil may be used as the lubricant. 14 (b), the surface treatment with the lubricating oil is performed on the surface of the second negative-tone mold 3 which is in contact with the material corresponding to the organ model structure 4 (Indicated by a thick solid line).

Referring to FIGS. 12 and 14 (b), a protruding portion 36a protruding in the outer radial direction may be formed at the upper end of the stamp unit 36.

The thickness of the base portion 41 of the organ model structure 4 can be determined by the height of the stamp unit 36 and the thickness of the second negative-tone mold 3 in step S142a.

A technically important factor in the production of the stamp unit 36 may be said to be the setting of the height of the stamp unit 36 (the height including the thickness of the second negative-tone mold 3). The thickness of the base portion 42 of the long term simulation structure 4 (the height of the lower end of the second relief pattern 41) can be determined according to the setting of the height of the stamp unit 36. [ In a case where the long term simulation structure 4 is exemplarily obtained by three-dimensionally simulating the small intestine of the human body, the height of the lower end of the villi structure of the small intestine corresponds to the thickness of the small intestine according to the height setting of the stamp unit 36 Can be set. In other words, the small intestinal thickness in the range of 0.5 mm to 2 mm can be accurately (imitated) according to the height setting of the second negative-tone mold 3 where stamping is performed.

In step S142a, the projecting portion 36a can be seated on the upper circumference 37a of the hollow portion of the well-like unit 37. [ The stamp unit 36 may also be designed so that the base portion 42 of the organ model structure 4 is formed to a desired thickness when the projection 36a is seated on the top periphery 37a of the hollow portion.

Another important factor in mimicking the thickness of an organ, such as the small intestine in a human body, is the amount of material (such as hydrogel) corresponding to the organ model structure 4 filled in the well-like unit 37. Illustratively, if a well type unit 37 based on the Transwell 3470 standard is used, up to 50 μl of hydrogel can be injected.

Step S143a is a step of incubating the material corresponding to the organ model structure 4 to prepare the organ model structure 4 and then separating the stamp unit 36 from the organ model structure 4 ).

Illustratively, a well unit 37 (Transwell 3470) with a stamp unit 36 coupled is incubated at 36 占 폚 for 1 hour and a 1.0% glutaraldehyde solution is passed through the column of the stamp unit 36 (36b) and incubated at 36 < 0 > C for 6 hours or more.

Next, the stamp unit 36 is slowly separated from the well-shaped unit 37 (Transwell 3470) by using a tweezers or the like. After that, the glutaraldehyde solution is removed, and 7% L-glutamic acid solution is injected, followed by incubation at 36 ° C for 24 hours. Next, the 7% L-glutamic acid solution was removed, and DPBS was injected. Then, the DPBS was replaced with a new DPBS once every 24 hours to remove the remaining 7 (hydrolyzed) Remove% L-Glutamic acid.

FIG. 16 is a conceptual diagram for explaining one function of the master unit of the stamp unit used in the steps S130 and S140 of the method of manufacturing a three-dimensional organ model structure according to an embodiment of the present invention.

11 and 16, the second indentation 31 of the second negative-tone mold 3 may be provided in a hole shape which is perforated in the vertical direction. 14 to 16, the stamp unit 36 may be formed with a passage 36b in the vertical direction so as to communicate with the second engraved shape 31. As shown in FIG. As described above, another important factor in the design of the stamp unit 36 of the present invention is that the stamp unit 36 has an empty space (passage) formed in the vertical direction.

15 (a), in step S143a, when the material corresponding to the organ model structure 4 is incubated, the long-term simulation structure (hereinafter, referred to as " 4) can be injected. That is, the empty space of the stamp unit 36 can be effectively utilized as a space for injecting a chemical substance (chemical agent) in a chemical treatment process necessary for crosslinking a substance such as a hydrogel.

16, when separating the stamp unit 36 from the organ model structure 4 in step S143a, gas can be supplied through the above-described passage 36b into the second indented shape 31 have. In other words, gas such as air can be introduced into the holes (second intaglio shape) of the second negative-tone mold 3 located at the lower end of the stamp unit 36 through the passage 36b, The pressure generated in the step (4) (hydrogel) separation can be minimized.

The pressure generating reduction action of the through-hole 36b can be achieved by organically linking the second indentation shape of the second negative-tone mold 3 to a hole shape vertically penetrated. Specifically, for example, a PDMS stamp is formed by attaching a PDMS in which a second indented shape is vertically penetrated to a metal holder (stamp unit). Through the second indentation formed above and below, the hydrogel component can easily flow into the second indentation shape to more clearly form the microstructure. Further, the surface treatment can be performed using a lubricant or a surfactant so that the hydrogel easily flows into the PDMS stamp and air bubbles are not formed.

As described above, according to the second embodiment (stamping method) of steps S130 and S140, the stamp unit stably fixes the microstructure (second relief shape) of the long-term simulation structure (hydrogel support) Can be faithfully reproduced.

In addition, the method for manufacturing a three-dimensional organ structure (S100) may include a step of culturing a cell on the organ model structure (4). Such a cell culturing step may be appropriately applied or applied to a known cell culturing method known to those of ordinary skill in the art to be simulated, so that a detailed description thereof will be omitted.

According to the present invention, it is possible to reproduce the tissue structure of a complex organs three-dimensionally more accurately than in the prior art, and it is also possible to reproduce the above-described precise cells through a cell-friendly substance such as a hydrogel having soft properties have. Also, according to the present application, a three-dimensional organs-like structure capable of maintaining high compatibility with existing experimental techniques and experimental instruments can be manufactured. For example, an observable level of a three-dimensional organs mimetic structure can be produced by a general microscope used for cell observation.

In addition, the present invention can provide a three-dimensional organ model structure manufactured by any one of various embodiments of the above-described three-dimensional organ model structure manufacturing method (S100).

Illustratively, the three-dimensional organ model structure may be a structure that simulates a field of the human body. More specifically, in the case of a human field, the height of the villi may be 0.5 mm to 1.6 mm, the average thickness of the duodenum and the plant may be about 1.5 mm, and the 3-dimensional organ structure of the present invention may be manufactured .

Further, as described above, the three-dimensional organs-like structure can be made of a cell-friendly material. Illustratively, the 3D long-term structure may be provided by preparing a desired shape of the hydrogel using a mold of various materials, but the material of the 3D long-term structure is not limited to a hydrogel, May be a material.

It will be understood by those of ordinary skill in the art that the foregoing description of the embodiments is for illustrative purposes and that those skilled in the art can easily modify the invention without departing from the spirit or essential characteristics thereof. It is therefore to be understood that the above-described embodiments are illustrative in all aspects and not restrictive. For example, each component described as a single entity may be distributed and implemented, and components described as being distributed may also be implemented in a combined form.

The scope of the present invention is defined by the appended claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be interpreted as being included in the scope of the present invention.

1: 1st negative angle mold
11: 1st indentation shape
12: Base member
13: Photoresist layer
14: Photomask
14a: pattern
2: Embossed mold
21: First embossed shape
22: bottom frame
23: middle frame
24: Top frame
25: Positive mold seating part
3: 2nd negative mold
31: 2nd engraved shape
32: Cover gasket
32a: hole of cover gasket
33: Membrane
34: Perforated plate
35: upper gasket
36: Stamp unit
36a:
However,
36c: Adhesive material
37: Well type unit
37a: upper circumference of the hollow portion
37b: permeable member
37c: Chemicals for crosslinking
4: long term simulated structure
41: second embossed shape
42: Base portion

Claims

As a method for manufacturing a three-dimensional organ model structure,
(a) fabricating a first negative-tone mold having a first engraved shape;
(b) fabricating a positive embossed mold having a first relief shape corresponding to the first relief pattern using the first relief mold;
(c) fabricating a second negative-tone mold having a second engraved shape corresponding to the first embossed shape using the positive mold; And
(d) fabricating an organ model structure having a second relief shape corresponding to the second relief shape using the second relief mold,
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The step (c)
(c1) pouring and hardening the silicone solution on the positive mold and separating the mold from the positive mold to produce the second negative mold; And
(c2) attaching the second negative-angle mold to the lower end of the stamp unit,
The step (d)
(d1) filling a hollow portion of a well-shaped unit having an upper portion opened with a material corresponding to the organ model structure;
(d2) stamping the material corresponding to the organ model structure with a second negative-shape mold by inserting a stamp unit with the second negative-angle mold on the hollow portion; And
(d3) incubating the organism corresponding to the organ model structure to produce the organ model structure, and then separating the stamp unit from the organ model structure.

The method according to claim 1,
Wherein the first indentation shape includes a three-dimensional solid shape of a groove or a hole shape whose width changes according to the depth,
Wherein in the step (a), the three-dimensional solid shape is formed by a photolithography process.

3. The method of claim 2,
Wherein in the step (a), the three-dimensional solid shape is formed by using at least one of overexposure and exposure by light refraction.

The method according to claim 1,
The step (a)
(a1) forming a photoresist layer on the base member;
(a2) disposing and exposing a photomask having a pattern corresponding to the first intaglio shape on the photoresist layer to selectively crosslink the photoresist layer according to the pattern; And
(a3) developing the photoresist layer to remove the non-crosslinked portion to form the first negative-type mold having the first intaglio shape,
Wherein the method comprises the steps of:

5. The method of claim 4,
Wherein the step (a2) is a step of exposing the photoresist layer to a crosslinking treatment corresponding to a three-dimensional solid shape of a groove or a hole shape varying in width depending on the depth, Gt;

5. The method of claim 4,
In the step (a2), a diffuser for guiding light refraction is disposed on the photomask and exposed to light so that a bridge corresponding to a groove or a hole-like three-dimensional solid shape having a width varying in depth is formed Wherein the method comprises the steps of:

5. The method of claim 4,
In the step (a1) and the step (a2), at least two of a three-dimensional solid shape of a groove or a hole shape varying in width depending on the depth and a three- And wherein the step of forming the three-dimensional organs is repeated.

The method according to claim 1,
In the step (b), the positive mold is manufactured by pouring and hardening a silicon solution on the first negative-angle mold, and then separating the first negative-angle mold from the first negative-angle mold.

delete

The method according to claim 1,
A protrusion protruding in an outer radial direction is formed on an upper end of the stamp unit,
In the step (d2)
Wherein the projection is seated on an upper periphery of the hollow portion,
Wherein the base unit thickness of the organ model structure is determined by the height of the stamp unit and the thickness of the second negative-angle mold.

The method according to claim 1,
The second indentation shape of the second negative-tone mold is vertically penetrated,
Wherein the stamp unit is vertically formed to communicate with the second engraved shape.

15. The method of claim 14,
In the step (d3)
Wherein when the stamp unit is detached from the organ model structure, gas is supplied into the second intaglio shape through the through-hole.

15. The method of claim 14,
In the step (d3)
Wherein when the material corresponding to the organ model structure is incubated, chemicals for crosslinking the material corresponding to the organ model structure in the second intaglio shape are injected through the through-hole, .

The method according to claim 1,
(e) culturing the cells on the organ model construct.

A three-dimensional organs mimetic structure produced by the method of manufacturing a three-dimensional organ structure according to claim 1.