KR102037595B1 - Hydrogel membrane fixed vertically in microfluidic chip and preparation method thereof - Google Patents

Abstract

The present invention relates to a hydrogel film fixed vertically in a microfluidic chip and a method of manufacturing the same. In detail, the hydrogel membrane was grafted to allow the free control and control of cell-cell interaction, material diffusion, and exchange using microchannels in the microfluidic chip. It relates to a culture system.

Description

Hydrogel membrane fixed vertically in microfluidic chip and preparation method according to microfluidic chip

The present invention relates to a hydrogel film fixed vertically in a microfluidic chip and a method of manufacturing the same.

Tissue engineering aims to improve human life by restoring, maintaining and improving the function of human organs and tissues.

In order to rebuild new tissues, four elements are required: biomaterials and scaffolds, cells, growth factors, and extracellular matrix (ECM).

Therefore, researches and studies on creating a three-dimensional in vivo environment by incorporating biomaterials have been conducted.

As a method of simulating a living body, a lab-on-a-chip means that a laboratory in a three-dimensional chip and an applied research for analyzing interactions between materials mean that the existing system is miniaturized and simplified. ) Is gaining importance.

This is a chip capable of separating, mixing, synthesizing and quantitatively analyzing analytes using micromachining technology, and may be given functionality such as a pump, a valve, a reactor and a separation system.

Cells present in the human body are exposed to a three-dimensional environment and exchange materials with various factors, thereby proliferating and behaving by them.

The microfluidic chip is composed of polydimethylsiloxane (PDMS) as a main material, which is a kind of silicon and is commonly used as a material in tissue engineering because it has advantages of thermal stability, chemical stability, inertness, and low cost.

By culturing the cells in the micro-fluidic chip of micro-units, there is an advantage that can control and analyze the interaction between the cells, the behavior of the cells and the like that were not observed in the conventional two-dimensional culture system.

KR 10-2016-0110740 A, 2016. 09. 22. KR 10-2016-0111683 A, 2016. 09. 27. KR 10-1776187 B1, 2017. 09. 01. KR 10-2017-0053288 A, 2017.05.16.

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The two-dimensional culture system used in the prior art is easy to observe the growth of the cell and the experimental process is relatively simple, but can not imitate the human body system in three dimensions, only the horizontal culture analysis of the cell, vertical culture Analysis was not possible and cultured in culture dishes such as glass and petri dishes, indicating poor biocompatibility in terms of materials.

In addition, the prior art used a method of controlling the flow of the microfluid by opening or blocking a channel including a separate pump or air valve, but this method requires a separate air hole layer, which makes it difficult to finely control the air pressure.

For example, in the "hydrogel-based cell co-culture microfluidic chip" disclosed in Patent Document 1 (KR 10-2016-0110740 A, September 22, 2016), it is possible to co-culture cancer cells and vascular endothelial cells. Although the point is significant, the horizontal culture assay was used, so there was no description of the vertical culture assay.

In addition, "3D cell culture scaffold and coculture method using the same" disclosed in Patent Document 2 (KR 10-2016-0111683 A, 2016. 09. 27.) is the same as the present invention by providing a 3D cell culture method. Or may be seen as an invention of a similar purpose.

However, the invention of the patent document 2 in the microfluidic device-based in vitro model is co-cultured by permanently arranging cells exhibiting the paracrine effect, so that the environment given to the cells at the initial stage until the end of the experiment Cultured auxiliary cells that secrete paracrine factors other than the cell of interest in analysis may be misleading in response to drugs, pointing out the problem as an irreversible design that cannot be maintained or altered or eliminated. The purpose of the present invention is to provide a three-dimensional cell culture scaffold that traps a hydrogel containing cells in a membrane form using surface tension in an O-shaped loop as a deformable co-culture technology. However, the invention of Patent Document 2 did not reach the vertical culture assay by breaking from the horizontal culture assay on the microfluidic chip. In addition, it has been disclosed to provide a method of dipping the scaffold into a microfluidic device and co-culturing the same, and a method of manufacturing a vascular module through the same, but this has the significance of creating a dynamic microenvironment in the microfluidic device. It is not only described, but also does not enable the vertical culture assay to be achieved by the present invention.

In addition, Patent Document 3 (KR 10-1776187 B1, 2017. 09. 01.) relates to a "microfluidic chip and a method for manufacturing the same." In the conventional cell culture microfluidic device, the bottom surface of the microchannel is mainly glass or silicon. It is formed of a series of materials, and there is a great gap between the bottom stiffness of the physiological range of most tissues, and some of the hydrophobic soluable factors that exist around the cell or in the medium are absorbed and confined to the bottom of the cell. It affects the signaling system and points out the problem that cells in the channel cannot be observed under a normal microscope through the thick PDMS bottom, and the bottom surface is selected as a transparent material and the base surface on which the cells are implanted is formed into a hydrogel thin film. It forms and controls the stiffness of the basal plane within the physiological range to simulate the organ environment such as blood vessels through which fluid flows. It was possible to observe in real time. However, the Patent Document 3 invention maintains the vertical culture analysis method as it is, and has not been disclosed for the vertical culture analysis.

In addition, in the patent document 4 (KR 10-2017-0053288 A, 2017. 05. 16.), in the invention, "microchip and its manufacturing method, enzyme detection system and method using the same" in the invention nanofibers at the bottom of the channel of the microfluidic chip Pointed out that the nanofibers could not be effectively fixed to the entire channel in the case of forming the nanofibers, and that the application of the nanofibers to the sensor was difficult because it was difficult to control the amount of the inserted nanofibers. The present invention provides a microchip which can be used for immunoassay or enzyme detection more effectively by forming a support using hydrogel lithography and then fixing the fiber in a microchip. However, there was no description of the vertical culture assay on microfluidic chip.

The present invention to solve the problem, the first step of preparing a plate coated with alginate solution; Injecting a solution containing a divalent cation into the dish; A third step of inducing alginate in the solution applied to the dish by the divalent cation to be ion crosslinked; A fourth step of removing moisture remaining in the dish; A fifth step of drying the dish at room temperature; It provides a hydrogel film prepared through.

In addition, in order to solve the above problems, the present invention, the hydrogel film in the channel in the microfluidic chip is fixed to the channel by electrostatic attraction without a separate process and partially cured through a very small amount of polydimethylsiloxane (PDMS) solution Provides a fluid chip.

Through this, in the present invention, it is formed as a micro-thick hydrogel film through an alginate crosslinking process, which is a kind of hydrogel, and is vertically fixed to the channel wall in the microchip to impart a valve role to the microfluidic channel. It compensates for the shortcomings and presents the functionality.

The three-dimensional culture system through the vertical culture analysis method of the present invention is capable of diffusion, filtration of the material in the micro-channel, excellent biomimetic, and can implement an environment suitable for cell research through the induction of various reactions, micro-channel Through this, it is possible to give effective mass transfer ability, and cell culture media exchange is advantageous.

In addition, the alginate hydrogel membrane of the present invention can completely control the diffusion and permeation between cells and cells or cells and materials in the channel, and enables the symbiotic culture of cells with cell compatible materials and facilitates cell culture analysis. .

In addition, the alginate hydrogel film was crosslinked with calcium chloride, which induces the natural decomposition of alginate by inactivating the binding of the alginate solution with the calcium chloride solution due to the property of binding to calcium ions, which are divalent cations of the triscationic citrate (TSC) solution. can do.

By utilizing the decomposition property of the alginate, the alginate hydrogel film used in the present invention can be decomposed in the channel as necessary.

1 is an optical microscope photograph showing the length and width of a microfluidic chip channel in which an alginate hydrogel film is vertically fixed.
2 is an enlarged photograph of a microfluidic chip in which an alginate hydrogel film is vertically fixed.
3 is an optical microscope photograph showing the width of an alginate hydrogel film.
Figure 4 is an optical microscope photograph showing the height of the alginate hydrogel film.
5 is a photograph of a microfluidic chip in which an alginate hydrogel film attached to a glass plate is vertically fixed.
FIG. 6 shows a photograph of a diffusion test of a fluorescent dye in a microfluidic chip in which an alginate hydrogel film is vertically fixed.
FIG. 7 shows a swelling test photograph of an alginate hydrogel film fixed vertically in a microfluidic chip.
FIG. 8 is an optical microscope photograph of the decomposition of an alginate hydrogel film by injecting a TSC solution into a microfluidic chip channel in which an alginate hydrogel film is vertically fixed.

Hereinafter, a hydrogel film vertically fixed in the microfluidic chip of the present invention and a manufacturing method thereof will be described in detail with reference to the accompanying drawings. The drawings introduced below are provided as examples to ensure that the spirit of the present invention can be fully conveyed to those skilled in the art. Therefore, the present invention is not limited to the drawings presented below and may be embodied in other forms.

In this case, unless there is another definition in the technical terms and scientific terms used, it has the meaning commonly understood by those of ordinary skill in the art to which the invention belongs, and the gist of the invention in the following description and the accompanying drawings. The description of well-known functions and configurations that may unnecessarily obscure them will be omitted.

Manufacturing process of hydrogel membrane

An embodiment according to an aspect of the present invention provides a hydrogel film.

Preferably, the present invention to solve the above problems, the first step of preparing a plate coated with alginate solution; Injecting a solution containing a divalent cation into the dish; A third step of inducing alginate in the solution applied to the dish by the divalent cation to be ion crosslinked; A fourth step of removing moisture remaining in the dish; A fifth step of drying the dish at room temperature; It provides a hydrogel film prepared through.

More preferably, the alginate solution in the first step is a sodium alginate solution, the concentration of which is 1.0 to 5.0 wt%, more preferably the concentration of 2.0 to 4.0 wt%.

More preferably, in the third step, the alginate is reacted with calcium chloride (CaCl ₂ ) to form ionic crosslinks by calcium cations (Ca ²⁺ ).

However, the divalent cation that causes the ion crosslinking is not limited to the calcium cation.

The third step may be 12 hours at room temperature.

In addition, more preferably, an ethanol solution may be used to remove moisture remaining in the fourth step.

Also preferably, the fifth step is to dry for 5-7 hours at room temperature, more preferably to dry for 6 hours.

Polydimethylsiloxane (PDMS) Microfluidic Chip Manufacturing Process

An embodiment according to an aspect of the present invention provides a PDMS-based microfluidic chip.

Preferably, a first step of coating the photosensitive agent by rotating and stirring the silicon wafer coated with the photosensitive agent; After placing a mask film on an aligner, the photosensitive agent-coated silicon wafer is irradiated with an ultraviolet light source to induce a reaction of the coated photosensitive agent so that the pattern of the mask film is coated with the photosensitive agent. A second step of being engraved on the surface of the wafer; A third step of removing residual impurities and the remaining photoresist after heat-treating the silicon wafer having the pattern of the mask film formed on the surface thereof; A fourth step of hard-baking the silicon wafer from which the residual impurities and the residual photoresist are removed; and polymimethylsiloxane (PDMS) including a silicon wafer manufactured through a photolithography process. ) Based microfluidic chip.

In addition, preferably, a solution in which polydimethylsiloxane (PDMS) is mixed with a curing agent and a ratio of 10: 1 by weight (weight ratio) is applied to a silicon wafer manufactured through the photolithography process, and the resulting bubbles are removed and then heat treated. The present invention provides a polymimethylsiloxane (PDMS) -based microfluidic chip, which is processed by a soft lithography process.

Specifically, it is as follows.

A photolithograpy process is performed to fabricate a silicon wafer, which is the basis for PDMS-based microfluidic chips.

The photoexposure process is a process of making a mold of a silicon wafer, and is a process of transferring a geometric pattern formed on a mask by a photoresist covering the semiconductor wafer surface to the wafer surface.

The photosensitizer is a material that reacts to light when it receives light, but the present invention uses, but is not limited to, SU-8.

After fixing the 4-inch silicon wafer by vacuum on the chuck in the spin coater equipment, apply SU-8, a photoresist, on the wafer surface, 15 seconds at 500 rpm, 30 seconds at 2300 rpm. Establish coating conditions.

After transferring the silicon wafer to a hot plate equipment, the coating of the photoresist was hardened by heat treatment at 65 ° C. for 12 minutes and 95 ° C. for 2 hours, and then cooled sufficiently at room temperature for 30 minutes.

Next, a mask film engraved with a microfluidic chip pattern was placed in an aligner device, the silicon wafer was fixed to the chuck of the lower part, and then the reaction of the photoresist was induced through a UV light source. Engraved on the wafer surface.

The silicon wafer is then placed on the hotplate equipment and heat treated at 65 ° C. for 5 minutes and 95 ° C. for 30 minutes.

To completely remove impurities and unnecessary photoresist remaining in the pattern, a development process was carried out. After the silicon wafer was sufficiently immersed in a rinse solution, impurities were removed for at least 10 minutes.

After removing the impurities by isopropyl alcohol (IPA), and finally hard bake for 15 minutes on a hot plate of 150 ℃.

A biocompatible polymer polydimethylsiloxane (PDMS) is used to form an embossed pattern on the fabricated silicon wafer surface.

The PDMS solution is mixed with a curing agent in a ratio of 10: 1 (PDMS: curing agent), applied 35ml on a silicon wafer, and then subjected to a soft lithography process in which an air bubble is removed and cured in an oven at 80 ° C. for 2 hours. .

In the PDMS mixed solution, when the ratio (weight ratio) of the curing agent is 10: 1.5 (PDMS: curing agent) or more, curing occurs rapidly and the viscosity of the solution becomes very high, so that the ratio (weight ratio) with the curing agent is 10: 1. To prepare.

Desorption of the hardened PDMS on the wafer to form an injection hole through which a solution can be injected using a bio punch to finally produce a microfluidic chip as shown in FIG. 1.

Each inlet has a size of 1.5π, 4π, and the length and width of the channel is 11.1 mm. The length and width of the channel are not limited thereto, and may be designed to different values.

Vertical Fixing Process of Hydrogel Film in Microfluidic Chips

An embodiment according to an aspect of the present invention provides a microfluidic chip in which a hydrogel film is vertically fixed.

Preferably, the hydrogel film is an alginate hydrogel film, and the microfluidic chip has a central channel, so that the alginate hydrogel film is vertically fixed to the lower end of the microfluidic chip central channel. Cutting is performed according to the height of the microfluidic chip channel.

Specifically, the inventors fix the alginate hydrogel film of about 3 cm in width and about 1 cm in length to a glass plate, and then prepare a cut of about 200-250 μm in width and about 1 cm in length by using a mes through an optical microscope.

This corresponds to the trapezoidal column height and channel length present in the channel in the microfluidic chip in which the alginate hydrogel film is to be fixed vertically.

Thereafter, the cut alginate hydrogel film is fixed perpendicularly to the bottom of the microfluidic chip channel using tweezers.

The fixation is permanently maintained at the bottom of the microfluidic chip channel through electrostatic attraction and therefore does not require any other materials or other processes.

Since alginate hydrogel membranes were used to physically control mass exchange and cell behavior between channels in the microfluidic chip, there is no space between the bottom of the vertical membrane and the surface of the microfluidic chip when the alginate hydrogel membrane is fixed. Manufacture to avoid.

A very small amount of pure polydimethylsiloxane (PDMS) solution is used for firm fixation of the alginate hydrogel film and the microfluidic chip, and an uncured polydimethylsiloxane (PDMS) solution is placed at each edge of the channel in the microfluidic chip. It is applied to the point and the center point to induce the fixation between the alginate hydrogel film and the microfluidic chip.

The microfluidic chip channel in which the alginate film is vertically fixed is shown in FIGS. 1 and 2.

The length and width of the channel is about 11.0 mm for the straight channel and about 1.0 mm for the width of the channel, as described in FIG.

The hydrogel film is erected vertically at the lower end of the center of the microchip channel and is stably fixed. There is no space between the bottom of the hydrogel film and the surface of the microfluidic chip, and it has a constant thickness and height. .

Details of the height and width of the alginate membrane are shown in FIGS. 1, 3 and 4 with a length of about 9.1 mm, an average height of about 243 μm and a width of about 43.5 μm on average.

The length, height, and width of the alginate membrane are not limited thereto, and may be changed and applied according to the purpose.

Swelling Analysis of Hydrogel Gel Membrane

The hydrogel, which is fixed perpendicular to the wall in the channel, the top surface of the microfluidic chip including the alginate film and the top surface of the glass plate are subjected to surface treatment through 60 W intensity and atmospheric oxygen oxygen plasma treatment for 1 minute.

It is possible to attach the microfluidic chip and the glass plate by the plasma surface treatment, and the photo of the entire microfluidic chip attached to the glass plate is shown in FIG. 5.

When all the channels of the microfluidic chip are emptied and red dye is injected into the lower channel through the formed injection hole, the red dye is not immediately diffused into the intermediate channel under physical control of the alginate hydrogel film and is completely trapped in the lower channel. (See FIG. 6).

Since the alginate hydrogel film has a property of containing water in the three-dimensional network structure, swelling occurs when the dye touches, and the volume increases and swells over time (see FIG. 7).

As a result, the boundary line between the blue dye and the red dye becomes unclear and the two dyes are diffused.

Degradability Test of Hydrogel Gel Membrane

Injecting a 4% TSC solution into the chip channel through the formed injection port inactivates the binding of the alginate, which is the main component of the hydrogel film, and the calcium chloride used together for the crosslinking of the hydrogel film, thereby inducing the natural decomposition of the alginate film. .

After injecting 60 μl of a 4% TSC solution into the vertically fixed channel of the alginate hydrogel membrane, it can be seen that the hydrogel membrane decomposes over time for about 1 minute, which is shown in FIG. 8. It is possible to analyze that the vertically fixed alginate membrane becomes unclear after about 12 seconds and naturally decomposes after about 28 seconds.

Alginate Properties

Alginate is a type of hydrogel and is a major component of the cell membrane of marine brown algae such as seaweed and seaweed.

The alginate structure has the form of a block copolymer consisting of α-L-guluronic aicd (gluronic acid) and β-D-mannuronic aicd (manneuronic acid).

Since it contains a large number of anionic carboxyl groups (-COOH), since the gluronic acid of alginate induces cross liking and gelation when bound with divalent cations, it becomes a major factor in determining physical properties. Can be.

As such, when the alginate is formed of a hydrogel, it has a three-dimensional network structure and may contain a complex such as a drug or a polysaccharide in the gel.

In addition, alginate has excellent biocompatibility, does not induce cytotoxicity, immune response, biodegradability, and hydrophilicity.

This is an essential field used in the smart medical devices and diagnostic devices of the microchip, the present invention to vertically fix the hydrogel film with excellent biocompatibility in the conventional microchip process can ensure the expansion of various diagnostic technologies in the microchip for diagnostic devices Can be.

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Claims (4)

A first step of applying an alginate solution in which sodium alginate is dissolved in water at 2 to 4 wt% in a dish;
Injecting a solution containing a divalent cation into a dish to which the alginate solution is applied;
A third step of preparing an alginate hydrogel membrane by performing an alginate ion crosslinking reaction using the divalent cation at room temperature for 12 hours;
A fourth step of removing water remaining on the alginate hydrogel film by the alginate ion crosslinking reaction using ethanol;
A fifth step of drying the alginate hydrogel film from which the moisture is removed for 5 to 7 hours at room temperature;
Method for producing an alginate hydrogel film comprising a. In a polydimethylsiloxane (PDMS) microfluidic chip in which an alginate hydrogel film is vertically fixed,
A first step of manufacturing a photosensitive agent-coated silicon wafer by applying a photosensitive agent to the silicon wafer, rotating stirring, and performing heat treatment;
A second step of manufacturing a mask film engraved with a microfluidic chip pattern;
A third step of manufacturing the microfluidic chip pattern silicon wafer having the microfluidic chip pattern engraved by fixing the mask film on the silicon wafer coated with the photosensitive agent and irradiating the photosensitive agent with ultraviolet light
Performing a heat treatment on the microfluidic chip pattern silicon wafer to remove impurities and photoresist remaining in the pattern;
A fifth step of hard-baking the microfluidic chip pattern silicon wafer from which the impurities and the photoresist are removed;
A sixth step of preparing a PDMS mixed solution by mixing a polydimethylsiloxane (PDMS) solution and a curing agent in a weight ratio of 10: 1 (PDMS: hardener);
The PDMS mixed solution is applied onto the hard-baked microfluidic chip pattern silicon wafer, bubbles are removed, and then cured in an oven to form a plurality of trapezoidal pillars in a central channel where a plurality of channels and a plurality of channels are merged. PDMS) a seventh step of manufacturing a microfluidic chip;
An alginate hydrogel film prepared with an average thickness of 43.5 μm is cut into a rectangular shape, and the depth of the central channel of the polydimethylsiloxane (PDMS) microfluidic chip corresponds to the length of the polydimethylsiloxane (PDMS) microfluidic chip. An eighth step of cutting the horizontal length of the center channel to correspond to the horizontal length;
A ninth step of vertically fixing the cut alginate hydrogel film vertically through an electrostatic attraction to an edge of a central channel of the polydimethylsiloxane (PDMS) microfluidic chip and to one surface of a plurality of trapezoidal pillars formed in the central channel; And
A tenth step of applying and fixing the polydimethylsiloxane pure solution between the vertically fixed alginate hydrogel film and the edge of the central channel and one side of a plurality of trapezoidal pillars formed in the central channel;
Characterized in that manufactured by a manufacturing method comprising a,
The vertically fixed alginate hydrogel film is a polydimethylsiloxane (PDMS) microfluidic chip in which an alginate hydrogel film is vertically fixed, which delays diffusion of a material between channels and stores the composite therein and gradually diffuses it. delete delete

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