KR20200132741A - Dual-crosslinkable two component type composition of bio-ink and Method for Producing Tissue-Like Structure Using the Same - Google Patents

Abstract

The present invention relates to a double cross-linkable two-component bio ink composition and to a manufacturing method of a tissue-like structure using the same and, more specifically, to a two-component type bio ink composition comprising: a first liquid including methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell carrier, a viscosity enhancer, and a lubricant; and a second liquid including thrombin, and to a manufacturing method of a tissue-like structure using the same.

Description

Dual-crosslinkable two component type composition of bio-ink and Method for Producing Tissue-Like Structure Using the Same}

The present invention relates to a dual-crosslinkable two-component bio-ink composition and a method for manufacturing a tissue-like structure using the same, and more particularly, to contain methacrylated low-molecular collagen as a cell transport material in the first solution, and meta- The present invention relates to a dual crosslinkable two-component bio-ink composition including both methacrylated gelatin and fibrinogen, but including thrombin in a second solution, and a method of manufacturing a tissue-like structure using the same.

The three-dimensional cell culture technology is developed as a technology capable of manufacturing cells and tissues in an environment similar to living tissues in vitro, and is applied to various research fields related to cell growth and differentiation, tissue and organ formation. Has become. These tissue-like organs can be usefully used in drug toxicity and pharmacokinetic studies in place of actual tissues or organs, and thus, it is expected that direct experimental application to human specimens 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.

The 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 closely mimic real human tissues. However, biomaterials that carry living cells during bioprinting, that is, bio-inks, have many limitations in their utilization. As a characteristic of bio-ink required to be applied to bio-printing, excellent biocompatibility is required, and it must have excellent printing properties 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 role of a mechanical support while providing cell-specific signals after printing. Although naturally derived or synthetic hydrogel bioinks have been developed and used in the field of 3D bioprinting, bioinks based on these existing hydrogels have physical and biological aspects such as biocompatibility, printing compatibility, geometric precision, and precision. Shows significant limitations.

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

Thus, in the case of including methacrylated low-molecular collagen as a cell carrier in the conventional bio-ink composition, and including both methacrylated gelatin, fibrinogen, and thrombin as structural materials, surprisingly improved structural stability and cells In addition to having proliferative properties, it was found that it can have excellent printability, and the present invention was completed.

An object of the present invention is a first liquid containing methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer and a lubricant; It is to provide a two-component bio-ink composition comprising a second liquid containing thrombin.

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

In order to achieve the above object, the present invention comprises methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer and a lubricant. The first amount; It provides a two-component bio-ink composition comprising a second liquid containing thrombin.

In a preferred embodiment of the present invention, methacrylated gelatin and fibrinogen as structural materials contained in the first solution may be 25 to 35 mg/ml and 16 to 24 mg/ml, respectively.

In another preferred embodiment of the present invention, the methacrylated low-molecular collagen as a cell transport material contained in the first solution may be 0.1 to 10 w/v%.

In another preferred embodiment of the present invention, the viscosity enhancing agent may be hyaluronic acid or dextran, and the lubricant may be glycerol.

In another preferred embodiment of the present invention, the first liquid may further contain a photoinitiator.

In another preferred embodiment of the present invention, the first solution is stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, Glioblast, germ cell, hepatocyte, renal cell, sertoli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte (keratinocyte), smooth muscle cell, cardiomyocyte, glial cell, endothelial cell, hormone secreting cell, immune cell, pancreatic islet cell, and nerve cell ( neuron) may further include one or more cells selected from the group consisting of.

The thrombin may be 10 to 100 IU/mL, and the concentration of thrombin contained in the second solution may be 0.1 to 1 mL/mL.

The volume ratio of the first liquid and the second liquid may be 1: 0.5 to 1: 10.

In order to achieve another object of the present invention, the present invention provides (a) methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell carrier, and viscosity. 3D printing a first solution containing an enhancer and a lubricant; And (b) photo-crosslinking the 3D printed first solution, and then naturally crosslinking by adding a second solution containing thrombin to double crosslinking.

In a preferred embodiment of the present invention, in the step (a), 3D printing is performed using a 3D bioprinter having a nozzle having a diameter of 100 to 500 μm, under a pressure of 10 to 100 kPa, and 100 to 500%. It can be carried out with an extrusion amount of.

In another preferred embodiment of the present invention, the photocrosslinking in step (b) may be performed by exposing light having a wavelength of 300 to 800 nm at a distance of 1 to 20 cm for 1 to 1000 seconds.

In another preferred embodiment of the present invention, the natural crosslinking in step (b) may be performed by allowing it to stand at a temperature of 4 to 30° C. for 10 minutes to 1 hour.

In order to achieve another object of the present invention, the present invention provides a tissue-like structure manufactured according to the above method.

The two-pack bio-ink composition according to the present invention comprises methacrylated low-molecular collagen as a cell transport material in the first solution, and includes both methacrylated gelatin and fibrinogen as structural materials. Bar, not only has excellent structural stability and cell proliferation, but also has excellent printability, so it can be very usefully used in the production of a tissue-like structure using a 3D bioprinter. In particular, when thrombin is included in the second solution, natural crosslinking can be performed together with photocrosslinking to perform double crosslinking, so that the physical properties of the tissue-like structure can be further improved.

FIG. 1 shows a 3D bioprinter used in Example 1, and a change in the shape of a structure according to 3D printing and thrombin processing using the same.
2 is a photograph comparing the printability of the structure when using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4%, and CFB 5%) prepared in Example 1.
3 is a picture comparing the structural stability of the structure when using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1.
Figure 4 is a picture comparing the cell proliferation properties of the structure when using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1. .

Hereinafter, the present invention will be described in detail.

2 component bio ink composition

The present invention relates to a first solution comprising methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer and a lubricant; It provides a two-component bio-ink composition comprising a second liquid containing thrombin.

In the present specification, the term “liquid bio-ink composition” refers to a bio-ink composition in which a first liquid and a second liquid are separately present, and the first liquid is a target of 3D printing and photocrosslinking, The second liquid is added after 3D printing for natural crosslinking.

First, the first solution according to the present invention contains methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer, and a lubricant. .

The structural material is preferably selected from materials capable of forming rapid crosslinking and maintaining mechanical rigidity, and among them, it is characterized in that it contains both methacrylated gelatin and fibrinogen.

At this time, the methacrylated gelatin has a feature of photocrosslinking. In particular, the fibrinogen is not only suitable as a structural material in terms of structural stability, but also can be selected as a preferred structural material in that it creates a microenvironment suitable for attachment and differentiation of cells after the cells are printed. In particular, the fibrinogen Since silver is capable of natural crosslinking with thrombin, the mechanical properties of the final structure can be further improved.

As structural substances contained in the first solution, methacrylated gelatin and fibrinogen may be 25 to 35 mg/mL and 16 to 24 mg/mL, respectively, most preferably 28 to 32 mg/mL and 18 to 22. It may be mg/mL, but is not limited thereto.

The cell carrier material is characterized in that it contains methacrylated low molecular weight collagen.

At this time, the methacrylated low-molecular collagen has undergone a methacrylate reaction to increase the structural safety of the low-molecular collagen used for food, and replaces expensive high-molecular collagen to adhere, grow, migrate, and differentiate cells. By helping the functional aspects of cells such as the back, it can be applied in many fields other than tissue regeneration. The methacrylated low-molecular collagen means a modified form of low-molecular collagen to be crosslinked through chemical modification, and preferably has a molecular weight of 500 to 4000 Da.

The methacrylated low-molecular collagen can serve as a structural material as well as a role as a cell transport material. That is, the methacrylated low-molecular collagen is a material that can provide an environment suitable for cells to survive in a bio-printed structure, and must exhibit biocompatibility and provide stability to the structure after bio-printing. Appropriate physical stiffness as possible should also be shown. Therefore, when methacrylated low-molecular collagen is used as the cell transport material, the structure stability after printing is remarkably excellent and more precise printing properties can be exhibited compared to the conventional bio ink composition using only gelatin as the cell transport material.

In addition, any one or more materials selected from the group consisting of methacrylated low-molecular collagen as the cell transport material, gelatin, collagen, alginate, agar, agarose, pluronic, and polyvinyl alcohol Can be used by mixing.

The methacrylated low-molecular collagen as a cell transport material contained in the first solution may be 0.1 to 10 w/v%, preferably 1 to 10 w/v%, and more preferably 3 to 10 It may be w/v%, most preferably 4 to 5 w/v%, but is not limited thereto.

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

The viscosity enhancing agent contained in the first solution may be 2 to 10 mg/mL, most preferably 2 to 10 mg/mL, but is not limited thereto.

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

The lubricant included in the first solution may be 0.01 to 1 mL/mL, preferably 0.05 to 0.5 mL/mL, but is not limited thereto.

The first liquid is a viscosity enhancing agent and a structural material, a step of mixing and stirring methacrylated gelatin and fibrinogen; Adding and stirring methacrylated low molecular weight collagen as a cell carrier material to the stirring solution; And it can be prepared by a method comprising the step of stirring by adding a lubricant to the stirring solution.

Therefore, the first solution is characterized in that it contains both methacrylated gelatin and fibrinogen as a structural material while containing methacrylated low-molecular collagen as a cell transport material, excellent structural stability and cell Since it has not only proliferative properties, but also has excellent printability, it can be very usefully used in manufacturing a tissue-like structure using a 3D bioprinter.

Meanwhile, the first liquid may further include a crosslinking agent or a photoinitiator for promoting photocrosslinking of 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, aluminum metasilicate magnesium, aluminum acetate, and magnesium aluminum silicate.

The optical initiator refers to a material that causes rapid crosslinking when exposed 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 with ultraviolet (UV) rays or an optical initiator in which a crosslinking reaction occurs by irradiation with visible light may be used. Non-limiting examples of suitable optical initiators 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 exposed light.

In addition, the first solution may contain 0.05 ~ 60 × 10 ⁶ /mL cells.

The cells are preferably stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glial cells, glioblasts, and germ cells. cell), hepatocyte, renal cell, seroli cell, chondrocyte, epithelial cell, cardiovascular cell, keratinocyte, smooth muscle cell ), cardiomyocytes, glial cells, endothelial cells, hormone secreting cells, immune cells, pancreatic islet cells, and neurons selected from the group consisting of However, it is not limited thereto.

The cells and cells contained within 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, and The contents thereof 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.

Cells may also be cultured with cell differentiation substances that induce differentiation of cells along the desired cell line. For example, stem cells are incubated in contact with a differentiation medium to produce 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 intestinal cell differentiation medium (e.g. Epidermis) and incubated with a differentiation medium.

The cells can be bioprinted by depositing or extruding the first liquid from a three-dimensional bioprinter. The first liquid may be in the form of a liquid, semi-solid, or solid composition including a plurality of cells. The first liquid contains a liquid or semi-solid cell solution, a cell suspension, or a cell concentrate. The first solution includes 1) preparing a first solution by mixing a plurality of cells or cell aggregates and a biocompatible liquid or gel at a predetermined ratio, and 2) densifying the first solution to have a desired cell density and viscosity. It may be prepared by the step of preparing the first liquid. Specifically, the densification of the first liquid is realized by centrifugation, tangential flow filtration ("TFF"), or a combination thereof, and the densification of the first liquid produces an extrudable composition to form multicellular aggregates or multicellular bodies. The term “extrudable” means that it can be molded by passing (eg, under pressure) a nozzle or orifice (eg, one or more holes or tubes). Further, the densification of the first liquid is induced by growing cells at an appropriate density. The cell density required for the first solution varies depending on the cells to be used and the tissues or organs to be manufactured.

Next, the second solution according to the present invention contains thrombin. Since the thrombin is capable of natural crosslinking with the fibrinogen, it is possible to further improve the mechanical properties of the final structure.

The thrombin is 10 to 100 IU/mL (most preferably 20 IU/mL to 100 IU/mL), and the concentration of thrombin contained in the second solution may be 0.1 to 1 mL/mL, but is limited thereto. It is not.

Meanwhile, the volume ratio of the first liquid and the second liquid may be 1: 0.5 to 1: 10, and the second liquid is preferably a volume enough to sufficiently immerse the photocrosslinked product, but is not limited thereto. .

Tissue-like structure and its manufacturing method

The present invention comprises (a) a first solution comprising methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer, and a lubricant. 3D printing the object; And (b) photo-crosslinking the 3D printed first solution, and then naturally crosslinking by adding a second solution containing thrombin to double crosslinking.

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

In addition, if necessary, the tissue-like structure can be prepared by 3D printing the first solution containing cells, and after the structure is prepared by 3D printing the first solution containing cells, the cells are added to the structure. Can be cultured.

First, the method of manufacturing a tissue-like structure according to the present invention includes methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer and a lubricant. It includes the step of filling the 3D printer with the first liquid containing [Step (a)].

Since the first amount has been described above, a redundant description will be omitted.

The 3D printing may be performed continuously and/or substantially continuously. An example of continuous 3D printing is to spray a first liquid from a bioprinter through a dispense tip (eg, a syringe, a capillary tube, etc.) connected to a reservoir of the first liquid. Continuous 3D printing is the spraying of a first liquid 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 geometries. In addition, the repeating pattern of the three-dimensional printed functional units includes layers, and a plurality of layers may be three-dimensionally printed (eg, stacked) adjacent to each other 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 can be 3D printed adjacent to form an engineered tissue or organ .

The functional units printed in 3D may be repeated in a tessellated pattern. A "lattice pattern" is a planar figure that does not overlap and fills a plane without gaps. Advantages of continuous and/or lattice bioprinting may include increased productivity of three-dimensional printed tissue. Another non-limiting potential advantage is that it can eliminate the need to align the bioprinter with the previously deposited elements of the first liquor. Continuous bioprinting may also facilitate printing larger tissues from large reservoirs of the first liquid, optionally using a syringe mechanism.

Proper and/or optimal spray distance from the three-dimensional bioprinter does not produce material flattening or adhesion to the spray needle. The 3D bioprinter jet tip has an inner diameter of about 5, 10, 20, 50, 100 μm or more and increments within this range. In addition, the first liquid reservoir of the 3D bioprinter 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 have 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 can depend on the ratio between the cross-sectional area of the reservoir and the injection needle, with higher ratios requiring lower pump speeds.

The 3D printing is preferably performed with an extrusion amount of 100% to 500% under a pressure of 10 kPa to 100 kPa using a 3D bioprinter equipped with a nozzle of 100 μm to 500 μm, but is limited thereto. It doesn't work.

Next, the method of manufacturing a tissue-like structure according to the present invention is a step of photocrosslinking the three-dimensional printed first solution, and then naturally crosslinking by adding a second solution containing thrombin to double crosslinking [(b ) Step].

The photo-crosslinking is performed after the first liquid is sprayed and laminated by a 3D printer, through which it can be completed into a rigid structure.

The light (ultraviolet or visible light) can be directly exposed to the surface of the 3D printed first liquid, for example, using a wavelength of 300 nm to 800 nm generated from a light (ultraviolet or visible light) generator Exposure may be performed for 1 second to 1000 seconds at a distance of 1 to 20 cm from the 3D printed first solution, or may be exposed for 20 seconds to 500 seconds, or 40 seconds to 240 seconds. These light (ultraviolet and visible) exposure distances and times are those of ordinary skill in the art, that a short distance and strong wavelength can form sufficient crosslinking even with exposure for a short time. You will be able to recognize it.

The wavelength of the preferred light (ultraviolet or visible light) for the photo-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 ultraviolet light. 355 nm to 375 nm for visible light, 400 nm to 500 nm for visible light, most preferably 360 nm to 370 nm for ultraviolet light, and 400 nm for visible light It may be ~ 480nm.

Since the second amount has been described above, a redundant description will be omitted.

The natural crosslinking is due to the reaction between the thrombin and the fibrinogen, and may be performed by allowing it to stand at a temperature of 4 to 30° C. for 10 minutes to 1 hour. Accordingly, since double crosslinking can be finally performed, the physical properties of the tissue-like structure can be further improved.

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

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

Example 1: Preparation of a two-component bio-ink composition and a tissue-like structure using the same

Methacrylic stylized low molecular collagen is reacted with methacrylic anhydride (methacrylic anhydride) to about low molecular weight collagen (Gelita, D-69412) having a molecular weight of 3500Da, was prepared in accordance with known methods (Ravichandran R. et al., Journal of Materials Chemistry B , 4(2):318-326, 2016).

A two-pack bio-ink composition was prepared at room temperature, and 4 mg/mL hyaluronic acid (Sigma-Aldrich) as a viscosity enhancer was first added to 0.7 mL/mL DMEM (biowest) and mixed for 1 hour. Then, 30 mg/mL methacrylated gelatin (GelMA, self-made) and 20 mg/ml fibrinogen (green cross, 030A18005) were added as structural materials, and stirred for 1 hour, and then methacrylated as a cell carrier. Low-molecular collagen was added at 1 w/v%, 2 w/v%, 3 w/v%, 4 w/v%, and 5 w/v%, respectively, and stirred for 1 hour. Thereafter, 0.1 mL/mL glycerol (Sigma-Aldrich) was added and stirred for 30 minutes to prepare a bioink composition CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%). Meanwhile, 0.2 w/v% 2-hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone optical initiator was added to prepare the first solution, and 0.6 mL/mL thrombin 20 IU in 0.7 mL/mL DMEM (biowest). /mL was added and stirred for 30 minutes to prepare a second solution (see Table 1).

First amount
10 mL standard Second amount
10 mL standard Structural material Cell carrier Viscosity enhancer slush Thrombin
20 UI/mL
(mL) Gel-MA
(mg) Fibrinogen
(mg) Small molecule
Col-MA
(mg) HA
(mg) Glee
Serol
(ml) Example 1 (CFB 1%) 300 200 100 40 One 6 Example 1 (CFB 2%) 200 Example 1 (CFB 3%) 300 Example 1 (CFB 4%) 400 Example 1 (CFB 5%) 500

Using the 3D bio printer shown in FIG. 1 (nozzle diameter = 300 μm), the prepared first solution was 3D printed in a grid patterned structure. In this case, the 3D bioprinter moves in three axes, and is composed of an injection-type reservoir for filling the first liquid, a dispensing module and a nozzle for injection in the form of extrusion. In addition, 3D bioprinting was performed under a pressure of 50-80 kPa, with an extrusion amount of 250%.

Thereafter, the 3D printed first solution was photo-crosslinked by UV irradiation at 365 nm wavelength for 2 minutes, and then the prepared second solution was added to the extent that the photo-crosslinked product was immersed, and naturally crosslinked at a temperature of 25°C for 30 minutes or more A double crosslinked structure was prepared. This was immersed in 5 mL of media (Aprotinin solution 20ug/mL) in a 37°C incubator (DAIHAN Scientific ThermoStable IG-50), and then incubated for 21 days and observed (see FIG. 1).

Comparative Example 1

It was carried out in the same manner as in the preparation of the first solution in Example 1, but using 3 mg/mL hyaluronic acid (Sigma-Aldrich) as a viscosity enhancing agent, and 20 mg/mL methacrylated gelatin (GelMA, itself) as a structural material. Preparation) was used, and a one-pack bio ink composition (Gel4cell) was prepared using 30 mg/mL gelatin (Sigma, P1002776296) instead of methacrylated low-molecular collagen as a cell carrier. On the other hand, the use of the second solution and natural crosslinking were omitted (see Table 2).

First amount
10 mL standard Structural material Cell carrier Viscosity enhancer slush Gel-MA
(mg) gelatin
(mg) HA
(mg) Glycerol
(ml) Comparative Example 1 200 300 30 One

Comparative Example 2

It was carried out in the same manner as in the preparation of the first solution in Example 1, but only 50 mg/mL methacrylated gelatin (GelMA, manufactured by itself) was used as a structural material as a structural material, and a methacrylated small molecule as a cell carrier One-component bio-ink compositions (CB 1% and CB 3%) were prepared using 1 w/v% and 3 w/v% of collagen, respectively. Meanwhile, the use of the second solution and natural crosslinking were omitted (see Table 3).

First amount
10 mL standard Structural material Cell carrier Viscosity enhancer slush Gel-MA
(mg) Small molecule
Col-MA
(mg) HA
(mg) Glycerol
(ml) Comparative Example 1 (CFB 1%) 500 100 40 One Comparative Example 3 (CFB 3%) 300

Experimental Example 1: Evaluation of printability of structure

When using the two-component bio-ink composition prepared in Example 1 (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%), the printability of the structure was evaluated.

As a result, as shown in Figure 2, when using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1, methacrylated low molecular weight As the concentration of collagen increases, it is confirmed to have excellent printability, and in particular, in the case of 3 to 5% of CFB, it is confirmed that the printability of the structure is excellent.

On the other hand, in the case of using the one-component bio-ink composition (Gel4cell) prepared in Comparative Example 1 and the one-component bio-ink composition (CB 1% and CB 3%) prepared in Comparative Example 2, the structure is significantly reduced in printability. Confirmed. Specifically, in the case of the one-component bio-ink composition (Gel4cell) prepared in Comparative Example 1, it was confirmed that even the skeleton of the structure was not properly formed because printing was not performed properly, and the one-part bio-ink composition prepared in Comparative Example 2 In the case of using (CB 1% and CB 3%), it was confirmed that the composition was spread sideways during printing, and thus the hole of the pattern in the structure was not properly formed.

Experimental Example 2: Evaluation of structural stability of the structure

When using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1, the structural stability of the structure was evaluated. Specifically, after immersing in 5 mL of media (Aprotinin solution 20ug/mL) in a 37°C incubator (DAIHAN Scientific ThermoStable IG-50), it was incubated for 7 days to observe the volume change (see Table 4).

0 days 1 day 3 days 7 days Comparative Example 1 100 99 64 44 Comparative Example 2 (CB 1%) 100 87 76 71 Example 1 (CFB 1%) 100 92 86 76 Example 1 (CFB 2%) 100 91 87 77 Example 1 (CFB 3%) 100 87 86 75 Example 1 (CFB 4%) 100 88 87 82 Example 1 (CFB 5%) 100 93 92 86

As a result, as shown in Table 4 and Figure 3, when using the two-component bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1, meta Regardless of the concentration of the acrylated low-molecular collagen, it is confirmed that the structural stability is excellent for 7 days, which can be seen as a result of natural crosslinking with thrombin contained in the second solution using fibrinogen as a structural material. In particular, in the case of 4 to 5% of CFB, it is confirmed that the structural safety of the structure is excellent as of the 7th day.

On the other hand, it was confirmed that the structural stability of the one-component bio-ink composition (Gel4cell) prepared in Comparative Example 1 and the one-component bio-ink composition (CB 1%) prepared in Comparative Example 2 significantly decreased after 3 days.

Experimental Example 3: Cell proliferation evaluation of construct

When using the two-component bio-ink composition prepared in Example 1 (CFB 1%, CFB 2%, CFB 3%, CFB 4%, and CFB 5%), the cell proliferation properties of the construct were evaluated. Specifically, cell proliferation was evaluated by immersing in media containing C2C12 (mouse myoblast cell line) 1×10 ⁶ cells/mL in a 37°C incubator (Thermo Scientific 3111) and incubating for 7 days.

As a result, as shown in Figure 4, when using the two-pack type bio-ink composition (CFB 1%, CFB 2%, CFB 3%, CFB 4% and CFB 5%) prepared in Example 1, methacrylated low molecular weight Regardless of the concentration of collagen, it is confirmed that cell proliferation is excellent for 7 days. This can be seen as a result of using methacrylated low-molecular collagen as a cell carrier. In particular, in the case of 4 to 5% of CFB, it is confirmed that the structure has excellent printability and excellent cell proliferation.

On the other hand, it was confirmed that the one-component bio-ink composition (Gel4cell) prepared in Comparative Example 1 and the one-component bio-ink composition (CB 3%) prepared in Comparative Example 2 significantly reduced cell proliferation.

Claims (13)

A first liquid comprising methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as a cell transport material, a viscosity enhancer and a lubricant;
Containing a second solution containing thrombin
Two-component bio ink composition.
The two-pack bio ink of claim 1, wherein the concentrations of methacrylated gelatin and fibrinogen as structural materials contained in the first solution are 25 to 35 mg/mL and 16 to 24 mg/mL, respectively. Composition.
The two-pack bio-ink composition according to claim 1, wherein the concentration of methacrylated low-molecular collagen as a cell carrier material contained in the first solution is 0.1 to 10 w/v%.
The two-component bio-ink composition of claim 1, wherein the viscosity enhancing agent is hyaluronic acid or dextran, and the lubricant is glycerol.
The bio-ink composition of claim 1, wherein the first liquid further contains a crosslinking agent or a photoinitiator.
The method of claim 1, wherein the first fluid is stem cells, osteoblasts, myoblasts, tenocytes, neuroblasts, fibroblasts, glioblasts, Germ cells, hepatocytes, renal cells, seroli cells, chondrocytes, epithelial cells, cardiovascular cells, keratinocytes, smooth muscle cells (smooth muscle cell), cardiomyocyte, glial cell, endothelial cell, hormone secreting cell, immune cell, pancreatic islet cell, and neuron A two-component bio-ink composition, characterized in that it further comprises at least one selected cell.
The two-pack bio-ink composition of claim 1, wherein the thrombin is 10 to 100 IU/mL, and the concentration of thrombin in the second solution is 0.1 to 1 mL/mL.
The two-component bio-ink composition of claim 1, wherein a volume ratio of the first liquid and the second liquid is 1: 0.5 to 1: 10.
(a) The first solution containing methacrylated gelatin and fibrinogen as structural materials, methacrylated low molecular weight collagen as cell carriers, viscosity enhancers and lubricants 3 Dimensional printing; And
(b) After photo-crosslinking the 3D printed first solution, a second solution containing thrombin is added to naturally crosslink, thereby double crosslinking.
The method of claim 9, wherein the 3D printing in step (a) is performed by using a 3D bioprinter equipped with a nozzle having a diameter of 100 to 500 μm, under a pressure of 10 to 100 kPa, with an extrusion amount of 100 to 500%. A method for producing a tissue-like structure, characterized in that it is performed.
The method of claim 9, wherein the photo-crosslinking in step (b) is performed by exposing light having a wavelength of 300 to 800 nm at a distance of 1 to 20 cm for 1 to 1000 seconds. Way.
The method of claim 9, wherein the natural crosslinking in step (b) is performed by allowing it to stand at a temperature of 4 to 30° C. for 10 minutes to 1 hour.
A tissue-like structure manufactured according to the method according to any one of claims 9 to 12.

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