KR20200066218A - 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 three dimensional (3D) printing bio-ink composition, and to a production method thereof and, more specifically, to a bio-ink composition having a synergistic effect of cell differentiation into specific tissues according to the tissue specificity of human-derived components contained in the bio-ink composition, and to a production method thereof.

Description

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

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

Tissue engineering is the creation of human tissues or organs based on biology, engineering, and materials engineering to replace lost or damaged tissue. The number of patients whose human tissue is lost or damaged due to industrialization and aging is increasing, and thus the scope of the field of tissue engineering for supplementing lost or damaged tissue is expanding. Among them, 3D bio printing is an area that has recently attracted attention.

3D bio printing is to obtain images by scanning a target structure in three dimensions, and to make an image scanned through cells and bio ink into a three-dimensional structure. Common 3D printing techniques include extrusion-based processes, inkjet-based processes, and laser-based processes. Among them, extrusion-based processes are mainly used as bio-printing to create a three-dimensional structure with cells. Since bioprinting is the key to including living cells, bioink, a material that is mixed with cells to form a three-dimensional structure, is the most important part of 3D bioprinting.

Bio inks should have both physicochemical properties that can maintain a fine 3D structure and biological safety levels that can be used with living cells. As a physicochemical property, it must have printabilitiy, in which bio-ink can be extruded from a 3D printer, and rheological characteristics that can maintain a 3D structure even after printing. Because of its biosafety properties, it is essential to have cell affinity because bio-ink has to serve as a scaffold for cell growth and differentiation. In addition, since the printout output by 3D bioprinting contains the materials constituting the bioink, and becomes a 3D printout of an implantable property in the body, all of the material elements constituting the bioink are to be implanted in the body. It must be a biocompatible material, and it must have properties that can control the growth and differentiation of cells in an in vitro culture or in vivo transplantation of the output. In addition, the material elements constituting the bio-ink should show a tendency to decompose the output in the body to a level consistent with the regeneration tendency of the new tissue, while the new tissue is regenerated at the transplant site through the output after transplantation in the body.

Biocompatible materials that can make up bio inks include agarose, alginate, chitosan, collagen, decellularized extracellular matrix, and fibrin/fibrinogen. /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 F 127. Among them, alginate is a divalent cation It has the characteristic of crosslinking and gelling through the chelation of cation, and this property satisfies the printability of bio ink and the mechanical properties after printing, but alginic acid alone has low cell affinity, It does not provide a suitable environment for cells to adhere and grow.

Micronized human tissue decellularizes allogeneic tissues such as skin, bones, ligaments, tendons, blood vessels, cartilage, heart valves, amniotic membranes, fascia, nerves and pericardium, which are donated after death, and then lyophilized and crushed. The process results in particles of tens to hundreds of micrometers in size. Since the micro-particulated human tissue is a human-derived material that has undergone only the decellularization process, the extracellular matrix (ECM) is the main component. The extracellular matrix component is collagen, which is more than 90% structural protein, and the remaining 10% is glycoprotein, fibronectin, laminin, and glycosaminoglycan (GAG). And other tissue-specific growth factors (Growth factor) and some cytokines (Cytokine) are known to promote cell growth and tissue-specific differentiation of undifferentiated cells. In particular, components such as laminin, fibronectin, and glycosaminoglycan in the microparticulated human tissue act as a surface binding protein, and extracellular matrix such as cytokines or growth factors secreted from cells. It is known to attach to the structure to help the growth and differentiation of cells. Therefore, when the bio ink containing the extracellular matrix is cultured together with the cells, the cytokines and growth factors necessary for cell growth and differentiation are more easily transferred, thereby making the structure more tissue-like. (Non-Patent Document 1). Moreover, the extracellular matrix extracted from different tissues has tissue specificity, which has characteristics of each tissue, so that the cells are alive and suitable for constructing each tissue by attaching cytokines or growth factors suitable for the tissue. Is formulated (non-patent document 2).

In addition, the micro-particulated human tissue is a micro-unit particle type, and when mixed with a bio-derived polymer and a hydrogel to hydrate, it is physically evenly mixed and has the advantage of controlling the mechanical strength of the bio-ink according to the content. . Since the extracellular matrix previously used for bio-ink is used in a state dissolved in pepsin and acetic acid or hydrochloric acid to produce a liquid hydrogel, there is a limit in increasing the mechanical properties of the extracellular matrix itself. Therefore, the bio-ink containing micro-particulated human tissue has higher mechanical properties than the extracellular matrix solution bio-ink, and the micro-particulated human tissue itself is a component that has the form of tissue and is a more suitable environment for cells to live. This can be created.

Lastly, the extracellular matrix extracted from heterologous tissues such as cows, pigs, horses, etc. may be an animal-derived virus and cross-infection, whereas the human-derived extracellular matrix deviates from the safety problems of heterogeneous substances in the body. It can be used as the most suitable material for human body application. Therefore, the microparticulated human tissue has a very high cell affinity, and since it is an extracellular matrix derived from the human body, it has the advantage of being able to be transplanted into the body with little or no immune rejection, foreign body reaction, and inflammatory reaction.

[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 3D bio printing bio ink composition 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 in a 3D printed structure by applying various human tissue-derived components to a bio ink composition and a method for manufacturing the same.

The present invention provides a bio-ink composition for 3D printing that includes microparticulated human tissue-derived components and biocompatible polymers.

The present invention also provides a method of manufacturing a structure comprising extruding a bioink composition for 3D printing comprising a microparticulated human tissue-derived component and a biocompatible polymer.

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

The bio-ink composition according to the present invention has cell affinity and thus exhibits excellent effects in cell growth and differentiation, 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 micro-particulated human-derived tissue components.

By applying the bio ink composition according to the present invention to 3D bio printing, cells in the output 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 bio printing contains micro-particulated human tissue components, and thus, the cell affinity is high and cells can survive for a long time, and human tissue is a cell growth factor and Since it contains differentiation factors, it can regulate the function and differentiation of cells.

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

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

Figure 1 (A) is a cell-free allogeneic dermal powder (M-ADM) mixed with a biocompatible polymer and a state of cross-linking by printing through a 3D printer and M-ADM-based bio ink (left) filled in a syringe (Woo). (B) is a cross-linked structure of a cell-free allogeneic cartilage powder (M-AC) mixed with a biocompatible polymer and printed through an M-AC based bio ink (top left) filled with a syringe and an extrusion-based 3D printer ( Idol). The cross-linked structure after printing maintains the structure against external pressure (below).
Figure 2 (A) is a result of confirming the viability of cells after 1, 7, 21, 28 days by culturing M-ADM-based bio ink containing human dermal fibroblasts under a fluorescence microscope at 100 magnification. Green indicates living cells, red indicates dead cells, and the graph shows the number of living cells quantified by culture time.
Figure 2 (B) is a result of confirming the viability of cells after 1, 7, 21, 28 days by culturing M-AC-based bio ink containing human chondrocytes with a fluorescence microscope at 100 magnification. The graph is a result of quantifying the number of living cells expressed in green by culture time.
FIG. 3(A) shows the results of culturing M-ADM-based bio ink containing adipose-derived stem cells, and confirming whether the cells survive after 7, 14, 21, and 28 days with a fluorescence microscope at 100 magnification. The graph is a result of quantifying the number of living cells expressed in green by culture time.
FIG. 3(B) shows the results of culturing M-AC-based bio ink containing adipose-derived stem cells and confirming whether the cells survive with a fluorescence microscope at 100 magnification. The graph is a result of quantifying the number of living cells expressed in green by culture time.
FIG. 4(A) is a photograph obtained by culturing M-ADM-based bio ink containing adipose-derived stem cells and staining tissue sections after 21 days with DAPI, Collagen type 1 and alpha-SMA antibodies. (B) is a result of quantifying the mRNA expression level of FSP1 after 3 weeks and 5 weeks by qRT-PCR analysis by culturing M-AC based bio ink containing adipose-derived stem cells.
4C and 4D are photographs of human chondrocytes and adipose-derived stem cells cultured and stained with tissue fragments of DAPI, Aggrecan, and CD44 antibodies. (C) is a result of confirming cartilage differentiation of adipose-derived stem cells by expressing each specific marker in human chondrocytes and adipose-derived stem cells and incubating for 4 weeks in M-AC-based bio ink in (D).
Figure 4 (E) is a result of quantifying the mNA expression levels of Collagen type II and SOX-9 after 1 week and 2 weeks by qRT-PCR analysis by culturing an M-AC-based bio ink containing adipose-derived stem cells. .

The present invention relates to a bio-ink composition for 3D printing that includes microparticled human tissue-derived components and biocompatible polymers.

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 Stem cell viability was analyzed to confirm the cell affinity of the bio ink composition of the present invention. In addition, the bio-ink composition of the present invention was confirmed by confirming the differentiation of adipose-derived stem cells into fibroblasts in M-ADM-based bioinks and the differentiation of adipose-derived stem cells into chondrocytes in M-AC-based bioinks. The activity of the 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 bio-ink) according to the present invention includes microparticulated human tissue-derived components and biocompatible polymers.

In the present invention, a component derived from human tissue refers to a decellularized tissue-derived component, and provides conditions for creating a microenvironment similar to the original tissue. Since it is a structure formed through interaction with cells in the human body rather than artificially generated substances, it has a tissue-specific component and phase, and can provide an advantageous environment for cell growth and differentiation.

In the present invention, the micro-particulated human tissue-derived component means that the human tissue is subjected to a decellularization step and crushed to a size of 10 to 1000 μm. The microparticulated human tissue-derived component may be used for the purpose of improving biocompatibility, cell adhesion, and physical properties of bioink.

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

In one embodiment, the components derived from human tissue are decellularized human tissues, Acellular Dermal Matrix (ADM), Acellular Cartilage (AC), Acellular Adipose (AA) , Acellular Bone (AB) and Acellular Bone Derived Mineral (ABDM). In this case, the allogeneic cartilage may be vitreous cartilage, elastic cartilage, fibrous cartilage, or a mixture thereof. In particular, the microparticle-derived human tissue-derived component derived from skin and cartilage can enhance the tissue specificity of the bio ink as an extracellular matrix.

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 particle size range, the bio-ink can implement the input form through the micro-unit 3D printing nozzle, and can give strength to maintain the structure of the structure. In addition, unlike the bio ink produced by hydrogel type by treating the enzyme with the extracellular matrix, the original structure of the tissue is preserved to provide an environment favorable for cell growth and differentiation.

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

In one embodiment, the microparticulated human tissue-derived component (S1) decellularizing human tissue; And

(S2) The decellularized human tissue may be prepared through microparticleization.

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

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

In the above step, for example, a defatting process, a decellularization process, and/or a hair removal process may be performed. The defatting process may be performed using a polar solution such as alcohol and/or hexane and a non-polar solution, 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, in order to easily perform the microparticulation described later, the decellularized human tissue may be freeze-dried to remove moisture.

Step (S2) is a step of microparticleizing the decellularized human tissue.

The microparticulation may be performed by physically pulverizing human tissue or chemically granulating the tissue.

The type of the physical process is not particularly limited, for example, pulverized using a pulverizer, particularly a micro pulverizer, or pulverized by rotating the ball, or pulverized by rotating a ball or rod at cryogenic temperature to be finely granulated. Can be. In addition, the type of the chemical process is not particularly limited, for example, it can be microparticles by performing a process of decellularization from tissues through an organic/inorganic solvent, a basic alkaline solvent, or the like.

In one embodiment, the microparticulated human tissue-derived component may include a differentiation factor. Differentiation factor refers to a substance that can regulate cell growth and function. The differentiation factor may act as a growth factor, and in particular, when applied to stem cells, it can 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. (Vascular Endothelial Growth Factor, VEGF), insulin-like growth factor (Insulin-like Growth Factor, IGF) and bone morphogenetic protein (Bone Morphogenic Protein, BMP).

In one embodiment, the content of the micro-particulated human tissue-derived component may be 2 to 20 parts by weight relative to the total weight of the bio ink composition (100 parts by weight). In addition, the micro-particulated human tissue-derived component may be present in the bio-ink composition at 5-10% (w/v).

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

In one embodiment, the type of biocompatible polymer is not particularly limited, for example, agarose, alginate, chitosan, collagen, decellularized extracellular matrix, fibrin/fibrinogen, gelatin, graphene, hyaluronic acid, hydroxyapatite, polycaprolactone (PCL), polylactic acid acid, PLA), poly(DL-lactic-co-glycolic acid (PLGA) and pluronic F 127). Can be.

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 bioink, and is easy to crosslink. Specifically, alginate (alginate) is cross-linked through the chelation of a divalent cation (divalent cation), so as to gel, it is possible to satisfy the printability as a bio ink (printability) and mechanical properties after printing through the gelation properties have.

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

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), keratinocytes (Keratinocyte), endothelial cells (Endothelial cell) and one or more selected from the group consisting of neurons (Neuron) can be used.

In addition, the differentiation factor may be included in the microparticle-derived human tissue-derived component itself, but may be additionally added to the ink composition to maximize the effect of the differentiation factor, that is, cell growth and function control.

For example, the differentiation factor may use the above-described types, specifically, transformation growth factor (beta), 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) One or more can be used.

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 of the ink composition (100 parts by weight). The cells may be included in 10 to 20 parts by weight, differentiation factor may be included in 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 of manufacturing the above-described bio ink composition.

The bio-ink composition according to the present invention may be prepared by mixing the aforementioned components, that is, microparticulated human tissue-derived components and biocompatible polymers. Since the microparticle-derived human tissue-derived component is in the form of a micro-unit particle, it can be physically and evenly mixed without a separate mixing process when hydrated to a bio-derived polymer. In addition, in the present invention, it is possible to control the mechanical strength of the bio-ink according to the content of components derived from micro-particulated human tissue.

In addition, the present invention provides a method for manufacturing a structure comprising extruding a bio-ink composition for 3D printing comprising a microparticulated human tissue-derived component 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 means a process of scanning a target structure in three dimensions to obtain an image, and making a 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, output, or scaffold. The bio-ink composition according to the present invention can serve as a support for cell growth and differentiation, has biocompatibility, and has biocompatibility. Therefore, it can be easily transplanted into the body, and it is possible to control the growth and differentiation of cells in an in vitro culture or in vivo transplantation environment.

In the present invention, the structure may be manufactured by extruding a bio ink composition using a 3D printer. Specifically, a structure may be manufactured by printing 3D drawing data according to a program pre-designed with a 3D computer.

In the present invention, according to the purpose to be produced, the extrusion force, and the extrusion speed can be appropriately adjusted to produce a structure.

In the present invention, the step of crosslinking the extruded structure may be further included.

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

In one embodiment, when using alginate as a biocompatible polymer, the alginic acid is crosslinked and gelled through the chelation of a divalent cation, so crosslinking 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 biotissue regeneration. Specifically, the structure made of bio-ink has biocompatibility, and can be usefully used in biotissue regeneration, tissue engineering, and regenerative medicine because it can induce tissue regeneration by containing cells on a support and attaching to a 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 those skilled in the art can understand that various modifications, modifications, or applications are possible without departing from the technical matters derived by the matters described in the appended claims. will be.

Example

Example 1 Preparation of bio-inks comprising microparticleized Acellular Dermal Matrix (M-ADM) tissue

(1) M-ADM powder production

Skin tissue (collected from a body donated for treatment of a non-profit patient from a tissue bank) was acellularized.

First, the skin tissue was defatted using alcohol (alcohol) and hexane (Hexane). Thereafter, 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 passing through 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 crushed using a micro grinder. During the pulverization, dermal tissue powder of 500 μm or less was produced by passing through a sieve having a diameter of 250 to 500 μm.

The prepared powder-form tissue was sterilized.

(2) M-ADM based bio ink manufacturing

The hydrogel type bio ink composition was prepared by mixing the M-ADM powder prepared in (1) with an aqueous alginate solution.

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

M-ADM 5-10% (w/v) was hydrated in the aqueous alginate solution. The two syringes were connected to a Luer adapter and uniformly hydrated to prepare the composition.

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

Example 2. Preparation of bio-inks comprising microparticleized Acellular Cartilage (M-AC) tissue

(1) M-AC powder production

Cartilage tissue (collected from a body donated for treatment of a non-profit patient from a tissue bank) was crushed using a micro grinder. The grinding was passed through a sieve having a diameter of 250 to 500 um to obtain cartilage tissue powder of 500 um or less.

Thereafter, cartilage tissue powder was obtained and acellularized.

First, the cartilage tissue was defatted using alcohol (alcohol) and hexane (Hexane). Thereafter, cells were removed using sodium hydroxide (NaOH), and washed with alcohol (Ethanol) and distilled water. After freeze-drying to remove moisture, the powdered tissue was sterilized by electron beam.

(2) M-AC based bio ink production

A bio-gel composition in the form of a hydrogel was prepared by mixing the M-AC powder prepared in (1) with an aqueous alginate solution.

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

M-AC 5-10% (w/v) was hydrated in an alginate aqueous solution. The two syringes were connected to a Luer adapter and uniformly hydrated to prepare the composition.

1(B) is a state in which the M-AC-based bio ink is filled in a syringe.

Experimental Example 1. Cell affinity analysis of bio ink

(1) Fibroblast or human chondrocyte viability analysis 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 the Luer adapter. The bio ink mixed with the cells was extruded with a 22G injection needle to produce a grid-like structure having a width of 10 mm, a height of 10 mm, and a spacing of 2.5 mm.

When the lattice pattern structure was completed, the structure was treated with 1% calcium chloride solution for 5-10 minutes for crosslinking. The cross-linked structure was washed with phosphate buffer (PBS), immersed in a cell culture medium, and cultured in a cell culture medium.

Cell affinity analysis was conducted as follows.

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

On the other hand, the cell affinity of the M-AC based bio ink prepared in Example 2 was confirmed using human chondrocytes (Human Chondrocyte).

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

When the lattice-like structure was completed, a 1% calcium chloride solution was treated on the structure for 7-10 minutes for crosslinking. The cross-linked structure was washed with phosphate buffer (PBS), immersed in a cell culture medium, and cultured in a cell culture medium.

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

② Result

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 as time goes by, there are more living cells. Through this, it can be confirmed that the cells not only survive in the bio ink, but also proliferate.

In addition, the graph of Fig. (A) is a result of quantifying the results. After 4 weeks of culture, it can be seen that the number of cells increased more than 4 times after 1 day of culture.

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

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

(2) Fat-derived stem cell viability 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 ul of cell turbidity solution containing 1×10 ⁷ cells of fat-derived stem cells. Mixing was uniformly hydrated by connecting two syringes to the Luer adapter. The bio ink mixed with the cells was extruded with a 22G needle to produce a grid-like structure having a width of 10 mm, a height of 10 mm, and a spacing of 2.5 mm.

When the grid-shaped structure was completed, a 1% calcium chloride solution was treated on the structure for 5 to 10 minutes for crosslinking. After washing the crosslinked structure with PBS, it was immersed in a cell culture medium and cultured in a cell culture medium.

Cell affinity analysis was conducted as follows.

Cells living structures were stained with Live/dead cell viability assay kit (Life Technology, USA) after 1, 2, 3, 4 weeks of culture and confirmed through confocal microscopy (LSM 700, Carl Zeiss, Germany).

On the other hand, 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 200 ul of cell turbidity solution containing 2×10 ⁷ cells of fat-derived stem cells. Mixing was uniformly hydrated by connecting two syringes to the Luer adapter. The bio ink mixed with the cells was extruded with a 22G needle to produce a grid-like structure having a width of 10 mm, a height of 10 mm, and a spacing of 2.5 mm.

When the lattice-like structure was completed, a 1% calcium chloride solution was treated on the structure for 7-10 minutes for crosslinking. After washing the crosslinked structure with PBS, it was immersed in a cell culture medium and cultured in a cell culture medium.

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

② Result

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

3(A), it can be confirmed that the amount of cells increases in the bio-ink structure as the culture time passes. In addition, it can be confirmed that by appropriately adjusting the ratio of M-ADM and alginic acid in the bio-ink composition, it is possible to improve cell suitability and cell affinity to survive and proliferate adipose-derived stem cells.

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

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

3(B), it can be confirmed that the amount of cells increases in the bio-ink structure as the culture time passes. In addition, it can be confirmed that by appropriately adjusting the ratio of M-AC and alginic acid in the bio-ink composition, it is possible to improve cell suitability and cell affinity to survive and proliferate adipose-derived stem cells.

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

Experimental Example 2. Confirmation of the activity of bio ink

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

① Method

Through Experimental Example 1 described above, it was confirmed that adipose-derived stem cells survive 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 can control not only viability but also cell function.

The evaluation was conducted through confirmation of the differentiation of adipose-derived stem cells into fibroblasts in the bioink composition.

Whether fibroblasts differentiated was performed through tissue analysis using specific markers of fibroblasts. Structures in which M-ADM-based bio ink and adipose-derived stem cells were cultured together were collected. Frozen specimens were prepared using OCT Compound, and sections were prepared with a thickness of 4 to 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 then the expression of collagen type 1 and alpha-SMA was confirmed with a fluorescence microscope.

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

② Result

The evaluation results are shown in FIG. 4.

It can be seen from FIG. 4(A) that adipose-derived stem cells express collagen type 1 and alpha-SMA, which are fibroblast markers, in the support of the M-ADM-based bio ink.

In addition, it can be seen through FIG. 4(B) that the expression of FSP-1 increases when adipose-derived stem cells are cultured up to 5 weeks with M-ADM-based bio ink. 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 the differentiation of adipose-derived stem cells into chondrocytes in bio ink

① Method

It was confirmed whether adipose-derived stem cells in M-AC-based bioink differentiate into chondrocytes.

Specifically, whether adipose-derived stem cells differentiated into chondrocytes was performed through tissue analysis using specific markers of chondrocytes. A structure in which M-AC-based bio ink and adipose-derived stem cells were cultured together was collected. Frozen specimens were prepared using OCT Compound, and sections were prepared on slide glass with a thickness of 4 to 20 μm through a frozen tissue cutter. The tissue sections on the slide glass were stained with the antibody of Aggrecan, a specific marker of chondrocytes, and the antibody of CD44, a specific marker of adipose-derived stem cells, and then the expression of Aggrecan and CD44 was confirmed by a fluorescence microscope.

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

② Result

The evaluation results are shown in FIG. 4.

4(C) is a staining of human chondrocytes and adipose-derived stem cells, and shows the expressions of Aggrecan and CD44, respectively. In addition, (D) was stained with adipose-derived stem cells in the M-AC based bio ink support to confirm the expression of each specific marker. Through this, CD44, a marker of adipose-derived stem cells, was expressed in the early stage of culture in the M-AC-based bio-ink support, but it was confirmed that Aggrecan, a marker of chondrocytes, was expressed after culture.

In addition, it can be seen through FIG. 4(E) that when the adipose-derived stem cells were cultured with M-AC-based bio ink for up to 2 weeks, 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 amounts of Collagen type II and SOX-9 expressed by human chondrocytes.

Experimental Example 3. Whether to use bio ink for 3D bio printing

To check whether the M-ADM-based bio ink can be output to a 3D printer, printing was performed using a 3D bio printer.

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

Fig. 1(A) shows the result of printing with M-ADM based bio ink. It was confirmed that the structure was completed in a shape that did not collapse according to the planned shape, and that it was possible to maintain the shape when crosslinked with a calcium chloride solution for 5-10 minutes after printing. A structure having a strength that did not collapse when the structure was picked up with tweezers was confirmed.

On the other hand, Fig. 1 (B) shows the result of printing with M-AC based bio ink. M-AC, like M-ADM-based bio ink, was confirmed that the structure was completed in a shape that does not fall within the planned shape and does not collapse, and it is possible to maintain the shape. In particular, it was confirmed that it is a form that expands again after bending the structure in a folded shape and maintains the structure. As a result of the above, bio-inks containing micro-particulated human tissues can be printed in various structures and shapes through a 3D printer, and can be used as bio-inks for bio-printing and the influence of the environment provided by bio-inks It was verified that the stem cells differentiate according to the specificity of human tissue in the bio ink.

Claims (15)

A bio-ink composition for 3D printing that includes micro-particulated human tissue-derived components and biocompatible polymers.
According to claim 1,
Components derived from human tissue 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 Bone Derived Mineral (ABDM).
According to claim 1,
The particle size of the microparticle-derived human tissue-derived component is 10 to 1000 um.
According to claim 1,
Components derived from micro-particulated human tissue
(S1) decellularizing human tissue; And
(S2) The bio-ink composition is prepared through the step of micro-particulating the decellularized human tissue.
The method of claim 4,
The step of micro-particulation is a bio-ink composition that physically crushes or chemically particles human tissue.
According to claim 1,
The micro-particle-derived human tissue-derived component comprises a differentiation factor.
The method of claim 6,
Differentiation factors include transforming growth factor (beta, TGF-β), fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor , VEGF), insulin-like growth factor (Insulin-like Growth Factor, IGF) and bone morphogenetic protein (Bone Morphogenic Protein, BMP).
According to claim 1,
The microparticle-derived human tissue-derived component is included in 2 to 20 parts by weight based on the total weight of the bioink composition.
According to claim 1,
Biocompatible polymers include agarose, alginate, chitosan, collagen, decellularized extracellular matrix, fibrin/fibrinogen, and 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 a bio ink composition comprising at least one selected from the group consisting of pluronic (pluronic F 127).
According to claim 1,
The biocompatible polymer is a bio ink composition that is included in an amount of 0.5 to 10 parts by weight based on the total weight of the bio ink composition.
According to claim 1,
A bio ink composition further comprising at least one selected from the group consisting of cells and differentiation factors.
The method of claim 11,
Cells include stem cells, fibroblasts, chondrocytes, osteoblasts, epithelial cells, keratinocytes, endothelial cells and nerve cells ( Neuron) comprises one or more selected from the group consisting of,
Differentiation factors include transforming growth factor (beta, TGF-β), fibroblast growth factor (FGF), epidermal growth factor (EGF), and vascular endothelial growth factor , VEGF), insulin-like growth factor (Insulin-like Growth Factor, IGF) and bone morphogenetic protein (Bone Morphogenic Protein, BMP).
The method of claim 11,
The at least one content 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.
Method of manufacturing a structure comprising the step of preparing a structure by extruding a bio-ink composition for 3D printing comprising a component derived from micro-particulated human tissue and a biocompatible polymer.
The method of claim 14,
A method of making a structure further comprising the step of crosslinking the extruded structure.

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