KR102345698B1 - Composition of Bio-ink Comprising Methacrylated Low Molecular Weight Collagen and Method for Producing Tissue-Like Structure Using the Same - Google Patents

Abstract

The present invention relates to a bio-ink composition containing methacrylated low-molecular collagen and a method for manufacturing a tissue-like organ using the same, and more particularly, to a bio-ink composition containing methacrylated low-molecular collagen as a cell transport material in a specific content or more. It relates to an ink composition and a method for manufacturing a tissue-like structure using the same.
The bio-ink composition comprising low molecular weight collagen methacrylated as a cell transport material of the present invention exhibits high viscoelasticity, strong structural stability, excellent printability, biocompatibility, and appropriate mechanical properties after printing. It can be very usefully used in the manufacture of similar structures.

Description

Bio-ink composition comprising methacrylated low molecular weight collagen and method for manufacturing a tissue-like structure using the same

The present invention relates to a bio-ink composition containing methacrylated low-molecular collagen and a method for manufacturing a tissue-like structure using the same, and more particularly, to a bio-ink composition containing methacrylated low-molecular collagen as a cell transport material in a specific content or more It relates to an ink composition and a method for manufacturing a tissue-like structure using the same.

Three-dimensional cell culture technology has developed into a technology that can manufacture cells and tissues in an environment similar to living tissue in vitro, and is applied to various research fields related to cell growth and differentiation, and formation of tissues and organs. have. These tissue-like organs can be usefully used in the study of drug toxicity and pharmacokinetics instead of actual tissues or organs, and accordingly, it is thought that direct experimental application to human samples and other mammals can be reduced. In addition, it is contributing to the engineering design of tissues and organs as an important element of tissue engineering techniques for the purpose of replacing or treating damaged tissues and organs.

3D bioprinting technology has become a useful device for precisely manufacturing such tissue-like organs and implantable structures. This technology makes it possible to create microscopic and large tissue structures that mimic real human tissues almost as they are. However, biomaterials that transport living cells during bioprinting, that is, bio-ink, have many limitations in their utilization. As the characteristics of bio-ink required to be applied to bio-printing, excellent biocompatibility is required, and it must have excellent printability that can be printed in a desired pattern by smoothly passing through a micro-diameter dispensing nozzle, It should have structural stability that can maintain the mechanical support role while providing cell-specific signals after printing. Although, naturally-derived or synthetic hydrogel bio-inks have been developed and used in the field of 3D bio-printing, bio-inks based on such existing hydrogels are biocompatible, printing compatibility, geometrical precision, and precision in physical and biological aspects. It has significant limitations.

The inventors of the present invention have disclosed a bio-ink composition comprising a specific content of a cell carrier material, a viscosity enhancer, a lubricant, and a structural material through prior research (Korean Patent Application Laid-Open No. 10-2017-0012099). Through this, some of the problems of the prior art described above could be solved, but structural stability not suitable for tissue engineering for the purpose of replacing or treating damaged tissues and organs, printing precision for simulating sophisticated biological tissues, etc. After discovering that there are limitations, intensive research was repeated to solve them.

Accordingly, when the conventional bio-ink composition contains methacrylated low molecular weight collagen as a cell carrier material in a specific content or more, a bio-ink having surprisingly improved structural stability and printing precision can be manufactured. was found, and the present invention was completed.

It is an object of the present invention to provide a bio-ink composition comprising methacrylated low molecular weight collagen as a cell carrier material.

Another object of the present invention is to provide a method for producing a tissue-like structure using the bio-ink composition and a tissue-like structure prepared by the method.

In order to achieve the above purpose,

The present invention includes a cell carrier material, a viscosity enhancer, a lubricant and a structural material,

The cell transport material provides a bio-ink composition comprising methacrylated low molecular weight collagen.

In a preferred embodiment of the present invention, the molecular weight of the methacrylated low molecular weight collagen may be 500 ~ 4000Da.

In another preferred embodiment of the present invention, the methacrylated low molecular weight collagen contained in the bio-ink composition may be 0.1 to 10 w/v%.

In another preferred embodiment of the present invention, the complex viscosity of the bio-ink composition before crosslinking may be 8 to 1000 Pa·s, and the viscosity after crosslinking may be 20 to 3000 Pa·s.

In another preferred embodiment of the present invention, when the frequency (Frequency) of the bio-ink composition is 1Hz, the viscosity before crosslinking may be 60 to 140 Pa·s, and the viscosity after crosslinking may be 130 to 350 Pa·s.

In another preferred embodiment of the present invention, the storage modulus before crosslinking of the bio-ink composition is 250 to 1000 Pa, the loss modulus is 80 to 160 Pa, and the storage modulus after crosslinking is 700 ~ 2300 Pa, the loss modulus may be 160 ~ 400 Pa.

In another preferred embodiment of the present invention, when the frequency (Frequency) of the bio-ink composition is 1Hz, the storage modulus before crosslinking is 400 to 850 Pa, the loss modulus is 85 to 200 Pa, and the storage modulus after crosslinking is 800 ~ 2200 Pa, the loss modulus may be 180 ~ 250 Pa.

In another preferred embodiment of the present invention, the bio-ink composition contains 0.01 to 1 w/v% of the viscosity enhancer, 1 to 30 w/v% of the lubricant, and 0.1 to 10 w/v% of the structural material. can

In another preferred embodiment of the present invention, the viscosity increasing agent may be hyaluronic acid or dextran, the lubricant may be glycerol, and the structural material may be fibrinogen or methacrylated gelatin.

In another preferred embodiment of the present invention, gelatin or collagen may be additionally included in the cell transport material.

In another preferred embodiment of the present invention, the bio-ink composition may further include cells ^{of 0.05 ~ 60 × 10 6 /mL.}

In another preferred embodiment of the present invention, the cells are stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, and glioblasts. Glioblast, germ cell, hepatocyte, renal cell, Sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte ), smooth muscle cells, cardiomyocytes, glial cells, endothelial cells, hormone-secreting cells, immune cells, pancreatic islet cells and neurons It may be at least one selected from the group consisting of.

In another preferred embodiment of the present invention, the composition may further include an optical initiator (photoinitiator).

In order to achieve another object of the present invention,

The present invention comprises the steps of (a) filling the bio-ink composition in a three-dimensional printer; (b) three-dimensional printing of the desired three-dimensional structure; And (c) provides a method for producing a three-dimensional structure for tissue regeneration comprising the step of crosslinking the three-dimensional printed bio-ink composition.

In addition, the present invention comprises the steps of (a) filling the bio-ink composition in a three-dimensional printer; (b) three-dimensional printing of the desired tissue-like structure; (c) provides a method for preparing a tissue-like structure comprising the step of crosslinking the three-dimensionally printed bio-ink composition.

In a preferred embodiment of the present invention, the method may further comprise (d) culturing the tissue-like construct in a culture medium.

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

The bio-ink composition comprising methacrylated low molecular weight collagen of the present invention exhibits high viscoelasticity, strong structural stability, excellent printability, biocompatibility, and appropriate mechanical properties after printing. can be very useful for

1 is data comparing the viscosities before (A) and after (B) cross-linking of a bio-ink composition containing methacrylated low molecular weight collagen (ColMA bio-ink) and a bio-ink composition containing gelatin (Cel4cell) .
2 is data comparing the viscosity of the bio-ink composition before and after cross-linking based on a frequency of 1 Hz. Each sample number was as follows: 1) Gel4cell, 2) ColMA 1%, 3) ColMA 2%, 4) ColMA 3%, 5) ColMA 4%, 6) ColMA 5%.
3 is data comparing the modulus of ColMA bio-ink and Cel4cell bio-ink before (A) and after (B) cross-linking.
4 is data comparing the modulus of the bio-ink composition before and after cross-linking based on a frequency of 1 Hz. Each sample number was as follows: 1) Gel4cell, 2) ColMA 1%, 3) ColMA 2%, 4) ColMA 3%, 5) ColMA 4%, 6) ColMA 5%.

Hereinafter, the present invention will be described in detail.

The present invention in one aspect relates to a bio-ink composition comprising a cell carrier material, a viscosity enhancing agent, a lubricant and a structural material, wherein the cell carrier material comprises methacrylated low molecular weight collagen .

In the present invention, the methacrylated low-molecular collagen is a methacrylate reaction to increase the structural safety of low-molecular-weight collagen used for food, and replaces high-cost high-molecular-weight collagen for cell adhesion, growth, migration, By helping functional aspects of cells such as differentiation, it can be applied to many fields other than tissue regeneration.

In the present invention, the methacrylated low-molecular collagen refers to a low-molecular-weight collagen modified to be cross-linked through chemical modification, and preferably has a molecular weight of 500 to 4000 Da.

The methacrylated low molecular weight collagen may be used as a cell transport material or a structural material. The cell transport material is a material that can provide an environment suitable for cells to survive in the bioprinted structure, and should exhibit biocompatibility as well as adequate physical rigidity to provide stability to the structure after bioprinting. It should also be indicated.

Therefore, when methacrylated low-molecular collagen is used as a cell carrier material, it is possible to prepare a bio-ink composition that has significantly superior structural stability after printing and more precise printability compared to a conventional bio-ink composition using only gelatin as a cell carrier material. can

In addition, any one or more substances selected from the group consisting of gelatin, collagen, alginate, agar, agarose, pluronic and polyvinyl alcohol and methacrylated low molecular weight collagen are added to the cell transport material. It can be used by mixing.

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

The lubricant is preferably selected from materials capable of minimizing shear rate and improving dispensing speed, and non-limiting examples thereof include glycerol.

The structural material is preferably selected from materials capable of forming rapid crosslinking and maintaining mechanical rigidity, and non-limiting examples thereof include fibrinogen, chemically crosslinkable hyaluronic acid (eg, methacrylate). hyaluronic acid, thiolated hyaluronic acid, etc.), chemically crosslinkable gelatin (eg gelatin methacrylate, thiolated gelatin, etc.), alginate, methyl cellulose, chitosan, chitin, synthetic peptide And it may be any one or more selected from the group consisting of a polyethylene glycol-based hydrogel.

The fibrinogen is not only suitable as a structural material in terms of gel stability, but also can be selected as a preferable structural material in that it creates a microenvironment suitable for cell adhesion 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 nozzle clogging prevention effect when applying three-dimensional bioprinting, respectively.

In the bio-ink composition of the present invention, the methacrylated low molecular weight collagen is 0.01 to 10 w/v%, 0.05 to 10 w/v%, 0.5 to 10 w/v%, 1 to 10 w/v%, 0.1 to 8 w/v%, 0.5 to 8 w/v%, 1 to 8 w/v%, 0.1 to 6 w/v%, 0.5 to 6 w/v%, 1 to 6 w/v%, 0.1 to 5 w/ v%, 0.5 to 5 w/v% or 1 to 5 w/v% may be included, and most preferably 1 to 4 w/v% may be included, but is not limited thereto.

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

In the bio-ink composition of the present invention, the viscosity enhancer is 0.01 to 1 w/v%, 0.05 to 1 w/v%, 0.1 to 1 w/v%, 0.2 to 1 w/v%, 0.01 to 0.8 w/v% , 0.05 to 0.8 w/v%, 0.1 to 0.8 w/v%, 0.2 to 0.8 w/v%, 0.01 to 0.5 w/v%, 0.05 to 0.5 w/v%, or 0.1 to 0.5 w/v% and most preferably 0.2 to 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 to 20 w/v%, 5 to 20 w/v%, 10 to 20 w/v%, 0.1 to 15 w/v%, 1 to 15 w/v%, 5 to 15 w/v%, or 10 to 15 w/v% may be included, and most preferably 10 to 13 w/v% may be included, but is not limited thereto.

In the bio-ink composition of the present invention, the structural material is 0.1 to 10 w/v%, 0.5 to 10 w/v%, 1 to 10 w/v%, 0.1 to 8 w/v%, 0.5 to 8 w/v% , 1 to 8 w/v%, 0.1 to 10 w/v%, 0.5 to 10 w/v% or 3 to 10 w/v% may be included, and most preferably 3 to 7 w/v% may be included. , but is not limited thereto.

The inventors of the present invention tested the physical and biological properties of compositions having various combinations of compositions in order to develop a bio-ink composition that exhibits physical and biological properties suitable for application to three-dimensional bioprinting, and as a result, the combination and the content It has been found that a bio-ink composition composed of a combination of components exhibits high structural stability, excellent printing performance, and appropriate mechanical properties after printing, making it particularly suitable for three-dimensional bioprinting for the production of tissue-like structures.

The bio-ink composition comprises the steps of: (a) mixing and stirring a viscosity increasing agent and a structural material; (b) adding and stirring methacrylated low molecular weight collagen to the stirring solution; and (c) adding a lubricant to the stirring solution and stirring.

The bio-ink composition may have a complex viscosity of 8 to 1000 Pa·s before crosslinking, and a viscosity of 20 to 3000 Pa·s after crosslinking. More preferably, under the condition of a frequency of 1 Hz, the viscosity before crosslinking may be 60 to 140 Pa·s, and the viscosity after crosslinking may be 130 to 350 Pa·s.

The storage modulus before crosslinking of the bio-ink composition is 250 to 1000 Pa, the loss modulus is 80 to 160 Pa, and the storage modulus after crosslinking is 700 to 2300 Pa, and the loss modulus is 160 to 400 Pa. can More preferably, when the frequency (Frequency) is 1 Hz, the storage modulus before crosslinking is 400 to 850 Pa, the loss modulus is 85 to 200 Pa, and the storage modulus after crosslinking is 800 to 2200 Pa, and the loss modulus may be 180 to 250 Pa. .

In a specific embodiment of the present invention, in order to confirm that low molecular weight collagen methacrylated as a cell carrier material is suitable as a bioink composition, 1 w/v%, 2 w/v% 3 w/ A bio-ink composition (ColMA) was prepared to be v%, 4 w/v%, and 5 w/v%, respectively, and a bio-ink composition (Gel4cell) containing gelatin instead of methacrylated low-molecular collagen as a cell carrier material was compared. used as an example.

As a result of comparing the physical properties of the ColMA bio-ink composition and the Gel4cell bio-ink composition prepared in the present invention, it was confirmed that the ColMA bio-ink before and after cross-linking showed similar or higher viscosity (FIG. 1) and modulus (FIG. 3) compared to the Gel4cell bio-ink In particular, it was confirmed that the viscosity was the best when the methacrylated low molecular weight collagen content was 1 w/v%.

In addition, as a result of comparing the viscosity and modulus based on the frequency of 1 Hz, it was confirmed that the viscosity, storage modulus, and loss modulus all showed maximum values when 1 w/v% of methacrylated low-molecular collagen was included before and after cross-linking. .

That is, it was confirmed that the mechanical properties of the bio-ink composition (ColMA 1%) containing methacrylated low-molecular collagen at 1 w/v% were superior to those of the bio-ink composition (Gel4cell) containing jellytin. .

Therefore, it was confirmed that the bio-ink composition including methacrylated low-molecular collagen of the present invention can produce a structure having superior physical properties compared to the conventional bio-ink composition (Gel4cell) using only gelatin as a cell carrier material.

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

In the present invention, the cells are preferably stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, embryos. Cells (germ cells), hepatocytes (hepatocytes), renal cells (renal cells), atrial cells (Sertoli cells), chondrocytes (chondrocytes), epithelial cells (epithelial cells), cardiovascular cells, keratinocytes, smooth muscle cells ( smooth muscle cells), cardiomyocytes, glial cells, endothelial cells, hormone-secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of It may be any one or more, but is not limited thereto.

The cells used in the present invention and the cells contained in the bioprinted tissue-like construct may 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 thereof are incorporated herein by reference. Common mammalian cell culture techniques, cell lines, and cell culture systems that may 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 for this information.

Cells may also be cultured with a cell differentiation material that induces differentiation of cells along a desired cell line. For example, stem cells are incubated in contact with a differentiation medium to generate a range of cell types. Many types of differentiation media are suitable. The stem cells include, but are not limited to, osteogenic differentiation medium, chondrogenic differentiation medium, adipogenic differentiation medium, neural differentiation medium, cardiomyocyte differentiation medium, and enterocyte differentiation medium (eg, intestinal epidermis) and incubated in contact with a differentiation medium containing

In the present invention, the cells may be bioprinted by depositing or extruding bio-ink 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 including a plurality of cells. Bio-inks include liquid or semi-solid cell solutions, cell suspensions, or cell concentrates. The bio-ink composition is prepared by 1) mixing a plurality of cells or cell aggregates and a biocompatible liquid or gel in a predetermined ratio to prepare a bio-ink, and 2) densifying the bio-ink to have a desired cell density and viscosity. It can be prepared by the step of preparing and the like. Specifically, densification of bio-inks is realized by centrifugation, tangential flow filtration (“TFF”), or a combination thereof, and densification of bio-inks produces extrudable compositions to form multicellular aggregates or multicellular bodies. By "extrudable" is meant capable of being shaped by passing (eg, under pressure) through a nozzle or orifice (eg, one or more holes or tubes). In addition, the densification of the bio-ink is derived from growing cells to an appropriate density. The cell density required for a bio-ink depends on the cells to be used and the tissue or organ to be manufactured.

The bio-ink composition of the present invention may further include a cross-linking agent or an optical initiator (photoinitiator) for promoting cross-linking 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 an aluminum compound, a calcium compound and a magnesium compound. For example, it may be one or more selected from the group consisting of aluminum hydroxide, hydrous aluminum silicate, calcium chloride, magnesium chloride, aluminum chloride, magnesium alumina metasilicate, aluminum acetate, and magnesium aluminum silicate.

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

The present invention from another point of view,

(a) filling the bio-ink composition in a three-dimensional printer;

(b) three-dimensional printing of the desired three-dimensional structure; and

(c) relates to a method for producing a three-dimensional structure for tissue regeneration comprising the step of crosslinking the three-dimensionally printed bio-ink composition.

In another aspect, the present invention

(a) filling the bio-ink composition in a three-dimensional printer;

(b) three-dimensional printing of the desired tissue-like structure; and

(c) relates to a method for producing a tissue-like structure comprising the step of crosslinking the three-dimensionally printed bio-ink composition.

The method may further include the step of (d) culturing the tissue-like structure in a culture medium, and a tissue-like organoid may be prepared through the culturing of the tissue-like structure.

In addition, if necessary, the tissue-like structure can be prepared by printing a bio-ink composition containing cells, and after preparing the structure by printing a bio-ink composition containing no cells, cells can be added to the structure and cultured. have.

The bio-ink composition provided in the present invention is easy to manufacture various three-dimensional structures for tissue regeneration, tissue-like structures, or tissue-like organs. Bioprinting technology is being developed in the art as a useful tool for manufacturing three-dimensional structures or tissue-like structures. On the other hand, conventional bio-ink compositions for use in bio-printing have a problem in that it is difficult to have sufficient physical rigidity because they are in a liquid form, or the survival of cells during the printing process is not sufficiently ensured due to excessive physical rigidity. The bio-ink composition of the present invention is applied to three-dimensional bioprinting to maintain sufficient physical rigidity in preparing a tissue-like structure, and has the advantage that the survival of cells is sufficiently guaranteed when sprayed by a bio-printer. It exhibits very suitable physical and biological properties for In addition, according to an embodiment of the present invention, it was confirmed that the bio-ink composition of the present invention exhibits sophisticated printing performance compared to the conventional bio-ink composition using only gelatin as a cell carrier material.

The bio-ink composition in the present invention enables the lamination of three-dimensional structures. The bio-ink composition of the present invention containing a sufficient amount of cells can be used to form a tissue-like structure by being laminated by a three-dimensional bio-printer.

In the present invention, "bioprinting" means accurate three-dimensional cell deposition (e.g., cell solution, cell-containing gel, cell suspension, 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 eject bio-ink from a bio-printer through a dispense tip (eg, syringe, capillary, etc.) connected to a reservoir of bio-ink. The continuous bioprinting method is to eject bio-ink in a repeating pattern of functional units. The repeating functional unit may have any suitable geometry including, for example, circles, squares, rectangles, triangles, polygons, and irregular geometries. In addition, a repeating pattern of bioprinted functional units can be bioprinted (eg, laminated) contiguously to include layers and multiple layers to form an engineered tissue or organ. Specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more layers may be contiguously bioprinted to form an engineered tissue or organ.

The bioprinted functional units may be repeated in a tessellated pattern. A "lattice pattern" is a planar figure that fills a non-overlapping, gap-free plane. Advantages of continuous and/or grid bioprinting may include increased productivity of bioprinted tissue. Another non-limiting potential advantage is that it may eliminate the need to align the bioprinter with elements of the previously deposited bioink. Continuous bioprinting may also facilitate printing of larger tissues from large reservoirs of bio-ink, optionally using a syringe mechanism.

A suitable and/or optimal jet distance from the bioprinter does not produce flattening of the material or adhesion to the jet needle. Bioprinter jet tips are approximately 1, 5, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 μm or greater, and increments within this range. Also, the bio-ink reservoir of the bio printer is about 0.5, 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 volumes in increments within this range. The pump speed may be moderate and/or optimal when the residual pressure rise in the system is low. A good pump speed may depend on the ratio between the dispense needle and the cross-sectional area of the reservoir, with higher ratios requiring lower pump speeds.

Various types of three-dimensional structures for tissue regeneration or tissue-like structures may be generated by the above-described method. The pattern or stacking arrangement for laminating the bio-ink composition may be determined by the size and diameter of the tissue-like structure to be manufactured. In addition, the number of cells included in the bio-ink used to prepare the three-dimensional structure or tissue-like structure may be adjusted according to the type of cell, the content of cell nutrients included 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 the three-dimensional structure or tissue-like structure to be manufactured according to the above method. Those of 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 three-dimensional structure or tissue-like structure to be manufactured through three-dimensional bioprinting.

After the bio-ink composition is sprayed and laminated by a three-dimensional bio-printer, cross-linking of the bio-ink composition can be promoted by exposing it to light (ultraviolet or visible light) or adding a cross-linking solution. This cross-linking allows the laminated bio-ink composition to be completed into a more rigid structure. On the other hand, the light (ultraviolet or visible light) can be directly exposed to the surface of the stacked 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 light and visible light) exposure distance and time is that those skilled in the art to which the present invention pertains can form sufficient crosslinks even with short exposures for short distances and strong wavelengths. you will be able to recognize

A preferred wavelength of light (ultraviolet or visible light) for crosslinking may be 300 nm to 800 nm, preferably 350 nm to 380 nm for ultraviolet light, 400 nm to 600 nm for visible light, more preferably UV light 355 nm to 375 nm, 400 nm to 500 nm for visible light, most preferably 360 nm to 370 nm for ultraviolet light, 400 nm for visible light ~ 480 nm.

In another aspect, the present invention relates to an implantable tissue-like organ or tissue construct manufactured according to the method.

3D bioprinting technology has the potential to produce clinically applicable tissue or organ structures in terms of shape and size. Through the combination of tissue-specific cell types and bioinks applied to three-dimensional structures, it is possible to utilize the reproductive ability inherent in cells, and through this, it may be possible to produce necessary tissues or organs. Accordingly, tissue structures bioprinted using the bio-ink composition according to the present invention can provide structures having anatomical and functional similarity to mammalian tissues and organs.

By using the composition of the present invention, it is possible to provide a single structure of a tissue-like organ or a tissue that can be transplanted into the body, as well as a tissue structure with significantly improved functionality that can reproduce the linkage between tissues by printing various cells at the same time. can also be manufactured.

Hereinafter, the present invention will be described in more detail through examples.

These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples.

Preparation of bio-ink composition

Methacrylated low molecular weight collagen was prepared according to a known method by reacting methacrylic anhydride with low molecular weight collagen having a molecular weight of about 3500 Da (R. Ravichandran et al. , Journal of Materials Chemistry B , 4(2). ):318-326, 2016).

The bio-ink composition was prepared at room temperature, and 4 mg/mL hyaluronic acid (Sigma-Aldrich) was first added to 0.7 mL/mL DMEM (biowest) and mixed for 1 hour. Then, 50 mg/mL methacrylated gelatin (GelMA, manufactured in-house) was added and stirred for 1 hour, followed by 1 w/v%, 2 w/v%, and 3 w of methacrylated low molecular weight collagen, respectively. /v%, 4 w/v% and 5 w/v% were added and stirred for 1 hour. Then, 0.1 mL/mL glycerol (Sigma-Aldrich) was added and stirred for 30 minutes to prepare a bio-ink composition.

As a comparative example, a bio-ink composition (Gel4cell) containing gelatin instead of methacrylated low molecular weight collagen was prepared as described above.

Confirmation of physicochemical properties of bio-ink composition - Viscosity

The viscosity of the bio-ink composition prepared in Example 1 was confirmed.

The measurements were divided into Before curing and After curing, and the complex viscosity was measured using a Rheometer (mcr 301, Anton paar). Viscosity measurement was performed under the conditions of a frequency range of 0.1 to 10Hz (Frequency range; logarithm uniformly 11 pts), a temperature of 25°C, and an amplitude of 0.1　 to 1000%.

Comparison of viscosity before and after crosslinking according to the content of methacrylated low molecular weight collagen Viscosity before crosslinking (Pa·s) Viscosity after crosslinking (Pa·s) Gel4cell 7 to 500 10 to 1000 ColMA 1% 16 to 1000 40 to 3000 ColMA 2% 10 to 600 20 to 1300 ColMA 3% 8 to 400 20 to 1000 ColMA 4% 8 to 400 30 to 1800 ColMA 5% 8 to 400 30 to 1800

Viscosity comparison before and after crosslinking at a frequency of 1 Hz Viscosity before crosslinking (Pa·s) Viscosity after crosslinking (Pa·s) Gel4cell 62 109 ColMA 1% 136 324 ColMA 2% 96 166 ColMA 3% 70 138 ColMA 4% 64 229 ColMA 5% 70 219

As a result, as shown in FIG. 1 and Table 1, it was confirmed that ColMA bio-ink showed similar viscosity or high viscosity compared to Gel4cell bio-ink both before and after cross-linking, especially when the methacrylated low molecular weight collagen content was 1% It was confirmed that the viscosity was the best.

In addition, as a result of comparing the viscosity based on a frequency of 1 Hz, as shown in FIG. 2 and Table 2, it was confirmed that the viscosity of the bio-ink composition containing methacrylated low molecular collagen instead of gelatin was excellent both before and after cross-linking, and methacrylic It was confirmed that the viscosity was highest when the low molecular weight collagen content was 1%.

Confirmation of physicochemical properties of bio-ink composition - modulus

The modulus of the bio-ink composition prepared in Example 1 was confirmed. The measurements were divided into Before curing and After curing.

The modulus measurement was performed under the same conditions as those of the viscosity measurement in Example 2.

Comparison of modulus before and after crosslinking according to the content of methacrylated low molecular weight collagen Modulus before crosslinking (Pa) Modulus after crosslinking (Pa) storage modulus loss modulus storage modulus loss modulus Gel4cell 350 to 400 14 to 48 680 to 690 47 to 48 ColMA 1% 650 to 1000 150 to 160 1700 to 2300 380 to 400 ColMA 2% 400 to 700 100 to 120 800 to 1300 220 to 270 ColMA 3% 300 to 550 80 to 100 700 to 1100 160 to 250 ColMA 4% 250 to 500 70 to 90 1100 to 1700 220 ~ 330 ColMA 5% 300 to 550 80 to 95 1100 to 1600 260 to 320

Comparison of modulus before and after crosslinking at a frequency of 1 Hz Modulus before crosslinking (Pa) Modulus after crosslinking (Pa) storage modulus loss modulus storage modulus loss modulus Gel4cell 390 25 680 30 ColMA 1% 830 170 2000 200 ColMA 2% 590 120 1000 150 ColMA 3% 430 95 850 140 ColMA 4% 400 85 1400 200 ColMA 5% 430 90 1360 190

As a result, as shown in Fig. 3 and Table 3, it was confirmed that the ColMA bio-ink showed a similar or higher modulus compared to the Gel4cell bio-ink both before and after cross-linking, and especially after cross-linking, and especially after cross-linking, like the viscosity. It was confirmed that the modulus was the best when the collagen content was 1 w/v%.

In addition, as a result of comparing viscosity and modulus at a frequency of 1 Hz, it was confirmed that both storage modulus and loss modulus showed maximum values when 1 w/v% of methacrylated low-molecular collagen was included before and after crosslinking.

That is, it was confirmed that the mechanical properties of the bio-ink composition (ColMA 1%) containing methacrylated low-molecular collagen at 1 w/v% were superior to those of the bio-ink composition containing jellytin (Gel4cell). .

Therefore, it was confirmed that the bio-ink composition containing the methacrylated low molecular weight collagen of the present invention can produce a structure having superior physical properties compared to the conventional bio-ink composition (Gel4cell) using only gelatin as a cell carrier material.

Claims (12)

As the cell carrier material, methacrylated low molecular weight collagen;
As a viscosity enhancing agent, hyaluronic acid;
As a lubricant, glycerol; and
As a structural material, a bio-ink composition comprising methacrylated gelatin,
The methacrylated low molecular weight collagen contained in the bio-ink composition is 1 to 5 w/v%,
When the frequency (Frequency) of the bio-ink composition is 1Hz, the storage modulus before crosslinking is 400 to 850 Pa and the loss modulus before crosslinking is 85 to 200 Pa, the storage modulus after crosslinking is 800 to 2200 Pa and the loss modulus after crosslinking is 180 ~ 250 Pa, characterized in that the bio-ink composition.
The bio-ink composition according to claim 1, wherein the molecular weight of the methacrylated low molecular weight collagen is 500 to 4000 Da.
delete The bio-ink composition according to claim 1, wherein the bio-ink composition has a viscosity of 8 to 1000 Pa·s before crosslinking, and a viscosity of 20 to 3000 Pa·s after crosslinking.
The bio-ink composition according to claim 1, wherein, when the bio-ink composition has a frequency of 1 Hz, the viscosity before crosslinking is 60 to 140 Pa·s, and the viscosity after crosslinking is 130 to 350 Pa·s. .
delete delete delete The method of claim 1, wherein the bio-ink composition comprises stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, Germ cells, hepatocytes, renal cells, Sertoli cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells From the group consisting of smooth muscle cells, cardiomyocytes, glial cells, endothelial cells, hormone-secreting cells, immune cells, pancreatic islet cells and neurons Bio-ink composition, characterized in that it further comprises one or more selected cells.
The bio-ink composition of claim 1, wherein the bio-ink composition further comprises a photoinitiator.
(a) claim 1, claim 2, claim 4, claim 5, claim 9 and claim 10 comprising the steps of filling any one of the bio-ink composition in a three-dimensional printer;
(b) three-dimensional printing of the desired tissue-like structure; and
(c) a method for producing a tissue-like structure comprising the step of crosslinking the three-dimensionally printed bio-ink composition.
A tissue-like construct prepared according to the method of claim 11 .

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