KR102640278B1 - Bio ink composition and Method for producing three-dimensional adipose and skin complex Tissue-like structure using the same - Google Patents

Abstract

The present invention relates to a bio-ink composition, a method of manufacturing a three-dimensional fat and skin composite tissue-like structure using the bio-ink composition, and a tissue-like structure manufactured by the method.
The bio-ink composition according to the present invention has improved cell survival rate and proliferation ability, so it can be very useful in the production of tissue-like structures using 3D bioprinting, and can be used to provide artificial tissues with morphological and functional characteristics similar to living bodies. It can be.

Description

Bio ink composition and method for producing three-dimensional fat and skin complex tissue-like structure using the same {Bio ink composition and Method for producing three-dimensional adipose and skin complex Tissue-like structure using the same}

The present invention relates to a bio-ink composition, a method of manufacturing a three-dimensional fat and skin composite tissue-like structure using the bio-ink composition, and a tissue-like structure manufactured by the method.

Three-dimensional (3D) bioprinting is a technology that produces tissues or organs by layering living cells in a desired shape or pattern, and is connected to a cyber-physical system (CPS) that combines computing technology with physical objects. It is a technology that realizes living tissue.

3D bioprinting technology is receiving great attention in the field of tissue engineering for the purpose of artificial tissue or organ regeneration. The organs or tissues that make up the human body are composed of various types of cells and biomaterials that make up the extracellular matrix. Research is being actively conducted to regenerate necessary functional tissues through the production of tissue-like cell structures that make up the human body using 3D bioprinting technology. Recently, this technology has also received great attention for the development of artificial biological models for testing the toxicity and efficacy of new drugs. In this 3D bioprinting process, bio-ink is the most important key element in precise patterning of cells and ensuring survival rate, and the characteristics of the ink are directly linked to the process.

'Bio-ink' is a general term for materials that contain living cells or bio molecules and can be applied to bio-printing technology to produce required structures. Bio-ink must meet the physical properties for three-dimensional processing and the biological environment for cells to perform their intended function. The characteristics that the ink required for 3D bio-printing technology using living cells are very diverse and complexly intertwined. Therefore, the development of excellent bio-ink technology can be said to be the key to developing excellent bio-printing technology. In this regard, various materials have been used to construct bioinks, and bioinks made using each material are affected by various factors such as microstructure, composition, and application process, so an approach to rheological research is also necessary. do.

In order to be applied to 3D bio-printing, bio-ink requires various characteristics such as excellent biocompatibility and the structure manufactured after printing must have structural stability, so there are many limitations in its usability to date.

Under this background, the inventors of the present invention completed the present invention by manufacturing a bio-ink in which GelMA and decellularized extracellular matrix were mixed with cells, and confirming that a tissue-like structure could be manufactured using the bio-ink.

Republic of Korea Patent Publication No. 10-2018-0122288

One exemplary purpose of the present invention is to provide a bio-ink composition containing a decellularized extracellular matrix (dECM), a biocompatible polymer compound, and cells.

Another exemplary object of the present invention is to provide a method for manufacturing a tissue-like structure, comprising the following steps.

(a) injecting the bio-ink composition into a nozzle for 3D bio printing and crosslinking; and

(b) 3D bio-printing by spraying the bio-ink composition crosslinked in step (a) onto a support.

Another exemplary object of the present invention is to provide a tissue-like structure prepared by the above manufacturing method.

The technical problem to be achieved according to the technical idea of the invention disclosed in this specification is not limited to the problem to solve the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below. There will be.

This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present application may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in this application fall within the scope of this application. Additionally, the scope of the present application cannot be considered limited by the specific description described below.

As an aspect for achieving the above object, the present invention provides a bio-ink composition containing a decellularized extracellular matrix (dECM), a biocompatible polymer compound, and cells.

The term "bio-ink" of the present invention includes living cells or biomolecules and refers collectively to materials that can be applied to bioprinting technology to produce required structures. Bio-ink is a material that provides physical properties for three-dimensional processing and a biological environment for cells to perform their intended functions, and has excellent cell compatibility. Additionally, the bio-ink composition may be for 3D bio-printing.

As used herein, the term "bio printing" refers to precise cell deposition (e.g., cells) in three dimensions through methods commonly used with automated, computer-assisted three-dimensional prototyping devices (e.g., bioprinters). solutions, cell-laden gels, cell suspensions, cell concentrates, multicellular aggregates, multicellular bodies, etc. Bioprinting inventions may be continuous. Non-limiting examples of continuous bioprinting methods include those connected to a reservoir of bioink. Bio-ink is dispensed from the bioprinter through a dispense tip (e.g., syringe, capillary tube, etc.). The continuous bioprinting method is to dispense bio-ink in a repeating pattern of functional units. The repeating function The units can have any suitable geometry, including, for example, circles, squares, rectangles, triangles, polygons, and irregular geometries.In one embodiment of the invention, the bioprinting may be 3D bioprinting.

The “3D bio printing (three-dimensional bio printing)” refers to creating artificial biological tissues or organ-like structures using 3D printing. Bio-ink containing cells extracted from human tissues is placed in a 3D printer, and the cells are printed in a three-dimensional space. This means building a structure.

In the present invention, the composition may be used for 3D bio printing.

In the present invention, the composition may be for manufacturing a three-dimensional fat and skin composite tissue-like structure.

The term "decellularized extracellular matrix" of the present invention is a matrix surrounding the outside of cells, occupies between cells, and has a network structure mainly composed of proteins and polysaccharides. Components of the extracellular matrix include structural components such as collagen and elastin, adhesive proteins such as fibronectin, vitronectin, laminin, and tenascin, and chondroitin sulfate or heparan sulfate, the main protein. There are hyaluronic acid, which consists only of proteoglycans and polysaccharides produced from .

The “decellularization” is a process of separating the extracellular matrix components contained in biological tissue through chemical treatment. The tissue extracted from the body is washed with phosphate buffered saline (PBS) and immersed in the decellularization solution to remove cell nuclei. This refers to a process that removes and leaves only the extracellular matrix.

In the present invention, the "decellularized extracellular matrix" is liver tissue, heart tissue, cartilage tissue, bone tissue, fat tissue, muscle tissue, skin tissue, mucosal epithelial tissue, amniotic tissue, or It may be obtained by decellularizing corneal tissue, but is not limited thereto.

The bio-ink composition of the present invention may additionally include one or more of cells or cell culture medium.

The cells include fibroblast, stem cell, osteoblast, myoblast, tenocyte, neuroblast, glioblast, germ cell, and hepatocyte. (hepatocyte), renal cell, Sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell, cardiomyocyte It may be one or more selected from the group consisting of (cardiomyocyte), glial cell, endothelial cell, hormone secreting cell, immune cell, pancreatic islet cell, and neuron, Specifically, it may be fibroblasts, and more specifically, it may be human dermal fibroblasts, but is not limited thereto.

The cells can be cultured in any manner, including general mammalian cell culture techniques known in the art.

The cell culture medium includes, but is not limited to, any medium suitable for the desired cells, and any medium suitable for cell lines known in the art, for example, MEM medium, RPMI1640 medium, DMEM medium, It may be an IMDM badge, etc.

In the present invention, the density of cells included in the bio-ink composition may be 1 x 10 ⁵ cells/ml to 1 x 10 ⁶ cells/ml, specifically 1 x 10 ⁵ cells/ml to 9 x 10 ⁵ cells. /ml, 1 x 10 ⁵ cells/ml to 8 x 10 ⁵ cells/ml, 1 x 10 ⁵ cells/ml to 7 x 10 ⁵ cells/ml, 1 x 10 ⁵ cells/ml to 6 x 10 ⁵ cells/ml , 1 x 10 ⁵ cells/ml to 5 x 10 ⁵ cells/ml, 2 x 10 ⁵ cells/ml to 9 x 10 ⁵ cells/ml, 3 x 10 ⁵ cells/ml to 8 x 10 ⁵ cells/ml or 4 It may be x 10 ⁵ cells/ml to 6 x 10 ⁵ cells/ml, and more specifically, 5 x 10 ⁵ cells/ml, but is not limited thereto.

In the present invention, the biocompatible polymer compound is gelatin methacryloyl (GelMA), polyethylene glycol (PEG), neophenyl glycol diacrylate (NPGDA), polyethylene oxide (PEO), and polyacrylamide. (PAAm), polyhydroxyethyl methacrylate (PHEMA), hyaluronic acid methacrylate (HAMA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), poly(N-isopropyl) Acrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), gelatin, alginate, carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar It may be one or more selected from the group consisting of carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, and polyvinyl chloride, a copolymer thereof, or a mixture thereof, and may specifically be gelatin methacryloyl, but is limited thereto. It doesn't work.

In the present invention, the biocompatible polymer compound may be included in an amount of 1 to 10 parts by weight per 100 parts by weight of the total bio-ink composition, specifically 2 to 9 parts by weight, 3 to 8 parts by weight, 4 to 7 parts by weight, or 5 to 5 parts by weight. It may be included in 6 parts by weight, and more specifically, it may be included in 5 parts by weight.

In the present invention, the bio-ink may additionally contain physiologically active substances.

The bioactive substances can, for example, proliferate cells, promote cell differentiation, and promote secretion of extracellular matrix, and include transforming growth factor-beta (TGF-β) and fibroblast growth factor. growth factor (FGF), bone morphogenic protein (BMP), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), insulin-like growth factor (insulin-like) growth factor (IGF), platelet-derived growth factor (PDGF), nerve growth factor (NGF), hepatocyte growth factor (HGF), placental growth factor , PIGF), granulocyte colony stimulating factor (GCSF), ascorbate 2-phosphate, dexamethasone (Dex), and β-glycerophosphate. Not limited.

As another aspect for achieving the above object, the present invention provides a method for producing a tissue-like structure, comprising the following steps.

(a) injecting the bio-ink composition into a spray nozzle for 3D bio printing and crosslinking; and

(b) 3D bio-printing by spraying the bio-ink composition crosslinked in step (a) onto a support.

In the present invention, the spacing between adjacent printing lines of the same printing layer in the support may be 1000 μm to 2000 μm, specifically 1200 μm to 1800 μm, 1300 μm to 1700 μm, 1400 μm to 1600 μm, or 1500 μm. It may be from μm to 1600 μm, more specifically 1600 μm, but is not limited thereto.

In the present invention, the support is polycaprolactone (PCL), poly(lactate-co-glycolate) (PLGA), poly lactic acid (PLA), poly It may be selected from the group consisting of urethane (polyurethane, PU) and poly(lactide-co-caprolactone) (PLCL), but is not limited thereto.

In the present invention, the tissue may be skin tissue, liver tissue, heart tissue, cartilage tissue, bone tissue, fat tissue, muscle tissue, mucosal epithelial tissue, amniotic membrane tissue, or corneal tissue, and may specifically be skin tissue. , but is not limited to this.

The term “tissue-like structure” of the present invention refers to a structure made by printing bio-ink containing animal-derived cells or stem cells using a 3D bioprinting method. The tissue-like structure can reproduce specific functions of tissue and can be spatially organized in a form similar to actual tissue, so it can be used for tissue and organ regeneration and the production of biological models for new drug development.

Specifically, in an embodiment of the present invention, the bio-ink containing GelMA, human dermal fibroblasts, and decellularized extracellular matrix is applied to a support made of polycaprolactone with an interval between adjacent printing lines of the same printing layer of 1600 μm. It was confirmed that a tissue-like structure similar to skin was produced by spraying it on.

As another aspect for achieving the above object, the present invention provides a tissue-like structure manufactured by the above manufacturing method.

In the present invention, the tissue-like structure may include one or more spheroids.

In the present invention, the size of the spheroid may be 100 μm to 1000 μm, specifically 200 μm to 900 μm, 300 μm to 800 μm, 400 μm to 800 μm, 500 μm to 800 μm, or 600 μm to 800 μm. It may be μm, more specifically 700 μm to 800 μm, but is not limited thereto.

In the present invention, the spacing between the spheroids may be 0.1 mm to 2 mm, specifically 0.3 mm to 1.8 mm or 0.5 mm to 1.5 mm, and more specifically 0.6 mm to 1.4 mm, but is limited thereto. It doesn't work.

In the present invention, the tissue may be skin tissue, liver tissue, heart tissue, cartilage tissue, bone tissue, fat tissue, muscle tissue, mucosal epithelial tissue, amniotic membrane tissue, or corneal tissue, and may specifically be skin tissue. , but is not limited to this.

In the present invention, the tissue-like structure may include a dermal-like layer at the top and a fat-like layer at the bottom.

The bio-ink composition according to the present invention has improved cell survival rate and proliferation ability, so it can be very useful in the production of tissue-like structures using 3D bioprinting, and can be used to provide artificial tissues with morphological and functional characteristics similar to living bodies. It can be.

Figure 1 shows the results of analyzing the survival and proliferation rates of bio-ink prepared by mixing GelMA and human dermal fibrotic cells.
Figure 2 shows the cell proliferation and survival rate results of the bio-ink of the present invention confirmed through LIVE/DEAD assay and CCK-8 analysis.
Figure 3 shows the 3D printing process using the bio-ink of the present invention.
Figure 4 shows the results of analyzing the change in viscosity of the bio-ink of the present invention as the temperature increases.
Figure 5 shows the results of modulus analysis of the bio-ink of the present invention.
Figure 6 is a schematic diagram of a support for manufacturing a tissue-like structure.
Figure 7 shows spraying the bio-ink of the present invention through a nozzle.
Figure 8 is a schematic diagram of a tissue-like structure manufactured with the bio-ink of the present invention.
Figure 9 shows the appearance of the manufactured tissue-like structure before and after ultraviolet irradiation.
Figure 10 shows fine control of the spheroid size by controlling the injection pneumatic pressure of the tissue-like structure produced by bioprinting.
Figure 11 shows fine control of the spheroid distance by controlling the XY position of the tissue-like structure produced by bioprinting.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1: Bio-ink preparation

Mix 0.25 parts by weight (0.25%) of decellularized extracellular matrix compared to 100 parts by weight of bio ink, 5 parts by weight (5%) of GelMA and human dermal fibrosis cells at a density of 5x10 ⁵ cells/mL compared to 100 parts by weight of total bio ink. Bio-ink was manufactured.

First, human dermal fibrotic cells were mixed in DMEM/HG medium (including 10% FBS and penicillin-streptomycin) containing 5% GelMA at a density of ^5x105 cells/mL, and 3D printed using an extrusion-based three-dimensional printing method. As a result of checking the survival and proliferation rates for days, it was confirmed that the survival and proliferation rates of cells increased over time (Figure 1).

Afterwards, decellularized extracellular matrix (0.25%) and human dermal fibroblasts were grown in DMEM/HG medium (10% FBS, penicillin-streptomycin) containing GelMA (5%) at a density of ^5x105 cells/mL. After mixing as much as possible, GelMA was cross-linked by ultraviolet light induction, and then the decellularized extracellular matrix was cross-linked at 37°C to prepare bio-ink. As a control, bio-ink was prepared by mixing human dermal fibrosis cells with GelMA.

Example 2: Evaluation of cell viability and functionality of bio-ink

To evaluate the cell viability and functionality of the bio-ink prepared in Example 1, it was printed using an extrusion-based 3D printing method and then subjected to LIVE/DEAD assay (LIVE/DEAD ^TM Viability/Cytotoxicity Kit, Thermofisher) and CCK-8 analysis ( Quanti-Max WST-8 Cell Viability Assay Kit, Biomax) was performed according to the manufacturer's manual.

As a result, compared to the control group in which only GelMA was mixed with human dermal fibrotic cells, it was confirmed that when decellularized extracellular matrix and GelMA were mixed with cells, higher cell proliferation and survival rates occurred over time (Figure 2, A: Survival rate analysis by LIVE/DEAD assay, B: Proliferation rate analysis by CCK-8 analysis), these results suggest that cytokines and growth factors contained in the decellularized extracellular matrix affected cell proliferation. .

Example 3: Evaluation of physicochemical properties of bio-ink

In order to evaluate the physicochemical properties of the bio-ink prepared in Example 1, 3D printing was performed using an extrusion-based three-dimensional printing method (FIG. 3), and then viscoelasticity (Rheological) analysis was performed using a rheometer (TA instrument Co.). Viscosity and modulus according to temperature were measured.

As a result, the bio-ink (experimental group) containing decellularized extracellular matrix, GelMA, and human dermal fibrotic cells showed a significantly lower rate of decrease in viscosity with increase in temperature compared to the control group excluding the decellularized extracellular matrix. (Figure 4), the modulus was found to be higher than the control group excluding the decellularized extracellular matrix (Figure 5), confirming that the mechanical properties were superior to the control group.

Example 4: Preparation of tissue-like structure

Polycaprolactone (PCL) polymer was put into the heating syringe barrel of the printer and heated at 80°C for more than 10 minutes to melt it, and then printed at a pressure of 300 to 550 kPa to prepare a support (FIG. 6).

Afterwards, 3D bio-printing was performed using the bio-ink of Example 1. The printing temperature was in the range of 80 to 100°C, the pressure was 450 to 550 kPa, the printing head and printing bead were at 10°C, and the internal spacing of the nozzle was 1600 um. The printing process of spraying bio-ink through a nozzle is shown in FIG. 7, and a schematic diagram of the tissue-like structure produced as a result of printing is shown in FIG. 8.

The spheroids forming the prepared tissue-like structures had a size of 700 μm to 800 μm, and when the tissue-like structures were irradiated with ultraviolet rays, it was confirmed that the structures were well maintained without structural deformation even after irradiation (FIG. 9).

Fine control of the spheroid size by controlling the injection pneumatic pressure of the tissue-like structure produced by bioprinting is shown in Figure 10, and fine control of the spheroid distance by controlling the X-Y position is shown in Figure 11. The fine control of the size and fine distance was 3D printed at a speed of 0.3 seconds per printing spheroid.

Since the characteristics of tissues and cells within the human body are different, it is very important to manufacture spheroids with appropriate sizes and spacing by adjusting the size of the spheroids or the spacing between spheroids according to the human body simulation environment.

As shown in Figure 10, when the nozzle pressure during 3D printing was adjusted to 8 kPa, 10 kPa, 12 kPa, and 14 kPa, respectively, the sizes of the spheroids were 200 um, 400 um, 600 um, and 800 um, respectively, so that the pressure was adjusted to It was confirmed that spheroid size control was possible.

In addition, as shown in Figure 11, the pressure of the nozzle during 3D printing is set to 14 kPa, and the spacing between spheroids in the tissue-like structure bioprinted by specifying the moving distance is 0.6 mm, 0.8 mm, 1 mm, 1.2 mm, or 1.4 mm. It was confirmed that the spheroid spacing could be adjusted.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.

Claims (21)

delete delete delete delete delete delete delete delete delete delete delete Method for producing a tissue-like construct comprising the following steps:
(a) A nozzle for 3D bio printing using a bio-ink composition containing decellularized extracellular matrix (dECM), gelatin methacryloyl (GelMA), and human dermal fibroblast. Injecting and crosslinking; and
(b) 3D bio-printing by spraying the bio-ink composition crosslinked in step (a) onto a support,
The gelatin methacryloyl in step (a) is cross-linked by ultraviolet rays, and the decellularized extracellular matrix is cross-linked by heat,
The gelatin methacryloyl is contained in an amount of 5 to 6 parts by weight per 100 parts by weight of the total bio ink composition,
The decellularized extracellular matrix is included in an amount of 0.25 parts by weight per 100 parts by weight of the total bio-ink composition,
A method of producing a tissue-like structure, wherein the spacing between adjacent printed lines of the same printed layer in the support is 1500 μm to 1600 μm.
delete According to clause 12,
The support includes polycaprolactone (PCL), poly(lactate-co-glycolate) (PLGA), polylactic acid (PLA), and polyurethane (PU). ) and polylactate-co-caprolactone (poly(lactide-co-caprolactone), PLCL).
According to clause 12,
The production method, wherein the tissue is skin tissue, liver tissue, heart tissue, cartilage tissue, bone tissue, fat tissue, muscle tissue, mucosal epithelial tissue, amniotic tissue, or corneal tissue.
A tissue-like structure manufactured by the manufacturing method of claim 12.
delete delete delete delete delete