KR101847303B1 - Microwell substrate and fabrication method thereof - Google Patents

Abstract

Disclosed are a microwell substrate and a method for manufacturing the same. The method for manufacturing a microwell substrate comprises the following steps: manufacturing a plate assembly arranged with a plurality of trap holes by combining an upper plate and a lower plate formed with a hole array; stacking the plate assembly on a magnetic array; filling the trap holes with steel beads; manufacturing a microwell mold by removing a top plate from the plate assembly; and manufacturing the microwell substrate using the resulting microwell mold.

Description

Microwell substrate and fabrication method thereof < RTI ID = 0.0 >

The present invention relates to a microwell substrate and a method of making the same.

Cells that grow in the form of spheroids are more similar to the actual tissues of the body than to 2-dimensional planar cultures. With these advantages, cell spheres have been used to improve the study of cell interactions, immune-activation, drug screening, and differentiation. In addition, cellular organisms, including multiple cell types, have recently been applied to organoids (physiological three-dimensional tissues). Organogens are very useful in studying human development and disease.

There are several methods for producing such cell spheres. The simplest of these involves using surfaces that do not stick together to form cells spheroids that are joined together. For example, a petridish can be treated with bovine serum albumin, pluronic f-127 or a hydrophobic polymer to have a non-stick surface.

And, a spinner-flask method is a well-known means of producing a large amount of cell spheroids. In this method, suspension of the cells is stopped by agitation in order to prevent the cells from sticking to the flask surface. Instead, floating cells are combined to form cell spheres.

Both the non-stick surface method and the spinner flask method can produce large amounts of cell spheroids. However, these methods have difficulties in controlling the size of the cell spheroid as well as tracking and monitoring each cell spheroid.

In order to solve this problem, a hanging drop method can be used as another cell spheroid forming method. This method involves dropping the deposited cell suspension onto the inside surface of the lid of the culture dish. These drops are usually 15 to 30 μL and contain about 300 to 3000 cells. When the lid is turned over, the dropped droplets do not fall to the surface tension. A subtle gravitational environment enriches the cells in each droplet and forms a single cellular globe at the free liquid air interface. The advantage of the droplet culture is that it is easy to track and monitor the cell spheroids and the size distribution is well controlled as compared with the method using a non-stick surface and the method using a spinner flask. However, this method has a drawback that mass production and generation of cell spheroids are labor intensive.

On the other hand, a microwell array is a flat plate with many micro-sized wells. Each microwell has a diameter in the range of 100 to 1000 mu m. The principle of cell spheroid formation using such microwells is similar to the non-stick surface method. This method has the advantage of providing a space between microwells for separating cells or cell spheres. This facilitates the monitoring of each cell spheroid and facilitates controlling the size of the cell spheroid. A large number of microwells allow a large number of cell spheroids to be generated. As another advantage, the microwell can be formed in a form according to the user's own experimental purpose. For example, the microwell can be formed in the form of a hexahedron, a cylinder, a triangular pole, or the like. However, in general, a three-dimensional concave or hemispherical shape may be most suitable for producing a cell spheroid having a uniform size.

A microwell with a flat bottom may be used to include multiple cellular spheres. Therefore, the usefulness of the concave-shaped microwells is due to the fact that in embryonic stem cells, cardiomyocyte differentiation of embryonic stem cells, insulin secretion of islet cell clusters, and enzymatic activity of hepatocytes hepatocytes, and drug resistance of tumor spheroids have been reported in many cell biology studies.

However, microwell fabrication often requires specialized micropatterning equipment. Existing photolithography based methods require exposure and developing facilities, and reactive ion etching based methods require plasma and ion etching equipment. Such equipment is expensive and has a complex manufacturing process, making it a high entry barriers for biologists who do not have access to micro-technologies.

To solve this problem, other cost-effective methods have been proposed. The proposed method includes a membrane, a substrate with a through-hole formed therein, a flexible membrane method using a vacuum, and ice lithography using frozen droplets. However, these methods also have serious problems. That is, there is a problem that it is difficult to control the pattern size, achieve a high aspect ratio, and generate a larger microwell.

The present invention relates to a microwell substrate in which a steel bead is fixed to a hole of a plate by a magnetic force using a hole-formed plate, a steel bead and a magnet array, So as to provide a manufacturing method.

According to an aspect of the invention, a method of making a microwell substrate in which a microwell is formed is disclosed.

A method of fabricating a microwell substrate according to an embodiment of the present invention includes assembling a plate assembly in which a plurality of trap holes are arranged by coupling an upper plate and a lower plate having a hole array, Forming a microwell mold by removing the top plate from the plate assembly; and forming a micro well mold by removing the top plate from the plate assembly. And forming the microwell substrate using a well mold.

The upper plate and the lower plate are overlapped with each other such that the center of the upper hole formed in the upper plate and the lower hole formed in the lower plate coincide with each other. The diameter is larger than the hole.

The step of filling the trap hole with a steel bead includes the steps of placing a steel bead in an amount exceeding the total number of the trap holes on the plate assembly and inserting the steel bead into a non- And sweeping the steel beads overlying the plate assembly.

The step of sweeping the steel beads placed on the plate assembly includes filling and fixing the steel beads in the respective trap holes by the magnetic force of the magnet array and removing remaining steel beads remaining on the plate holes from the plate assembly Lt; / RTI >

When the upper plate is removed from the plate assembly, the microwell mold having the steel bead embedded in each lower hole of the lower plate is formed, and the magnet array is formed by inserting the steel bead into each lower hole of the lower plate, And is positioned below the microwell mold so as to be fixed by the microwell.

The step of fabricating the microwell substrate includes placing the microwell mold on the magnet array in a bowl, pouring a substrate material in a liquid state into the microwell mold to immerse the microwell mold, Separating the steel bead embedded micro-well substrate from the lower plate when the substrate material is cured, and heating the steel bead embedded in the microwell substrate with a magnet .

The size and shape of the microwell are determined according to the diameter of the steel bead and the diameter of the lower hole formed in the lower plate.

According to another aspect of the present invention, a microwell substrate is disclosed.

In the microwell substrate according to the embodiment of the present invention, a concave microwell having an opening is formed so as to correspond to a microwell mold having a steel bead embedded in each hole of a plate on which a hole array is formed, Is determined according to the diameter of the steel bead and the diameter of the hole of the plate.

The absolute size of the microwell is proportional to the size of the steel bead, and the shape of the microwell is determined by the difference between the diameter of the lower hole and the diameter of the steel bead.

A method of manufacturing a microwell substrate according to an embodiment of the present invention includes the steps of fixing a steel bead to a hole of a plate by a magnetic force using a hole formed plate, a steel bead, and a magnet array By forming a concave spherical microwell, an inexpensive and uncomplicated facility is used, and many high-level technologies are not required, so that even an untrained person can be easily manufactured.

1 illustrates a method of fabricating a microwell substrate in accordance with an embodiment of the present invention.
2 to 9 are views showing an example of fabrication of a microwell substrate according to an embodiment of the present invention.
10 is a diagram illustrating a cell culture method using a microwell substrate according to an embodiment of the present invention.

As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. In this specification, the terms "comprising ", or" comprising "and the like should not be construed as necessarily including the various elements or steps described in the specification, Or may be further comprised of additional components or steps. Also, the terms "part," " module, "and the like described in the specification mean units for processing at least one function or operation, which may be implemented in hardware or software or a combination of hardware and software .

Hereinafter, various embodiments of the present invention will be described in detail with reference to the accompanying drawings.

FIG. 1 is a view showing a method of manufacturing a microwell substrate according to an embodiment of the present invention, and FIGS. 2 to 9 are views showing an example of manufacturing a microwell substrate according to an embodiment of the present invention. Hereinafter, a method of manufacturing a microwell substrate according to an embodiment of the present invention will be described with reference to FIG. 1, with reference to FIGS. 2 to 9. FIG.

In step S110, the plate assembly 10 is manufactured. The plate assembly 10 includes an upper plate 11 and a lower plate 12 in which a hole array is formed such that centers of the upper holes 21 formed in the upper plate 11 and the lower holes 22 formed in the lower plate 12 are aligned with each other, And the upper hole 21 formed in the upper plate 11 is formed to be larger in diameter than the lower hole 22 formed in the lower plate 12. Thus, the plate assembly 10 has the trap hole 20 whose upper diameter is larger than the lower diameter to which the steel bead 40 is filled.

For example, referring to FIG. 2, the top plate 21 and the bottom plate 22 may be aluminum plates each having a size of 50 x 50 mm or more and a thickness of 0.3 mm. The upper plate 11 and the lower plate 22 are formed such that the diameter of the holes is 750 占 퐉 for the upper plate 21, 550 占 퐉 for the lower plate 22, 1 mm between the centers of the holes, And a hole array may be formed in each of the first and second contact holes 12a and 12b. The upper plate 21 and the lower plate 22 are formed with alignment holes each having a diameter of 3 mm at each of four corners and in the state where the upper plate 21 and the lower plate 22 are overlapped with each other, The upper plate 21 and the lower plate 22 can be fixed through the use of the bolts and the nuts. The top plate 21 and the bottom plate 22 may be immersed in 15% sulfuric acid to clean the surface before being engaged and fixed. Since the thin layer of aluminum oxide on the aluminum plate surface provides corrosion resistance, the diameter of the holes and the plate thickness do not change by the acid treatment.

In step S120, the plate assembly 10 is stacked on the magnet array 30.

For example, the magnet array 30 may be formed by arranging 30 x 30 neodymium magnets of 1 x 1 x 1 mm, and each magnet may have a magnetic field strength of 0.363N. Each magnet is arranged so that its polarity is opposite to its neighboring magnet. And, the magnet array 30 can be attached using a double-sided tape on an aluminum plate of 30 x 30 mm in size to prevent the magnet array from being broken or disturbed.

In step S130, the trap hole 20 of the plate assembly 10 is filled with the steel bead 40.

3 and 4, a sufficient amount of steel beads 40 having a diameter of 600 mu m exceeding the total number of the trap holes 20 is placed on the plate assembly 10, The steel beads 40 placed on the plate assembly 10 are swept away by the non-metallic acrylic plate 50 so that the steel beads 40 are filled in the steel plate 20. At this time, the magnetic beads 40 can be filled and fixed to the respective trap holes 20 by the magnetic force of the magnet array 30. As a result, the steel beads 40 are filled in the respective trap holes 20, and the remaining steel beads 40 filled in the trap holes 20 are removed from the plate assembly 10.

In step S140, the upper plate 11 is removed from the plate assembly 10 to produce the microwell mold 100. [

5, when the upper plate 11 is removed from the plate assembly 10, the micro beads 40 having the steel beads 40 embedded in the respective lower holes 22 of the lower plate 12 are formed, The well mold 100 can be produced. At this time, the magnet array 30 can be continuously positioned below the microwell mold 100 so that the steel beads 40 are fixed to the respective lower holes 22 of the lower plate 12 by magnetic force.

In step S150, a microwell substrate 200 on which microwells 210 are formed is fabricated by using the microwell mold 100 thus formed.

6, a microwell mold 100 placed on a magnet array 30 is placed in a bowl 60 and a substrate material 70 in a liquid state is immersed in the microwell mold 100, Is poured into the vessel (60), and then baked to harden the substrate material (70) in the liquid state. Here, the vessel 60 may be a Petri-dish, and the substrate material 70 may be a polydimethylsiloxane (PDMS) mixture. The polydimethylsiloxane mixture can be produced by mixing a polydimethylsiloxane monomer and a curing agent in a ratio of 10: 1. To remove the bubbles contained in the resulting polydimethylsiloxane mixture, the polydimethylsiloxane mixture may be degassed using a desiccator and a vacuum pump, and the polydimethylsiloxane mixture may be removed from the microwell mold 100, The gas can be removed again in the same manner. The polydimethylsiloxane mixture can then be baked at 80 ° C on a hotplate.

Thereafter, when the liquid state substrate material 70 is cured, the microwell substrate 200 with the steel beads 40 embedded therein can be produced. The microwell substrate 200 having the steel bead 40 embedded therein is separated from the lower plate 12 as shown in Fig. The steel beads 40 of the microwell substrate 200 having the steel beads 40 embedded therein can be removed using the magnets 80. Here, the magnet 80 has a size of? 15 mm x 2 mm and can have a magnetic force strong enough to remove the steel bead 40 embedded in the microwell substrate 200.

When the steel bead 40 of the microwell substrate 200 having the steel bead 40 embedded therein is removed, the space where the steel bead 40 of the microwell substrate 200 is embedded becomes the microwell 210 .

Since the microwell 210 is a space in which the steel bead 40 is embedded in the microwell substrate 200, the size (i.e., diameter) of the steel bead 40 and the size The size and / or shape thereof can be determined according to the size (i.e., diameter) of the substrate 22.

For example, referring to FIG. 9, when the microwell mold 100 is composed of a steel bead 40 having a diameter of 600 μm and a lower plate 12 having a lower hole 22 having a diameter of 300 μm, (210) may be formed corresponding to the size and shape of the steel bead (40) having a diameter of 600 mu m. When the microwell mold 100 is composed of a steel bead 40 having a diameter of 600 μm and a lower plate 12 having a lower hole 22 having a diameter of 550 μm, The microwells 210 can be formed corresponding to the shape and size of the hemispheres of the steel beads 40 having a diameter of 600 mu m. When the microwell mold 100 is composed of a steel bead 40 having a diameter of 800 μm and a lower plate 12 having a lower hole 22 having a diameter of 550 μm, the microwell 210 has a diameter of 800 μm And may be formed corresponding to the size and shape of the steel bead 40.

The absolute size of the microwell 210 may be proportional to the size of the steel bead 40 and the shape of the microwell 210 may be determined by the diameter of the lower hole 22 of the lower plate 12 and the diameter of the steel bead 40 ) Of the diameter. That is, as the diameter of the steel bead 40 is larger than the diameter of the lower hole 22 of the lower plate 12, the shape of the microwell 210 can be formed close to the shape of the steel bead 40, The opening of the microwell 210 may be formed to be more sharp and may become narrower. This can prevent planted cells from flowing out of the microwell 210 during cell culture in the microwell 210.

10 is a diagram illustrating a cell culture method using a microwell substrate according to an embodiment of the present invention.

Referring to FIG. 10, in step (a), a cell is planted in a microwell substrate 200 on which a microwell 210 is formed.

For example, a polydimethylsiloxane substrate on which microwells 210 are formed is cut out by using a punch for collecting a living tissue having a diameter of 14 mm. The resulting 14 mm diameter polydimethylsiloxane substrate is then sterilized in an autoclave sterilizer. Next, the entire polydimethylsiloxane substrate was coated with 4% pluronic F-127 overnight to prevent cells from adhering to the surface of the microwell. The air bubbles contained in the microwells 210 during the coating procedure can be removed using pipetting or an ultrasonic cleaner. Next, Pluronic F-127 is washed 3 times with phosphate-buffered saline (PBS). Subsequently, 1 mL of a cell medium containing 2 x 10 ⁶ cells is planted on the polydimethylsiloxane substrate. The seeding density may vary depending on the target cell spheroid and / or the target cell type. For cell-mediated solutions, adipose-derived stem cells (ASCs) may be used.

In step (b), 1 mL of medium is sucked with a 1000 μL pipette to remove the unfilled cells in the microwell 210. In this way, cells can be filled even in a space where the cells of the microwell 210 are not filled.

In step (c), the microwell substrate 200 is refilled with the cell medium.

In step (d), when the cells are cultured for a certain period of time, cells grow in the form of spheroids.

It will be apparent to those skilled in the art that various modifications, additions and substitutions are possible, without departing from the spirit and scope of the invention as defined by the appended claims. Should be regarded as belonging to the following claims.

10: Plate assembly
20: Trap hole
30: magnet array
40: Steel bead
50: Acrylic plate
60: bowl
70: Substrate material
100: microwell mold
200: Microwell substrate

Claims (10)

1. A method for manufacturing a microwell substrate in which a microwell is formed,
Assembling a plate assembly in which a plurality of trap holes are arranged by assembling an upper plate and a lower plate in which a hole array is formed;
Arranging a plurality of neodymium magnets in such a manner that each magnet is opposite to a neighboring magnet;
Stacking the plate assembly on the magnet array;
Filling the trap hole with a steel bead;
Removing the top plate from the plate assembly to produce a microwell mold; And
And fabricating the microwell substrate using the microwell mold,
When the upper plate is removed from the plate assembly, the microwell mold having the steel bead embedded in each lower hole of the lower plate is generated,
Wherein the magnet array is positioned below the microwell mold so that the steel bead is fixed to each lower hole of the lower plate by a magnetic force,
Wherein the size and shape of the microwell are determined according to the diameter of the steel bead and the diameter of the lower hole formed in the lower plate.
The method according to claim 1,
The upper plate and the lower plate are overlapped with each other such that the center of the upper hole formed in the upper plate and the lower hole formed in the lower plate coincide with each other. And the diameter of the hole is larger than that of the hole.
The method according to claim 1,
The step of filling the trap hole with a steel bead comprises:
Placing a steel bead in an amount exceeding the total number of the trap holes on the plate assembly; And
And sweeping the steel beads placed on the plate assembly with a non-metallic acrylic plate so that each trap hole is filled with steel beads.
The method of claim 3,
The step of sweeping the steel beads overlying the plate assembly comprises:
Filling and fixing the steel beads in the respective trap holes by the magnetic force of the magnet array; And
And filling remaining trap holes with remaining steel beads on the plate assembly. ≪ RTI ID = 0.0 > 15. < / RTI >
delete The method according to claim 1,
The step of fabricating the microwell substrate comprises:
Pouring the microwell mold on the magnet array into a bowl, pouring the substrate material in a liquid state to immerse the microwell mold in the vessel, and baking the substrate material to cure the substrate material;
Separating the microwell substrate with the steel bead from the lower plate when the substrate material is cured; And
And removing the steel beads embedded in the microwell substrate using a magnet.
delete delete A concave microwell having an opening is formed so as to correspond to a microwell mold having a steel bead embedded in each hole of the plate on which the hole array is formed,
The size and shape of the microwell are determined by the diameter of the steel bead and the diameter of the hole of the plate,
Wherein the absolute size of the microwell is proportional to the size of the steel bead and the shape of the microwell is determined by the difference between the diameter of the hole and the diameter of the steel bead.



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