KR20200052467A - Functional bio-ink composition comprising transforming growth factor β for inducing production of cartilage - Google Patents

Abstract

The present invention relates to a functional bio-ink composition comprising a transforming growth factor-β (TGF-β) for inducing production of cartilage, and more particularly, to a bio-ink composition comprising TGF-β which has cytocompatibility, high viscosity and elasticity for easy printing, excellent structural stability, and appropriate mechanical properties after printing, thereby having an excellent effect of inducing the production of cartilage. The bio-ink composition of the present invention shows an effect of promoting differentiation from stem cell into cartilage cell along with high visco-elasticity, strong structural stability, excellent printability, biocompatibility and appropriate mechanical properties after printing, and thus may be very valuably used in preparing tissue-like organs and treating cartilage-related diseases by using a three-dimensional bio-printing.

Description

Functional bio-ink composition comprising transforming growth factor β for inducing production of cartilage

The present invention relates to a functional bio-ink composition that includes transforming growth factor-β (transforming growth factor, TGF-β) and induces cartilage production, more specifically, high viscosity and elasticity with cellular compatibility and easy printing. It relates to a functional bio-ink composition having excellent structural stability and suitable mechanical properties after printing, and having an excellent effect of inducing cartilage production including conversion growth factors.

The three-dimensional cell culture technology has been developed as a technology capable of manufacturing cells and tissues in an environment similar to biological tissues in vitro, and has been applied to various research fields related to cell growth and differentiation, and tissue and organ formation. ought. These tissue-like organs can be usefully used in drug toxicity and pharmacokinetic studies on behalf of actual tissues or organs, and thus are expected to reduce direct experimental application to human specimens and other mammals. In addition, it contributes to the engineering design of tissues and organs as an important element of tissue engineering techniques that are intended to replace or treat damaged tissues and organs.

Three-dimensional bioprinting technology has become a useful device for precisely manufacturing such tissue-like organs and implantable structures. This technique makes it possible to create microscopic and large tissue structures that mimic real human tissue. However, biomaterials that transport live cells during bioprinting, that is, bio-inks, exhibit many limitations in their utilization. The bio-ink for application to bio-printing requires excellent biocompatibility, and must have excellent printing properties that can be printed in a desired pattern by smoothly passing through a fine-diameter dispensing nozzle, and after cell-specific printing It must have structural stability to maintain a mechanical support role while providing an enemy signal. Although natural-derived or synthetic hydrogel bio-inks have been developed and used in the field of three-dimensional bio-printing, bio-inks based on these existing hydrogels have physical and biological aspects such as biocompatibility, printing suitability, geometric precision, and precision. It has significant limitations.

On the other hand, the present inventor has disclosed a bio ink composition including a specific content of a cell transport material, a viscosity enhancer, a lubricant, and a structural material through prior research (Korean Patent Publication No. 10-2017-0012099). Through this, it was possible to obtain a bio ink composition with improved physical and biological properties by solving the above-mentioned problems of the prior art, but this is not suitable for tissue engineering that is intended to replace or treat damaged tissues and organs. There are still limitations in terms of printing precision to simulate.

Accordingly, the present inventor has repeated research to develop a functional bio-ink composition that can be used for the treatment of damaged tissues and organs. As a result, when the conversion growth factor of a specific content is added to a bio ink composition containing a specific component in a specific content, physical and physiological properties are improved, cartilage production is induced, and extracellular matrix production is promoted. It has been discovered that bio-inks can be produced and the present invention has been completed.

Accordingly, an object of the present invention is a bio ink composition comprising a cell transport material, a viscosity enhancer, a lubricant, and a structural material, wherein the bio ink further includes transforming growth factor-β (TGF-β). It is to provide a composition.

Another object of the present invention (a) filling the bio-ink composition in a three-dimensional printer; (b) three-dimensional printing the desired tissue-like organs; And (c) crosslinking the three-dimensional printed bio ink composition.

Another object of the present invention is to provide an organoid prepared according to the method prepared according to the above method.

In order to achieve the above object, the present invention in a bio ink composition comprising a cell transport material, a viscosity enhancer, a lubricant and a structural material, further comprising a transforming growth factor-β (TGF-β) Provided is a bio ink composition.

In order to achieve another object of the present invention, the present invention (a) filling the bio-ink composition in a three-dimensional printer; (b) three-dimensional printing the desired tissue-like organs; And (c) cross-linking the three-dimensional printed bio ink composition.

In order to achieve another object of the present invention, the present invention provides a tissue-like organ (organoid) prepared according to the method prepared according to the above method.

Hereinafter, the present invention will be described in detail.

The present invention provides a bio-ink composition further comprising a transforming growth factor-β TGF-β in a bio-ink composition comprising a cell transport material, a viscosity enhancer, a lubricant, and a structural material.

In the present invention, the transforming growth factor-β (Transforming growth factor-β) is a variety of physiological processes such as cell proliferation, differentiation, apoptosis, migration, and production of extracellular matrix (ECM), angiogenesis, and development in vivo. It is a cytokine that regulates. The active form of TGF-β is present in the form of a 25 kDa dimer. When secreted and activated by cells, it binds to the serine / threonine receptor kinase of the cell membrane, whereby signal is transmitted. The TGF-β receptor on the cell membrane consists of type I and type II. When TGF-β is bound, these two types of receptors form a heterotetrameric complex, and the always active type II receptor phosphorylates the type I receptor. Kinase activates. In the signaling of TGF-β, it is a transcription factor called Smad that acts as a mediator that transmits extracellular signals to the nucleus. The type I receptor phosphorylated by the type II receptor phosphorylates and activates the Smad protein. Phosphorylated Smad protein enters the nucleus and regulates the expression of various genes in cooperation with other transcription factors (Masague J, Seoane J, Wotton D. Genes Dev 19: 2783-2810, 2005). In addition, the TGF-β promotes the proliferation of normal cells to transformed cells, thereby inducing cartilage production and also promoting the production of extracellular matrix such as collagen.

The TGF-β exists in four isoforms of TGF-β1, TGF-β2, TGF-β3 and TGF-β4, and the TGF-β in the present invention may preferably be TGF-β3.

In the present invention, the amino acid sequence of the TGF-β can be easily confirmed by a person skilled in the art through a known database such as Genebank, and not only the TGF-β currently known in the art, but also a new TGF-β to be revealed in the future. It is apparent to those skilled in the art that the present invention may be included in the TGF-β.

The TGF-β of the present invention may be derived from nature or may be synthesized using known peptide synthesis methods. The peptides of the present invention can be prepared by chemical synthesis known in the art (Creighton, Proteins; Structures and Molecular Principles, W. H. Freeman and Co., NY, 1983). Representative methods include, but are not limited to, liquid or solid phase synthesis, fragment condensation, F-MOC or T-BOC chemistry (ChemicalApproaches to the Synthesis of Peptides and Proteins, Williams et al., Eds., CRC Press, Boca Raton Florida, 1997; A Practical Approach, Athert on & Sheppard, Eds., IRL Press, Oxford, England, 1989).

In addition, the TGF-β of the present invention can be produced by genetic engineering methods. First, a DNA sequence encoding the protein is constructed according to a conventional method. DNA sequences can be constructed by PCR amplification using appropriate primers. Alternatively, DNA sequences may be synthesized by standard methods known in the art, for example, using an automatic DNA synthesizer (sold by Biosearch or Applied Biosystems). The produced DNA sequence is a vector that includes one or more expression control sequences (eg, promoters, enhancers, etc.) that are operatively linked to the DNA sequence and regulate the expression of the DNA sequence. , And transformed the host cell with the recombinant expression vector formed therefrom. The resulting transformant is cultured under medium and conditions suitable for allowing the DNA sequence to be expressed, thereby recovering substantially pure protein encoded by the DNA sequence from the culture. The recovery can be performed using methods known in the art (eg, chromatography). In the above, the term "substantially pure protein" means that the protein according to the present invention does not substantially contain any other protein derived from the host. The genetic engineering method for protein synthesis of the present invention can refer to the following documents: (Maniatis et al., Molecular Cloning; A laboratory Manual, Cold Spring Harbor laboratory, 1982; Sambrooket al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, NY, Second (1998) and Third (2000) Edition; Gene Expression Technology, Method in Enzymology, Genetics and Molecular Biology, Method in Enzymology, Guthrie & Fink (eds.)).

Proteins produced by the genetic engineering method or chemically synthesized proteins include, for example, extraction methods, recrystallization methods, various chromatographs (gel filtration, ion exchange, precipitation, adsorption, reverse phase), electrophoresis, countercurrent distribution, etc. It can be isolated and purified by methods known in the art.

TGF-β of the composition according to the invention can be used by itself or in the form of a salt, preferably a pharmaceutically acceptable salt. 'Pharmaceutically acceptable' in the present invention refers to a physiologically acceptable, and when administered to a human, usually does not cause an allergic reaction or a similar reaction, and the salt is a pharmaceutically acceptable free acid. The acid addition salt formed by) is preferred. Organic acids and inorganic acids can be used as the free acid. The organic acid is not limited thereto, citric acid, acetic acid, lactic acid, tartaric acid, maleic acid, fumaric acid, formic acid, propionic acid, oxalic acid, trifluoroacetic acid, benzoic acid, gluconic acid, metasulfonic acid, glycolic acid, succinic acid, 4-toluenesulfonic acid, Glutamic acid and aspartic acid. In addition, the inorganic acids include, but are not limited to, hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid.

Preferably, in the present invention, the TGF-β may be obtained from a mammal, and a method for obtaining a desired protein from a mammal is well known in the art.

Most preferably, the TGF-β may be prepared according to a method comprising the following steps:

(a) preparing TGF-β solution by mixing TGF-β with distilled water and bovine serum albumin;

(b) filtering the solution obtained in (a) using a 0.45 micro filter; And

(c) lyophilizing the solution obtained in (b) to powder it.

On the other hand, in the bio-ink composition of the present invention, the cell transport material may be selected to be suitable for the preparation of a uniform cell suspension and excellent printing tendency, and non-limiting examples thereof include gelatin, collagen, alginate, and agar. , Agarose, pluronic, sub-mucosal tissue, sub-mucosal tissue derivative and polyvinyl alcohol may be any one or more selected from the group consisting of, preferably, sub-mucosal tissue, sub-mucosal tissue derivative, gelatin or collagen It may be, and most preferably, it may be a small intestinal mucosal tissue or a derivative thereof.

In the present invention, the small intestinal mucosal tissue is a tissue without cells, and an immune response hardly occurs, and more than 90% is composed of collagen Ⅰ and Ⅱ in the skin, and a small amount of collagen Ⅴ, Ⅵ, etc. exists. . In addition, sub-mucosal tissues are extracellular matrix (ECM), glycosaminoglycan and fibronectin, chondroitin sulfate, heparin, heparin sulfate, hyaluronic acid and basic fibroblast growth factor-2 (FGF-2). ), Nerve growth factor (NGF), transforming growth factor-β (TGF-β), epidermal growth factor (EGF), vascular endothelial It contains a large amount of various cytokines such as growth factor (VEGF) and insulin-like growth factor-1 (IGF-1). As such, since ECM and cytokines exist in the small intestinal mucosal tissue, it has been applied to many fields other than tissue regeneration by helping the functional aspects of cells such as cell adhesion, growth, migration, and differentiation.

In the present invention, the small intestinal submucosal tissue can be used without limitation as long as it is obtained from mammals other than humans. Preferably, the small intestinal submucosal tissue can be used.

The small intestinal mucosal tissue used in the present invention can be used without limitation as long as it is manufactured according to the manufacturing method used in the art, for example, Korean Registered Patent No. 1288088, Korean Published Patent No. 20060100430, Korean Registered Patent Powdered ones may be used with reference to No. 1364591 or the like. Preferably, the small intestinal mucosal tissue can be prepared according to a method comprising the following steps:

(1) preparing a small intestinal mucosal tissue solution by mixing the small intestinal mucosal tissue with an acidic solution and pepsin; (2) adjusting the solution obtained in (1) to a biocompatible pH using a basic solution; And (3) lyophilizing the solution obtained in (2) to form a powder.

The acidic solution can be any solution that can be adjusted to a pH of 2.5 to 4.5, and preferably, an aqueous solution containing an acid selected from acetic acid, paratoluenesulphonic acid, and maleic acid at a concentration of 1 to 5% by weight is preferable.

The basic solution is used to titrate the acidity and reduce the inflammatory reaction of surrounding cells when used as a tissue engineering support, for example, sodium hydroxide (NaOH), sodium carbonate (Na2CO3), sodium hydrogen carbonate (NaHCO3) , Disodium hydrogen phosphate (Na2HPO4), calcium hydrogen carbonate (Ca (HCO3) 2), calcium hydroxide (Ca (OH) 2), calcium hydroxide nitrate (Ca (OH) NO3), calcium hydroxide (Ca (OH) Cl), hydroxide Cyanium (Ca (OH) CN), potassium hydroxide (KOH), ammonium hydroxide (NH4OH) and sodium acetate (CH3COONa) may be used. The basic solution is titrated to adjust the pH similar to the in vivo pH of 5.5 to 7.8.

On the other hand, before the step (1) (a) pre-treatment step of removing the mesentery, mucosal layer and muscle layer in the sub-mucosal tissue obtained from mammals other than humans; And (b) pre-treatment of the pre-intestinal submucosal tissue, including lyophilization and pulverization of the pre-treated sub-intestinal submucosal tissue.

In the present invention, the small intestinal mucosal tissue derivative means a small intestinal mucosal tissue in a form modified to be crosslinkable through chemical modification. If the derivative is capable of inducing a crosslinking reaction through a crosslinking agent or an optical initiator, the type is particularly limited. Although not, preferably, it may be methacrylated small intestinal mucosal tissue (methacrylated SIS), acrylated small intestinal mucosal tissue (acrylated SIS), or may be a thiolated small intestinal mucosal tissue (thiolated SIS), most preferably methacrylicized small intestinal mucosal tissue. (methacrylated SIS).

In the bio-ink composition of the present invention, the cell transport material is characterized by including the small intestinal mucosal tissue or a derivative thereof, preferably, the cell transport material is 30 to 100 w / w%, more preferably, the small intestinal submucosal tissue derivative. 50 to 100 w / w%, more preferably 70 to 100 w / w%, and most preferably 100 w / w%.

When the small intestinal mucosal tissue derivative is contained in the cell carrier material in an amount of less than 30%, it is not preferable in that the structural stability of the structure printed with the bioink composition may be lowered.

In the bio ink composition of the present invention, the viscous enhancer may be selected to be suitable for maintaining excellent printing tendency and initial strength of the bio ink, and non-limiting examples thereof may be hyaluronic acid or dextran.

In the bioink composition of the present invention, the lubricant is preferably selected from materials capable of minimizing shear rate and improving dispensing speed, and non-limiting examples thereof include glycerol. Can be lifted.

In the bioink composition of the present invention, the structural material is preferably selected from materials capable of maintaining rapid crosslinking and mechanical stiffness, and non-limiting examples include fibrinogen and hyaluronic acid modified to be chemically crosslinkable. (Eg, methacrylated hyaluronic acid, thiolated hyaluronic acid, etc.), gelatin modified to be chemically crosslinkable (e.g. gelatin methacrylate, thiolated gelatin, etc.), collagen, alginate, It may be any one or more selected from the group consisting of methyl cellulose, chitosan, chitin, synthetic peptides and polyethylene glycol based hydrogels.

The fibrinogen is not only suitable as a structural material in terms of stability of the gel, but also can be selected as a preferred structural material in that it creates a microenvironment suitable for cell attachment and differentiation after the cells are printed. Hyaluronic acid and glycerol may be suitably used in the composition of the present invention in terms of dispensing uniformity and prevention of clogging of the nozzle when applying three-dimensional bio printing, respectively.

In the bio ink composition of the present invention, the cell transport material is 0.01-10 w / v%, 0.05-10 w / v%, 0.5-10 w / v%, 1-10 w / v%, 1.5-10 w / v %, 2-10 w / v%, 0.1-8 w / v%, 0.5-8 w / v%, 1-8 w / v%, 2-8 w / v%, 0.1-6 w / v%, 0.5-6 w / v%, 1-6 w / v%, 2-6 w / v%, 0.1-5 w / v%, 0.5-5 w / v%, 1-5 w / v% or 2- 5 w / v% may be included, most preferably 2-4 w / v%, but is not limited thereto.

In the bio ink composition of the present invention, the viscosity enhancer is 0.01-1 w / v%, 0.05-1 w / v%, 0.1-1 w / v%, 0.2-1 w / v%, 0.01-0.8 w / v% , 0.05-0.8 w / v%, 0.1-0.8 w / v%, 0.2-0.8 w / v%, 0.01-0.5 w / v%, 0.05-0.5 w / v% or 0.1-0.5 w / v% And most preferably 0.2-0.4 w / v%, but is not limited thereto.

In the bio ink composition of the present invention, the lubricant is 0.1-30 w / v%, 1-30 w / v%, 5-30 w / v%, 10-30 w / v%, 0.1-20 w / v%, 1-20 w / v%, 5-20 w / v%, 10-20 w / v%, 0.1-15 w / v%, 1-15 w / v%, 5-15 w / v% or 10- 15 w / v% may be included, most preferably 10-13 w / v%, but is not limited thereto.

In the bio ink composition of the present invention, the structural material is 0.1-10 w / v%, 0.5-10 w / v%, 1-10 w / v%, 0.1-8 w / v%, 0.5-8 w / v% , 1-8 w / v%, 0.1-5 w / v%, 0.5-5 w / v% or 1-5 w / v%, most preferably 1-3 w / v%, , But is not limited thereto.

The inventors of the present invention tested the physical and biological properties of the composition having various combinations of configurations to develop a bio-ink composition that exhibits physical and biological properties suitable for application to three-dimensional bioprinting through conventional research. It has been confirmed that the bio-ink composition consisting of the combination of the composition and the components of the above content is particularly suitable for three-dimensional bio-printing for the production of tissue-like organs by exhibiting high structural stability, excellent printing performance and appropriate mechanical properties after printing.

In the bio ink composition of the present invention, the TGF-β is 0.01-1 w / v%, 0.05-1 w / v%, 0.1-1 w / v%, 0.2-1 w / v%, 0.01-0.8 w / v %, 0.05-0.8 w / v%, 0.1-0.8 w / v%, 0.2-0.8 w / v%, 0.01-0.5 w / v%, 0.05-0.5 w / v% or 0.1-0.5 w / v% May be, and most preferably, may include 0.01-0.03 w / v%, but is not limited thereto.

On the other hand, the bio-ink composition of the present invention may include cells 0.05-60 × 10 ⁶ / mL.

In the present invention, the cells are preferably, stem cells, osteoblasts, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, embryos Cells (germ cells), hepatocytes, renal cells, Sertoli cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells ( selected from the group consisting of smooth muscle cells, cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells and neurons. It may be any one or more, preferably stem cells or osteoblasts, and most preferably stem cells.

The cells used in the present invention and the cells included in the bioprinted tissue-like construct can be cultured in any manner known in the art. Cell and tissue culture methods are known in the art and are described, for example, in Cell & Tissue Culture: Laboratory Procedures; Freshney (1987), Culture of Animal Cells: A Manual of Basic Techniques, The contents of this are incorporated herein by reference. General mammalian cell culture techniques, cell lines, and cell culture systems that can be used with the present invention are also described in Doyle, A., Griffiths, JB, Newell, DG, (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley (1998), the contents of which are incorporated herein by reference.

In the present invention, the cells can be bioprinted by depositing or extruding bioink from a three-dimensional bioprinter. The bio ink of the present invention may be in the form of a liquid, semi-solid, or solid composition comprising a plurality of cells. Bio inks include liquid or semi-solid cell solutions, cell suspensions, or cell concentrates. The bio-ink composition comprises: 1) mixing a plurality of cells or cell aggregates with a biocompatible liquid or gel at a predetermined ratio to prepare a bio-ink, and 2) densifying the bio-ink to obtain a bio-ink having a desired cell density and viscosity. It may be manufactured by the steps of manufacturing. Specifically, the densification of the bio ink is realized by centrifugation, tangential flow filtration ("TFF"), or a combination thereof, and the densification of the bio ink produces an extrudable composition to form multicellular aggregates or multicellular bodies. The term “extrudable” means that it can be molded by passing a nozzle or orifice (eg, one or more holes or tubes) (eg under pressure). In addition, densification of bio-inks is derived from growing cells at a suitable density. The cell density required for bioinks depends on the cell to be used and the tissue or organ to be manufactured.

In the present invention, the bio-ink composition may further include a tissue-derived component.

The tissue-derived component means that specific tissues of animals, such as cartilage, kidney, heart, liver, and muscle, are decellularized and gelled of a substance having an extracellular matrix as a main component, which indicates the tissue specificity of the bio-ink composition. Can be included to enhance.

In order for cells included in the bioink composition to grow and differentiate normally in a three-dimensional printed tissue-like organ, it is necessary to create a natural environment similar to the microenvironment of the original tissue in the body. It is virtually impossible to recreate all of the properties of natural tissue-derived components, such as extracellular matrix, in any artificially produced composition, so by adding decellularized tissue-derived components to the bioink composition, the cells Can create a microenvironment that can grow and differentiate normally.

Since the decellularized tissue-derived component has each tissue-specific component and phase formed through interaction with cells in the human body, it can provide an advantageous environment for cell growth and differentiation. Therefore, by adding a tissue-derived component to the bio-ink composition of the present invention, it is possible to maintain the normal shape of the cell and to smoothly preserve its function.

The tissue capable of obtaining the tissue-derived component in the present invention is not particularly limited, and may be selected and decellularized according to a tissue-like organ to be produced and then gelled. Methods for decellularization of tissues and methods for gelation are known in the art and will be readily carried out by those skilled in the art to which the present invention pertains.

In the present invention, the bio-ink composition may additionally include a cell culture medium. The cell culture medium is a concept that includes any medium suitable for the desired cell, and non-limiting examples of the cell culture medium include Dulbecco's phosphate buffered saline, Ear Balancing Salt, Hank Balancing Salt, Tyrod Salt, and Alciver solution. , Gay Balancing Salt Solution, Crab-Henzelite Denatured Buffer, Crab-Ringer Bicarbonate Buffer, Puck Saline, Dulbecco's Modified Eagle's Medium, Dulbecco's Modified Eagle's Medium / Nutrient F-12 Ham, Nutrient Mixture F-10Ham (Ham's F-10 ), Medium 199, Eagle Minimum Essential Medium, RPMI-1640 Medium, Ames Medium, BGJb Medium (Fitton-JacksonModification), Click Medium, CMRL-1066 Medium, Fisher Medium, Glascow Minimum Essential Medium (GMEM), Iscov Denaturation Two Beco medium (IMDM), L-15 medium (Leibovitz), McCoy 5A denaturation medium, NCTC medium, Swim S-77 medium, Weimouth medium, William medium E, or combinations thereof, but are not limited thereto. In addition, the cell culture medium may further include albumin, selenium, transferrin, fetuin, sugar, amino acids, vitamins, growth factors, cytokines, hormones, antibiotics, lipids, lipid carriers, cyclodextrins, or combinations thereof. have.

In the present invention, the bio ink composition may further include a substance and / or antioxidant that promotes cell adhesion.

In addition, the bio-ink composition of the present invention may further include a substance that inhibits cell death (eg, necrosis, cell death, or autonomous absorption). Non-limiting examples of substances that inhibit cell death include small molecules, antibodies, peptides, peptibodies, anti-TNF substances, substances that inhibit the activity of interleukins, substances that inhibit the activity of interferons, and GCSF (granulocyte colony-stimulating factor). Substances that inhibit activity, substances that inhibit the activity of macrophage inflammatory proteins, substances that inhibit the activity of TGF-B (transformation growth factor B), substances that inhibit the activity of MMP (matrix metalloproteinase), Substances that inhibit the activity of the carspace, substances that inhibit the activity of the MAPK / JNK signaling cascade, substances that inhibit the activity of the Src kinase, substances that inhibit the activity of the JAK (janus kinase), or combinations thereof. have.

The present invention also, the bio-ink composition may further include a crosslinking agent or a photoinitiator to promote crosslinking of the components.

The crosslinking agent may be a compound containing a polyvalent metal ion used in a conventional hydrogel composition. The polyvalent metal ion compound is preferably selected from the group consisting of aluminum compounds, calcium compounds and magnesium compounds. For example, it may be one or more selected from the group consisting of aluminum hydroxide, aluminum hydrous silicate, calcium chloride, magnesium chloride, aluminum chloride, magnesium metasilicate, aluminum acetate and magnesium silicate.

The optical initiator refers to a material that causes rapid crosslinking upon exposure to light. In the present invention, the type of the optical initiator is not particularly limited, but an optical initiator that cross-links by irradiation of ultraviolet (UV) light or an optical initiator that cross-links by irradiation of visible light may be used. Non-limiting examples of suitable photoinitiators include acetophenone, benzoin methyl ether, diethoxyacetophenone, benzoyl phosphine oxide and 1-hydroxycyclohexyl phenyl ketone, eosin, and the like. The amount of the optical initiator added may vary depending on the wavelength and time of the light to be exposed.

The bio-ink composition of the present invention described above may exhibit the effect of promoting differentiation of stem cells into chondrocytes by including TGF-β in the composition in a specific content.

Stem cells in which the bioink composition of the present invention can induce differentiation into chondrocytes may be mesenchymal stem cells of an animal, preferably mammalian, more preferably human mesenchymal stem cells. have. In addition, the mesenchymal stem cells of the present invention may be derived from bone marrow, adipose tissue, peripheral blood, and liver.

Mesenchymal stem cells are present in small amounts in the bone marrow and the like, but the process of isolating and culturing them is well known in the art, and for example, refer to US Patent No. 5,486,359. In addition, in the present invention, the mesenchymal stem cells can be obtained by separating from the hematopoietic stem cells of the bone marrow according to a known method and then propagating in a state in which the differentiation ability is not lost.

The present invention also comprises the steps of (a) filling the bio-ink composition of the present invention in a three-dimensional bio printer; (b) three-dimensional printing the desired tissue-like organs; And (c) cross-linking the three-dimensional printed bio ink composition.

The bio-ink composition provided in the present invention is easy to manufacture various tissue-like organs. Bio-printing technology has been developed in the art as a useful tool for manufacturing tissue-like organs. On the other hand, conventional bio-ink compositions for use in bio-printing have a problem that it is difficult to have sufficient physical stiffness, or excessive physical stiffness, so that cell survival is not sufficiently ensured in the printing process. The bio-ink composition of the present invention is applied to three-dimensional bio-printing to produce a tissue-like organ, and has the advantage of maintaining sufficient physical stiffness and ensuring sufficient cell survival when sprayed by a bio-printer, thereby producing a tissue-like organ. It is very suitable for physical and biological properties. In addition, the bio ink composition of the present invention was confirmed to exhibit sophisticated printing performance compared to a bio ink composition using only gelatin as a cell carrier.

In addition, as compared to the conventional bio-ink composition using only gelatin as the cell transport material, it was confirmed that the structural stability after printing is remarkably excellent and more precise printing property when the subcarrier tissue is included as a cell transport material.

The bio ink composition in the present invention enables stacking of three-dimensional structures. The bioink composition of the present invention containing a sufficient amount of cells can be used to form a tissue-like organ by being stacked by a three-dimensional bioprinter.

“Bioprinting” in the present invention means accurate cell deposition in three dimensions (eg, cell solutions, cell-containing gels, cell suspensions) through methodologies compatible with automated, computer-aided, three-dimensional prototyping devices (eg, bioprinters), It means using cell concentrates, multicellular aggregates, multicellular bodies, etc.).

The bioprinting method in the present invention is continuous and / or substantially continuous. A non-limiting example of a continuous bioprinting method is to jet bioink from a bio printer through a dispensing tip (eg, syringe, capillary tube, etc.) connected to a reservoir of bioink. The continuous bioprinting method is to spray bioink in a repeating pattern of functional units. The repeating functional unit has any suitable geometry, including, for example, circular, square, rectangular, triangular, polygonal, and irregular geometry. In addition, repeat patterns of bioprinted functional units include layers and multiple layers can be bioprinted adjacently to form engineered tissue or organs (eg, stacked). Specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more layers can be bioprinted adjacent to form engineered tissue or organs.

The bioprinted functional units can be repeated in a tessellated pattern. The "lattice pattern" is a planar shape that fills non-overlapping and gap-free planes. The benefits of continuous and / or lattice bioprinting can include increased productivity of bioprinted tissue. Another non-limiting potential advantage is that it eliminates the need to align the bio printer with elements of previously deposited bio ink. Continuous bioprinting can also facilitate printing larger tissue from a large reservoir of bioink, optionally using a syringe mechanism.

Proper and / or optimal jetting distance from the bio printer does not result in material flattening or adhesion to the jetting needle. The bio printer spray tips are about, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 , Has an inner diameter of 1000 µm or more and an increment within this range. In addition, the bio ink storage of the bio printer is about .5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 , 60, 65, 70, 75, 80, 85, 90, 95, 100 cubic centimeters or more and incremental volumes within this range. The pump speed may be suitable and / or optimal if the residual pressure rise in the system is low. Good pump speeds can depend on the ratio between the cross-sectional area of the reservoir and the injection needle, and higher rates require lower pump speeds.

Various types of tissue-like organs can be created by the methods described above. The pattern or stacking arrangement for stacking the bioink composition may be determined by the size and diameter of tissue-like organs to be manufactured. In addition, the number of cells included in the bio-ink used to manufacture tissue-like organs can be adjusted according to the type of cells, the content of the cell nutrients contained in the bio-ink composition, and the like. In addition, the type of cells included in the bio-ink composition can be variously changed according to the type of tissue-like organ to be manufactured according to the above method. Any person having ordinary skill in the art to which the present invention pertains will be able to select and apply appropriate cells according to the type of tissue-like organs to be manufactured through three-dimensional bioprinting.

After the bio-ink composition is sprayed and stacked by a three-dimensional bio-printer, cross-linking of the bio-ink composition may be promoted by exposing it to light (ultraviolet or visible light) or by adding a crosslinking solution. This crosslinking allows the layered bio ink composition to be completed with a more rigid structure. On the other hand, the light (ultraviolet or visible light) can be directly exposed to the surface of the laminated bio-ink composition, for example, using a wavelength of 300 nm to 800 nm generated from a light (ultraviolet or visible light) generator It may be exposed for 1 second to 1000 seconds at a distance of 1 to 20 cm from the stacked bio ink composition, or may be exposed for 20 seconds to 500 seconds, or 40 seconds to 240 seconds. Such light (ultraviolet and visible light) exposure distances and times are those skilled in the art to which the present invention pertains, short distances and strong wavelengths can form sufficient crosslinking even with short exposure times. Will be recognizable.

The present invention also provides an implantable tissue-like organ or tissue construct made in accordance with the above method.

The 3D bioprinting technology has the potential to produce tissue or organ structures that are clinically applicable in shape and size. Through the combination of tissue-specific cell types and bio-ink applied to the three-dimensional structure, it is possible to utilize the reproducing ability inherent in the cells, and it may be possible to produce necessary tissues or organs. Thus, bioprinted tissue constructs using the bioink composition according to the present invention provide constructs having anatomically and functionally similarities to mammalian tissues and organs.

By using the composition of the present invention, not only can a tissue-like organ or a single structure of tissue transplantable in the body be provided, but also a tissue structure having significantly improved functionality, capable of reproducing the connection between tissue and tissue by simultaneously printing several cells, It can also be manufactured.

Since the bio-ink composition of the present invention exhibits an effect of promoting differentiation of stem cells into chondrocytes with high viscoelasticity, strong structural stability, excellent printing properties, biocompatibility and proper mechanical properties after printing, three-dimensional bioprinting is used. It can be very useful in the production of tissue-like organs and in the treatment of cartilage related diseases.

Hereinafter, the present invention will be described in detail with reference to specific examples.

However, the following examples are merely illustrative of the present invention, and the contents of the present invention are not limited to the following examples.

<Example 1>

Preparation of bio ink composition

The bio-ink composition of the present invention has sufficient viscosity to allow printing to be performed in a uniformly mixed form of cells, and is sensitive to temperature (35 mg / mL, Sigma-Aldrich, St. Louis, MO, USA), during printing Hyaluronic acid (3 mg / mL, Sigma-Aldrich), which can enhance the viscosity of the bio ink and improve the uniformity of the pattern, glycerol (10 v / v%, Sigma-Aldrich) as a lubricant to reduce nozzle clogging And chemically modified gelatin (20 mg / mL methacrylated gelatin) that provides structural stability by crosslinking after printing. Each composition was uniformly mixed by stirring at 37 ° C for one day. The completely mixed and dissolved bio ink composition was sterilized using a 0.45 um injection filter. In order to achieve crosslinking after printing, methylpropiophenone (1%, Sigma-Aldrich), an optical initiator, was used. TGF-β3 (0.1mg / mL, GenScript), which is used when inducing bioprinted stem cells to differentiate into chondrocytes, was included in the bioink composition through chemical bonding.

Claims (7)

A bio ink composition comprising a cell transport material, a viscosity enhancer, a lubricant, and a structural material, wherein the bio ink composition further comprises a transforming growth factor (TGF-β).
The bioink composition of claim 1, wherein the conversion growth factor-β is a conversion growth factor-β3.
The method according to claim 1, wherein the cell transport material is 0.1-10 w / v%, the viscosity enhancer is 0.01-1 w / v%, the lubricant is 1-30 w / v%, and the structural material is 0.1-10 w / v%. And a conversion growth factor-β of 0.01-1 w / v%.
According to claim 1, wherein the bio-ink composition, characterized in that the cell further contains 0.05-60 × 10 ⁶ / mL.
The method of claim 1, wherein the cell transport material is small intestinal mucosal tissue, small intestinal mucosal tissue derivatives, gelatin or collagen, the viscosity enhancer is hyaluronic acid or dextran, the lubricant is glycerol and the structural material is fibrinogen or gelatin methacrylate. Composition characterized in that.
(A) filling the composition of any one of claims 1 to 5 in a three-dimensional printer;
(b) three-dimensional printing the desired tissue-like organs; And
(c) A method for manufacturing a tissue-like organ comprising cross-linking a three-dimensional printed bio ink composition.
An organoid made according to the method of claim 6.