KR20230083677A - A Method for 3-Dimensionally Aligned Culture of Cells - Google Patents

Abstract

The present invention relates to a method for three-dimensionally aligning and cultivating cells. Providing is the method for three-dimensionally aligning and cultivating cells comprising: a step of preparing bio-ink by mixing the cells with hydrogels; a step of outputting the bio-ink while moving a nozzle of a discharging device and forming an output with at least a part of a parallel pattern; a step of hardening the output; and a step of culturing a hardened product. According to the present invention, since the cells are cultivated at a high density in three-dimensional alignment depending on a parallel pattern, the present invention simulates or reproduces a texture of tissue in which the cells are formed, such as a muscle tissue-specific texture.

Description

Method for 3-Dimensionally Aligned Culture of Cells {A Method for 3-Dimensionally Aligned Culture of Cells}

The present invention relates to a method for arranging and culturing cells in three dimensions.

Alignment of cells means that cells are elongated to a certain level or higher and have a directionality. In vivo, cells are naturally aligned by not only microscopic factors such as the surface structure of extracellular matrix, but also macroscopic factors such as curves and shapes of tissues. It is not easy to reproduce the phenomenon in an in vitro culture process.

Conventionally, cell culture vessels have been improved for sorted culture of cells as described above. It was allowed to be cultured as it was. However, since the cultured surface as described above is manufactured using photolithography, E-beam lithography, etc., it is expensive to manufacture and has limitations in mass production, as well as a three-dimensional complex biological environment It was also not an easy problem to form a cultured surface that could mimic it with high precision.

Therefore, it is no longer necessary to research and develop a technology capable of sorting and culturing cells using a conventional culture vessel through methodological improvement rather than improvement of the culture vessel.

An object of the present invention is to provide a method for arranging and culturing cells in three dimensions.

In order to achieve the above object, the present invention provides a step of preparing bio-ink by mixing cells with hydrogel, and outputting the bio-ink while moving a nozzle of an ejection device to form an output of a pattern at least partially parallel to each other. , It provides a three-dimensional array culture method of cells comprising the steps of curing the output and culturing the cured product.

According to the present invention, since the cells can be cultured up to high density in a state aligned according to parallel patterns, there is an advantage in that the texture of the tissue formed by the cells, for example, the texture unique to muscle tissue can be simulated or reproduced. .

However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the following description.

1 shows a path in which a nozzle of a bio-ink ejection device is moved in an embodiment of the present invention.
FIG. 2 is a photograph of a cured product formed by outputting bio-ink while moving a nozzle of a bio-ink ejection device along the path shown in FIG. 1 in an embodiment of the present invention and curing an output product.
3 is a photograph of a process of culturing and further differentiating a cured product obtained by curing an output product in an embodiment of the present invention.
Figure 4 is a photograph showing the result of confirming the alignment state of the cells in the culture by immunostaining the differentiated culture in Example of the present invention.

Hereinafter, the present invention will be described in detail.

The present invention provides a method for arranging and culturing cells in three dimensions.

The three-dimensional sorting and culturing method of the cells of the present invention includes preparing bioink by mixing cells with a hydrogel, outputting the bioink while moving a nozzle of an ejection device to form an output of at least a part of a parallel pattern A step of curing the output product and culturing the cured product.

First, prepare bio-ink.

The bioink is a generic term for materials that can be applied to bioprinting technology to produce necessary structures, including materials derived from living organisms and materials for providing a biological environment in which the biologically derived materials can perform desired functions. It is done. In the present invention, the bio-ink may be prepared by mixing cells and hydrogel, and other additives including cell culture medium or physiologically active substances may be further included as needed.

The cells are cells to be cultured by arranging them in a certain direction, and may be cells before differentiation, cells in differentiation, or cells after differentiation. In particular, the cells may be cells constituting textured tissue, for example muscle cells, in particular myoblasts, myotubes, etc., but are not limited thereto.

The hydrogel is a gel using an aqueous liquid containing water as a dispersion medium, wherein the dispersoid may be a hydrophilic polymer. The hydrophilic polymer may be crosslinked by covalent or non-covalent bonds to form a three-dimensional network structure, and absorbs water in an aqueous environment due to the excellent absorbency inherent in the hydrophilic polymer and swells, but is not dissolved by the crosslinked structure. It may indicate a characteristic that does not exist. The hydrogel as described above serves as a support for maintaining an aligned state of the cells after being cured, and may be derived from nature such as microorganisms, animals, or plants, or artificially synthesized. For example, the hydrogel may contain a dispersoid such as gelatin, gellan gum, collagen, agar, alginic acid, hyaluronic acid, carrageenan, gum arabic, ghatti gum, pullulan gum, mannan gum, locust bean gum, xanthan gum, or the like alone or in combination with two or more. It may be mixed and dispersed in a dispersion medium containing water. The hydrogel is 0.1% to 15% by weight, such as 0.5% to 13% by weight, 1.0% to 11% by weight, 3.0% to 10% by weight, 4.0% to 9.0% by weight or 5.0% to 8.0% by weight. It can be a concentration of % by weight.

In addition, the cells are 1 × 10 ⁴ to 1 × 10 ¹² per 1 mL of the hydrogel, for example, 1 × 10 ⁵ to 1 × 10 ¹¹ , 1 × 10 ⁶ to 1 × 10 ¹⁰ or 1 × 10 It may be mixed in a ratio of ⁷ to 1×10 ⁹ and mixed with a hydrogel preheated to a temperature of 30 to 40 °C, for example, 35 °C to 38 °C.

In addition, the bio-ink prepared as described above is bioprinted to form an output product.

The bioprinting refers to the formation of a three-dimensional deposit through an automated or computer-assisted means equipped with a bioink ejection device such as a bioprinter, for example, ejection connected to a reservoir of bioink. The ejection of bioink from the bioprinter through a device to form a deposit having any suitable geometry, including circular, square, rectangular, triangular, polygonal, and irregular geometries. In particular, forming a three-dimensional deposit with bioink through bioprinting as described above is called 3D-bioprinting. Therefore, the bioprinted output may be a three-dimensional deposit, and within the output having a three-dimensional structure as described above, the cells may also be three-dimensionally arranged or arranged accordingly.

The bioprinting may be performed by outputting the bioink while moving a nozzle of the ejection device. In particular, the bioink may be continuously ejected while the nozzle of the ejection device moves, and the nozzle of the ejection device may move in a certain direction, particularly in one direction. Accordingly, an output formed by outputting bio-ink while the nozzle of the ejection device moves as described above may have a pattern having at least a part of a parallel shape. In a specific embodiment of the present invention, bio-ink was output while moving the nozzle of the ejection device along the path shown in FIG. 1, and as a result, an output having a parallel pattern as shown in FIG. there was.

In the bioprinting process as described above, the nozzle of the ejection device may be 20 gauge (G) to 30 gauge (G), for example, 21G to 29G, 22G to 28G, 23G to 27G, or 24G to 26G. The bioink may be ejected at a pressure of 30 kPa to 100 kPa, 40 kPa to 80 kPa, 45 kPa to 75 kPa, 50 kPa to 70 kPa, or 55 kPa to 65 kPa through a nozzle of the size to form an output product.

Then, the three-dimensional output product formed as described above may be cured.

The curing refers to a process in which the hydrogel in the print is crosslinked by a covalent or non-covalent bond, and a material that induces or promotes such crosslinking may be used as a curing agent, if necessary, depending on the type of the hydrogel. Examples of the curing agent include transglutaminase, genipin, glutaraldehyde, diisocyanates, carbodiimides, calcium salts (eg, calcium chloride), Thrombin and the like may be used, and an appropriate curing agent may be selectively used according to the type of the hydrogel. For example, when the hydrogel is gelatin, transglutaminase may be used as the curing agent, and when the hydrogel is alginic acid, a calcium salt such as calcium chloride may be used as the curing agent.

The curing may be performed by treating the printed object with a solution containing the curing agent, at an appropriate temperature (eg, 30 to 40 ° C, particularly 35 ° C to 38 ° C) for a sufficient time (eg, 5 to 60 minutes). , 10 minutes to 50 minutes, 20 minutes to 40 minutes, particularly 25 minutes to 35 minutes).

Through the curing process as described above, the hydrogel in the print is crosslinked to form a three-dimensional network structure, and cells can be stably placed using the three-dimensional network structure as a support, and at least some formed on the print The cells can be fixed in a three-dimensionally aligned state according to a parallel pattern.

The cured product cured as described above may be cultured.

The culturing is a process of proliferating cells included in the cured material, and through this, the size and number of cells included in the cured material are increased. The culturing may be performed using a medium suitable for culturing according to the type of the cell, and an appropriate culture medium according to the type of the cell may be appropriately selected and used by a person skilled in the art. On the other hand, if the cells are fully differentiated cells such as myotubes, sorted culture of the cells is possible through the above-described culture process.

On the other hand, when the cells are before differentiation or are in the process of differentiation, a process of further differentiating the cultured culture as described above may be further performed. When the cells are simply cultured before or during differentiation, only the size and number of cells may be increased in a state before or during differentiation, and thus completely differentiated cells in the culture through the above differentiation process. can make it For example, when the cells are myoblasts, they can be differentiated into myotubes through the above process. Differentiation as described above may be performed by further culturing the culture using a differentiation induction medium, and the differentiation induction medium may also be appropriately selected and used by a person skilled in the art according to the type of cell.

When the final culture or additional differentiation is performed through the sorted culture method of the present invention as described above, cells with increased size and number are arranged at high density along the three-dimensional structure of the hydrogel in the cured product. Depending on the method, it is possible to simulate the texture of the tissue formed by the cells. For example, when myotube cells are cultured according to the present invention or myoblasts are cultured according to the present invention and then further differentiated, muscle cells are arranged at high density according to a pattern in which at least some of the myotubes are parallel to each other in the hardened material. Therefore, it is possible to reproduce the texture inherent in muscle tissue.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

Formation of an output in which cells are arranged in three dimensions

After preheating 1 mL of DPBS (Dulbecco's phosphate-buffered saline) containing 0.67% gellan gum and 6.00% gelatin at 37 ° C, a pellet of 1 × 10 ⁸ cells of C2C12 cells, which are mouse myoblasts, was added and suspended. Thus, bio-ink was prepared. The bioink prepared as described above is mounted on the output barrel of a 3D bioprinter, and the bioink is output at a pressure of 60 kPa while moving a 25 gauge (G) nozzle along the path shown in FIG. 1 to a culture dish ( After forming an output having a pattern as shown in FIG. 1 on a culture dish), the output was treated with DPBS containing 5% of transglutaminase and reacted at 37° C. for 30 minutes. As a result, as shown in FIG. 2, a cured product having a thickness of 0.25 to 0.5 mm having a pattern having the same shape as the path of FIG. 1 was obtained.

Then, the gel-like cured material was removed from the bottom of the culture dish and placed on a low-attachment plate containing 10% FBS (fetal bovine serum) and 1% antibiotic antimycotic solution. Using DMEM (Dulbecco's modified eagle medium), 5% CO ₂ was supplied for 5 days at 37 ° C. for 5 days, as shown in FIG. 3, the suspension culture was carried out in three dimensions. Then, it was cultured again for 8 days in the same manner by replacing the differentiation induction medium of DMEM containing 2% HS (horse serum) and 1% antibiotic antibacterial solution.

Check cell alignment

The culture differentiated in Example 1 was washed three times with PBS (phosphate buffer saline), treated with a 4% paraformaldehyde solution, fixed at 37° C. for 15 minutes, and then washed three times with PBS. did Then, PBS containing 0.5% Triton X-100 was treated, reacted at room temperature for 15 minutes, washed three times with PBS, and then treated with PBS containing 2% BSA (bovine serum albumin) and treated at room temperature for 1 minute. reacted over time. In addition, the MyHC antibody (primary) was mixed with PBS containing 0.1% BSA at a ratio of 1:400, reacted overnight at 4 ° C, washed three times with PBS, and then the secondary antibody and DAPI were mixed with 0.1% BSA. was mixed in a PBS solution containing the mixture at a ratio of 1:200, followed by reacting at room temperature for 2 hours. Then, the reaction was repeated three times by treating with PBS containing 0.1% of Tween 20 for 5 minutes, and observing the culture under a fluorescence microscope.

As a result, as shown in FIG. 4 , it was confirmed that myotubes differentiated from myoblasts were cultured in a neatly aligned state according to the path of FIG. 1 .

In the above, preferred embodiments of the present invention have been described by way of example, but the scope of the present invention is not limited to the specific embodiments as described above, and those skilled in the art are within the scope described in the claims of the present invention. will be able to change accordingly.

Claims (8)

Preparing bioink by mixing cells with hydrogel;
outputting the bio-ink while moving a nozzle of an ejection device to form an output product of a pattern at least partially parallel to each other;
curing the output; and
culturing the cured product;
Including, three-dimensional alignment culture method of cells. The method of claim 1,
The cell is a three-dimensional array culture method of cells that are myoblasts or muscle cells. The method of claim 1,
The hydrogel is preheated to a temperature of 30 to 40 ℃, three-dimensional alignment culture method of cells. The method of claim 1,
In the hydrogel, the dispersoid is at least one selected from the group consisting of gelatin, gellan gum, collagen, agar, alginic acid, hyaluronic acid, carrageenan, gum arabic, ghatti gum, pullulan gum, mannan gum, locust bean gum, and xanthan gum. That is, a three-dimensional alignment culture method of cells. The method of claim 1,
Wherein the bio-ink is output at a pressure of 30 kPa to 100 kPa from a nozzle of 20 gauge (G) to 30 gauge (G), a three-dimensional alignment culture method of cells. The method of claim 1,
The curing is performed by treating the output with a curing agent-containing solution, a three-dimensional alignment culture method of cells. The method of claim 6,
The curing agent is a group consisting of transglutaminase, genipin, glutaraldehyde, diisocyanates, carbodiimides, calcium chloride and thrombin At least one selected from, three-dimensional array culture method of cells. The method of claim 1,
Further culturing the culture with a differentiation induction medium;
Which further comprises, a three-dimensional alignment culture method of cells.

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