Method for manufacturing 3D-printed artificial tissues based on extracellular matrix-induced self-organizing bodies, and the artificial tissues produced therefrom.

Abstract

The present invention relates to a method for producing a 3D printed artificial tissue based on an autologous tissue induced by an extracellular matrix and an artificial tissue produced therefrom, and provides a method for producing an artificial tissue that can be finely patterned in a width of micrometers and can embody the morphological outline of a primary tissue by applying an autologous tissue formed by inducing differentiation of stem cells using an extracellular matrix-derived biomaterial to 3D printing, and an artificial tissue that is printed in the form of a mature tissue rather than a mixture of cells and biomaterials from the time of printing. The artificial tissue produced through the production method of the present invention can provide an artificial tissue and an artificial organ that are very similar to the actual tissue of origin by mimicking the biological characteristics of the target organ due to the origin of the extracellular matrix.

Description

Technical Field

[0001] The present invention relates to a method for manufacturing an extracellular matrix-induced self-assembled substrate 3D printed artificial tissue and an artificial tissue manufactured therefrom. By applying a self-assembled body formed by inducing the differentiation of stem cells using an extracellular matrix-derived biomaterial to 3D printing, it is possible to perform micro-patterning with a width in the micrometer unit and to embody the morphological outer shape of the primary tissue. The present invention provides a method for manufacturing an artificial tissue and an artificial tissue printed in the form of a mature tissue rather than a cell-biomaterial mixture from the time of printing. The artificial tissue manufactured through the manufacturing method of the present invention can mimic the biological characteristics of the target organ according to the origin of the extracellular matrix and can provide an artificial tissue and an artificial organ that are very similar to the actual derived tissue.

Background Art

[0002] Recently, in the field of tissue engineering, after collecting allogeneic or xenogeneic organs and tissues, a technique of removing cells (decellularization) and using them in various forms of tissue engineering preparations has attracted attention. To date, various tissue-derived biomaterials such as small intestinal submucosal tissue, bladder, skin, amnion, bone, ligament, cartilage, etc. have been commercialized or research is underway. Methods for fabricating tissue-engineered artificial organs using tissue-derived biomaterials reported to date include salt leaching, electrospraying, 3D printing, etc.

[0003] Among them, 3D printing is under extensive research because it can utilize various materials and cells and can embody the desired form. In particular, liquefying the above-mentioned tissue-derived biomaterials or mixing them in powder form and printing them together with cells has confirmed the possibility of induction into tissues such as increasing the activity of cells and expressing specific genes and proteins of the primary tissue. However, there are still limitations in terms of tissue induction and maturity, such as using synthetic materials that can generate by-products harmful to cells during degradation or forming non-uniform cell-biomaterial composites.

[0004] For example, Korean Published Patent No. 10-2020-0066218 discloses a bio-ink composition containing finely particulated human tissue and a technology for manufacturing structures using the same. However, it requires separate cell growth factors and differentiation factors to regulate cell function and differentiation, and structures fabricated through 3D printing must undergo a cross-linking step to satisfy bio-ink printability and post-printing mechanical properties.

[0005] Against this backdrop, the present inventors aimed to present an improved method for manufacturing artificial tissue compared to conventional techniques by providing a method for printing self-organized tissue that has undergone tissue-specific differentiation and matured at the time of printing, without using differentiation factors, by utilizing cells and biomaterials derived from extracellular matrix. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, the present invention aims to provide a technology for producing artificial tissues similar in biochemical properties to primary tissues by utilizing self-organizing cell-biomaterial complexes (cell-decellularized extracellular matrix self-assemblies) that have excellent tissue differentiation and maturation induction capabilities in printing.

[0007] Specifically, the object of the present invention is to provide a method for producing 3D-printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly material.

[0008] Another object of the present invention is to provide 3D-printed artificial tissues and artificial organs based on cell-decellularized extracellular matrix self-assembly materials produced by the method described above. [Means for solving the problem]

[0009] To solve the aforementioned problems, the present invention provides a method for producing a cell-decellularized extracellular matrix self-assembly-based 3D-printed artificial tissue, comprising the following steps: (a) The step of decellularizing and pulverizing tissue-derived extracellular matrix (ECM) to produce decellularized extracellular matrix (DECM) powder; (b) Add the decellularized extracellular matrix powder to the culture medium containing the cells, and then culture to form a cell-decellularized extracellular matrix autoorganism; (c) The step of homogenizing the cell-decellularized extracellular matrix self-assembly and producing tissue strand ink; and (d) The step of applying the homogenized tissue strand ink to a 3D printing equipment to manufacture a 3D printed artificial tissue.

[0010] According to one preferred embodiment of the present invention, the tissue in step (a) may be bone, ligament, muscle, fibrocartilage, or cartilage.

[0011] According to another preferred embodiment of the present invention, in step (b), the cells may be stem cells.

[0012] According to another preferred embodiment of the present invention, the stem cells may be one or more selected from the group consisting of mesenchymal stem cells, embryonic stem cells, and reverse-differentiated stem cells.

[0013] According to another preferred embodiment of the present invention, the decellularized extracellular matrix powder in step (b) can be added at a concentration of 0.05 to 3 mg / ml.

[0014] According to another preferred embodiment of the present invention, the cell-decellularized extracellular matrix self-assembly can be formed in vitro in step (b).

[0015] According to another preferred embodiment of the present invention, a cell-decellularized extracellular matrix powder self-assembly can be formed by inducing cell proliferation or cell differentiation in step (b).

[0016] According to another preferred embodiment of the present invention, the (b) step may further include adding a solubilized and decellularized extracellular matrix solution.

[0017] According to another preferred embodiment of the present invention, the solubilized and decellularized extracellular matrix solution may be added at a concentration of 50 to 500 μg / ml.

[0018] According to another preferred embodiment of the present invention, step (b) may be performed for 2 to 9 days after the cells and the decellularized extracellular matrix powder have begun to fuse.

[0019] According to another preferred embodiment of the present invention, homogenization of the cell-decellularized extracellular matrix self-assemblies in step (c) may be performed by passing the cell-decellularized extracellular matrix self-assemblies obtained after step (b) through a molecular sieve or by blending them through a syringe connector connected by a nozzle.

[0020] According to yet another preferred embodiment of the present invention, the mesh diameter of the molecular sieve may be 50 to 800 μm, and the diameter of the nozzle connected to the syringe connector may be 1 to 3 mm.

[0021] According to another preferred embodiment of the present invention, the tissue strand ink produced in step (d) above may be injected into a 3D printing syringe and 3D printing may be performed with a nozzle size of 200 μm or more, an air pressure of 20 to 150 kPa or less, and a print speed of 0.1 to 3 mm / second.

[0022] The present invention also provides 3D-printed artificial tissues and artificial organs based on cell-decellularized extracellular matrix self-assembly substrates produced by the method described above.

[0023] According to a preferred embodiment of the present invention, the artificial tissue and artificial organ can exhibit the biochemical properties of the primary tissue.

Advantages of the Invention

[0024] The method for manufacturing a 3D printed artificial tissue of the present invention enables fine patterning with a width in the micrometer unit, and can not only embody the morphological outer shape of the original tissue, but also enable the maturation of a tissue that mimics the biological characteristics of the target organ according to the origin of the extracellular matrix. Also, different from the conventional methods, it is possible to print in a tissue form that is a mature self-tissue body rather than a composite of cells and biomaterials at the time of printing. As a result, the artificial tissue manufactured by the method of the present invention can be utilized for the development of medical products required for regenerative medicine, such as, for example, the repair of bone, ligament, muscle, cartilage or meniscus cartilage damage. In addition, it can produce tissue engineering products suitable for the anatomical location, characteristics and physicochemical requirements of the target tissue, and thus broad utilization can be expected.

Brief Description of the Drawings

[0025] [Figure 1a-b] Figures 1a to 1b are diagrams showing the results of visual and biochemical analyses before and after the decellularization process of each tissue (bone, ligament, muscle, fibrocartilage and cartilage tissue). Specifically, Figure 1a is the result of visual analysis of the native tissue before the decellularization process and after the decellularization process, and Figure 1b is the result of analysis of the biochemical substance content of the native tissue before the decellularization process and after the decellularization process. [Figure 2a-d] Figures 2a to 2d are diagrams showing the results for the production of cell / DECM self-tissue bodies. Specifically, Figure 2a is a photograph of high-density culture of porcine synovial membrane-derived stem cells and a photograph after DECM treatment, Figure 2b is a photograph showing the condensation of cell / DECM self-tissue bodies, Figure 2c is the result of visual and live / dead assay analysis according to the concentration of the extracellular matrix of cell / DECM self-tissue bodies, and Figure 2d is the result of live / dead assay-based quantitative analysis. [Figure 3a-b]Figures 3a and 3b show the results of the enhancement of the cartilage tissue differentiation ability of autologous tissue by solubilized cartilage DECM treatment. Specifically, Figure 3a is a graph showing the results of the analysis of the increase or decrease in cartilage-related genes in autologous tissue after solubilized cartilage DECM treatment, and Figure 3b is a photograph showing the results of the histological analysis of autologous tissue after solubilized cartilage DECM treatment. [Figure 4a-c] Figures 4a to 4c show the results of the tissue strand ink production process through the homogenization of cell / DECM self-assemblies. Figure 4a is a graph comparing the shape of the 3D printed structure of the tissue strand ink and cell viability based on the culture period of the self-assemblies. Figure 4b is a graph comparing the printability of the tissue strand ink based on the mesh diameter of the molecular sieve used in the homogenization process of the self-assemblies. Figure 4c is a graph comparing the printability of the tissue strand ink based on the diameter of the syringe nozzle used in the homogenization process of the self-assemblies. [Figure 5a-b] Figures 5a and 5b show the results of the characterization analysis of artificial tissues printed with cell / DECM self-organizing substrate strand ink. Specifically, Figure 5a is a photograph showing the process of fabricating artificial tissue using cell / DECM self-organizing substrate strand ink and 3D printing, and Figure 5b is a graph comparing the cell viability of artificial tissue using 3D printing with that of cell / DECM self-organizing substrate strand ink. [Figure 6a-d]Figures 6a to 6d show the results of the biochemical property analysis of artificial tissues printed with tissue-specific DECM-based cell / DECM self-assembly tissue strand ink. Specifically, Figure 6a shows the visual observation results of the artificial tissue printed with tissue-specific cell / DECM self-assembly tissue strand ink, Figure 6b shows the protein profile analysis results of the artificial tissue printed with tissue-specific cell / DECM self-assembly tissue strand ink, Figure 6c shows the collagen analysis results of the artificial tissue printed with tissue-specific cell / DECM self-assembly tissue strand ink, and Figure 6d shows the sGAG analysis results of the artificial tissue printed with tissue-specific cell / DECM self-assembly tissue strand ink. [Figure 7a-e] Figures 7a to 7e show the results of evaluating the degree of tissue differentiation of artificial tissues printed with tissue-specific cell / DECM self-organization base tissue strand ink, respectively, showing the results of evaluating the degree of tissue differentiation of artificial tissues printed with cartilage (Figure 7a), fibrocartilage (Figure 7b), bone (Figure 7c), ligament (Figure 7d), and muscle (Figure 7e) tissue strand inks. [Figure 8] Figure 8 is a schematic diagram illustrating the method for producing a 3D-printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly material according to the present invention. [Modes for carrying out the invention]

[0026] The present invention will be described in more detail below.

[0027] Furthermore, each description and embodiment disclosed in this application is applicable to each other description and embodiment. In other words, all combinations of the various elements disclosed in this application fall within the scope of the present invention. Moreover, the scope of the present invention is not limited by the specific descriptions described later.

[0028] Furthermore, a person with ordinary skill in the art can recognize or confirm numerous equivalents to the specific aspects of the present invention described in this application by conventional experimentation alone. Such equivalents are intended to be included in the present invention.

[0029] As mentioned above, 3D printing is the subject of much research due to its ability to utilize various materials and cells and to embody desired forms. However, it still has limitations in terms of tissue induction and maturation, such as the use of synthetic materials that can produce harmful byproducts to cells during degradation, or the formation of heterogeneous cell-biomaterial complexes. Therefore, the inventors sought to solve the aforementioned problems by forming a self-organized tissue with a homogeneous distribution of cells and extracellular matrix without using separate synthetic materials or growth factors, and applying this to 3D printing technology to derive an optimized 3D printing method. The method for manufacturing 3D printed artificial tissue according to the present invention enables fine patterning with a width in the micrometer range, not only embodying the morphological outline of the primary tissue, but also enabling maturation into a tissue that mimics the biological characteristics of the target organ based on the origin of the extracellular matrix.

[0030] Therefore, the first aspect of the present invention relates to a method for producing 3D-printed artificial tissues based on cell-decellularized extracellular matrix self-assembly.

[0031] Specifically, the manufacturing method includes the following steps: (a) The step of decellularizing and pulverizing tissue-derived extracellular matrix (ECM) to produce decellularized extracellular matrix (DECM) powder; (b) Add the decellularized extracellular matrix powder to the culture medium containing the cells, and then culture to form a cell-decellularized extracellular matrix autoorganism; (c) The step of homogenizing the cell-decellularized extracellular matrix self-assembly and preparing it into tissue strand ink; and (d) The step of applying the homogenized tissue strand ink to a 3D printing equipment to manufacture a 3D printed artificial tissue.

[0032] In the manufacturing method of the present invention, step (a) is a step of producing a decellularized extracellular matrix powder, where decellularization is performed to eliminate the immune response to the cellular components of a different tissue. For effective decellularization to be achieved, the cellular components of the tissue must be completely removed, and the biochemical characteristics must be preserved to the greatest extent possible so that the extracellular matrix can be used as a tissue support in the field of tissue engineering while maintaining the physical properties of the tissue, and various cleaning agents and chemicals used in the processing must be completely removed.

[0033] The decellularization process in step (a) above can be carried out without limitation by methods known in the art. For example, a portion of the target tissue can be obtained from animal or human tissue or organs, washed, freeze-dried, and freeze-crushed to produce a powder, which can then be dissolved in a hypotonic solution for a certain period of time and treated with a solution containing a surfactant to promote decellularization. Alternatively, the extracellular matrix derived from the tissue can be decellularized first and then powdered.

[0034] The tissue-derived extracellular matrix may be derived from the artificial tissue or artificial organ to be ultimately manufactured, and may, but is not limited to, fat, muscle, cartilage, fibrocartilage, heart, bone, ligament, skin, blood vessels, lungs, cornea, brain, mucosal epithelial tissue, bladder, liver, kidney, esophagus, testes, uterus, placenta, nerves, spinal cord, pancreas, spleen, intestines, etc.

[0035] The surfactants used may include, but are not limited to, anionic surfactants such as sodium dodecyl sulfate (SDS) and nonionic surfactants such as Triton X-100. A preferred concentration of SDS is 0.1-0.5%, and a preferred concentration of Triton X-100 is 0.5-1%. The hypotonic solution is used with the surfactant to increase the decellularization efficiency. A preferred hypotonic solution may include, but is not limited to, 5-10 mM Tris-HCl (pH 7.4).

[0036] The decellularization can be carried out by treating the tissue powder in a hypotonic solution for 2 to 6 hours, followed by treatment in a solution containing a surfactant for 1 to 4 hours, and the process is carried out at 4°C to room temperature (e.g., 4 to 35°C).

[0037] Finally, to remove the genetic material present in the tissue powder, it is treated with DNA-degrading enzymes and stirred for 10-12 hours.

[0038] After removing the genetic material, decellularized extracellular matrix powder is finally produced by freeze-drying, and the powder can be manufactured to a fine particle size of 25-100 μm or less. If fine particles larger than the above range are used, the biological and physical properties of the cells may change, and ultimately affect the regulation of the degree of differentiation. If fine particles smaller than the above range are used, the yield will be low and the process will take longer due to the limitations of the internal manufacturing process, and the use will be restricted.

[0039] In a specific embodiment of the present invention, the collagen, sGAG, and DNA content of the decellularized extracellular matrix powder produced through the process described above was analyzed. As a result, as shown in Figure 1b, the collagen and sGAG content of the tissue was well maintained even after decellularization, and more than 97% of the DNA was removed, confirming that the decellularization process proceeded successfully.

[0040] In the manufacturing method of the present invention, step (b) is a step of forming a cell-decellularized extracellular matrix self-assembly, which is carried out by adding the decellularized extracellular matrix powder produced in step (a) to a culture medium containing cells, and then culturing.

[0041] In step (b) above, the cells are stem cells, which may be autologous or heterologous stem cells, and may be, but not limited to, one or more selected from the group consisting of mesenchymal stem cells, embryonic stem cells, and reverse-differentiated stem cells. The cells are preferably seeded in a number of 1.5x10 to 4x10 cells and cultured until a culture rate of 90% or more is observed. If fewer cells are used than the above range, the accumulation of extracellular matrix may be limited, and problems may occur in which self-organization does not proceed. Also, if more cells than the above range are used, the supply of oxygen and nutrients to some cells may not be smooth, which may affect the viability of the cells.

[0042] Subsequently, the decellularized extracellular matrix powder produced in step (a) above can be added to the culture medium and cultured for a certain period of time. At this time, the decellularized extracellular matrix powder may be added at a concentration of 0.05 to 3 mg / ml, preferably 1 to 2.5 mg / ml, but is not limited to this. The culture time may be up to 48 hours, preferably 12 to 24 hours.

[0043] In one specific embodiment of the present invention, the optimal concentration of decellularized extracellular matrix powder was established by analyzing the cell viability of cell-decellularized extracellular matrix self-assembly substrate strand inks based on the concentration of the decellularized extracellular matrix powder. When chondrocyte extracellular matrix powder was used in a concentration range of 0 to 2.5 mg / ml, as can be seen in Figures 2c and 2d, a cell viability of approximately 72% was observed when treated with 2.5 mg / ml or higher, and significant cell death was observed compared to the control group and groups treated with other concentrations of extracellular matrix powder.

[0044] The decellularized extracellular matrix powder in step (b) above can not only act as a chemoattractant to attract cells, but also has a strong ability to bind to cells and promote proliferation and differentiation. Since the decellularized extracellular matrix induces differentiation depending on the type of tissue from which it is derived, it can create a variety of biomimetic structures. Therefore, the decellularized extracellular matrix powder is effective for cell adhesion and proliferation, and can have a significant impact in the differentiation of stem cells into specific cells.

[0045] The cell-decellularized extracellular matrix self-assembly in step (b) above can be formed in vitro.

[0046] Furthermore, in step (b) above, cell-decellularized extracellular matrix self-assemblies can be formed by inducing cell proliferation or cell differentiation. After the cells and decellularized extracellular matrix begin to fuse, further cultivation for a certain period produces cell-decellularized extracellular matrix self-assemblies that gradually condense through self-assembly.

[0047] In step (b) above, a solubilized decellularized extracellular matrix solution may be further added to enhance the tissue differentiation ability of the cell-decellularized extracellular matrix self-organization. The solubilized decellularized extracellular matrix solution can be prepared, for example, by stirring decellularized extracellular matrix powder with pepsin in a 0.01 M to 0.5 M aqueous hydrochloric acid solution or a 0.1 M to 0.5 M aqueous acetic acid solution at 4°C to 36°C, and neutralizing the pH with an NaOH solution. A dialysis membrane (MWCO: 1,000 to 3,000 Da) may be used to remove the salt (NaCl) generated during the neutralization process of the solubilized decellularized extracellular matrix solution, and phosphate buffer solution (PBS) may be added to adjust the pH, ion concentration, and osmotic pressure. The solution may also contain, but is not limited to, extracellular matrix derived from the same tissue as the decellularized extracellular matrix powder prepared in step (a), and may be added at a concentration of 50 to 500 μg / ml. It is preferable that the solubilized and decellularized extracellular matrix solution be added together at the time of autotissue formation and at the time of subsequent replacement of the culture medium within the autotissue.

[0048] In the manufacturing method of the present invention, step (c) is a step of manufacturing the cell-decellularized extracellular matrix self-assembly obtained in step (b) into tissue strand ink for application to 3D printing equipment, which is manufactured through a homogenization process into tissue strand ink optimized for use in 3D printing.

[0049] In this invention, the term "tissue strand ink" refers to a three-dimensional tissue culture obtained by homogeneously blending heterogeneous self-organized tissues.

[0050] In the manufacturing method of the present invention, step (c), that is, the process of producing tissue strand ink from self-organized material, includes a step of homogeneously blending early, immature self-organized material having physical properties suitable for printing.

[0051] The self-assembled material obtained in step (b) above is in a physically / biochemically heterogeneous state and has poor printability, thus limiting its direct application to 3D printing.

[0052] In a specific embodiment of the present invention, tissue strand inks were produced by adjusting the culture period and / or blending process of cell-decellularized extracellular matrix self-organizations, and their printability and cell viability were confirmed.

[0053] First, after the cells and decellularized extracellular matrix began to fuse, the tissue strand inks were cultured for 1, 3, 7, and 10 days, respectively, and then the printability and cell viability of the tissue strand inks produced through the blending process were checked. As can be seen in Figure 4a, when the culture period of the self-tissue was less than 3 days, proper fusion between the cells and extracellular matrix did not occur, resulting in poor adhesion and difficulty in forming a three-dimensional structure. When the culture period exceeded 10 days, the binding between the cells and extracellular matrix was very strong, making it difficult to ensure the physical homogeneity of the ink, increasing the physical stress during the blending process, and resulting in significant cell death.

[0054] Therefore, the optimal culture period for self-organized tissues for use in tissue strand ink may be 2 to 9 days, more preferably 3 to 8 days, and most preferably 3 to 7 days, after the cells and decellularized extracellular matrix have begun to fuse.

[0055] Furthermore, the yield, cell viability, and printability of tissue strand inks obtained through a blending process of cultured self-organic tissues were confirmed. The blending process of self-organic tissues for the production of homogeneous tissue strand inks can be carried out, for example, by passing cultured self-organic tissues through a molecular sieve or syringe nozzle.

[0056] First, blending was performed using molecular sieves with various mesh diameters ranging from 50 to 800 μm (50, 100, 200, 400, and 800 μm, respectively). The yield, cell viability, and printability of the tissue strand ink were then examined based on the mesh diameter. As can be seen in Figure 4b, the yield and cell viability of the tissue strand ink increased with increasing mesh diameter, while print resolution (printability) decreased. Therefore, considering the yield, cell viability, and printability of the tissue strand ink, it is suitable, but not limited to, blending the self-assembled material using molecular sieves with mesh diameters of 50 to 800 μm, more preferably 100 to 600 μm, and most preferably 200 to 400 μm. The blending process using the molecular sieves may be repeated several times until the particle size of the tissue strand ink becomes homogeneous, for example, 1 to 5 times, but is not limited to these.

[0057] Secondly, the cultured autotissues were repeatedly passed through syringe nozzles with various diameters ranging from 1.2 mm to 2.4 mm (1.2, 1.4, and 2.4 mm, respectively) to perform blending, and the yield, cell viability, and printability of the tissue strand ink were then checked. As can be seen from Figure 4c, the yield and printability improved as the diameter of the syringe nozzle decreased, and the cell viability remained at a constant level regardless of the diameter of the syringe nozzle. Therefore, considering the yield, cell viability, and printability of the tissue strand ink as a whole, it is suitable, but not limited, to blend the autotissues using syringes with nozzle diameters of 1.0 to 3.0 mm, more preferably 1.0 to 2.7 mm, and most preferably 1.2 to 2.4 mm. The blending step using the molecular sieve may be repeated several times until the particle size of the tissue strand ink becomes homogeneous, for example, 1 to 5 times, but is not limited to this.

[0058] In the manufacturing method of the present invention, step (d) is a step of manufacturing a 3D printed artificial tissue by applying the tissue strand ink homogenized in step (c) to a 3D printing device. Since the cell-decellularized extracellular matrix self-assembly base tissue strand ink applied to the 3D printing device in step (d) contains living cells and extracellular matrix, it is preferable to carry out the 3D printing under conditions that maximize cell viability. For example, it is preferable to inject the homogenized tissue strand ink obtained in step (c) into a 3D printing syringe and perform 3D printing with a nozzle size of 200 μm or larger, an air pressure of 20 to 150 kPa or less, and a printing speed of 0.1 to 3 mm / second.

[0059] In one specific embodiment of the present invention, the shape of artificial tissue manufactured by 3D printing under the above conditions was observed, and its cell viability was confirmed. As a result, as can be seen from Figure 5a, not only fine cross-shaped structures of 500 μm size but also larger structures of 1 cm or more could be manufactured, and as can be seen from Figure 5b, the tissue strand ink retained a cell viability of 85% or more even after printing.

[0060] As a result, the method for producing 3D-printed artificial tissues based on cell-decellularized extracellular matrix self-assembly of the present invention makes it possible to produce tissue strand inks with conditions optimized for use in 3D printing through the homogenization process described above, and to apply this to 3D printing to obtain finely patterned artificial tissues and artificial organs with widths in the micrometer range (e.g., 200-700 μm).

[0061] Therefore, a second aspect of the present invention relates to 3D-printed artificial tissues and artificial organs based on cell-decellularized extracellular matrix self-assembly substrates produced by the method described above.

[0062] Cell-decellularized extracellular matrix self-assembly-based 3D-printed artificial tissues and organs produced by the aforementioned method can embody the morphological outline of the primary tissue and, depending on the origin of the extracellular matrix, can mature into tissues that mimic the biological characteristics of the target organ.

[0063] The present invention will be described in more detail below with reference to the following examples. However, the following examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto. [Examples]

[0064] [Example 1] Preparation of decellularized extracellular matrix (DECM) powders derived from bone, ligaments, muscles, fibrocartilage, and cartilage tissue. Pig bones were harvested from the tibia and femoral condyle, ligaments from the patellar tendon, muscles from the quadriceps, and fibrocartilage and cartilage tissue from the knee, using surgical blades and saws. Each tissue was washed three times with distilled water, and then obtained as powder through freeze-drying and freeze-crushing.

[0065] The obtained tissue powder was treated with a 10 mM Tris-HCl, pH 7.4 hypotonic solution at room temperature for 4 hours, and then treated with TBS buffer containing 0.1% SDS (sodium dodecyl sulfate) for 2 hours to advance decellularization. Subsequently, it was washed six times with distilled water to remove SDS, a surfactant component. Finally, to remove genetic material present in the extracellular matrix powder of the tissue cells, a solution containing DNA-degrading enzymes (DNAase) was added and stirred for 12 hours. After that, it was washed six more times with distilled water to complete the decellularization process.

[0066] Finally, as shown in Figure 1a, this was freeze-dried to produce decellularized extracellular matrix (DECM) powder, which was then sieved to produce fine powder with a particle size of 100 μm or less.

[0067] [Example 2] Analysis of the biochemical properties of DECM powders derived from bone, ligaments, muscles, fibrocartilage, and cartilage tissue. 2-1. Analysis of collagen and sGAG content in decellularized bone, ligament, muscle, fibrocartilage, and cartilage tissue-derived DECM powders. To quantitatively analyze the collagen and sGAG components maintained after the decellularization process, we performed the Sircol collagen assay and the Blyscan Glycosaminoglycan assay.

[0068] As a result, as shown in Figure 1b, we confirmed that collagen content was well maintained after decellularization in four types of tissue, excluding muscle tissue, and in particular, we confirmed that the collagen composition increased after decellularization in fibrocartilage and cartilage tissue.

[0069] In the case of sGAG content, more than 50% was maintained compared to native tissue even after the decellularization process in four types of tissue excluding bone tissue, and in particular, there was no statistically significant difference compared to native tissue even after the decellularization process in ligament tissue.

[0070] 2-2. Analysis of DNA content of decellularized bone, ligament, muscle, fibrocartilage, and cartilage tissue-derived DECM powders The dsDNA content of bone, ligament, muscle, fibrocartilage, and cartilage tissue extracellular matrix powders after the decellularization process was completed was analyzed using the Picogreen assay.

[0071] As a result, as shown in Figure 1b, more than 97% of the DNA was removed from the five tissues, and the absolute amount was less than 50 ng per 1 mg of tissue. Therefore, it was confirmed that decellation proceeded successfully in the five tissues.

[0072] The results above confirm that the decellularization process performed on the five types of tissue efficiently reduces genetic material while maintaining key substances such as sGAG and collagen.

[0073] [Example 3] Fabrication of stem cell / DECM autoorganisms for 3D printing 3-1. Production of Cell / DECM Autoorganizations Cell / DECM self-assemblies using DECM powders from each tissue were manufactured through the following process.

[0074] As shown in Figure 2a, porcine synovial membrane-derived stem cells (pSYMSCs) were seeded at a density of 2.5 x 10⁶ in a 60 mm diameter culture dish, and then cultured in an incubator for up to 48 hours to achieve a culture rate (confluency) of over 90%. Subsequently, the prepared DECM powders derived from each tissue were suspended in the cell culture medium at a concentration of 1 mg / ml and cultured for up to 48 hours. Once the stem cells and DECM began to fuse, they were separated from the culture dish using a cell scraper, transferred to a 6-well plate, and 5 ml of culture medium was added. The culture medium was replaced with fresh cell culture medium every 3 days.

[0075] As shown in Figure 2b, we were able to confirm that, after stem cell / DECM fusion, a stem cell / DECM self-assembly structure is produced that gradually condenses through self-assembly over a week.

[0076] 3-2. Optimization of DECM concentration in cell / DECM self-organizations for 3D printing To analyze the cell viability of stem cell / DECM auto-organization substrate inks based on extracellular matrix concentration, auto-organizations were prepared by suspending 0-2.5 mg / ml of chondrocyte extracellular matrix powder in cell culture medium, and a live / dead assay was performed on day 7 of culture.

[0077] As a result, as shown in Figures 2c and 2d, it was confirmed that the volume of self-organized tissue increased with increasing concentration of DECM powder. Furthermore, when treated with DECM powder at concentrations of 2.5 mg / ml or higher, a cell viability rate of approximately 72% was observed, and significant cell death was observed compared to the control group and groups treated with other concentrations of DECM.

[0078] [Example 4] Enhancement of tissue differentiation ability of cells / DECM autoorgans by solubilized cartilage DECM treatment. To analyze whether treatment with solubilized DECM (DECM-Sol) enhances the tissue differentiation potential of cell / DECM autoorganizations, RT-qPCR and histological analysis were performed on day 14 of autoorganization culture. For this purpose, DECM-Sol was administered at a concentration of 250 μg / ml for two weeks at each culture medium change, starting from the day when stem cells / DECM began to form self-organizations.

[0079] As a result, as shown in Figure 3a, we observed that the DECM-Sol-treated autoorgan group showed a significant increase in the expression of cartilage-specific markers (COL2, SOX9, and ACAN) compared to the stem cell-only and stem cell / DECM autoorgan groups.

[0080] Furthermore, as shown in Figure 3b, the H&E staining results of the auto-tissue tissues confirmed that the distribution of cells and extracellular matrix was most homogeneous in the group treated with DECM powder + DECM-Sol compared to the stem cell-only group and the group treated with DECM powder only. Safranin-O staining analysis also confirmed that sGAG expression in the stem cell / DECM auto-tissue tissues treated with DECM-Sol was significantly enhanced compared to the other two groups.

[0081] [Example 5] Optimization of the homogenization process for cell / DECM self-organized substrate tissue strand inks. The process of preparing tissue strand ink from self-organized tissues involves a blending step of early, immature self-organized tissues cultured from day 1 to day 10 that possess print-ready physical properties. During this process, as the culture period lengthens, the cells and ECM in the self-organized tissues become more aggregated, increasing their stickiness and physical strength. The stress generated during the blending process can reduce cell viability in the tissue strand ink. Therefore, in this example, the yield, cell viability, and printability of the tissue strand ink were evaluated by adjusting the culture period of the self-organized tissues and the blending process to optimize the homogenization process of the cell / DECM self-organized tissue strand ink.

[0082] 5-1. Optimization of the culture period of autologous tissues After the cells and decellularized extracellular matrix began to fuse, they were cultured for 1, 3, 7, and 10 days, respectively. The printability and cell viability of the tissue strand inks produced through the blending process were then checked, and the results are shown in Figure 4a.

[0083] As shown in the left-hand photograph of Figure 4a, tissue strand ink produced from self-organized tissue on day 1 of culture did not exhibit sufficient aggregation between cells and the ECM, resulting in weak adhesion. This made it difficult to maintain the shape during printing, and the structure easily collapsed. However, tissue strand ink produced from self-organized tissue on days 3 and 7 of culture showed improved print resolution compared to the day 1 group, enabling the production of stable three-dimensional structures. Nevertheless, tissue strand ink produced from self-organized tissue on day 10 of culture had high physical rigidity due to the strong cohesive force between cells and the ECM, and even after the blending process, the ink's homogeneity was poor, making it unsuitable for printing.

[0084] In the case of cell viability, the viability of cells after the blending process to prepare tissue strand ink was evaluated compared to the viability before the process. To observe the change in cell viability before and after the blending process, the same weight of self-tissue and tissue strand ink after the blending process were prepared, treated with collagenase for 4 hours to separate into single cells, and then treated with trypan blue solution to measure the number of viable cells using a cell counter. Here, the cell viability was calculated as a percentage with the viability of the self-tissue before the blending process set to 100%. As can be seen from the graph on the right in Figure 4a, the cell viability was highest in the group on day 1 of culture at 85.3%, and tended to decrease as the culture period increased. In the groups on day 3 and day 7 of culture, all showed viability of 70% or higher, and no statistical difference was observed between the two groups. Finally, the group on day 10 of culture showed the lowest cell viability at 49.8%.

[0085] From the results above, it can be seen that the optimal culture period for self-organized tissues for use in tissue strand ink is 2 to 9 days after the cells and decellularized extracellular matrix begin to fuse.

[0086] 5-2. Optimization of the blending process for cultured autoorgans The blending process of self-assembled materials can be performed using a molecular sieve with a mesh diameter in the micrometer range, or by repeatedly piercing the self-assembled materials with a syringe connector connected to a nozzle with a millimeter diameter.

[0087] Therefore, to optimize the self-assembly blending process, the cultured self-assemblies were passed through a molecular sieve or syringe nozzle, and the yield, cell viability, and printability of the tissue strand ink were evaluated.

[0088] First, in the blending process using molecular sieves, the self-organized tissue was placed on a sterilized sieve, and then the tissue was pierced through the sieve while being moved from side to side using a cell scraper. Molecular sieves with mesh diameters of 50, 100, 200, 400, and 800 μm were used, and the yield of tissue strand ink, cell viability, and printability were confirmed based on the mesh diameter. The yield of tissue strand ink was evaluated by calculating the weight of the tissue strand ink obtained after the blending process as a percentage, with 1 g of self-organized tissue as the baseline (100%). As can be seen from the left-hand graph in Figure 4b, the yield of tissue strand ink tended to decrease as the mesh diameter of the molecular sieve decreased. In particular, there was a significant decrease in mesh diameters of 100 μm or less, and no meaningful difference in mesh diameters of 200 μm or more (50 μm: 38.1%, 100 μm: 48.2%, 200 μm: 69.7%, 400 μm: 73.5%, 800 μm: 80.5%).

[0089] Cell viability was confirmed using the same method as in Example 4-1. As can be seen from the intermediate graph in Figure 4b, cell viability tended to increase as the mesh diameter of the molecular sieve increased. When a mesh diameter of 50 μm was used, a significant cell death of approximately 44.7% was observed, but with diameters of 100 μm or more, a cell viability of over 70% was observed, and in the 800 μm group, a cell viability of approximately 83.8% was observed.

[0090] Printability was assessed by linearly printing a 4x4 mm square structure, then analyzing the area of ​​the actually formed structure relative to the designed pore size using image analysis software, and calculating the printing precision as a percentage. As can be seen from the right-hand graph in Figure 4b, the best printability was 88.5% when using a molecular sieve with a mesh diameter of 50 μm, and gradually decreased as the mesh diameter increased, falling to 7.8% when using a molecular sieve with a mesh diameter of 800 μm. Overall, it can be observed that while yield and cell viability increase with increasing mesh diameter, there is a tendency for printability to decrease.

[0091] In the blending process using syringe connectors connected by nozzles, homogeneous tissue strand ink was produced by repeatedly moving the tissue strand through the syringe connector nozzles. Nozzles with diameters of 1.2, 1.4, and 2.4 mm were used, and the yield, cell viability, and printability of the tissue strand ink were confirmed based on the nozzle diameter.

[0092] The yield, cell viability, and printability of the tissue strand ink were evaluated in the same manner as described above. As can be seen in Figure 4c, the yield of the tissue strand ink tended to decrease as the nozzle diameter increased (see the left graph in Figure 4c), while cell viability remained above approximately 70% regardless of the nozzle diameter (see the middle graph in Figure 4c). Furthermore, as shown in the right graph in Figure 4c, the printability showed a value of 69.2% at a diameter of 1.2 mm, which was confirmed to be the highest resolution compared to the remaining groups. Thus, it can be seen that printability tends to decrease as the nozzle diameter increases (1.4 mm: 62.8%, 2.4 mm: 54.5%). Overall, it can be seen that cell viability increases with increasing nozzle diameter, but yield and printability tend to decrease.

[0093] [Example 6] Fabrication of 3D printed artificial structures using stem cell / DECM autoorganism-based tissue strand ink. The tissue strand ink produced in Example 5 was ultimately filled into a printing syringe and fabricated into an artificial tissue with a three-dimensional shape (Figure 5a). To utilize the tissue strand ink in a 3D printer and fabricate a structure with the desired shape, the printing conditions were set as follows.

[0094] The stem cell / DECM autoorganized substrate tissue strand ink produced in Example 5 contains living cells and extracellular matrix. Therefore, to maximize cell viability during printing, a nozzle size of 200 μm or larger, air pressure of less than 80 kPa, and a printing speed of 1 mm / sec were used.

[0095] As a result, as shown in Figure 5a, we were able to fabricate not only fine cross-shaped structures of 500 μm size, but also larger structures of 1 cm or more, and as shown in Figure 5b, we confirmed that the cells retained a viability rate of over 80% even after printing.

[0096] [Example 7] Biochemical properties of tissue-specific artificial tissues printed with stem cell / DECM autoorganism-based tissue strand ink. 7-1. Visual observation of artificial tissue printed with tissue-specific tissue strand ink. Visual observation was conducted to confirm whether the artificial tissue printed with stem cell / DECM autologous tissue base ink was printed to the specified size.

[0097] As can be seen in Figure 6a, the structure was successfully fabricated in the shape of a circular disc with a diameter of 5 mm, as designed.

[0098] 7-2. Analysis of the biochemical properties of artificial tissues printed with tissue-specific tissue strand inks. SDS-PAGE analysis was performed to analyze the protein cargo profile of artificial tissues printed with tissue-specific tissue strand inks.

[0099] As a result, as shown in Figure 6b, the expression of major protein bands found in natural tissues was also found in the printed artificial tissue, confirming that the artificial tissue has similar biochemical properties to natural tissues.

[0100] Furthermore, the degree of natural tissue realism of the artificial tissue was evaluated by quantitative assessment of collagen and GAG, which are major components of the musculoskeletal tissue ECM. As shown in Figure 6c, the collagen content of the printed artificial tissue was confirmed to be at a level of 60-170% compared to natural tissue (cartilage: 172%, crescent cartilage: 124%, bone: 96%, ligament: 100%, muscle: 61% compared to natural tissue). As shown in Figure 6b, in the case of sGAG, it was also confirmed to be at a level of 30-100% compared to natural tissue (cartilage: 76%, crescent cartilage: 46%, bone: 32%, ligament: 103%, muscle: 57% compared to natural tissue).

[0101] [Example 8] Analysis of tissue differentiation of tissue-specific artificial tissues printed with stem cell / DECM autoorganism-based tissue strand ink. To evaluate whether the artificial tissue possesses biological properties similar to natural tissue after differentiation induction, the tissue was cultured in differentiation medium for 4 weeks after printing, followed by biochemical and histological analysis of the structures.

[0102] As confirmed in Figure 7a, RT-qPCR results of artificial tissues prepared using cartilage tissue-derived DECM powder showed a significant increase in the expression of cartilage-specific markers SOX9 and COL2 compared to the control group prepared using only cells. Furthermore, immunofluorescence staining revealed an increase in the expression of type II collagen (collagen type 2), which appeared red, compared to the control group.

[0103] As confirmed in Figure 7b, RT-qPCR results of artificial tissues prepared using fibrocartilage DECM powder showed a significant increase in gene expression of type II collagen, which is abundant in fibrocartilage, compared to the control group prepared using only cells. Immunofluorescence staining also confirmed an increase in protein expression.

[0104] As confirmed in Figure 7c, RT-qPCR results of artificial tissue prepared using bone tissue-derived DECM powder showed a significant increase in gene expression of type 1 collagen, a major component of bone tissue, and an increase in ALP compared to the control group prepared using only cells. Histological analysis via H&E staining confirmed that DECM powder and stem cells were homogeneously distributed to form the artificial tissue, and that the expression of alizarin red, which indicates calcium accumulation, was increased compared to the control group.

[0105] As confirmed in Figure 7d, RT-qPCR results of artificial tissues prepared using ligament-derived DECM powder showed a significant increase in the expression of type 1 collagen and SCX genes, which are major ECM components of ligaments, compared to the control group prepared using only cells. Observation of the interior of the artificial tissue through H&E staining revealed that the DECM powder and cells were homogeneously distributed to form a single artificial tissue, and immunochemical staining also confirmed the accumulation of type 1 collagen within the artificial tissue.

[0106] Finally, as confirmed in Figure 7e, RT-qPCR results from artificial tissues prepared using muscle DECM powder showed a significant increase in the expression of MYF5, a muscle-specific protein, compared to the control group prepared using only cells. Immunofluorescence staining analysis confirmed an increase in the expression of desmin protein, another muscle-specific protein, compared to the control group.

[0107] From the above description, those skilled in the art in which the present invention pertains will understand that the invention can be implemented in other specific forms without altering the technical idea or essential features of the invention. In this regard, the embodiments described above should be understood to be illustrative and not limiting in all respects. The scope of the present invention should be interpreted as encompassing all modified or altered forms derived from the meaning and scope of the claims and their equivalent concepts described below, rather than from the above detailed description.

[0108] The following are the South Korean national research and development projects that supported this invention: [Project-specific number] 1711187902 [Issue Number] 2023R1A2C100720011 [Department Name] Ministry of Science and ICT, Korea [Project Management (Specialized) Institution Name] Korea Research Foundation [Research project name] (Type 1-1) Mid-career research) [Research Project Title] Development of next-generation artificial tissue injection-type cartilage tissue strand bioink for clinical application, as a replacement for existing cell-biomaterial mixture bioinks. [Contribution Rate] 1 / 1 [Name of organization carrying out the project] Ajou University Industry-Academia Cooperation Group [Research Period] March 1, 2023 - February 28, 2027

Claims

1. A method for manufacturing 3D-printed artificial tissues based on cell-decellularized extracellular matrix self-assemblies, including the following steps: (a) A step of decellularizing and pulverizing tissue-derived extracellular matrix (ECM) to produce decellularized extracellular matrix (DECM) powder; (b) Adding the decellularized extracellular matrix powder to the culture medium containing the cells at a concentration of 1 to 2.5 mg / ml, and culturing the cells for 2 to 9 days after the cells and the decellularized extracellular matrix powder begin to fuse, in order to form cell-decellularized extracellular matrix self-assemblies; (c) A step of homogenizing the cell-decellularized extracellular matrix self-assemblies obtained after step (b) by passing them through a molecular sieve with a mesh diameter of 100 to 600 μm, or by passing them through a syringe connector connected with a nozzle with a diameter of 1.0 to 3.0 mm and blending them to produce tissue strand ink; and (d) The step of applying the homogenized tissue strand ink to a 3D printing equipment to manufacture a 3D printed artificial tissue.

2. The method for producing a 3D printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly substrate according to claim 1, wherein the tissue in step (a) is bone, ligament, muscle, fibrocartilage, or cartilage.

3. The method for producing a 3D printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly substrate according to claim 1, wherein the cells in step (b) are stem cells.

4. The method for producing a cell-decellularized extracellular matrix self-organized 3D printed artificial tissue according to claim 3, wherein the stem cells are one or more selected from the group consisting of mesenchymal stem cells, embryonic stem cells, and reverse-differentiated stem cells.

5. The method for producing a 3D-printed artificial tissue based on a cell-decellularized extracellular matrix self-organization, wherein the cell-decellularized extracellular matrix self-organization is formed in vitro in the step (b) described above.

6. A method for producing a 3D printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly, according to claim 1, wherein a cell-decellularized extracellular matrix powder self-assembly is formed by inducing cell proliferation or cell differentiation in step (b) above.

7. A method for producing a cell-decellularized extracellular matrix self-assembly-based 3D-printed artificial tissue according to claim 1, further comprising adding a solubilized decellularized extracellular matrix solution to step (b) above.

8. The method for producing a 3D printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly substrate according to claim 7, wherein the solubilized and decellularized extracellular matrix solution is added at a concentration of 50 to 500 μg / ml.

9. A method for producing a cell-decellularized extracellular matrix self-organized 3D printed artificial tissue according to claim 1, wherein the tissue strand ink produced in step (d) is injected into a 3D printing syringe, and 3D printing is performed with a nozzle size of 200 μm or more, an air pressure of 20 to less than 150 kPa, and a printing speed of 0.1 to 3 mm / second.

10. A 3D-printed artificial tissue based on a cell-decellularized extracellular matrix self-assembly material, manufactured by the method described in any one of claims 1 to 9.

11. The artificial tissue exhibits the biochemical properties of the primary tissue, as described in claim 10, for a cell-decellularized extracellular matrix self-assembly-based 3D-printed artificial tissue.

12. A 3D-printed artificial organ based on a cell-decellularized extracellular matrix self-assembly substrate, manufactured by the method described in any one of claims 1 to 9.

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