KR102302770B1 - 3D printing bio-ink composed of human-derived elements and having tissue-specific cell differentiation effect, and methods of making the same - Google Patents

Abstract

The present invention relates to a bio-ink composition for 3D (Three dimensional, 3D) printing and a manufacturing method thereof.
In the present invention, it is possible to provide a bio-ink composition having a synergistic effect on cell differentiation into a specific tissue according to the tissue specificity of a human-derived component contained in the bio-ink composition, and a method for producing the same.

Description

3D printing bio-ink composition containing human-derived components and having a tissue-specific cell differentiation effect, and a method for manufacturing the same }

The present invention relates to a bio-ink composition for 3D (Three dimensional) printing and a method for manufacturing the same, and more particularly, has a synergistic effect on cell differentiation into specific tissues according to the tissue specificity of human tissue-derived components contained in the bio-ink composition. It relates to a bio-ink composition and a method for manufacturing the same.

Tissue engineering is the replacement of lost or damaged tissues by creating human tissues or organs based on biology, engineering, and materials engineering. Due to industrialization and aging, the number of patients with loss or damage to human tissue is increasing, and as a result, the scope of the tissue engineering field for replenishing the lost or damaged tissue is expanding. Among them, 3D bioprinting is a field that has recently received attention.

3D bioprinting is to obtain an image by scanning a target structure in three dimensions, and making the scanned image through cells and bio-ink in the form of a three-dimensional structure. Common 3D printing technologies include an extrusion-based process, an inkjet-based process, and a laser-based process. Among them, the extrusion-based process is mainly used as bioprinting that creates a three-dimensional structure with cells. Since bioprinting is the key to including living cells, bioink, a material that forms a three-dimensional structure by mixing with cells, is the most important part of 3D bioprinting.

Bio-ink should have both physicochemical properties that can maintain a microscopic 3D structure and a level of biological safety that can be used with living cells. As physicochemical properties, the bio-ink should have printability that can be extruded from a 3D printer, and rheological characteristics that can maintain the 3D structure even after printing. Because bio-ink has to play a role as a scaffold for cell growth and differentiation due to its biological safety characteristics, it must have cell affinity basically. In addition, the output output by 3D bioprinting contains the materials constituting the bio-ink as it is, and at the same time becomes a 3D output that can be implanted in the body. It should be a biocompatible material, and it should have properties that can control the growth and differentiation of cells in the in vitro culture or transplantation environment of the output. In addition, the material elements constituting the bio-ink should show the decomposition tendency of the printed matter in the body at a level consistent with the regeneration tendency of the new tissue, while the new tissue is regenerated at the implantation site through the printout after implantation in the body.

Biocompatible materials that can constitute the bio-ink include agarose, alginate, chitosan, collagen, decellularized extracellular matrix, and fibrin/fibrinogen. /fibrinogen), gelatin, graphene, hyaluronic acid, hydroxyapatite, polycaprolactone (PCL), polylactic acid (PLA), poly (DL) -Lact-co-glycolic acid (poly-D, L-lactic-co-glycolic acid, PLGA) and pluronic (pluronic F 127), etc. Among them, alginic acid (alginate) is a divalent cation (divalent cation), it has the characteristic of gelation by crosslinking, and this property satisfies the printability of bio-ink and mechanical properties after printing.However, alginic acid alone has low cell affinity, It does not provide an environment suitable for cells to attach and grow.

Micronized human tissue is decellularized and then freeze-dried and crushed allogeneic tissues such as skin, bone, ligament, tendon, blood vessel, cartilage, heart valve, amnion, fascia, nerve, and pericardium, which are donated after death. Through the process, it is made into particles with a size of tens to hundreds of micrometers. Since microparticulated human tissue is a human-derived material that has only undergone a decellularization process, the extracellular matrix (ECM) is the main component. More than 90% of the extracellular matrix component is collagen, a structural protein, and the remaining 10% is glycoprotein, fibronectin, laminin, and glycosaminoglycan (GAG). In addition, it is known that tissue-specific growth factors and some cytokines exist to promote cell growth and tissue-specific differentiation of undifferentiated cells. In particular, components such as laminin, fibronectin, and glycosaminoglycan in microparticulated human tissue act as a surface binding protein and convert cytokines or growth factors secreted from cells into extracellular matrix. It is known to aid in cell growth and differentiation by attaching to the structure. Therefore, when the bio-ink containing the extracellular matrix is cultured with cells, the delivery of cytokines and growth factors necessary for cell growth and differentiation is easier, and the structure can be changed into a more tissue-like form. (Non-Patent Document 1). Moreover, the extracellular matrix extracted from different tissues has tissue specificity that has the characteristics of each tissue, so cytokines or growth factors suitable for the tissue are attached to the environment suitable for cells to survive and form each tissue. to create (Non-Patent Document 2).

In addition, the fine-grained human tissue is in the form of micro-unit particles, and when it is hydrated by mixing it with a bio-derived polymer in the form of a hydrogel, it is physically uniformly mixed, and the mechanical strength of the bio-ink can be adjusted according to the content. . Since the extracellular matrix used in the existing bio-ink is dissolved in pepsin, acetic acid or hydrochloric acid to produce a liquid hydrogel, there is a limit to improving the mechanical properties of the extracellular matrix itself. Therefore, bio-ink including fine-grained human tissue has higher mechanical properties than extracellular matrix solution bio-ink, and the fine-grained human tissue itself is a component that has the shape of a tissue and is a more suitable environment for cells to live. This can be formulated.

Finally, while the extracellular matrix extracted from xenogeneic tissues such as cattle, pig, and horse has concerns about animal-derived viruses and factor cross-infection, the human-derived extracellular matrix is free from the safety problem of heterogeneous substances in the body. It can be used as the most suitable material for application to the human body. Therefore, the microparticulate human tissue has a very high cell affinity, and since it is an extracellular matrix derived from the human body, it has the advantage that it can be transplanted into the body without almost any immune rejection reaction, foreign body reaction, inflammatory reaction, and the like.

[1] D. Choudhury et al. Trend in Biotechnology. 2018 [2] R. Londono et al. Annals of Biomedical Engineering. 2015.

An object of the present invention is to provide a bio-ink composition for 3D bioprinting containing a component derived from human tissue.

Specifically, an object of the present invention is to provide a bio-ink composition capable of cell growth and tissue-specific differentiation within a 3D printed structure by applying various components derived from human tissue to the bio-ink composition, and a method for manufacturing the same.

The present invention provides a bio-ink composition for 3D printing comprising a microparticulated human tissue-derived component and a biocompatible polymer.

The present invention also provides a method of manufacturing a structure comprising the step of manufacturing a structure by extruding a bio-ink composition for 3D printing comprising a component derived from a microparticulated human tissue and a biocompatible polymer.

The present invention provides a bio-ink composition containing microparticulated human tissue.

The bio-ink composition according to the present invention exhibits excellent effects in cell growth and differentiation due to cell affinity, and can provide tissue-specific differentiation according to the origin of human tissue in the bio-ink. In addition, the bio-ink composition can improve mechanical properties because it contains microparticulated human tissue components.

Cells in the output output by applying the bio-ink composition according to the present invention to 3D bio-printing can be differentiated according to the origin of the human tissue of the bio-ink. When the bio-ink composition contains living cells, the output output by 3D bioprinting contains microparticulate human tissue components, so the cell affinity is high and the cells can survive for a long time. It contains differentiation factors and can regulate cell function and differentiation.

In addition, the bio-ink composition of the present invention can be printed in a form that is extruded through a 3D printer for bio-printing, maintains a 3D structure after printing, and can be used as a structure for tissue regeneration.

In addition, in the present invention, the bio-ink composition containing various human tissue-derived components can be applied to various fields by applying the bio-ink composition together with the cells of each tissue as a biomaterial through 3D bio-printing.

Figure 1 (A) is a cross-linked structure printed by 3D printer with M-ADM-based bio-ink (left) filled in a syringe by mixing acellular allogeneic dermal powder (M-ADM) with a biocompatible polymer It is (right). (B) shows the cross-linked structure by mixing M-AC-based cartilage powder (M-AC) with a biocompatible polymer and filling a syringe with M-AC-based bio-ink (top left) and printing through an extrusion-based 3D printer ( idol). The crosslinked structure after printing maintains the structure against external pressure (below).
Figure 2 (A) is the result of culturing the M-ADM-based bio-ink containing human dermal fibroblasts and confirming the survival of the cells after 1, 7, 21, and 28 days with a fluorescence microscope at 100 magnification. Green indicates live cells, red indicates dead cells, and the graph is the result of quantifying the number of living cells for each culture period.
2B is a result of culturing the M-AC-based bio-ink containing human chondrocytes and confirming the survival of the cells after 1, 7, 21, and 28 days with a fluorescence microscope at 100 magnification. The graph is the result of quantifying the number of living cells expressed in green for each culture period.
Figure 3 (A) is the result of culturing the M-ADM-based bio-ink containing adipose-derived stem cells and confirming the survival of the cells after 7, 14, 21, and 28 days with a fluorescence microscope at 100x magnification. The graph is the result of quantifying the number of living cells expressed in green for each culture period.
Figure 3 (B) is the result of confirming the survival of the cells after culturing the M-AC-based bio-ink containing adipose-derived stem cells with a fluorescence microscope at 100x magnification. The graph is the result of quantifying the number of living cells expressed in green for each culture period.
Figure 4 (A) is a photograph of the tissue section after 21 days of culturing M-ADM-based bio-ink containing adipose-derived stem cells and staining with DAPI, Collagen type 1, and alpha-SMA antibody. (B) is the result of quantifying the mRNA expression level of FSP1 by qRT-PCR analysis after 3 weeks and 5 weeks by culturing M-AC-based bio-ink containing adipose-derived stem cells.
4 (C), (D) are photographs of culturing human chondrocytes and adipose-derived stem cells and staining the tissue sections with DAPI, Aggrecan, and CD44 antibodies. (C) is the result of confirming the chondrogenic differentiation of adipose-derived stem cells by expressing human chondrocytes and adipose-derived stem cells, respectively, and culturing them for 4 weeks in an M-AC-based bio-ink in (D).
4(E) is the result of quantifying the mNA expression levels of Collagen type II and SOX-9 by qRT-PCR analysis after 1 week and 2 weeks after culturing M-AC-based bio-ink containing adipose-derived stem cells. .

The present invention relates to a bio-ink composition for 3D printing comprising a microparticulated human tissue-derived component and a biocompatible polymer.

In an embodiment of the present invention, fibroblast viability in M-ADM-based bio-ink, human chondrocyte viability in M-AC-based bio-ink, and fat-derived in M-ADM-based and M-AC-based bio-ink The cell affinity of the bio-ink composition of the present invention was confirmed by analyzing the stem cell viability. In addition, by confirming the differentiation of adipose-derived stem cells into fibroblasts in the M-ADM-based bio-ink and the differentiation of adipose-derived stem cells into chondrocytes in the M-AC-based bio-ink, the bio-ink composition of the present invention activity was confirmed. In addition, the applicability of the bio-ink composition to 3D bio-printing was confirmed.

Hereinafter, the bio-ink composition for 3D printing of the present invention will be described in more detail.

The bio-ink composition (or referred to as bio-ink) according to the present invention includes a micro-particulated human tissue-derived component and a biocompatible polymer.

In the present invention, human tissue-derived components refer to decellularized tissue-derived components, and provide conditions for creating a microenvironment similar to the original tissue. Since it is not an artificially generated material, but a structure formed through interaction with cells in the human body, it has tissue-specific components and phases, and can provide an environment favorable for cell growth and differentiation.

In the present invention, the microparticulated human tissue-derived component means that the human tissue is decellularized and pulverized to a size of 10 to 1000 μm. The microparticulated human tissue-derived component can be used for the purpose of improving biocompatibility, cell adhesion, and physical properties of the bio-ink.

In one embodiment, the human tissue may be obtained from allogeneic tissues such as skin, bone, ligament, tendon, blood vessel, cartilage, heart valve, amniotic membrane, fascia, nerve, and pericardium, which are donated after death.

In one embodiment, the component derived from human tissue is a decellularized human tissue, including Acellular Dermal Matrix (ADM), Acellular Cartilage (AC), and Acellular Adipose (AA). , and may include at least one selected from the group consisting of Acellular Bone (AB) and Acellular Bone Derived Mineral (ABDM). In this case, the allogeneic cartilage may be hyaline cartilage, elastic cartilage, fibrous cartilage, or a mixture thereof. In particular, the extracellular matrix is the main component of microparticulated human tissue-derived components derived from skin and cartilage, thereby enhancing the tissue specificity of the bio-ink.

In one embodiment, the human tissue-derived component is microparticulated, and the average particle diameter of the microparticulated human tissue-derived component may be 10 to 1000 um, 10 to 700 um, or 10 to 500 um. In the above particle size range, the bio-ink can implement an input shape through a micro-unit 3D printing nozzle, and can impart strength to maintain the structure of the structure. In addition, unlike the bio-ink produced in a hydrogel type by treating the extracellular matrix with an enzyme, the original structure of the tissue is preserved, thereby providing a favorable environment for cell growth and differentiation.

In one embodiment, when the bio-ink composition contains acellular dermal matrix (ADM) as a component derived from microparticulate human tissue, it can be expressed as an M-ADM-based bio-ink, and acellular allogeneic cartilage (Acellular). Cartilage, AC) can be expressed as M-AC-based bio-ink.

In one embodiment, the microparticulate human tissue-derived component comprises the steps of (S1) decellularizing human tissue; and

(S2) It can be prepared through the step of microparticles of the decellularized human tissue.

The order of the steps (S1) and (S2) is not limited, and the steps (S1) and (S2) may be sequentially performed, or (S1) may be performed after receiving the steps (S2).

Step (S1) is a step of decellularizing the human tissue, and the decellularization may be performed by a general method in the art.

In the above step, for example, a delipidation process, a decellularization process and/or a hair removal process may be performed. The delipidation process may be performed using a polar solution and a non-polar solution such as alcohol and/or hexane, and the decellularization process may be performed using a basic solution such as sodium hydroxide (NaOH). In addition, the hair removal process may be performed using an alkaline solution such as _{Na 2 S.} The process may be appropriately selected and performed according to the human tissue used.

After the decellularization, a bleaching process and/or a neutralization process may be additionally performed.

In addition, moisture may be removed by freeze-drying the decellularized human tissue in order to easily perform microparticle formation, which will be described later.

Step (S2) is a step of fine-graining the decellularized human tissue.

The fine particle formation may be performed by physically pulverizing human tissue or chemically particleizing the human tissue.

The type of physical process is not particularly limited, and for example, it can be pulverized by using a pulverizer, particularly a micro pulverizer, pulverized by rotating a ball, or pulverized by pulverizing a ball or rod at a cryogenic temperature. can In addition, the type of chemical process is not particularly limited, and for example, a process of decellularization from tissue through an organic/inorganic solvent, a basic alkaline solvent, etc. may be performed to form microparticles.

In one embodiment, the microparticulated human tissue-derived component may include a differentiation factor. Differentiation factors refer to substances that can regulate the growth and function of cells. The differentiation factor may act as a growth factor, and in particular, when applied to stem cells, may induce differentiation into other cells.

For example, the differentiation factor is Transforming Growth Factor-beta (TGF-β), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), vascular endothelial growth factor It may be at least one selected from the group consisting of (Vascular Endothelial Growth Factor, VEGF), insulin-like growth factor (IGF), and bone morphogenic protein (BMP).

In one embodiment, the content of the microparticulated human tissue-derived component may be 2 to 20 parts by weight based on the total weight (100 parts by weight) of the bio-ink composition. In addition, the microparticulate human tissue-derived component may be present in an amount of 5 to 10% (w/v) in the bio-ink composition.

In the present invention, the biocompatible polymer imparts biocompatibility to the fabricated structure.

In one embodiment, the type of biocompatible polymer is not particularly limited, for example, agarose, alginic acid (alginate), chitosan (chitosan), collagen (collagen), decellularized extracellular matrix (decellularized extracellular) matrix), fibrin/fibrinogen, gelatin, graphene, hyaluronic acid, hydroxyapatite, polycaprolactone (PCL), polylactic acid acid, PLA), poly (DL-lactic-co-glycolic acid, poly-D, L-lactic-co-glycolic acid, PLGA) and at least one selected from the group consisting of pluronic F 127 may be used can

In one embodiment, alginic acid may be used as the biocompatible polymer. The alginic acid may include calcium, sodium, potassium, ammonium, or magnesium alginic acid. The alginic acid has excellent biodegradability, improves physical properties of the bio-ink, and is easy to cross-link. Specifically, alginic acid (alginate) is crosslinked through chelation of divalent cations and gels, so it is possible to satisfy printability as a bio-ink and mechanical properties after printing through the gelling property. have.

In one embodiment, the content of the biocompatible polymer may be 0.5 to 10 parts by weight based on the total weight (100 parts by weight) of the bio-ink composition. In the range of parts by weight, desired physical properties may be satisfied when manufacturing the support.

In the present invention, the bio-ink composition may further include one or more selected from the group consisting of cells and differentiation factors.

In one embodiment, cells to be used for tissue regeneration may be used as cells, for example, stem cells, fibroblasts, chondrocytes, osteoblasts, epithelial cells. (Epithelial cell), keratinocyte (Keratinocyte), endothelial cell (Endothelial cell), and one or more selected from the group consisting of neurons (Neuron) may be used.

In addition, the differentiation factor may be included in the microparticulate human tissue-derived component itself, but by additionally adding it to the ink composition, the effect of the differentiation factor, ie, cell growth and function control, can be maximized.

For example, the differentiation factor may be any of the above-described types, specifically, Transforming Growth Factor-beta (TGF-β), Fibroblast Growth Factor (FGF), epidermal growth factor ( Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor (VEGF), Insulin-like Growth Factor (IGF) and Bone Morphogenic Protein (BMP) selected from the group consisting of You can use more than one.

In one embodiment, the content of one or more selected from the group consisting of cells and differentiation factors may be 0.001 to 20 parts by weight based on the total weight (100 parts by weight) of the ink composition. The cells may be included in an amount of 10 to 20 parts by weight, and the differentiation factor may be included in an amount of 0.001 parts by weight or more.

In addition, the ink composition of the present invention may contain water.

In addition, the present invention provides a method for preparing the above-described bio-ink composition.

The bio-ink composition according to the present invention can be prepared by mixing the above-mentioned components, that is, a component derived from microparticulated human tissue and a biocompatible polymer. Since the microparticulate human tissue-derived component is in the form of micro-sized particles, it can be physically mixed evenly without a separate mixing process when hydrated with a bio-derived polymer. In addition, in the present invention, the mechanical strength of the bio-ink can be adjusted according to the content of the microparticulated human tissue-derived component.

In addition, the present invention provides a method of manufacturing a structure comprising the step of manufacturing a structure by extruding a bio-ink composition for 3D printing comprising a component derived from a microparticulated human tissue and a biocompatible polymer.

In one embodiment, the bio-ink composition according to the present invention can be applied to 3D bio-printing.

The 3D bioprinting refers to a process of three-dimensionally scanning a target structure to obtain an image, and converting the scanned image through bio-ink into a three-dimensional structure. In the present invention, it can be applied to an extrusion-based process.

In the present invention, the extrudate of the bio-ink composition for 3D printing may be expressed as a structure, an output, or a scaffold. The bio-ink composition according to the present invention can serve as a support for cell growth and differentiation, and has biocompatibility and biocompatibility. Therefore, it can be easily transplanted into the body, and the growth and differentiation of cells can be controlled in an in vitro culture or in vivo transplantation environment.

In the present invention, the structure can be manufactured by extruding the bio-ink composition using a 3D printer. Specifically, it is possible to manufacture a structure by printing 3D drawing data according to a program designed in advance with a 3D computer.

In the present invention, the structure can be manufactured by appropriately adjusting the extrusion force and the extrusion speed according to the purpose of manufacture.

The present invention may further include a step of crosslinking the extruded structure.

The crosslinking may be performed through a process generally used in the art.

In one embodiment, when alginic acid is used as the biocompatible polymer, the alginic acid is cross-linked through chelation of a divalent cation to gel, so cross-linking can be performed using this property.

In the present invention, by providing a bio-ink composition for 3D bio-printing, the structure completed through bio-printing can be used for regeneration of living tissue. Specifically, a construct made of bio-ink has biocompatibility, and can be usefully used in bio-tissue regeneration, tissue engineering, and regenerative medicine because it can induce tissue regeneration by containing cells in the support and attaching to the surface.

The present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited to the following examples, and it will be understood by those skilled in the art that various changes, modifications, or applications are possible within the scope without departing from the technical matters derived from the matters described in the appended claims. will be.

Example

Example 1. Preparation of bio-ink containing micro-particulated acellular dermal matrix (M-ADM) tissue

(1) M-ADM powder preparation

Skin tissue (collected from cadavers donated by a tissue bank for non-profit patient care) was decellularized.

First, the skin tissue was subjected to a delipidation process using alcohol and hexane. Thereafter, the epidermis and cells were removed using sodium hydroxide (NaOH), and hair attached to the tissue was removed using _{sodium sulfide (Na 2 S).} Next, after hydrogen peroxide (H ₂ O ₂ ), neutralization and washing were performed using distilled water. The washed skin tissue was lyophilized to remove moisture, and the tissue was pulverized using a micro grinder. During the pulverization, a dermal tissue powder of 500 μm or less was prepared by passing it through a sieve having a diameter of 250 μm to 500 μm.

The prepared powdery tissue was sterilized.

(2) M-ADM-based bio-ink manufacturing

A bio-ink composition in the form of a hydrogel was prepared by mixing the M-ADM powder prepared in (1) and an aqueous alginic acid solution.

Alginic acid 1-2% (w/v) aqueous solution was prepared. Alginic acid aqueous solution was used after filtration with a 0.22 μm syringe filter.

M-ADM 5-10% (w/v) was hydrated in an aqueous alginic acid solution. Two syringes were connected to a Luer adapter to uniformly hydrate to prepare a composition.

Figure 1 (A) is a state in which the M-ADM-based bio-ink is filled in the syringe.

Example 2. Preparation of Bio-Ink Containing Micronized Acellular Cartilage (M-AC) Tissue

(1) Preparation of M-AC powder

Cartilage tissue (collected from a cadaver donated by a tissue bank for patient treatment for non-profit purposes) was pulverized using a micro grinder. The pulverization was passed through a sieve having a caliber of 250 to 500 um to obtain a cartilage tissue powder of 500 μm or less.

Thereafter, the cartilage tissue powder was obtained and then decellularized.

First, the cartilage tissue was subjected to a delipidation process using alcohol and hexane. Then, cells were removed using sodium hydroxide (NaOH), and washed with alcohol (Ethanol) and distilled water. After removing moisture by freeze-drying, the tissue in powder form was sterilized with an electron beam.

(2) M-AC-based bio-ink manufacturing

A bio-ink composition in the form of a hydrogel was prepared by mixing the M-AC powder prepared in (1) and an aqueous alginic acid solution.

Alginic acid 1-2% (w/v) aqueous solution was prepared. Alginic acid aqueous solution was used after filtration with a 0.22 μm syringe filter.

M-AC 5-10% (w/v) was hydrated in an aqueous alginic acid solution. Two syringes were connected to a Luer adapter to uniformly hydrate to prepare a composition.

1(B) is a view of filling a syringe with M-AC-based bio-ink.

Experimental Example 1. Cell affinity analysis of bio-ink

(1) Analysis of viability of fibroblasts or human chondrocytes in bio-ink

① Method

The cell affinity of the M-ADM-based bio-ink prepared in Example 1 was confirmed using Human Dermal Fibroblasts.

Specifically, 2×10 ⁷ cells of human dermal fibroblasts were mixed with 1 ml of M-ADM-based bio-ink in 100 μl of medium. Mixing was uniformly hydrated by connecting two syringes to a Luer adapter. The bio-ink mixed with the cells was extruded with a 22G injection needle to form a grid pattern structure having a width of 10 mm, a length of 10 mm, and an interval of 2.5 mm.

When the grid-shaped structure was completed, 1% calcium chloride solution was applied to the structure for 5 to 10 minutes for cross-linking. The cross-linked structure was washed with phosphate buffer (PBS), immersed in a cell culture medium, and cultured in a cell incubator.

Cell affinity analysis was performed as follows.

After 1 day, 1 week, 2 weeks, 3 weeks, and 4 weeks of culturing the live cell structure, analysis was performed using a Live/dead cell viability assay kit (Life Technology, USA). A medium in which 0.5 μl/ml Calcein-AM and 2 ul/ml Ethidium homodimer-1 were dissolved was immersed in the construct and reacted for 30 minutes. After the reaction, it is confirmed through a confocal microscope (LSM 700, Carl Zeiss, Germany). Specifically, cell survival inside the construct was confirmed by focusing at intervals of 10 μm to a depth of about 200 μm.

Meanwhile, the cell affinity of the M-AC-based bio-ink prepared in Example 2 was confirmed using human chondrocytes.

Specifically, 2×10 ⁷ cells of human chondrocytes were mixed with 1.8 ml of M-AC-based bio-ink in 200 μl of medium. Mixing was uniformly hydrated by connecting two syringes to a Luer adapter. The bio-ink mixed with the cells was extruded with a 22G injection needle to form a grid pattern structure having a width of 10 mm, a length of 10 mm, and an interval of 2.5 mm.

When the grid pattern structure was completed, 1% calcium chloride solution was applied to the structure for 7 to 10 minutes for cross-linking. The cross-linked structure was washed with phosphate buffer (PBS), immersed in a cell culture medium, and cultured in a cell incubator.

Cell affinity analysis was performed in the same manner as the M-ADM analysis method.

② Results

The results of cell affinity analysis of the M-ADM-based bio-ink are shown in FIG. 2(A).

2(A), it can be seen that the number of living cells increases as time passes. Through this, it can be confirmed that cells can not only survive in the bio-ink but also proliferate.

In addition, the graph of FIG. (A) is the result of quantifying the said result. After 4 weeks of culture, it can be seen that the number of cells increased 4 times or more compared to after 1 day of culture.

Meanwhile, the cell affinity analysis result of the M-AC-based bio-ink is shown in FIG. 2(B).

2(B), as in the M-ADM-based bio-ink, it can be confirmed that the number of living cells increases over time. In addition, it can be seen that the cells not only survive in the bio-ink but also proliferate.

(2) Viability of adipose-derived stem cells in bio-ink

① Method

The cell affinity of the M-ADM-based bio-ink prepared in Example 1 was confirmed using adipose tissue-derived stem cells.

Specifically, 1 ml of M-ADM-based bio-ink was mixed with 100 μl of a cell suspension containing 1×10 ^{7 cells of adipose-derived stem cells.} Mixing was uniformly hydrated by connecting two syringes to a Luer adapter. The bio-ink mixed with the cells was extruded with a 22G needle to form a grid-like structure with a width of 10 mm, a length of 10 mm, and an interval of 2.5 mm.

When the grid-shaped structure was completed, 1% calcium chloride solution was applied to the structure for 5 to 10 minutes for cross-linking. The cross-linked structure was washed with PBS, immersed in a cell culture medium, and cultured in a cell incubator.

Cell affinity analysis was performed as follows.

After 1, 2, 3, and 4 weeks of culture, the cells in which the cells were alive were stained with a Live/dead cell viability assay kit (Life Technology, USA) and confirmed through a confocal microscope (LSM 700, Carl Zeiss, Germany).

Meanwhile, the cell affinity of the M-AC-based bio-ink was confirmed using adipose tissue-derived stem cells.

Specifically, 1.8 ml of M-AC-based bio-ink was mixed with 200ul of a cell suspension containing 2×10 ^{7 cells of adipose-derived stem cells.} Mixing was uniformly hydrated by connecting two syringes to a Luer adapter. The bio-ink mixed with the cells was extruded with a 22G needle to form a grid-like structure with a width of 10 mm, a length of 10 mm, and an interval of 2.5 mm.

When the grid-shaped structure was completed, 1% calcium chloride solution was applied to the structure for 7 to 10 minutes for cross-linking. The cross-linked structure was washed with PBS, immersed in a cell culture medium, and cultured in a cell incubator.

Cell affinity analysis was performed in the same manner as in the M-ADM.

② Results

The results of cell affinity analysis of the M-ADM-based bio-ink are shown in FIG. 3(A).

Referring to FIG. 3(A) , it can be seen that the amount of cells increases in the bio-ink structure as the culture time elapses. In addition, it can be confirmed that by appropriately adjusting the ratio of M-ADM and alginic acid in the bio-ink composition, cell compatibility and cell affinity for survival and proliferation of adipose-derived stem cells can be improved.

The graph in FIG. 3(A) is the result of quantifying the living cells expressed in green in the photograph. Through this, it can be confirmed that cell proliferation increases with time.

The results of cell affinity analysis of the M-AC-based bio-ink are shown in FIG. 3(B).

Referring to FIG. 3(B) , it can be seen that the amount of cells increases in the bio-ink structure as the culture time elapses. In addition, it can be confirmed that by appropriately adjusting the ratio of M-AC and alginic acid in the bio-ink composition, cell compatibility and cell affinity for survival and proliferation of adipose-derived stem cells can be improved.

The graph in FIG. 3(B) is the result of quantifying the living cells expressed in green in the photograph. Through this, it can be confirmed that cell proliferation increases with time.

Experimental Example 2. Confirmation of the activity of bio-ink

(1) Confirmation of differentiation of adipose-derived stem cells into fibroblasts in bio-ink

① Method

Through the aforementioned Experimental Example 1, it was confirmed that the adipose-derived stem cells survived in the structure of the bio-ink composition. In this experimental example, it was evaluated whether the bio-ink composition according to the present invention could regulate cell function as well as viability.

The evaluation was conducted by checking whether or not the adipose-derived stem cells were differentiated into fibroblasts in the bio-ink composition.

Whether to differentiate into fibroblasts was carried out through tissue analysis using specific markers of fibroblasts. Constructs in which M-ADM-based bio-ink and adipose-derived stem cells were co-cultured were collected. Frozen specimens were prepared using OCT Compound, and sections were fabricated with a thickness of 4-20 μm through a frozen tissue cutter. The tissue sections on the slide glass were stained with antibodies of collagen type 1 and alpha-SMA, which are specific markers of fibroblasts, and the expression of collagen type 1 and alpha-SMA was confirmed by fluorescence microscopy.

In addition, the mRNA expression of FSP-1 (Fibroblast specific protein-1), a marker of fibroblasts, was quantitatively analyzed through qRT-PCR to determine whether adipose-derived stem cells differentiated into fibroblasts.

② Results

The evaluation results are shown in FIG. 4 .

4(A), it can be confirmed that the adipose-derived stem cells express collagen type 1 and alpha-SMA, markers of fibroblasts, in the support of the M-ADM-based bio-ink.

In addition, through FIG. 4(B), when the adipose-derived stem cells were cultured with the M-ADM-based bio-ink for up to 5 weeks, it can be confirmed that the expression of FSP-1 is increased. It can be confirmed that the expression level at this time is similar to or superior to the amount of FSP-1 expressed by fibroblasts.

(2) Confirmation of differentiation of adipose-derived stem cells into chondrocytes in bio-ink

① Method

It was confirmed whether adipose-derived stem cells in the M-AC-based bio-ink were differentiated into chondrocytes.

Specifically, whether the adipose-derived stem cells differentiated into chondrocytes was determined through tissue analysis using specific markers of chondrocytes. Constructs in which M-AC-based bio-ink and adipose-derived stem cells were co-cultured were collected. Frozen specimens were prepared using OCT Compound, and sections with a thickness of 4-20 μm were prepared on a slide glass through a frozen tissue cutter. The tissue sections on the slide glass were stained with Aggrecan antibody, a specific marker for chondrocytes, and CD44, a specific marker for adipose-derived stem cells.

In addition, the mRNA expression of collagen type II and SOX-9, which are chondrocyte markers, was quantitatively analyzed through qRT-PCR to determine whether adipose-derived stem cells differentiated into chondrocytes.

② Results

The evaluation results are shown in FIG. 4 .

4(C) shows the expression of Aggrecan and CD44, which are markers, by staining human chondrocytes and adipose-derived stem cells. In addition, in (D), the expression of each specific marker was confirmed by staining the adipose-derived stem cells in the M-AC-based bio-ink support. Through this, it can be confirmed that CD44, a marker of adipose-derived stem cells, was expressed in the M-AC-based bio-ink support at the beginning of culture, but Aggrecan, a marker of chondrocytes, was expressed after culture.

In addition, through FIG. 4(E), when the adipose-derived stem cells were cultured with the M-AC-based bio-ink for up to 2 weeks, it was confirmed that the expression of collagen type II and SOX-9 increased. It can be confirmed that the expression level at this time is similar to or superior to the amount of collagen type II and SOX-9 expressed by human chondrocytes.

Experimental Example 3. Whether bio-ink is used for 3D bio-printing

In order to check whether the M-ADM-based bio-ink can be printed with a 3D printer, printing was performed using a 3D bio-printer.

Bio-ink was placed in a 10 ml syringe, and printing was performed at an extrusion rate of 4 mm/s through a 22G needle. The structure was completed with a size of 10 mm x 10 mm x 2 mm (width x length x height) under the condition of porosity of 12%.

1(A) shows the result of printing with M-ADM-based bio-ink. The structure was completed in a form that did not collapse according to the planned shape, and it was confirmed that the shape could be maintained when cross-linked with calcium chloride solution for 5 to 10 minutes after printing. A structure with strength that did not collapse even when the structure was picked up with tweezers was confirmed.

Meanwhile, FIG. 1(B) shows the result of printing with M-AC-based bio-ink. Like the M-ADM-based bio-ink, the M-AC structure was completed in a form that did not deviate from the planned shape and did not collapse, and it was confirmed that the shape could be maintained. In particular, it was confirmed that after bending the structure into a folding shape, it is unfolded again and maintains the structure. As a result of the above, bio-ink containing fine-grained human tissue can be printed in various structures and shapes through a 3D printer, and can be used as bio-ink for bio-printing and due to the influence of the environment provided by bio-ink. It was verified that the stem cells were differentiated according to the specificity of the human tissue in the bio-ink.

Claims (15)

Contains microparticulate human tissue-derived components and biocompatible polymers,
A bio-ink composition for 3D printing wherein the particle diameter of the micro-particulated human tissue-derived component is 10 to 1000 um.
The method of claim 1,
Human tissue-derived components include Acellular Dermal Matrix (ADM), Acellular Cartilage (AC), Acellular Adipose (AA), Acellular Bone (AB) and A bio-ink composition comprising at least one selected from the group consisting of acellular allogeneic bone-derived minerals (Acellular Bone Derived Mineral, ABDM).
delete The method of claim 1,
Ingredients derived from micro-particulated human tissue
(S1) decellularizing the human tissue; and
(S2) The bio-ink composition produced through the step of microparticles of the decellularized human tissue.
5. The method of claim 4,
The step of microparticulating is a bio-ink composition that physically pulverizes human tissue or chemically particleizes the human tissue.
The method of claim 1,
The microparticled human tissue-derived component is a bio-ink composition comprising a differentiation factor.
7. The method of claim 6,
Differentiation factors include Transforming Growth Factor-beta (TGF-β), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor , VEGF), insulin-like growth factor (Insulin-like Growth Factor, IGF), and bone morphogenic protein (Bone Morphogenic Protein, BMP), the bio-ink composition comprising at least one selected from the group consisting of.
The method of claim 1,
The bio-ink composition that the micro-particulated human tissue-derived component is included in an amount of 2 to 20 parts by weight based on the total weight of the bio-ink composition.
The method of claim 1,
Biocompatible polymers include agarose, alginate, chitosan, collagen, decellularized extracellular matrix, fibrin/fibrinogen, gelatin. , graphene, hyaluronic acid, hydroxyapatite, polycaprolactone (PCL), polylactic acid (PLA), poly (DL-lactic-co-glycolic acid) (poly-D, L-lactic-co-glycolic acid, PLGA), and pluronic (pluronic F 127), the bio-ink composition comprising at least one selected from the group consisting of.
The method of claim 1,
The biocompatible polymer is included in an amount of 0.5 to 10 parts by weight based on the total weight of the bio-ink composition.
The method of claim 1,
A bio-ink composition further comprising one or more selected from the group consisting of cells and differentiation factors.
12. The method of claim 11,
Cells are stem cells, fibroblasts, chondrocytes, osteoblasts, epithelial cells, keratinocytes, endothelial cells, and nerve cells ( Neuron) comprising at least one selected from the group consisting of,
Differentiation factors include Transforming Growth Factor-beta (TGF-β), Fibroblast Growth Factor (FGF), Epidermal Growth Factor (EGF), Vascular Endothelial Growth Factor , VEGF), insulin-like growth factor (Insulin-like Growth Factor, IGF), and bone morphogenic protein (Bone Morphogenic Protein, BMP), the bio-ink composition comprising at least one selected from the group consisting of.
12. The method of claim 11,
The content of one or more selected from the group consisting of cells and differentiation factors is 0.001 to 20 parts by weight based on the total weight of the bio-ink composition.
A method of manufacturing a structure, comprising the step of manufacturing a structure by extruding a bio-ink composition for 3D printing comprising a microparticulated human tissue-derived component and a biocompatible polymer.
15. The method of claim 14,
A method for producing a structure further comprising the step of crosslinking the extruded structure.

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